Papel de las actividades superóxido dismutasa y catalasa en la ...
Papel de las actividades superóxido dismutasa y catalasa en la ...
Papel de las actividades superóxido dismutasa y catalasa en la ...
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FACULTAD DE CIENCIAS<br />
DEPARTAMENTO DE MICROBIOLOGÍA<br />
<strong>Papel</strong> <strong>de</strong> <strong><strong>la</strong>s</strong> activida<strong>de</strong>s <strong>superóxido</strong> <strong>dismutasa</strong><br />
y cata<strong><strong>la</strong>s</strong>a <strong>en</strong> <strong>la</strong> virul<strong>en</strong>cia <strong>de</strong> Photobacterium<br />
damse<strong>la</strong>e subsp. piscicida. Estrategias para <strong>la</strong><br />
estimu<strong>la</strong>ción <strong>de</strong>l estallido respiratorio <strong>en</strong><br />
fagocitos <strong>de</strong> l<strong>en</strong>guados cultivados<br />
PATRICIA DÍAZ ROSALES<br />
Tesis doctoral<br />
2006
FACULTAD DE CIENCIAS<br />
DEPARTAMENTO DE MICROBIOLOGÍA<br />
<strong>Papel</strong> <strong>de</strong> <strong><strong>la</strong>s</strong> activida<strong>de</strong>s <strong>superóxido</strong> <strong>dismutasa</strong><br />
y cata<strong><strong>la</strong>s</strong>a <strong>en</strong> <strong>la</strong> virul<strong>en</strong>cia <strong>de</strong> Photobacterium<br />
damse<strong>la</strong>e subsp. piscicida. Estrategias para <strong>la</strong><br />
estimu<strong>la</strong>ción <strong>de</strong>l estallido respiratorio <strong>en</strong><br />
fagocitos <strong>de</strong> l<strong>en</strong>guados cultivados<br />
Memoria pres<strong>en</strong>tada por Dña. Patricia Díaz Rosales<br />
para optar al grado <strong>de</strong> Doctora <strong>en</strong> Biología<br />
con M<strong>en</strong>ción <strong>de</strong> Doctorado Europeo
FACULTAD DE CIENCIAS<br />
DEPARTAMENTO DE MICROBIOLOGÍA<br />
D. ANTONIO DE VICENTE MORENO, Director <strong>de</strong>l Departam<strong>en</strong>to <strong>de</strong><br />
Microbiología <strong>de</strong> <strong>la</strong> Universidad <strong>de</strong> Má<strong>la</strong>ga.<br />
INFORMA QUE:<br />
Dña. Patricia Díaz Rosales ha realizado <strong>en</strong> los <strong>la</strong>boratorios d e<br />
este Departam<strong>en</strong>to el trabajo experim<strong>en</strong>tal conduc<strong>en</strong>te a <strong>la</strong> e<strong>la</strong>boración<br />
<strong>de</strong> <strong>la</strong> pres<strong>en</strong>te memoria <strong>de</strong> Tesis Doctoral<br />
Y para que así conste, expido el pres<strong>en</strong>te informe,<br />
Má<strong>la</strong>ga, 11 <strong>de</strong> Septiembre <strong>de</strong> 2006<br />
Fdo. Antonio <strong>de</strong> Vic<strong>en</strong>te Mor<strong>en</strong>o
Esta Tesis ha sido realizada <strong>en</strong> el Departam<strong>en</strong>to <strong>de</strong> Microbiología <strong>de</strong> <strong>la</strong><br />
Universidad <strong>de</strong> Má<strong>la</strong>ga, bajo <strong>la</strong> dirección <strong>de</strong>l Dr. Miguel Ángel Moriñigo Gutiérrez y <strong>la</strong><br />
Dra. Mª Carm<strong>en</strong> Balebona Accino. Durante <strong>la</strong> realización <strong>de</strong> este trabajo <strong>de</strong><br />
investigación se ha llevado a cabo el apr<strong>en</strong>dizaje <strong>de</strong> técnicas útiles para dicha tesis <strong>en</strong><br />
los sigui<strong>en</strong>tes <strong>la</strong>boratorios :<br />
- School of Biological Sci<strong>en</strong>ces, University of Aber<strong>de</strong><strong>en</strong> (Aber<strong>de</strong><strong>en</strong>, Escocia,<br />
Reino Unido), bajo <strong>la</strong> supervisión <strong>de</strong>l Dr. C.J. Secombes (<strong>de</strong> Octubre a Diciembre<br />
<strong>de</strong> 2003).<br />
- Departam<strong>en</strong>to <strong>de</strong> Biología Celu<strong>la</strong>r, Facultad <strong>de</strong> Biología, Universidad <strong>de</strong><br />
Murcia (Murcia, España), bajo <strong>la</strong> supervisión <strong>de</strong>l Dr. J. Meseguer (<strong>de</strong> Septiembre a<br />
Diciembre <strong>de</strong> 2004).<br />
- Laboratory of Microbiology, Agrotechnology and Food Sci<strong>en</strong>ces, University<br />
of Wag<strong>en</strong>ing<strong>en</strong> (Wag<strong>en</strong>ing<strong>en</strong>, Ho<strong>la</strong>nda), bajo <strong>la</strong> supervisión <strong>de</strong>l Dr. H. Smidt (<strong>de</strong><br />
Septiembre a Diciembre <strong>de</strong> 2005).<br />
El Dr. Miguel Ángel Moriñigo Gutiérrez, Profesor Titu<strong>la</strong>r <strong>de</strong> Microbiología <strong>de</strong> <strong>la</strong><br />
Universidad <strong>de</strong> Má<strong>la</strong>ga, y <strong>la</strong> Dra. Mª Carm<strong>en</strong> Balebona Accino, Profesora Titu<strong>la</strong>r <strong>de</strong><br />
Microbiología <strong>de</strong> <strong>la</strong> Universidad <strong>de</strong> Má<strong>la</strong>ga, dan su conformidad a <strong>la</strong> Memoria <strong>de</strong> <strong>la</strong><br />
Tesis titu<strong>la</strong>da: <strong>Papel</strong> <strong>de</strong> <strong><strong>la</strong>s</strong> activida<strong>de</strong>s <strong>superóxido</strong> <strong>dismutasa</strong> y cata<strong><strong>la</strong>s</strong>a <strong>en</strong> <strong>la</strong><br />
virul<strong>en</strong>cia <strong>de</strong> Photobacterium damse<strong>la</strong>e subsp. piscicida. Estrategias para <strong>la</strong><br />
estimu<strong>la</strong>ción <strong>de</strong>l estallido respiratorio <strong>en</strong> fagocitos <strong>de</strong> l<strong>en</strong>guados cultivados,<br />
pres<strong>en</strong>tada por <strong>la</strong> Doctoranda Dña. Patricia Díaz Rosales para optar al Título <strong>de</strong> Doctor<br />
<strong>en</strong> Biología con M<strong>en</strong>ción <strong>de</strong> Doctorado Europeo por <strong>la</strong> Universidad <strong>de</strong> Má<strong>la</strong>ga.<br />
En Má<strong>la</strong>ga, a 11 <strong>de</strong> Septiembre <strong>de</strong> 2006.<br />
Dr. Miguel Ángel Moriñigo Gutiérrez<br />
Dra. Mª Carm<strong>en</strong> Balebona Accino
Los <strong>en</strong>sayos que constituy<strong>en</strong> esta Tesis han sido subv<strong>en</strong>cionados principalem<strong>en</strong>te a<br />
través <strong>de</strong> difer<strong>en</strong>tes proyectos <strong>de</strong>l Ministerio <strong>de</strong> Ci<strong>en</strong>cia y Tecnología (España),<br />
concretam<strong>en</strong>te los proyectos con <strong><strong>la</strong>s</strong> refer<strong>en</strong>cias AGL2002-01488 y PETRI 95-0657<br />
subv<strong>en</strong>cionaron los trabajos realizados sobre <strong>la</strong> virul<strong>en</strong>cia <strong>de</strong> Photobacterium damse<strong>la</strong>e<br />
subsp. piscicida. Los experim<strong>en</strong>tos realizados con Porphyridium cru<strong>en</strong>tum fueron<br />
sufragados fundam<strong>en</strong>talm<strong>en</strong>te con cargo al proyecto AGL2002-01488, así como los<br />
proyectos AGL2005-02655 y RNM-295 (Junta <strong>de</strong> Andalucía) que subv<strong>en</strong>cionaron <strong>la</strong> parte<br />
re<strong>la</strong>cionada con el cultivo <strong>de</strong> <strong><strong>la</strong>s</strong> algas. Por último, con cargo al proyecto AGL2005-<br />
07454-CO2-O2 se realizaron los <strong>en</strong>sayos con bacterias pot<strong>en</strong>cialm<strong>en</strong>te probióticas.<br />
La Doctoranda ha sido becaria <strong>de</strong>l p<strong>la</strong>n <strong>de</strong> Formación <strong>de</strong> Profesorado Universitario<br />
(F.P.U.) <strong>de</strong>l Ministerio <strong>de</strong> Educación, Cultura y Deporte.<br />
Parte <strong>de</strong> los resultados expuestos <strong>en</strong> esta Tesis han sido publicados y comunicados<br />
<strong>en</strong> <strong><strong>la</strong>s</strong> sigui<strong>en</strong>tes revistas y congresos:<br />
Publicaciones:<br />
- Díaz-Rosales, P, Chabrillón, M, Arijo, S, Martínez-Manzanares, E, Moriñigo,<br />
MA & Balebona, MC (2006). Production of superoxi<strong>de</strong> dismutase and cata<strong><strong>la</strong>s</strong>e activities<br />
in Photobacterium damse<strong>la</strong>e subsp. piscicida and ability to survive in contact with sole<br />
phagocytes. Journal of Fish Diseases 29, 1-10.<br />
- Díaz-Rosales, P, Chabrillón, M, Moriñigo, MA & Balebona, MC (2003). Survival<br />
to exog<strong>en</strong>ous hydrog<strong>en</strong> peroxi<strong>de</strong> of Photobacterium damse<strong>la</strong>e subsp. piscicida un<strong>de</strong>r<br />
differ<strong>en</strong>t culture conditions. Journal of Fish Diseases 26, 305-308.<br />
Congresos internacionales:<br />
- Díaz-Rosales, P, Chabrillón, M, Smidt, H, Salinas, I, Arijo, S, Cuesta, A,<br />
Meseguer, J, Esteban, MA, Balebona, MC & Moriñigo, MA. Study of the intestinal<br />
microbiota of gilthead seabream (Sparus aurata, L.) and sole (Solea s<strong>en</strong>egal<strong>en</strong>sis, Kaup<br />
1858) by DGGE. Society of Applied Microbiology. Summer confer<strong>en</strong>ce “Living<br />
Together: polymicrobial communities”. Edinburgh, Scot<strong>la</strong>nd, U.K. 2006.
- Díaz-Rosales, P, Rico, RM, Arijo, S, Chabrillón, M, Balebona, MC, Sá<strong>en</strong>z <strong>de</strong><br />
Rodrigáñez, M, A<strong>la</strong>rcón, FJ & Moriñigo, MA. Effect of two probiotics on respiratory<br />
burst of phagocytes from sole (Solea s<strong>en</strong>egal<strong>en</strong>sis, Kaup 1858). Aquaculture Europe<br />
2006. “Linking Tradition and Technology. Highest Quality for the Consumer”.<br />
Flor<strong>en</strong>ce, Italy. 2006.<br />
- Balebona, MC, Díaz, P, Chabrillón, M, Zorril<strong>la</strong>, I, Arijo, S & Martínez, E.<br />
Determination of superoxi<strong>de</strong> dismutase and cata<strong><strong>la</strong>s</strong>e activity in Photobacterium<br />
damse<strong>la</strong>e subsp. piscicida un<strong>de</strong>r differ<strong>en</strong>t culture conditions. 10 th International<br />
Confer<strong>en</strong>ce of the European Association of Fish Pathologists. Dublin, Ire<strong>la</strong>nd, U.K.<br />
2001.<br />
Congresos nacionales:<br />
- Díaz-Rosales, P, León-Rubio, JM, Rico, RM, Decara, J, Balebona, MC, Abda<strong>la</strong>,<br />
R, Figueroa, FL & Moriñigo, MA. Efecto inmunoestimu<strong>la</strong>nte <strong>de</strong>l alga Porphyridium<br />
cru<strong>en</strong>tum sobre <strong>la</strong> respuesta inmune <strong>de</strong>l l<strong>en</strong>guado (Solea s<strong>en</strong>egal<strong>en</strong>sis) tras su<br />
administración por vía oral fr<strong>en</strong>te a <strong>la</strong> infección por Photobacterium damse<strong>la</strong>e subsp.<br />
piscicida. X Congreso Nacional <strong>de</strong> Acuicultura. Gandía. 2005.<br />
- Díaz-Rosales, P, Martínez-Manzanares, E, Moriñigo, MA & Balebona, MC.<br />
Efecto inmunoestimu<strong>la</strong>nte <strong>de</strong>l alga Porphyridium cru<strong>en</strong>tum sobre el estallido<br />
respiratorio <strong>en</strong> fagocitos <strong>de</strong> l<strong>en</strong>guado (Solea s<strong>en</strong>egal<strong>en</strong>sis). V Congreso <strong>de</strong><br />
Microbiología <strong>de</strong>l Medio Acuático <strong>de</strong> <strong>la</strong> Sociedad Españo<strong>la</strong> <strong>de</strong> Microbiología.<br />
Tarragona. 2004.<br />
- Díaz-Rosales, P, Arijo, S, Moriñigo, MA & Balebona, MC. Resist<strong>en</strong>cia <strong>de</strong><br />
Photobacterium damse<strong>la</strong>e subsp. piscicida al estallido respiratorio <strong>de</strong> fagocitos <strong>de</strong><br />
l<strong>en</strong>guado (Solea s<strong>en</strong>egal<strong>en</strong>sis). V Congreso <strong>de</strong> Microbiología <strong>de</strong>l Medio Acuático <strong>de</strong> <strong>la</strong><br />
Sociedad Españo<strong>la</strong> <strong>de</strong> Microbiología. Tarragona. 2004.
- Díaz-Rosales, P, Arijo, S, Chabrillón, M, Castán, J, Martínez-Manzanares, E &<br />
Balebona, MC. <strong>Papel</strong> <strong>de</strong> <strong>la</strong> <strong>superóxido</strong> <strong>dismutasa</strong> y <strong>de</strong> <strong>la</strong> cata<strong><strong>la</strong>s</strong>a <strong>en</strong> <strong>la</strong> virul<strong>en</strong>cia <strong>de</strong><br />
Photobacterium damse<strong>la</strong>e subsp. piscicida. IV Congreso <strong>de</strong> Microbiología <strong>de</strong>l Medio<br />
Acuático <strong>de</strong> <strong>la</strong> Sociedad Españo<strong>la</strong> <strong>de</strong> Microbiología. Sevil<strong>la</strong>. 2002.
A Raúl Díaz Rosales
Sabe esperar, aguarda que <strong>la</strong> marea fluya<br />
–así <strong>en</strong> <strong>la</strong> costa un barco– sin que el partir te inquiete,<br />
todo el que aguarda sabe que <strong>la</strong> victoria es suya;<br />
porque <strong>la</strong> vida es <strong>la</strong>rga y el arte es un juguete.<br />
Y si <strong>la</strong> vida es corta<br />
y no llega el mar a tu galera,<br />
aguarda sin partir y siempre espera,<br />
que el arte es <strong>la</strong>rgo y, a<strong>de</strong>más, no importa.<br />
ANTONIO MACHADO
Í ND I C E<br />
I<br />
ND E X
ÍNDICE / INDEX<br />
<strong>Papel</strong> <strong>de</strong> <strong><strong>la</strong>s</strong> activida<strong>de</strong>s <strong>superóxido</strong> <strong>dismutasa</strong> y cata<strong><strong>la</strong>s</strong>a <strong>en</strong> <strong>la</strong> virul<strong>en</strong>cia <strong>de</strong><br />
Photobacterium damse<strong>la</strong>e subsp. piscicida. Estrategias para <strong>la</strong> estimu<strong>la</strong>ción <strong>de</strong>l<br />
estallido respiratorio <strong>en</strong> fagocitos <strong>de</strong> l<strong>en</strong>guados cultivados<br />
PÁGINA / PAGE<br />
RESUMEN 1<br />
INTRODUCCIÓN 5<br />
1. La acuicultura. El cultivo <strong>de</strong>l l<strong>en</strong>guado<br />
(Solea s<strong>en</strong>egal<strong>en</strong>sis, Kaup 1858) 7<br />
2. Photobacterium damse<strong>la</strong>e subsp. piscicida 8<br />
2.1. Características morfológicas,<br />
bioquímicas y serológicas 9<br />
2.2. Sintomatología <strong>de</strong> <strong>la</strong> pseudotuberculosis 11<br />
2.3. Modo <strong>de</strong> transmisión 12<br />
2.4. Mecanismos <strong>de</strong> virul<strong>en</strong>cia 13<br />
3. Las activida<strong>de</strong>s <strong>superóxido</strong> <strong>dismutasa</strong><br />
y cata<strong><strong>la</strong>s</strong>a como factores <strong>de</strong> virul<strong>en</strong>cia 16<br />
3.1. Estallido respiratorio 17<br />
3.2. Actividad <strong>superóxido</strong> <strong>dismutasa</strong> 19<br />
3.3. Actividad cata<strong><strong>la</strong>s</strong>a 19<br />
3.4. Las activida<strong>de</strong>s <strong>superóxido</strong> <strong>dismutasa</strong><br />
y cata<strong><strong>la</strong>s</strong>a <strong>en</strong> Photobacterium damse<strong>la</strong>e<br />
subsp. piscicida 21
ÍNDICE / INDEX<br />
4. Estimu<strong>la</strong>ción <strong>de</strong>l estallido respiratorio por difer<strong>en</strong>tes<br />
microorganismos fr<strong>en</strong>te a <strong>la</strong> infección por<br />
Photobacterium damse<strong>la</strong>e subsp. piscicida 22<br />
4.1. Prev<strong>en</strong>ción y tratami<strong>en</strong>to <strong>de</strong> <strong>la</strong> pseudotuberculosis 22<br />
4.2. Inmunomodu<strong>la</strong>ción. Inmunoestimu<strong>la</strong>ción 24<br />
4.3. Uso <strong>de</strong> <strong><strong>la</strong>s</strong> algas como inmunoestimu<strong>la</strong>ntes 26<br />
4.3.1. Porphyridium cru<strong>en</strong>tum 28<br />
4.4. Efecto inmunoestimu<strong>la</strong>nte <strong>de</strong> bacterias<br />
pot<strong>en</strong>cialm<strong>en</strong>te probióticas 29<br />
OBJETIVOS 35<br />
MATERIAL Y MÉTODOS 39<br />
RESULTADOS Y DISCUSIÓN 43<br />
CONCLUSIONES 59<br />
Role of superoxi<strong>de</strong> dismutase and cata<strong><strong>la</strong>s</strong>e activities in Photobacterium<br />
damse<strong>la</strong>e subsp. piscicida virul<strong>en</strong>ce. Strategies for respiratory burst activity<br />
stimu<strong>la</strong>tion in sole phagocytes<br />
ABSTRACT 65<br />
INTRODUCTION 69<br />
1. Aquaculture. The culture of sole<br />
(Solea s<strong>en</strong>egal<strong>en</strong>sis, Kaup 1858) 71
ÍNDICE / INDEX<br />
2. Photobacterium damse<strong>la</strong>e subsp. piscicida 72<br />
2.1. Transmission mo<strong>de</strong> 73<br />
2.2. Virul<strong>en</strong>ce mechanisms 74<br />
3. Superoxi<strong>de</strong> dismutase and cata<strong><strong>la</strong>s</strong>e activities<br />
as virul<strong>en</strong>ce factors 74<br />
3.1. Respiratory burst 75<br />
3.2. Superoxi<strong>de</strong> dismutase activity 76<br />
3.3. Cata<strong><strong>la</strong>s</strong>e activity 76<br />
3.4. Superoxi<strong>de</strong> dismutase and cata<strong><strong>la</strong>s</strong>e<br />
activities in Photobacterium damse<strong>la</strong>e<br />
subsp. piscicida 78<br />
4. Stimu<strong>la</strong>tion of respiratory burst activity by<br />
differ<strong>en</strong>t microorganisms after Photobacterium<br />
damse<strong>la</strong>e subsp. piscicida infection 79<br />
4.1. Prev<strong>en</strong>tion and treatm<strong>en</strong>t of pseudotuberculosis 79<br />
4.2. Immunomodu<strong>la</strong>tion. Immunostimu<strong>la</strong>tion 80<br />
4.3. Use of algae as immunostimu<strong>la</strong>nts 81<br />
4.3.1. Porphyridium cru<strong>en</strong>tum 82<br />
4.4. Immunostimu<strong>la</strong>nt effect of pot<strong>en</strong>tial probiotic bacteria 84<br />
AIMS 87<br />
MATERIALS AND METHODS 91<br />
RESULTS AND DISCUSSION 95<br />
CONCLUSIONS 107<br />
REFERENCIAS / REFERENCES 111<br />
SECCIÓN DE ARTÍCULOS / ARTICLE SECTION 133
R<br />
E S U M E N
RESUMEN<br />
Photobacterium damse<strong>la</strong>e subsp. piscicida es una bacteria gram negativa capaz <strong>de</strong><br />
sobrevivir como patóg<strong>en</strong>o intracelu<strong>la</strong>r <strong>en</strong> el interior <strong>de</strong> fagocitos <strong>de</strong> l<strong>en</strong>guado, gracias a<br />
<strong>la</strong> acción protectora <strong>de</strong> <strong><strong>la</strong>s</strong> activida<strong>de</strong>s <strong>superóxido</strong> <strong>dismutasa</strong> y cata<strong><strong>la</strong>s</strong>a. Estas <strong>en</strong>zimas<br />
confier<strong>en</strong> resist<strong>en</strong>cia al patóg<strong>en</strong>o fr<strong>en</strong>te a los radicales reactivos <strong>de</strong> oxíg<strong>en</strong>o producidos<br />
<strong>en</strong> el interior <strong>de</strong> los fagocitos durante el <strong>de</strong>nominado estallido respiratorio. Por tanto,<br />
ambas activida<strong>de</strong>s <strong>en</strong>zimáticas pue<strong>de</strong>n ser consi<strong>de</strong>radas importantes factores <strong>de</strong><br />
virul<strong>en</strong>cia <strong>de</strong> este patóg<strong>en</strong>o, facilitando su invasión y el establecimi<strong>en</strong>to <strong>de</strong> <strong>la</strong><br />
<strong>en</strong>fermedad, <strong>la</strong> pseudotuberculosis. La estrategia <strong>de</strong>sarrol<strong>la</strong>da para <strong>la</strong> prev<strong>en</strong>ción <strong>de</strong><br />
dicha <strong>en</strong>fermedad se ha <strong>en</strong>focado hacia <strong>la</strong> búsqueda <strong>de</strong> microorganismos con capacidad<br />
estimu<strong>la</strong>nte <strong>de</strong>l estallido respiratorio <strong>de</strong> fagocitos <strong>de</strong> l<strong>en</strong>guado. Los microorganismos<br />
<strong>en</strong>sayados fueron <strong>la</strong> microalga Porphyridium cru<strong>en</strong>tum y dos bacterias pot<strong>en</strong>cialm<strong>en</strong>te<br />
probióticas. Los resultados obt<strong>en</strong>idos son prometedores ya que tanto <strong>la</strong> microalga como<br />
una <strong>de</strong> <strong><strong>la</strong>s</strong> bacterias <strong>en</strong>sayadas –Pdp11– son capaces <strong>de</strong> estimu<strong>la</strong>r el estallido respiratorio<br />
y, <strong>de</strong> esta manera, contribuir a <strong>la</strong> resist<strong>en</strong>cia a <strong>la</strong> <strong>en</strong>fermedad. Se abre, por tanto, un<br />
nuevo campo <strong>en</strong> <strong>la</strong> lucha contra <strong>la</strong> pseudotuberculosis: <strong>la</strong> aplicación <strong>de</strong> sustancias<br />
proce<strong>de</strong>ntes <strong>de</strong> algas, así como <strong>de</strong> compon<strong>en</strong>tes bacterianos, que pudieran ser<br />
consi<strong>de</strong>rados probióticos.<br />
3
I N T R O D U C C I Ó N
INTRODUCCIÓN<br />
1. LA ACUICULTURA. EL CULTIVO DEL LENGUADO<br />
(Solea s<strong>en</strong>egal<strong>en</strong>sis, KAUP 1858)<br />
El increm<strong>en</strong>to <strong>de</strong>mográfico mundial junto con el estancami<strong>en</strong>to <strong>de</strong> <strong>la</strong> actividad<br />
extractiva y cambios prefer<strong>en</strong>ciales <strong>en</strong> <strong>la</strong> dieta, <strong>en</strong> <strong>la</strong> que se valora cada vez más el<br />
pescado como un alim<strong>en</strong>to muy saludable, son factores que han conducido a que <strong>la</strong><br />
producción global <strong>de</strong> pesca no satisfaga <strong><strong>la</strong>s</strong> <strong>de</strong>mandas creci<strong>en</strong>tes <strong>de</strong>l mercado. Según <strong>la</strong><br />
Organización <strong>de</strong> <strong><strong>la</strong>s</strong> Naciones Unidas para <strong>la</strong> Agricultura y <strong>la</strong> Alim<strong>en</strong>tación (FAO),<br />
nuestro p<strong>la</strong>neta está habitado por alre<strong>de</strong>dor <strong>de</strong> 6500 millones <strong>de</strong> personas, y se prevée<br />
que para el año 2050, <strong>la</strong> pob<strong>la</strong>ción mundial alcance <strong>la</strong> cifra <strong>de</strong> 9000 a 10000 millones.<br />
(FAO, 2005 ). Esta situación<br />
preocupa a <strong>la</strong> hora <strong>de</strong> p<strong>la</strong>ntear cómo alim<strong>en</strong>tar a <strong>la</strong> pob<strong>la</strong>ción mundial <strong>en</strong> un futuro<br />
próximo. En respuesta a esta situación surge <strong>la</strong> acuicultura como una bu<strong>en</strong>a alternativa<br />
para el abastecimi<strong>en</strong>to <strong>de</strong> <strong>la</strong> pob<strong>la</strong>ción.<br />
La acuicultura, según <strong>la</strong> <strong>de</strong>finición <strong>de</strong> <strong>la</strong> FAO, es el cultivo <strong>de</strong> organismos<br />
acuáticos, incluy<strong>en</strong>do peces, moluscos, crustáceos y p<strong>la</strong>ntas acuáticas. Actualm<strong>en</strong>te es<br />
uno <strong>de</strong> los sectores productores <strong>de</strong> alim<strong>en</strong>to con mayor pot<strong>en</strong>cial <strong>de</strong> crecimi<strong>en</strong>to y<br />
juega, por tanto, un papel es<strong>en</strong>cial <strong>en</strong> el futuro <strong>de</strong> <strong>la</strong> alim<strong>en</strong>tación humana.<br />
En los últimos años <strong>la</strong> actividad productiva <strong>de</strong> <strong>la</strong> industria acuíco<strong>la</strong>, tanto <strong>de</strong> agua<br />
marina como <strong>de</strong> agua dulce, ha experim<strong>en</strong>tado un crecimi<strong>en</strong>to expon<strong>en</strong>cial muy<br />
importante, especialm<strong>en</strong>te <strong>en</strong> lo que se refiere a <strong>la</strong> acuicultura marina int<strong>en</strong>siva <strong>de</strong><br />
peces. Hasta ahora <strong>la</strong> acuicultura marina <strong>en</strong> países mediterráneos se ha c<strong>en</strong>trado<br />
fundam<strong>en</strong>talm<strong>en</strong>te <strong>en</strong> <strong>la</strong> producción <strong>de</strong> dos especies –dorada (Sparus aurata, L.) y<br />
lubina (Dic<strong>en</strong>trarchus <strong>la</strong>brax, L.)– lo que ha provocado una saturación <strong>en</strong> el mercado.<br />
En los últimos años, para increm<strong>en</strong>tar <strong><strong>la</strong>s</strong> oportunida<strong>de</strong>s <strong>de</strong> mercado, se han realizado<br />
investigaciones <strong>en</strong>caminadas a <strong>la</strong> búsqueda <strong>de</strong> nuevas especies pot<strong>en</strong>cialm<strong>en</strong>te aptas<br />
para el cultivo. El l<strong>en</strong>guado s<strong>en</strong>egalés (Solea s<strong>en</strong>egal<strong>en</strong>sis, Kaup 1858) es actualm<strong>en</strong>te<br />
una <strong>de</strong> <strong><strong>la</strong>s</strong> especies cuyo cultivo se ha increm<strong>en</strong>tado <strong>de</strong> forma significativa (JACUMAR,<br />
2005) <strong>en</strong> el área atlántica y mediterránea (Dinis, 1999). El l<strong>en</strong>guado s<strong>en</strong>egalés es una<br />
especie acuíco<strong>la</strong> que <strong>de</strong>s<strong>de</strong> <strong>la</strong> década <strong>de</strong> los set<strong>en</strong>ta ha sido objeto <strong>de</strong> interés <strong>en</strong><br />
7
INTRODUCCIÓN<br />
acuicultura, <strong>de</strong>bido a su alto precio <strong>en</strong> el mercado, <strong>la</strong> posibilidad <strong>de</strong> reproducción <strong>en</strong><br />
cautividad, los resultados obt<strong>en</strong>idos <strong>en</strong> el cultivo <strong>de</strong> <strong><strong>la</strong>s</strong> <strong>la</strong>rvas y a que cu<strong>en</strong>ta con un<br />
mercado pot<strong>en</strong>cial (Dinis y Reis, 1995; Magalhaes y Dinis, 1996).<br />
Actualm<strong>en</strong>te no es posible afirmar que esta especie esté consolidada a nivel <strong>de</strong> una<br />
producción industrial, ya que <strong>la</strong> inci<strong>de</strong>ncia <strong>de</strong> patologías limita <strong>la</strong> producción <strong>de</strong><br />
<strong>en</strong>gor<strong>de</strong> <strong>de</strong>l l<strong>en</strong>guado. Se han realizado estudios sobre los microorganismos patóg<strong>en</strong>os<br />
que afectan a esta especie (Rodríguez et al., 1997; Zorril<strong>la</strong> et al., 1999; Magariños et al.,<br />
2003; Arijo et al., 2005), y aunque <strong>en</strong>tre otros patóg<strong>en</strong>os se han ais<strong>la</strong>do Vibrio harveyi y<br />
T<strong>en</strong>acibaculum maritimum (Zorril<strong>la</strong> et al., 1999; Cepeda y Santos, 2003),<br />
Photobacterium damse<strong>la</strong>e subsp. piscicida, causante <strong>de</strong> <strong>la</strong> pasteurelosis, o<br />
pseudotuberculosis, es el principal patóg<strong>en</strong>o responsable <strong>de</strong> importantes pérdidas <strong>en</strong> el<br />
cultivo <strong>de</strong>l l<strong>en</strong>guado s<strong>en</strong>egalés, llegando a ser el principal factor limitante <strong>en</strong> <strong>la</strong><br />
producción <strong>de</strong> esta especie (Zorril<strong>la</strong> et al., 1999; Magariños et al., 2003; Arijo et al.,<br />
2005).<br />
2. Photobacterium damse<strong>la</strong>e subsp. piscicida<br />
La bacteria marina P. damse<strong>la</strong>e subsp. piscicida es el ag<strong>en</strong>te etiológico <strong>de</strong> <strong>la</strong><br />
septicemia bacteriana <strong>de</strong>nominada pseudotuberculosis (Kubota et al., 1970) <strong>de</strong>bido a que<br />
<strong>en</strong> los casos crónicos, los peces <strong>en</strong>fermos muestran gránulos b<strong>la</strong>ncos promin<strong>en</strong>tes <strong>en</strong> los<br />
órganos internos, consist<strong>en</strong>tes <strong>en</strong> una acumu<strong>la</strong>ción <strong>de</strong> célu<strong><strong>la</strong>s</strong> bacterianas. Esta<br />
<strong>en</strong>fermedad fue <strong>de</strong>scrita por primera vez <strong>en</strong> pob<strong>la</strong>ciones salvajes <strong>de</strong> perca (Morone<br />
americanus) y lubina estriada (Morone saxatilis) <strong>en</strong> Estados Unidos (Snieszko et al.,<br />
1964). Sin embargo, actualm<strong>en</strong>te los hospedadores naturales <strong>de</strong>l patóg<strong>en</strong>o incluy<strong>en</strong> una<br />
amplia variedad <strong>de</strong> especies piscíco<strong><strong>la</strong>s</strong> marinas, tanto <strong>de</strong> aguas cálidas como frías,<br />
provocando importantes pérdidas económicas <strong>en</strong> pob<strong>la</strong>ciones salvajes y cultivadas <strong>de</strong><br />
Japón, don<strong>de</strong> afecta principalm<strong>en</strong>te a <strong>la</strong> serio<strong>la</strong> (Serio<strong>la</strong> quinqueradiata) (Kusuda y<br />
Sa<strong>la</strong>ti, 1993), <strong>de</strong> Estados Unidos y <strong>de</strong> Europa, don<strong>de</strong> causa estragos <strong>en</strong> cultivos <strong>de</strong><br />
dorada (Sparus aurata) (Ceshia et al., 1991; Toranzo et al., 1991), lubina (Dic<strong>en</strong>trarchus<br />
<strong>la</strong>brax) (Baudin-Laur<strong>en</strong>cin et al., 1991; Balebona et al., 1992), lubina estriada (Morone<br />
8
INTRODUCCIÓN<br />
saxatilis) (Hawke et al., 1987) y l<strong>en</strong>guado (Solea s<strong>en</strong>egal<strong>en</strong>sis), como se ha <strong>de</strong>scrito<br />
reci<strong>en</strong>tem<strong>en</strong>te (Zorril<strong>la</strong> et al., 1999; Magariños et al., 2003; Arijo et al., 2005).<br />
2.1. CARACTERÍSTICAS MORFOLÓGICAS, BIOQUÍMICAS Y SEROLÓGICAS<br />
P. damse<strong>la</strong>e subsp. piscicida es una bacteria halófi<strong>la</strong>, Gram negativa, <strong>de</strong> forma<br />
baci<strong>la</strong>r (0,8-1,3 x 1,4-4 µm <strong>de</strong> tamaño). Se caracteriza por su tinción bipo<strong>la</strong>r y su<br />
pleomorfismo, <strong>de</strong>p<strong>en</strong>di<strong>en</strong>te <strong>de</strong> <strong><strong>la</strong>s</strong> condiciones <strong>de</strong> cultivo. Las características f<strong>en</strong>otípicas<br />
están resumidas <strong>en</strong> <strong>la</strong> Tab<strong>la</strong> 1.<br />
Siempre se ha consi<strong>de</strong>rado que P. damse<strong>la</strong>e subsp. piscicida constituía un taxón<br />
morfológico, bioquímico, fisiológico, f<strong>en</strong>otípico y serológicam<strong>en</strong>te homogéneo<br />
(Magariños et al., 1992). Sin embargo, <strong>la</strong> aplicación <strong>de</strong> nuevas técnicas molecu<strong>la</strong>res para<br />
el análisis g<strong>en</strong>ético, como ribotipado y RAPD (Random Amplification of Polymorphic<br />
DNA) ha mostrado <strong>la</strong> exist<strong>en</strong>cia <strong>de</strong> dos linajes clonales o g<strong>en</strong>ogrupos <strong>en</strong> P. damse<strong>la</strong>e<br />
subsp. piscicida, uno <strong>en</strong> cepas proce<strong>de</strong>ntes <strong>de</strong> Europa y otro <strong>en</strong> ais<strong>la</strong>dos <strong>de</strong> Japón<br />
(Magariños et al, 1997; Thyss<strong>en</strong> et al., 1999; Magariños et al., 2000; Kvitt et al., 2002;<br />
Romal<strong>de</strong>, 2002; Juíz-Río et al., 2005). En cualquier caso, se pue<strong>de</strong> observar una<br />
homog<strong>en</strong>eidad <strong>de</strong>ntro <strong>de</strong>l linaje clonal <strong>en</strong>tre cepas ais<strong>la</strong>das <strong>de</strong> especies piscíco<strong><strong>la</strong>s</strong><br />
difer<strong>en</strong>tes; confirmándose este hecho <strong>en</strong> los estudios g<strong>en</strong>éticos realizados a cepas <strong>de</strong><br />
este microorganismo ais<strong>la</strong>das a partir <strong>de</strong> l<strong>en</strong>guados cultivados <strong>en</strong> nuestro país,<br />
revelándose que estas cepas pert<strong>en</strong>ec<strong>en</strong> al g<strong>en</strong>ogrupo europeo (Magariños et al., 2003).<br />
Este hecho podría indicar una posible transmisión horizontal <strong>de</strong>l patóg<strong>en</strong>o <strong>en</strong>tre peces<br />
cultivados <strong>en</strong> <strong><strong>la</strong>s</strong> mismas áreas (Magariños et al., 2003).<br />
9
INTRODUCCIÓN<br />
Tab<strong>la</strong> 1 Características g<strong>en</strong>erales <strong>de</strong> Photobacterium damse<strong>la</strong>e subsp. piscicida<br />
Tinción <strong>de</strong> Gram - Tinción bipo<strong>la</strong>r +<br />
Cata<strong><strong>la</strong>s</strong>a + Oxidasa +<br />
Movilidad - Rojo Metilo +<br />
Producción H 2 S - O/F +/+<br />
Producción gas (glucosa) - Crecimi<strong>en</strong>to TCBS -<br />
Crecimi<strong>en</strong>to a:<br />
Crecimi<strong>en</strong>to <strong>en</strong> NaCl:<br />
4 ºC - 0% -<br />
10 ºC + 3% +<br />
20 ºC + 6% -<br />
30 ºC + 8% -<br />
37 ºC - Producción <strong>de</strong> ácidos:<br />
β ga<strong>la</strong>ctosidasa (ONPG) - arabinosa -<br />
Ge<strong>la</strong>tinasa - maltosa -<br />
Ureasa - sacarosa -<br />
Caseinasa - rhamnosa -<br />
Ami<strong><strong>la</strong>s</strong>a - amigdalina -<br />
Fosfolipasa + inositol -<br />
Lipasa (Twe<strong>en</strong> 80) + manosa +<br />
Arginina dihidro<strong><strong>la</strong>s</strong>a + manitol -<br />
Lisina <strong>de</strong>scarboxi<strong><strong>la</strong>s</strong>a - sorbitol -<br />
Ornitina <strong>de</strong>scarboxi<strong><strong>la</strong>s</strong>a - glicerol -<br />
Hidrólisis <strong>de</strong> esculina - <strong>la</strong>ctosa -<br />
Voges-Proskauer + melobiosa -<br />
Indol - glucosa +<br />
Nitrato - ga<strong>la</strong>ctosa +<br />
Citrato - fructosa +<br />
Resist<strong>en</strong>cia a: S<strong>en</strong>sibilidad a:<br />
Estreptomicina Ampicilina Novobiocina<br />
Eritromicina Cloranf<strong>en</strong>icol Tetraciclina<br />
Kanamicina Oxitetraciclina Ácido oxolínico<br />
Nitrofurantoína Ag<strong>en</strong>te vibriostático O/129<br />
Trimetoprim-sulfametoxazol<br />
10
INTRODUCCIÓN<br />
2.2. SINTOMATOLOGÍA DE LA PSEUDOTUBERCULOSIS<br />
Los signos patológicos externos son, g<strong>en</strong>eralm<strong>en</strong>te, poco l<strong>la</strong>mativos y, por lo<br />
g<strong>en</strong>eral, los peces afectados no suel<strong>en</strong> mostrar lesiones externas. En algunos casos <strong>de</strong><br />
doradas <strong>en</strong>fermas se pue<strong>de</strong> apreciar una pigm<strong>en</strong>tación anormal <strong>en</strong> <strong>la</strong> piel así como leves<br />
zonas hemorrágicas <strong>en</strong> cabeza y branquias (Toranzo et al., 1991). En lubina<br />
(Dic<strong>en</strong>trarchus <strong>la</strong>brax) se ha llegado a observar hinchazón <strong>en</strong> <strong>la</strong> cavidad abdominal<br />
(Balebona et al., 1992), a<strong>de</strong>más <strong>de</strong> lesiones ulcerativas <strong>en</strong> <strong>la</strong> piel y ext<strong>en</strong>sas hemorragias,<br />
especialm<strong>en</strong>te <strong>en</strong> <strong>la</strong> boca, ojos y muscu<strong>la</strong>tura (Fouz et al., 2000).<br />
Internam<strong>en</strong>te los peces <strong>en</strong>fermos muestran septicemia hemorrágica y necrosis <strong>en</strong> <strong>la</strong><br />
mayoría <strong>de</strong> los órganos, apareci<strong>en</strong>do los tubérculos típicos <strong>de</strong> <strong>la</strong> <strong>en</strong>fermedad. Estos no<br />
son sino acumu<strong>la</strong>ciones <strong>de</strong> bacterias, fagocitos necróticos y granulomas.<br />
Histopatológicam<strong>en</strong>te esas l<strong>la</strong>mativas lesiones necróticas con gran<strong>de</strong>s masas<br />
bacterianas, <strong>de</strong> forma ext<strong>en</strong>siva, aguda y multifocal observadas <strong>en</strong> los órganos internos,<br />
sugier<strong>en</strong> que <strong>la</strong> <strong>en</strong>fermedad se <strong>de</strong>sarrol<strong>la</strong> como un proceso septicémico agudo. Dichas<br />
lesiones granulomatosas aparec<strong>en</strong> como una reacción <strong>de</strong> <strong><strong>la</strong>s</strong> célu<strong><strong>la</strong>s</strong> epiteliales cuando <strong>la</strong><br />
viabilidad <strong>de</strong> <strong>la</strong> bacteria <strong>de</strong>crece por medicación. A<strong>de</strong>más <strong>de</strong> <strong>la</strong> necrosis y <strong>la</strong> exist<strong>en</strong>cia<br />
<strong>de</strong> granulomas <strong>en</strong> bazo, riñón e hígado, don<strong>de</strong> se pue<strong>de</strong> <strong>de</strong>tectar bacterias <strong>en</strong> los<br />
sinusoi<strong>de</strong>s y vasos hepáticos, es posible el <strong>de</strong>sarrollo <strong>de</strong> una espl<strong>en</strong>omegalia (Toranzo et<br />
al., 1991) <strong>de</strong>bida a <strong>la</strong> infiltración <strong>de</strong> célu<strong><strong>la</strong>s</strong> sanguíneas junto con grupos <strong>de</strong> bacterias que<br />
tapan los capi<strong>la</strong>res y espacios intersticiales, así como <strong>la</strong> aparición <strong>de</strong> zonas<br />
b<strong>la</strong>nquecinas, o pali<strong>de</strong>z, <strong>en</strong> bazo y riñón (Kubota et al., 1970; Wolke, 1975; Tung et al.,<br />
1985; Hawke et al., 1987; Toranzo et al., 1991; Balebona et al., 1992; Noya et al., 1995a).<br />
En l<strong>en</strong>guados (Solea s<strong>en</strong>egal<strong>en</strong>sis) afectados por <strong>la</strong> pseudotuberculosis <strong>la</strong><br />
pigm<strong>en</strong>tación oscura <strong>en</strong> <strong>la</strong> piel y <strong>la</strong> hinchazón <strong>en</strong> <strong>la</strong> cavidad abdominal pue<strong>de</strong>n ser dos<br />
síntomas externos que po<strong>de</strong>mos <strong>de</strong>tectar. Algunas muestras pue<strong>de</strong>n también pres<strong>en</strong>tar<br />
exoftalmia hemorrágica, pequeñas úlceras <strong>en</strong> <strong>la</strong> piel y pali<strong>de</strong>z branquial (Zorril<strong>la</strong> et al.,<br />
1999). En cuanto a los órganos internos se pue<strong>de</strong> apreciar espl<strong>en</strong>omegalia, pali<strong>de</strong>z <strong>en</strong><br />
hígado y riñón, así como tubérculos <strong>de</strong> 1-2 mm <strong>de</strong> diámetro <strong>en</strong> el bazo.<br />
11
INTRODUCCIÓN<br />
2.3. MODO DE TRANSMISIÓN<br />
P. damse<strong>la</strong>e subsp. piscicida es una bacteria altam<strong>en</strong>te patóg<strong>en</strong>a que no parece<br />
t<strong>en</strong>er especificidad por el hospedador. Por tanto, <strong>la</strong> pseudotuberculosis pue<strong>de</strong> ser un<br />
riesgo para especies piscíco<strong><strong>la</strong>s</strong> marinas <strong>en</strong> <strong><strong>la</strong>s</strong> que aún no se ha <strong>de</strong>scrito. Algunos<br />
autores seña<strong>la</strong>n <strong>la</strong> exist<strong>en</strong>cia <strong>de</strong> difer<strong>en</strong>cias <strong>en</strong> <strong>la</strong> susceptibilidad a <strong>la</strong> pseudotuberculosis,<br />
<strong>en</strong> doradas y lubinas, basadas <strong>en</strong> <strong>la</strong> edad y el tamaño <strong>de</strong>l pez (Noya et al., 1995b). Esto<br />
podría <strong>de</strong>berse a <strong>la</strong> funcionalidad <strong>de</strong> macrófagos y neutrófilos que <strong>en</strong> doradas mayores<br />
<strong>de</strong> 20-30 g pue<strong>de</strong>n fagocitar efici<strong>en</strong>tem<strong>en</strong>te y matar a <strong><strong>la</strong>s</strong> bacterias (Noya et al., 1995b;<br />
Skarmeta et al., 1995), <strong>en</strong> tanto que <strong>en</strong> doradas <strong>de</strong> m<strong>en</strong>os <strong>de</strong> 1 g <strong>de</strong>b<strong>en</strong> existir<br />
<strong>de</strong>fici<strong>en</strong>cias <strong>en</strong> compon<strong>en</strong>tes <strong>de</strong>l suero implicados <strong>en</strong> <strong>la</strong> fagocitosis y posterior muerte<br />
<strong>de</strong> Photobacterium por los fagocitos, haciéndo<strong><strong>la</strong>s</strong> más susceptibles a <strong>la</strong> infección.<br />
El modo <strong>de</strong> transmisión y <strong>la</strong> ruta <strong>de</strong> infección implicadas <strong>en</strong> esta <strong>en</strong>fermedad aún<br />
se <strong>de</strong>sconoc<strong>en</strong> con <strong>de</strong>talle (Magariños et al., 1995). Los datos exist<strong>en</strong>tes apuntan a que <strong>la</strong><br />
pseudotuberculosis es una <strong>en</strong>fermedad <strong>de</strong> mayor preval<strong>en</strong>cia <strong>en</strong> los meses <strong>de</strong> verano<br />
(Frerichs y Roberts, 1989) con altas temperaturas <strong>de</strong>l agua (mayores <strong>de</strong> 23ºC) y alta<br />
salinidad (20-30%) (Hawke et al., 1987). En cuanto a <strong>la</strong> superviv<strong>en</strong>cia <strong>en</strong> el agua, se ha<br />
<strong>de</strong>mostrado que este patóg<strong>en</strong>o sobrevive <strong>en</strong> ambi<strong>en</strong>tes acuáticos marinos como célu<strong><strong>la</strong>s</strong><br />
viables pero no cultivables durante periodos prolongados (Magariños et al., 1994) pero<br />
mant<strong>en</strong>i<strong>en</strong>do prácticam<strong>en</strong>te <strong>la</strong> misma infectividad pot<strong>en</strong>cial para los peces que <strong><strong>la</strong>s</strong><br />
célu<strong><strong>la</strong>s</strong> viables y cultivables (Magariños et al., 1994). Esto sugiere que el medio acuático<br />
podría constituir un reservorio y un vehículo <strong>de</strong> transmisión para este patóg<strong>en</strong>o,<br />
contribuy<strong>en</strong>do el aum<strong>en</strong>to <strong>de</strong> <strong><strong>la</strong>s</strong> temperaturas al <strong>de</strong>sarrollo <strong>de</strong> <strong>la</strong> epizootia (Toranzo et<br />
al., 1991; Magariños et al., 2001).<br />
Se han hecho difer<strong>en</strong>tes estudios para valorar <strong><strong>la</strong>s</strong> distintas vías <strong>de</strong> <strong>en</strong>trada <strong>de</strong>l<br />
patóg<strong>en</strong>o. Por un <strong>la</strong>do, parece ser que <strong>la</strong> infección pue<strong>de</strong> iniciarse por ingestión <strong>de</strong>l<br />
patóg<strong>en</strong>o (Magariños et al., 1995). Por otro <strong>la</strong>do, evaluando el papel <strong>de</strong> <strong>la</strong> piel como<br />
puerta <strong>de</strong> <strong>en</strong>trada <strong>de</strong>l microorganismo, se ha observado que P. damse<strong>la</strong>e subsp.<br />
piscicida es resist<strong>en</strong>te a <strong>la</strong> acción <strong>de</strong>l mucus <strong>de</strong> piel <strong>de</strong> dorada y <strong>de</strong> lubina, aunque<br />
s<strong>en</strong>sible al <strong>de</strong> rodaballo (Magariños et al., 1995), lo que podría ser una razón por <strong>la</strong> que<br />
12
INTRODUCCIÓN<br />
<strong><strong>la</strong>s</strong> epi<strong>de</strong>mias surgidas <strong>en</strong> Europa nunca hayan afectado al rodaballo, y sí a <strong>la</strong> dorada y a<br />
<strong>la</strong> lubina.<br />
De mom<strong>en</strong>to, no se ha podido <strong>de</strong>mostrar <strong>la</strong> exist<strong>en</strong>cia <strong>de</strong> un portador <strong>de</strong> esta<br />
<strong>en</strong>fermedad, ya que no se ha podido ais<strong>la</strong>r <strong>la</strong> bacteria <strong>de</strong> peces supervivi<strong>en</strong>tes a una<br />
exposición experim<strong>en</strong>tal (Toranzo et al., 1991), sin po<strong>de</strong>r <strong>de</strong>scartar, por otro <strong>la</strong>do, que <strong>la</strong><br />
bacteria se <strong>en</strong>cu<strong>en</strong>tre <strong>en</strong> estos casos a una conc<strong>en</strong>tración por <strong>de</strong>bajo <strong>de</strong> los límites <strong>de</strong><br />
<strong>de</strong>tección clásicos, o <strong>en</strong> estado viable no cultivable.<br />
2.4. MECANISMOS DE VIRULENCIA<br />
La virul<strong>en</strong>cia <strong>de</strong> los microorganismos patóg<strong>en</strong>os es un complejo proceso<br />
multifactorial. En el caso <strong>de</strong> P. damse<strong>la</strong>e subsp. piscicida aún se <strong>de</strong>sconoc<strong>en</strong> muchos<br />
aspectos re<strong>la</strong>tivos a su virul<strong>en</strong>cia, sobre todo a nivel molecu<strong>la</strong>r.<br />
La capacidad <strong>de</strong> adher<strong>en</strong>cia e invasión es es<strong>en</strong>cial <strong>en</strong> los primeros estadios <strong>de</strong> <strong>la</strong><br />
infección. Una vez <strong>en</strong> el interior <strong>de</strong>l hospedador, <strong>la</strong> adhesión a los tejidos promueve <strong>la</strong><br />
liberación <strong>de</strong> toxinas y prece<strong>de</strong> a <strong>la</strong> p<strong>en</strong>etración <strong>en</strong> <strong><strong>la</strong>s</strong> célu<strong><strong>la</strong>s</strong> diana por parte <strong>de</strong> los<br />
microorganismos. Aunque P. damse<strong>la</strong>e subsp. piscicida pres<strong>en</strong>ta una débil adhesión a<br />
difer<strong>en</strong>tes líneas celu<strong>la</strong>res <strong>de</strong> peces, sí ha mostrado una elevada capacidad adhesiva a<br />
intestino <strong>de</strong> dorada, lubina y rodaballo (Magariños et al., 1996a). Se ha <strong>de</strong>tectado su<br />
capacidad para invadir líneas celu<strong>la</strong>res <strong>de</strong> peces (Magariños et al., 1996a; Elkamel y<br />
Thune, 2003) y permanecer viable, así como <strong>de</strong> proliferar <strong>en</strong> el interior <strong>de</strong> los<br />
macrófagos sin sufrir cambios morfológicos apar<strong>en</strong>tes, liberándose microorganismos al<br />
medio que inva<strong>de</strong>n célu<strong><strong>la</strong>s</strong> adyac<strong>en</strong>tes (Magariños et al., 1996a; Elkamel et al., 2003).<br />
Este hecho pue<strong>de</strong> ser relevante in vivo ya que garantiza el mant<strong>en</strong>imi<strong>en</strong>to <strong>de</strong>l patóg<strong>en</strong>o<br />
durante cierto periodo <strong>de</strong> tiempo <strong>en</strong> el tejido infectado, lo que contribuye a un estado <strong>de</strong><br />
infección crónica y <strong>de</strong> portador por parte <strong>de</strong>l hospedador. Incluso Elkamel et al. (2003)<br />
concluy<strong>en</strong> <strong>en</strong> su estudio que P. damse<strong>la</strong>e subsp. piscicida es un patóg<strong>en</strong>o intracelu<strong>la</strong>r<br />
muy efici<strong>en</strong>te, que pue<strong>de</strong> sobrevivir y multiplicarse <strong>en</strong> el interior <strong>de</strong> macrófagos <strong>de</strong><br />
peces como <strong>la</strong> lubina.<br />
La importancia como factores <strong>de</strong> virul<strong>en</strong>cia <strong>de</strong> los productos extracelu<strong>la</strong>res (ECPs)<br />
secretados por P. damse<strong>la</strong>e subsp. piscicida está bi<strong>en</strong> docum<strong>en</strong>tada (Balebona et al.,<br />
13
INTRODUCCIÓN<br />
1992; Magariños et al., 1992; Noya et al., 1995a y b; Romal<strong>de</strong>, 2002; Bakopoulos et al.,<br />
2004). Los ECPs <strong>de</strong> P. damse<strong>la</strong>e subsp. piscicida son fuertem<strong>en</strong>te tóxicos por vía<br />
intraperitoneal (Noya et al., 1995a; Bakopoulos et al., 2004) y llegan a ser letales para<br />
difer<strong>en</strong>tes especies piscíco<strong><strong>la</strong>s</strong> y para ratón (Magariños et al., 1992). Las principales<br />
activida<strong>de</strong>s <strong>de</strong>mostradas son <strong>la</strong> hemolítica, fosfolipasa y citotóxica (Magariños et al.,<br />
1992). Estudios histológicos han implicado estas activida<strong>de</strong>s, <strong>en</strong> particu<strong>la</strong>r –<strong><strong>la</strong>s</strong><br />
fosfolipasas– <strong>en</strong> <strong>la</strong> patogénesis <strong>de</strong> <strong>la</strong> pseudotuberculosis (Noya et al., 1995b).<br />
La implicación <strong>de</strong>l material polisacarídico capsu<strong>la</strong>r ha sido c<strong>la</strong>ram<strong>en</strong>te <strong>de</strong>mostrada<br />
<strong>en</strong> <strong>la</strong> virul<strong>en</strong>cia <strong>de</strong> P. damse<strong>la</strong>e subsp. piscicida (Bonet et al., 1994; Magariños et al.,<br />
1996b; Romal<strong>de</strong> y Magariños, 1997; Acosta et al., 2006). Aunque todas <strong><strong>la</strong>s</strong> cepas <strong>de</strong> P.<br />
damse<strong>la</strong>e subsp. piscicida sintetizan una estructura externa adicional <strong>en</strong> un medio<br />
<strong>en</strong>riquecido <strong>en</strong> glucosa, sólo <strong><strong>la</strong>s</strong> cepas virul<strong>en</strong>tas sintetizan constitutivam<strong>en</strong>te una fina<br />
cápsu<strong>la</strong> (Magariños et al., 1996b) que les confiere resist<strong>en</strong>cia a <strong>la</strong> inactivación por suero,<br />
e increm<strong>en</strong>ta el grado <strong>de</strong> virul<strong>en</strong>cia (Magariños et al., 1996b; Acosta et al., 2006),<br />
a<strong>de</strong>más <strong>de</strong> reducir <strong>la</strong> fagocitosis por parte <strong>de</strong> los macrófagos (Arijo et al., 1998). Por lo<br />
tanto, <strong>la</strong> pres<strong>en</strong>cia <strong>de</strong> cápsu<strong>la</strong> juega un importante papel <strong>en</strong> <strong>la</strong> patogénesis <strong>de</strong> P.<br />
damse<strong>la</strong>e subsp. piscicida, como prueba el hecho <strong>de</strong> que <strong><strong>la</strong>s</strong> cepas no virul<strong>en</strong>tas son<br />
eliminadas <strong>de</strong>l pez <strong>en</strong> corto tiempo, aunque <strong><strong>la</strong>s</strong> cepas no virul<strong>en</strong>tas, <strong>en</strong> <strong><strong>la</strong>s</strong> que se induce<br />
<strong>la</strong> síntesis <strong>de</strong> cápsu<strong>la</strong>, pres<strong>en</strong>tan también resist<strong>en</strong>cia al suero (Magariños et al., 1997;<br />
Arijo et al., 1998). A<strong>de</strong>más, esta inducción <strong>de</strong> <strong>la</strong> expresión capsu<strong>la</strong>r <strong>en</strong> cepas no<br />
virul<strong>en</strong>tas increm<strong>en</strong>ta su resist<strong>en</strong>cia a <strong>la</strong> acción bactericida <strong>de</strong>l suero y disminuye su<br />
DL 50 alre<strong>de</strong>dor <strong>de</strong> 2-3 unida<strong>de</strong>s logarítmicas (Magariños et al., 1996b). De todas formas,<br />
in vivo, <strong><strong>la</strong>s</strong> condiciones limitantes <strong>en</strong> hierro hac<strong>en</strong> que P. damse<strong>la</strong>e subsp. piscicida no<br />
pres<strong>en</strong>te una cápsu<strong>la</strong> <strong>de</strong> tamaño importante (Acosta et al., 2003), lo que podría suponer<br />
una mayor exposición <strong>de</strong> <strong><strong>la</strong>s</strong> adhesinas a <strong>la</strong> superficie, si<strong>en</strong>do esto un aspecto útil para <strong>la</strong><br />
colonización (Magariños et al., 1996b).<br />
La capacidad <strong>de</strong> conseguir hierro es primordial para el crecimi<strong>en</strong>to <strong>de</strong> bacterias<br />
patóg<strong>en</strong>as <strong>en</strong> el interior <strong>de</strong>l hospedador, si<strong>en</strong>do, por tanto, es<strong>en</strong>cial para causar<br />
infección. A<strong>de</strong>más, se ha constatado que este microorganismo muestra un elevado<br />
número <strong>de</strong> activida<strong>de</strong>s <strong>de</strong> sus ECPs bajo condiciones limitantes <strong>de</strong> hierro (Bakopoulos<br />
14
INTRODUCCIÓN<br />
et al., 1997). En P. damse<strong>la</strong>e subsp. piscicida se han <strong>de</strong>scrito difer<strong>en</strong>tes estrategias para<br />
conseguir hierro, un sistema <strong>de</strong> incorporación <strong>de</strong> hierro codificado a nivel<br />
cromosómico, consist<strong>en</strong>te <strong>en</strong> un si<strong>de</strong>róforo química y biológicam<strong>en</strong>te re<strong>la</strong>cionado con <strong>la</strong><br />
multocidina producida por Pasteurel<strong>la</strong> multocida y, al m<strong>en</strong>os, tres proteínas <strong>de</strong><br />
membrana externa <strong>de</strong> alto peso molecu<strong>la</strong>r regu<strong>la</strong>das por hierro (Magariños et al., 1994).<br />
También se ha <strong>de</strong>scrito <strong>la</strong> capacidad <strong>de</strong> P. damse<strong>la</strong>e subsp. piscicida <strong>de</strong> utilizar<br />
directam<strong>en</strong>te el grupo hemo como única fu<strong>en</strong>te <strong>de</strong> hierro y que <strong>la</strong> inyección<br />
intraperitoneal <strong>de</strong> hemina antes <strong>de</strong> <strong>la</strong> infección experim<strong>en</strong>tal increm<strong>en</strong>ta <strong>la</strong> letalidad <strong>de</strong><br />
este patóg<strong>en</strong>o (Magariños et al., 1994). Igualm<strong>en</strong>te se ha <strong>de</strong>mostrado que <strong>en</strong> rodaballo el<br />
sistema génico <strong>de</strong> transporte <strong>de</strong>l grupo hemo se expresa in vivo, durante <strong>la</strong> infección <strong>de</strong><br />
P. damse<strong>la</strong>e subsp. piscicida (Juíz-Río, 2006). La base g<strong>en</strong>ética <strong>de</strong> estos sistemas <strong>de</strong><br />
captación <strong>de</strong> hierro por si<strong>de</strong>róforos muestra una pat<strong>en</strong>te diversidad (Juíz-Río, 2006).<br />
Se ha <strong>de</strong>mostrado <strong>la</strong> re<strong>la</strong>ción <strong>en</strong>tre <strong>la</strong> cápsu<strong>la</strong> y <strong>la</strong> capacidad <strong>de</strong> adquirir hierro (do<br />
Vale et al., 2001; Romal<strong>de</strong>, 2002), jugando los polisacáridos capsu<strong>la</strong>res un papel<br />
secundario <strong>en</strong> <strong>la</strong> unión <strong>de</strong> <strong>la</strong> hemina. La expresión <strong>de</strong>l material capsu<strong>la</strong>r es <strong>de</strong>p<strong>en</strong>di<strong>en</strong>te<br />
<strong>de</strong> <strong>la</strong> disponibilidad <strong>de</strong> hierro y <strong>de</strong> <strong>la</strong> fase <strong>de</strong> crecimi<strong>en</strong>to, así <strong><strong>la</strong>s</strong> célu<strong><strong>la</strong>s</strong> crecidas bajo<br />
condiciones limitantes <strong>de</strong> hierro manifiestan m<strong>en</strong>or cantidad <strong>de</strong> material capsu<strong>la</strong>r que<br />
<strong><strong>la</strong>s</strong> célu<strong><strong>la</strong>s</strong> suplem<strong>en</strong>tadas con hierro (do Vale et al., 2001). Esto pue<strong>de</strong> explicar <strong>la</strong><br />
necesidad <strong>de</strong> <strong>la</strong> bacteria <strong>de</strong> expresar su si<strong>de</strong>róforo y/o receptores <strong>de</strong> hierro durante el<br />
tiempo que recorre el sistema circu<strong>la</strong>torio <strong>de</strong>l hospedador. Una vez que el<br />
microorganismo alcanza los difer<strong>en</strong>tes tejidos, <strong>la</strong> cantidad <strong>de</strong> material capsu<strong>la</strong>r<br />
probablem<strong>en</strong>te se increm<strong>en</strong>ta <strong>en</strong> respuesta a los mecanismos <strong>de</strong> <strong>de</strong>f<strong>en</strong>sa celu<strong>la</strong>r <strong>de</strong>l<br />
hospedador. A<strong>de</strong>más el papel <strong>de</strong>l hierro <strong>en</strong> <strong>la</strong> expresión <strong>de</strong> activida<strong>de</strong>s <strong>en</strong>zimáticas ha<br />
sido también <strong>de</strong>scrito, y algunas <strong>en</strong>zimas proteolíticas, como <strong>la</strong> ge<strong>la</strong>tinasa y caseinasa<br />
son sólo sintetizadas cuando <strong><strong>la</strong>s</strong> cepas son cultivadas bajo condiciones restrictivas <strong>de</strong><br />
hierro (Magariños et al., 1994; Romal<strong>de</strong>, 2002).<br />
Reci<strong>en</strong>tem<strong>en</strong>te se ha <strong>de</strong>scrito que difer<strong>en</strong>tes cepas virul<strong>en</strong>tas <strong>de</strong> este patóg<strong>en</strong>o<br />
produc<strong>en</strong> una exotoxina, <strong>la</strong> AIP56, codificada p<strong><strong>la</strong>s</strong>mídicam<strong>en</strong>te, y que ti<strong>en</strong>e <strong>la</strong> capacidad<br />
<strong>de</strong> inducir apoptosis <strong>en</strong> leucocitos <strong>de</strong> lubina (do Vale et al., 2005).<br />
15
INTRODUCCIÓN<br />
En resum<strong>en</strong>, los mecanismos <strong>de</strong> invasión y superviv<strong>en</strong>cia <strong>de</strong> <strong>la</strong> bacteria<br />
Photobacterium damse<strong>la</strong>e subsp. piscicida <strong>en</strong> el interior <strong>de</strong>l hospedador aún no se<br />
conoc<strong>en</strong>: mi<strong>en</strong>tras que unos autores confirman <strong>la</strong> pres<strong>en</strong>cia <strong>de</strong> bacterias intactas <strong>en</strong> el<br />
interior <strong>de</strong> <strong><strong>la</strong>s</strong> célu<strong><strong>la</strong>s</strong> <strong>de</strong>l pez, sugiri<strong>en</strong>do <strong>la</strong> capacidad <strong>de</strong> <strong>la</strong> bacteria <strong>de</strong> sobrevivir como<br />
patóg<strong>en</strong>o intracelu<strong>la</strong>r (Noya et al., 1995b; López-Dóriga et al., 2000); otros autores han<br />
observado que este patóg<strong>en</strong>o es altam<strong>en</strong>te susceptible a los radicales oxigénicos<br />
g<strong>en</strong>erados durante el estallido respiratorio <strong>en</strong> el interior <strong>de</strong> los fagocitos (Skarmeta et<br />
al., 1995; Arijo et al., 1998; Barnes et al., 1999a).<br />
Es obvio, por tanto, que <strong>la</strong> patogénesis <strong>de</strong> P. damse<strong>la</strong>e subsp. piscicida es un<br />
proceso complejo y multifactorial, no <strong>en</strong>t<strong>en</strong>dido por completo. En esta Tesis Doctoral se<br />
int<strong>en</strong>ta profundizar y ac<strong>la</strong>rar, <strong>en</strong> <strong>la</strong> medida <strong>de</strong> lo posible, parte <strong>de</strong> ese proceso, si P.<br />
damse<strong>la</strong>e subsp. piscicida es capaz, o no, <strong>de</strong> sobrevivir al estallido respiratorio g<strong>en</strong>erado<br />
<strong>en</strong> el interior <strong>de</strong> <strong><strong>la</strong>s</strong> célu<strong><strong>la</strong>s</strong> fagocíticas <strong>de</strong> l<strong>en</strong>guado s<strong>en</strong>egalés, asimismo, se realiza un<br />
estudio <strong>de</strong> difer<strong>en</strong>tes estrategias <strong>de</strong> prev<strong>en</strong>ción <strong>de</strong> esta <strong>en</strong>fermedad bacteriana, mediante<br />
<strong>la</strong> aplicación <strong>de</strong> inmunoestimu<strong>la</strong>ntes y probióticos al hospedador.<br />
3. LAS ACTIVIDADES SUPERÓXIDO DISMUTASA Y<br />
CATALASA COMO FACTORES DE VIRULENCIA<br />
La inactivación bacteriana <strong>en</strong> el interior <strong>de</strong> los fagocitos se efectúa mediante dos<br />
tipos <strong>de</strong> mecanismos: in<strong>de</strong>p<strong>en</strong>di<strong>en</strong>tes <strong>de</strong> oxíg<strong>en</strong>o, mediados por los constituy<strong>en</strong>tes <strong>de</strong> los<br />
gránulos <strong>de</strong> los fagocitos (<strong>en</strong>zimas lisosomales, catepsinas, <strong>de</strong>f<strong>en</strong>sinas, <strong>la</strong>ctoferrina,<br />
<strong>en</strong>zimas proteolíticas), y <strong>de</strong>p<strong>en</strong>di<strong>en</strong>tes <strong>de</strong> oxíg<strong>en</strong>o. En estos últimos se da <strong>la</strong> formación<br />
<strong>de</strong> compuestos oxig<strong>en</strong>ados como peróxido <strong>de</strong> hidróg<strong>en</strong>o (H 2 O 2 ), radicales como el<br />
anión <strong>superóxido</strong> (O 2·- ) y radical hidroxilo (OH - ), productos que se forman durante el<br />
<strong>de</strong>nominado estallido respiratorio que sigue a <strong>la</strong> activación <strong>de</strong> <strong>la</strong> <strong>en</strong>zima nicotinamidaa<strong>de</strong>nín-dinucleótido-fosfato-hidróg<strong>en</strong>o<br />
(NADPH) oxidasa <strong>de</strong> <strong>la</strong> membrana tras <strong>la</strong><br />
fagocitosis.<br />
16
INTRODUCCIÓN<br />
3.1. ESTALLIDO RESPIRATORIO<br />
Tras <strong>la</strong> fagocitosis –proceso por el cual los fagocitos interiorizan a los<br />
microorganismos– los leucocitos liberan al interior <strong>de</strong> los fagosomas el cont<strong>en</strong>ido <strong>de</strong> sus<br />
gránulos citop<strong><strong>la</strong>s</strong>máticos, <strong>en</strong>tre los que <strong>de</strong>stacan diversos factores citotóxicos tales como<br />
metabolitos oxig<strong>en</strong>ados y <strong>en</strong>zimas lisosomales, con el fin <strong>de</strong> matar y digerir a los<br />
microorganismos.<br />
La producción <strong>de</strong> dichos metabolitos <strong>de</strong> oxíg<strong>en</strong>o se lleva a cabo <strong>en</strong> el proceso<br />
conocido como estallido respiratorio, o explosión respiratoria, que se produce <strong>en</strong> los<br />
fagocitos ante <strong>la</strong> pres<strong>en</strong>cia <strong>de</strong> bacterias, experim<strong>en</strong>tando un rápido increm<strong>en</strong>to <strong>en</strong> el<br />
consumo <strong>de</strong> oxíg<strong>en</strong>o. Actualm<strong>en</strong>te, el término <strong>de</strong> estallido respiratorio se consi<strong>de</strong>ra<br />
ina<strong>de</strong>cuado ya que dicho increm<strong>en</strong>to <strong>en</strong> el consumo <strong>de</strong> oxíg<strong>en</strong>o no se <strong>de</strong>be a un<br />
increm<strong>en</strong>to <strong>en</strong> <strong>la</strong> tasa respiratoria, sino que se produce <strong>en</strong> <strong>la</strong> superficie celu<strong>la</strong>r don<strong>de</strong> se<br />
usa el oxíg<strong>en</strong>o extracelu<strong>la</strong>r para g<strong>en</strong>erar radicales reactivos <strong>de</strong> oxíg<strong>en</strong>o, los<br />
<strong>de</strong>nominados ROS (reactive oxyg<strong>en</strong> species) (O 2·- , H 2 O 2 , OH - ). La pres<strong>en</strong>cia <strong>de</strong> dichos<br />
radicales libres se asocia al <strong>en</strong>vejecimi<strong>en</strong>to celu<strong>la</strong>r, sin embargo, su toxicidad ha<br />
<strong>en</strong>contrado utilidad <strong>en</strong> los fagocitos como mecanismo <strong>de</strong> <strong>de</strong>f<strong>en</strong>sa fr<strong>en</strong>te a bacterias<br />
<strong>de</strong>bido a su gran actividad microbiocida.<br />
El estallido respiratorio se <strong>de</strong>s<strong>en</strong>ca<strong>de</strong>na por <strong>la</strong> estimu<strong>la</strong>ción <strong>de</strong> <strong>la</strong> membrana <strong>de</strong>l<br />
fagocito. Tras dicha estimu<strong>la</strong>ción, <strong>la</strong> <strong>en</strong>zima NADPH oxidasa, pres<strong>en</strong>te <strong>en</strong> <strong>la</strong> membrana<br />
celu<strong>la</strong>r, es capaz <strong>de</strong> reducir el O 2 <strong>en</strong> anión <strong>superóxido</strong> (O 2·- ) (Roos et al., 2003). De<br />
forma secu<strong>en</strong>cial, por <strong>la</strong> reducción unival<strong>en</strong>te <strong>de</strong>l O 2 , se g<strong>en</strong>era toda una serie <strong>de</strong><br />
especies reactivas altam<strong>en</strong>te tóxicas: los <strong>de</strong>nominados radicales reactivos <strong>de</strong>l oxíg<strong>en</strong>o.<br />
Los primeros <strong>en</strong> producirse son el radical <strong>superóxido</strong> (O 2·- ) y el peróxido <strong>de</strong> hidróg<strong>en</strong>o<br />
(H 2 O 2 ) por acción <strong>de</strong> <strong>la</strong> <strong>superóxido</strong> <strong>dismutasa</strong> (SOD) sobre el O 2·- . El anión <strong>superóxido</strong><br />
ti<strong>en</strong>e un alto po<strong>de</strong>r bactericida, así que es prob<strong>la</strong>ble que este radical por sí solo sea<br />
capaz <strong>de</strong> eliminar microorganismos. El peróxido <strong>de</strong> hidróg<strong>en</strong>o pue<strong>de</strong> reaccionar con el<br />
<strong>superóxido</strong>, g<strong>en</strong>erando radicales hidroxilo (OH - ) y oxíg<strong>en</strong>o singleto ( 1 O 2 ), ambos<br />
altam<strong>en</strong>te reactivos y tóxicos.<br />
Por otro <strong>la</strong>do el anión <strong>superóxido</strong> pue<strong>de</strong> también reaccionar con óxido <strong>de</strong> nitróg<strong>en</strong>o<br />
(NO), que es <strong>de</strong>rivado <strong>de</strong> L-arginina y O 2 molecu<strong>la</strong>r, <strong>en</strong> una reacción catalizada por <strong>la</strong><br />
17
INTRODUCCIÓN<br />
óxido nítrico sintasa (NOS) <strong>en</strong> <strong>la</strong> que se produce peroxinitrito, un intermediario <strong>de</strong>l<br />
nitróg<strong>en</strong>o muy reactivo.<br />
El oxíg<strong>en</strong>o singleto pue<strong>de</strong> ser convertido <strong>en</strong> un compuesto simi<strong>la</strong>r al ozono (O 3 ) <strong>en</strong><br />
una reacción catalizada por <strong>la</strong> unión <strong>de</strong> anticuerpos con microorganismos o neutrófilos.<br />
El peróxido <strong>de</strong> hidróg<strong>en</strong>o, junto con el cloruro, pue<strong>de</strong> ser sustrato <strong>de</strong> <strong>la</strong> <strong>en</strong>zima<br />
mieloperoxidasa (MPO) g<strong>en</strong>erándose ácido hipoclórico (HClO), muy tóxico para <strong>la</strong><br />
mayor parte <strong>de</strong> los microorganismos. El hipoclórico reacciona con aminas secundarias,<br />
formando cloraminas secundarias, que son igual <strong>de</strong> microbiocidas que el ácido, pero<br />
mucho más estables.<br />
Por lo tanto, un gran número <strong>de</strong> reacciones químicas se produce <strong>en</strong> el pequeño<br />
espacio <strong>en</strong>tre <strong>la</strong> bacteria ingerida y <strong>la</strong> membrana <strong>de</strong>l fagosoma. Para comp<strong>en</strong>sar <strong>la</strong> carga<br />
electrónica <strong>de</strong>bida a <strong>la</strong> reducción <strong>de</strong>l oxíg<strong>en</strong>o molecu<strong>la</strong>r <strong>en</strong> anión <strong>superóxido</strong>, se da un<br />
flujo <strong>de</strong> protones (H + ) o <strong>de</strong> otros cationes, como K + . Si todos los electrones bombeados<br />
al interior <strong>de</strong>l fagosoma fueran comp<strong>en</strong>sados por el flujo <strong>de</strong> protones, el pH <strong>de</strong>l<br />
fagosoma permanecería neutro; sin embargo, se aum<strong>en</strong>ta hasta 8, a pesar <strong>de</strong> <strong>la</strong> liberación<br />
<strong>de</strong> ácidos proce<strong>de</strong>ntes <strong>de</strong> los gránulos citop<strong><strong>la</strong>s</strong>máticos que se fusionan con el fagosoma.<br />
Esto indica que otros cationes, como el potasio (K + ), pue<strong>de</strong>n <strong>en</strong>trar <strong>en</strong> el fagosoma <strong>en</strong><br />
lugar <strong>de</strong> los protones (Reeves et al., 2002). Si se da ese flujo <strong>de</strong> iones potasio, estos<br />
cationes mediarían <strong>la</strong> solubilización <strong>de</strong> proteasas que están unidas a <strong>la</strong> matriz <strong>de</strong><br />
proteoglucano <strong>de</strong> los gránulos. Por tanto, el increm<strong>en</strong>to <strong>de</strong>l pH intrafagosomal alcanza<br />
los valores óptimos <strong>de</strong> <strong>la</strong> acción <strong>de</strong> proteasas, pudi<strong>en</strong>do afirmarse que <strong>la</strong> NADPH<br />
oxidasa, a<strong>de</strong>más <strong>de</strong> matar a los microorganismos por medio <strong>de</strong> sus radicales oxigénicos,<br />
actúa liberando proteasas lisosomales. De este modo, <strong>la</strong> NADPH oxidasa leucocitaria<br />
induce <strong>la</strong> muerte microbiana directa, vía productos oxidativos, e indirectam<strong>en</strong>te, vía<br />
liberación <strong>de</strong> proteasas.<br />
El estallido respiratorio conduce a <strong>la</strong> inactivación <strong>de</strong> proteínas y a <strong>la</strong> oxidación <strong>de</strong><br />
ácidos nucleicos y otras molécu<strong><strong>la</strong>s</strong> es<strong>en</strong>ciales, lo que repres<strong>en</strong>ta una estrategia<br />
importante <strong>de</strong>l sistema inmunitario <strong>en</strong> <strong>la</strong> lucha contra <strong><strong>la</strong>s</strong> infecciones. Para competir con<br />
los radicales libres g<strong>en</strong>erados, los microorganismos patóg<strong>en</strong>os se han visto obligados a<br />
<strong>de</strong>sarrol<strong>la</strong>r estrategias <strong>en</strong> un doble fr<strong>en</strong>te: por un <strong>la</strong>do, <strong>la</strong> protección fr<strong>en</strong>te a los<br />
18
INTRODUCCIÓN<br />
radicales g<strong>en</strong>erados <strong>en</strong> su propio metabolismo aerobio y por otro, <strong>la</strong> <strong>de</strong>f<strong>en</strong>sa fr<strong>en</strong>te al<br />
contacto con estos radicales producidos <strong>en</strong> célu<strong><strong>la</strong>s</strong> fagocíticas. A esta resist<strong>en</strong>cia fr<strong>en</strong>te a<br />
los ROS contribuy<strong>en</strong> <strong>en</strong>zimas antioxidantes tales como <strong><strong>la</strong>s</strong> <strong>superóxido</strong> <strong>dismutasa</strong>s,<br />
cata<strong><strong>la</strong>s</strong>as y peroxidasas.<br />
Estas <strong>en</strong>zimas repres<strong>en</strong>tan un arma <strong>de</strong>f<strong>en</strong>siva fr<strong>en</strong>te al ataque <strong>de</strong> célu<strong><strong>la</strong>s</strong><br />
fagocíticas, contribuy<strong>en</strong>do al pot<strong>en</strong>cial virul<strong>en</strong>to <strong>de</strong>l microorganismo patóg<strong>en</strong>o <strong>en</strong> su<br />
interacción con el hospedador.<br />
3.2. LA ACTIVIDAD SUPERÓXIDO DISMUTASA<br />
La <strong>superóxido</strong> <strong>dismutasa</strong> repres<strong>en</strong>ta <strong>la</strong> primera línea <strong>de</strong> <strong>de</strong>f<strong>en</strong>sa <strong>de</strong> <strong><strong>la</strong>s</strong> célu<strong><strong>la</strong>s</strong><br />
fr<strong>en</strong>te al estrés oxidativo. Cataliza <strong>la</strong> conversión <strong>de</strong> los radicales anión <strong>superóxido</strong> <strong>en</strong><br />
peróxido <strong>de</strong> hidróg<strong>en</strong>o y oxíg<strong>en</strong>o (ecuación 1).<br />
(1) O 2·- + 2H + H 2 O 2 + O 2<br />
La actividad SOD ha sido <strong>de</strong>tectada <strong>en</strong> una amplia variedad <strong>de</strong> seres vivos, <strong>de</strong>s<strong>de</strong><br />
bacterias a humanos, implicada como <strong>de</strong>f<strong>en</strong>sa es<strong>en</strong>cial fr<strong>en</strong>te a <strong>la</strong> toxicidad pot<strong>en</strong>cial <strong>de</strong>l<br />
oxíg<strong>en</strong>o. Cualquier célu<strong>la</strong> que utilice el oxíg<strong>en</strong>o ti<strong>en</strong>e el pot<strong>en</strong>cial <strong>de</strong> producir anión<br />
<strong>superóxido</strong> y, por tanto, <strong>de</strong>be cont<strong>en</strong>er alguna forma <strong>de</strong> <strong>superóxido</strong> <strong>dismutasa</strong><br />
(Fridovich, 1974).<br />
Las <strong>superóxido</strong> <strong>dismutasa</strong>s constituy<strong>en</strong> una familia <strong>de</strong> metalo<strong>en</strong>zimas que se<br />
c<strong><strong>la</strong>s</strong>ifican <strong>en</strong> cuatro grupos según el metal que actúe <strong>de</strong> cofactor: FeSOD, MnSOD,<br />
CuZnSOD y NiSOD, esta última <strong>de</strong>scrita reci<strong>en</strong>tem<strong>en</strong>te <strong>en</strong> Streptomyces<br />
(Lynch y Kuramitsu, 2000).<br />
3.3. LA ACTIVIDAD CATALASA<br />
Las cata<strong><strong>la</strong>s</strong>as también forman parte <strong>de</strong> <strong>la</strong> <strong>de</strong>f<strong>en</strong>sa <strong>de</strong> <strong><strong>la</strong>s</strong> bacterias fr<strong>en</strong>te al estrés<br />
oxidativo. Catalizan <strong>la</strong> <strong>de</strong>scomposición <strong>de</strong>l peróxido <strong>de</strong> hidróg<strong>en</strong>o, transformándolo <strong>en</strong><br />
agua y oxíg<strong>en</strong>o (ecuación 2).<br />
19
INTRODUCCIÓN<br />
(2) 2H 2 O 2 2H 2 O + O 2<br />
Algunas cata<strong><strong>la</strong>s</strong>as ti<strong>en</strong><strong>en</strong> a<strong>de</strong>más actividad peroxidasa, un donador orgánico <strong>de</strong><br />
electrones, o a veces un ión haluro, es empleado <strong>en</strong> <strong>la</strong> reducción <strong>de</strong>l peróxido <strong>de</strong><br />
hidróg<strong>en</strong>o (ecuación 3).<br />
(3) RH 2 + H 2 O 2 2H 2 O + R<br />
Las cata<strong><strong>la</strong>s</strong>as se pue<strong>de</strong>n dividir <strong>en</strong> tres grupos (Loew<strong>en</strong>, 1997): cata<strong><strong>la</strong>s</strong>as<br />
monofuncionales con grupo hemo (FeCat), cata<strong><strong>la</strong>s</strong>as bifuncionales con grupo hemo<br />
(cata<strong><strong>la</strong>s</strong>as-peroxidasas) y pseudocata<strong><strong>la</strong>s</strong>as sin grupo hemo (MnCat), estas últimas se<br />
<strong>de</strong>nominan pseudocata<strong><strong>la</strong>s</strong>as porque son resist<strong>en</strong>tes a los inhibidores comunes para <strong><strong>la</strong>s</strong><br />
cata<strong><strong>la</strong>s</strong>as, como <strong>la</strong> azida y el cianuro.<br />
Las activida<strong>de</strong>s <strong>superóxido</strong> <strong>dismutasa</strong> y cata<strong><strong>la</strong>s</strong>a proteg<strong>en</strong> no sólo <strong>de</strong> forma directa<br />
eliminando aniones supéroxido y peróxido <strong>de</strong> hidróg<strong>en</strong>o, respectivam<strong>en</strong>te, sino que<br />
también impi<strong>de</strong>n <strong>la</strong> formación <strong>de</strong>l radical hidroxilo OH·, <strong>la</strong> especie reactiva <strong>de</strong>rivada <strong>de</strong>l<br />
oxíg<strong>en</strong>o con mayor po<strong>de</strong>r oxidante.<br />
De esta forma, el papel <strong>de</strong> dichas <strong>en</strong>zimas pue<strong>de</strong> ser fundam<strong>en</strong>tal a <strong>la</strong> hora <strong>de</strong><br />
proteger a bacterias patóg<strong>en</strong>as durante el estallido respiratorio que sigue a <strong>la</strong> fagocitosis<br />
y por ello se asocian a mecanismos <strong>de</strong> virul<strong>en</strong>cia (Tab<strong>la</strong> 2). Así, <strong>la</strong> habilidad <strong>de</strong> un<br />
organismo <strong>de</strong> infectar a su hospedador es <strong>de</strong>bida, al m<strong>en</strong>os <strong>en</strong> parte, a su resist<strong>en</strong>cia<br />
fr<strong>en</strong>te a <strong>la</strong> producción <strong>de</strong> ROS por <strong><strong>la</strong>s</strong> célu<strong><strong>la</strong>s</strong>, principalm<strong>en</strong>te monocitos/macrófagos y<br />
polimorfonucleados. Irónicam<strong>en</strong>te <strong>la</strong> evolución ha seleccionado organismos que utilizan<br />
dichas célu<strong><strong>la</strong>s</strong> como diana, así que <strong>la</strong> posesión <strong>de</strong> <strong>en</strong>zimas como SOD y cata<strong><strong>la</strong>s</strong>a<br />
contribuirá a <strong>la</strong> resist<strong>en</strong>cia fr<strong>en</strong>te al hospedador y, por tanto, al establecimi<strong>en</strong>to <strong>de</strong> <strong>la</strong><br />
infección.<br />
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INTRODUCCIÓN<br />
Tab<strong>la</strong> 2 Ejemplos <strong>de</strong> microorganismos patóg<strong>en</strong>os <strong>en</strong> los que <strong><strong>la</strong>s</strong> activida<strong>de</strong>s SOD y cata<strong><strong>la</strong>s</strong>a<br />
juegan un papel importante <strong>en</strong> <strong>la</strong> virul<strong>en</strong>cia<br />
Patóg<strong>en</strong>o Factor <strong>de</strong> virul<strong>en</strong>cia Refer<strong>en</strong>cia<br />
Listeria monocytog<strong>en</strong>es FeSOD Welch et al., 1979<br />
Shigel<strong>la</strong> flexneri FeSOD Franzon et al., 1990<br />
Pseudomonas syringae Cata<strong><strong>la</strong>s</strong>as (no <strong>de</strong>terminadas) Klotz y Hutcheson,<br />
1992<br />
Caulobacter cresc<strong>en</strong>tus CuZnSOD Schnell y Steinman,<br />
1995<br />
Aeromonas salmonicida FeSOD Barnes et al., 1996<br />
Pseudomonas aeruginosa MnSOD Po<strong>la</strong>ck et al., 1996<br />
Legionel<strong>la</strong> pneumophi<strong>la</strong> CuZnSOD St. John y Steinman,<br />
1996<br />
A. salmonicida subsp. MnSOD y FeCata<strong><strong>la</strong>s</strong>a Barnes et al., 1999b<br />
salmonicida<br />
Mycobacterium tuberculosis Cata<strong><strong>la</strong>s</strong>a-Peroxidasa Manca et al., 1999<br />
Streptococcus pneumoniae MnSOD Yesilkaya et al., 2000<br />
Vibrio harveyi Cata<strong><strong>la</strong>s</strong>a monofuncional Vattanaviboon y<br />
Mongkolsuk, 2001<br />
Salmonel<strong>la</strong> <strong>en</strong>terica CuZnSOD Uzzau et al., 2002<br />
Vibrio shiloi SOD (no <strong>de</strong>terminada) Banin et al., 2003<br />
3.4. LAS ACTIVIDADES SUPERÓXIDO DISMUTASA Y CATALASA EN<br />
Photobacterium damse<strong>la</strong>e subsp. piscicida<br />
Como ya se citó <strong>en</strong> el apartado 2.4., algunos autores confirman <strong>la</strong> pres<strong>en</strong>cia <strong>de</strong><br />
bacterias intactas <strong>en</strong> el interior <strong>de</strong> célu<strong><strong>la</strong>s</strong> <strong>de</strong>l pez, sugiri<strong>en</strong>do <strong>la</strong> habilidad <strong>de</strong> P.<br />
damse<strong>la</strong>e subsp. piscicida <strong>de</strong> sobrevivir como patóg<strong>en</strong>o intracelu<strong>la</strong>r (Noya et al., 1995a;<br />
López-Dóriga et al., 2000; Elkamel et al., 2003), mi<strong>en</strong>tras que otros autores han<br />
observado que este patóg<strong>en</strong>o es altam<strong>en</strong>te susceptible a los radicales oxidativos<br />
g<strong>en</strong>erados durante el estallido respiratorio <strong>en</strong> los fagocitos (Skarmeta et al., 1995; Arijo<br />
et al., 1998; Barnes et al., 1999a).<br />
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INTRODUCCIÓN<br />
Para esc<strong>la</strong>recer este último punto es imprescindible profundizar <strong>en</strong> el estudio <strong>de</strong> <strong><strong>la</strong>s</strong><br />
activida<strong>de</strong>s <strong>superóxido</strong> <strong>dismutasa</strong> y cata<strong><strong>la</strong>s</strong>a manifestadas por P. damse<strong>la</strong>e subsp.<br />
piscicida. Una mayor información sobre el papel <strong>de</strong> dichas activida<strong>de</strong>s <strong>en</strong> <strong>la</strong> virul<strong>en</strong>cia<br />
<strong>de</strong>l patóg<strong>en</strong>o podría contribuir a <strong>en</strong>t<strong>en</strong><strong>de</strong>r <strong><strong>la</strong>s</strong> interacciones <strong>en</strong>tre P. damse<strong>la</strong>e subsp.<br />
piscicida y su hospedador.<br />
Barnes et al. (1999a) <strong>de</strong>terminaron <strong>la</strong> exist<strong>en</strong>cia <strong>en</strong> P. damse<strong>la</strong>e subsp. piscicida <strong>de</strong><br />
una SOD con hierro <strong>en</strong> su c<strong>en</strong>tro activo, localizada <strong>en</strong> el espacio periplásmico, y <strong>de</strong> una<br />
cata<strong><strong>la</strong>s</strong>a, sin <strong>de</strong>terminar, localizada <strong>en</strong> el citop<strong><strong>la</strong>s</strong>ma. La actividad SOD se veía reducida<br />
por el crecimi<strong>en</strong>to <strong>en</strong> condiciones restrictivas <strong>de</strong> hierro, así como <strong>en</strong> bajas<br />
conc<strong>en</strong>traciones <strong>de</strong> oxíg<strong>en</strong>o, mi<strong>en</strong>tras que <strong>la</strong> cata<strong><strong>la</strong>s</strong>a era expresada constitutivam<strong>en</strong>te,<br />
aunque <strong>en</strong> los geles se apreciaron difer<strong>en</strong>cias <strong>en</strong> los niveles <strong>de</strong> <strong>la</strong> actividad <strong>de</strong> <strong><strong>la</strong>s</strong><br />
difer<strong>en</strong>tes cepas analizadas.<br />
4. ESTIMULACIÓN DEL ESTALLIDO RESPIRATORIO POR<br />
DIFERENTES MICROORGANISMOS FRENTE A LA<br />
INFECCIÓN POR Photobacterium damse<strong>la</strong>e subsp. piscicida<br />
4.1. PREVENCIÓN Y TRATAMIENTO DE LA PSEUDOTUBERCULOSIS<br />
El principal método utilizado <strong>en</strong> <strong><strong>la</strong>s</strong> piscifactorías para el control <strong>de</strong> <strong>la</strong> <strong>en</strong>fermedad<br />
son los ag<strong>en</strong>tes quimioterapéuticos: los antibióticos. Hasta finales <strong>de</strong> los och<strong>en</strong>ta eran<br />
muy efectivos <strong>en</strong> el tratami<strong>en</strong>to <strong>de</strong> esta infección, pero <strong>la</strong> aparición <strong>de</strong> cepas resist<strong>en</strong>tes<br />
(Aoki et al., 1981; Miranda y Zemelman, 2002; Radu et al., 2003; Zorril<strong>la</strong> et al., 2003) y<br />
<strong>la</strong> contaminación <strong>de</strong>l medio acuático (Kautsky et al., 2000; Sivaram et al., 2004) han<br />
llegado a constituir un grave problema <strong>en</strong> <strong>la</strong> acuicultura. La prev<strong>en</strong>ción <strong>de</strong> <strong><strong>la</strong>s</strong><br />
<strong>en</strong>fermeda<strong>de</strong>s infecciosas es una alternativa conv<strong>en</strong>i<strong>en</strong>te, <strong>de</strong> ahí que el <strong>de</strong>sarrollo <strong>de</strong><br />
sustancias inmunoestimu<strong>la</strong>ntes, capaces <strong>de</strong> activar el sistema inmune <strong>de</strong>l pez e inducir<br />
una mejor respuesta fr<strong>en</strong>te a los patóg<strong>en</strong>os, suponga una bu<strong>en</strong>a alternativa al uso <strong>de</strong><br />
antibióticos <strong>en</strong> acuicultura (Sakai, 1999). A<strong>de</strong>más <strong>de</strong> <strong>la</strong> posible aparición <strong>de</strong><br />
resist<strong>en</strong>cias, otro inconv<strong>en</strong>i<strong>en</strong>te que explica <strong>la</strong> ineficacia <strong>de</strong>l tratami<strong>en</strong>to con antibióticos<br />
es el hecho <strong>de</strong> que P. damse<strong>la</strong>e subsp. piscicida pue<strong>de</strong> pasar por un periodo intracelu<strong>la</strong>r<br />
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INTRODUCCIÓN<br />
<strong>de</strong> parasitismo <strong>en</strong> el interior <strong>de</strong>l macrófago durante <strong>la</strong> infección (Kusuda y Sa<strong>la</strong>ti, 1993),<br />
lo que le evitaría estar <strong>en</strong> contacto con el antibiótico. Se comprueba, por tanto, que <strong>la</strong><br />
inmunoprofi<strong>la</strong>xis sería <strong>la</strong> mejor vía para prev<strong>en</strong>ir <strong>la</strong> pseudotuberculosis.<br />
A lo <strong>la</strong>rgo <strong>de</strong> los últimos veinte años, ha salido a <strong>la</strong> luz una gran variedad <strong>de</strong><br />
estudios que han analizado <strong>la</strong> eficacia <strong>de</strong> <strong>la</strong> inmunización mediante vacunación a <strong>la</strong> hora<br />
<strong>de</strong> prev<strong>en</strong>ir <strong>la</strong> pseudotuberculosis (Romal<strong>de</strong> y Magariños, 1997). La mayoría <strong>de</strong> <strong><strong>la</strong>s</strong><br />
vacunas probadas consistieron <strong>en</strong> célu<strong><strong>la</strong>s</strong> inactivadas por calor o por formalina (Fukuda<br />
y Kusuda, 1981; Kusuda y Hamaguchi, 1987; Kusuda y Hamaguchi, 1988; Hamaguchi y<br />
Kusuda, 1989). Aunque se alcanzó un cierto grado <strong>de</strong> protección, los mejores resultados<br />
fueron obt<strong>en</strong>idos empleando formu<strong>la</strong>ciones basadas <strong>en</strong> los lipopolisacáridos y <strong>en</strong> <strong><strong>la</strong>s</strong><br />
fracciones ribosomales <strong>de</strong> <strong><strong>la</strong>s</strong> bacterias (Fukuda y Kusuda, 1985; Kusuda et al., 1988;<br />
Kawakami et al., 1997). Sin embargo, estas formu<strong>la</strong>ciones pres<strong>en</strong>taron no sólo<br />
problemas <strong>de</strong> reproducibilidad, sino también dificulta<strong>de</strong>s <strong>en</strong> su producción a gran<br />
esca<strong>la</strong>. La inmunización pasiva también ha sido evaluada (Fukuda y Kusuda, 1981), pero<br />
los resultados mostraron un tiempo muy corto <strong>de</strong> protección. Uno <strong>de</strong> los porc<strong>en</strong>tajes <strong>de</strong><br />
protección más altos fr<strong>en</strong>te a <strong>la</strong> pseudotuberculosis se obtuvo con una bacterina<br />
<strong>en</strong>riquecida con productos extracelu<strong>la</strong>res (ECPs) (Magariños et al., 1994, 1997, 1999).<br />
Esta vacuna <strong>en</strong> <strong>la</strong> actualidad está disponible comercialm<strong>en</strong>te y ha sido empleada con<br />
éxito <strong>en</strong> varios países europeos, incluy<strong>en</strong>do España, Portugal y Grecia.<br />
La investigación <strong>en</strong> busca <strong>de</strong> vacunas más efectivas se ha dirigido también hacia el<br />
uso <strong>de</strong> bacterias vivas at<strong>en</strong>uadas (Kusuda y Hamaguchi, 1988), cuya utilización todavía<br />
no está permitida, y el uso <strong>de</strong> proteínas <strong>de</strong> <strong>la</strong> <strong>en</strong>vuelta celu<strong>la</strong>r (Magariños et al., 1994)<br />
como antíg<strong>en</strong>os protectores.<br />
Tal y como m<strong>en</strong>cionamos anteriorm<strong>en</strong>te, Magariños et al. (2000) <strong>de</strong>mostraron por<br />
medio <strong>de</strong> <strong>la</strong> técnica RAPD <strong>la</strong> exist<strong>en</strong>cia <strong>de</strong> dos linajes clonales según su proce<strong>de</strong>ncia:<br />
uno que incluiría a <strong><strong>la</strong>s</strong> cepas <strong>de</strong> orig<strong>en</strong> europeo y otro que <strong>en</strong>globaría a <strong><strong>la</strong>s</strong> <strong>de</strong><br />
proce<strong>de</strong>ncia japonesa y norteamericana. Más reci<strong>en</strong>tem<strong>en</strong>te, Juíz-Río et al. (2005),<br />
aplicando <strong>la</strong> técnica <strong>de</strong> hibridación subtractiva, concluyeron que este patóg<strong>en</strong>o pres<strong>en</strong>ta<br />
una alta heterog<strong>en</strong>eidad g<strong>en</strong>ética. Sin embargo, P. damse<strong>la</strong>e subsp. piscicida ha<br />
mostrado ser un microorganismo bioquímica y antigénicam<strong>en</strong>te homogéneo, incluy<strong>en</strong>do<br />
23
INTRODUCCIÓN<br />
<strong><strong>la</strong>s</strong> cepas <strong>de</strong>l patóg<strong>en</strong>o ais<strong>la</strong>das <strong>de</strong> l<strong>en</strong>guados cultivados <strong>en</strong> España (Bakopoulos et al.,<br />
1995; Magariños et al., 1996c, 2003). Por lo tanto, <strong>la</strong> inmunización con vacunas<br />
comerciales, originalm<strong>en</strong>te <strong>de</strong>sarrol<strong>la</strong>das para otros peces cultivados tales como dorada<br />
y lubina (Romal<strong>de</strong> y Magariños, 1997; Magariños et al., 1999), podría ser consi<strong>de</strong>rada<br />
como una medida efectiva para prev<strong>en</strong>ir <strong>la</strong> pseudotuberculosis <strong>en</strong> l<strong>en</strong>guado. Es<br />
necesario reseñar, sin embargo, que el l<strong>en</strong>guado es una especie totalm<strong>en</strong>te difer<strong>en</strong>te a<br />
aquel<strong><strong>la</strong>s</strong> especies piscíco<strong><strong>la</strong>s</strong> <strong>en</strong> <strong><strong>la</strong>s</strong> que estas vacunas se vi<strong>en</strong><strong>en</strong> aplicando, y, por tanto,<br />
es necesaria una a<strong>de</strong>cuación <strong>de</strong> su empleo <strong>en</strong> el l<strong>en</strong>guado. Hay <strong>de</strong>scritos diseños<br />
vacunales dival<strong>en</strong>tes que incluy<strong>en</strong> bacterina y ECPs inactivados por formol <strong>de</strong> Vibrio<br />
harveyi y P. damse<strong>la</strong>e subsp. piscicida y que se han aplicado específicam<strong>en</strong>te a<br />
l<strong>en</strong>guado obt<strong>en</strong>iéndose resultados prometedores (Arijo et al., 2005) aunque su<br />
efectividad es limitada <strong>en</strong> el tiempo. Por lo tanto, no hay que <strong>de</strong>scartar otros aspectos <strong>de</strong><br />
<strong>la</strong> profi<strong>la</strong>xis como es el empleo <strong>de</strong> los inmunoestimu<strong>la</strong>ntes y los probióticos.<br />
Los inmunoestimu<strong>la</strong>ntes son más seguros que los antibióticos, y su rango <strong>de</strong><br />
eficacia es más amplio que el <strong>de</strong> <strong><strong>la</strong>s</strong> vacunas, aunque su acción es <strong>de</strong> corta duración.<br />
Así, <strong>la</strong> estrategia más efectiva para prev<strong>en</strong>ir y combatir posibles <strong>en</strong>fermeda<strong>de</strong>s<br />
infecciosas <strong>de</strong> peces pue<strong>de</strong> ser el uso combinado <strong>de</strong> los métodos <strong>de</strong>scritos (Sakai, 1999).<br />
De hecho, <strong>en</strong> <strong>la</strong> actualidad, el uso <strong>de</strong> inmunoestimu<strong>la</strong>ntes, junto a ag<strong>en</strong>tes<br />
quimioterapéuticos o vacunas, ha sido ampliam<strong>en</strong>te aceptado por parte <strong>de</strong> los<br />
acuicultores. Sin embargo, es necesaria <strong>la</strong> búsqueda <strong>de</strong> nuevos ag<strong>en</strong>tes<br />
inmunoestimu<strong>la</strong>ntes que abarat<strong>en</strong> los costes <strong>de</strong> producción y result<strong>en</strong> efectivos fr<strong>en</strong>te a<br />
los patóg<strong>en</strong>os.<br />
La aplicación <strong>de</strong> los probióticos <strong>en</strong> acuicultura surge también por <strong>la</strong> necesidad <strong>de</strong><br />
contar con estrategias <strong>de</strong>stinadas al control <strong>de</strong> <strong>en</strong>fermeda<strong>de</strong>s que afectan a <strong><strong>la</strong>s</strong> especies<br />
cultivadas.<br />
4.2. INMUNOMODULACIÓN. INMUNOESTIMULACIÓN<br />
La inmunomodu<strong>la</strong>ción es <strong>la</strong> capacidad que ti<strong>en</strong><strong>en</strong> <strong>de</strong>terminadas sustancias y<br />
ag<strong>en</strong>tes <strong>de</strong> regu<strong>la</strong>r el sistema inmunitario, pudiéndose hab<strong>la</strong>r <strong>de</strong> inmunoestimu<strong>la</strong>ción o<br />
inmuno<strong>de</strong>presión si se estimu<strong>la</strong> o <strong>de</strong>prime dicho sistema, respectivam<strong>en</strong>te. La principal<br />
24
INTRODUCCIÓN<br />
razón <strong>de</strong> <strong>la</strong> búsqueda <strong>de</strong> nuevos ag<strong>en</strong>tes inmunoestimu<strong>la</strong>ntes es el gran <strong>de</strong>sarrollo <strong>de</strong> <strong>la</strong><br />
acuicultura y el increm<strong>en</strong>to <strong>de</strong> situaciones <strong>de</strong> estrés y <strong>en</strong>fermeda<strong>de</strong>s causadas por los<br />
cultivos int<strong>en</strong>sivos, que suel<strong>en</strong> producir un increm<strong>en</strong>to <strong>de</strong> <strong>la</strong> susceptibilidad a <strong><strong>la</strong>s</strong><br />
infecciones. Se han llevado a cabo algunos estudios <strong>en</strong> los que este efecto negativo fue<br />
superado por el uso <strong>de</strong> inmunoestimu<strong>la</strong>ntes (Siwicki et al., 1994; An<strong>de</strong>rson, 1996), ya<br />
que increm<strong>en</strong>tan los mecanismos <strong>de</strong> <strong>de</strong>f<strong>en</strong>sa inespecíficos. Facilitan <strong>la</strong> función <strong>de</strong> <strong><strong>la</strong>s</strong><br />
célu<strong><strong>la</strong>s</strong> fagocíticas e increm<strong>en</strong>ta su actividad bactericida, si<strong>en</strong>do los mecanismos<br />
implicados los ya m<strong>en</strong>cionados in<strong>de</strong>p<strong>en</strong>di<strong>en</strong>tes y <strong>de</strong>p<strong>en</strong>di<strong>en</strong>tes (estallido respiratorio) <strong>de</strong><br />
oxíg<strong>en</strong>o (An<strong>de</strong>rson et al., 1992; Sakai, 1999).<br />
El empleo <strong>de</strong> inmunoestimu<strong>la</strong>ntes ti<strong>en</strong>e un valor principalm<strong>en</strong>te prev<strong>en</strong>tivo,<br />
pue<strong>de</strong>n ser capaces <strong>de</strong> comp<strong>en</strong>sar <strong><strong>la</strong>s</strong> limitaciones <strong>de</strong> los quimioterapéuticos y <strong>de</strong> <strong><strong>la</strong>s</strong><br />
vacunas (Tab<strong>la</strong> 3). Los inmunoestimu<strong>la</strong>ntes son más seguros que los quimioterapéuticos<br />
y su rango <strong>de</strong> eficacia es más amplio que el <strong>de</strong> <strong><strong>la</strong>s</strong> vacunas (Sakai, 1999). Su principal<br />
inconv<strong>en</strong>i<strong>en</strong>te es <strong>la</strong> corta duración <strong>de</strong> su acción ya que estas sustancias actúan sobre el<br />
sistema inmunitario inespecífico el cual carece <strong>de</strong> memoria (An<strong>de</strong>rson, 1996; Sakai,<br />
1999). Sakai (1999) afirma que, como reg<strong>la</strong> g<strong>en</strong>eral, <strong>la</strong> estrategia más efectiva para<br />
prev<strong>en</strong>ir y combatir posibles <strong>en</strong>fermeda<strong>de</strong>s infecciosas <strong>de</strong> peces es el uso combinado <strong>de</strong><br />
<strong>la</strong> vacunación y <strong>la</strong> administración <strong>de</strong> immunoestimu<strong>la</strong>ntes. De esta manera, con un<br />
conocimi<strong>en</strong>to <strong>de</strong>tal<strong>la</strong>do <strong>de</strong> <strong>la</strong> eficacia y limitaciones, el inmunoestimu<strong>la</strong>nte pue<strong>de</strong> llegar<br />
a ser una herrami<strong>en</strong>ta po<strong>de</strong>rosa <strong>en</strong> el control <strong>de</strong> <strong>en</strong>fermeda<strong>de</strong>s <strong>en</strong> peces.<br />
Aunque se han estudiado muchas sustancias naturales y sintéticas, con resultados<br />
que <strong>de</strong>muestran una pot<strong>en</strong>ciación <strong>de</strong>l sistema inmune <strong>de</strong> peces y un increm<strong>en</strong>to <strong>de</strong> <strong>la</strong><br />
resist<strong>en</strong>cia a <strong>la</strong> <strong>en</strong>fermedad, <strong>la</strong> búsqueda <strong>de</strong> nuevos inmunoestimu<strong>la</strong>ntes continúa hacia<br />
<strong>la</strong> mejora <strong>de</strong> <strong><strong>la</strong>s</strong> condiciones <strong>en</strong> los cultivos int<strong>en</strong>sivos. Estos nuevos productos <strong>de</strong>b<strong>en</strong><br />
poseer dos características: proporcionar una estimu<strong>la</strong>ción g<strong>en</strong>eral y ser económicam<strong>en</strong>te<br />
asequibles. En los últimos años, los estudios <strong>de</strong>stinados a tal fin se han c<strong>en</strong>trado<br />
principalm<strong>en</strong>te <strong>en</strong> el empleo <strong>de</strong> sustancias <strong>de</strong> orig<strong>en</strong> natural cuyas v<strong>en</strong>tajas principales<br />
respecto a <strong><strong>la</strong>s</strong> <strong>de</strong> orig<strong>en</strong> sintético radican <strong>en</strong> el hecho <strong>de</strong> ser sustancias no tóxicas,<br />
bio<strong>de</strong>gradables y biocompatibles con <strong>la</strong> salud humana.<br />
25
INTRODUCCIÓN<br />
Tab<strong>la</strong> 3 Comparación <strong>de</strong> <strong><strong>la</strong>s</strong> características <strong>de</strong> quimioterapéuticos, vacunas e<br />
inmunoestimu<strong>la</strong>ntes (Sakai, 1999)<br />
QUIMIOTERAPÉUTICOS VACUNAS INMUNOESTIMULANTES<br />
Cuándo Terapéutico Profiláctico Profiláctico<br />
Eficacia Excel<strong>en</strong>te Excel<strong>en</strong>te Bu<strong>en</strong>a<br />
Espectro <strong>de</strong><br />
actividad<br />
Medio Limitado Amplio<br />
Duración Corta Larga Corta<br />
4.3. USO DE LAS ALGAS COMO INMUNOESTIMULANTES<br />
En los últimos años se ha c<strong>en</strong>trado <strong>la</strong> at<strong>en</strong>ción <strong>en</strong> organismos marinos como fu<strong>en</strong>te<br />
<strong>de</strong> sustancias <strong>de</strong> interés terapéutico. En este s<strong>en</strong>tido, <strong>la</strong> capacidad <strong>de</strong> <strong><strong>la</strong>s</strong> algas para<br />
producir metabolitos secundarios <strong>de</strong> interés farmacéutico, como antibióticos, antivirales,<br />
antitumorales y antiinf<strong>la</strong>matorios ha sido ext<strong>en</strong>sam<strong>en</strong>te docum<strong>en</strong>tada (Scheuer, 1990;<br />
Faulkner, 1993; González <strong>de</strong>l Val et al., 2001). Sin embargo, los estudios <strong>en</strong>focados<br />
hacia <strong>la</strong> <strong>de</strong>tección <strong>de</strong> propieda<strong>de</strong>s inmunomodu<strong>la</strong>doras <strong>de</strong> extractos proce<strong>de</strong>ntes <strong>de</strong><br />
algas son todavía muy escasos (Blinkova et al., 2001; Castro et al., 2004, 2006). En esta<br />
Memoria nos hemos c<strong>en</strong>trado <strong>en</strong> el estudio <strong>de</strong> <strong>la</strong> microalga roja Porphyridium cru<strong>en</strong>tum<br />
como posible fu<strong>en</strong>te <strong>de</strong> sustancias inmunoestimu<strong>la</strong>ntes para l<strong>en</strong>guados cultivados. El<br />
alga cumpliría con los requisitos que hoy día se p<strong>la</strong>ntean <strong>en</strong> <strong>la</strong> búsqueda <strong>de</strong> nuevas<br />
sustancias pot<strong>en</strong>cialm<strong>en</strong>te inmunoestimu<strong>la</strong>ntes, ya que es una sustancia natural y su<br />
cultivo no suele ser costoso, tanto <strong>de</strong>s<strong>de</strong> el punto <strong>de</strong> vista económico, como <strong>en</strong> cuanto a<br />
tiempo y esfuerzo necesarios.<br />
Las algas ti<strong>en</strong><strong>en</strong> difer<strong>en</strong>tes compuestos con efecto sobre el sistema inmunitario <strong>de</strong><br />
los peces. Muchas algas son fu<strong>en</strong>te importante <strong>de</strong> los <strong>de</strong>nominados ácidos grasos<br />
poliinsaturados, PUFAs, es<strong>en</strong>ciales como requerimi<strong>en</strong>to dietético <strong>de</strong> muchos teleósteos<br />
(Bell et al., 1985; Kov<strong>en</strong> et al., 2001). A<strong>de</strong>más, algunos <strong>de</strong> ellos, como el ácido<br />
26
INTRODUCCIÓN<br />
araquidónico, están implicados <strong>en</strong> <strong>la</strong> síntesis <strong>de</strong> eicosanoi<strong>de</strong> y, por tanto, <strong>en</strong> <strong>la</strong><br />
producción <strong>de</strong> prostag<strong>la</strong>ndinas, implicadas <strong>en</strong> los procesos <strong>de</strong> estrés a través <strong>de</strong> <strong>la</strong><br />
modu<strong>la</strong>ción <strong>en</strong> <strong>la</strong> liberación <strong>de</strong>l cortisol y, por consigui<strong>en</strong>te, <strong>de</strong> <strong>la</strong> inmunidad celu<strong>la</strong>r<br />
(Vil<strong>la</strong>lta et al., 2005).<br />
Otro <strong>de</strong> los compon<strong>en</strong>tes que <strong>en</strong>contramos <strong>en</strong> <strong><strong>la</strong>s</strong> algas son los carot<strong>en</strong>oi<strong>de</strong>s, como<br />
el β-carot<strong>en</strong>o, <strong>la</strong> astaxantina, <strong>la</strong> cataxantina o <strong><strong>la</strong>s</strong> xantofilinas. Amar et al. (2004)<br />
<strong>de</strong>mostraron que el β-carot<strong>en</strong>o ais<strong>la</strong>do <strong>de</strong>l alga Dunaliel<strong>la</strong> salina es capaz <strong>de</strong> modu<strong>la</strong>r,<br />
tras <strong>la</strong> administración oral, algunos <strong>de</strong> los mecanismos <strong>de</strong> <strong>de</strong>f<strong>en</strong>sa innata <strong>en</strong> trucha<br />
arcoiris (Oncorhynchus mykiss), como <strong>la</strong> actividad alternativa <strong>de</strong>l complem<strong>en</strong>to y <strong>la</strong><br />
lisozima <strong>en</strong> el suero, así como <strong>la</strong> fagocitosis. Los carot<strong>en</strong>oi<strong>de</strong>s increm<strong>en</strong>tan igualm<strong>en</strong>te<br />
<strong>la</strong> actividad fagocítica y <strong>la</strong> producción <strong>de</strong> citoquinas (B<strong>en</strong>dich, 1989; Chew, 1993).<br />
Las algas son también una fu<strong>en</strong>te natural <strong>de</strong> vitaminas, algunas <strong>de</strong> <strong><strong>la</strong>s</strong> cuales ti<strong>en</strong><strong>en</strong><br />
posibles efectos estimu<strong>la</strong>ntes sobre el sistema inmune <strong>de</strong> peces, como es el caso <strong>de</strong> <strong>la</strong><br />
vitamina C (Hardie et al., 1991; Cuesta et al., 2002; J<strong>en</strong>ey y J<strong>en</strong>ey, 2002; Lin y Shiau,<br />
2005), <strong>la</strong> vitamina E (Hardie et al., 1990; Cuesta et al., 2001) y otras <strong>de</strong>l grupo B (Miles<br />
et al., 2001).<br />
La utilización <strong>de</strong> polisacáridos como inmunoestimu<strong>la</strong>ntes está ampliam<strong>en</strong>te<br />
ext<strong>en</strong>dida <strong>en</strong> <strong>la</strong> acuicultura, pudiéndose adquirir comercialm<strong>en</strong>te (Siwicki et al., 1994;<br />
Cook et al., 2003; Couso et al., 2003; Bagni et al., 2005), si<strong>en</strong>do los glucanos los más<br />
estudiados <strong>en</strong> peces (Kumar et al., 2005). Los β-glucanos consist<strong>en</strong> <strong>en</strong> una serie <strong>de</strong><br />
residuos <strong>de</strong> β-1,3-glucopiranosil <strong>de</strong>rivados <strong>de</strong> levaduras y micelios <strong>de</strong> hongos. Estos<br />
azúcares parec<strong>en</strong> t<strong>en</strong>er un pot<strong>en</strong>te efecto inmunoestimu<strong>la</strong>nte, fundam<strong>en</strong>talm<strong>en</strong>te sobre<br />
los mecanismos <strong>de</strong> <strong>de</strong>f<strong>en</strong>sa inespecíficos, induci<strong>en</strong>do resist<strong>en</strong>cia a infecciones. La<br />
mayor parte <strong>de</strong> estos estudios se c<strong>en</strong>tra <strong>en</strong> β-glucanos ais<strong>la</strong>dos <strong>de</strong> <strong>la</strong> levadura<br />
Saccharomyces cerevisiae (Santarém et al., 1997; Castro et al., 1999; Kumari y Sahoo,<br />
2006; Marqués et al., 2006).<br />
Otro polisacárido pot<strong>en</strong>cialm<strong>en</strong>te inmunoestimu<strong>la</strong>nte, proce<strong>de</strong>nte <strong>de</strong> difer<strong>en</strong>tes<br />
macro-y micro- algas pardas, es el ácido algínico. El alginato es conocido <strong>en</strong> <strong>la</strong><br />
acuicultura hace mucho tiempo, utilizándose <strong>en</strong> <strong>la</strong> fabricación <strong>de</strong> pi<strong>en</strong>so como<br />
estabilizador <strong>de</strong> <strong>la</strong> estructura. Las propieda<strong>de</strong>s inmunomodu<strong>la</strong>doras fueron <strong>de</strong>terminadas<br />
27
INTRODUCCIÓN<br />
<strong>en</strong> extractos <strong>de</strong> Phyophaecaetes, como Laminaria digitata (Dalmo et al., 1998;<br />
Gabriels<strong>en</strong> y Austr<strong>en</strong>g, 1998) y otros (Miles et al., 2001; Peddie et al., 2002; Skjermo y<br />
Bergh, 2004; Bagni et al., 2005).<br />
4.3.1. Porphyridium cru<strong>en</strong>tum<br />
Porphyridium cru<strong>en</strong>tum es una microalga roja, pert<strong>en</strong>eci<strong>en</strong>te a <strong>la</strong> familia<br />
Rodophyta, or<strong>de</strong>n Porphyridiales. Sus célu<strong><strong>la</strong>s</strong> se caracterizan por ser esféricas y sin<br />
pared celu<strong>la</strong>r. Acumu<strong>la</strong> gran<strong>de</strong>s cantida<strong>de</strong>s <strong>de</strong> ácidos grasos, que llegan a alcanzar <strong>en</strong>tre<br />
9 y 14% <strong>de</strong>l peso seco, especialm<strong>en</strong>te el ácido araquidónico (36% <strong>de</strong>l total <strong>de</strong> los ácidos<br />
grasos), y cantida<strong>de</strong>s consi<strong>de</strong>rables <strong>de</strong> ácido eicosap<strong>en</strong>ta<strong>en</strong>oico. El cont<strong>en</strong>ido proteico<br />
está <strong>en</strong> el rango <strong>de</strong>l 28 al 39%, y los carbohidratos disponibles varían <strong>en</strong>tre un 40 y un<br />
57%. La biomasa conti<strong>en</strong>e tocoferol, vitamina K y una gran cantidad <strong>de</strong> carot<strong>en</strong>os<br />
(Rebolloso et al., 2000).<br />
Una propiedad característica <strong>de</strong> P. cru<strong>en</strong>tum es que sus célu<strong><strong>la</strong>s</strong> son capaces <strong>de</strong><br />
excretar un polisacárido sulfatado, un heteropolímero acídico compuesto por xilosa,<br />
glucosa, ga<strong>la</strong>ctosa y ésteres <strong>de</strong> sulfato (You y Barneu, 2004). Este polisacárido es <strong>de</strong><br />
gran importancia dado que pue<strong>de</strong> ser usado comercialm<strong>en</strong>te como espesante,<br />
estabilizante y emulsionante (Arad et al., 1985, 1988; Adda et al., 1986).<br />
El alga P. cru<strong>en</strong>tum conti<strong>en</strong>e sustancias pres<strong>en</strong>tes <strong>en</strong> otros microorganismos <strong>de</strong> los<br />
cuales se ha <strong>de</strong>mostrado su efecto inmunoestimu<strong>la</strong>nte <strong>en</strong> fagocitos <strong>de</strong> peces. Estas<br />
sustancias incluy<strong>en</strong> ácido araquidónico (Kov<strong>en</strong> et al., 2001), carbohidratos (Kumar et<br />
al., 2005), vitaminas (Hardie et al. 1990, 1991; Ortuño et al., 1999, 2003; J<strong>en</strong>ey y J<strong>en</strong>ey,<br />
2002), carot<strong>en</strong>oi<strong>de</strong>s (Amar et al., 2004) y polisacáridos (Siwicki et al., 1994; Santarém et<br />
al., 1997; Castro et al., 1999; Bagni et al., 2000, 2005; Esteban et al., 2001; J<strong>en</strong>ey y J<strong>en</strong>ey,<br />
2002; Cook et al., 2003; Couso et al., 2003), pudi<strong>en</strong>do producir una respuesta inmune más<br />
g<strong>en</strong>eral al poseer varias sustancias ya probadas como inmunoestimu<strong>la</strong>ntes (Ortuño et<br />
al., 2002).<br />
P. cru<strong>en</strong>tum posee <strong><strong>la</strong>s</strong> principales características para po<strong>de</strong>r ser consi<strong>de</strong>rada como<br />
un pot<strong>en</strong>cial inmunoestimu<strong>la</strong>nte. Su cultivo no es costoso, ni <strong>de</strong>s<strong>de</strong> el punto <strong>de</strong> vista <strong>de</strong><br />
esfuerzo necesario, ni <strong>en</strong> términos económicos. A nivel industrial es conv<strong>en</strong>i<strong>en</strong>te<br />
28
INTRODUCCIÓN<br />
simplificar <strong>la</strong> administración <strong>de</strong>l inmunoestimu<strong>la</strong>nte, proporcionándo<strong>la</strong> por vía oral,<br />
incluyéndo<strong>la</strong> <strong>en</strong> el pi<strong>en</strong>so y con el m<strong>en</strong>or grado <strong>de</strong> manipu<strong>la</strong>ción posible. De hecho,<br />
durante los últimos años exist<strong>en</strong> cada vez más trabajos <strong>en</strong> los que se <strong>en</strong>sayan<br />
organismos completos, como levaduras (Siwicki et al., 1994; Ortuño et al., 2002;<br />
Rodríguez et al., 2003), hongos (Rodríguez et al., 2004) y probióticos (Verschuere et al.,<br />
2000; Irianto y Austin, 2003; Salinas et al., 2005; Díaz-Rosales et al., 2006). Sin embargo,<br />
a pesar <strong>de</strong> que son numerosos los estudios realizados con extractos o <strong>de</strong>terminados<br />
compuestos <strong>de</strong>rivados <strong>de</strong> algas (Kov<strong>en</strong> et al., 2001; Castro et al., 2004; Skjermo y Bergh,<br />
2004; Díaz-Rosales et al., 2005; Hou y Ch<strong>en</strong>, 2005; Vil<strong>la</strong>lta et al., 2005), son escasos los<br />
estudios que p<strong>la</strong>ntean el empleo <strong>de</strong> algas completas (Blinkova et al., 2001; Val<strong>en</strong>te et al.,<br />
2006).<br />
4.4. EFECTO INMUNOESTIMULANTE DE BACTERIAS<br />
POTENCIALMENTE PROBIÓTICAS<br />
La <strong>de</strong>finición <strong>de</strong> probiótico ha ido cambiando a lo <strong>la</strong>rgo <strong>de</strong>l tiempo, proponiéndose<br />
como probióticos a bacterias vivas o inactivadas o a alguno <strong>de</strong> sus compon<strong>en</strong>tes<br />
celu<strong>la</strong>res que también pue<strong>de</strong>n ejercer ciertos efectos b<strong>en</strong>eficiosos (Ouwehand y<br />
Salmin<strong>en</strong>, 1998; Iso<strong>la</strong>uri et al., 2002). Así, Salmin<strong>en</strong> et al. (1999) han separado <strong>la</strong><br />
<strong>de</strong>finición <strong>de</strong> probiótico <strong>de</strong>l alim<strong>en</strong>to y <strong>de</strong> su característica <strong>de</strong> ser microorganismos<br />
vivos con lo que se ha dado paso al sigui<strong>en</strong>te concepto: “un probiótico es cualquier<br />
preparación microbiana (no necesariam<strong>en</strong>te viva), o los compon<strong>en</strong>tes <strong>de</strong> célu<strong><strong>la</strong>s</strong><br />
microbianas, que ti<strong>en</strong><strong>en</strong> un efecto b<strong>en</strong>eficioso <strong>en</strong> <strong>la</strong> salud <strong>de</strong>l hospedador”. Igualm<strong>en</strong>te,<br />
Schrez<strong>en</strong>meir y <strong>de</strong> Vrese (2001) hac<strong>en</strong> refer<strong>en</strong>cia a los probióticos como “una<br />
preparación o producto que conti<strong>en</strong>e microorganismos <strong>de</strong>finidos <strong>en</strong> número sufici<strong>en</strong>te,<br />
capaces <strong>de</strong> alterar <strong>la</strong> microbiota, por imp<strong>la</strong>ntación o colonización, <strong>en</strong> un compartim<strong>en</strong>to<br />
<strong>de</strong>l hospedador y por el que ejerce efectos b<strong>en</strong>eficiosos sobre <strong>la</strong> salud <strong>de</strong>l hospedador”.<br />
Según <strong>la</strong> FAO, el término probiótico hace refer<strong>en</strong>cia a un complem<strong>en</strong>to microbiano <strong>de</strong><br />
<strong>la</strong> dieta que afecta b<strong>en</strong>eficiosam<strong>en</strong>te a <strong>la</strong> fisiología <strong>de</strong>l hospedador mediante modu<strong>la</strong>ción<br />
<strong>de</strong> <strong>la</strong> inmunidad sistémica y local, a<strong>de</strong>más <strong>de</strong> mejorar el ba<strong>la</strong>nce microbiano mediante <strong>la</strong><br />
29
INTRODUCCIÓN<br />
prev<strong>en</strong>ción <strong>de</strong> <strong>la</strong> colonización gastrointestinal por bacterias no <strong>de</strong>seables. Los avances<br />
<strong>en</strong> el empleo <strong>de</strong> los probióticos <strong>en</strong> gana<strong>de</strong>ría y medicina humana han conducido a<br />
consi<strong>de</strong>rar su aplicación también <strong>en</strong> <strong>la</strong> práctica acuíco<strong>la</strong> ya que pue<strong>de</strong>n ser una<br />
alternativa <strong>en</strong> <strong>la</strong> lucha contra <strong>la</strong> infección microbiana (Sakai, 1999). La investigación <strong>de</strong><br />
<strong>la</strong> aplicación <strong>de</strong> los probióticos <strong>en</strong> <strong>la</strong> industria acuíco<strong>la</strong> se ha increm<strong>en</strong>tado <strong>en</strong> los<br />
últimos años por <strong>la</strong> <strong>de</strong>manda <strong>de</strong> una industria acuíco<strong>la</strong> que, <strong>en</strong>tre otros aspectos, int<strong>en</strong>ta<br />
respetar el medio ambi<strong>en</strong>te. Los probióticos surg<strong>en</strong> <strong>de</strong> <strong>la</strong> necesidad <strong>de</strong> contar con<br />
estrategias <strong>de</strong>stinadas al biocontrol <strong>de</strong> <strong>en</strong>fermeda<strong>de</strong>s que afectan a <strong><strong>la</strong>s</strong> especies<br />
cultivadas <strong>en</strong> acuicultura. La mayoría <strong>de</strong> los trabajos realizados con probióticos <strong>en</strong><br />
peces se ha c<strong>en</strong>trado <strong>en</strong> el grado <strong>de</strong> protección <strong>de</strong>l pez fr<strong>en</strong>te a <strong>en</strong>fermeda<strong>de</strong>s infecciosas<br />
por su capacidad para inhibir el crecimi<strong>en</strong>to <strong>de</strong>l patóg<strong>en</strong>o. Actualm<strong>en</strong>te muchos <strong>de</strong> los<br />
estudios que se están realizando para dilucidar los mecanismos responsables <strong>de</strong> los<br />
efectos <strong>de</strong> los probióticos se están c<strong>en</strong>trando <strong>en</strong> <strong><strong>la</strong>s</strong> propieda<strong>de</strong>s inmunomodu<strong>la</strong>doras <strong>de</strong><br />
<strong><strong>la</strong>s</strong> cepas (Nikoske<strong>la</strong>in<strong>en</strong> et al., 2003; Irianto y Austin, 2003; Panigrahi et al., 2004;<br />
Salinas et al., 2005, 2006; Díaz-Rosales, 2006). De hecho, muchos <strong>de</strong> los<br />
inmunoestimu<strong>la</strong>ntes probados <strong>en</strong> acuicultura son compon<strong>en</strong>tes <strong>de</strong> célu<strong><strong>la</strong>s</strong> microbianas,<br />
como los glucanos, lipopolisacáridos y muramil dipéptido (An<strong>de</strong>rson, 1992). Sin<br />
embargo, <strong>la</strong> mayoría <strong>de</strong> ellos están c<strong>en</strong>trados <strong>en</strong> especies <strong>de</strong> agua dulce, si<strong>en</strong>do escasos,<br />
por el contrario, los llevados a cabo <strong>en</strong> especies marinas, y nulos los que se refier<strong>en</strong> a<br />
l<strong>en</strong>guado s<strong>en</strong>egalés (Tab<strong>la</strong> 4).<br />
Los animales acuáticos son muy difer<strong>en</strong>tes a los terrestres por lo que el concepto<br />
<strong>de</strong> probiótico cambia a <strong>la</strong> hora <strong>de</strong> su aplicación <strong>en</strong> acuicultura (Verschuere et al., 2000).<br />
La microbiota intestinal <strong>de</strong> <strong><strong>la</strong>s</strong> especies cultivadas interactúa <strong>de</strong> forma constante con el<br />
ambi<strong>en</strong>te, el cual ti<strong>en</strong>e una influ<strong>en</strong>cia mucho mayor sobre <strong>la</strong> salud <strong>de</strong> los peces que <strong>en</strong> el<br />
caso <strong>de</strong> los humanos o animales terrestres. Por lo tanto, <strong>de</strong>bido a que existe un flujo<br />
continuo <strong>de</strong> agua pasando a través <strong>de</strong>l tracto digestivo, <strong>la</strong> microbiota intestinal <strong>de</strong> los<br />
peces es <strong>de</strong>p<strong>en</strong>di<strong>en</strong>te <strong>de</strong>l ambi<strong>en</strong>te externo. De hecho, se han realizado estudios sobre <strong>la</strong><br />
microbiota <strong>de</strong>l pez y se ha visto que <strong>la</strong> variación es sustancial y que fluctúa diariam<strong>en</strong>te<br />
(Spanggaard et al., 2000). Por lo tanto, <strong>la</strong> mayoría <strong>de</strong> <strong><strong>la</strong>s</strong> bacterias son transitorias <strong>en</strong> el<br />
intestino <strong>de</strong>l pez, con intrusiones continuas <strong>de</strong> bacterias proce<strong>de</strong>ntes <strong>de</strong>l agua y <strong>de</strong> <strong>la</strong><br />
comida. De esta manera, no sólo se hab<strong>la</strong> <strong>de</strong> probiótico cuando se adiciona al alim<strong>en</strong>to,<br />
30
INTRODUCCIÓN<br />
sino también cuando se aña<strong>de</strong>n al medio, tanque o <strong>la</strong>guna <strong>de</strong> cultivo. Aquí el concepto<br />
<strong>de</strong> probiótico se amplía, <strong>de</strong>nominándose biocontrol cuando el tratami<strong>en</strong>to es con<br />
microorganismos antagonistas al patóg<strong>en</strong>o, o biorremediación cuando <strong>la</strong> calidad <strong>de</strong>l<br />
agua es mejorada.<br />
Verschuere et al. (2000) propon<strong>en</strong> una <strong>de</strong>finición modificada para el término<br />
probiótico aplicada <strong>en</strong> acuicultura: “Complem<strong>en</strong>to microbiano vivo que ti<strong>en</strong>e un efecto<br />
b<strong>en</strong>eficioso sobre el hospedador modificando <strong>la</strong> comunidad microbiana re<strong>la</strong>cionada con<br />
el hospedador o con el ambi<strong>en</strong>te, asegurando un uso mejorado <strong>de</strong>l alim<strong>en</strong>to o<br />
aum<strong>en</strong>tando su valor nutricional, favoreci<strong>en</strong>do <strong>la</strong> respuesta <strong>de</strong>l hospedador a <strong><strong>la</strong>s</strong><br />
<strong>en</strong>fermeda<strong>de</strong>s, o mejorando <strong>la</strong> calidad <strong>de</strong>l ambi<strong>en</strong>te”.<br />
Entre los mecanismos propuestos para explicar el modo <strong>en</strong> el que los probióticos<br />
pue<strong>de</strong>n interactuar con los patóg<strong>en</strong>os t<strong>en</strong>emos: (1) Exclusión competitiva por <strong>la</strong><br />
producción <strong>de</strong> compuestos antimicrobianos <strong>en</strong>tre los que se han <strong>de</strong>scrito bacteriocinas,<br />
lisozimas y proteasas (Austin et al., 1995; Sugita et al., 1997; Gatesoupe, 1999; Gram et<br />
al., 1999; Verschuere et al., 2000). Tan importante se ha consi<strong>de</strong>rado <strong>la</strong> capacidad <strong>de</strong><br />
inhibir el crecimi<strong>en</strong>to <strong>de</strong> bacterias patóg<strong>en</strong>as <strong>de</strong> peces, que esta característica se ha<br />
convertido <strong>en</strong> uno <strong>de</strong> los criterios más empleados para <strong>la</strong> selección <strong>de</strong> pot<strong>en</strong>ciales<br />
probióticos <strong>en</strong> acuicultura; (2) Competición por los nutri<strong>en</strong>tes y <strong>en</strong>ergía disponible<br />
(Smith y Davey, 1993; Pybus et al., 1994; Gatesoupe et al., 1997; Gram et al., 1999) y (3)<br />
Interfer<strong>en</strong>cia adhesiva <strong>en</strong> el hospedador (Olsson et al., 1992; Jöborn et al., 1997;<br />
Nikoske<strong>la</strong>in<strong>en</strong> et al., 2001; Chabrillón et al., 2005a y b).<br />
31
INTRODUCCIÓN<br />
Tab<strong>la</strong> 4 Efectos b<strong>en</strong>eficiosos <strong>de</strong> probióticos sobre el sistema inmune <strong>de</strong> peces<br />
PROBIÓTICO HOSPEDADOR INCREMENTO RESPUESTA REFERENCIA<br />
INMUNE<br />
Bacteria Gram Oncorhynchus Número eritrocitos y leucocitos, Irianto y Austin,<br />
positiva, no mykiss (trucha actividad lisozima, fagocitosis 2003<br />
i<strong>de</strong>ntificada arcoiris)<br />
Vibrio fluvialis Oncorhynchus Número eritrocitos y leucocitos, Irianto y Austin,<br />
mykiss<br />
actividad lisozima, fagocitosis 2003<br />
Aeromonas Oncorhynchus Número eritrocitos y leucocitos, Irianto y Austin,<br />
hydrophi<strong>la</strong> mykiss<br />
actividad lisozima, fagocitosis 2003<br />
Carnobacterium Oncorhynchus Número eritrocitos y leucocitos, Irianto y Austin,<br />
mykiss<br />
actividad lisozima, fagocitosis 2003<br />
Lactobacillus Oncorhynchus Estallido respiratorio, actividad Nikoske<strong>la</strong>in<strong>en</strong> et al.,<br />
rhamnosus mykiss<br />
bactericida suero, niveles Ig suero 2003<br />
Bacillus<br />
P<strong>en</strong>aeus<br />
Índice inmune (hemograma, Gullian et al., 2004<br />
vannamei producción anión <strong>superóxido</strong>,<br />
actividad f<strong>en</strong>oloxidasa, actividad<br />
antibacteriana, conc<strong>en</strong>tración<br />
proteica p<strong><strong>la</strong>s</strong>ma)<br />
Lactobacillus Oncorhynchus Activida<strong>de</strong>s lisozima y complem<strong>en</strong>to Panigrahi et al., 2004<br />
rhamnosus mykiss<br />
suero, fagocitosis<br />
Lactobacillus Sparus aurata Fagocitosis, actividad citotóxica Salinas et al., 2005<br />
<strong>de</strong>lbrüeckii subsp. (dorada)<br />
<strong>la</strong>ctis<br />
Bacillus subtilis Sparus aurata Fagocitosis, actividad citotóxica Salinas et al., 2005<br />
Aeromonas sobria<br />
Alteromonadaceae,<br />
G. Shewanel<strong>la</strong><br />
(Pdp11)<br />
Alteromonadaceae,<br />
G. Shewanel<strong>la</strong><br />
(51M6)<br />
Oncorhynchus Número leucocitos, fagocitosis,<br />
mykiss<br />
estallido respiratorio<br />
Sparus aurata Peroxidasa suero, actividad<br />
complem<strong>en</strong>to, fagocitosis<br />
Sparus aurata Peroxidasa suero, actividad<br />
complem<strong>en</strong>to, fagocitosis, actividad<br />
citotóxica<br />
Brunt y Austin, 2005<br />
Díaz-Rosales et al.,<br />
2006<br />
Díaz-Rosales et al.,<br />
2006<br />
32
INTRODUCCIÓN<br />
La necesidad <strong>de</strong> mejorar <strong>la</strong> resist<strong>en</strong>cia fr<strong>en</strong>te a <strong><strong>la</strong>s</strong> <strong>en</strong>fermeda<strong>de</strong>s, así como <strong>de</strong><br />
aum<strong>en</strong>tar <strong>la</strong> efici<strong>en</strong>cia <strong>en</strong> <strong>la</strong> alim<strong>en</strong>tación y <strong>en</strong> el <strong>de</strong>sarrollo <strong>de</strong>l crecimi<strong>en</strong>to son aspectos<br />
fundam<strong>en</strong>tales <strong>en</strong> varios sectores <strong>de</strong> esta industria <strong>en</strong> el empeño <strong>de</strong> lograr una reducción<br />
<strong>de</strong> los costes <strong>de</strong> producción. La microbiota gastrointestinal <strong>de</strong>sempeña una función<br />
importante <strong>en</strong> <strong>la</strong> nutrición y salud <strong>de</strong>l organismo hospedador. En este s<strong>en</strong>tido, los<br />
probióticos también pue<strong>de</strong>n <strong>de</strong>sempeñar un papel interesante, <strong>de</strong> esta forma <strong>en</strong> humanos<br />
y <strong>en</strong> <strong>la</strong> gana<strong>de</strong>ría terrestre se han investigado distintas formas <strong>de</strong> alterar <strong>la</strong> microbiota<br />
gastrointestinal por el empleo <strong>de</strong> probióticos con vistas a lograr unos efectos favorables,<br />
tales como mejora <strong>de</strong>l crecimi<strong>en</strong>to, <strong>de</strong> <strong>la</strong> digestión, <strong>de</strong> <strong>la</strong> inmunidad y <strong>de</strong> <strong>la</strong> resist<strong>en</strong>cia a<br />
<strong>la</strong> <strong>en</strong>fermedad <strong>de</strong>l hospedador. Si bi<strong>en</strong> <strong>en</strong> el campo <strong>de</strong> <strong>la</strong> acuicultura hay numerosos<br />
trabajos que han caracterizado <strong>la</strong> microbiota gastrointestinal <strong>de</strong> distintos peces<br />
cultivados, fundam<strong>en</strong>talm<strong>en</strong>te salmónidos (Spanggaard et al., 2000; Huber et al., 2004,<br />
J<strong>en</strong>s<strong>en</strong> et al., 2004; Burr et al., 2005), estos estudios son nulos <strong>en</strong> lo que se refiere a<br />
algunos <strong>de</strong> los peces marinos más cultivados <strong>en</strong> nuestra área, así como <strong>en</strong> <strong>la</strong> valoración<br />
<strong>de</strong> los efectos que sobre <strong>la</strong> microbiota gastrointestinal <strong>de</strong> estos peces pue<strong>de</strong> t<strong>en</strong>er <strong>la</strong><br />
aplicación <strong>de</strong> microorganismos probióticos. Este tipo <strong>de</strong> estudios son muy interesantes<br />
<strong>de</strong>s<strong>de</strong> el punto <strong>de</strong> vista <strong>de</strong> <strong>la</strong> información que pue<strong>de</strong>n aportar para una mejor aplicación<br />
<strong>de</strong> microorganismos <strong>en</strong> estrategias profilácticas y <strong>de</strong> biocontrol <strong>de</strong> <strong>en</strong>fermeda<strong>de</strong>s.<br />
33
O BJ E T I V O S
OBJETIVOS<br />
El trabajo p<strong>la</strong>nteado <strong>en</strong> esta Memoria <strong>de</strong> Tesis Doctoral consiste <strong>en</strong> <strong>la</strong><br />
profundización <strong>en</strong> el conocimi<strong>en</strong>to <strong>de</strong> ciertos aspectos <strong>de</strong> <strong>la</strong> virul<strong>en</strong>cia <strong>de</strong><br />
Photobacterium damse<strong>la</strong>e subsp. piscicida, así como <strong>la</strong> optimización <strong>de</strong> estrategias<br />
dirigidas a <strong>la</strong> prev<strong>en</strong>ción <strong>de</strong> <strong>la</strong> <strong>en</strong>fermedad que este patóg<strong>en</strong>o causa. En base a ello, se<br />
han p<strong>la</strong>nteado los sigui<strong>en</strong>tes objetivos:<br />
1. Estudio <strong>de</strong>l papel <strong>de</strong> <strong><strong>la</strong>s</strong> activida<strong>de</strong>s <strong>en</strong>zimáticas <strong>superóxido</strong> <strong>dismutasa</strong> y<br />
cata<strong><strong>la</strong>s</strong>a pres<strong>en</strong>tes <strong>en</strong> Photobacterium damse<strong>la</strong>e subsp. piscicida, evaluando<br />
su contribución a <strong>la</strong> resist<strong>en</strong>cia <strong>de</strong>l patóg<strong>en</strong>o fr<strong>en</strong>te a <strong>la</strong> acción bactericida <strong>de</strong><br />
los fagocitos <strong>de</strong> l<strong>en</strong>guado.<br />
2. Evaluación <strong>de</strong>l posible efecto inmunoestimu<strong>la</strong>nte sobre el estallido<br />
respiratorio <strong>de</strong> fagocitos <strong>de</strong> l<strong>en</strong>guados cultivados que pueda ejercer <strong>la</strong><br />
utilización <strong>de</strong> <strong>la</strong> microalga Porphyridium cru<strong>en</strong>tum y <strong>de</strong> microorganismos<br />
probióticos.<br />
37
M<br />
A T E R I A L Y M É T O D O S
MATERIA L Y MÉTODOS<br />
La metodología, así como el material empleado, <strong>en</strong> <strong>la</strong> realización <strong>de</strong> los difer<strong>en</strong>tes<br />
experim<strong>en</strong>tos que conforman esta Tesis Doctoral, se <strong>de</strong>tal<strong>la</strong>n <strong>en</strong> cada uno <strong>de</strong> los<br />
artículos incluidos <strong>en</strong> <strong>la</strong> Sección <strong>de</strong> artículos.<br />
41
R<br />
E S U L T A D O S Y D I S C U S I Ó N
RESULTADO S Y DISCUSIÓN<br />
El primer objetivo abordado <strong>en</strong> esta Tesis Doctoral ha consistido <strong>en</strong> el estudio <strong>de</strong>l<br />
papel <strong>de</strong> <strong><strong>la</strong>s</strong> activida<strong>de</strong>s <strong>en</strong>zimáticas <strong>superóxido</strong> <strong>dismutasa</strong> y cata<strong><strong>la</strong>s</strong>a <strong>en</strong> <strong>la</strong> virul<strong>en</strong>cia <strong>de</strong><br />
Photobacterium damse<strong>la</strong>e subsp. piscicida. En concreto, se ha evaluado <strong>la</strong> contribución<br />
<strong>de</strong> estas activida<strong>de</strong>s <strong>en</strong> <strong>la</strong> resist<strong>en</strong>cia <strong>de</strong>l patóg<strong>en</strong>o fr<strong>en</strong>te a <strong>la</strong> formación <strong>de</strong> radicales<br />
oxigénicos g<strong>en</strong>erados durante el estallido respiratorio <strong>de</strong> fagocitos <strong>de</strong> l<strong>en</strong>guado y, por<br />
tanto, su posible papel como factores <strong>de</strong> virul<strong>en</strong>cia (artículos 1.1. y 1.2., Sección <strong>de</strong><br />
artículos).<br />
Previam<strong>en</strong>te, Barnes et al. (1999a) <strong>de</strong>mostraron que este microorganismo in vitro<br />
conti<strong>en</strong>e sufici<strong>en</strong>te actividad <strong>superóxido</strong> <strong>dismutasa</strong> para <strong>de</strong>scomponer los aniones<br />
<strong>superóxido</strong> g<strong>en</strong>erados fotoquímicam<strong>en</strong>te, y que <strong>la</strong> susceptibilidad <strong>de</strong> <strong>la</strong> bacteria vi<strong>en</strong>e<br />
dada por <strong>la</strong> acumu<strong>la</strong>ción <strong>de</strong> peróxido <strong>de</strong> hidróg<strong>en</strong>o, ya que al adicionar cata<strong><strong>la</strong>s</strong>a <strong>la</strong><br />
superviv<strong>en</strong>cia se increm<strong>en</strong>ta. Estos hechos muestran <strong>la</strong> importancia <strong>de</strong>l peróxido <strong>de</strong><br />
hidróg<strong>en</strong>o <strong>en</strong> <strong>la</strong> inactivación <strong>de</strong> esta bacteria y, por consigui<strong>en</strong>te, <strong>la</strong> importancia <strong>de</strong> <strong>la</strong><br />
actividad cata<strong><strong>la</strong>s</strong>a <strong>en</strong> <strong>la</strong> resist<strong>en</strong>cia a este radical, cuyo papel no había sido <strong>de</strong>terminado<br />
anteriorm<strong>en</strong>te. En el pres<strong>en</strong>te trabajo, los resultados obt<strong>en</strong>idos sobre el papel <strong>de</strong> <strong>la</strong><br />
cata<strong><strong>la</strong>s</strong>a están <strong>de</strong>sarrol<strong>la</strong>dos <strong>en</strong> los artículos 1.1. y 1.2. (Sección <strong>de</strong> artículos). El primer<br />
artículo evalúa el papel in vitro <strong>de</strong> dicha <strong>en</strong>zima antioxidante <strong>en</strong> <strong>la</strong> protección <strong>de</strong> P.<br />
damse<strong>la</strong>e subsp. piscicida fr<strong>en</strong>te al peróxido <strong>de</strong> hidróg<strong>en</strong>o, <strong>de</strong>mostrándose <strong>la</strong> relevancia<br />
<strong>de</strong> <strong>la</strong> actividad cata<strong><strong>la</strong>s</strong>a <strong>en</strong> <strong>la</strong> resist<strong>en</strong>cia <strong>de</strong>l patóg<strong>en</strong>o fr<strong>en</strong>te a este radical oxig<strong>en</strong>ado.<br />
Los resultados obt<strong>en</strong>idos muestran que <strong>la</strong> resist<strong>en</strong>cia que confiere <strong>la</strong> actividad cata<strong><strong>la</strong>s</strong>a<br />
fr<strong>en</strong>te al peróxido <strong>de</strong> hidróg<strong>en</strong>o varía según <strong>la</strong> condición <strong>de</strong> cultivo a <strong>la</strong> que <strong>la</strong> bacteria<br />
se vea sometida. Así, cuando se ejerce un estrés oxidativo, por adición <strong>de</strong> peróxido <strong>de</strong><br />
hidróg<strong>en</strong>o, esa resist<strong>en</strong>cia se increm<strong>en</strong>ta; <strong>en</strong> cambio, cuando <strong><strong>la</strong>s</strong> condiciones son<br />
limitantes <strong>de</strong> hierro, los porc<strong>en</strong>tajes <strong>de</strong> superviv<strong>en</strong>cia disminuy<strong>en</strong>. A<strong>de</strong>más, según el<br />
grado <strong>de</strong> virul<strong>en</strong>cia esa resist<strong>en</strong>cia variará, si<strong>en</strong>do mayor <strong>en</strong> <strong>la</strong> cepa virul<strong>en</strong>ta que <strong>en</strong> <strong>la</strong><br />
no virul<strong>en</strong>ta. Por otro <strong>la</strong>do, <strong>en</strong> el artículo 1.2. (Sección <strong>de</strong> artículos) se <strong>de</strong>terminó <strong>la</strong><br />
pres<strong>en</strong>cia <strong>de</strong> actividad cata<strong><strong>la</strong>s</strong>a <strong>en</strong> difer<strong>en</strong>tes cepas <strong>de</strong> P. damse<strong>la</strong>e subsp. piscicida y se<br />
cuantificó espectrofotométricam<strong>en</strong>te dicha actividad <strong>en</strong> bacterias sometidas a difer<strong>en</strong>tes<br />
condiciones <strong>de</strong> cultivo. Los resultados obt<strong>en</strong>idos seña<strong>la</strong>n que los mayores porc<strong>en</strong>tajes <strong>de</strong><br />
superviv<strong>en</strong>cia coinci<strong>de</strong>n con <strong><strong>la</strong>s</strong> condiciones <strong>en</strong> <strong><strong>la</strong>s</strong> que los niveles <strong>de</strong> actividad cata<strong><strong>la</strong>s</strong>a<br />
<strong>de</strong>tectados son también superiores. Así, <strong>la</strong> cepa virul<strong>en</strong>ta Lg h41/01 pres<strong>en</strong>ta mayor<br />
45
RESULTADO S Y DISCUSIÓN<br />
resist<strong>en</strong>cia al estallido respiratorio <strong>de</strong> fagocitos <strong>de</strong> l<strong>en</strong>guado que <strong>la</strong> no virul<strong>en</strong>ta EPOY-<br />
8803-II, mi<strong>en</strong>tras que <strong>la</strong> adición <strong>de</strong> peróxido <strong>de</strong> hidróg<strong>en</strong>o a los cultivos increm<strong>en</strong>ta <strong>la</strong><br />
superviv<strong>en</strong>cia <strong>de</strong> ambos. La aus<strong>en</strong>cia <strong>de</strong> cápsu<strong>la</strong> podría ser un factor que haría disminuir<br />
<strong>la</strong> superviv<strong>en</strong>cia fr<strong>en</strong>te a los ROS, pero los bajos niveles <strong>de</strong> actividad cata<strong><strong>la</strong>s</strong>a obt<strong>en</strong>idos<br />
para EPOY-8803-II, sugier<strong>en</strong> que <strong>la</strong> falta <strong>de</strong> virul<strong>en</strong>cia podría v<strong>en</strong>ir <strong>de</strong>terminada, <strong>en</strong> gran<br />
parte, por <strong>la</strong> baja actividad <strong>en</strong>zimática <strong>de</strong> dicha proteína antioxidante, es <strong>de</strong>cir, <strong>la</strong><br />
cata<strong><strong>la</strong>s</strong>a ejerce un papel importante <strong>en</strong> <strong>la</strong> virul<strong>en</strong>cia <strong>de</strong> P. damse<strong>la</strong>e subsp. piscicida. En<br />
muchas bacterias ya se ha <strong>de</strong>mostrado que <strong>la</strong> exposición previa a conc<strong>en</strong>traciones<br />
subletales <strong>de</strong> un oxidante pue<strong>de</strong> inducir <strong>la</strong> protección fr<strong>en</strong>te a conc<strong>en</strong>traciones letales<br />
<strong>de</strong>l mismo (Mongkolsuk et al., 1996). Un ejemplo es el <strong>de</strong>scrito por Barnes et al. (1999b)<br />
<strong>en</strong> el caso <strong>de</strong> A. salmonicida, <strong>la</strong> cual, tras los pulsos <strong>de</strong> peróxido <strong>de</strong> hidróg<strong>en</strong>o, es capaz<br />
<strong>de</strong> resistir <strong>la</strong> conc<strong>en</strong>tración <strong>de</strong> peróxido <strong>de</strong> 100 mM, letal <strong>en</strong> aus<strong>en</strong>cia <strong>de</strong> pretratami<strong>en</strong>to<br />
con peróxido <strong>de</strong> hidróg<strong>en</strong>o. Es interesante seña<strong>la</strong>r que para EPOY-8803-II <strong><strong>la</strong>s</strong><br />
difer<strong>en</strong>cias, <strong>en</strong> cuanto a los porc<strong>en</strong>tajes <strong>de</strong> superviv<strong>en</strong>cia, son significativas <strong>en</strong>tre los<br />
cultivos crecidos hasta fase estacionaria y aquellos adicionados con peróxido, si<strong>en</strong>do<br />
estos últimos mayores. Sin embargo, para <strong>la</strong> cepa Lg h41/01 no ocurre lo mismo, ya que no<br />
se aprecian difer<strong>en</strong>cias significativas <strong>en</strong> <strong>la</strong> superviv<strong>en</strong>cia <strong>de</strong> los cultivos con o sin<br />
peróxido.<br />
Por otro <strong>la</strong>do, <strong>la</strong> superviv<strong>en</strong>cia disminuye <strong>de</strong> forma significativa <strong>en</strong> ambas cepas<br />
cuando se somet<strong>en</strong> a condiciones limitantes <strong>de</strong> hierro. No po<strong>de</strong>mos olvidar que estamos<br />
ante una cata<strong><strong>la</strong>s</strong>a <strong>de</strong> tipo férrica (artículo 1.2., Sección <strong>de</strong> artículos), por lo que <strong>la</strong><br />
car<strong>en</strong>cia <strong>de</strong> este metal limita su síntesis, y, por tanto, su actividad.<br />
Tanto <strong>superóxido</strong> <strong>dismutasa</strong> como cata<strong><strong>la</strong>s</strong>a son metalo<strong>en</strong>zimas, pudi<strong>en</strong>do poseer<br />
difer<strong>en</strong>tes metales <strong>en</strong> su c<strong>en</strong>tro activo. En <strong>la</strong> bibliografía se han <strong>de</strong>scrito<br />
microorganismos capaces <strong>de</strong> producir difer<strong>en</strong>tes iso<strong>en</strong>zimas <strong>de</strong> <strong>superóxido</strong> <strong>dismutasa</strong> y<br />
cata<strong><strong>la</strong>s</strong>a inducibles según <strong><strong>la</strong>s</strong> condiciones <strong>de</strong> cultivo a <strong><strong>la</strong>s</strong> que el microorganismo es<br />
sometido, como altos niveles <strong>de</strong> oxíg<strong>en</strong>o, bajos niveles <strong>de</strong> hierro o crecimi<strong>en</strong>to hasta <strong>la</strong><br />
fase estacionaria (Storz et al., 1990; Privalle y Fridovich, 1992; Crockford et al., 1995;<br />
Schnell y Steinman, 1995; Barnes et al., 1996; Po<strong>la</strong>ck et al., 1996; St. John y Steinman,<br />
46
RESULTADO S Y DISCUSIÓN<br />
1996; Lynch y Kuramitsu, 2000; Yesilkaya et al., 2000; Geslin et al., 2001; Vattanaviboon<br />
y Mongkolsuk, 2001).<br />
Sin embargo, aún es escasa <strong>la</strong> información sobre <strong><strong>la</strong>s</strong> activida<strong>de</strong>s <strong>superóxido</strong><br />
<strong>dismutasa</strong> y cata<strong><strong>la</strong>s</strong>a y <strong>la</strong> posible inducción <strong>de</strong> <strong><strong>la</strong>s</strong> difer<strong>en</strong>tes isoformas <strong>en</strong> P. damse<strong>la</strong>e<br />
subsp. piscicida. Así <strong>en</strong> este trabajo se <strong>de</strong>terminó si este patóg<strong>en</strong>o podría expresar<br />
difer<strong>en</strong>tes iso<strong>en</strong>zimas <strong>de</strong> <strong>superóxido</strong> <strong>dismutasa</strong> y <strong>de</strong> cata<strong><strong>la</strong>s</strong>a cuando es cultivado bajo<br />
difer<strong>en</strong>tes condiciones <strong>de</strong> cultivo. Los resultados obt<strong>en</strong>idos <strong>en</strong> este trabajo muestran que<br />
ninguna <strong>de</strong> <strong><strong>la</strong>s</strong> condiciones <strong>de</strong> cultivo <strong>en</strong>sayadas induc<strong>en</strong> <strong>la</strong> síntesis <strong>de</strong> difer<strong>en</strong>tes<br />
isoformas <strong>de</strong> <strong>superóxido</strong> <strong>dismutasa</strong> o cata<strong><strong>la</strong>s</strong>a <strong>en</strong> ninguna <strong>de</strong> <strong><strong>la</strong>s</strong> cepas <strong>de</strong> P. damse<strong>la</strong>e<br />
subsp. piscicida.<br />
Todas y cada una <strong>de</strong> <strong><strong>la</strong>s</strong> cepas <strong>en</strong>sayadas pres<strong>en</strong>tan una so<strong>la</strong> banda <strong>de</strong> actividad<br />
<strong>superóxido</strong> <strong>dismutasa</strong>, banda <strong>de</strong> simi<strong>la</strong>r movilidad electroforética a <strong>la</strong> <strong>superóxido</strong><br />
<strong>dismutasa</strong> férrica <strong>de</strong>scrita por Barnes et al. (1999a). Incluso bajo condiciones <strong>de</strong> estrés<br />
oxidativo, tras <strong>la</strong> adición <strong>de</strong> peróxido <strong>de</strong> hidróg<strong>en</strong>o o <strong>de</strong>l g<strong>en</strong>erador <strong>de</strong> radicales<br />
oxigénicos paraquat, no se induce una iso<strong>en</strong>zima distinta, estando <strong>de</strong>scrito que<br />
condiciones aeróbicas induc<strong>en</strong> <strong>la</strong> síntesis <strong>de</strong> CuZnSOD y <strong>de</strong> MnSOD <strong>en</strong> Escherichia<br />
coli (Hassan y Fridovich, 1977; Privalle y Fridovich, 1992; B<strong>en</strong>ov y Fridovich, 1994;<br />
Geslin et al., 2001), así como <strong>de</strong> esta última <strong>en</strong> A. salmonicida (Barnes et al., 1996;<br />
Barnes et al., 1999b), Pseudomonas aeruginosa (Po<strong>la</strong>ck et al., 1996) y Streptococcus<br />
pneumoniae (Yesilkaya et al., 2000). Ni tan siquiera condiciones restrictivas <strong>de</strong> hierro<br />
que induc<strong>en</strong> <strong>la</strong> síntesis <strong>de</strong> MnSOD (Privalle y Fridovich, 1992; Barnes et al., 1999b)<br />
induc<strong>en</strong> <strong>la</strong> síntesis <strong>de</strong> una iso<strong>en</strong>zima distinta. Aunque serían necesarios más estudios, <strong>la</strong><br />
falta <strong>de</strong> inducción <strong>de</strong> una SOD nueva podría ser <strong>de</strong>bida a <strong>la</strong> exist<strong>en</strong>cia <strong>de</strong> un único g<strong>en</strong><br />
codificador <strong>de</strong> <strong>la</strong> <strong>superóxido</strong> <strong>dismutasa</strong> férrica, el sodB (Lynch y Kuramitsu, 2000).<br />
De igual modo, todas <strong><strong>la</strong>s</strong> cepas, bajo todas <strong><strong>la</strong>s</strong> condiciones <strong>de</strong> cultivo <strong>en</strong>sayadas,<br />
pres<strong>en</strong>tan una so<strong>la</strong> banda <strong>de</strong> actividad cata<strong><strong>la</strong>s</strong>a <strong>de</strong> simi<strong>la</strong>r movilidad electroforética a <strong>la</strong><br />
ya <strong>de</strong>scrita por Barnes et al. (1999a). Esta <strong>en</strong>zima fue caracterizada, mediante el uso <strong>de</strong><br />
inhibidores, no pudi<strong>en</strong>do ser <strong>de</strong>tectada <strong>en</strong> geles tras <strong>la</strong> exposición a <strong>la</strong> azida sódica, y<br />
viéndose reducida ligeram<strong>en</strong>te tras el tratami<strong>en</strong>to con cianuro potásico, por lo que<br />
estamos ante una cata<strong><strong>la</strong>s</strong>a férrica, ya que <strong><strong>la</strong>s</strong> cata<strong><strong>la</strong>s</strong>as con manganeso <strong>en</strong> su c<strong>en</strong>tro<br />
47
RESULTADO S Y DISCUSIÓN<br />
activo reti<strong>en</strong><strong>en</strong> su actividad tras tratami<strong>en</strong>to con azida y cianuro, y son inhibidas con<br />
cloruro <strong>de</strong> mercurio (Kono y Fridovich, 1983; Allgood y Perry, 1986; Barnes et al.,<br />
1999b).<br />
A pesar <strong>de</strong> que ninguna <strong>de</strong> <strong><strong>la</strong>s</strong> condiciones <strong>de</strong> cultivo <strong>en</strong>sayadas induc<strong>en</strong> <strong>la</strong> síntesis<br />
<strong>de</strong> más <strong>de</strong> un tipo <strong>de</strong> iso<strong>en</strong>zima <strong>superóxido</strong> <strong>dismutasa</strong> o cata<strong><strong>la</strong>s</strong>a, sí se aprecian<br />
difer<strong>en</strong>cias <strong>en</strong> <strong>la</strong> int<strong>en</strong>sidad <strong>de</strong> <strong><strong>la</strong>s</strong> bandas <strong>de</strong>tectadas por electroforesis nativa, así como<br />
<strong>en</strong> los niveles <strong>de</strong> actividad, tras su cuantificación espectrofotométricam<strong>en</strong>te. Estos<br />
resultados concuerdan con los obt<strong>en</strong>idos por Barnes et al. (1999a) que <strong>de</strong>tectaron<br />
difer<strong>en</strong>cias <strong>en</strong> cultivos sometidos a difer<strong>en</strong>tes conc<strong>en</strong>traciones <strong>de</strong> hierro y distintos<br />
niveles <strong>de</strong> oxíg<strong>en</strong>o. Los niveles más bajos <strong>de</strong> actividad <strong>superóxido</strong> <strong>dismutasa</strong> y cata<strong><strong>la</strong>s</strong>a<br />
se obti<strong>en</strong><strong>en</strong> <strong>en</strong> cultivos sometidos a condiciones limitantes <strong>de</strong> hierro, hecho atribuible a<br />
<strong>la</strong> naturaleza férrica <strong>de</strong> ambas <strong>en</strong>zimas. Es interesante seña<strong>la</strong>r que, tanto para <strong>la</strong><br />
actividad <strong>superóxido</strong> <strong>dismutasa</strong>, como cata<strong><strong>la</strong>s</strong>a, bajo condiciones restrictivas <strong>de</strong> hierro,<br />
<strong>la</strong> cepa virul<strong>en</strong>ta pres<strong>en</strong>ta mayores niveles <strong>de</strong> actividad. Esto nos indica <strong>la</strong> importancia<br />
<strong>de</strong> <strong>la</strong> pres<strong>en</strong>cia <strong>de</strong> mecanismos <strong>de</strong> captación <strong>de</strong> hierro tanto para <strong>la</strong> expresión <strong>de</strong> SOD<br />
como <strong>de</strong> cata<strong><strong>la</strong>s</strong>a. De esta forma, los microorganismos capaces <strong>de</strong> obt<strong>en</strong>er hierro a partir<br />
<strong>de</strong>l hospedador serían capaces <strong>de</strong> expresar niveles más elevados <strong>de</strong> <strong><strong>la</strong>s</strong> <strong>en</strong>zimas<br />
antioxidantes y podrían <strong>de</strong>scomponer los radicales <strong>superóxido</strong> y peróxido <strong>de</strong> hidróg<strong>en</strong>o<br />
g<strong>en</strong>erados por dicho hospedador. Sin embargo, si bi<strong>en</strong> <strong>en</strong> otras especies bacterianas<br />
como Listeria monocytog<strong>en</strong>es (Welch et al., 1979), Shigel<strong>la</strong> flexneri (Franzon et al.,<br />
1990) o A. salmonicida (Barnes et al., 1999b) se ha <strong>de</strong>mostrado <strong>la</strong> importancia <strong>de</strong> SOD<br />
como <strong>en</strong>zima antioxidante y su contribución a <strong>la</strong> patogénesis, <strong>en</strong> el caso <strong>de</strong> P. damse<strong>la</strong>e<br />
subsp. piscicida este papel no está tan c<strong>la</strong>ro. Pero <strong>la</strong> cepa EPOY-8803-II, que no es<br />
virul<strong>en</strong>ta para l<strong>en</strong>guados, ti<strong>en</strong>e niveles <strong>de</strong> actividad cercanos a los <strong>de</strong> <strong>la</strong> cepa virul<strong>en</strong>ta.<br />
Estos resultados pue<strong>de</strong>n atribuirse a unos niveles <strong>de</strong>fici<strong>en</strong>tes <strong>de</strong> otras activida<strong>de</strong>s<br />
antioxidantes tales como cata<strong><strong>la</strong>s</strong>a, lo que haría que se dieran oxidaciones como<br />
consecu<strong>en</strong>cia <strong>de</strong> <strong>la</strong> acumu<strong>la</strong>ción <strong>de</strong> otros radicales <strong>de</strong>rivados <strong>de</strong> <strong>la</strong> <strong>de</strong>scomposición <strong>de</strong><br />
<strong>superóxido</strong>. Otra posible explicación a <strong>la</strong> no virul<strong>en</strong>cia <strong>de</strong> <strong>la</strong> cepa EPOY-8803-II es su<br />
car<strong>en</strong>cia <strong>de</strong> cápsu<strong>la</strong>, que <strong>la</strong> haría más susceptible al reconocimi<strong>en</strong>to por el sistema<br />
inmune <strong>de</strong>l hospedador.<br />
48
RESULTADO S Y DISCUSIÓN<br />
Una vez <strong>de</strong>terminada in vitro <strong>la</strong> importancia tanto <strong>de</strong> SOD (Barnes et al., 1999a),<br />
como <strong>de</strong> cata<strong><strong>la</strong>s</strong>a (artículo 1.2., Sección <strong>de</strong> artículos), se analizó el papel <strong>de</strong> estas<br />
activida<strong>de</strong>s in vivo, mediante incubación <strong>de</strong> <strong>la</strong> bacteria con fagocitos. Como se ha<br />
m<strong>en</strong>cionado anteriorm<strong>en</strong>te, <strong>la</strong> interacción, el modo <strong>de</strong> invadir y sobrevivir <strong>en</strong> el interior<br />
<strong>de</strong>l hospedador, <strong>en</strong>tre los fagocitos <strong>de</strong> l<strong>en</strong>guado y P. damse<strong>la</strong>e subsp. piscicida aún no<br />
se conoc<strong>en</strong> bi<strong>en</strong>. Mi<strong>en</strong>tras que algunos autores seña<strong>la</strong>n <strong>la</strong> pres<strong>en</strong>cia <strong>de</strong> célu<strong><strong>la</strong>s</strong> <strong>de</strong> P.<br />
damse<strong>la</strong>e subsp. piscicida intactas <strong>en</strong> el interior <strong>de</strong> célu<strong><strong>la</strong>s</strong> <strong>de</strong> dorada, sugiri<strong>en</strong>do <strong>la</strong><br />
capacidad <strong>de</strong> <strong>la</strong> bacteria para sobrevivir como patóg<strong>en</strong>o intracelu<strong>la</strong>r <strong>en</strong> dorada (Noya et<br />
al., 1995b; López-Dóriga et al., 2000), incluso <strong>de</strong> multiplicarse <strong>en</strong> el interior <strong>de</strong><br />
macrófagos <strong>de</strong>l pez (Kubota et al., 1970; Hawke et al., 1987; Noya et al., 1995a; Elkamel<br />
et al., 2003), otros han observado que este patóg<strong>en</strong>o es altam<strong>en</strong>te susceptible a los<br />
radicales oxidativos g<strong>en</strong>erados durante el estallido respiratorio <strong>en</strong> los fagocitos <strong>de</strong><br />
trucha, lubina y dorada (Skarmeta et al., 1995). Barnes et al. (1999a) confirmaron que P.<br />
damse<strong>la</strong>e subsp. piscicida es incapaz <strong>de</strong> respon<strong>de</strong>r al ataque oxidativo g<strong>en</strong>erado durante<br />
el estallido respiratorio, ya que <strong>en</strong> dicho trabajo <strong><strong>la</strong>s</strong> cepas <strong>en</strong>sayadas mostraron alta<br />
susceptibilidad a radicales <strong>de</strong> oxíg<strong>en</strong>o g<strong>en</strong>erados in vitro.<br />
Los resultados incluidos <strong>en</strong> el artículo 1.2. (Sección <strong>de</strong> artículos) muestran que P.<br />
damse<strong>la</strong>e subsp. piscicida es capaz <strong>de</strong> sobrevivir al m<strong>en</strong>os cinco horas <strong>en</strong> contacto con<br />
fagocitos <strong>de</strong> l<strong>en</strong>guado, si<strong>en</strong>do los porc<strong>en</strong>tajes <strong>de</strong> superviv<strong>en</strong>cia mayores <strong>en</strong> <strong>la</strong> cepa<br />
virul<strong>en</strong>ta (62%) que <strong>en</strong> <strong>la</strong> no virul<strong>en</strong>ta (19%), y correspondi<strong>en</strong>do <strong><strong>la</strong>s</strong> condiciones <strong>de</strong><br />
cultivo con aquel<strong><strong>la</strong>s</strong> <strong>en</strong> <strong><strong>la</strong>s</strong> que <strong>la</strong> actividad cata<strong><strong>la</strong>s</strong>a muestra también un increm<strong>en</strong>to, lo<br />
que indica un importante papel <strong>de</strong> <strong>la</strong> cata<strong><strong>la</strong>s</strong>a <strong>en</strong> <strong>la</strong> superviv<strong>en</strong>cia bacteriana. Estos<br />
resultados sugier<strong>en</strong> que <strong>la</strong> inactivación bacteriana podría ser <strong>de</strong>bida a <strong>la</strong> acumu<strong>la</strong>ción <strong>de</strong><br />
peróxido <strong>de</strong> hidróg<strong>en</strong>o, precursor <strong>de</strong>l radical hidroxilo. Mi<strong>en</strong>tras que Barnes et al.<br />
(1999a) mostraron que tanto <strong>la</strong> cepa virul<strong>en</strong>ta como <strong>la</strong> no virul<strong>en</strong>ta eran susceptibles a<br />
los radicales g<strong>en</strong>erados fotoquímicam<strong>en</strong>te, <strong>en</strong> este trabajo se <strong>de</strong>muestra que <strong>la</strong> cepa no<br />
virul<strong>en</strong>ta EPOY-8803-II, es significativam<strong>en</strong>te más susceptible que <strong>la</strong> virul<strong>en</strong>ta, Lg h41/01 ,<br />
al estallido respiratorio <strong>de</strong> fagocitos <strong>de</strong> l<strong>en</strong>guado. A<strong>de</strong>más <strong>de</strong> los niveles bajos <strong>de</strong><br />
actividad cata<strong><strong>la</strong>s</strong>a, esa susceptibilidad podría también ser causada por <strong>la</strong> aus<strong>en</strong>cia <strong>de</strong><br />
cápsu<strong>la</strong>, <strong>la</strong> cápsu<strong>la</strong> podría proteger a <strong><strong>la</strong>s</strong> bacterias <strong>de</strong> los radicales oxig<strong>en</strong>ados o prev<strong>en</strong>ir<br />
<strong>la</strong> activación <strong>de</strong> los fagocitos (Miller y Britigan, 1997; Arijo et al., 1998).<br />
49
RESULTADO S Y DISCUSIÓN<br />
Finalm<strong>en</strong>te, se ha constatado <strong>la</strong> importancia <strong>de</strong>l hierro <strong>en</strong> <strong>la</strong> resist<strong>en</strong>cia <strong>de</strong> <strong>la</strong><br />
bacteria P. damse<strong>la</strong>e subsp. piscicida, tal y como se ha <strong>de</strong>scrito <strong>en</strong> otros<br />
microorganismos (Miller y Britigan, 1997; Weinberg, 2000). P. damse<strong>la</strong>e subsp.<br />
piscicida es más susceptible a los fagocitos <strong>de</strong> l<strong>en</strong>guado cuando <strong><strong>la</strong>s</strong> célu<strong><strong>la</strong>s</strong> bacterianas<br />
son cultivadas bajo condiciones limitantes <strong>de</strong> hierro. La bacteria requiere hierro para su<br />
crecimi<strong>en</strong>to, replicación y síntesis <strong>de</strong> <strong>en</strong>zimas tales como <strong>superóxido</strong> <strong>dismutasa</strong> y<br />
cata<strong><strong>la</strong>s</strong>a, habiéndose <strong>de</strong>scrito <strong>la</strong> pres<strong>en</strong>cia <strong>de</strong> un si<strong>de</strong>róforo <strong>en</strong> P. damse<strong>la</strong>e subsp.<br />
piscicida (Magariños et al., 1994; Naka et al., 2005). Sin embargo, a pesar <strong>de</strong> su<br />
capacidad para obt<strong>en</strong>er hierro, varios autores han observado que <strong><strong>la</strong>s</strong> célu<strong><strong>la</strong>s</strong> cultivadas<br />
<strong>en</strong> condiciones limitantes <strong>de</strong> hierro, reduc<strong>en</strong> su material capsu<strong>la</strong>r (do Vale et al., 2001).<br />
Cabría p<strong>en</strong>sar que estas célu<strong><strong>la</strong>s</strong> con cápsu<strong>la</strong> reducida, serían más susceptibles a <strong>la</strong><br />
fagocitosis y al estrés oxidativo. Los resultados obt<strong>en</strong>idos (artículos 1.1. y 1.2., Sección<br />
<strong>de</strong> artículos) indican que el hierro juega un importante papel <strong>en</strong> <strong>la</strong> superviv<strong>en</strong>cia <strong>de</strong> P.<br />
damse<strong>la</strong>e subsp. piscicida <strong>en</strong> contacto con los fagocitos, sugiriéndose que ello es<br />
atribuible a <strong>la</strong> contribución <strong>de</strong>l material capsu<strong>la</strong>r, o a <strong>la</strong> síntesis <strong>de</strong> SOD y cata<strong><strong>la</strong>s</strong>a. En<br />
conclusión, P. damse<strong>la</strong>e subsp. piscicida es capaz <strong>de</strong> sobrevivir <strong>en</strong> contacto con<br />
fagocitos <strong>de</strong> l<strong>en</strong>guado, si<strong>en</strong>do los porc<strong>en</strong>tajes <strong>de</strong> superviv<strong>en</strong>cia mayores <strong>en</strong> <strong>la</strong> cepa<br />
virul<strong>en</strong>ta que <strong>en</strong> <strong>la</strong> no virul<strong>en</strong>ta. El hecho <strong>de</strong> que los niveles <strong>de</strong> cata<strong><strong>la</strong>s</strong>a también se vean<br />
increm<strong>en</strong>tados sugiere un posible papel <strong>de</strong> <strong>la</strong> <strong>en</strong>zima cata<strong><strong>la</strong>s</strong>a <strong>en</strong> <strong>la</strong> superviv<strong>en</strong>cia<br />
bacteriana.<br />
Una vez <strong>de</strong>terminados los papeles <strong>de</strong> <strong><strong>la</strong>s</strong> activida<strong>de</strong>s <strong>superóxido</strong> <strong>dismutasa</strong> y<br />
cata<strong><strong>la</strong>s</strong>a <strong>en</strong> <strong>la</strong> protección <strong>de</strong> P. damse<strong>la</strong>e subsp. piscicida fr<strong>en</strong>te al estallido respiratorio<br />
<strong>de</strong> fagocitos <strong>de</strong> l<strong>en</strong>guado, el sigui<strong>en</strong>te problema a abordar fue <strong>la</strong> búsqueda <strong>de</strong> estrategias<br />
para <strong>la</strong> prev<strong>en</strong>ción <strong>de</strong> <strong>la</strong> <strong>en</strong>fermedad que dicha bacteria produce. Como ya hemos<br />
m<strong>en</strong>cionado anteriorm<strong>en</strong>te, una <strong>de</strong> <strong><strong>la</strong>s</strong> líneas a <strong>de</strong>sarrol<strong>la</strong>r <strong>en</strong> <strong>la</strong> prev<strong>en</strong>ción y tratami<strong>en</strong>to<br />
<strong>de</strong> <strong>la</strong> pseudotuberculosis es <strong>la</strong> aplicación <strong>de</strong> inmunoestimu<strong>la</strong>ntes, que increm<strong>en</strong>tan <strong>la</strong><br />
respuesta inmune <strong>de</strong>l hospedador, fr<strong>en</strong>te a una infección. El parámetro inmunológico <strong>en</strong><br />
el que se ha c<strong>en</strong>trado esta Memoria es el estallido respiratorio <strong>en</strong> fagocitos <strong>de</strong> l<strong>en</strong>guado<br />
y como posibles immunoestimu<strong>la</strong>ntes, dos tipos <strong>de</strong> microorganismos: una micralga roja,<br />
Porphyridium cru<strong>en</strong>tum y dos bacterias pot<strong>en</strong>cialm<strong>en</strong>te probióticas, cuyas activida<strong>de</strong>s<br />
50
RESULTADO S Y DISCUSIÓN<br />
inmunoestimu<strong>la</strong>ntes han sido <strong>de</strong>mostradas <strong>en</strong> fagocitos <strong>de</strong> mamíferos (Morris et al.,<br />
2000) y <strong>de</strong> dorada (Díaz-Rosales et al., 2006; Salinas et al., 2006), respectivam<strong>en</strong>te.<br />
En primer lugar se evaluó <strong>la</strong> posible actividad inmunoestimu<strong>la</strong>nte <strong>de</strong>l estallido<br />
respiratorio <strong>de</strong> fagocitos <strong>de</strong> l<strong>en</strong>guado ejercido por P. cru<strong>en</strong>tum. Para ello se realizaron<br />
experim<strong>en</strong>tos in vitro con los extractos acuosos y etanólicos obt<strong>en</strong>idos a partir <strong>de</strong>l alga,<br />
ya que exist<strong>en</strong> numerosos datos acerca <strong>de</strong> <strong>la</strong> capacidad <strong>de</strong> estimu<strong>la</strong>ción <strong>de</strong> difer<strong>en</strong>tes<br />
extractos algales sobre el sistema inmune <strong>de</strong> peces (Fujiki et al., 1992; Castro et al.,<br />
2004; Díaz-Rosales et al., 2005; Hou y Ch<strong>en</strong>, 2005; Castro et al., 2006). Los resultados<br />
obt<strong>en</strong>idos (artículo 2.1., Sección <strong>de</strong> artículos) muestran que ninguno <strong>de</strong> los dos extractos<br />
estimu<strong>la</strong> <strong>la</strong> producción <strong>de</strong>l anión <strong>superóxido</strong>, mi<strong>en</strong>tras que el control positivo que se<br />
<strong>en</strong>sayó, β-glucano comercial extraído <strong>de</strong>l alga Eugl<strong>en</strong>a gracilis, sí es capaz <strong>de</strong><br />
increm<strong>en</strong>tar el estallido respiratorio tras 30 min <strong>en</strong> contacto con los fagocitos pero sólo a<br />
<strong>la</strong> conc<strong>en</strong>tración más alta <strong>en</strong>sayada (10 mg ml -1 ). Estos resultados concuerdan con los<br />
<strong>de</strong>scritos por Castro et al. (1999), que observó increm<strong>en</strong>to <strong>de</strong>l estallido respiratorio <strong>en</strong><br />
fagocitos <strong>de</strong> rodaballo (Psetta maxima) y dorada tras <strong>en</strong>sayar difer<strong>en</strong>tes conc<strong>en</strong>traciones<br />
<strong>de</strong> β-glucanos proce<strong>de</strong>ntes <strong>de</strong> hongos y levaduras. Por otro <strong>la</strong>do, Castro et al. (2004)<br />
<strong>en</strong>contraron gran<strong>de</strong>s variaciones <strong>en</strong> <strong><strong>la</strong>s</strong> capacida<strong>de</strong>s inmunoestimu<strong>la</strong>ntes <strong>de</strong> los extractos<br />
algales <strong>de</strong>p<strong>en</strong>di<strong>en</strong>do, no sólo <strong>de</strong>l orig<strong>en</strong>, sino también <strong>de</strong> <strong><strong>la</strong>s</strong> conc<strong>en</strong>traciones <strong>en</strong>sayadas<br />
y <strong>de</strong> los tiempos <strong>de</strong> incubación.<br />
P. cru<strong>en</strong>tum pres<strong>en</strong>ta varias v<strong>en</strong>tajas <strong>en</strong> su cultivo, pues ti<strong>en</strong>e un crecimi<strong>en</strong>to<br />
rápido y con bajo coste, lo que hace que sea muy fácil trabajar con el<strong>la</strong>. A<strong>de</strong>más, el<br />
hecho <strong>de</strong> ser una sustancia natural permite consi<strong>de</strong>rar<strong>la</strong> a priori como biocompatible,<br />
bio<strong>de</strong>gradable y segura para el medio ambi<strong>en</strong>te y <strong>la</strong> salud humana, características que<br />
permit<strong>en</strong> consi<strong>de</strong>rar<strong>la</strong> una bu<strong>en</strong>a sustancia inmunoestimu<strong>la</strong>nte. Por ello, y una vez<br />
<strong>en</strong>sayados in vitro los extractos acuosos y etanólicos <strong>de</strong> <strong>la</strong> microalga, se realizó un<br />
experim<strong>en</strong>to in vivo <strong>en</strong> el que se administró por vía oral el alga completa (artículo 2.1.,<br />
Sección <strong>de</strong> artículos). Hasta ahora, los trabajos realizados administrando<br />
microorganismos completos a peces han sido, principalm<strong>en</strong>te, con bacterias,<br />
consi<strong>de</strong>radas probióticas (Verschuere et al., 2000; Nikoske<strong>la</strong>in<strong>en</strong> et al., 2001; Irianto y<br />
Austin, 2003; Salinas et al., 2005; Balcázar et al., 2006; Díaz-Rosales et al., 2006; Salinas<br />
51
RESULTADO S Y DISCUSIÓN<br />
et al., 2006) pero son muy escasos los estudios sobre algas completas (Blinkova et al.,<br />
2001; Val<strong>en</strong>te et al., 2006). Por otro <strong>la</strong>do, P. cru<strong>en</strong>tum acumu<strong>la</strong> gran<strong>de</strong>s cantida<strong>de</strong>s <strong>de</strong><br />
lípidos, como ácido araquidónico o eicosap<strong>en</strong>tanoico (Kinsel<strong>la</strong> et al., 1990; Kov<strong>en</strong> et al.,<br />
2001); carbohidratos (Fujiki et al., 1992; Santarém et al., 1997; Bagni et al., 2000; Morris<br />
et al., 2000; Esteban et al., 2001; J<strong>en</strong>ey y J<strong>en</strong>ey, 2002; Cook et al., 2003; Couso et al.,<br />
2003; Castro et al., 2004; Bagni et al., 2005; Kumar et al., 2005); carot<strong>en</strong>os (Tachinaba et<br />
al., 1997; Amar et al., 2004); vitaminas (Hardie et al., 1990, 1991; Ortuño et al., 1999;<br />
J<strong>en</strong>ey y J<strong>en</strong>ey, 2002). Debido al hecho <strong>de</strong> cont<strong>en</strong>er difer<strong>en</strong>tes sustancias<br />
inmunoestimu<strong>la</strong>ntes, su uso podría g<strong>en</strong>erar una respuesta inmune más g<strong>en</strong>eral como ya<br />
ha sido propuesta para otros microorganismos como levaduras (Ortuño et al., 2002;<br />
Rodríguez et al., 2003).<br />
Así, durante cuatro semanas, se alim<strong>en</strong>taron tres grupos <strong>de</strong> l<strong>en</strong>guados con pi<strong>en</strong>so<br />
suplem<strong>en</strong>tado con <strong>la</strong> microalga liofilizada, pi<strong>en</strong>so normal, o con un inmunoestimu<strong>la</strong>nte<br />
comercial, Sanostim. A<strong>de</strong>más, para evaluar el posible efecto sinérgico <strong>de</strong>l<br />
inmunoestimu<strong>la</strong>nte con una vacuna, tras dos semanas <strong>de</strong>l inicio <strong>de</strong>l experim<strong>en</strong>to, un<br />
conjunto <strong>de</strong> peces fue inocu<strong>la</strong>do intraperitonealm<strong>en</strong>te con una bacterina compuesta por<br />
célu<strong><strong>la</strong>s</strong> <strong>de</strong> P. damse<strong>la</strong>e subsp. piscicida inactivadas con formol. Los resultados<br />
obt<strong>en</strong>idos muestran que tras cuatro semanas <strong>de</strong> administración <strong>de</strong>l alga como<br />
suplem<strong>en</strong>to <strong>en</strong> <strong>la</strong> dieta normal <strong>de</strong> los peces, <strong>la</strong> producción <strong>de</strong> anión <strong>superóxido</strong> se<br />
increm<strong>en</strong>ta <strong>en</strong> aquellos l<strong>en</strong>guados que han sido inmunizados con <strong>la</strong> vacuna. Ese<br />
increm<strong>en</strong>to es estadísticam<strong>en</strong>te significativo, no sólo respecto a los peces alim<strong>en</strong>tados<br />
con una dieta normal, sino también respecto a los peces alim<strong>en</strong>tados con el<br />
inmunoestimu<strong>la</strong>nte comercial. La acción conjunta <strong>de</strong> un inmunoestimu<strong>la</strong>nte y una<br />
vacuna ha sido <strong>de</strong>scrita por numerosos autores que concluy<strong>en</strong> que <strong>la</strong> combinación <strong>de</strong><br />
vacunación y administración <strong>de</strong> inmunoestimu<strong>la</strong>nte increm<strong>en</strong>ta <strong>la</strong> pot<strong>en</strong>cia <strong>de</strong> <strong>la</strong> vacuna<br />
(J<strong>en</strong>ey y An<strong>de</strong>rson, 1993; RØsrstad et al., 1993; Aakre et al., 1994; Sakai et al., 1995;<br />
Baulny et al., 1996; Sakai, 1999).<br />
Seguidam<strong>en</strong>te, y una vez <strong>de</strong>mostrado el efecto inmunoestimu<strong>la</strong>nte <strong>de</strong>l alga, se<br />
valoró el efecto inmunoestimu<strong>la</strong>nte <strong>de</strong> <strong>la</strong> fracción polisacarídica extracelu<strong>la</strong>r <strong>de</strong> P.<br />
52
RESULTADO S Y DISCUSIÓN<br />
cru<strong>en</strong>tum sobre el estallido respiratorio <strong>de</strong> fagocitos <strong>de</strong> l<strong>en</strong>guado, tanto in vitro como in<br />
vivo (artículo 2.2., Sección <strong>de</strong> artículos).<br />
Los resultados obt<strong>en</strong>idos indican que in vitro, <strong>de</strong>spués <strong>de</strong> treinta minutos <strong>de</strong><br />
contacto con <strong><strong>la</strong>s</strong> célu<strong><strong>la</strong>s</strong> <strong>de</strong>l pez, ninguna <strong>de</strong> <strong><strong>la</strong>s</strong> conc<strong>en</strong>traciones <strong>en</strong>sayadas <strong>de</strong>l<br />
polisacárido extracelu<strong>la</strong>r <strong>de</strong> P. cru<strong>en</strong>tum estimu<strong>la</strong> el estallido respiratorio <strong>de</strong> fagocitos<br />
<strong>de</strong> l<strong>en</strong>guado. Estos resultados contrastan con los obt<strong>en</strong>idos por Castro et al. (2004, 2006)<br />
qui<strong>en</strong>es sugier<strong>en</strong> que <strong>la</strong> estimu<strong>la</strong>ción <strong>de</strong>l estallido respiratorio <strong>en</strong> fagocitos <strong>de</strong> rodaballo<br />
(Psetta maxima), <strong>en</strong> pres<strong>en</strong>cia <strong>de</strong> extractos <strong>de</strong> algas, es <strong>de</strong>bida a los polisacáridos<br />
algales. Por otro <strong>la</strong>do, los resultados obt<strong>en</strong>idos por Castro et al. (2004) indican que <strong>la</strong><br />
capacidad inmunoestimu<strong>la</strong>nte varía <strong>en</strong> gran medida según <strong>la</strong> especie <strong>de</strong> alga. Por lo que<br />
<strong>la</strong> falta <strong>de</strong> estimu<strong>la</strong>ción <strong>de</strong>l estallido respiratorio in vitro por parte <strong>de</strong> los polisacáridos<br />
<strong>de</strong> P. cru<strong>en</strong>tum pue<strong>de</strong> ser <strong>de</strong>bida, por un <strong>la</strong>do, a que simplem<strong>en</strong>te no t<strong>en</strong>gan capacidad<br />
inmunoestimu<strong>la</strong>nte, <strong>la</strong> conc<strong>en</strong>tración a <strong>la</strong> que se <strong>en</strong>cu<strong>en</strong>tra sea baja, o el tiempo <strong>de</strong><br />
contacto con los fagocitos sea insufici<strong>en</strong>te. Sin embargo, el control positivo que<br />
cont<strong>en</strong>ía β-glucano comercial sí induce un increm<strong>en</strong>to <strong>en</strong> el estallido respiratorio,<br />
aunque únicam<strong>en</strong>te a <strong>la</strong> conc<strong>en</strong>tración mayor <strong>en</strong>sayada (10 mg ml -1 ).<br />
Adicionalm<strong>en</strong>te, los l<strong>en</strong>guados se inocu<strong>la</strong>ron intraperitonealm<strong>en</strong>te con 500 µg <strong>de</strong> <strong>la</strong><br />
fracción extracelu<strong>la</strong>r polisacarídica. Como <strong>en</strong> el trabajo anterior, posteriorm<strong>en</strong>te se<br />
inmunizaron con una bacterina compuesta por célu<strong><strong>la</strong>s</strong> <strong>de</strong> P. damse<strong>la</strong>e subsp. piscicida<br />
inactivadas con formol. Se tomaron muestras a <strong><strong>la</strong>s</strong> 24 h y a los 7 días. Los resultados<br />
obt<strong>en</strong>idos indican que a <strong>la</strong> conc<strong>en</strong>tración y tiempos <strong>en</strong>sayados, 1 y 7 días tras <strong>la</strong><br />
inocu<strong>la</strong>ción <strong>de</strong> <strong>la</strong> fracción polisacarídica, no se produce un increm<strong>en</strong>to <strong>en</strong> el estallido<br />
respiratorio <strong>de</strong> fagocitos <strong>de</strong> l<strong>en</strong>guado, ni siquiera <strong>en</strong> los peces inmunizados. A<strong>de</strong>más, se<br />
observa que 24 h post-inocu<strong>la</strong>ción, el estallido respiratorio se reduce <strong>en</strong> los fagocitos <strong>de</strong><br />
los peces inocu<strong>la</strong>dos con <strong>la</strong> fracción polisacarídica o con <strong>la</strong> bacterina. Esta disminución<br />
<strong>de</strong>l estallido respiratorio podría ser atribuida a una inmunosupresión causada por estrés<br />
tras <strong>la</strong> manipu<strong>la</strong>ción (Thompson et al., 1993; Pulsford et al., 1995), <strong>de</strong>sapareci<strong>en</strong>do este<br />
efecto tras siete días <strong>de</strong>l inicio <strong>de</strong>l experim<strong>en</strong>to.<br />
En resum<strong>en</strong>, se pue<strong>de</strong> concluir que <strong>la</strong> fracción polisacarídica <strong>de</strong> P. cru<strong>en</strong>tum <strong>en</strong> <strong><strong>la</strong>s</strong><br />
condiciones <strong>de</strong>scritas no increm<strong>en</strong>tó el estallido respiratorio <strong>de</strong> fagocitos <strong>de</strong> l<strong>en</strong>guado.<br />
53
RESULTADO S Y DISCUSIÓN<br />
Estos resultados sugier<strong>en</strong> que <strong>la</strong> estimu<strong>la</strong>ción observada tras <strong>la</strong> administración por vía<br />
oral <strong>de</strong>l alga completa liofilizada sería <strong>de</strong>bida a otro <strong>de</strong> los compon<strong>en</strong>tes <strong>de</strong>l alga, con<br />
propieda<strong>de</strong>s inmunoestimu<strong>la</strong>ntes. Por otro <strong>la</strong>do, <strong>la</strong> acción inmunoestimu<strong>la</strong>nte <strong>de</strong>l<br />
polisacárido pue<strong>de</strong> estar dirigida a otro parámetro inmunológico, no necesariam<strong>en</strong>te al<br />
estallido respiratorio, así exist<strong>en</strong> numerosos trabajos que <strong>de</strong>scrib<strong>en</strong> <strong>la</strong> capacidad<br />
inmunoestimu<strong>la</strong>nte <strong>de</strong> polisacáridos <strong>en</strong> peces, increm<strong>en</strong>tando <strong>la</strong> actividad lisozima <strong>en</strong> el<br />
suero, <strong>la</strong> actividad <strong>de</strong>l complem<strong>en</strong>to, <strong>la</strong> actividad citotóxica <strong>de</strong> fagocitos o <strong>la</strong> actividad<br />
fagocítica, <strong>en</strong>tre otras (Santarém et al., 1997; Esteban et al., 2001; Chang et al., 2003;<br />
Bagni et al., 2005; Kumari y Sahoo, 2006).<br />
Otra <strong>de</strong> <strong><strong>la</strong>s</strong> estrategias <strong>de</strong>sarrol<strong>la</strong>das <strong>en</strong> esta Memoria para el tratami<strong>en</strong>to <strong>de</strong> <strong>la</strong><br />
pseudotuberculosis fue el empleo <strong>de</strong> bacterias pot<strong>en</strong>cialm<strong>en</strong>te probióticas. En el trabajo<br />
realizado (artículo 2.3., Sección <strong>de</strong> artículos) <strong><strong>la</strong>s</strong> bacterias pot<strong>en</strong>cialm<strong>en</strong>te probióticas<br />
fueron administradas por vía oral, suplem<strong>en</strong>tándose el pi<strong>en</strong>so con el que eran<br />
alim<strong>en</strong>tados los l<strong>en</strong>guados. En este caso, a<strong>de</strong>más <strong>de</strong> evaluar el estallido respiratorio <strong>de</strong><br />
fagocitos <strong>de</strong> riñón, se llevó a cabo una infección experim<strong>en</strong>tal con P. damse<strong>la</strong>e subsp.<br />
piscicida, para <strong>de</strong>terminar el grado <strong>de</strong> protección que pudieran aportar los probióticos.<br />
Al mismo tiempo, se estudió <strong>la</strong> microbiota intestinal, con objeto <strong>de</strong> <strong>de</strong>tectar posibles<br />
cambios que produjeran los probióticos incorporados <strong>en</strong> <strong>la</strong> dieta.<br />
Las bacterias seleccionadas para este experim<strong>en</strong>to fueron <strong><strong>la</strong>s</strong> cepas Pdp11 y Pdp13,<br />
ais<strong>la</strong>das <strong>de</strong> piel <strong>de</strong> dorada (Chabrillón, 2003). Su i<strong>de</strong>ntificación nos lleva a situar<strong><strong>la</strong>s</strong><br />
<strong>de</strong>ntro <strong>de</strong> <strong>la</strong> familia Alteromonadaceae, género Shewanel<strong>la</strong>. Es el primer <strong>en</strong>sayo in vivo<br />
que se realiza con Pdp13, <strong>en</strong> cambio con <strong>la</strong> cepa Pdp11 ya exist<strong>en</strong> varios trabajos<br />
publicados, así Chabrillón et al. (2005a) estudiaron <strong>la</strong> interacción con el patóg<strong>en</strong>o Vibrio<br />
harveyi, mostrando <strong>la</strong> capacidad <strong>de</strong> Pdp11 <strong>de</strong> adherirse al mucus intestinal <strong>de</strong> dorada, el<br />
efecto antagonista fr<strong>en</strong>te a una cepa patóg<strong>en</strong>a <strong>de</strong> V. harveyi, <strong>la</strong> capacidad <strong>de</strong> inhibir <strong>la</strong><br />
unión <strong>de</strong>l patóg<strong>en</strong>o y, por último, <strong>de</strong> conferir protección fr<strong>en</strong>te una infección<br />
experim<strong>en</strong>tal. También se han publicado resultados obt<strong>en</strong>idos con Pdp11 y <strong>la</strong> bacteria<br />
patóg<strong>en</strong>a P. damse<strong>la</strong>e subsp. piscicida, así Chabrillón et al. (2005b) <strong>de</strong>mostraron el<br />
efecto antagonista <strong>de</strong> Pdp11 fr<strong>en</strong>te a una cepa <strong>de</strong> P. damse<strong>la</strong>e subsp. piscicida y <strong>la</strong><br />
inhibición <strong>de</strong> <strong>la</strong> unión <strong>de</strong> dicho patóg<strong>en</strong>o al mucus intestinal. Estos resultados obt<strong>en</strong>idos<br />
54
RESULTADO S Y DISCUSIÓN<br />
permit<strong>en</strong> consi<strong>de</strong>rar a <strong>la</strong> cepa Pdp11 como una bu<strong>en</strong>a candidata para ser usada como<br />
probiótico.<br />
En cuanto a <strong>la</strong> posible estimu<strong>la</strong>ción <strong>de</strong>l estallido respiratorio se ha observado que<br />
mi<strong>en</strong>tras <strong>la</strong> producción <strong>de</strong> anión <strong>superóxido</strong> por parte <strong>de</strong> los fagocitos ais<strong>la</strong>dos <strong>de</strong> peces<br />
alim<strong>en</strong>tados con <strong>la</strong> cepa Pdp11 se ve increm<strong>en</strong>tada significativam<strong>en</strong>te transcurridos dos<br />
meses <strong>de</strong>s<strong>de</strong> el inicio <strong>de</strong>l experim<strong>en</strong>to, los peces alim<strong>en</strong>tados con <strong>la</strong> cepa Pdp13 no<br />
muestran esta respuesta. El efecto <strong>de</strong> <strong>la</strong> cepa Pdp13 se observa tras <strong>la</strong> infección<br />
experim<strong>en</strong>tal, ya que el grupo <strong>de</strong> peces alim<strong>en</strong>tados con esta cepa alcanza un porc<strong>en</strong>taje<br />
<strong>de</strong> superviv<strong>en</strong>cia más elevado tras <strong>la</strong> inocu<strong>la</strong>ción <strong>de</strong>l patóg<strong>en</strong>o P. damse<strong>la</strong>e subsp.<br />
piscicida.<br />
Aunque exist<strong>en</strong> numerosos trabajos <strong>en</strong> los que se muestra <strong>la</strong> inducción <strong>de</strong>l estallido<br />
respiratorio por probióticos (Nikoske<strong>la</strong>in<strong>en</strong> et al., 2003; Gullian et al., 2004; Aubin et al.,<br />
2005; Brunt y Austin, 2005), el que el pot<strong>en</strong>cial probiótico Pdp13 no haya increm<strong>en</strong>tado<br />
<strong>la</strong> producción <strong>de</strong> anión <strong>superóxido</strong>, no significa que no sea capaz <strong>de</strong> inducir algún otro<br />
parámetro inmunológico. De hecho, el porc<strong>en</strong>taje <strong>de</strong> superviv<strong>en</strong>cia, tras <strong>la</strong> infección<br />
experim<strong>en</strong>tal, sí se ve increm<strong>en</strong>tado. Varios autores han mostrado el efecto <strong>de</strong><br />
probióticos sobre parámetros inmunológicos difer<strong>en</strong>tes al estallido respiratorio, como<br />
<strong><strong>la</strong>s</strong> activida<strong>de</strong>s fagocítica (Irianto y Austin, 2003; Panigrahi et al., 2004; Brunt y Austin,<br />
2005; Salinas et al., 2005; Díaz-Rosales et al., 2006), <strong>de</strong>l complem<strong>en</strong>to (Panigrahi et al.,<br />
2004; Díaz-Rosales et al., 2006), <strong>de</strong> <strong>la</strong> lisozima (Irianto y Austin, 2003; Panigrahi et al.,<br />
2004) o <strong>la</strong> citotóxica (Salinas et al., 2005; Díaz-Rosales et al., 2006). A<strong>de</strong>más ha sido<br />
<strong>de</strong>scrito el efecto <strong>de</strong> los probióticos no sólo sobre <strong>la</strong> respuesta inmune inespecífica, sino<br />
también sobre <strong>la</strong> específica, increm<strong>en</strong>tando los niveles <strong>de</strong> inmunoglobulinas <strong>en</strong> el suero<br />
(Nikoske<strong>la</strong>in<strong>en</strong> et al., 2003; Aubin et al., 2005). Por estas razones, <strong>la</strong> cepa Pdp13 pue<strong>de</strong><br />
ser consi<strong>de</strong>rado también como probiótico, aunque sería necesaria <strong>la</strong> evaluación <strong>de</strong> otros<br />
parámetros inmunológicos.<br />
Por otro <strong>la</strong>do, <strong><strong>la</strong>s</strong> bacterias probióticas ti<strong>en</strong><strong>en</strong> que ser administradas a una dosis<br />
óptima, que <strong>de</strong>p<strong>en</strong><strong>de</strong>rá <strong>de</strong>l tamaño <strong>de</strong>l pez y <strong>de</strong> <strong>la</strong> cepa (Nikoske<strong>la</strong>in<strong>en</strong> et al., 2003), por<br />
esta razón pue<strong>de</strong> que el efecto <strong>de</strong> <strong>la</strong> cepa Pdp11 sea difer<strong>en</strong>te al <strong>de</strong> Pdp13, y que los<br />
55
RESULTADO S Y DISCUSIÓN<br />
resultados obt<strong>en</strong>idos con Pdp11 y l<strong>en</strong>guado sean difer<strong>en</strong>tes a los obt<strong>en</strong>idos previam<strong>en</strong>te<br />
con Pdp11 y dorada (Díaz-Rosales et al., 2006).<br />
Por último, a<strong>de</strong>más <strong>de</strong>l efecto inmunológico <strong>de</strong> los probióticos sobre <strong>la</strong> respuesta<br />
inmune <strong>de</strong> l<strong>en</strong>guado, se han evaluado posibles cambios <strong>en</strong> <strong>la</strong> microbiota intestinal<br />
provocados por los probióticos adicionados a <strong>la</strong> dieta. A pesar <strong>de</strong>l gran número <strong>de</strong><br />
trabajos publicados que estudian <strong><strong>la</strong>s</strong> comunida<strong>de</strong>s microbianas <strong>de</strong> peces (Spanggaard et<br />
al., 2000; Holb<strong>en</strong> et al., 2002; Sandaa et al., 2003; Al-Harbi y Naim Uddin, 2004; Hjelm et<br />
al., 2004; Huber et al., 2004; J<strong>en</strong>s<strong>en</strong> et al., 2004), ninguno evalúa los posibles cambios <strong>en</strong><br />
esa microbiota tras <strong>la</strong> administración <strong>de</strong> probióticos.<br />
En este trabajo se ha empleado <strong>la</strong> técnica <strong>de</strong> electroforesis <strong>en</strong> geles <strong>de</strong> gradi<strong>en</strong>te<br />
<strong>de</strong>snaturalizante (DGGE) (Muyzer et al., 1993) para estudiar <strong>la</strong> microbiota intestinal <strong>de</strong><br />
l<strong>en</strong>guados alim<strong>en</strong>tados con probióticos. Se <strong>en</strong>sayaron dos pares <strong>de</strong> cebadores<br />
universales (Nübel et al., 1996; J<strong>en</strong>s<strong>en</strong> et al., 2004), y tras el análisis <strong>de</strong>l patrón <strong>de</strong> bandas<br />
por el coefici<strong>en</strong>te <strong>de</strong> Pearson, el <strong>de</strong> Nübel et al. (1996) fue seleccionado como el mejor<br />
para el estudio <strong>de</strong> <strong><strong>la</strong>s</strong> comunida<strong>de</strong>s con <strong><strong>la</strong>s</strong> que se estaba trabajando.<br />
El patrón <strong>de</strong> bandas obt<strong>en</strong>ido fue muy simple, con pocas bandas predominantes, lo<br />
que podría concordar con lo <strong>de</strong>scrito por Muyzer et al. (1993) qui<strong>en</strong>es afirman que<br />
comunida<strong>de</strong>s con pocas especies dominantes producirán patrones más simples y que <strong><strong>la</strong>s</strong><br />
especies m<strong>en</strong>os abundantes no estarán repres<strong>en</strong>tadas a<strong>de</strong>cuadam<strong>en</strong>te <strong>en</strong> dicho patrón.<br />
A<strong>de</strong>más <strong>la</strong> técnica pres<strong>en</strong>ta limitaciones, y es posible que algunas bandas no repres<strong>en</strong>t<strong>en</strong><br />
especies individuales, como sería <strong>en</strong> teoría, sino que grupos <strong>de</strong> especies pue<strong>de</strong>n t<strong>en</strong>er el<br />
mismo cont<strong>en</strong>ido re<strong>la</strong>tivo <strong>de</strong> G+C, comigrando (Simpson et al., 1999; Temmerman et<br />
al., 2003). Estas limitaciones conducirían a un <strong>de</strong>sc<strong>en</strong>so <strong>en</strong> el número <strong>de</strong> bandas<br />
pres<strong>en</strong>tes, pudi<strong>en</strong>do t<strong>en</strong>er una influ<strong>en</strong>cia <strong>en</strong> <strong>la</strong> apar<strong>en</strong>te diversidad, así como <strong>en</strong> los<br />
valores <strong>de</strong> similitud (McCrak<strong>en</strong> et al., 2001).<br />
Los resultados obt<strong>en</strong>idos no <strong>de</strong>muestran que los probióticos induzcan cambios<br />
significativos <strong>en</strong> <strong>la</strong> microbiota intestinal, ya que <strong><strong>la</strong>s</strong> bandas que aparec<strong>en</strong> <strong>en</strong> los grupos<br />
que recib<strong>en</strong> el probiótico también están pres<strong>en</strong>tes <strong>en</strong> los grupos control.<br />
Por otro <strong>la</strong>do, no pue<strong>de</strong> confirmarse que <strong><strong>la</strong>s</strong> bandas pres<strong>en</strong>tes correspondan a <strong>la</strong><br />
cepa Pdp11 o a Pdp13, at<strong>en</strong>di<strong>en</strong>do simplem<strong>en</strong>te a <strong>la</strong> movilidad electroforética, por tanto,<br />
56
RESULTADO S Y DISCUSIÓN<br />
harían falta estudios <strong>de</strong> i<strong>de</strong>ntificación filog<strong>en</strong>ética, secu<strong>en</strong>ciación <strong>de</strong> los productos <strong>de</strong><br />
PCR. Lo que sí po<strong>de</strong>mos afirmar es que ninguna <strong>de</strong> <strong><strong>la</strong>s</strong> cepas es capaz, a <strong>la</strong> dosis (10 9 ufc<br />
g -1 ) y tiempo <strong>en</strong>sayados (dos meses), <strong>de</strong> inducir cambios significativos <strong>en</strong> <strong>la</strong> microbiota<br />
intestinal. De acuerdo con Ouwehand et al. (2002), para consi<strong>de</strong>rar una bacteria como<br />
probiótico no es necesario que induzca cambios <strong>en</strong> <strong>la</strong> microbiota intestinal, para ejercer<br />
un efecto local o durante el tránsito a través <strong>de</strong>l sistema gastrointestinal, <strong>de</strong> hecho, <strong>la</strong><br />
variación <strong>en</strong> <strong>la</strong> microbiota <strong>en</strong> peces es sustancial y fluctúa diariam<strong>en</strong>te (Spanggaard et<br />
al., 2000; Al-Harbi y Naim Uddin, 2004; Panigrahi et al., 2004).<br />
57
C ON C L U S I O N E S
CONCLUSIONES<br />
Tras los estudios realizados sobre el papel <strong>de</strong> <strong><strong>la</strong>s</strong> activida<strong>de</strong>s <strong>superóxido</strong> <strong>dismutasa</strong><br />
y cata<strong><strong>la</strong>s</strong>a <strong>en</strong> <strong>la</strong> virul<strong>en</strong>cia <strong>de</strong>l patóg<strong>en</strong>o Photobacterium damse<strong>la</strong>e subsp. piscicida, así<br />
como <strong>de</strong> <strong><strong>la</strong>s</strong> estrategias <strong>de</strong>sarrol<strong>la</strong>das <strong>en</strong> el control <strong>de</strong> dicho patóg<strong>en</strong>o, se obti<strong>en</strong><strong>en</strong> <strong><strong>la</strong>s</strong><br />
sigui<strong>en</strong>tes conclusiones:<br />
1. Photobacterium damse<strong>la</strong>e subsp. piscicida sintetiza una so<strong>la</strong> iso<strong>en</strong>zima con<br />
actividad <strong>superóxido</strong> <strong>dismutasa</strong>, caracterizada por <strong>la</strong> pres<strong>en</strong>cia <strong>de</strong> hierro <strong>en</strong> su<br />
c<strong>en</strong>tro activo.<br />
2. Photobacterium damse<strong>la</strong>e subsp. piscicida conti<strong>en</strong>e una so<strong>la</strong> iso<strong>en</strong>zima con<br />
actividad cata<strong><strong>la</strong>s</strong>a, con hierro <strong>en</strong> su c<strong>en</strong>tro activo.<br />
3. La actividad cata<strong><strong>la</strong>s</strong>a es <strong>de</strong> gran importancia <strong>en</strong> <strong>la</strong> resist<strong>en</strong>cia <strong>de</strong> P. damse<strong>la</strong>e<br />
subsp. piscicida fr<strong>en</strong>te al peróxido <strong>de</strong> hidróg<strong>en</strong>o, pues cuando <strong><strong>la</strong>s</strong> célu<strong><strong>la</strong>s</strong><br />
bacterianas pres<strong>en</strong>tan niveles elevados <strong>de</strong> esta actividad, resist<strong>en</strong> más<br />
efici<strong>en</strong>tem<strong>en</strong>te a estos radicales reactivos <strong>de</strong> oxíg<strong>en</strong>o.<br />
4. El hierro juega un importante papel <strong>en</strong> <strong>la</strong> superviv<strong>en</strong>cia <strong>de</strong> P. damse<strong>la</strong>e subsp.<br />
piscicida <strong>en</strong> pres<strong>en</strong>cia <strong>de</strong> radicales oxidantes ya que bajo condiciones<br />
limitantes <strong>de</strong> este metal se <strong>de</strong>tectan niveles más bajos <strong>de</strong> actividad tanto<br />
<strong>superóxido</strong> <strong>dismutasa</strong> como cata<strong><strong>la</strong>s</strong>a, así como mayor susceptibilidad al<br />
peróxido <strong>de</strong> hidróg<strong>en</strong>o.<br />
5. Photobacterium damse<strong>la</strong>e subsp. piscicida es capaz <strong>de</strong> sobrevivir como<br />
patóg<strong>en</strong>o intracelu<strong>la</strong>r <strong>en</strong> el interior <strong>de</strong> fagocitos <strong>de</strong> l<strong>en</strong>guado durante, al<br />
m<strong>en</strong>os, 5 h.<br />
6. La administración por vía oral <strong>de</strong> <strong>la</strong> microalga roja Porphyridium cru<strong>en</strong>tum, <strong>en</strong><br />
combinación con <strong>la</strong> inocu<strong>la</strong>ción intraperitoneal <strong>de</strong> una bacterina fr<strong>en</strong>te a P.<br />
damse<strong>la</strong>e subsp. piscicida, increm<strong>en</strong>ta el estallido respiratorio <strong>de</strong> los fagocitos<br />
<strong>de</strong> l<strong>en</strong>guado.<br />
7. La administración oral <strong>de</strong> <strong><strong>la</strong>s</strong> cepas <strong>de</strong> Shewanel<strong>la</strong> Pdp11 y Pdp13, propuestas<br />
como probióticos, increm<strong>en</strong>ta el estallido respiratorio <strong>de</strong> los fagocitos <strong>de</strong><br />
61
CONCLUSIONES<br />
l<strong>en</strong>guado y confiere protección fr<strong>en</strong>te a <strong>la</strong> infección experim<strong>en</strong>tal con P.<br />
damse<strong>la</strong>e subsp. piscicida, respectivam<strong>en</strong>te.<br />
8. La técnica DGGE no ha permitido <strong>de</strong>tectar posibles cambios que se hayan<br />
podido efectuar <strong>en</strong> <strong>la</strong> microbiota intestinal <strong>de</strong> l<strong>en</strong>guado, tras <strong>la</strong> administración<br />
oral <strong>de</strong> <strong><strong>la</strong>s</strong> cepas Shewanel<strong>la</strong> Pdp11 y Pdp13.<br />
62
FACULTAD DE CIENCIAS<br />
DEPARTAMENTO DE MICROBIOLOGÍA<br />
Role of superoxi<strong>de</strong> dismutase and cata<strong><strong>la</strong>s</strong>e<br />
activities in Photobacterium damse<strong>la</strong>e subsp.<br />
piscicida virul<strong>en</strong>ce. Strategies for respiratory<br />
burst activity stimu<strong>la</strong>tion in sole phagocytes<br />
PATRICIA DÍAZ ROSALES<br />
Tesis doctoral<br />
2006
A B S T R A C T
ABSTRACT<br />
Photobacterium damse<strong>la</strong>e subsp. piscicida is a gram negative bacterium, capable<br />
to survive as intracellu<strong>la</strong>r pathog<strong>en</strong> within sole phagocytes, thanks to the protective<br />
action of superoxi<strong>de</strong> dismutase and cata<strong><strong>la</strong>s</strong>e activities. These <strong>en</strong>zymes confer the<br />
pathog<strong>en</strong> resistance against oxyg<strong>en</strong> reactive radicals produced within phagocytes during<br />
the respiratory burst. Therefore, both of these <strong>en</strong>zymes can be consi<strong>de</strong>red important<br />
virul<strong>en</strong>ce factors for P. damse<strong>la</strong>e subsp. piscicida, facilitating its invasion and disease<br />
establishm<strong>en</strong>t. Research on disease prev<strong>en</strong>tion has be<strong>en</strong> focused on the use of<br />
microorganisms capable to stimu<strong>la</strong>te the respiratory burst activity of sole phagocytes.<br />
Assayed microorganisms inclu<strong>de</strong> the microalga Porphyridium cru<strong>en</strong>tum and two<br />
pot<strong>en</strong>tial probiotic bacteria strains. Results obtained are promising, since the microalga<br />
and one strain of the assayed bacteria, Pdp11, are capable to stimu<strong>la</strong>te the respiratory<br />
burst activity and, therefore, confer resistance against the disease. A new research field<br />
is op<strong>en</strong>ed in the fight against pseudotuberculosis, applying substances from algae or<br />
bacterial cells that may be consi<strong>de</strong>red as probiotics.<br />
67
I N T R O D U C T I O N
INTRODUCTION<br />
1. AQUACULTURE. THE CULTURE OF SOLE<br />
(Solea s<strong>en</strong>egal<strong>en</strong>sis, Kaup, 1858)<br />
According to Food and Agriculture Organization of the United Nations (FAO) the<br />
Earth is inhabited by nearly 6.5 billion people and the popu<strong>la</strong>tion will increase to<br />
betwe<strong>en</strong> 9 and 10 billion by 2050. This concerns, particu<strong>la</strong>rly people p<strong>la</strong>nning on how to<br />
feed the future world (FAO, 2005<br />
).<br />
Fish is one of the most wi<strong>de</strong>ly used low-cost protein sources in many parts of the<br />
world. However, it is clear that the avai<strong>la</strong>bility of fish harvested from capture fisheries<br />
to support the growing <strong>de</strong>mand for fish protein will be ina<strong>de</strong>quate. Thus, the world<br />
needs to turn to producing fish, i.e. aquaculture. FAO <strong>de</strong>fines aquaculture as the<br />
culture of aquatic organisms, including fish, mollusks, crustaceans and aquatic p<strong>la</strong>nts.<br />
Nowadays aquaculture industry p<strong>la</strong>ys an ess<strong>en</strong>tial role in feeding future world.<br />
In southern European countries, aquaculture production is conc<strong>en</strong>trated on shore<br />
based cultivation of gilthead seabream (Sparus aurata, L.) and seabass (Dic<strong>en</strong>trarchus<br />
<strong>la</strong>brax, L.). Due to high production, markets have begun to be saturated. Investigation<br />
of pot<strong>en</strong>tial new species for aquaculture is one of the strategies to increase market<br />
opportunities. S<strong>en</strong>egalese sole (Solea s<strong>en</strong>egal<strong>en</strong>sis, Kaup 1858) is a common high-value<br />
f<strong>la</strong>tfish in Southern Europe, is well adapted to warm climates and is commonly raised in<br />
the ext<strong>en</strong>sive earth<strong>en</strong> ponds along the south coasts of Portugal and Spain (Dinis et al.,<br />
1999; JACUMAR, 2005).<br />
Regardless of the pot<strong>en</strong>tial economic importance of the culture of this fish species<br />
(Dinis et al., 1999; Ims<strong>la</strong>nd et al., 2003) data about the susceptibility of captive<br />
S<strong>en</strong>egalese sole to fish pathog<strong>en</strong>s are still scarce. The control and prev<strong>en</strong>tion of<br />
infectious diseases is a major goal in farmed sole. Several authors have reported<br />
differ<strong>en</strong>t diseases and pathog<strong>en</strong>ic microorganisms (Rodríguez et al., 1997; Zorril<strong>la</strong> et al.,<br />
1999; Magariños et al., 2003; Arijo et al., 2005). Although, some studies reported Vibrio<br />
harveyi and T<strong>en</strong>acibaculum maritimum iso<strong>la</strong>tion from diseased fish (Zorril<strong>la</strong> et al., 1999;<br />
Cepeda and Santos, 2003), pseudotuberculosis, caused by Photobacterium damse<strong>la</strong>e<br />
71
INTRODUCTION<br />
subsp. piscicida, is the disease responsible for higher mortalities (Zorril<strong>la</strong> et al., 1999),<br />
becoming the main limiting factor for sole production.<br />
2. Photobacterium damse<strong>la</strong>e subsp. piscicida<br />
Photobacterium damse<strong>la</strong>e subsp. piscicida, an obligate halophilic bacterium, is the<br />
causal ag<strong>en</strong>t of psedotuberculosis (Kubota et al., 1970), because in chronic cases,<br />
affected fish show promin<strong>en</strong>t white tubercles in several internal organs. This disease<br />
was first <strong>de</strong>scribed in wild popu<strong>la</strong>tions of white perch (Morone americanus) and<br />
stripped bass (Morone saxatilis) in the United States (Snieszko et al., 1964). Curr<strong>en</strong>tly<br />
natural hosts of the pathog<strong>en</strong> inclu<strong>de</strong> a wi<strong>de</strong> variety of marine fish. This disease has<br />
great economic impact in Japan, where affects mainly yellowtail (Serio<strong>la</strong><br />
quinqueradiata) cultures (Kusuda and Sa<strong>la</strong>ti, 1993), in the United States and in the<br />
European Mediterranean area, causing important losses in gilthead seabream (Sparus<br />
aurata, L.) (Ceshia et al., 1991; Toranzo et al., 1991), stripped seabass (Hauwke et al.,<br />
1987), seabass (Dic<strong>en</strong>trarchus <strong>la</strong>brax, L.) (Baudin-Laur<strong>en</strong>cin et al., 1991; Balebona et<br />
al., 1992) and, rec<strong>en</strong>tly, in sole (Solea s<strong>en</strong>egal<strong>en</strong>sis, Kaup) (Zorril<strong>la</strong> et al., 1999;<br />
Magariños et al., 2003; Arijo et al., 2005).<br />
External pathological signs of the pseudotuberculosis are usually inconspicuous,<br />
surface lesions being usually abs<strong>en</strong>t in affected fish. Occasionally, diseased gilthead<br />
seabream and seabass disp<strong>la</strong>y abnormal skin pigm<strong>en</strong>tation or slight haemorrhagic areas<br />
in the head and gills and swelling of the abdominal cavity (Toranzo et al., 1991;<br />
Balebona et al. 1992; Fouz et al., 2000).<br />
Internally, diseased fish show haemorrhagic septicaemia and necrosis in the<br />
majority of the organs, disp<strong>la</strong>ying the typical tubercles consisting in accumu<strong>la</strong>tion of<br />
bacteria, necrotic phagocytes and granulomes. Moreover the necrosis and the exist<strong>en</strong>ce<br />
of granulomata in sple<strong>en</strong>, kidney and liver, where bacteria are visible in the sinusoids<br />
and within the hepatic vessels, have be<strong>en</strong> reported (Kubota et al., 1970; Wolke, 1975;<br />
Tung et al., 1985; Hawke et al., 1987; Toranzo et al., 1991; Balebona et al., 1992; Noya et<br />
al., 1995a).<br />
72
INTRODUCTION<br />
With regard to Solea s<strong>en</strong>egal<strong>en</strong>sis, diseased fish show no appar<strong>en</strong>t lesions except<br />
for dark skin pigm<strong>en</strong>tation and swelling in the abdominal cavity. In some cases,<br />
haemorrhagic exophthalmia, small ulcers on the skin and gill pal<strong>en</strong>ess have also be<strong>en</strong><br />
observed. Internally, affected specim<strong>en</strong>s show pal<strong>en</strong>ess of liver and kidney, and the<br />
typical white tubercles of 1-2 mm in diameter in the sple<strong>en</strong> (Zorril<strong>la</strong> et al., 1999).<br />
2.1. TRANSMISSION MODE<br />
P. damse<strong>la</strong>e subsp. piscicida is a highly pathog<strong>en</strong>ic bacterium appar<strong>en</strong>tly without<br />
host specificity. Therefore, pseudotuberculosis could be a risk for new fish species<br />
where the disease has not be<strong>en</strong> <strong>de</strong>scribed yet. Noya et al. (1995b) reported that the<br />
resistance in seabream and seabass is re<strong>la</strong>ted to the size and age of the fish. This maybe<br />
due to macrophage and neutrophyl functionality, since effici<strong>en</strong>t phagocytosis and killing<br />
of the bacteria have be<strong>en</strong> observed in ol<strong>de</strong>r seabream (Noya et al., 1995b; Skarmeta et<br />
al., 1995). Also, it is possible that some <strong>de</strong>fici<strong>en</strong>cies in serum compon<strong>en</strong>ts may have<br />
some influ<strong>en</strong>ce on phagocytosis and killing of P. damse<strong>la</strong>e subsp. piscicida by<br />
phagocytes, making younger seabream more s<strong>en</strong>sitive to infection.<br />
The transmission route of infection involved in these diseases is still uncertain<br />
(Magariños et al., 1995). Some authors suggest that Photobacterium may survive in the<br />
aquatic <strong>en</strong>vironm<strong>en</strong>t as unculturable viable cells and an increase in water temperature<br />
and salinity (20-30%) could contribute to the <strong>de</strong>velopm<strong>en</strong>t of the epizootic outbreak<br />
(Hawke et al., 1987; Toranzo et al., 1991; Magariños et al., 2001).<br />
The pathways of <strong>en</strong>try may vary <strong>de</strong>p<strong>en</strong>ding on the host. With some fish, infection<br />
may follow ingestion of the pathog<strong>en</strong> (Magariños et al., 1995). Studies to <strong>de</strong>termine the<br />
importance of skin as a portal of <strong>en</strong>try for P. damse<strong>la</strong>e subsp. piscicida have be<strong>en</strong><br />
carried out (Magariños et al., 1995), and the results obtained show that all iso<strong>la</strong>tes are<br />
s<strong>en</strong>sitive to the antibacterial action of turbot (Scophthalmus maximus) mucus but<br />
resistant to gilthead seabream and seabass skin mucus. These observations may exp<strong>la</strong>in<br />
the fact that all P. damse<strong>la</strong>e subsp. piscicida outbreaks <strong>de</strong>scribed in Europe affected<br />
seabream and seabass, but never turbot.<br />
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INTRODUCTION<br />
2.2. VIRULENCE MECHANISMS<br />
Several virul<strong>en</strong>ce mechanisms have be<strong>en</strong> <strong>de</strong>scribed in P. damse<strong>la</strong>e subsp. piscicida<br />
that inclu<strong>de</strong> production of extracellu<strong>la</strong>r products (ECPs) with haemolytic, phospholipase<br />
and cytotoxic activities (Balebona et al., 1992; Magariños et al., 1992; Noya et al., 1995a<br />
and b; Romal<strong>de</strong>, 2002; Bakopoulos et al., 2004). In addition, virul<strong>en</strong>t strains constitutively<br />
synthesize a thin <strong>la</strong>yer of capsu<strong>la</strong>r material that confers resistance to serum killing and<br />
<strong>de</strong>creases macrophage phagocytosis (Magariños et al., 1996b; Arijo et al., 1998; Acosta<br />
et al., 2006).<br />
A close re<strong>la</strong>tionship has be<strong>en</strong> observed betwe<strong>en</strong> capsule production and iron<br />
avai<strong>la</strong>bility. Thus, do Vale et al. (2001) observed that cells grown un<strong>de</strong>r iron-limiting<br />
conditions always have significantly lower amounts of capsu<strong>la</strong>r material. This thinner<br />
capsule probably results in a better exposure of the adhesins and iron receptors at the<br />
bacterial surface during passage through circu<strong>la</strong>tory system. Once the microorganism<br />
reaches the differ<strong>en</strong>t host tissues, the amount of capsu<strong>la</strong>r material probably increases in<br />
response to host cellu<strong>la</strong>r <strong>de</strong>f<strong>en</strong>ce mechanisms such as phagocytosis<br />
3. SUPEROXIDE DISMUTASE AND CATALASE<br />
ACTIVITIES AS VIRULENCE FACTORS<br />
Bacterial inactivation within phagocytes takes p<strong>la</strong>ce by two mechanisms: oxyg<strong>en</strong><br />
in<strong>de</strong>p<strong>en</strong><strong>de</strong>nt mechanisms through phagocyte granule constitu<strong>en</strong>ts (lysosomal <strong>en</strong>zymes,<br />
catepsines, <strong>de</strong>f<strong>en</strong>sines, <strong>la</strong>ctoferrine, proteolytic <strong>en</strong>zymes) and oxyg<strong>en</strong> <strong>de</strong>p<strong>en</strong><strong>de</strong>nt<br />
mechanisms. In the <strong>la</strong>tter mechanisms oxyg<strong>en</strong>ic compounds such as hydrog<strong>en</strong> peroxi<strong>de</strong><br />
(H 2 O 2 ), superoxi<strong>de</strong> anion (O 2·- ) and hydroxyl radical (OH - ) takes p<strong>la</strong>ce. These products<br />
are g<strong>en</strong>erated during the called respiratory burst, due to the activation after phagocytosis<br />
of nicotinami<strong>de</strong>-a<strong>de</strong>nin-dinucleoti<strong>de</strong>-phosphate-hydrog<strong>en</strong> (NADPH) oxidase located in<br />
the membranes.<br />
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INTRODUCTION<br />
3.1. RESPIRATORY BURST<br />
After phagocytosis, a process that involves the ingestion of microorganisms,<br />
phagocytic cells liberate the cont<strong>en</strong>t of their lysosomes and a phago-lysosome is<br />
formed. Lysosomes contain several cytotoxic factors, such as oxyg<strong>en</strong> metabolites and<br />
hydrolytic <strong>en</strong>zymes capable to kill and digest microorganisms.<br />
Production of these oxyg<strong>en</strong> metabolites occurs during the respiratory burst, within<br />
phagocytes in the pres<strong>en</strong>ce of bacteria, increasing oxyg<strong>en</strong> rate consumption.<br />
Extracellu<strong>la</strong>r oxyg<strong>en</strong> is used to g<strong>en</strong>erate reactive oxyg<strong>en</strong> species (ROS), O 2·- , H 2 O 2 , OH -<br />
in the cellu<strong>la</strong>r surface. The pres<strong>en</strong>ce of these free radicals is associated with cellu<strong>la</strong>r<br />
ageing; however, their toxicity is useful as a <strong>de</strong>f<strong>en</strong>ce mechanism against bacteria, due to<br />
their great microbiocidal activity.<br />
The respiratory burst starts by stimu<strong>la</strong>tion of the NADPH oxidase located in the<br />
phagocytic membrane. This <strong>en</strong>zymatic activity is able to reduce O 2 in superoxi<strong>de</strong> anion<br />
(O 2·- ) (Roos et al., 2003). Sequ<strong>en</strong>tially, by unival<strong>en</strong>t reduction of O 2 , highly toxig<strong>en</strong>ic<br />
reactive species, are g<strong>en</strong>erated. Within the phagosome, superoxi<strong>de</strong> is, spontaneously or<br />
by superoxi<strong>de</strong> dismutase (SOD), converted to hydrog<strong>en</strong> peroxi<strong>de</strong> (H 2 O 2 ), which may<br />
th<strong>en</strong> react with superoxi<strong>de</strong> to g<strong>en</strong>erate hydroxyl radicals (OH - ) and singlet oxyg<strong>en</strong> ( 1 O 2 ),<br />
both highly reactive and toxic compounds. Superoxi<strong>de</strong> can also react with nitrog<strong>en</strong><br />
oxi<strong>de</strong> (NO), g<strong>en</strong>erated by inducible NO synthase (NOS), to yield peroxynitrite, a very<br />
reactive nitrog<strong>en</strong> intermediate. There are ev<strong>en</strong> indications that single oxyg<strong>en</strong> may be<br />
converted to a ozone-like (O 3 ) compound in a reaction catalyzed by antibodies bound to<br />
microbes or neutrophils. H 2 O 2 may also, together with chlori<strong>de</strong>, be used as a substrate<br />
by myeloperoxidase released from granules to g<strong>en</strong>erate hypochlorous acid, a very toxic<br />
compound for almost all microbes. Subsequ<strong>en</strong>tly, the short-lived hypochlorous acid can<br />
react with secondary amines to form secondary chloramines, which are as microbiocidal<br />
as hypochlorous acid but more stable.<br />
The respiratory burst, due to the g<strong>en</strong>eration of a great amount of free radicals<br />
highly toxic, inactivating proteins and oxidizing nucleic acids and other ess<strong>en</strong>tial<br />
molecules, repres<strong>en</strong>ts an immune system strategy in the fight against infections.<br />
Pathog<strong>en</strong>ic microorganisms have had to <strong>de</strong>velop a fight against free radicals, in two<br />
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INTRODUCTION<br />
si<strong>de</strong>s, on one hand, protection against free radicals that are g<strong>en</strong>erated by their own<br />
metabolism, and, on the other hand, <strong>de</strong>f<strong>en</strong>ce against the contact with phagocytic cells.<br />
Enzymes, such as superoxi<strong>de</strong> dismutases, cata<strong><strong>la</strong>s</strong>es and peroxidases, contribute to the<br />
resistance against ROS.<br />
These <strong>en</strong>zymes are consi<strong>de</strong>red as virul<strong>en</strong>ce factors; they are <strong>de</strong>f<strong>en</strong>sive weapons<br />
against phagocytic cell attack and, therefore, contribute to the virul<strong>en</strong>ce of pathog<strong>en</strong>s.<br />
3.2. SUPEROXIDE DISMUTASE ACTIVITY<br />
Superoxi<strong>de</strong> dismutase is the first <strong>de</strong>f<strong>en</strong>ce line of the cells against oxidative stress.<br />
This <strong>en</strong>zyme catalyzes the conversion of superoxi<strong>de</strong> anion radicals to hydrog<strong>en</strong><br />
peroxi<strong>de</strong> and oxyg<strong>en</strong> (equation 4).<br />
(4) O 2·- + 2H + H 2 O 2 + O 2<br />
Superoxi<strong>de</strong> dismutase activity has be<strong>en</strong> <strong>de</strong>tected in a wi<strong>de</strong> variety of living<br />
organisms, from bacteria to humans. Any cell that utilizes oxyg<strong>en</strong> has the capacity to<br />
produce superoxi<strong>de</strong> anion (O 2·- ) and so should contain some form of superoxi<strong>de</strong><br />
dismutase (Fridovich, 1974).<br />
Superoxi<strong>de</strong> dismutases constitute a family of metallo<strong>en</strong>zymes, c<strong><strong>la</strong>s</strong>sified into four<br />
groups, <strong>de</strong>p<strong>en</strong>ding on the metal cofactor: FeSOD, MnSOD, CuZnSOD and NiSOD,<br />
this <strong>la</strong>tter <strong>de</strong>scribed rec<strong>en</strong>tly in Streptomyces (Lynch and Kuramitsu, 2000).<br />
3.3. CATALASE ACTIVITY<br />
Cata<strong><strong>la</strong>s</strong>es participate in the <strong>de</strong>composition of hydrog<strong>en</strong> peroxi<strong>de</strong> into water (H 2 O)<br />
and oxyg<strong>en</strong> (O 2 ) (equation 5).<br />
(5) 2H 2 O 2 2H 2 O + O 2<br />
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INTRODUCTION<br />
Some cata<strong><strong>la</strong>s</strong>es have also peroxidase activity and in this case an organic electron<br />
donor, or sometimes an hali<strong>de</strong> ion, is employed in the reduction of hydrog<strong>en</strong> peroxi<strong>de</strong><br />
(equation 6).<br />
(6) RH 2 + H 2 O 2 2H 2 O + R<br />
Cata<strong><strong>la</strong>s</strong>es have be<strong>en</strong> divi<strong>de</strong>d into three groups (Loew<strong>en</strong>, 1997): monofunctional<br />
cata<strong><strong>la</strong>s</strong>e with heme group (FeCat); bifunctional cata<strong><strong>la</strong>s</strong>e with heme group (cata<strong><strong>la</strong>s</strong>eperoxidase)<br />
and pseudocata<strong><strong>la</strong>s</strong>e without heme group (MnCat), referred to as<br />
pseudocata<strong><strong>la</strong>s</strong>e because they are not inhibited by the common catalytic inhibitors, azi<strong>de</strong><br />
and cyani<strong>de</strong>.<br />
Superoxi<strong>de</strong> dismutase and cata<strong><strong>la</strong>s</strong>e activities protect the cells also by prev<strong>en</strong>ting<br />
the g<strong>en</strong>eration of hydroxyl radical (OH·), a more toxic reactive species.<br />
The role of these <strong>en</strong>zymes can be ess<strong>en</strong>tial to protect pathog<strong>en</strong>ic bacteria during<br />
respiratory burst after phagocytosis, therefore superoxi<strong>de</strong> dismutase and cata<strong><strong>la</strong>s</strong>e are<br />
re<strong>la</strong>ted to virul<strong>en</strong>ce mechanisms (Table 5). The ability of many microorganisms to<br />
infect the host <strong>de</strong>p<strong>en</strong>ds on their resistance to ROS production by cells, mainly<br />
monocytes, macrophages and polymorphonuclears. Ironically, evolution has selected<br />
organisms that use these cells as targets, so pres<strong>en</strong>ce of SOD and cata<strong><strong>la</strong>s</strong>e in bacteria<br />
will contribute to resistance against host and, therefore, infection establishm<strong>en</strong>t.<br />
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INTRODUCTION<br />
Table 5 Examples of pathog<strong>en</strong>s in which a role in the virul<strong>en</strong>ce has be<strong>en</strong> <strong>de</strong>monstrated for<br />
SOD and cata<strong><strong>la</strong>s</strong>e activities<br />
Pathog<strong>en</strong> Virul<strong>en</strong>ce factor Refer<strong>en</strong>ce<br />
Listeria monocytog<strong>en</strong>es FeSOD Welch et al., 1979<br />
Shigel<strong>la</strong> flexneri FeSOD Franzon et al., 1990<br />
Pseudomonas syringae Cata<strong><strong>la</strong>s</strong>es (not<br />
<strong>de</strong>termined)<br />
Klotz and Hutcheson, 1992<br />
Caulobacter cresc<strong>en</strong>tus CuZnSOD Schnell and Steinman, 1995<br />
Aeromonas salmonicida FeSOD Barnes et al., 1996<br />
Pseudomonas aeruginosa MnSOD Po<strong>la</strong>ck et al., 1996<br />
Legionel<strong>la</strong> pneumophi<strong>la</strong> CuZnSOD St. John and Steinman,<br />
1996<br />
Aeromonas<br />
subsp. salmonicida<br />
salmonicida<br />
MnSOD and FeCata<strong><strong>la</strong>s</strong>e<br />
Barnes et al., 1999b<br />
Mycobacterium tuberculosis Cata<strong><strong>la</strong>s</strong>e-Peroxidase Manca et al., 1999<br />
Streptococcus pneumoniae MnSOD Yesilkaya et al., 2000<br />
Vibrio harveyi<br />
Monofunctional<br />
Vattanaviboon<br />
and<br />
cata<strong><strong>la</strong>s</strong>e<br />
Mongkolsuk, 2001<br />
Salmonel<strong>la</strong> <strong>en</strong>terica CuZnSOD Uzzau et al., 2002<br />
Vibrio shiloi SOD (not <strong>de</strong>termined) Banin et al., 2003<br />
3.4. SUPEROXIDE DISMUTASE AND CATALASE ACTIVITIES IN<br />
Photobacterium damse<strong>la</strong>e subsp. piscicida<br />
Processes involved in the invasion and survival of P. damse<strong>la</strong>e subsp. piscicida<br />
insi<strong>de</strong> the host are still unknown and while authors have reported the pres<strong>en</strong>ce of intact<br />
bacteria insi<strong>de</strong> fish cells, suggesting the ability of the bacterium to survive as an<br />
intracellu<strong>la</strong>r pathog<strong>en</strong> (Noya et al., 1995a; López-Dóriga et al., 2000), others have<br />
observed that this pathog<strong>en</strong> is highly susceptible to oxidative radicals g<strong>en</strong>erated during<br />
the macrophage respiratory burst (Skarmeta et al., 1995; Barnes et al., 1999a). Due to the<br />
protective role attributed to SOD and cata<strong><strong>la</strong>s</strong>e activities in a variety of bacterial<br />
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INTRODUCTION<br />
pathog<strong>en</strong>s, study of these activities in P. damse<strong>la</strong>e subsp. piscicida could contribute to<br />
the un<strong>de</strong>rstanding of the interactions betwe<strong>en</strong> this bacterium and its host.<br />
Barnes et al. (1999a) <strong>de</strong>termined the exist<strong>en</strong>ce of a perip<strong><strong>la</strong>s</strong>mic FeSOD and a<br />
citop<strong><strong>la</strong>s</strong>mic cata<strong><strong>la</strong>s</strong>e, not characterized. The SOD activity is repressed un<strong>de</strong>r iron<br />
restricted or low oxyg<strong>en</strong> conditions. Cata<strong><strong>la</strong>s</strong>e activity is constituvely expressed,<br />
although there are differ<strong>en</strong>ces in int<strong>en</strong>sity gel bands <strong>de</strong>p<strong>en</strong>ding on strains. In this PhD.<br />
Thesis we have tried to <strong>de</strong>ep more in the study of the role of superoxi<strong>de</strong> dismutase and<br />
cata<strong><strong>la</strong>s</strong>e activities in P. damseale subsp. piscicida virul<strong>en</strong>ce.<br />
4. STIMULATION OF RESPIRATORY BURST ACTIVITY BY<br />
DIFFERENT MICROORGANISMS AFTER Photobacterium<br />
damse<strong>la</strong>e subsp. piscicida INFECTION<br />
4.1. PREVENTION AND TREATMENT OF PSEUDOTUBERCULOSIS<br />
Over the <strong><strong>la</strong>s</strong>t <strong>de</strong>ca<strong>de</strong>, inci<strong>de</strong>nce of drug-resistant strains, carrying a transferable R-<br />
p<strong><strong>la</strong>s</strong>mid have increased, making treatm<strong>en</strong>t with antimicrobial chemotherapeutics less<br />
successful. Moreover, a period of intracellu<strong>la</strong>r parasitism within macrophages has be<strong>en</strong><br />
<strong>de</strong>scribed for P. damse<strong>la</strong>e subsp. piscicida (Kusuda and Sa<strong>la</strong>ti, 1993); this finding can<br />
exp<strong>la</strong>in the ineffectiv<strong>en</strong>ess of chemotherapy in the treatm<strong>en</strong>t of some disease outbreaks.<br />
Therefore, immunoprophy<strong>la</strong>xis has become the best way to prev<strong>en</strong>t pseudotuberculosis.<br />
Throughout the <strong><strong>la</strong>s</strong>t 20 years, there have be<strong>en</strong> a variety of studies analyzing the<br />
effectiv<strong>en</strong>ess of immunization in prev<strong>en</strong>ting pseudotuberculosis (Romal<strong>de</strong> and<br />
Magariños, 1997). Most vaccine formu<strong>la</strong>tions tested consisted of heat- or formalin-killed<br />
cells (Fukuda and Kusuda, 1981; Kusuda and Hamaguchi, 1987; Kusuda and Hamaguchi,<br />
1988; Hamaguchi and Kusuda, 1989). Best results were obtained using formu<strong>la</strong>tions<br />
based on lipopolysacchari<strong>de</strong>s (LPS) and ribosomal fractions of the bacteria (Fukuda and<br />
Sukuda, 1985; Kusuda et al., 1988; Kawakami et al., 1997). However, these formu<strong>la</strong>tions<br />
pres<strong>en</strong>ted not only problems of reproducibility, but also difficulties on <strong>la</strong>rge scale<br />
production. Passive immunization has also be<strong>en</strong> evaluated (Fukuda and Kusuda, 1981),<br />
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INTRODUCTION<br />
but the results showed short-term protection. The best protection against<br />
pseudotuberculosis was obtained with an ECP-<strong>en</strong>riched bacterin (Magariños et al., 1994,<br />
1997, 1999). This vaccine is curr<strong>en</strong>tly commercially avai<strong>la</strong>ble and has be<strong>en</strong> successfully<br />
employed in several European countries including Spain, Portugal and Greece. In<br />
addition, a dival<strong>en</strong>t vaccine with bacterin and formaline-inactivated ECPs of V. harveyi<br />
and P. damse<strong>la</strong>e subsp. piscicida has shown promising results in sole (Arijo et al.,<br />
2005).<br />
Future tr<strong>en</strong>ds in vaccine formu<strong>la</strong>tions against P. damse<strong>la</strong>e subsp. piscicida inclu<strong>de</strong><br />
the use of proteins from cellu<strong>la</strong>r <strong>en</strong>velope (Magariños et al., 1994) and iron-regu<strong>la</strong>ted<br />
OMPs as protective antig<strong>en</strong>s.<br />
The combination of vaccination and immunostimu<strong>la</strong>nts appears as the most<br />
effective strategy to prev<strong>en</strong>t and fight against infectious diseases in fish (Sakai, 1999).<br />
At pres<strong>en</strong>t, the use of immunostimu<strong>la</strong>nts, in addition to chemotherapeutic ag<strong>en</strong>ts and<br />
vaccines, has be<strong>en</strong> wi<strong>de</strong>ly accepted by fish farmers. However it is necessary search for<br />
new immunostimu<strong>la</strong>nt ag<strong>en</strong>ts effective against pathog<strong>en</strong>s and with reduced production<br />
costs.<br />
Finally, probiotics, microbial cells orally administered capable to induce positive<br />
effects on host health repres<strong>en</strong>t another alternative to combat diseases affecting farmed<br />
fish.<br />
4.2. IMMUNOMODULATION. IMMUNOSTIMULATION<br />
Immunomodu<strong>la</strong>tion is the ability of certain substances to regu<strong>la</strong>te the immune<br />
system, may be immunostimu<strong>la</strong>tion or immuno<strong>de</strong>pression, stimu<strong>la</strong>ting or <strong>de</strong>pressing the<br />
immune system, respectively. The main reason to search for new immunostimu<strong>la</strong>nt<br />
ag<strong>en</strong>ts is the great <strong>de</strong>velopm<strong>en</strong>t of the aquaculture and the increase of stress situations<br />
and diseases caused by int<strong>en</strong>sive cultures. Some studies have reported that the negative<br />
effects associated to immuno<strong>de</strong>pression were overcome by immunostimu<strong>la</strong>nts, since the<br />
immunostimu<strong>la</strong>nts increase resistance against infectious diseases, by <strong>en</strong>hancing nonspecific<br />
<strong>de</strong>f<strong>en</strong>ce mechanisms. Immunostimu<strong>la</strong>nts facilitate phagocytic cells function and<br />
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INTRODUCTION<br />
increase their bactericidal activity, oxyg<strong>en</strong> <strong>de</strong>p<strong>en</strong><strong>de</strong>nt and in<strong>de</strong>p<strong>en</strong><strong>de</strong>nt mechanisms<br />
being involved (Siwicki et al., 1994; An<strong>de</strong>rson, 1996).<br />
Immunostimu<strong>la</strong>nts may be able to comp<strong>en</strong>sate some chemotherapeutic and vaccine<br />
limitations. Immunostimu<strong>la</strong>nts are safer than chemotherapeutics and their range of<br />
efficacy is wi<strong>de</strong>r than vaccination. However, we cannot expect the marked or long-term<br />
effects observed with vaccines to occur with immunostimu<strong>la</strong>nts because these<br />
substances act on non-specific immune system, without a memory compon<strong>en</strong>t<br />
(An<strong>de</strong>rson, 1996; Sakai, 1999). Sakai (1999) suggests that the most effective strategy to<br />
prev<strong>en</strong>t and combate possible infectious diseases in fish, is the combination of<br />
immuostimu<strong>la</strong>nts and vaccines. Thus, with a <strong>de</strong>tailed un<strong>de</strong>rstanding of the efficacy and<br />
limitations of immunostimu<strong>la</strong>nts, they may become powerful tools to control fish<br />
diseases.<br />
4.3. USE OF ALGAE AS IMMUNOSTIMULANTS<br />
During <strong><strong>la</strong>s</strong>t years searching for therapeutically active substances has focused on<br />
marine organisms. Ability of algae to produce secondary metabolites, such as<br />
antibiotics, antivirals, antitumorals and antiinf<strong>la</strong>mmatories, with pot<strong>en</strong>tial<br />
pharmaceutical interest, has be<strong>en</strong> well docum<strong>en</strong>ted (Scheuer, 1990; Faulkner, 1993;<br />
González <strong>de</strong>l Val et al., 2001). However, studies on immunomodu<strong>la</strong>tory properties of<br />
extracts and whole cells from algae are still scarce. In this Thesis we have studied the<br />
red microalga Porphyridium cru<strong>en</strong>tum as a possible source of immunostimu<strong>la</strong>nts in<br />
cultured sole.<br />
A great number of algae constitute an important source of polyunsaturated fatty<br />
acids (PUFAs), which are the ess<strong>en</strong>tial dietary requirem<strong>en</strong>ts for many marine teleosts<br />
(Bell et al., 1985; Kov<strong>en</strong> et al., 2001). Some of these fatty acids, such as the arachidonic<br />
acid, AA (20:4(n-6)) participate in eicosanoid synthesis, therefore, in prostag<strong>la</strong>ndin<br />
production, involved in stress through modu<strong>la</strong>tion of cortisol release and, therefore in<br />
cellu<strong>la</strong>r immunity (Vil<strong>la</strong>lta et al., 2005).<br />
Algae also contain carot<strong>en</strong>oids, β-carot<strong>en</strong>e, astaxanthin, cataxanthin or<br />
xanthofilins. Amar et al. (2004) <strong>de</strong>monstrated that β-carot<strong>en</strong>e, iso<strong>la</strong>ted from Dunaliel<strong>la</strong><br />
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INTRODUCTION<br />
salina, is able to modu<strong>la</strong>te, after oral administration, some compon<strong>en</strong>ts of the innate<br />
<strong>de</strong>f<strong>en</strong>ce mechanisms in rainbow trout (Oncorhynchus mykiss) such as the complem<strong>en</strong>t<br />
alternative way, serum lysozyme and phagocytosis. The carot<strong>en</strong>oids increase the<br />
phagocytic activity and citoquine production (B<strong>en</strong>dich, 1989; Chew, 1993).<br />
Algae are also a natural source of vitamins, some of them have possible<br />
immunostimu<strong>la</strong>nt effects on fish immune system, such as vitamin C (Hardie et al., 1991;<br />
Cuesta et al., 2002; J<strong>en</strong>ey and J<strong>en</strong>ey, 2002; Lin and Shiau, 2005), vitamin E (Hardie et al.,<br />
1990; Cuesta et al., 2001) and vitamins B (Miles et al., 2001).<br />
The use of polysacchari<strong>de</strong>s as immunostimu<strong>la</strong>nts is wi<strong>de</strong>ly ext<strong>en</strong><strong>de</strong>d in aquaculture<br />
(Kumar et al., 2005), the most studied in fish are the glucans. β-glucans are polyglucoses<br />
that consist in series of residues of β-1,3-glucopiranosyl <strong>de</strong>rived from yeast and fungus<br />
micellium. These sugars seem to have a pot<strong>en</strong>t immunostimu<strong>la</strong>nt effect, mainly on<br />
unspecific <strong>de</strong>f<strong>en</strong>ce mechanisms, inducing resistance against infections. Numerous<br />
studies confirm the use of β,1-3,1-6 glucans from yeasts and fungus cell walls as<br />
immunostimu<strong>la</strong>nts in aquaculture. Greater part of these studies has focused on β-<br />
glucans from the yeast specie Saccharomyces cerevisiae (Santarém et al., 1997; Castro<br />
et al., 1999; Kumari and Sahoo, 2006; Marqués et al., 2006).<br />
The use of these polysacchari<strong>de</strong>s as immunostimu<strong>la</strong>nts in aquaculture industry is<br />
wi<strong>de</strong>ly ext<strong>en</strong><strong>de</strong>d and there are commercial products avai<strong>la</strong>ble (Siwicki et al., 1994; Cook<br />
et al., 2003; Couso et al., 2003; Bagni et al., 2005).<br />
Another pot<strong>en</strong>tial immunostimu<strong>la</strong>nt polysacchari<strong>de</strong> <strong>de</strong>rived from brown<br />
macroalgae and microalgae, is the alginic acid. The alginate is known in aquaculture as<br />
stabilizer of the structure of pellet diets. The immunostimu<strong>la</strong>nt properties were<br />
<strong>de</strong>termined in phyophaecaetes extracts of species such as Laminaria digitata (Dalmo et<br />
al., 1998; Gabriels<strong>en</strong> and Austr<strong>en</strong>g, 1998) and others (Miles et al., 2001; Peddie et al.,<br />
2002; Skjermo and Bergh, 2004; Bagni et al., 2005).<br />
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INTRODUCTION<br />
4.3.1. Porphyridium cru<strong>en</strong>tum<br />
Porphyridium cru<strong>en</strong>tum is a red microalga belonging to Rodophyta family and<br />
Porphyridiales or<strong>de</strong>r, with spherical cells that <strong>la</strong>ck of cell wall. This alga accumu<strong>la</strong>tes<br />
<strong>la</strong>rge amounts of fatty acids (9-14% dry weight), specially araquidonic acid (36%) and<br />
noticeable amounts of eicosap<strong>en</strong>ta<strong>en</strong>oic acid. The protein cont<strong>en</strong>t ranges from 28 to 39%,<br />
and avai<strong>la</strong>ble carbohydrates vary betwe<strong>en</strong> 40 and 57%. The biomass cont<strong>en</strong>ts tocopherol,<br />
vitamin K and a <strong>la</strong>rge amount of carot<strong>en</strong>es (Rebolloso et al., 2000).<br />
P. cru<strong>en</strong>tum cells are capable to excrete a sulphated polysacchari<strong>de</strong>, an acidic<br />
heteropolymer composed of xylose, glucose, ga<strong>la</strong>ctose and sulphate esters (You and<br />
Barneu, 2004). This polysacchari<strong>de</strong> is commercially used as thick<strong>en</strong>er, stabilizer and<br />
emulsifier (Arad et al., 1985, 1988; Adda et al., 1986).<br />
Substances such as araquidonic acid (Kov<strong>en</strong> et al., 2001), carbohydrates (Kumar et<br />
al., 2005), vitamins (Hardie et al., 1990, 1991; Ortuño et al., 1999, 2003; J<strong>en</strong>ey and J<strong>en</strong>ey,<br />
2002), carot<strong>en</strong>oids (Amar et al., 2004) and polysacchari<strong>de</strong>s (Siwicki et al., 1994;<br />
Santarém et al., 1997; Castro et al., 1999; Bagni et al., 2000, 2005; Esteban et al., 2001;<br />
J<strong>en</strong>ey and J<strong>en</strong>ey, 2002; Cook et al., 2003; Couso et al., 2003) pres<strong>en</strong>t in differ<strong>en</strong>t<br />
organisms, have be<strong>en</strong> <strong>de</strong>monstrated their immunostimu<strong>la</strong>nt effects on fish. All of these<br />
compounds have be<strong>en</strong> <strong>de</strong>termined in P. cru<strong>en</strong>tum.<br />
In addition, the fact that P. cru<strong>en</strong>tum culture is not costly makes this alga a good<br />
candidate as a source of immunostimu<strong>la</strong>nt active substances. However, the<br />
polysacchari<strong>de</strong> extraction is a <strong>la</strong>borious process; moreover intraperitoneal<br />
administration is not advisable due to stress by handling. For this reason it is conv<strong>en</strong>i<strong>en</strong>t<br />
to simplify the immunostimu<strong>la</strong>nt administration, providing whole and oral,<br />
supplem<strong>en</strong>ting feed. Last years the number of works, that study whole organisms is<br />
increasing, such as yeasts (Siwicki et al., 1994; Ortuño et al., 2002; Rodríguez et al.,<br />
2003), fungus (Rodríguez et al., 2004) and probiotics (Verschuere et al., 2000; Irianto and<br />
Austin, 2003; Salinas et al., 2005; Díaz-Rosales et al., 2006). However, in spite of the<br />
<strong>la</strong>rge number of studies carried out with extracts or compounds <strong>de</strong>rived from algae<br />
(Kov<strong>en</strong> et al., 2001; Castro et al., 2004; Skjermo and Bergh, 2004; Díaz-Rosales et al.,<br />
83
INTRODUCTION<br />
2005; Hou and Ch<strong>en</strong>, 2005; Vil<strong>la</strong>lta et al., 2005), the works using whole alga cells are still<br />
scarce (Blinkova et al., 2001; Val<strong>en</strong>te et al., 2006).<br />
As <strong>de</strong>scribed above, the combination of vaccination and immunostimu<strong>la</strong>nt<br />
administration could increase vaccine pot<strong>en</strong>cy. Thus, one objective is to evaluate a<br />
possible fish immunostimu<strong>la</strong>tion by administration of the alga P. cru<strong>en</strong>tum jointly with<br />
a vaccine formu<strong>la</strong>tion, evaluating a possible synergetic effect of both prophy<strong>la</strong>ctic<br />
methods against P. damse<strong>la</strong>e subsp. piscicida infection.<br />
4.4. IMMUNOSTIMULANT EFFECT OF POTENTIAL PROBIOTIC BACTERIA<br />
Definition of probiotics has changed along time. Thus, Salmin<strong>en</strong> et al. (1999)<br />
consi<strong>de</strong>r probiotics as “every microbiane preparation (not alive necessary) or microbial<br />
cellu<strong>la</strong>r compounds that have b<strong>en</strong>eficial effect on host health”. According to FAO<br />
probiotics consist of a microbial complem<strong>en</strong>t that affects b<strong>en</strong>eficially to host physiology<br />
by modu<strong>la</strong>tion of local and systemic immunity, moreover to improve microbial ba<strong>la</strong>nce<br />
by prev<strong>en</strong>tion of gastrointestinal colonization by non <strong>de</strong>sired bacteria. The advances in<br />
probiotic employm<strong>en</strong>t in cattle and human medicine have lead to consi<strong>de</strong>r their<br />
application in aquaculture, as an alternative in the fight against microbial infection<br />
(Sakai, 1999).<br />
Aquatic animals are very differ<strong>en</strong>t to terrestrial animals, therefore probiotic<br />
concept changes in aquaculture application (Verschuere et al., 2000). In aquatic animals<br />
there is a constant interaction betwe<strong>en</strong> the intestinal microbiota and <strong>en</strong>vironm<strong>en</strong>t. For<br />
this reason, in aquaculture systems the immediate <strong>en</strong>vironm<strong>en</strong>t has <strong>la</strong>rger influ<strong>en</strong>ce on<br />
the health status than in the case of terrestrial animals or humans. Due to the exist<strong>en</strong>ce<br />
of a continuous flux of water trough digestive tract, the fish intestinal microbiota is<br />
highly <strong>de</strong>p<strong>en</strong><strong>de</strong>nt on external <strong>en</strong>vironm<strong>en</strong>t.<br />
Verschuere et al. (2000) proposed a modified <strong>de</strong>finition: “as a live microbial<br />
adjunct which has a b<strong>en</strong>eficial effect on the host by modifying the host-associated or<br />
ambi<strong>en</strong>t microbial community, by <strong>en</strong>suring improved use of the feed or <strong>en</strong>hancing its<br />
nutritional value, by <strong>en</strong>hancing the host response towards disease, or by improving the<br />
quality of its ambi<strong>en</strong>t <strong>en</strong>vironm<strong>en</strong>t”.<br />
84
INTRODUCTION<br />
Differ<strong>en</strong>t mechanisms have be<strong>en</strong> proposed to exp<strong>la</strong>in the types of interactions<br />
betwe<strong>en</strong> probiotics and pathog<strong>en</strong>s: (1) Competitive exclussion, production of<br />
antimicrobial compounds such as bacteriocines, lysozimes and proteases (Austin et al.,<br />
1995; Sugita et al., 1997; Gatesoupe, 1999; Gram et al., 1999; Verschuere et al., 2000); (2)<br />
competition for avai<strong>la</strong>ble <strong>en</strong>ergy and nutri<strong>en</strong>ts (Smith and Davey, 1993; Pybus et al.,<br />
1994; Gatesoupe et al., 1997; Gram et al., 1999); (3) Adhesive interfer<strong>en</strong>ce in the host<br />
(Olsson et al., 1992; Jöborn et al., 1997; Nikoske<strong>la</strong>in<strong>en</strong> et al., 2001; Chabrillón et al.,<br />
2005a and b). Rec<strong>en</strong>tly, research have focused also on the immunomodu<strong>la</strong>tory properties<br />
of probiotic microorganisms (Irianto and Austin, 2003; Nikoske<strong>la</strong>in<strong>en</strong> et al., 2003;<br />
Panigrahi et al., 2004; Salinas et al., 2005, 2006; Díaz-Rosales, 2006) (Table 6).<br />
The gastrointestinal microbiota p<strong>la</strong>ys an important role in host nutrition and<br />
health. Therefore, probiotics could perform an interesting function, modifying<br />
gastrointestinal microbiota to induce favourable effects, such as improvem<strong>en</strong>t of<br />
growth, digestion, immunity or resistance against a pathog<strong>en</strong>. There are numerous<br />
authors that have characterized gastrointestinal microbiota of differ<strong>en</strong>t farmed fish,<br />
fundam<strong>en</strong>tally salmonids (Spanggaard et al., 2000; Huber et al., 2004, J<strong>en</strong>s<strong>en</strong> et al., 2004;<br />
Burr et al., 2005), however studies on gastrointestinal microbiota characterization or<br />
evaluation of possible effects of probiotics are null in fish from Mediterranean area.<br />
These studies are very interesting since could provi<strong>de</strong> information for a better<br />
application of microorganisms in prophy<strong>la</strong>ctic strategies and diseases biocontrol.<br />
85
INTRODUCTION<br />
Table 6 B<strong>en</strong>eficial effects of probiotics on fish immune system<br />
PROBIOTIC HOST INCREASE OF IMMUNE<br />
REFERENCE<br />
RESPONSE<br />
Bacteria Gram Oncorhynchus Erythrocytes and leucocytes number, Irianto and Austin,<br />
positive, not mykiss (rainbow lysozyme activity, phagocytosis 2003<br />
i<strong>de</strong>ntified<br />
trout)<br />
Vibrio fluvialis Oncorhynchus Erythrocytes and leucocytes number, Irianto and Austin,<br />
mykiss<br />
lysozyme activity, phagocytosis 2003<br />
Aeromonas<br />
Oncorhynchus Erythrocytes and leucocytes number, Irianto and Austin,<br />
hydrophi<strong>la</strong><br />
mykiss<br />
lysozyme activity, phagocytosis 2003<br />
Carnobacterium Oncorhynchus Erythrocytes and leucocytes number, Irianto and Austin,<br />
mykiss<br />
lysozyme activity, phagocytosis 2003<br />
Lactobacillus<br />
rhamnosus<br />
Oncorhynchus<br />
mykiss<br />
Respiratory burst, serum bactericidal<br />
activity, Ig serum levels<br />
Nikoske<strong>la</strong>in<strong>en</strong> et al.,<br />
2003<br />
Bacillus P<strong>en</strong>aeus vannamei Immune in<strong>de</strong>x (hemograme, Gullian et al., 2004<br />
superoxi<strong>de</strong> anion production,<br />
ph<strong>en</strong>oloxidase activity, antibacterial<br />
activity, p<strong><strong>la</strong>s</strong>ma protein conc<strong>en</strong>tration)<br />
Lactobacillus Oncorhynchus Lysozyme, serum complem<strong>en</strong>t and Panigrahi et al., 2004<br />
rhamnosus<br />
mykiss<br />
phagocytic activities<br />
Lactobacillus Sparus aurata Phagocytosis, cytotoxic activity Salinas et al., 2005<br />
<strong>de</strong>lbrüeckii subsp. (gilthead<br />
<strong>la</strong>ctis<br />
seabream)<br />
Bacillus subtilis Sparus aurata Phagocytosis, cytotoxic activity Salinas et al., 2005<br />
Aeromonas sobria<br />
Alteromonadaceae,<br />
G. Shewanel<strong>la</strong><br />
(Pdp11)<br />
Alteromonadaceae,<br />
G. Shewanel<strong>la</strong><br />
(51M6)<br />
Oncorhynchus Leucocytes number, phagocytosis,<br />
mykiss<br />
respiratory burst<br />
Sparus aurata Serum peroxidase, complem<strong>en</strong>t<br />
activity, phagocytosis<br />
Sparus aurata Serum peroxidase, complem<strong>en</strong>t<br />
activity, phagocytosis, cytotoxic<br />
activity<br />
Brunt and Austin,<br />
2005<br />
Díaz-Rosales et al.,<br />
2006<br />
Díaz-Rosales et al.,<br />
2006<br />
86
A<br />
I M S
AIMS<br />
The work pres<strong>en</strong>ted in this Thesis contributes to the knowle<strong>de</strong>ge of<br />
Photobacterium damse<strong>la</strong>e subsp. piscicida virul<strong>en</strong>ce, and <strong>de</strong>velops new strategies for<br />
prev<strong>en</strong>tion of the disease caused by this pathog<strong>en</strong>. The aims proposed are the following:<br />
1. Study of the role of superoxi<strong>de</strong> dimutase and cata<strong><strong>la</strong>s</strong>e <strong>en</strong>zymatic activities in<br />
Photobacterium damse<strong>la</strong>e subsp. piscicida, evaluating the pot<strong>en</strong>tial resistance<br />
of the pathog<strong>en</strong> against bactericidal action of sole phagocytes.<br />
2. Evaluation of possible immunostimu<strong>la</strong>nt effect on respiratory burst activity of<br />
sole phagocytes, that could be exert the use of the microalgae Porphyridium<br />
cru<strong>en</strong>tum and probiotics microorganisms.<br />
89
M<br />
A T E R I A L S A N D M E T H O D S
MATERIA L AND METHODS<br />
Materials and methodology followed to carry out the experim<strong>en</strong>ts inclu<strong>de</strong>d in this<br />
PhD. Thesis are <strong>de</strong>tailed in the papers attached in Article section.<br />
93
R<br />
E S U L T S A N D D I S C U S S I O N
RESULTS AND DISCUSSION<br />
The first objective of this PhD. Thesis consisted in the study of the contribution of<br />
superoxi<strong>de</strong> dismutase and cata<strong><strong>la</strong>s</strong>e activities to Photobacterium damse<strong>la</strong>e subsp.<br />
piscicida resistance against oxyg<strong>en</strong>ic radicals g<strong>en</strong>erated during respiratory burst in sole<br />
phagocytes and, therefore, their possible role as virul<strong>en</strong>ce factors (articles 1.1. and 1.2.,<br />
Article section).<br />
Previously, Barnes et al. (1999a) <strong>de</strong>monstrated in vitro that this microorganism<br />
contains <strong>en</strong>ough superoxi<strong>de</strong> dismutase activity to disproportionate photochemically<br />
g<strong>en</strong>erated superoxi<strong>de</strong> anions, and that the bacterial susceptibility is due to the action of<br />
hydrog<strong>en</strong> peroxi<strong>de</strong>. In fact, the addition of exog<strong>en</strong>ous cata<strong><strong>la</strong>s</strong>e to the medium protected<br />
the bacteria from inactivation by superoxi<strong>de</strong> anions. The role of cata<strong><strong>la</strong>s</strong>e and superoxi<strong>de</strong><br />
dismutase activities in P. damse<strong>la</strong>e subsp. piscicida virul<strong>en</strong>ce has be<strong>en</strong> studied in this<br />
work and results are inclu<strong>de</strong>d in articles 1.1. and 1.2. (Article section).<br />
In vitro role of cata<strong><strong>la</strong>s</strong>e activity in P. damse<strong>la</strong>e subsp. piscicida protection against<br />
exog<strong>en</strong>ous hydrog<strong>en</strong> peroxi<strong>de</strong> has be<strong>en</strong> evaluated in the first article. Results obtained<br />
show that resistance to hydrog<strong>en</strong> peroxi<strong>de</strong> increases wh<strong>en</strong> bacterial cultures are pulsed<br />
with hydrog<strong>en</strong> peroxi<strong>de</strong>. On the contrary, survival perc<strong>en</strong>tages <strong>de</strong>crease wh<strong>en</strong> bacteria<br />
grow un<strong>de</strong>r iron-limiting conditions. Quantification of cata<strong><strong>la</strong>s</strong>e activity in a set of P.<br />
damse<strong>la</strong>e subsp. piscicida strains shows that highest survival perc<strong>en</strong>tages are pres<strong>en</strong>t in<br />
bacterial cultures with higher cata<strong><strong>la</strong>s</strong>e activity levels. Moreover, resistance to oxidative<br />
stress varied <strong>de</strong>p<strong>en</strong>ding on the virul<strong>en</strong>ce, being higher in more virul<strong>en</strong>t strains. Thus, the<br />
virul<strong>en</strong>t strain Lg h41/01 shows higher resistance to respiratory burst of sole phagocytes<br />
than the non virul<strong>en</strong>t strain EPOY-8803-II, and hydrog<strong>en</strong> peroxi<strong>de</strong> addition into cultures<br />
increases survival. The <strong>la</strong>ck of cell capsule could contribute to the <strong>de</strong>crease of survival<br />
against reactive oxyg<strong>en</strong> species, but the low levels of cata<strong><strong>la</strong>s</strong>e activity obtained in<br />
EPOY-8803-II, suggest that its abs<strong>en</strong>ce of virul<strong>en</strong>ce could be <strong>de</strong>termined, in great part,<br />
by low cata<strong><strong>la</strong>s</strong>e activity. Therefore, cata<strong><strong>la</strong>s</strong>e p<strong>la</strong>ys an important role in P. damse<strong>la</strong>e<br />
subsp. piscicida virul<strong>en</strong>ce. It has be<strong>en</strong> <strong>de</strong>monstrated, in a great number of bacteria, that<br />
previous exposition to an oxidant in sublethal conc<strong>en</strong>trations can induce protection<br />
against lethal conc<strong>en</strong>trations of the oxidant (Mongkolsuk et al., 1996). Barnes et al.<br />
(1999b) reported that after being pulsed with hydrog<strong>en</strong> peroxi<strong>de</strong> A. salmonicida is able to<br />
97
RESULTS AND DISCUSSION<br />
resist 100 mM of peroxi<strong>de</strong>, a lethal conc<strong>en</strong>tration without peroxi<strong>de</strong> pretreatm<strong>en</strong>t. In the<br />
case of P. damse<strong>la</strong>e subsp. piscicida, it is interesting to note that a significant increase<br />
in survival rates of the non-virul<strong>en</strong>t strain was observed wh<strong>en</strong> cultures were pulsed with<br />
hydrog<strong>en</strong> peroxi<strong>de</strong> compared to cells cultured until stationary phase. In contrast, this<br />
increase has not be<strong>en</strong> observed for the virul<strong>en</strong>t strain, which always showed higher<br />
survival regardless of the growth phase, or the pulse with hydrog<strong>en</strong> peroxi<strong>de</strong>.<br />
On the other hand, culture un<strong>de</strong>r iron-restricted conditions results in a significant<br />
<strong>de</strong>crease in survival of both virul<strong>en</strong>t and avirul<strong>en</strong>t strains. The fact that this bacterial<br />
species contains a ferric cata<strong><strong>la</strong>s</strong>e (article 1.2., Article section), whose activity is reduced<br />
un<strong>de</strong>r iron limiting conditions, may exp<strong>la</strong>in this result.<br />
Superoxi<strong>de</strong> dismutase and cata<strong><strong>la</strong>s</strong>e are metallo<strong>en</strong>zymes that can possess differ<strong>en</strong>t<br />
metals in their active c<strong>en</strong>tre. Microorganisms capable to produce differ<strong>en</strong>t superoxi<strong>de</strong><br />
dismutase and cata<strong><strong>la</strong>s</strong>e iso<strong>en</strong>zymes, <strong>de</strong>p<strong>en</strong>ding on culture conditions, have be<strong>en</strong><br />
<strong>de</strong>scribed. These culture conditions inclu<strong>de</strong> oxyg<strong>en</strong> levels, low iron levels or growth<br />
until stationary phase (Storz et al., 1990; Privalle and Fridovich, 1992; Crockford et al.,<br />
1995; Schnell and Steinman, 1995; Barnes et al., 1996; Po<strong>la</strong>ck et al., 1996; St. John and<br />
Steinman, 1996; Lynch and Kuramitsu, 2000; Yesilkaya et al., 2000; Geslin et al., 2001;<br />
Vattanaviboon and Mongkolsuk, 2001).<br />
Results obtained in the pres<strong>en</strong>t work show that P. damse<strong>la</strong>e subsp. piscicida does<br />
not synthetize a new form of superoxi<strong>de</strong> dismutase or cata<strong><strong>la</strong>s</strong>e wh<strong>en</strong> cultured un<strong>de</strong>r<br />
assayed conditions. Thus, all strains show only one band of superoxi<strong>de</strong> dismutase<br />
activity, with simi<strong>la</strong>r electrophoretic mobility to ferric superoxi<strong>de</strong> dismutase <strong>de</strong>scribed<br />
by Barnes et al. (1999a). Ev<strong>en</strong> oxidative stress, by hydrog<strong>en</strong> peroxi<strong>de</strong> or paraquat (an<br />
oxyg<strong>en</strong>ic radical g<strong>en</strong>erator) addition, did not induce the synthesis of a differ<strong>en</strong>t<br />
iso<strong>en</strong>zyme, unlike CuZnSOD and MnSOD <strong>de</strong>scribed in Escherichia coli (Hassan and<br />
Fridovich, 1977; Privalle and Fridovich, 1992; B<strong>en</strong>ov and Fridovich, 1994; Geslin et al.,<br />
2001), MnSOD in A. salmonicida (Barnes et al., 1996; Barnes et al., 1999b),<br />
Pseudomonas aeruginosa (Po<strong>la</strong>ck et al., 1996) and Streptococcus pneumoniae<br />
(Yesilkaya et al., 2000). Neither restrictive iron conditions induce MnSOD synthesis<br />
(Privalle and Fridovich, 1992; Barnes et al., 1999b). Although further studies would be<br />
98
RESULTS AND DISCUSSION<br />
necessary, this <strong>la</strong>ck of a new superoxi<strong>de</strong> dismutase induction could be due to the<br />
pres<strong>en</strong>ce of only one sod g<strong>en</strong>e, sodB, <strong>en</strong>coding ferric superoxi<strong>de</strong> dismutase (Lynch and<br />
Kuramitsu, 2000).<br />
In the same way all strains, un<strong>de</strong>r all assayed culture conditions, show only one<br />
band of cata<strong><strong>la</strong>s</strong>e activity, with simi<strong>la</strong>r electrophoretic mobility to the band <strong>de</strong>scribed by<br />
Barnes et al. (1999a). Treatm<strong>en</strong>t of cata<strong><strong>la</strong>s</strong>e gels with inhibitors indicates that this<br />
bacterium contains an iron-cofactored <strong>en</strong>zyme, because cata<strong><strong>la</strong>s</strong>es with manganese retain<br />
its activity after treatm<strong>en</strong>t with azida and cyani<strong>de</strong>, but they are inhibited with mercuric<br />
chlori<strong>de</strong> (Kono and Fridovich, 1983; Allgood and Perry, 1986; Barnes et al., 1999b).<br />
Despite none assayed culture condition induced synthesis of more than one<br />
superoxi<strong>de</strong> dismutase or cata<strong><strong>la</strong>s</strong>e iso<strong>en</strong>zyme, differ<strong>en</strong>ces in the int<strong>en</strong>sity of the bands<br />
and activity levels, after spectrophotometrically quantification, are observed. These<br />
results are in agreem<strong>en</strong>t with those obtained by Barnes et al. (1999a), who also <strong>de</strong>tected<br />
differ<strong>en</strong>ces in cultures carried out un<strong>de</strong>r iron replete and <strong>de</strong>pleted conditions and highand<br />
low- aerated broths. The quantification of both superoxi<strong>de</strong> dismutase and cata<strong><strong>la</strong>s</strong>e<br />
activities carried out in this study corroborated that differ<strong>en</strong>t band int<strong>en</strong>sities<br />
correspon<strong>de</strong>d to variations in the levels of activity. The lowest levels of superoxi<strong>de</strong><br />
dismutase activity and cata<strong><strong>la</strong>s</strong>e are <strong>de</strong>tected in bacteria grown un<strong>de</strong>r iron-restricted<br />
conditions, attributable to the ferric nature of P. damse<strong>la</strong>e subsp. piscicida superoxi<strong>de</strong><br />
dismutase and cata<strong><strong>la</strong>s</strong>e. Un<strong>de</strong>r iron-limiting conditions the virul<strong>en</strong>t strain shows higher<br />
activity levels of superoxi<strong>de</strong> dismutase and cata<strong><strong>la</strong>s</strong>e, than the non virul<strong>en</strong>t strain. This<br />
result indicates the relevant role of iron uptake mechanisms for superoxi<strong>de</strong> dismutase<br />
and cata<strong><strong>la</strong>s</strong>e activity. Thus, a microorganism capable to obtain iron from the host would<br />
be able to express higher levels of antioxidant <strong>en</strong>zymes and could <strong>de</strong>compose<br />
superoxi<strong>de</strong> and hydrog<strong>en</strong> peroxi<strong>de</strong> radicals g<strong>en</strong>erated by the host. The importance of<br />
superoxi<strong>de</strong> dismutase as antioxidant <strong>en</strong>zyme and its contribution to bacterial virul<strong>en</strong>ce<br />
have be<strong>en</strong> <strong>de</strong>monstrated in several bacterial species such as Listeria monocytog<strong>en</strong>es<br />
(Welch et al., 1979), Shigel<strong>la</strong> flexneri (Franzon et al., 1990) or A. salmonicida (Barnes et<br />
al., 1999a), however its role in P. damse<strong>la</strong>e subsp. piscicida is not very clear and in the<br />
99
RESULTS AND DISCUSSION<br />
pres<strong>en</strong>t work we have observed that the non virul<strong>en</strong>t strain, EPOY-8803-II, shows<br />
activity levels simi<strong>la</strong>r to the virul<strong>en</strong>t strain.<br />
As <strong>de</strong>scribed before, information concerning mechanisms involved in the invasion<br />
and survival of P. damse<strong>la</strong>e subsp. piscicida insi<strong>de</strong> the host is scarce and results<br />
regarding interaction of this pathog<strong>en</strong> with phagocytes have be<strong>en</strong> contradictory. For this<br />
reason, pathog<strong>en</strong> survival after contact with sole phagocytes was evaluated. Results<br />
inclu<strong>de</strong>d in article 1.2. (Article section) show that P. damse<strong>la</strong>e subsp. piscicida is able to<br />
survive insi<strong>de</strong> sole phagocytes at least for five hours, survival rates being higher for the<br />
virul<strong>en</strong>t strain (62%) than the non virul<strong>en</strong>t strain (19%). Also, higher survival rates were<br />
observed in cultures with higher cata<strong><strong>la</strong>s</strong>e activity. These results suggest that bacterial<br />
inactivation could be due to the accumu<strong>la</strong>tion of hydrog<strong>en</strong> peroxi<strong>de</strong>, the precursor of<br />
hydroxyl radicals, after <strong>de</strong>composition of superoxi<strong>de</strong> radicals by bacterial superoxi<strong>de</strong><br />
dismutase. Both virul<strong>en</strong>t and non-virul<strong>en</strong>t strains assayed by Barnes et al. (1999a)<br />
showed high susceptibility to cell-free g<strong>en</strong>erated superoxi<strong>de</strong> radicals, in contrast, we<br />
have observed that a non-virul<strong>en</strong>t strain, EPOY-8803-II, is significantly more<br />
susceptible to killing by sole phagocytes than a virul<strong>en</strong>t strain (Lg h41/01 ). Besi<strong>de</strong>s the<br />
lower cata<strong><strong>la</strong>s</strong>e activity pres<strong>en</strong>t in the non-virul<strong>en</strong>t strain, the <strong>la</strong>ck of a capsule in cells of<br />
EPOY-8803-II could contribute to the high inactivation rates observed. Thus, the capsule<br />
could protect bacterial cells from oxidative radicals or ev<strong>en</strong> prev<strong>en</strong>t activation of<br />
phagocytes (Miller and Britigan, 1997; Arijo et al., 1998).<br />
Finally, the important role of iron in microbial infections has be<strong>en</strong> pointed out by<br />
several authors (Miller and Britigan, 1997; Weinberg, 2000). P. damse<strong>la</strong>e subsp.<br />
piscicida is more susceptible to killing by sole phagocytes wh<strong>en</strong> bacterial cells have<br />
be<strong>en</strong> cultured un<strong>de</strong>r iron-<strong>de</strong>pleted conditions. Bacteria require iron for growth and<br />
replication and synthesize SOD and cata<strong><strong>la</strong>s</strong>e to <strong>de</strong>al with oxidizing anions. P. damse<strong>la</strong>e<br />
subsp. piscicida posses a high-affinity iron uptake system, a sy<strong>de</strong>rophore (Magariños et<br />
al. 1994; Naka et al., 2005). However, <strong>de</strong>spite its ability to obtain iron from high-affinity<br />
systems, several authors have reported that cells grown un<strong>de</strong>r iron-limited conditions<br />
have reduced amounts of capsu<strong>la</strong>r material covering the cells (do Vale et al., 2001).<br />
These cells with reduced capsule would be more susceptible to phagocytosis and<br />
100
RESULTS AND DISCUSSION<br />
oxidative stress. Results obtained (articles 1.1. and 1.2., Article section) show that iron<br />
p<strong>la</strong>ys an important role in survival of P. damse<strong>la</strong>e subsp. piscicida in contact with sole<br />
phagocytes; whether this is attributable to its contribution to capsu<strong>la</strong>r material or SOD<br />
and cata<strong><strong>la</strong>s</strong>e synthesis by the bacterium needs to be investigated. In conclusion, P.<br />
damse<strong>la</strong>e subsp. piscicida is able to survive in contact with sole phagocytes, survival<br />
rates being higher for a virul<strong>en</strong>t strain. The increased levels of cata<strong><strong>la</strong>s</strong>e activity <strong>de</strong>tected<br />
in the virul<strong>en</strong>t strain indicate a possible role for this <strong>en</strong>zyme in bacterial survival.<br />
Once <strong>de</strong>termined the role of superoxi<strong>de</strong> dismutase and cata<strong><strong>la</strong>s</strong>e activities in P.<br />
damse<strong>la</strong>e subsp. piscicida protection against respiratory burst of sole phagocyes, the<br />
following topic to study was the search for prev<strong>en</strong>tion tools such as immunostimu<strong>la</strong>nt<br />
application. This work has focused on the respiratory burst activity of sole phagocytes<br />
to evaluate the possible immunostimu<strong>la</strong>nt effect of two types of microorganisms: a red<br />
microalga, Porphyridium cru<strong>en</strong>tum, and two pot<strong>en</strong>tially probiotic bacteria strains,<br />
whose immunostimu<strong>la</strong>nt activity has be<strong>en</strong> reported in mammals (Morris et al., 2000) and<br />
gilthead seabream (Díaz-Rosales et al., 2006; Salinas et al., 2006) phagocytes,<br />
respectively.<br />
Evaluation of the pot<strong>en</strong>tial stimu<strong>la</strong>nt activity of aqueous and ethanolic extracts<br />
from P. cru<strong>en</strong>tum indicated that none of them stimu<strong>la</strong>ted superoxi<strong>de</strong> anion production,<br />
while the positive control, commercial β-glucan from alga Eugl<strong>en</strong>a gracilis, is able to<br />
increase respiratory burst after 30 min in contact with phagocytes, but only at the highest<br />
conc<strong>en</strong>tration assayed (10 mg ml -1 ). These results are in agreem<strong>en</strong>t with data reported by<br />
Castro et al. (1999), who observed increases of the respiratory burst activity of head<br />
kidney phagocytes of turbot (Psetta maxima) phagocytes and gilthead seabream with<br />
differ<strong>en</strong>t conc<strong>en</strong>trations of β-glucans obtained from fungus and yeasts. On the other<br />
hand, Castro et al. (2004) found great variations in the stimu<strong>la</strong>tive capacities of algal<br />
extracts <strong>de</strong>p<strong>en</strong>ding not only on their origin, but on the conc<strong>en</strong>trations used and time of<br />
incubation.<br />
P. cru<strong>en</strong>tum culture has several advantages, such as fast growth and low cost. On<br />
the other hand P. cru<strong>en</strong>tum could be a natural immunostimu<strong>la</strong>nt, which is<br />
biocompatible, bio<strong>de</strong>gradable and safe for the <strong>en</strong>vironm<strong>en</strong>t and human health. For this<br />
101
RESULTS AND DISCUSSION<br />
reason, once aqueous and ethanolic extracts were assayed in vitro, oral administration of<br />
whole alga cells was performed. In fish, as in other aquatic organisms, administration of<br />
whole microorganisms has focused mainly on bacterial species, such as probiotics<br />
(Verschuere et al., 2000; Nikoske<strong>la</strong>in<strong>en</strong> et al., 2001; Irianto and Austin, 2003; Salinas et<br />
al., 2005; Balcázar et al., 2006; Díaz-Rosales et al., 2006; Salinas et al., 2006), but studies<br />
about whole algae are still very scarce. On the other hand, P. cru<strong>en</strong>tum acumu<strong>la</strong>tes <strong>la</strong>rge<br />
amounts of lipids, specially arachidonic acid and noticeable amounts of<br />
eicosap<strong>en</strong>ta<strong>en</strong>oic acid (Kinsel<strong>la</strong> et al., 1990; Kov<strong>en</strong> et al., 2001); carbohydrates (Fujiki et<br />
al., 1992; Santarém et al., 1997; Bagni et al., 2000; Morris et al., 2000; Esteban et al.,<br />
2001; J<strong>en</strong>ey and J<strong>en</strong>ey, 2002; Cook et al., 2003; Couso et al., 2003; Castro et al., 2004;<br />
Bagni et al., 2005; Kumar et al., 2005); carot<strong>en</strong>es (Tachinaba et al., 1997; Amar et al.,<br />
2004); vitamins (Hardie et al., 1990, 1991; Ortuño et al., 1999; J<strong>en</strong>ey and J<strong>en</strong>ey, 2002).<br />
Due to the fact that this alga contains differ<strong>en</strong>t immunostimu<strong>la</strong>nt substances, its use<br />
could g<strong>en</strong>erate a more g<strong>en</strong>eral immune response as has be<strong>en</strong> proposed for other<br />
microorganisms such as yeasts (Ortuño et al., 2002; Rodríguez et al., 2003).<br />
Thus, three groups of sole specim<strong>en</strong>s received daily for four weeks one of the<br />
differ<strong>en</strong>t diets assayed on a daily basis: commercial diet supplem<strong>en</strong>ted with lyophilized<br />
alga, diet consisting on non-supplem<strong>en</strong>ted commercial diet (control group) or diet<br />
composed of commercial diet containing immunostimu<strong>la</strong>nt, Sanostim. Besi<strong>de</strong>s, to<br />
evaluate the possible synergic effect of immunostimu<strong>la</strong>nt with a vaccine, two weeks<br />
after the beginning of the feeding trial, a group of fish per treatm<strong>en</strong>t were<br />
intraperitoneally inocu<strong>la</strong>ted with a bacterin of P. damse<strong>la</strong>e subsp. piscicida, a formalinkilled<br />
aqueous vaccine. Results obtained show that after four weeks of algal<br />
administration, the superoxi<strong>de</strong> anion production increases in immunized fish. This<br />
increase is statistically significant, not only compared to fish fed with normal diet, but<br />
also compared to fish fed with commercial immunostimu<strong>la</strong>nt. The combined action of<br />
immunostimu<strong>la</strong>nt and vaccine has be<strong>en</strong> <strong>de</strong>scribed by numerous authors, who conclu<strong>de</strong>d<br />
that combination of vaccination and immunostimu<strong>la</strong>nt administration increases vaccine<br />
pot<strong>en</strong>cy (J<strong>en</strong>ey and An<strong>de</strong>rson, 1993; RØsrstad et al., 1993; Aakre et al., 1994; Sakai et al.,<br />
1995; Baulny et al., 1996; Sakai, 1999).<br />
102
RESULTS AND DISCUSSION<br />
Once immunostimu<strong>la</strong>nt effect of algal cells was <strong>de</strong>monstrated, the pot<strong>en</strong>tial effect<br />
of the extracellu<strong>la</strong>r polysaccharidic fraction of P. cru<strong>en</strong>tum on the respiratory burst<br />
activity of sole phagocytes was evaluated (article 2.2., Article section).<br />
Results obtained indicate that in vitro none of the assayed conc<strong>en</strong>trations of the<br />
extracellu<strong>la</strong>r polysacchari<strong>de</strong> from P. cru<strong>en</strong>tum stimu<strong>la</strong>te the respiratory burst of sole<br />
phagocytes, after 30 min contact with fish cells. These results are not in agreem<strong>en</strong>t with<br />
the data obtained by Castro et al. (2004, 2006), who suggest that stimu<strong>la</strong>tion of<br />
respiratory burst activity in turbot (Psetta maxima) phagocytes incubated with algal<br />
extracts, is due to algal polysacchari<strong>de</strong>s. On the other hand, according to Castro et al.<br />
(2004) the modu<strong>la</strong>tory ability of the respiratory burst activity of fish phagocytes varies<br />
greatly among algal species. Therefore, the non stimu<strong>la</strong>tion of respiratory burst in vitro<br />
by P. cru<strong>en</strong>tum polysacchari<strong>de</strong>s could be due to the abs<strong>en</strong>ce of immunostimu<strong>la</strong>nt<br />
activity in this fraction, pres<strong>en</strong>ce in low conc<strong>en</strong>tration or short incubation time with<br />
phagocytes. Whereas, the positive control, a commercial β-glucan, induced an increase<br />
in the respiratory burst, activity wh<strong>en</strong> applied at 10 mg ml -1 .<br />
Additionally, fish were intraperitoneally inocu<strong>la</strong>ted with 500 µg of extracellu<strong>la</strong>r<br />
polysaccharidic fraction. Later, fish were immunized with a bacterin, composed by P.<br />
damse<strong>la</strong>e subsp. piscicida formol-inactivated cells. Sampling time was carried out at 24<br />
h and sev<strong>en</strong> days post-vaccination. Results obtained indicate that the conc<strong>en</strong>tration and<br />
time assayed, 1 and 7 days after polysaccharidic fraction inocu<strong>la</strong>tion, do not produce an<br />
increase in the respiratory burst activity of sole phagocytes, not ev<strong>en</strong> immunized fish.<br />
Moreover, 24 h post-inocu<strong>la</strong>tion, the respiratory burst <strong>de</strong>creases in phagocytes from fish<br />
inocu<strong>la</strong>ted with polysaccharidic fraction or with the bacterin. This <strong>de</strong>crease may be due<br />
to an immunosupression by stress after handling (Thompson et al., 1993; Pulsford et al.,<br />
1995) as it is not observed after 7 days of the inocu<strong>la</strong>tion.<br />
To sum up, the polysaccharidic fraction of P. cru<strong>en</strong>tum, in assayed conditions,<br />
does not <strong>en</strong>hance the respiratory burst activity in sole phagocytes. These results suggest<br />
that stimu<strong>la</strong>tion observed after oral administration of alga cells would be due to other<br />
compounds with immunostimu<strong>la</strong>nt properties. However, we cannot rule out the<br />
possibility that the polysacchari<strong>de</strong> stimu<strong>la</strong>te another immunological parameter. Thus, a<br />
103
RESULTS AND DISCUSSION<br />
great number of works point out the immunostimu<strong>la</strong>nt capacity of polysacchari<strong>de</strong>s in<br />
fish, increasing serum lysozyme, complem<strong>en</strong>t, cytotoxic or phagocytic activities<br />
(Santarém et al., 1997; Esteban et al., 2001; Chang et al., 2003; Bagni et al., 2005; Kumari<br />
and Sahoo, 2006).<br />
Results obtained after oral administration of pot<strong>en</strong>tial probiotic bacteria are<br />
inclu<strong>de</strong>d in article 2.3. (Article section). In this work, the respiratory burst activity was<br />
evaluated and, in or<strong>de</strong>r to <strong>de</strong>termine the protection <strong>de</strong>gree that probiotics could provi<strong>de</strong>,<br />
a chall<strong>en</strong>ge with P. damse<strong>la</strong>e subsp. piscicida was carried out. On the other hand,<br />
intestinal microbiota of fish fed with probiotics was studied in or<strong>de</strong>r to <strong>de</strong>tect possible<br />
changes due to feeding treatm<strong>en</strong>t.<br />
The selected bacterial strains, Pdp11 and Pdp13, iso<strong>la</strong>ted from gilthead seabream<br />
skin (Chabrillón, 2003), belong to Alteromonadaceae family, Shewanel<strong>la</strong> g<strong>en</strong>us.<br />
Although this is the first assay in vivo with strain Pdp13, several works have be<strong>en</strong><br />
carried out previously with strain Pdp11. Thus, Chabrillón et al. (2005a) studied<br />
interactions with the pathog<strong>en</strong> Vibrio harveyi, showing Pdp11 capacity to adhere to<br />
gilthead seabream intestinal mucus, the antagonist effect against a pathog<strong>en</strong>ic strain of<br />
V. harveyi, the capacity to inhibit the pathog<strong>en</strong> union and confer protection against an<br />
experim<strong>en</strong>tal infection. With regard to interactions betwe<strong>en</strong> strain Pdp11 and P.<br />
damse<strong>la</strong>e subsp. piscicida, Chabrillón et al. (2005b) <strong>de</strong>monstrated the antagonistic effect<br />
of Pdp11 against one strain of P. damse<strong>la</strong>e subsp. piscicida and the inhibition of the<br />
adhesion to intestinal mucus of this pathog<strong>en</strong>. These results lead the authors to consi<strong>de</strong>r<br />
the Pdp11 strain as a good candidate to be used as probiotic.<br />
In the pres<strong>en</strong>t work, it has be<strong>en</strong> observed that superoxi<strong>de</strong> anion production is<br />
significantly increased in fish fed with strain Pdp11 after two months from the<br />
beginning of the feeding trial, but fish fed with strain Pdp13 do not show modify<br />
phagocyte respiratory burst. However, fish fed with strain Pdp13 showed higher survival<br />
perc<strong>en</strong>tages after inocu<strong>la</strong>tion of the pathog<strong>en</strong> P. damse<strong>la</strong>e subsp. piscicida.<br />
Although there are numerous works in which respiratory burst activity induction<br />
by probiotics is <strong>de</strong>monstrated (Nikoske<strong>la</strong>in<strong>en</strong> et al., 2003; Gullian et al., 2004; Aubin et<br />
al., 2005; Brunt and Austin, 2005) the fact that pot<strong>en</strong>tial probiotic strain Pdp13 does not<br />
104
RESULTS AND DISCUSSION<br />
increase superoxi<strong>de</strong> anion production, does not mean that this bacterial strains cannot<br />
stimu<strong>la</strong>te another immunological parameter. In fact, survival rates, after experim<strong>en</strong>tal<br />
infection, increased in fish fed with strain Pdp13. Several authors have shown that<br />
probiotics may stimu<strong>la</strong>te differ<strong>en</strong>t immunological parameters, such as phagocytic<br />
activity (Irianto and Austin, 2003; Panigrahi et al., 2004; Brunt and Austin, 2005; Salinas<br />
et al., 2005; Díaz-Rosales et al., 2006), complem<strong>en</strong>t activity (Panigrahi et al., 2004; Díaz-<br />
Rosales et al., 2006), lysozyme activity (Irianto and Austin, 2003; Panigrahi et al., 2004)<br />
or cytotoxic activity (Salinas et al., 2005; Díaz-Rosales et al., 2006). Moreover, effects of<br />
probiotics have be<strong>en</strong> <strong>de</strong>scribed on specific immune response, increasing serum<br />
immunoglobulins levels (Nikoske<strong>la</strong>in<strong>en</strong> et al., 2003; Aubin et al., 2005). For these<br />
reasons, strain Pdp13 can be consi<strong>de</strong>red as a probiotic also, although evaluation of other<br />
immunological parameters would be necessary.<br />
On the other hand, probiotic bacteria have to be administered at optimal doses,<br />
<strong>de</strong>p<strong>en</strong>ding on fish size and bacterial strain (Nikoske<strong>la</strong>in<strong>en</strong> et al., 2003), for this reason<br />
Pdp11 effect is differ<strong>en</strong>t to Pdp13 and the results obtained with Pdp11 and sole are<br />
differ<strong>en</strong>t to results previously obtained with Pdp11 and gilthead seabream (Díaz-Rosales<br />
et al., 2006).<br />
Finally, apart from the immunological effects of probiotics in sole immune<br />
response, possible changes in intestinal microbiota due to probiotics were evaluated. In<br />
spite of the great number of published works concerning microbial communities in fish<br />
(Spanggaard et al., 2000; Holb<strong>en</strong> et al., 2002; Sandaa et al., 2003; Al-Harbi and Naim<br />
Uddin, 2004; Hjelm et al., 2004; Huber et al., 2004; J<strong>en</strong>s<strong>en</strong> et al., 2004), none of them try<br />
to evaluate possible shifts in microbiota after probiotic administration.<br />
In this work DGGE, D<strong>en</strong>aturing Gradi<strong>en</strong>t Gel Electrophoresis, (Muyzer et al.,<br />
1993), was used to study the intestinal microbiota of soles fed with probiotics. Two sets<br />
of primers were evaluated (Nübel et al., 1996; J<strong>en</strong>s<strong>en</strong> et al., 2004). Primers <strong>de</strong>scribed by<br />
Nübel et al. (1996) being selected as the best to study of bacterial communities in the<br />
pres<strong>en</strong>t work after analysis of banding pattern by Pearson coeffici<strong>en</strong>t.<br />
The band pattern obtained was very simple, with few predominant bands, results<br />
alike those <strong>de</strong>scribed by Muyzer et al. (1993), who reported that communities with a few<br />
105
RESULTS AND DISCUSSION<br />
dominant species will produce simpler patterns and the less abundant species may not<br />
a<strong>de</strong>quately repres<strong>en</strong>t in the community pattern. Moreover, the technique has limitations,<br />
and it is possible that some of these bands may not be individual species, as<br />
theoretically is <strong>de</strong>fined, but rather groups which have the same re<strong>la</strong>tive G+C cont<strong>en</strong>t and<br />
have comigrated (Simpson et al., 1999; Temmerman et al., 2003). These limitations may<br />
account in part for the <strong>de</strong>creased band number and may also have influ<strong>en</strong>ced the<br />
appar<strong>en</strong>t diversity and simi<strong>la</strong>rity values (McCrak<strong>en</strong> et al., 2001).<br />
The obtained results do not <strong>de</strong>monstrate that probiotics induce significant shifts in<br />
intestinal microbiota, since bands that appear in groups fed with probiotics are also<br />
pres<strong>en</strong>t in control groups.<br />
On the other hand, it is not possible to confirm that observed bands correspond to<br />
Pdp11 or Pdp13, strains only att<strong>en</strong>ding to electrophoretic mobility, therefore,<br />
phylog<strong>en</strong>etic i<strong>de</strong>ntification or PCR products sequ<strong>en</strong>cing studies will be necessary. After<br />
this work it is possible to affirm that Pdp11 and Pdp13 strains are not capable, at doses<br />
(10 9 ufc g -1 ) and time assayed (two months), to induce significant shifts in intestinal<br />
microbiota. However to exert a local effect during transit through gastrointestinal<br />
system it is not necessary colonization to induce shifts on intestinal microbiota<br />
(Ouwehand et al., 2002). In fact, variation of fish microbiota is substantial and fluctuates<br />
daily (Spanggaard et al., 2000; Al-Harbi and Naim Uddin, 2004; Panigrahi et al., 2004).<br />
106
C ON C L U S I O N S
CONCLUSIONS<br />
Studies carried out on the role of superoxi<strong>de</strong> dismutase and cata<strong><strong>la</strong>s</strong>e activities in<br />
the virul<strong>en</strong>ce of Photobacterium damse<strong>la</strong>e subsp. piscicida, and the use of<br />
microorganisms for the control of this pathog<strong>en</strong>, have yiel<strong>de</strong>d the following<br />
conclusions:<br />
1. Photobacterium damse<strong>la</strong>e subsp. piscicida synthetizes only one iso<strong>en</strong>zyme<br />
with superoxi<strong>de</strong> dismutase activity, characterized by the pres<strong>en</strong>ce of iron in<br />
its active c<strong>en</strong>tre.<br />
2. Photobacterium damse<strong>la</strong>e subsp. piscicida contains only one iso<strong>en</strong>zyme with<br />
cata<strong><strong>la</strong>s</strong>e activity, with iron in its active c<strong>en</strong>tre.<br />
3. Cata<strong><strong>la</strong>s</strong>e activity p<strong>la</strong>ys an important role in P. damse<strong>la</strong>e subsp. piscicida<br />
resistance against hydrog<strong>en</strong> peroxi<strong>de</strong>. Thus, bacterial cells with higher<br />
cont<strong>en</strong>ts of this activity, are able to resist effici<strong>en</strong>tly to hydrog<strong>en</strong> peroxi<strong>de</strong>.<br />
4. Iron p<strong>la</strong>ys a significant role in P. damse<strong>la</strong>e subsp. piscicida survival in the<br />
pres<strong>en</strong>ce of oxidant radicals, because un<strong>de</strong>r iron limiting conditions, lower<br />
levels of superoxi<strong>de</strong> dismutase and cata<strong><strong>la</strong>s</strong>e activities are <strong>de</strong>tected, and higher<br />
susceptibility to hydrog<strong>en</strong> peroxi<strong>de</strong>.<br />
5. Photobacterium damse<strong>la</strong>e subsp. piscicida is able to survive as intracellu<strong>la</strong>r<br />
pathog<strong>en</strong> within sole phagocytes, at least for 5 h.<br />
6. Oral administration of red microalga Porphyridium cru<strong>en</strong>tum, in combination<br />
with intraperitoneal inocu<strong>la</strong>tion of a bacterin containing P. damse<strong>la</strong>e subsp.<br />
piscicida cells, increases respiratory burst activity.<br />
7. Oral administration of Shewanel<strong>la</strong> strains Pdp11 and Pdp13, proposed as<br />
probiotics, increases respiratory burst activity and confers protection against<br />
experim<strong>en</strong>tal infection with P. damse<strong>la</strong>e subsp. piscicida, respectively.<br />
109
CONCLUSIONS<br />
8. The technique DGGE has not allowed to <strong>de</strong>tect possible shifts of sole intestinal<br />
microbiota after oral administration of Shewanel<strong>la</strong> strains Pdp11 and Pdp13.<br />
110
R<br />
R<br />
E F E R E N C I A S<br />
E F E R E N C E S
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S<br />
E C C I Ó N D E A R T Í C U L O S<br />
A<br />
R T I C L E S E C T I O N
SECCIÓN D E ARTÍCU LOS / ARTICLE SECTION<br />
1.1. Díaz-Rosales, P, Chabrillón, M, Moriñigo, MA & Balebona, MC. Survival<br />
against exog<strong>en</strong>ous hydrog<strong>en</strong> peroxi<strong>de</strong> of Photobacterium damse<strong>la</strong>e subsp. piscicida<br />
un<strong>de</strong>r differ<strong>en</strong>t culture conditions. Journal of Fish Diseases 2003; 26, 305–308.<br />
1.2. Díaz-Rosales, P, Chabrillón, M, Arijo, S, Martínez-Manzanares, E, Moriñigo,<br />
MA, Balebona & MC. Superoxi<strong>de</strong> dismutase and cata<strong><strong>la</strong>s</strong>e activities in Photobacterium<br />
damse<strong>la</strong>e ssp. piscicida. Journal of Fish Diseases 2006; 29, 355–364.<br />
2.1. Díaz-Rosales, P, Felices, C, Chabrillón, M, Abda<strong>la</strong>, RT, Figueroa, FL,<br />
Balebona, MC & Moriñigo, MA. Effect of dietary administration of Porphyridium<br />
cru<strong>en</strong>tum on the respiratory burst activity of sole (Solea s<strong>en</strong>egal<strong>en</strong>sis, Kaup 1858)<br />
phagocytes. S<strong>en</strong>d to: Fish & Shellfish Immunology.<br />
2.2. Díaz-Rosales, P, Abda<strong>la</strong>, RT, Decara, J, Arijo, A, Figueroa, FL, Moriñigo,<br />
MA & Balebona, MC. Effect of the extracellu<strong>la</strong>r polysaccharidic fraction from the red<br />
microalga Porphyridium cru<strong>en</strong>tum on the respiratory burst activity of sole (Solea<br />
s<strong>en</strong>egal<strong>en</strong>sis, Kaup 1858) phagocytes. Manuscript in preparation.<br />
2.3. Díaz-Rosales, P, Chabrillón, M, Smidt, H, Arijo, A, León-Rubio, JM, Rico,<br />
RM, A<strong>la</strong>rcón, FJ, Sá<strong>en</strong>z <strong>de</strong> Rodrigáñez, MA, Balebona, MC & Moriñigo, MA. Effect of<br />
dietary administration of probiotics on respiratory burst activity of phagocytes and<br />
intestinal microbiota of S<strong>en</strong>egalese sole (Solea s<strong>en</strong>egal<strong>en</strong>sis, Kaup 1858). Manuscript in<br />
preparation.<br />
135
A RTÍCULO 1.1.<br />
A RTICLE 1.1.
Journal of Fish Diseases 2003, 26, 305–308<br />
Short communication<br />
Survival against exog<strong>en</strong>ous hydrog<strong>en</strong> peroxi<strong>de</strong> of<br />
Photobacterium damse<strong>la</strong>e subsp. piscicida un<strong>de</strong>r differ<strong>en</strong>t<br />
culture conditions<br />
P Díaz-Rosales, M Chabrillón, M A Moriæigo and M C Balebona<br />
Departm<strong>en</strong>t of Microbiology, Faculty of Sci<strong>en</strong>ces, University of Má<strong>la</strong>ga, Spain<br />
Keywords: culture, hydrog<strong>en</strong> peroxi<strong>de</strong>, Photobacterium<br />
damse<strong>la</strong>e subsp. piscicida, survival.<br />
Photobacterium damse<strong>la</strong>e subsp. piscicida is a fish<br />
pathog<strong>en</strong> responsible for important losses in aquaculture<br />
world-wi<strong>de</strong>. Several studies on its virul<strong>en</strong>ce<br />
mechanisms have be<strong>en</strong> carried out and outer<br />
membrane proteins involved in the acquisition of<br />
iron or production of extracellu<strong>la</strong>r products have<br />
be<strong>en</strong> suggested as the main <strong>de</strong>terminants of its<br />
virul<strong>en</strong>ce for fish (Magariños, Santos, Romal<strong>de</strong>,<br />
Rivas, Barja & Toranzo 1992; Magariños, Romal<strong>de</strong>,<br />
Lemos, Barja & Toranzo 1994). However, the actual<br />
methods of invasion and survival insi<strong>de</strong> the host are<br />
still unknown and while some authors have reported<br />
the pres<strong>en</strong>ce of intact bacteria insi<strong>de</strong> fish cells,<br />
suggesting the ability of the bacterium to survive<br />
as an intracellu<strong>la</strong>r pathog<strong>en</strong> (Noya, Magariños,<br />
Toranzo & Lamas 1995; López-Dóriga, Barnes,<br />
dos Santos & Ellis 2000), others have observed that<br />
this pathog<strong>en</strong> is highly susceptible to oxidative<br />
radicals g<strong>en</strong>erated during the macrophage respiratory<br />
burst (Skarmeta, Bandín, Santos & Toranzo<br />
1995; Barnes, Balebona, Horne & Ellis 1999a).<br />
Reactive oxyg<strong>en</strong> species (ROS) such as hydrog<strong>en</strong><br />
peroxi<strong>de</strong> and superoxi<strong>de</strong> are g<strong>en</strong>erated during the<br />
macrophage respiratory burst in response to microbial<br />
infection. Bacterial pathog<strong>en</strong>s must overcome<br />
the toxic effects of ROS to establish infections.<br />
Correspon<strong>de</strong>nce Dr M C Balebona, Departm<strong>en</strong>t of Microbiology,<br />
Faculty of Sci<strong>en</strong>ces, University of Ma<strong>la</strong>ga, Campus<br />
Teatinos, 29071 Ma<strong>la</strong>ga, Spain<br />
1 (e-mail: balebona@uma.es)<br />
Production of superoxi<strong>de</strong> dismutase and cata<strong><strong>la</strong>s</strong>e<br />
<strong>en</strong>zymes, which <strong>de</strong>compose superoxi<strong>de</strong> and peroxi<strong>de</strong><br />
radicals, respectively, have be<strong>en</strong> reported to<br />
contribute to the virul<strong>en</strong>ce of a number of<br />
pathog<strong>en</strong>s (Franzon, Aron<strong>de</strong>l & Sansonetti 1990;<br />
Lefebre & Valvano 2001; Uzzau, Bossi & Figueroa-<br />
Bossi 2002). Thus, the ability of cata<strong><strong>la</strong>s</strong>e to<br />
<strong>de</strong>compose peroxi<strong>de</strong> radicals increases survival of<br />
bacteria in the pres<strong>en</strong>ce of peroxi<strong>de</strong>.<br />
In addition, increased levels of cata<strong><strong>la</strong>s</strong>e activity<br />
wh<strong>en</strong> bacteria are cultured un<strong>de</strong>r certain conditions,<br />
such as the pres<strong>en</strong>ce of peroxi<strong>de</strong> radicals or until<br />
the stationary phase, have be<strong>en</strong> reported (Stortz,<br />
Tartaglia & Ames 1990; Loew<strong>en</strong> 1997). Moreover,<br />
the fact that most cata<strong><strong>la</strong>s</strong>es are iron-cofactored<br />
suggests that growth un<strong>de</strong>r differ<strong>en</strong>t iron conc<strong>en</strong>trations<br />
may have some effect on this <strong>en</strong>zyme activity.<br />
Cata<strong><strong>la</strong>s</strong>e activity has be<strong>en</strong> reported in P. damse<strong>la</strong>e<br />
subsp. piscicida (Barnes et al. 1999a), however, the<br />
role of this <strong>en</strong>zyme in the protection against<br />
peroxi<strong>de</strong> has not yet be<strong>en</strong> <strong>de</strong>termined. For this<br />
reason, the resistance to peroxi<strong>de</strong> radicals of<br />
P. damse<strong>la</strong>e subsp. piscicida cells grown un<strong>de</strong>r iron<br />
limited and replete conditions, and pulsed with<br />
hydrog<strong>en</strong> peroxi<strong>de</strong>, has be<strong>en</strong> evaluated in this study.<br />
Two strains of P. damse<strong>la</strong>e subsp. piscicida have<br />
be<strong>en</strong> inclu<strong>de</strong>d in this study. The virul<strong>en</strong>t strain<br />
(Lg41/01) (LD 50 ¼ 2.2 · 10 4 CFU g )1 ) was iso<strong>la</strong>ted<br />
from diseased sole, Solea s<strong>en</strong>egal<strong>en</strong>sis Kaup,<br />
showing typical signs of pseudotuberculosis, and<br />
the non-virul<strong>en</strong>t strain (Epoy) (LD 50 ><br />
1.0 · 10 8 CFU g )1 ; Magariños, Bonet, Romal<strong>de</strong>,<br />
Martínez, Congregado & Toranzo 1996) kindly<br />
supplied by Dr K. Muroga (Faculty of Applied<br />
Ó 2003<br />
B<strong>la</strong>ckwell Publishing Ltd<br />
305
Journal of Fish Diseases 2003, 26, 305–308<br />
PDíaz-Rosales et al. Resistance of Photobacterium damse<strong>la</strong>e to hydrog<strong>en</strong> peroxi<strong>de</strong><br />
Biological Sci<strong>en</strong>ce, Hiroshima University, Japan).<br />
Iso<strong>la</strong>tes were cultured in 250-mL f<strong><strong>la</strong>s</strong>ks containing<br />
100 mL of tryptic soya broth supplem<strong>en</strong>ted with 2%<br />
NaCl (TSBS) at 22° C until the early stationary<br />
phase (O.D. 600 nm ¼ 1.0). The effect of iron<br />
conc<strong>en</strong>tration on the cultures was evaluated in cells<br />
grown in TSBS supplem<strong>en</strong>ted with 2,2-dipyridyl<br />
(100 lm) or ferric chlori<strong>de</strong> (100 lm) according to the<br />
methodology <strong>de</strong>scribed by Barnes et al. (1999a).<br />
Bacterial survival against peroxi<strong>de</strong> after a pot<strong>en</strong>tial<br />
induction of cata<strong><strong>la</strong>s</strong>e by hydrog<strong>en</strong> peroxi<strong>de</strong> was tested<br />
according to Barnes, Bow<strong>de</strong>n, Horne & Ellis (1999b)<br />
by adding 20 lm hydrog<strong>en</strong> peroxi<strong>de</strong> to mid-expon<strong>en</strong>tial<br />
phase cultures and 2 mm hydrog<strong>en</strong> peroxi<strong>de</strong><br />
to early stationary phase cultures.<br />
Cells were harvested, washed and resusp<strong>en</strong><strong>de</strong>d in<br />
phosphate-buffered saline (PBS) to a <strong>de</strong>nsity of<br />
10 9 CFU mL )1 (O.D. 600 nm ¼ 1.00). Aliquots of<br />
100 lL were used to inocu<strong>la</strong>te 9.9 mL PBS containing<br />
hydrog<strong>en</strong> peroxi<strong>de</strong> at conc<strong>en</strong>trations of 0, 0.05,<br />
0.1, 0.5, 1 and 10 mm. Samples were incubated for<br />
1 h at 22° C and surviving bacteria were <strong>en</strong>umerated<br />
by viable counts on tryptic soya agar with 2% NaCl<br />
3 (TSAS) p<strong>la</strong>tes. The survival of H 2 O 2 -treated bacteria<br />
was expressed as the perc<strong>en</strong>tage of colony forming<br />
units recovered compared with untreated samples.<br />
An ANOVA test was performed to compare the<br />
results of the experim<strong>en</strong>ts.<br />
Previous studies with P. damse<strong>la</strong>e subsp. piscicida<br />
exposed to photochemically g<strong>en</strong>erated superoxi<strong>de</strong><br />
radicals show that bacterial inactivation is overcome<br />
wh<strong>en</strong> cata<strong><strong>la</strong>s</strong>e is ad<strong>de</strong>d to the medium (Barnes et al.<br />
1999b), thus indicating the important effect of<br />
hydrog<strong>en</strong> peroxi<strong>de</strong> on the inactivation of this<br />
bacterium. Results obtained in this study indicate<br />
that P. damse<strong>la</strong>e subsp. piscicida shows increased<br />
survival wh<strong>en</strong> exposed to peroxi<strong>de</strong> radicals wh<strong>en</strong><br />
cells have previously be<strong>en</strong> in contact with hydrog<strong>en</strong><br />
peroxi<strong>de</strong>. Both the virul<strong>en</strong>t and non-virul<strong>en</strong>t strains<br />
were inactivated after 1 h incubation with 10 mm<br />
H 2 O 2 , however, wh<strong>en</strong> <strong>de</strong>creasing conc<strong>en</strong>trations of<br />
peroxi<strong>de</strong> were used, a higher <strong>de</strong>gree of resistance to<br />
peroxi<strong>de</strong> was observed in the virul<strong>en</strong>t strain compared<br />
with the non-virul<strong>en</strong>t strain (Fig. 1).<br />
(a)<br />
Perc<strong>en</strong>tage of survival<br />
140<br />
120<br />
100<br />
80<br />
60<br />
40<br />
20<br />
(b)<br />
140<br />
120<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
Perc<strong>en</strong>tage of survival<br />
0 0.05 0.1 0.5 1 10<br />
Peroxi<strong>de</strong> conc<strong>en</strong>tration (m M)<br />
Figure 1 Survival of Photobacterium<br />
damse<strong>la</strong>e subsp. piscicida, strains Epoy (a)<br />
and Lg41/01 (b) to exog<strong>en</strong>ous peroxi<strong>de</strong>.<br />
( )Stationary phase cultures; ( ) cultures<br />
treated at the mid-expon<strong>en</strong>tial phase with<br />
20 lm peroxi<strong>de</strong> followed by 2 mm peroxi<strong>de</strong><br />
in the early stationary phase; ( ) cells grown<br />
in TSBS with 100 lm 2,2-dypiridyl; ( )<br />
cells grown in TSBS with 100 lm ferric<br />
chlori<strong>de</strong>.<br />
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PDíaz-Rosales et al. Resistance of Photobacterium damse<strong>la</strong>e to hydrog<strong>en</strong> peroxi<strong>de</strong><br />
A significant (P < 0.05) increase in the survival<br />
rates of the non-virul<strong>en</strong>t strain was observed wh<strong>en</strong><br />
cultures were pulsed with hydrog<strong>en</strong> peroxi<strong>de</strong><br />
compared with cells cultured until the stationary<br />
phase. In contrast, this increase has not be<strong>en</strong><br />
observed for the virul<strong>en</strong>t strain, which always<br />
showed higher survival regardless of the growth<br />
phase or the pulse with hydrog<strong>en</strong> peroxi<strong>de</strong>.<br />
Peroxi<strong>de</strong> induction of cata<strong><strong>la</strong>s</strong>e and increased cell<br />
survival have be<strong>en</strong> reported for several bacterial<br />
4 pathog<strong>en</strong>s (Loew<strong>en</strong>, Swita<strong>la</strong> & Triggs-Raine 1985;<br />
Barnes et al. 1999b; Vattanaviboon & Mongkolsuk<br />
2001). Results obtained in this study suggest that<br />
peroxi<strong>de</strong>-<strong>de</strong>composing <strong>en</strong>zymes induced in the<br />
strain Epoy only by peroxi<strong>de</strong> treatm<strong>en</strong>t could<br />
protect these cells from oxidation, whilst <strong>de</strong>creasing<br />
survival rates observed in cells grown in other<br />
conditions could be attributable to lower levels of<br />
cata<strong><strong>la</strong>s</strong>e and peroxidase activities. In contrast, the<br />
high survival rates observed in the virul<strong>en</strong>t strain in<br />
stationary phase cultures, and in cells cultured in<br />
the pres<strong>en</strong>ce of iron or pulsed with hydrog<strong>en</strong><br />
peroxi<strong>de</strong> suggest the pres<strong>en</strong>ce of higher levels of<br />
cata<strong><strong>la</strong>s</strong>e activity in the cells grown un<strong>de</strong>r these<br />
conditions, although a possible re<strong>la</strong>tionship with<br />
virul<strong>en</strong>ce remains to be <strong>de</strong>monstrated. Furthermore,<br />
the pres<strong>en</strong>ce of a capsule in the virul<strong>en</strong>t strain<br />
may have an important role in the protection of<br />
P. damse<strong>la</strong>e subsp. piscicida cells against peroxi<strong>de</strong>.<br />
This capsule would partially contribute to<br />
the increased survival of the virul<strong>en</strong>t strain compared<br />
with strain Epoy, a non-capsu<strong>la</strong>ted strain<br />
(Magariños et al. 1996).<br />
Wh<strong>en</strong> bacteria were cultured un<strong>de</strong>r iron limited<br />
conditions, a significant <strong>de</strong>crease (P < 0.05) in<br />
survival was observed for both strains compared<br />
with cells grown un<strong>de</strong>r iron replete conditions or<br />
pulsed with peroxi<strong>de</strong>. The <strong>de</strong>crease in bacterial<br />
survival in cultures grown un<strong>de</strong>r iron limited<br />
conditions suggests the pres<strong>en</strong>ce of an ironcofactored<br />
cata<strong><strong>la</strong>s</strong>e in P. damse<strong>la</strong>e subsp. piscicida.<br />
In this way, the ability to obtain iron from the<br />
host would <strong>de</strong>termine the ability to cope with the<br />
radicals g<strong>en</strong>erated during the respiratory burst. It<br />
should also be noted that <strong>de</strong>composition of<br />
superoxi<strong>de</strong> anions primarily g<strong>en</strong>erated during the<br />
phagocytic respiratory burst <strong>de</strong>p<strong>en</strong>ds on the activity<br />
of a ferric superoxi<strong>de</strong> dismutase in P. damse<strong>la</strong>e<br />
subsp. piscicida (Barnes et al. 1999a). Additional<br />
studies to <strong>de</strong>monstrate the pres<strong>en</strong>ce of iron as a<br />
cofactor in the cata<strong><strong>la</strong>s</strong>e, and the s<strong>en</strong>sitivity of<br />
P. damse<strong>la</strong>e subsp. piscicida to the radicals<br />
g<strong>en</strong>erated during the macrophage respiratory burst,<br />
are in progress.<br />
Refer<strong>en</strong>ces<br />
Barnes A.C., Balebona M.C., Horne M.T. & Ellis A.E. (1999a)<br />
Superoxi<strong>de</strong> dismutase and cata<strong><strong>la</strong>s</strong>e in Photobacterium damse<strong>la</strong>e<br />
subsp. piscicida and their roles in resistance to reactive oxyg<strong>en</strong><br />
species. Microbiology 145, 483–494.<br />
Barnes A.C., Bow<strong>de</strong>n T.J., Horne M.T. & Ellis A.E. (1999b)<br />
Peroxi<strong>de</strong>-inducible cata<strong><strong>la</strong>s</strong>e in Aeromonas salmonicida subsp.<br />
salmonicida protects against exog<strong>en</strong>ous hydrog<strong>en</strong> peroxi<strong>de</strong> and<br />
killing by activated rainbow trout, Oncorhynchus mykiss, L.,<br />
macrophages. Microbial Pathog<strong>en</strong>esis 26, 149–158.<br />
Franzon V.L., Aron<strong>de</strong>l I. & Sansonetti P.I. (1990) Contribution<br />
of superoxi<strong>de</strong> dismutase and cata<strong><strong>la</strong>s</strong>e activities to<br />
Shigel<strong>la</strong> flexneri pathog<strong>en</strong>esis. Infection and Immunity 58,<br />
529–535.<br />
Lefebre M.D. & Valvano M.A. (2001) In vitro resistance of<br />
Burkhol<strong>de</strong>ria cepacia complex iso<strong>la</strong>tes to reactive oxyg<strong>en</strong><br />
species in re<strong>la</strong>tion to cata<strong><strong>la</strong>s</strong>e and superoxi<strong>de</strong> dismutase<br />
production. Microbiology 147, 97–109.<br />
Loew<strong>en</strong> P.C. (1997) Bacterial cata<strong><strong>la</strong>s</strong>es. In: Oxidative Stress and<br />
the Molecu<strong>la</strong>r Biology of Antioxidant Def<strong>en</strong>ses (ed. by J.G.<br />
Scandalios ), pp. 273–308. Cold Spring Harbor Press,<br />
5 Woodbury, NY, USA.<br />
Loew<strong>en</strong> P.C., Swita<strong>la</strong> J. & Triggs-Raine B.L. (1985) Cata<strong><strong>la</strong>s</strong>es<br />
HPI and HPII in Escherichia coli are induced in<strong>de</strong>p<strong>en</strong><strong>de</strong>ntly.<br />
Archives in Biochemistry and Biophysics 243, 144–149.<br />
López-Dóriga M.V., Barnes A.C., dos Santos N.M.S. & Ellis<br />
A.E. (2000) Invasion of fish epithelial cells by Photobacterium<br />
damse<strong>la</strong>e subsp. piscicida : evi<strong>de</strong>nce for receptor specificity, and<br />
effect of capsule and serum. Microbiology 146, 21–30.<br />
Magariños B., Santos Y., Romal<strong>de</strong> J.L., Rivas C., Barja J.L. &<br />
Toranzo A.E. (1992) Pathog<strong>en</strong>ic activities of live cells and<br />
extracellu<strong>la</strong>r products of the fish pathog<strong>en</strong> Pasteurel<strong>la</strong> piscicida.<br />
Journal of G<strong>en</strong>eral Microbiology 138, 2491–2498.<br />
Magariños B., Romal<strong>de</strong> J.L., Lemos M.L., Barja J.L. & Toranzo<br />
A.E. (1994) Iron uptake by Pasteurel<strong>la</strong> piscicida and its role in<br />
pathog<strong>en</strong>icity for fish. Applied and Environm<strong>en</strong>tal Microbiology<br />
60, 2990–2998.<br />
Magariños B., Bonet R., Romal<strong>de</strong> J.L., Martínez M.J.,<br />
Congregado F. & Toranzo A.E. (1996) Influ<strong>en</strong>ce of the<br />
capsu<strong>la</strong>r <strong>la</strong>yer on the virul<strong>en</strong>ce of Pasteurel<strong>la</strong> piscicida for fish.<br />
Microbial Pathog<strong>en</strong>esis 21, 289–297.<br />
Noya M., Magariños B., Toranzo A.E. & Lamas J. (1995)<br />
Sequ<strong>en</strong>tial pathology of experim<strong>en</strong>tal pasteurellosis in gilthead<br />
sea bream, Sparus aurata. A light-and electron microscopic<br />
study. Diseases of Aquatic Organisms 21, 177–186.<br />
Skarmeta A.M., Bandín I., Santos Y. & Toranzo A.E. (1995)<br />
In vitro killing of Pasteurel<strong>la</strong> piscicida by fish macrophages.<br />
Diseases of Aquatic Organisms 23, 51–57.<br />
Stortz G., Tartaglia L.A. & Ames B.N. (1990) Transcriptional<br />
regu<strong>la</strong>tion of oxidative stress inducible g<strong>en</strong>es: direct activation<br />
by oxidation. Sci<strong>en</strong>ce 248, 189–194.<br />
Uzzau S., Bossi L. & Figueroa-Bossi N. (2002) Differ<strong>en</strong>tial<br />
accumu<strong>la</strong>tion of Salmonel<strong>la</strong> (Cu, Zn) superoxi<strong>de</strong> dismutases<br />
Ó 2003<br />
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Journal of Fish Diseases 2003, 26, 305–308<br />
PDíaz-Rosales et al. Resistance of Photobacterium damse<strong>la</strong>e to hydrog<strong>en</strong> peroxi<strong>de</strong><br />
SodCI and SodCII in intracellu<strong>la</strong>r bacteria: corre<strong>la</strong>tion with<br />
their re<strong>la</strong>tive contribution to pathog<strong>en</strong>icity. Molecu<strong>la</strong>r Microbiology<br />
46, 147–156.<br />
Vattanaviboon P. & Mongkolsuk S. (2001) Unusual adaptive,<br />
cross protection responses and growth phase resistance against<br />
peroxi<strong>de</strong> killing in a bacterial shrimp pathog<strong>en</strong>, Vibrio harveyi.<br />
FEMS Microbiology Letters 200, 111–116.<br />
Received: 18 November 2002<br />
Accepted: 23 January 2003<br />
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A RTÍCULO 1.2.<br />
A RTICLE 1.2.
Journal of Fish Diseases 2006, 29, 355–364<br />
Superoxi<strong>de</strong> dismutase and cata<strong><strong>la</strong>s</strong>e activities<br />
in Photobacterium damse<strong>la</strong>e ssp. piscicida<br />
P Díaz-Rosales, M Chabrillón, S Arijo, E Martinez-Manzanares, M A MoriÇigo<br />
and M C Balebona<br />
Departm<strong>en</strong>t of Microbiology, Faculty of Sci<strong>en</strong>ces, University of Má<strong>la</strong>ga, Ma<strong>la</strong>ga, Spain<br />
Abstract<br />
The ability of a set of Photobacterium damse<strong>la</strong>e ssp.<br />
piscicida strains iso<strong>la</strong>ted from differ<strong>en</strong>t fish species to<br />
produce differ<strong>en</strong>t superoxi<strong>de</strong> dismutase (SOD) and<br />
cata<strong><strong>la</strong>s</strong>e <strong>en</strong>zymes was <strong>de</strong>termined. Unlike other bacterial<br />
pathog<strong>en</strong>s, P. damse<strong>la</strong>e ssp. piscicida is not able<br />
to produce differ<strong>en</strong>t isoforms of SOD or cata<strong><strong>la</strong>s</strong>e<br />
containing differ<strong>en</strong>t metal cofactors wh<strong>en</strong> cultured<br />
un<strong>de</strong>r oxidative stress induced by hydrog<strong>en</strong> peroxi<strong>de</strong><br />
or methyl violog<strong>en</strong>, or un<strong>de</strong>r iron <strong>de</strong>pleted conditions.<br />
However, iron cont<strong>en</strong>t of the growth medium<br />
influ<strong>en</strong>ced the levels of SOD and cata<strong><strong>la</strong>s</strong>e activity in<br />
cells, these levels <strong>de</strong>creasing with iron avai<strong>la</strong>bility of<br />
the medium. Comparison of virul<strong>en</strong>t and non-virul<strong>en</strong>t<br />
strains of P. damse<strong>la</strong>e ssp. piscicida showed simi<strong>la</strong>r<br />
cont<strong>en</strong>ts of SOD, but higher levels of cata<strong><strong>la</strong>s</strong>e<br />
were <strong>de</strong>tected in cells of the virul<strong>en</strong>t strain. Incubation<br />
of bacteria with sole, Solea s<strong>en</strong>egal<strong>en</strong>sis (Kaup),<br />
phagocytes has shown that survival rates range from<br />
19% to 62%, these rates being higher for the virul<strong>en</strong>t<br />
strain. The increased levels of cata<strong><strong>la</strong>s</strong>e activity<br />
<strong>de</strong>tected in the virul<strong>en</strong>t strain indicates a possible role<br />
for this <strong>en</strong>zyme in bacterial survival.<br />
Keywords: cata<strong><strong>la</strong>s</strong>e, phagocyte, Photobacterium<br />
damse<strong>la</strong>e subsp, Solea s<strong>en</strong>egal<strong>en</strong>sis, superoxi<strong>de</strong><br />
dismutase, virul<strong>en</strong>ce.<br />
Introduction<br />
Photobacterium damse<strong>la</strong>e ssp. piscicida is a pathog<strong>en</strong><br />
responsible for important losses in fish<br />
Correspon<strong>de</strong>nce Prof. M C Balebona, Departm<strong>en</strong>t of<br />
Microbiology, Faculty of Sci<strong>en</strong>ces, University of Ma<strong>la</strong>ga, 29071<br />
Ma<strong>la</strong>ga, Spain<br />
(e-mail: balebona@uma.es)<br />
aquaculture worldwi<strong>de</strong>. The importance of extracellu<strong>la</strong>r<br />
products, the pres<strong>en</strong>ce of iron uptake<br />
mechanisms and the capsu<strong>la</strong>r material as virul<strong>en</strong>ce<br />
factors in P. damse<strong>la</strong>e ssp. piscicida are well<br />
docum<strong>en</strong>ted (Magariños, Romal<strong>de</strong>, Bandín, Fouz<br />
& Toranzo 1992; Magariños, Pazos, Santos,<br />
Romal<strong>de</strong> & Toranzo 1994; Magariños, Romal<strong>de</strong>,<br />
Lemos, Barja & Toranzo 1995; Arijo, Borrego,<br />
Zorril<strong>la</strong>, Balebona & Moriñigo 1998). However,<br />
information concerning mechanisms involved in<br />
the invasion and survival insi<strong>de</strong> the host is scarce<br />
and results regarding interaction of P. damse<strong>la</strong>e<br />
ssp. piscicida with phagocytes have be<strong>en</strong> contradictory.<br />
While some authors have reported the<br />
pres<strong>en</strong>ce of intact bacteria insi<strong>de</strong> fish cells,<br />
suggesting the ability of P. damse<strong>la</strong>e to survive as<br />
an intracellu<strong>la</strong>r pathog<strong>en</strong> (Kubota, Kimura &<br />
Egusa 1970; Nelson, Kawahara, Kawai & Kusuda<br />
1981; Kusuda & Sa<strong>la</strong>ti 1993; Noya, Magariños &<br />
Lamas 1995a; Noya, Magariños, Toranzo &<br />
Lamas 1995b), others have observed that this<br />
pathog<strong>en</strong> is highly susceptible to oxidative radicals<br />
g<strong>en</strong>erated during the macrophage respiratory burst<br />
(Skarmeta, Bandín, Santos & Toranzo 1995; Arijo<br />
et al. 1998).<br />
The reactive oxyg<strong>en</strong> species (ROS), such as<br />
hydrog<strong>en</strong> peroxi<strong>de</strong> (H 2 O 2 ) and superoxi<strong>de</strong> anion<br />
(O 2 ), are produced by phagocytes in response to<br />
microbial infection. ROS constitute an important<br />
compon<strong>en</strong>t of the innate active <strong>de</strong>f<strong>en</strong>ce response<br />
against invading microorganisms by fish phagocytic<br />
cells. Therefore, bacterial pathog<strong>en</strong>s must<br />
overcome the toxic effects of ROS to establish<br />
infections. Microorganisms have evolved systems<br />
to protect themselves from these highly toxic<br />
radicals. One of these protective pathways involves<br />
the production of <strong>de</strong>toxifying <strong>en</strong>zymes such as<br />
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Journal of Fish Diseases 2006, 29, 355–364<br />
PDíaz-Rosales et al. Superoxi<strong>de</strong> dismutase and cata<strong><strong>la</strong>s</strong>e in P. damse<strong>la</strong>e ssp. piscicida<br />
superoxi<strong>de</strong> dismutases (SODs) and cata<strong><strong>la</strong>s</strong>es. Production<br />
of SOD and cata<strong><strong>la</strong>s</strong>e <strong>en</strong>zymes, which<br />
<strong>de</strong>compose superoxi<strong>de</strong> and peroxi<strong>de</strong> radicals,<br />
respectively, have be<strong>en</strong> reported to contribute to<br />
the virul<strong>en</strong>ce of a great number of pathog<strong>en</strong>s<br />
(Franzon, Aron<strong>de</strong>l & Sansonetti 1990; Lynch &<br />
Kuramitsu 2000; Lefebre & Valvano 2001; Uzzau,<br />
Bossi & Figueroa-Bossi 2002).<br />
Superoxi<strong>de</strong> dismutases are a family of metallo<strong>en</strong>zymes<br />
including four types <strong>de</strong>p<strong>en</strong>ding on the<br />
metal cofactor, copper-zinc (Cu/Zn-SOD), manganese<br />
(Mn-SOD), iron (Fe-SOD) and nickel<br />
(Ni-SOD) (Lynch & Kuramitsu 2000). Three<br />
types of cata<strong><strong>la</strong>s</strong>e have be<strong>en</strong> <strong>de</strong>scribed: monofunctional<br />
cata<strong><strong>la</strong>s</strong>es, bifunctional cata<strong><strong>la</strong>s</strong>es or cata<strong><strong>la</strong>s</strong>e/<br />
peroxidase and pseudocata<strong><strong>la</strong>s</strong>es or non-haeme<br />
cata<strong><strong>la</strong>s</strong>es, with manganese as a metal cofactor<br />
(Loew<strong>en</strong> 1997).<br />
Microorganisms produce differ<strong>en</strong>t SOD and<br />
cata<strong><strong>la</strong>s</strong>e isozymes inducible un<strong>de</strong>r certain culture<br />
conditions such as high oxyg<strong>en</strong> t<strong>en</strong>sion, low levels<br />
of iron or stationary growth phase (Crockford,<br />
Davis & Williams 1995; Schnell & Steinman 1995;<br />
Barnes, Horne & Ellis 1996; Po<strong>la</strong>ck, Dacheux,<br />
Delic-Attree, Toussaint & Vignais 1996; St John &<br />
Steinman 1996; Lynch & Kuramitsu 2000; Vattanaviboon<br />
& Mongkolsuk 2001). However, information<br />
on the SOD and cata<strong><strong>la</strong>s</strong>e activities of<br />
P. damse<strong>la</strong>e ssp. piscicida is scarce.<br />
The aim of this work was to <strong>de</strong>termine whether<br />
P. damse<strong>la</strong>e ssp. piscicida can express differ<strong>en</strong>t SOD<br />
and cata<strong><strong>la</strong>s</strong>e activities wh<strong>en</strong> cultured un<strong>de</strong>r differ<strong>en</strong>t<br />
conditions, and whether these <strong>en</strong>zymatic activities<br />
may protect the bacterium in vitro from oxyg<strong>en</strong><br />
radicals g<strong>en</strong>erated during the macrophage respiratory<br />
burst.<br />
Materials and methods<br />
Bacteria<br />
Strains of P. damse<strong>la</strong>e ssp. piscicida used in this<br />
study are listed in Table 1. Strains B180, D 26/98 ,<br />
Pp8H, R45, R46, B51 and Lg h41/01 were iso<strong>la</strong>ted in<br />
our <strong>la</strong>boratory (Departm<strong>en</strong>t of Microbiology, Faculty<br />
of Sci<strong>en</strong>ces, University of Má<strong>la</strong>ga, Spain).<br />
Strains MT 1415, MT 1375, MT 1376 and MT<br />
1379 were kindly provi<strong>de</strong>d by Dr A.C. Barnes<br />
(Marine Laboratory, Aber<strong>de</strong><strong>en</strong>, UK); strain DI-21S<br />
by Dr A.E. Toranzo (Departm<strong>en</strong>t of Microbiology<br />
and Parasitology, Faculty of Chemistry, University<br />
of Santiago <strong>de</strong> Composte<strong>la</strong>, Spain) and EPOY-<br />
8803-II by Dr K. Muroga (Faculty of Applied<br />
Biological Sci<strong>en</strong>ces, Hiroshima University, Hiroshima,<br />
Japan). Strains 17911 and 29690 were<br />
obtained from the American Type Culture Collection<br />
(ATCC).<br />
Virul<strong>en</strong>ce assays were carried out with two<br />
selected strains: Lg h41/01 and EPOY-8803-II. Assays<br />
to <strong>de</strong>termine the lethal dose 50% (LD 50 ) for sole,<br />
Solea s<strong>en</strong>egal<strong>en</strong>sis (Kaup), were carried out following<br />
the methodology <strong>de</strong>scribed by Santos (1991).<br />
Groups of five fish (10–15 g body weight) maintained<br />
in tanks at 24 °C, were intraperitoneally<br />
inocu<strong>la</strong>ted with 0.1 mL of serial bacterial dilutions<br />
containing 10 3 –10 8 cfu. The same number of fish<br />
was inocu<strong>la</strong>ted with phosphate-buffered saline<br />
(PBS) and used as a control. Inocu<strong>la</strong>ted fish were<br />
observed daily for 14 days, and all mortalities were<br />
recor<strong>de</strong>d. Mortalities were consi<strong>de</strong>red to be due to<br />
the inocu<strong>la</strong>tion wh<strong>en</strong> the bacterial strain was<br />
iso<strong>la</strong>ted in pure culture from internal organs of<br />
<strong>de</strong>ad fish. Lethal dose 50% (LD 50 ) repres<strong>en</strong>ts the<br />
Strain Host Source<br />
17911 Roccus americanus ATCC<br />
29690 Serio<strong>la</strong> quinqueradiata ATCC<br />
B51 Dic<strong>en</strong>trarchus <strong>la</strong>brax UMA, Spain<br />
B180 Sparus aurata UMA, Spain<br />
D 26/98 S. aurata UMA, Spain<br />
Pp8H S. aurata UMA, Spain<br />
R45 S. aurata UMA, Spain<br />
R46 S. aurata UMA, Spain<br />
DI-21S S. aurata USC, Spain<br />
EPOY-8803-II Epinephelus akaara Japan<br />
Lg h41/01 Solea s<strong>en</strong>egal<strong>en</strong>sis UMA, Spain<br />
MT1415 D. <strong>la</strong>brax Marine Laboratory, Aber<strong>de</strong><strong>en</strong>, UK<br />
MT1375 D. <strong>la</strong>brax Marine Laboratory, Aber<strong>de</strong><strong>en</strong>, UK<br />
MT1376 S. aurata Marine Laboratory, Aber<strong>de</strong><strong>en</strong>, UK<br />
MT1379 S. aurata Marine Laboratory, Aber<strong>de</strong><strong>en</strong>, UK<br />
Table 1 Photobacterium damse<strong>la</strong>e subsp.<br />
piscicida strains used in this study<br />
ATCC, American Type Culture Collection; UMA, University of Má<strong>la</strong>ga; USC, University of<br />
Santiago <strong>de</strong> Composte<strong>la</strong>.<br />
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number of bacteria nee<strong>de</strong>d to kill 50% of the<br />
inocu<strong>la</strong>ted fish (Reed & Mü<strong>en</strong>ch 1938). Strain<br />
Lg h41/01 with an LD 50 ¼ 2.8 · 10 4 cfu g )1 fish<br />
was consi<strong>de</strong>red virul<strong>en</strong>t for sole and strain EPOY-<br />
8803-II with LD 50 > 7.7 · 10 6 cfu g )1 fish was<br />
consi<strong>de</strong>red non-virul<strong>en</strong>t.<br />
Bacterial growth conditions<br />
Bacteria were stored at )80 °C in tryptic soy broth<br />
(TSB; Oxoid Ltd., Basingstoke, UK) containing<br />
2% NaCl and 20% glycerol. Bacteria were cultured<br />
on tryptic soy agar (TSA; Oxoid) containing 2%<br />
NaCl and incubated at 22 °C for 48 h. One colony<br />
was used to inocu<strong>la</strong>te 5 mL TSBs and incubated for<br />
18 h at 22 °C with shaking. Aliquots (25 lL) of<br />
these cultures were used to inocu<strong>la</strong>te 250 mL TSBs<br />
which was incubated at 22 °C with shaking. The<br />
incubation time varied <strong>de</strong>p<strong>en</strong>ding on the culture<br />
condition and strain to be assayed.<br />
Differ<strong>en</strong>t growth conditions were assayed to<br />
<strong>de</strong>termine the pot<strong>en</strong>tial induction of SOD and<br />
cata<strong><strong>la</strong>s</strong>e activities. Thus, 250-mL culture f<strong><strong>la</strong>s</strong>ks were<br />
supplem<strong>en</strong>ted with an iron che<strong>la</strong>nt, dipyridyl<br />
(100 lm), FeCl 3 Æ6H 2 O (100 lm) or MnSO 4 Æ2H 2 O<br />
(250 lm) to <strong>de</strong>termine the influ<strong>en</strong>ce of iron and<br />
manganese avai<strong>la</strong>bility on <strong>en</strong>zymatic activity. In<br />
or<strong>de</strong>r to induce oxidative stress, methyl violog<strong>en</strong><br />
(0.2 mm), which g<strong>en</strong>erates superoxi<strong>de</strong> radicals, was<br />
ad<strong>de</strong>d to mid-expon<strong>en</strong>tial cultures, which were th<strong>en</strong><br />
incubated for 8 h before c<strong>en</strong>trifugation. The<br />
pot<strong>en</strong>tial induction of <strong>en</strong>zymatic activities by<br />
hydrog<strong>en</strong> peroxi<strong>de</strong> was tested in cultures after the<br />
addition of two pulses of hydrog<strong>en</strong> peroxi<strong>de</strong>, one of<br />
20 lm in the mid-expon<strong>en</strong>tial phase, and another of<br />
2mm in the early stationary phase. Cells were<br />
harvested after 1-h incubation. The influ<strong>en</strong>ce of the<br />
growth phase was investigated with bacteria harvested<br />
from mid-expon<strong>en</strong>tial (OD 600 ¼ 0.4–0.6) and<br />
early stationary (OD 600 ¼ 1–1.2) phase cultures.<br />
Preparation of cru<strong>de</strong> extracts<br />
Bacteria were harvested from cultures grown as<br />
<strong>de</strong>scribed above by c<strong>en</strong>trifugation at 2000 g for<br />
20 min at 4 °C and washing twice in 25 mm<br />
potassium phosphate buffer containing 1 mm disodium<br />
ethyl<strong>en</strong>e diamine tetraacetic acid (EDTA;<br />
Sigma-Aldrich, St. Louis, MO, USA), pH 7.2 and<br />
0.5 mm ph<strong>en</strong>yl methylsulphonyl fluori<strong>de</strong> (Sigma)<br />
followed by re-susp<strong>en</strong>sion in 1 mL of the same<br />
buffer. Susp<strong>en</strong>sions were sonicated on ice for 120 s<br />
(four pulses of 30 s with 15 s cooling betwe<strong>en</strong><br />
bursts). Lysates were c<strong>la</strong>rified twice by c<strong>en</strong>trifugation<br />
at 10 000 g for 20 min at 4 °C. Supernatants<br />
were assayed for the <strong>de</strong>tection of SOD and cata<strong><strong>la</strong>s</strong>e<br />
and quantification of <strong>en</strong>zymatic activity on acry<strong>la</strong>mi<strong>de</strong><br />
gels. Total protein conc<strong>en</strong>tration was <strong>de</strong>termined<br />
by the method of Bradford (1976) using<br />
bovine serum albumin as standard.<br />
Polyacry<strong>la</strong>mi<strong>de</strong> gel electrophoresis<br />
Electrophoresis was performed in non-<strong>de</strong>naturing<br />
discontinuous polyacry<strong>la</strong>mi<strong>de</strong> mini-gels using the<br />
Bio-Rad Mini Protean II System (Bio-Rad Laboratories,<br />
Richmond, CA, USA) with a 10% acry<strong>la</strong>mi<strong>de</strong>/bis<br />
separating gel (1.5 m Tris–HCl, pH 8.8)<br />
and a 4% acry<strong>la</strong>mi<strong>de</strong>/bis stacking gel (0.5 m Tris–<br />
HCl, pH 8.3). The extracts in the sample buffer<br />
were applied to the gel at a conc<strong>en</strong>tration of<br />
20–24 lg protein per <strong>la</strong>ne. Gels were th<strong>en</strong> stained<br />
for SOD or cata<strong><strong>la</strong>s</strong>e and peroxidase activities.<br />
Detection and quantification of SOD activity<br />
Superoxi<strong>de</strong> dismutase activity was visualized on gels<br />
by nitroblue tetrazolium (NBT; Sigma) negative<br />
staining (Beauchamp & Fridovich 1971). Briefly,<br />
gels were washed in distilled water, soaked in a<br />
solution of 2.45 mm NBT for 20 min, followed by<br />
10-min incubation in darkness in a solution<br />
containing 50 mm potassium phosphate buffer<br />
(pH 7.2), 0.028 mm ribof<strong>la</strong>vin (Sigma) and<br />
28 mm tetramethylethyl<strong>en</strong>ediamine (TEMED;<br />
Sigma). Gels were illuminated on a light box to<br />
<strong>de</strong>velop a dark background with achromatic bands<br />
corresponding to SOD activity, due to inhibition of<br />
the photochemical reduction of NBT to formazan<br />
blue.<br />
The method employed to quantify SOD activity<br />
is based on the ability of SOD to inhibit the<br />
reduction of NBT by superoxi<strong>de</strong> (Winterbourn,<br />
Hawkins, Brian & Correll 1975; Worthington<br />
Enzyme Manual 1993). One unit is <strong>de</strong>fined as the<br />
amount of <strong>en</strong>zyme causing half the maximum<br />
inhibition of NBT reduction. Differ<strong>en</strong>t volumes of<br />
extracts were ad<strong>de</strong>d to cuvettes containing 0.2 mL<br />
of a solution of 0.1 m EDTA, 0.3 mm sodium<br />
cyani<strong>de</strong> (NaCN; Sigma) and 0.1 mL of 1.5 mm<br />
NBT. Th<strong>en</strong>, 0.05 mL of 0.12 mm ribof<strong>la</strong>vin was<br />
ad<strong>de</strong>d at zero time and at timed intervals. All<br />
cuvettes were incubated in a light box for 12 min<br />
and absorbance at 560 nm was read at timed<br />
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intervals by a spectrophotometer (Hitachi U-2000:<br />
Hitachi, Tokyo, Japan). The amount of <strong>en</strong>zyme<br />
resulting in 50% of maximum inhibition of NBT<br />
reduction was <strong>de</strong>termined.<br />
Detection, characterization and quantification<br />
of cata<strong><strong>la</strong>s</strong>e activity<br />
Cata<strong><strong>la</strong>s</strong>e activity was visualized on non-<strong>de</strong>naturing<br />
acry<strong>la</strong>mi<strong>de</strong> gels following the methodology of<br />
Woodbury, Sp<strong>en</strong>cer & Stahmann (1971). After<br />
electrophoresis, gels were washed three times in<br />
distilled water for 20 min and soaked in a solution<br />
of 0.015% H 2 O 2 (30%) (Merck, Darmstadt,<br />
Germany). Th<strong>en</strong>, the activity was visualized by<br />
transferring the gels to a solution of 1% (w/v)<br />
ferric chlori<strong>de</strong> (Panreac Quimica, Barcelona,<br />
Spain) and 1% (w/v) potassium ferricyani<strong>de</strong><br />
(Sigma). Regions corresponding to cata<strong><strong>la</strong>s</strong>e activity<br />
were i<strong>de</strong>ntified as clear yellow bands on a dark<br />
gre<strong>en</strong> background.<br />
The metal cofactor of the cata<strong><strong>la</strong>s</strong>e produced by<br />
P. damse<strong>la</strong>e ssp. piscicida was <strong>de</strong>termined by<br />
<strong>en</strong>zymatic inhibition studies according to Barnes,<br />
Bow<strong>de</strong>n, Horne & Ellis (1999b). Lysates of<br />
P. damse<strong>la</strong>e ssp. piscicida strains were incubated<br />
for 1 h with either 100 and 50 mm potassium<br />
cyani<strong>de</strong> (KCN; Sigma), 1 and 0.5 mm mercuric<br />
chlori<strong>de</strong> (HgCl 2 ; Sigma), 25 and 12.5 mm sodium<br />
azi<strong>de</strong> (NaN 3 ; Sigma) or 50 mm phosphate buffer as<br />
control. Equal volumes of treated extracts were<br />
electrophoresed and gels stained for cata<strong><strong>la</strong>s</strong>e activity<br />
(Woodbury et al. 1971). Cata<strong><strong>la</strong>s</strong>es with manganese<br />
as metal cofactor are resistant against sodium azi<strong>de</strong><br />
and potassium cyani<strong>de</strong> and s<strong>en</strong>sitive to mercuric<br />
chlori<strong>de</strong> (Kono & Fridovich 1983; Allgood &<br />
Perry 1986; Barnes et al. 1999b). Control wells<br />
inocu<strong>la</strong>ted with extracts of Escherichia coli (ATCC<br />
13706) containing a ferric cata<strong><strong>la</strong>s</strong>e retained the<br />
activity after treatm<strong>en</strong>t with mercuric chlori<strong>de</strong> but<br />
not with sodium azi<strong>de</strong>.<br />
Cata<strong><strong>la</strong>s</strong>e activity was measured spectrophotometrically<br />
by monitoring the <strong>de</strong>crease in absorbance at<br />
240 nm due to <strong>de</strong>composition of hydrog<strong>en</strong> peroxi<strong>de</strong>.<br />
One unit of cata<strong><strong>la</strong>s</strong>e was <strong>de</strong>fined as the activity<br />
causing the hydrolysis of 1 lmol of hydrog<strong>en</strong><br />
peroxi<strong>de</strong> per minute (Aebi 1984). Briefly, bacterial<br />
extracts were diluted (1:100) in 50 mm potassium<br />
phosphate buffer, pH 7.0 and the absorbance of<br />
the sample containing 660 lL of lysate and<br />
340 lL of H 2 O 2 was measured against a b<strong>la</strong>nk<br />
with buffer. The <strong>de</strong>crease in absorbance at 240 nm<br />
(Hitachi U-2000) was monitored during a 10-min<br />
period.<br />
Bactericidal activity of sole phagocytes<br />
Mono<strong>la</strong>yers of sole phagocytes were prepared<br />
following the methodology of Secombes (1990).<br />
Briefly, the kidneys of 100–300 g sole were dissected<br />
and pressed through a 100 lm nylon mesh with<br />
L-15 medium (Gibco, Gaithersburg, MD, USA)<br />
containing 2% fetal calf serum (FCS; Sigma), 1%<br />
p<strong>en</strong>icillin/streptomycin (Sigma), 0.1% g<strong>en</strong>tamicin<br />
sulphate (50 mg mL )1 distilled water; Sigma) and<br />
10 U mL )1 sodium heparin. The resultant susp<strong>en</strong>sion<br />
was <strong>la</strong>yered onto a 30–51% (v/v) Percoll<br />
(Amersham Pharmacia, Piscataway, NJ, USA) <strong>de</strong>nsity<br />
gradi<strong>en</strong>t and the band of cells lying at the 30–<br />
51% interface was collected. The cell susp<strong>en</strong>sion<br />
was washed and adjusted to 10 7 cells mL )1 in L-15<br />
medium with antibiotics. The viability was <strong>de</strong>termined<br />
by the exclusion test with trypan blue<br />
(Sigma) (0.5% in PBS). A volume of 100 lL per<br />
well was ad<strong>de</strong>d to 96-well microtitre p<strong>la</strong>tes.<br />
Mono<strong>la</strong>yers were maintained at 22 °C overnight<br />
until bactericidal assays were performed.<br />
Bacterial culture conditions to <strong>de</strong>termine the<br />
ability to resist the bactericidal activity of phagocytes<br />
inclu<strong>de</strong>d growth until stationary phase, addition<br />
of two hydrog<strong>en</strong> peroxi<strong>de</strong> pulses and growth in<br />
replete or reduced iron medium as previously<br />
<strong>de</strong>scribed. The bacterial conc<strong>en</strong>tration was adjusted<br />
to 1 OD 600 , corresponding to 10 8 bacteria per mL.<br />
The methodology employed to test bacterial survival<br />
after contact with phagocytes was according to<br />
Secombes (1990).<br />
Phagocyte mono<strong>la</strong>yers were washed twice with<br />
L-15 and the cells were th<strong>en</strong> supplem<strong>en</strong>ted with<br />
100 lL L-15, 5% FCS per well. Bacterial susp<strong>en</strong>sions<br />
(20 lL) were ad<strong>de</strong>d to triplicate wells containing<br />
macrophages. The microtitre p<strong>la</strong>te was shak<strong>en</strong><br />
and c<strong>en</strong>trifuged at 150 g for 5 min to bring the<br />
bacteria into contact with cells and subsequ<strong>en</strong>tly<br />
incubated at 22 °C for 0 and 5 h. At the <strong>en</strong>d of the<br />
incubation period, the supernatants were removed<br />
and the killing stopped by lysing the phagocytes with<br />
50 lL of cold sterile distilled water. Subsequ<strong>en</strong>tly,<br />
100 lL of TSBs was ad<strong>de</strong>d to support the growth of<br />
the surviving bacteria for 18–20 h at 22 °C.<br />
The number of surviving bacteria was quantified<br />
colorimetrically following the methodology of Peck<br />
(1985) as modified by Graham, Jeffries &<br />
Secombes (1988). Briefly, 10 lL of 3 [4,5-di-<br />
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methylthiazoyl-2-yl] 2,5-diph<strong>en</strong>yltetrazolium bromi<strong>de</strong><br />
(MTT, Sigma) (5 mg mL )1 distilled water)<br />
was ad<strong>de</strong>d to the wells, p<strong>la</strong>tes were shak<strong>en</strong> and<br />
absorbance at 550 nm was read after 15-min<br />
incubation on a multiscan spectrophotometer<br />
(Microp<strong>la</strong>te Rea<strong>de</strong>r 2001; Whittaker Bioproducts<br />
Inc., Walkersville, MD, USA). The perc<strong>en</strong>tage of<br />
surviving bacteria was calcu<strong>la</strong>ted by dividing the<br />
absorbance obtained from the wells incubated with<br />
bacteria for 5 h by the values obtained from wells<br />
incubated with bacteria for 0 h.<br />
Statistical analysis<br />
Quantification of <strong>en</strong>zymatic activities was carried<br />
out in three in<strong>de</strong>p<strong>en</strong><strong>de</strong>nt experim<strong>en</strong>ts. Fish experim<strong>en</strong>ts<br />
were performed in triplicate, data corresponding<br />
to measurem<strong>en</strong>ts were carried out with<br />
phagocytes from three differ<strong>en</strong>t fish and three<br />
replicate wells for each fish. An anova test was<br />
performed to compare the results obtained.<br />
P < 0.05 was consi<strong>de</strong>red significant.<br />
Results<br />
(a)<br />
1 2 3 4 5 6 7 8 9 10<br />
(b)<br />
1 2 3 4 5 6 7 8 9 10<br />
Figure 1 Detection of superoxi<strong>de</strong> dismutase (a) and cata<strong><strong>la</strong>s</strong>e<br />
activity (b) in extracts of differ<strong>en</strong>t strains of Photobacterium<br />
damse<strong>la</strong>e subsp. piscicida grown until stationary phase. Lane 1:<br />
ATCC 17911; 2: B51; 3: B180; 4: DI-21S; 5: D 26/98 ; 6: Pp8H;<br />
7: R45; 8: Lg h41/01 ; 9: MT 1415 and 10: MT 1379.<br />
All the extracts of the strains of P. damse<strong>la</strong>e ssp.<br />
piscicida inclu<strong>de</strong>d in this study produced simi<strong>la</strong>r<br />
SOD and cata<strong><strong>la</strong>s</strong>e activity bands (Fig. 1). Thus, a<br />
single band with i<strong>de</strong>ntical mobility in native polyacry<strong>la</strong>mi<strong>de</strong><br />
gel electrophoresis gels was observed for<br />
all iso<strong>la</strong>tes and culture conditions assayed (Fig. 2).<br />
Simi<strong>la</strong>r protein conc<strong>en</strong>trations were loa<strong>de</strong>d in the<br />
gel <strong>la</strong>nes. However, differ<strong>en</strong>ces in the int<strong>en</strong>sity of<br />
the SOD and cata<strong><strong>la</strong>s</strong>e bands were observed. Thus,<br />
SOD and cata<strong><strong>la</strong>s</strong>e activity bands showed lower<br />
int<strong>en</strong>sity in the extracts from cultures carried out<br />
un<strong>de</strong>r iron-limiting conditions, whilst increased<br />
int<strong>en</strong>sity of SOD bands was observed in extracts<br />
from cultures un<strong>de</strong>r iron-supplem<strong>en</strong>ted conditions<br />
and in the pres<strong>en</strong>ce of the cytop<strong><strong>la</strong>s</strong>mic superoxi<strong>de</strong><br />
radical g<strong>en</strong>erator, methyl violog<strong>en</strong> (Fig. 2).<br />
Two iso<strong>la</strong>tes with differ<strong>en</strong>t <strong>de</strong>grees of virul<strong>en</strong>ce<br />
for sole were selected for further characterization:<br />
one virul<strong>en</strong>t, Lg h41/01 (LD 50 ¼ 2.8 · 10 4 cfu g )1<br />
fish) and one non-virul<strong>en</strong>t, EPOY-8803-II<br />
(LD 50 > 7 · 10 6 cfu g )1 fish).<br />
Cultures carried out until the early stationary<br />
phase of the non-virul<strong>en</strong>t iso<strong>la</strong>te contained significantly<br />
(P < 0.05) lower amounts of SOD than<br />
cultures of the virul<strong>en</strong>t strain. However, wh<strong>en</strong> iron<br />
was ad<strong>de</strong>d to the growth broth, EPOY-8803-II<br />
contained significantly higher amounts of SOD<br />
(Fig. 3).<br />
There was no significant hydrog<strong>en</strong> peroxi<strong>de</strong><br />
induction of SOD in any of the strains, and in<strong>de</strong>ed<br />
a <strong>de</strong>crease in activity in strain Lg h41/01 was <strong>de</strong>tected<br />
(Fig. 3). In contrast, cells of both strains cultured<br />
un<strong>de</strong>r iron limiting or replete conditions contained<br />
significantly differ<strong>en</strong>t amounts of SOD activity. In<br />
all the cases, growth un<strong>de</strong>r iron-limiting conditions<br />
resulted in a significant <strong>de</strong>crease in SOD activity<br />
compared with iron replete conditions, this <strong>de</strong>crease<br />
being more important in the non-virul<strong>en</strong>t strain<br />
than in the virul<strong>en</strong>t strain.<br />
Unlike SOD, cata<strong><strong>la</strong>s</strong>e activity in cultures of the<br />
non-virul<strong>en</strong>t strain was lower than in the virul<strong>en</strong>t<br />
strain (Fig. 4). Moreover, whilst no significant<br />
differ<strong>en</strong>ces were observed in cata<strong><strong>la</strong>s</strong>e cont<strong>en</strong>ts of<br />
Lg h41/01 cultures grown until stationary phase and<br />
those pulsed with hydrog<strong>en</strong> peroxi<strong>de</strong>, strain EPOY-<br />
8803-II showed a consi<strong>de</strong>rably greater amount of<br />
cata<strong><strong>la</strong>s</strong>e activity wh<strong>en</strong> cultures were pulsed with<br />
hydrog<strong>en</strong> peroxi<strong>de</strong>. A significant <strong>de</strong>crease of activity<br />
was also observed for cultures of both strains carried<br />
out un<strong>de</strong>r iron-limiting conditions compared with<br />
iron-overloa<strong>de</strong>d broths.<br />
Cata<strong><strong>la</strong>s</strong>e activity could not be <strong>de</strong>tected in the gels<br />
following exposure to 100 mm sodium azi<strong>de</strong> and<br />
treatm<strong>en</strong>t with potassium cyani<strong>de</strong> resulted in a<br />
slight reduction of activity, suggesting that this<br />
bacterium contains an iron-cofactored <strong>en</strong>zyme.<br />
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(a)<br />
(b)<br />
1 2 3 4 5 6 1 2 3 4 5 6<br />
Figure 2 Detection of superoxi<strong>de</strong> dismutase<br />
(a) and cata<strong><strong>la</strong>s</strong>e activity (b) in extracts of<br />
Photobacterium damse<strong>la</strong>e subsp. piscicida<br />
(strain EPOY-8803-II) grown un<strong>de</strong>r differ<strong>en</strong>t<br />
conditions. Lane 1: growth until expon<strong>en</strong>tial<br />
phase; 2: stationary growth phase;<br />
3: exposure to hydrog<strong>en</strong> peroxi<strong>de</strong> (20 lm<br />
H 2 O 2 mid-expon<strong>en</strong>tial phase and 2 mm<br />
H 2 O 2 early stationary phase); 4: addition of<br />
methyl violog<strong>en</strong> (0.2 mm) to the culture<br />
medium; 5: addition of 2,2¢-dipyridyl<br />
(100 lm) to the culture medium; 6: addition<br />
of FeCl 3 Æ6H 2 O (100 lm) to the culture<br />
medium.<br />
U SOD/mg prot.<br />
20<br />
15<br />
10<br />
5<br />
0<br />
EPOY-8803-II<br />
Lgh41/01<br />
Figure 3 Superoxi<strong>de</strong> dismutase activity (U mg )1 protein) of<br />
Photobacterium damse<strong>la</strong>e subsp. piscicida strains grown un<strong>de</strong>r<br />
differ<strong>en</strong>t culture conditions. ( ) Growth until stationary phase;<br />
( ) exposure to hydrog<strong>en</strong> peroxi<strong>de</strong> (20 lm mid-expon<strong>en</strong>tial<br />
phase and 2 mm early stationary phase); ( ) culture supplem<strong>en</strong>ted<br />
with FeCl 3 Æ6H 2 O 100 lm and (h) culture supplem<strong>en</strong>ted<br />
with the iron che<strong>la</strong>nt 2,2¢-dipyridyl 100 lm. Data repres<strong>en</strong>t the<br />
mean (SD) of three in<strong>de</strong>p<strong>en</strong><strong>de</strong>nt <strong>de</strong>terminations.<br />
U cat./mg prot.<br />
140000<br />
120 000<br />
100000<br />
80000<br />
60000<br />
40000<br />
20 000<br />
0<br />
EPOY<br />
Lgh41/01<br />
Figure 4 Cata<strong><strong>la</strong>s</strong>e activity (U mg )1 protein) of Photobacterium<br />
damse<strong>la</strong>e subsp. piscicida strains grown un<strong>de</strong>r differ<strong>en</strong>t culture<br />
conditions. ( ) Growth until stationary phase; ( ) exposure to<br />
hydrog<strong>en</strong> peroxi<strong>de</strong> (20 lm mid-expon<strong>en</strong>tial phase and 2 mm<br />
early stationary phase); ( ) culture supplem<strong>en</strong>ted with FeCl 3 Æ<br />
6H 2 O 100 lm and (h) culture supplem<strong>en</strong>ted with the iron<br />
che<strong>la</strong>nt 2,2¢-dipyridyl 100 lm. Data repres<strong>en</strong>t the mean (SD)<br />
of three in<strong>de</strong>p<strong>en</strong><strong>de</strong>nt <strong>de</strong>terminations.<br />
In or<strong>de</strong>r to <strong>de</strong>termine the influ<strong>en</strong>ce of the levels<br />
of SOD and cata<strong><strong>la</strong>s</strong>e activity on the resistance to the<br />
bactericidal activity of sole phagocytes, killing assays<br />
were carried out with a virul<strong>en</strong>t and non-virul<strong>en</strong>t<br />
strain of P. damse<strong>la</strong>e ssp. piscicida. The perc<strong>en</strong>tages<br />
of surviving bacteria after 5 h contact with sole<br />
phagocytes are shown in Fig. 5. It can be observed<br />
that survival of the virul<strong>en</strong>t strain in contact with<br />
phagocytes was significantly higher (P < 0.05) in<br />
all cases compared with the non-virul<strong>en</strong>t strain.<br />
Despite this differ<strong>en</strong>t survival rate, both strains<br />
showed a simi<strong>la</strong>r behaviour <strong>de</strong>p<strong>en</strong>ding on the<br />
bacterial culture condition with highest rates<br />
corresponding in both cases to growth in ironreplete<br />
broths and lowest to growth un<strong>de</strong>r ironlimiting<br />
conditions. In addition, a significant<br />
increase in the survival perc<strong>en</strong>tages was observed<br />
%Survival<br />
100<br />
90<br />
80<br />
70<br />
60<br />
50<br />
40<br />
30<br />
20<br />
10<br />
0<br />
EPOY-8803-II<br />
Lgh41/01<br />
Figure 5 Survival perc<strong>en</strong>tage of Photobacterium damse<strong>la</strong>e subsp.<br />
piscicida after 5 h in contact with sole phagocytes. ( ) Growth<br />
until stationary phase; ( ) exposure to hydrog<strong>en</strong> peroxi<strong>de</strong> (20 lm<br />
mid-expon<strong>en</strong>tial phase and 2 mm early stationary phase); ( )<br />
culture supplem<strong>en</strong>ted with FeCl 3 Æ6H 2 O 100 lm and (h) culture<br />
supplem<strong>en</strong>ted with 2,2¢-dipyridyl 100 lm. Data repres<strong>en</strong>t the<br />
mean (SD) of nine wells containing phagocytes from three fish<br />
specim<strong>en</strong>s.<br />
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Journal of Fish Diseases 2006, 29, 355–364<br />
PDíaz-Rosales et al. Superoxi<strong>de</strong> dismutase and cata<strong><strong>la</strong>s</strong>e in P. damse<strong>la</strong>e ssp. piscicida<br />
in both strains pulsed with hydrog<strong>en</strong> peroxi<strong>de</strong><br />
compared with stationary phase cultures.<br />
Discussion<br />
Enzymes such as SOD and cata<strong><strong>la</strong>s</strong>e, which neutralize<br />
ROS produced during aerobic metabolism or<br />
during respiratory burst in fish phagocytes are<br />
important virul<strong>en</strong>ce factors in many pathog<strong>en</strong>s<br />
(Barnes et al. 1996, 1999b; Yesilkaya, Kadioglu,<br />
Gingles, Alexan<strong>de</strong>r, Mitchell & Andrew 2000;<br />
Vattanaviboon & Mongkolsuk 2001; Uzzau et al.<br />
2002; Banin, Vassi<strong>la</strong>kos, Orr, Martínez & Ros<strong>en</strong>berg<br />
2003). In this study, all the strains of<br />
P. damse<strong>la</strong>e ssp. piscicida assayed showed a single<br />
band of SOD activity with i<strong>de</strong>ntical mobility on<br />
acry<strong>la</strong>mi<strong>de</strong> gels. A unique band simi<strong>la</strong>r in all the<br />
strains was also observed on cata<strong><strong>la</strong>s</strong>e activity gels.<br />
Simi<strong>la</strong>rly, Barnes, Balebona, Horne & Ellis<br />
(1999a), in a study that inclu<strong>de</strong>d a collection of<br />
P. damse<strong>la</strong>e ssp. piscicida strains iso<strong>la</strong>ted from<br />
gilthead seabream, Sparus aurata (L.), reported<br />
only one SOD located in the perip<strong><strong>la</strong>s</strong>mic space and<br />
one cytop<strong><strong>la</strong>s</strong>mic cata<strong><strong>la</strong>s</strong>e.<br />
Several studies have reported that microorganisms<br />
contain differ<strong>en</strong>t SOD and cata<strong><strong>la</strong>s</strong>e isozymes<br />
inducible un<strong>de</strong>r certain growth conditions (Storz,<br />
Tartaglia, Farr & Ames 1990; Privalle & Fridovich<br />
1992; Barnes et al. 1996; Yesilkaya et al. 2000;<br />
Geslin, L<strong>la</strong>nos, Prieur & Jeanthon 2001; Vattanaviboon<br />
& Mongkolsuk 2001). However, culture<br />
conditions assayed in this work have not induced<br />
new SOD or cata<strong><strong>la</strong>s</strong>e isozymes in P. damse<strong>la</strong>e ssp.<br />
piscicida. Mn-SOD activity has be<strong>en</strong> reported to be<br />
modu<strong>la</strong>ted by oxidative stress and iron-limiting<br />
conditions (Privalle & Fridovich 1992; Barnes<br />
et al. 1999b) but in the case of P. damse<strong>la</strong>e ssp.<br />
piscicida neither production of intracellu<strong>la</strong>r superoxi<strong>de</strong><br />
by methyl violog<strong>en</strong> nor culture un<strong>de</strong>r ironrestricted<br />
conditions induced the production of a<br />
differ<strong>en</strong>t type of SOD. Although further studies are<br />
necessary, this <strong>la</strong>ck of induction of a new SOD<br />
could be due to the pres<strong>en</strong>ce of only one sod g<strong>en</strong>e,<br />
i.e. sod B <strong>en</strong>coding Fe-SOD (Lynch & Kuramitsu<br />
2000).<br />
In contrast, differ<strong>en</strong>ces in the int<strong>en</strong>sity of the<br />
bands were observed in extracts obtained un<strong>de</strong>r<br />
differ<strong>en</strong>t culture conditions for both SOD and<br />
cata<strong><strong>la</strong>s</strong>e activities. As the amount of protein loa<strong>de</strong>d<br />
in the electrophoretic <strong>la</strong>nes was simi<strong>la</strong>r in all cases,<br />
the differ<strong>en</strong>t int<strong>en</strong>sities suggest variations in the<br />
levels of activity in the extracts <strong>de</strong>p<strong>en</strong>ding on the<br />
culture condition. These results are in agreem<strong>en</strong>t<br />
with those obtained by Barnes, Balebona, Horne &<br />
Ellis (1999a), who also <strong>de</strong>tected differ<strong>en</strong>ces in<br />
cultures carried out un<strong>de</strong>r iron replete and <strong>de</strong>pleted<br />
conditions and high- and low-aerated broths.<br />
The quantification of both SOD and cata<strong><strong>la</strong>s</strong>e<br />
activities carried out in this study corroborated that<br />
differ<strong>en</strong>t band int<strong>en</strong>sities correspon<strong>de</strong>d to variations<br />
in the levels of activity. The lowest levels of SOD<br />
activity were <strong>de</strong>tected wh<strong>en</strong> bacteria were grown<br />
un<strong>de</strong>r iron-restricted conditions. The ferric nature<br />
of P. damse<strong>la</strong>e ssp. piscicida SOD <strong>de</strong>scribed by<br />
Barnes et al. (1999a) could exp<strong>la</strong>in this lower<br />
activity in the pres<strong>en</strong>ce of an iron che<strong>la</strong>nt.<br />
Iron also influ<strong>en</strong>ced the levels of cata<strong><strong>la</strong>s</strong>e activity<br />
in P. damse<strong>la</strong>e ssp. piscicida. The role of iron as<br />
cofactor in this <strong>en</strong>zyme has be<strong>en</strong> <strong>de</strong>monstrated with<br />
inhibition studies. Thus, cata<strong><strong>la</strong>s</strong>e activity could not<br />
be <strong>de</strong>tected in the gels following exposure to<br />
sodium azi<strong>de</strong> and it was slightly reduced after<br />
treatm<strong>en</strong>t with potassium cyani<strong>de</strong>. These results<br />
suggest that the <strong>en</strong>zyme is an iron cofactored<br />
cata<strong><strong>la</strong>s</strong>e, as Mn-containing cata<strong><strong>la</strong>s</strong>es retain activity<br />
after treatm<strong>en</strong>t with azi<strong>de</strong> and cyani<strong>de</strong> and are<br />
inhibited by mercuric chlori<strong>de</strong> (Kono & Fridovich<br />
1983; Allgood & Perry 1986; Barnes et al. 1999b).<br />
This ferric nature of the cata<strong><strong>la</strong>s</strong>e may exp<strong>la</strong>in the<br />
lower cata<strong><strong>la</strong>s</strong>e activity observed in cultures with<br />
ad<strong>de</strong>d iron che<strong>la</strong>nt and lower survival with H 2 O 2<br />
observed by Díaz-Rosales, Chabrillón, Moriñigo &<br />
Balebona (2003).<br />
Lower survival of P. damse<strong>la</strong>e ssp. piscicida in sole<br />
phagocytes has be<strong>en</strong> observed for strain EPOY-<br />
8803-II compared with the virul<strong>en</strong>t strain. Contradictory<br />
results have be<strong>en</strong> reported on the ability of<br />
P. damse<strong>la</strong>e ssp. piscicida to survive insi<strong>de</strong> macrophages<br />
from several fish species. In a study using<br />
macrophages from sea bass, gilthead sea bream and<br />
rainbow trout, Skarmeta et al. (1995) conclu<strong>de</strong>d<br />
that head kidney macrophages from these fish<br />
species were able to kill the pathog<strong>en</strong>. However,<br />
Noya et al. (1995b) reported that whilst bacteria<br />
within granulocytes and macrophages from <strong>la</strong>rge<br />
gilthead sea bream were morphologically altered,<br />
bacteria insi<strong>de</strong> small fish remained unaffected. In<br />
addition, data on the ability of P. damse<strong>la</strong>e to<br />
survive insi<strong>de</strong> fish macrophages have be<strong>en</strong> reported<br />
by several authors who observed that bacteria can<br />
multiply insi<strong>de</strong> fish macrophages (Kubota et al.<br />
1970; Hawke, P<strong>la</strong>kas, Minton, McPherson, Zin<strong>de</strong>r<br />
& Guarino 1987; Noya et al. 1995a; Elkamel,<br />
Hawke, H<strong>en</strong>k & Thune 2003).<br />
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PDíaz-Rosales et al. Superoxi<strong>de</strong> dismutase and cata<strong><strong>la</strong>s</strong>e in P. damse<strong>la</strong>e ssp. piscicida<br />
Multiplication of P. damse<strong>la</strong>e ssp. piscicida insi<strong>de</strong><br />
several fish cell lines has also be<strong>en</strong> reported. Elkamel<br />
& Thune (2003) observed that the bacteria multiply<br />
in EPC, CCO, and FHM cells and López-<br />
Dóriga, Barnes, dos Santos & Ellis (2000) using<br />
EPC cells observed that both virul<strong>en</strong>t and avirul<strong>en</strong>t<br />
iso<strong>la</strong>tes were able to adhere to and inva<strong>de</strong> cells.<br />
Results obtained from this study show that<br />
P. damse<strong>la</strong>e ssp. piscicida is able to survive insi<strong>de</strong><br />
sole phagocytes at least for 5 h, the survival rates<br />
being higher for the virul<strong>en</strong>t iso<strong>la</strong>te. Although the<br />
bacterium was able to survive, the rates obtained<br />
always indicated a certain <strong>de</strong>gree of bacterial<br />
inactivation insi<strong>de</strong> phagocytes.<br />
Survival of the non-virul<strong>en</strong>t strain in contact with<br />
sole phagocytes was significantly lower compared<br />
with the virul<strong>en</strong>t strain. The non-virul<strong>en</strong>t strain also<br />
showed lower cata<strong><strong>la</strong>s</strong>e activity. These results suggest<br />
that bacterial inactivation could be due to the<br />
accumu<strong>la</strong>tion of hydrog<strong>en</strong> peroxi<strong>de</strong>, the precursor<br />
of hydroxyl radicals, after <strong>de</strong>composition of superoxi<strong>de</strong><br />
radicals by bacterial SOD. This accumu<strong>la</strong>tion<br />
would not take p<strong>la</strong>ce to such an ext<strong>en</strong>t in the virul<strong>en</strong>t<br />
strain, as levels of cata<strong><strong>la</strong>s</strong>e are higher. The important<br />
role of cata<strong><strong>la</strong>s</strong>e in the protection against oxidative<br />
damage in P. damse<strong>la</strong>e ssp. piscicida has be<strong>en</strong> pointed<br />
out by Barnes et al. (1999a), who observed that the<br />
addition of exog<strong>en</strong>ous cata<strong><strong>la</strong>s</strong>e to the medium<br />
protected the bacteria from inactivation by photochemically<br />
g<strong>en</strong>erated superoxi<strong>de</strong> anions.<br />
Both virul<strong>en</strong>t and non-virul<strong>en</strong>t strains assayed by<br />
Barnes et al. (1999a) showed high susceptibility to<br />
cell-free g<strong>en</strong>erated superoxi<strong>de</strong> radicals. In contrast,<br />
we have observed that a non-virul<strong>en</strong>t strain, EPOY-<br />
8803-II, is significantly more susceptible to killing<br />
by sole phagocytes than a virul<strong>en</strong>t strain (Lg h41/01 ).<br />
Besi<strong>de</strong>s the lower cata<strong><strong>la</strong>s</strong>e activity pres<strong>en</strong>t in the<br />
non-virul<strong>en</strong>t strain, the <strong>la</strong>ck of a capsule in cells of<br />
EPOY-8803-II could contribute to the high inactivation<br />
rates observed. Thus, the capsule could<br />
protect bacterial cells from oxidative radicals or ev<strong>en</strong><br />
prev<strong>en</strong>t activation of phagocytes (Miller & Britigan<br />
1997; Arijo et al. 1998).<br />
The important role of iron in microbial infections<br />
has be<strong>en</strong> pointed out by several authors<br />
(Miller & Britigan 1997; Weinberg 2000). The<br />
pathog<strong>en</strong> needs to obtain iron from the host, where<br />
this metal is linked to high-affinity proteins and<br />
iron avai<strong>la</strong>bility is very low; also, a transition metal<br />
catalyst such as iron p<strong>la</strong>ys an important role in the<br />
g<strong>en</strong>eration of hydroxyl radicals in vivo. In<strong>de</strong>ed, at<br />
physiological pH, g<strong>en</strong>eration of hydroxyl radical<br />
from hydrog<strong>en</strong> peroxi<strong>de</strong> and superoxi<strong>de</strong> anions is of<br />
little biological importance unless a metal such as<br />
ferric iron is pres<strong>en</strong>t (Haber–Weiss reaction) (Miller<br />
& Britigan 1997). Photobacterium damse<strong>la</strong>e ssp.<br />
piscicida is more susceptible to killing by sole<br />
phagocytes wh<strong>en</strong> bacterial cells have be<strong>en</strong> cultured<br />
un<strong>de</strong>r iron-<strong>de</strong>pleted conditions. This could be due<br />
to the lower levels of cata<strong><strong>la</strong>s</strong>e <strong>de</strong>tected in both the<br />
virul<strong>en</strong>t and avirul<strong>en</strong>t cells, the lowest rates corresponding<br />
to strain EPOY-8803-II. Thus, although<br />
the pres<strong>en</strong>ce of iron in <strong>en</strong>vironm<strong>en</strong>ts where superoxi<strong>de</strong><br />
and hydrog<strong>en</strong> peroxi<strong>de</strong> are g<strong>en</strong>erated, such as<br />
in phagocytes, may promote the g<strong>en</strong>eration of<br />
highly toxic hydroxyl radicals, it is also true that<br />
bacteria require iron for growth and replication and<br />
synthesize SOD and cata<strong><strong>la</strong>s</strong>e to <strong>de</strong>al with the<br />
oxidizing anions. Thus, the ability to obtain iron<br />
from the host seems to be crucial for P. damse<strong>la</strong>e<br />
ssp. piscicida. In<strong>de</strong>ed, it has be<strong>en</strong> <strong>de</strong>monstrated that<br />
immune-activated macrophages modify intracellu<strong>la</strong>r<br />
distribution and damp<strong>en</strong> iron influx in or<strong>de</strong>r to<br />
diminish iron avai<strong>la</strong>bility for inva<strong>de</strong>rs (Weinberg<br />
2000).<br />
Photobacterium damse<strong>la</strong>e ssp. piscicida posses a<br />
high-affinity iron uptake system (Magariños et al.<br />
1994; Naka, Hirono & Aoki 2005). However,<br />
<strong>de</strong>spite its ability to obtain iron from high-affinity<br />
systems, several authors have reported that cells<br />
grown un<strong>de</strong>r iron-limited conditions have a<br />
reduced amount of capsu<strong>la</strong>r material covering the<br />
cells (Do Vale, Ellis & Silva 2001). These cells with<br />
reduced capsule would be more susceptible to<br />
phagocytosis and oxidative stress. Our results show<br />
that iron p<strong>la</strong>ys an important role in survival of<br />
P. damse<strong>la</strong>e ssp. piscicida in contact with sole<br />
phagocytes; whether this is attributable to its<br />
contribution to capsu<strong>la</strong>r material or SOD and<br />
cata<strong><strong>la</strong>s</strong>e synthesis by the bacterium needs to be<br />
investigated.<br />
In conclusion, we have shown that P. damse<strong>la</strong>e<br />
ssp. piscicida is able to survive in contact with sole<br />
phagocytes, survival rates being higher for a virul<strong>en</strong>t<br />
strain. The increased levels of cata<strong><strong>la</strong>s</strong>e activity<br />
<strong>de</strong>tected in the virul<strong>en</strong>t strain indicate a possible<br />
role for this <strong>en</strong>zyme in bacterial survival.<br />
Acknowledgem<strong>en</strong>ts<br />
P. Díaz-Rosales thanks the Ministerio Español <strong>de</strong><br />
Educación y Ci<strong>en</strong>cia for a F.P.U. scho<strong>la</strong>rship. This<br />
research has be<strong>en</strong> supported in part by the Research<br />
Project AGL-2002-01488 and PETRI 95-0657.01.<br />
Ó 2006<br />
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Journal of Fish Diseases 2006, 29, 355–364<br />
PDíaz-Rosales et al. Superoxi<strong>de</strong> dismutase and cata<strong><strong>la</strong>s</strong>e in P. damse<strong>la</strong>e ssp. piscicida<br />
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Received: 17 November 2005<br />
Revision received: 20 March 2006<br />
Accepted: 23 March 2006<br />
Ó 2006<br />
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364
A RTÍCULO 2.1.<br />
A RTICLE 2.1.
Effect of dietary administration of Porphyridium cru<strong>en</strong>tum on the respiratory burst<br />
activity of sole (Solea s<strong>en</strong>egal<strong>en</strong>sis, Kaup 1858) phagocytes<br />
Patricia Díaz-Rosales a , Carm<strong>en</strong> Felices b , Mariana Chabrillón a , Roberto T. Abda<strong>la</strong> b ,<br />
Félix L. Figueroa b , M. Carm<strong>en</strong> Balebona a and M. Ángel Moriñigo a*<br />
a. Departm<strong>en</strong>t of Microbiology, Faculty of Sci<strong>en</strong>ces, University of Má<strong>la</strong>ga. 29071<br />
Má<strong>la</strong>ga. Spain<br />
b. Departm<strong>en</strong>t of Ecology and Geology, Group of Photobiology and Biotechnology of<br />
algae, Faculty of Sci<strong>en</strong>ces, University of Má<strong>la</strong>ga. 29071 Má<strong>la</strong>ga. Spain<br />
* Corresponding author. Tel. +34 952131862 ; fax: +34 952131889<br />
E-mail address: morinigo@uma.es (M. Ángel Moriñigo)<br />
1
Abstract<br />
The stimu<strong>la</strong>tory effect of the red microalga Porphyridium cru<strong>en</strong>tum on<br />
respiratory burst activity of sole phagocytes was evaluated in vitro and in vivo. Sole<br />
phagocytes incubated in vitro with aqueous and ethanolic extracts (10, 5, 2 and 1 mg ml -<br />
1 ) iso<strong>la</strong>ted from P. cru<strong>en</strong>tum did not show increased superoxi<strong>de</strong> anion production. By<br />
contrast, incubation of phagocytes with β-glucan from Eugl<strong>en</strong>a gracilis (10 mg ml -1 )<br />
increased respiratory burst activity. However, oral administration of a diet supplem<strong>en</strong>ted<br />
with lyophilized P. cru<strong>en</strong>tum cells (10 g kg -1 ) stimu<strong>la</strong>ted respiratory burst activity after<br />
4 weeks feeding only in sole vaccinated with Photobacterium damse<strong>la</strong>e subsp. piscicida<br />
bacterin. Results obtained are discussed in terms of the usefulness of the administration<br />
route of immunostimu<strong>la</strong>nt and synergistic effect with a vaccine.<br />
Keywords: Immunostimu<strong>la</strong>nts; Porphyridium cru<strong>en</strong>tum; Respiratory burst; Phagocytes;<br />
Sole (Solea s<strong>en</strong>egal<strong>en</strong>sis, Kaup 1858); Teleosts.<br />
2
1. Introduction<br />
Fish aquaculture is an expanding industry worldwi<strong>de</strong>. Marine aquaculture in<br />
southern Europe has focused on species such as gilthead seabream (Sparus aurata, L.)<br />
and sea bass (Dic<strong>en</strong>trarchus <strong>la</strong>brax, L.). However, the diversification of the species<br />
farmed is required and S<strong>en</strong>egalese sole (Solea s<strong>en</strong>egal<strong>en</strong>sis, Kaup) is a species with a<br />
high economic value, which is farmed in Portugal and Spain [1]. Diseases of bacterial<br />
origin constitute the most significant cause of economic losses suffered in the<br />
aquaculture industry [2]. The most frequ<strong>en</strong>tly iso<strong>la</strong>ted pathog<strong>en</strong> from outbreaks <strong>de</strong>tected<br />
in farmed S<strong>en</strong>egalese sole is Photobacterium damse<strong>la</strong>e subsp. piscicida, which is<br />
responsible for high mortalities in cultured fish [3]. Antibiotic treatm<strong>en</strong>t of bacterial<br />
diseases affecting farmed fish has be<strong>en</strong> applied for many years. However, the<br />
occurr<strong>en</strong>ce of antibiotic resistance in pathog<strong>en</strong>ic bacteria is limiting the usefulness of<br />
these substances [4-6] and therefore, several alternative strategies to the use of<br />
antimicrobials have be<strong>en</strong> proposed, including the use of immunostimu<strong>la</strong>nts which<br />
repres<strong>en</strong>t a promising tool in aquaculture. In<strong>de</strong>ed, many authors have reported that the<br />
injection of immunostimu<strong>la</strong>nts, such as glucans, <strong>en</strong>hances the function of leucocytes<br />
and protection against pathog<strong>en</strong>s [7-10]. Several authors have observed that P.<br />
damse<strong>la</strong>e subsp. piscicida is highly susceptible to oxidative radicals g<strong>en</strong>erated during<br />
the macrophage respiratory burst [11, 12]. However, other authors have reported the<br />
pres<strong>en</strong>ce of intact cells of this pathog<strong>en</strong> insi<strong>de</strong> fish cells, suggesting the ability of the<br />
bacterium to survive as an intracellu<strong>la</strong>r pathog<strong>en</strong> [13-17]. For this reason, the<br />
stimu<strong>la</strong>tion of the respiratory burst activity of the phagocytes of S<strong>en</strong>egalese sole could<br />
facilitate a more effective <strong>de</strong>struction of P. damse<strong>la</strong>e subsp. piscicida.<br />
Marine organisms constitute a pot<strong>en</strong>tial alternative source of substances for the<br />
prev<strong>en</strong>tion and treatm<strong>en</strong>t of infectious diseases [18]. In this connection, algae have be<strong>en</strong><br />
studied as dietary ingredi<strong>en</strong>ts for fish nutrition [19] and as a source of bioactive<br />
compounds such as pharmaceutical [20, 21] and immunostimu<strong>la</strong>nt [22-25] ag<strong>en</strong>ts.<br />
Several authors [26] have suggested a possible stimu<strong>la</strong>tion of the metabolic and<br />
functional action of phagocytic system cells from Balb/c mice after intraperitoneal<br />
administration of polysacchari<strong>de</strong>s iso<strong>la</strong>ted from cultures in the stationary phase of red<br />
3
alga Porphyridium cru<strong>en</strong>tum. For this reason, this alga has be<strong>en</strong> selected in this work to<br />
evaluate its pot<strong>en</strong>tial immunostimu<strong>la</strong>nt effect on farmed fish. However, most studies<br />
performed to examine the immunostimu<strong>la</strong>nt ability of algae have be<strong>en</strong> carried out by in<br />
vitro incubation of immune cells with algal extracts, and information on the in vivo<br />
effects of whole algal cells is still scarce [27]. In addition, algal extracts are inocu<strong>la</strong>ted<br />
intraperitoneally in theses studies. This route of administration, although very effective,<br />
is also very <strong>la</strong>borious, time-consuming, stressful for fish and difficult to apply to<br />
fingerlings [28, 29]. Oral administration of immunostimu<strong>la</strong>nts is a non-stressful method<br />
with minimum economic cost and effort and <strong>en</strong>ables mass administration regardless of<br />
the fish size [7], but studies addressing this route of administration are scarce and<br />
usually inclu<strong>de</strong> only algal extracts instead of whole cells [22].<br />
In this study the pot<strong>en</strong>tial immunostimu<strong>la</strong>nt effect of aqueous and ethanolic<br />
extracts obtained from P. cru<strong>en</strong>tum on the respiratory burst activity of S<strong>en</strong>egalese sole<br />
phagocytes has be<strong>en</strong> <strong>de</strong>termined. In addition, the pot<strong>en</strong>tial stimu<strong>la</strong>tion of the respiratory<br />
burst activity of phagocytes iso<strong>la</strong>ted from fish fed with a commercial diet supplem<strong>en</strong>ted<br />
with P. cru<strong>en</strong>tum cells has be<strong>en</strong> studied. In this case, pot<strong>en</strong>tial synergetic or antagonic<br />
effects resulting from the alga diet and vaccination against P. damse<strong>la</strong>e subsp. piscicida<br />
have be<strong>en</strong> evaluated.<br />
Materials and Methods<br />
2.1. Microorganisms<br />
The virul<strong>en</strong>t strain Lg h41/01 of Photobacterium damse<strong>la</strong>e subsp. piscicida<br />
iso<strong>la</strong>ted from diseased S<strong>en</strong>egalese sole [16] was selected to test the respiratory burst<br />
activity of sole phagocytes. The bacterial strain was cultured on tryptic soy agar (Oxoid)<br />
supplem<strong>en</strong>ted with 1.5% NaCl (TSAs) for 24 h at 22 ºC. Bacterial susp<strong>en</strong>sions for<br />
respiratory burst assays were obtained from tubes containing tryptic soy broth (Oxoid)<br />
ad<strong>de</strong>d with 1.5% NaCl (TSBs) inocu<strong>la</strong>ted with one colony from a TSAs p<strong>la</strong>te and<br />
incubated at 22 ºC for 24 h. Th<strong>en</strong>, the cultures were c<strong>en</strong>trifuged at 6000 xg for 15 min at<br />
4 ºC, and pellets were resusp<strong>en</strong><strong>de</strong>d in L-15 medium at an optical <strong>de</strong>nsity (600 nm) equal<br />
to 1(10 8 cells ml -1 ).<br />
4
2.2. Alga culture<br />
The red microalga Porphyridium cru<strong>en</strong>tum (S.F. Gray) Näegli was obtained<br />
from the collection at the C<strong>en</strong>tro <strong>de</strong> Investigaciones Marinas <strong>de</strong> Cádiz, Cádiz, Spain. It<br />
was grown in Porphyridium medium [30] in batch culture at 25ºC, with a 12h<br />
photoperiod for 7 days. The algal biomass was c<strong>en</strong>trifuged at 3000 xg, 15 min at 4ºC<br />
and the pellet was lyophilized.<br />
2.3. Obt<strong>en</strong>tion of aqueous and ethanolic extracts<br />
The preparation of water-soluble extract, aqueous extract, was carried out as<br />
follows: 10 g of lyophilized alga was resusp<strong>en</strong><strong>de</strong>d in 100 ml of HBSS (Hank’s Ba<strong>la</strong>nced<br />
Salt Solution) using a mortar and pestle. The extract was sonicated for 20 min and<br />
c<strong>en</strong>trifuged at 3000 xg, 5 min. The supernatant was separated from the pellet and<br />
lyophilized and 10 mg of the lyophilized extract was resusp<strong>en</strong><strong>de</strong>d in 1 ml of HBSS.<br />
Extraction of the non-soluble fraction of the alga, ethanolic extract, was carried out as<br />
<strong>de</strong>scribed above, but instead of HBSS, ethanol was used. Dilutions from both extracts<br />
were prepared in HBSS to achieve conc<strong>en</strong>trations of 10 mg ml -1 of lyophilized extract, 5<br />
mg ml -1 , 2 mg ml -1 and 1 mg ml -1 .<br />
Commercial β-1,3-glucan from Eugl<strong>en</strong>a gracilis (BioChemika Fluka, Sigma)<br />
was used as a positive control of stimu<strong>la</strong>tion of respiratory burst activity. β-1,3-glucan<br />
(10 mg) was dissolved following commercial instructions, and diluted in HBSS to<br />
achieve conc<strong>en</strong>trations of 10 mg ml -1 , 5 mg ml -1 , 2 mg ml -1 and 1 mg ml -1 .<br />
2.4. Fish and experim<strong>en</strong>tal <strong>de</strong>sign<br />
Experim<strong>en</strong>ts to test the in vitro effect of P. cru<strong>en</strong>tum on the respiratory burst<br />
activity of sole phagocytes were carried out. Sole of 200 g body weight, stocked in 250 l<br />
tanks with recircu<strong>la</strong>ting, aerated seawater at 20 ºC, 35‰ salinity, were used to iso<strong>la</strong>te<br />
kidney phagocytes and <strong>de</strong>termine the production of anion radicals in contact with<br />
aqueous and ethanolic extracts from P. cru<strong>en</strong>tum and the commercial β-1,3-glucan.<br />
5
Feeding assays were carried out with soles of 80 g mean body weight, which<br />
were randomly separated into six experim<strong>en</strong>tal groups, and stocked in six 250 l tanks<br />
(20 fish per tank) with simi<strong>la</strong>r culture conditions to those <strong>de</strong>scribed above.<br />
The diet assayed was prepared in the <strong>la</strong>boratory from the commercial pellet diet<br />
routinely used in fish farms (Skreeting, Trouw España, Nutreco, Burgos, Spain).<br />
Briefly, the commercial pellet diet was crushed and mixed with tap water before adding<br />
the lyophilized alga Porphyridium cru<strong>en</strong>tum at the <strong>de</strong>sired conc<strong>en</strong>tration (10 g kg -1 ), and<br />
th<strong>en</strong> ma<strong>de</strong> into pellets again. The re-ma<strong>de</strong> pellets were allowed to dry and stored at 4 ºC<br />
until use.<br />
The commercial pellet Sanostim (Skreeting, Trouw España, Nutreco, Burgos,<br />
Spain), containing β-glucans, was used to test the response of sole phagocytes. Two<br />
groups of fish received daily one of the differ<strong>en</strong>t diets assayed: diet consisting of nonsupplem<strong>en</strong>ted<br />
commercial diet (control group); diet composed of the commercial diet<br />
containing immunostimu<strong>la</strong>nt Sanostim; and finally, a commercial diet supplem<strong>en</strong>ted<br />
with lyophilized alga (1%). Fish were fed at a rate of 20 g dry diet kg -1 biomass (2 %)<br />
per day for 4 weeks. The biomass of the fish in each aquarium was measured before the<br />
experim<strong>en</strong>t and daily ration, being adjusted accordingly. No mortality was observed<br />
during the experim<strong>en</strong>t.<br />
2.5. Immunization assay<br />
Two weeks after beginning the feeding trial, fish from one tank per treatm<strong>en</strong>t<br />
were intraperitoneally inocu<strong>la</strong>ted with a bacterin of P. damse<strong>la</strong>e subsp. piscicida. The<br />
formalin-killed aqueous vaccine was prepared with a virul<strong>en</strong>t strain of P. damse<strong>la</strong>e<br />
subsp. piscicida (Lg h411/01 ) iso<strong>la</strong>ted from diseased sole [16] according to the following<br />
protocol. Briefly, bacteria were cultured on TSAs for 24 h and one colony was<br />
inocu<strong>la</strong>ted in tubes containing 5 ml of TSBs. After 18 h incubation at 22 ºC, an aliquot<br />
of the culture, 50 μl, was inocu<strong>la</strong>ted in f<strong><strong>la</strong>s</strong>ks with 50 ml TSBs and incubated at 22ºC<br />
with continuous shaking. After 18 h incubation the culture achieved O.D. 600 of 1.2.<br />
The total bacterial number was counted, obtaining a bacterial conc<strong>en</strong>tration of 6 x 10 8<br />
bacteria ml -1 . Th<strong>en</strong> bacterial cells were killed by addition of formal<strong>de</strong>hy<strong>de</strong> to achieve<br />
1% final conc<strong>en</strong>tration, and overnight incubation. Sterility tests were performed by<br />
6
spreading an aliquot of the bacterin on TSA p<strong>la</strong>tes and incubation for 2 days at 22 ºC.<br />
The vaccine was administered by intraperitoneal injection (0.1 ml per fish). Control fish<br />
were injected with phosphate buffer saline (PBS, pH 7.2).<br />
2.6. Iso<strong>la</strong>tion of head kidney phagocytes<br />
Sole phagocytes were iso<strong>la</strong>ted from the kidney following the technique<br />
<strong>de</strong>scribed by Secombes [31]. Briefly, the kidney was removed aseptically and pushed<br />
through a 100 μm nylon mesh with Leibovitz medium (L-15) containing 2% foetal calf<br />
serum (FCS, Sigma), 1% p<strong>en</strong>icillin-streptomycin (Sigma), 0.1 % (5 mg ml -1 )<br />
g<strong>en</strong>tamicine (Sigma) (P/S/G) and 10 U heparine ml -1 . This cell susp<strong>en</strong>sion was <strong>la</strong>yered<br />
on a 30 to 51% Percoll (Amersham) gradi<strong>en</strong>t and c<strong>en</strong>trifuged at 600 xg for 30 min.<br />
Th<strong>en</strong> the bands separated at the interface were resusp<strong>en</strong><strong>de</strong>d in L-15 medium<br />
supplem<strong>en</strong>ted with P/S/G. The viable cell conc<strong>en</strong>tration was <strong>de</strong>termined after staining<br />
with trypan blue and microscope counting. Aliquots of 100 μl containing 1x10 7 cells ml -<br />
1 in L-15 medium supplem<strong>en</strong>ted with P/S/G were ad<strong>de</strong>d to 96-well microtitre p<strong>la</strong>tes.<br />
After 3 h incubation at 22 ºC, non-adher<strong>en</strong>t cells were removed and medium was<br />
substituted by L-15 and P/S/G supplem<strong>en</strong>ted with 2% FCS. Mono<strong>la</strong>yers were incubated<br />
overnight at 22 ºC.<br />
2.7. Respiratory burst activity<br />
The g<strong>en</strong>eration of intracellu<strong>la</strong>r superoxi<strong>de</strong> radicals by sole phagocytes was<br />
<strong>de</strong>termined by the reduction of nitro-blue tetrazolium (NBT) according to the technique<br />
<strong>de</strong>scribed by Secombes [31] and Boes<strong>en</strong> et al. [32]. Phagocyte mono<strong>la</strong>yers were washed<br />
with L-15 medium and HBSS (Hank´s Ba<strong>la</strong>nced Salt Solution) to remove any trace of<br />
the antibiotic.<br />
In or<strong>de</strong>r to test the in vitro effects of algal extracts on the production of<br />
superoxi<strong>de</strong> radicals by sole phagocytes, a volume of 20 μl of the extracts (aqueous,<br />
ethanolic or β-glucan) were ad<strong>de</strong>d to the wells (15 wells from 5 fish) containing<br />
phagocyte mono<strong>la</strong>yers prepared as above. Th<strong>en</strong>, 100 μl of NBT dissolved at 1 mg ml -1<br />
in HBSS was ad<strong>de</strong>d to the wells and the phagocytes incubated at 22 ºC for 30 min.<br />
Wells containing phagocytes were infected with 20 μl of P. damse<strong>la</strong>e subsp. piscicida<br />
7
(10 8 bacterias ml -1 ) and used to <strong>de</strong>termine the response of the phagocytes to the fish<br />
pathog<strong>en</strong>. As a positive control phorbol myristate acetate (PMA, Sigma) (1 μg ml -1 ) was<br />
used to stimu<strong>la</strong>te the respiratory burst activity of non-infected phagocytes (data not<br />
shown). The specificity of the reaction was tested by adding superoxi<strong>de</strong> dismutase<br />
(SOD) (300 I.U. per well) to some wells containing PMA-stimu<strong>la</strong>ted phagocytes (data<br />
not shown). After incubation, cells were fixed in 70% methanol and the reduced<br />
formazan within phagocytes was solubilised by adding 120 μl 2M KOH and 140 μl<br />
dimethyl sulfoxi<strong>de</strong> (DMSO, Sigma). Finally, absorbance was read at 630 nm in a<br />
multiscan spectrophotometer (UV-1601 Spectrophotometer, Whitakker Bioproducts).<br />
In the P. cru<strong>en</strong>tum feeding experim<strong>en</strong>ts, the effect of oral administration of the<br />
alga or Sanostim was <strong>de</strong>termined on phagocyte mono<strong>la</strong>yers as <strong>de</strong>scribed above, but<br />
algal extracts or bacteria were not ad<strong>de</strong>d to the wells.<br />
2.8. Statistical analysis<br />
Results are expressed as the stimu<strong>la</strong>tion in<strong>de</strong>x (mean + standard error, SE),<br />
obtained by dividing each sample value by the mean control value. Values higher than 1<br />
reflect an increase and lower than 1 a <strong>de</strong>crease in each parameter compared to the<br />
control. Data were statistically analysed by one-way analysis of variance (ANOVA) and<br />
Tukey’s comparison of means using SPSS for Windows. Differ<strong>en</strong>ces were consi<strong>de</strong>red<br />
statistically significant wh<strong>en</strong> P< 0.05.<br />
3. Results<br />
3.1. In vitro assays<br />
Results have be<strong>en</strong> expressed as the ratio of the absorbance at 630 nm of treated<br />
phagocytes to the absorbance of non-treated phagocytes. The treatm<strong>en</strong>t of phagocytes<br />
consisted of incubation with aqueous (Figure 1a) or ethanolic (Figure 1b) algal extracts<br />
or β-glucan (Figure 1c), in the pres<strong>en</strong>ce or abs<strong>en</strong>ce of P. damse<strong>la</strong>e subsp. piscicida.<br />
Therefore the data repres<strong>en</strong>ted in the Figures are the result of a corre<strong>la</strong>tion betwe<strong>en</strong><br />
phagocytes incubated with the algal extract or β-glucan, with or without bacteria, and<br />
phagocytes incubated without the pot<strong>en</strong>tial immunostimu<strong>la</strong>nt (algal extract or β-glucan).<br />
8
The results obtained show that extract from P. cru<strong>en</strong>tum is not able to<br />
significantly increase the respiratory burst activity of sole phagocytes, both incubated<br />
with and without bacteria (Figures 1a and 1b). Only the commercial β-glucan (10 mg<br />
ml -1 ) significantly <strong>en</strong>hanced (P
are in agreem<strong>en</strong>t with the data reported by Castro et al. [35], who observed increases in<br />
the respiratory burst activity of the head kidney phagocytes of turbot and gilthead<br />
seabream incubated with differ<strong>en</strong>t conc<strong>en</strong>trations of β-glucans obtained from fungi and<br />
yeasts. The fact that several authors found significant variations in the stimu<strong>la</strong>tory<br />
capacities of algal extracts <strong>de</strong>p<strong>en</strong>ding not only on their origin, but on the conc<strong>en</strong>trations<br />
used and time of incubation [23] does not rule out a pot<strong>en</strong>tial in vitro modu<strong>la</strong>tion of<br />
respiratory burst activity by P. cru<strong>en</strong>tum extracts. Thus, higher conc<strong>en</strong>trations and<br />
longer incubation times need to be assayed.<br />
The results obtained in the in vivo assays showed that fish immunized with a<br />
bacterin against P. damse<strong>la</strong>e subsp. piscicida and receiving the diet supplem<strong>en</strong>ted with<br />
the alga P. cru<strong>en</strong>tum significantly increased the respiratory burst activity of their<br />
phagocytes after four weeks of the feeding treatm<strong>en</strong>t. This increase was higher than that<br />
obtained in the group of fish fed with the diet supplem<strong>en</strong>ted with Sanostim for four<br />
weeks. These stimu<strong>la</strong>ted phagocytes may be more effici<strong>en</strong>t in the inactivation of<br />
intracellu<strong>la</strong>r pathog<strong>en</strong>s such as P. damse<strong>la</strong>e subsp. piscicida than other phagocytes<br />
which had not be<strong>en</strong> in contact with the compon<strong>en</strong>ts of the alga assayed. However,<br />
experim<strong>en</strong>ts to test the bactericidal activity of phagocytes from sole specim<strong>en</strong>s fed with<br />
this diet should be consi<strong>de</strong>red in or<strong>de</strong>r to confirm this hypothesis.<br />
Evi<strong>de</strong>nce is increasing to support the ess<strong>en</strong>tiality of nutri<strong>en</strong>ts for maintaining a<br />
normal immune system in fish [22, 36]. These nutri<strong>en</strong>ts are proteins, ess<strong>en</strong>tial fatty<br />
acids, polysacchari<strong>de</strong>s, vitamins C and E, and some oligoelem<strong>en</strong>ts such as Se and Zn<br />
[37, 38]. P. cru<strong>en</strong>tum is a red microalga member of Rodophyta, of the or<strong>de</strong>r of<br />
Porphyridiales, with spherical cells that <strong>la</strong>ck a rigid cell wall. This alga accumu<strong>la</strong>tes<br />
<strong>la</strong>rge amounts of lipids, specially arachidonic acid and noticeable amounts of<br />
eicosap<strong>en</strong>ta<strong>en</strong>oic acid, these being substances which have be<strong>en</strong> reported as<br />
immunostimu<strong>la</strong>nts [39, 40]. The avai<strong>la</strong>ble carbohydrates ranged from 40 to 57% [41].<br />
Differ<strong>en</strong>t carbohydrates have be<strong>en</strong> reported previously as immunostimu<strong>la</strong>nt active on<br />
fish phagocytes [7, 23, 33, 40-48]. Morris et al. [26] suggested a pot<strong>en</strong>tial<br />
immunostimu<strong>la</strong>nt activity of the polysacchari<strong>de</strong>s iso<strong>la</strong>ted from cultures of P. cru<strong>en</strong>tum<br />
on the metabolic and functional action of mice phagocytes. This alga also contains <strong>la</strong>rge<br />
amounts of carot<strong>en</strong>es [41] and several studies have reported that the carot<strong>en</strong>oids<br />
10
increase the resistance to diseases [49] and they modu<strong>la</strong>te some of the innate <strong>de</strong>f<strong>en</strong>se<br />
mechanisms in fish such as rainbow trout [22]. Other substances pres<strong>en</strong>t in this alga are<br />
vitamins such as tocopherol, vitamins K and C, the <strong>la</strong>tter having be<strong>en</strong> reported as a very<br />
important immunostimu<strong>la</strong>nt [44, 50-52]. Due to the fact that this alga contains differ<strong>en</strong>t<br />
immunostimu<strong>la</strong>nt substances, its use could g<strong>en</strong>erate a more g<strong>en</strong>eral immune response as<br />
has be<strong>en</strong> proposed for other microorganisms such as yeasts [8, 53].<br />
Castro et al. [23] reported that wh<strong>en</strong> turbot phagocytes were pre-incubated with<br />
water–soluble extracts of Chondrus crispus and th<strong>en</strong> incubated with PMA, the extracts<br />
had a priming effect on fish cells. Supplem<strong>en</strong>tation of the diet with whole cells of P.<br />
cru<strong>en</strong>tum fed to the fish during a four-week period did not show a pot<strong>en</strong>tial priming<br />
effect on the respiratory burst activity of sole phagocytes. This finding could indicate<br />
that this alga does not have the ability to induce the priming effect reported for C.<br />
crispus or that the oral administration of the alga did not supply the phagocytes with<br />
levels of immunostimu<strong>la</strong>nt capable of facilitating this priming effect.<br />
Val<strong>en</strong>te et al. [19] suggested that the inclusion of dry pellet of the red<br />
macroalga Graci<strong>la</strong>ria bursa-pastoris and the gre<strong>en</strong> macroalga Ulva rigida, up to 10%<br />
can be consi<strong>de</strong>red as very interesting ingredi<strong>en</strong>ts in diets for sea bass juv<strong>en</strong>iles, as no<br />
negative consequ<strong>en</strong>ces on growth performance, nutri<strong>en</strong>t utilization or body composition<br />
were observed. On the other hand, the inclusion of the red macroalga Graci<strong>la</strong>ria cornea<br />
should be limited to 5% of the diet.<br />
The stimu<strong>la</strong>tion of sole phagocytes has be<strong>en</strong> observed only wh<strong>en</strong> fish with P.<br />
cru<strong>en</strong>tum were immunized with the P. damse<strong>la</strong>e subsp. piscicida bacterin. Sakai [28]<br />
indicates that the combination of vaccination and immunostimu<strong>la</strong>nt administration may<br />
increase the pot<strong>en</strong>cy of vaccines. J<strong>en</strong>ey and An<strong>de</strong>rson [54] reported that rainbow trout<br />
bathed in A. salmonicida O-antig<strong>en</strong> in combination with immunostimu<strong>la</strong>nts (levamisole,<br />
quaternary ammonium compound –QAC- and a polypepti<strong>de</strong> <strong>de</strong>rived from fish products<br />
–ISK-) <strong>en</strong>hanced phagocytosis by leucocytes and antibody titers against A. salmonicida,<br />
and showed adjuvant effects with vaccination. Sakai et al. [55] reported that R.<br />
salmoninarum-vaccinated rainbow trout receiving EF203, the ferm<strong>en</strong>ted product of<br />
chick<strong>en</strong> eggs, showed higher phagocytic activities and NBT responses in kidney<br />
leucocytes wh<strong>en</strong> compared to vaccinated fish without EF203 treatm<strong>en</strong>t or to<br />
11
unvaccinated fish. However, the serum agglutinating antibody titers of vaccinated fish<br />
did not show a significant increase compared to control groups, and vaccinated fish<br />
treated with EF203 showed slightly increased survival in comparison with the other<br />
groups following R. salmoninarum chall<strong>en</strong>ge. The adjuvant effects of yeast glucan have<br />
also be<strong>en</strong> <strong>de</strong>monstrated. Injection of the A. salmonicida vaccine and yeast glucan in<br />
At<strong>la</strong>ntic salmon <strong>en</strong>hanced antibody responses [56] and induced significantly increased<br />
protection against furunculosis over vaccines without yeast glucan [57]. The injection of<br />
yeast glucan alone did not afford protection. Baulny et al. [58] reported that oral<br />
administration of yeast glucan to turbot immersed in the V. anguil<strong>la</strong>rum bacterin also<br />
increased protection compared to the bacterin alone, but simi<strong>la</strong>r to the results obtained<br />
in this work with β-glucan from Eugl<strong>en</strong>a gracilis, yeast glucan alone did not <strong>en</strong>hance<br />
protection against V. anguil<strong>la</strong>rum infection.<br />
P. cru<strong>en</strong>tum has advantages in its culture, such as fast growth and low cost.<br />
This makes it easier to work with at known conc<strong>en</strong>tration, compared with using soluble<br />
substances such as vitamins, which exist as micronutri<strong>en</strong>ts in feed and are very s<strong>en</strong>sitive<br />
to differ<strong>en</strong>t factors (light, humidity, temperature). On the other hand P. cru<strong>en</strong>tum could<br />
be a natural immunostimu<strong>la</strong>nt, which is biocompatible, bio<strong>de</strong>gradable and safe for the<br />
<strong>en</strong>vironm<strong>en</strong>t and human health. In this way, these algae could be inclu<strong>de</strong>d in the groups<br />
of differ<strong>en</strong>t whole microorganisms, alive or not, such as yeast [8, 53], fungi [59] and<br />
bacteria [60-62] which increase disease resistance in mammals and fish. In fish, as in<br />
other aquatic organisms, the whole microorganisms administered have be<strong>en</strong> mainly<br />
bacterial species, such as probiotics [60-66], but algae can repres<strong>en</strong>t another promising<br />
alternative [18, 25], although studies about whole algae are still very scarce.<br />
In conclusion, this study provi<strong>de</strong>s a <strong>de</strong>scription of the stimu<strong>la</strong>tion of the<br />
respiratory burst activity of phagocytes from sole vaccinated with a bacterin against P.<br />
damse<strong>la</strong>e subsp. piscicida and fed with cells from P. cru<strong>en</strong>tum.<br />
Acknowledgem<strong>en</strong>ts<br />
This study has be<strong>en</strong> supported by a grant from the Spanish Governm<strong>en</strong>t<br />
(AGL2002-01488). P. Díaz-Rosales thanks the Ministerio Español <strong>de</strong> Educación y<br />
12
Ci<strong>en</strong>cia for a F.P.U. scho<strong>la</strong>rship. The authors thank the PROMAN (Promotora<br />
Alpujarreña <strong>de</strong> Negocios, S.L., Motril, Granada, Spain) fishery for its help and<br />
participation in this study; J.M. León-Rubio for his help in fish diet preparation and<br />
Tracey Coffey for her help in the English revision of the manuscript.<br />
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[38] Sealey, WM and Gatlin, DM III. Overview of nutritional strategies affecting health<br />
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the microalga Porphyridium cru<strong>en</strong>tum. Food Chemistry 2000; 70: 345-53.<br />
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[43] Bagni, M, Archetti, L, Amadori, M and Marino, G. Effect on long-term oral<br />
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(Dic<strong>en</strong>trarchus <strong>la</strong>brax). Journal of Veterinary Medicine 2000; 47: 745-51.<br />
[44] J<strong>en</strong>ey, G and J<strong>en</strong>ey, Zs. Application of immunostimu<strong>la</strong>nts for modu<strong>la</strong>tion of the<br />
non-specific <strong>de</strong>f<strong>en</strong>se mechanisms in sturgeon hybrid: Acip<strong>en</strong>ser ruth<strong>en</strong>us x A.<br />
baerii. Journal of Applied Ichthyology 2002; 18: 416-9.<br />
[45] Cook, MT, Hayball, P, Hutchinson, W, Nowak, BF and Hayball, JD.<br />
Administration of a commercial immunostimu<strong>la</strong>nt preparation, EcoActiva, as<br />
feed supplem<strong>en</strong>t <strong>en</strong>hances macrophage respiratory burst and the growth rate of<br />
snapper (Pagrus auratus, Sparidae (Bloch and Schnei<strong>de</strong>r)) in winter. Fish &<br />
Shellfish, 2003; 14: 333-45.<br />
[46] Couso, N, Castro, R, Magariños, B, Obach, A and Lamas, J. Effect of oral<br />
administration of glucans on the resistance of gilthead seabream to pasteurellosis.<br />
Aquaculture 2003; 219: 99-109.<br />
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and Marino, G. Short- and long- term effects of a dietary yeast β-glucan<br />
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(Macrogard) and alginic acid (Ergosan) preparation on immune response in sea<br />
bass (Dic<strong>en</strong>trarchus <strong>la</strong>brax). Fish & Shellfish Immunology 2005; 18: 311-25.<br />
[48] Kumar, S, Sahu, NP, Pal, AK, Choudhury, D, Y<strong>en</strong>gkokpam, S and Mukherjee, SC.<br />
Effect of dietary carbohydrate on haematology, respiratory burst activity and<br />
histological changes in L. rohita juv<strong>en</strong>iles. Fish & Shellfish Immunology 2005;<br />
19: 331-44.<br />
[49] Tachinaba, K, Yagi, M, Hara, K, Mishima, T and Tsuchimoto, M. Effects of<br />
feeding β-carot<strong>en</strong>e supplem<strong>en</strong>ted rotifers on survival and lymphocyte proliferation<br />
reaction of fish <strong>la</strong>rvae of Japanese parrotfish (Oplegnathus fasciatus) and Spotted<br />
parrot fish (Oplegnathus punctatus): preliminary trials. Hydrobiologia 1997; 358:<br />
313-6.<br />
[50] Hardie, LJ, Fletcher, TC and Secombes, CJ. The effect of vitamin E on the immune<br />
response of the At<strong>la</strong>ntic Salmon (Salmo sa<strong>la</strong>r L.). Aquaculture 1990; 87: 1-13.<br />
[51] Hardie, LJ, Fletcher, TC and Secombes, CJ. The effect of dietary vitamin C on the<br />
immune response of the At<strong>la</strong>ntic Salmon (Salmo sa<strong>la</strong>r L.). Aquaculture 1991; 95:<br />
201-14.<br />
[52] Ortuño, J, Esteban, MA and Meseguer, J. Effect of high dietary intake vitamin C on<br />
non-specific immune response of gilthead seabream (Sparus aurata L.). Fish &<br />
Shellfish Immunology 1999; 9: 429-43<br />
[53] Rodríguez, A, Cuesta, A, Ortuño, J, Esteban, MA and Meseguer, J.<br />
Immunostimu<strong>la</strong>nt properties of a cell wall-modified whole Saccharomyces<br />
cerevisiae strain administered by diet to seabream (Sparus aurata L.). Veterinary<br />
Immunology & Immunopathology 2003; 96: 183-92.<br />
[54] J<strong>en</strong>ey, G and An<strong>de</strong>rson, DP. Enhanced immune response and protection in rainbow<br />
trout to Aeromonas salmonicida bacterin following prior immersion in<br />
immunostimu<strong>la</strong>nts. Fish & Shellfish Immunology 1993; 3: 51-8.<br />
[55] Sakai, M, Yoshida, T and Kobayashi, M. Influ<strong>en</strong>ce of the immunostimu<strong>la</strong>nt,<br />
EF203, on the immune responses of rainbow trout, Oncorhynchus mykiss, to<br />
R<strong>en</strong>ibacterium salmoninarum. Aquaculture 1995; 138:61-7.<br />
[56] Aakre, R, Werge<strong>la</strong>nd, HI, Aasjord, PM and En<strong>de</strong>rs<strong>en</strong>, C. Enhanced antibody<br />
response in At<strong>la</strong>ntic salmon (Salmo sa<strong>la</strong>r L.) to Aeromonas salmonicida cell wall<br />
18
antig<strong>en</strong>s using a bacterin containing β-1,3-M-glucan as adjuvant. Fish & Shellfish<br />
Immunology 1994; 4: 47-61.<br />
[57] RØsrstad, G, Aasjord, PM and Roberts<strong>en</strong>, B. Adjuvant effect of a yeast glucan in<br />
vaccines against furunculosis in At<strong>la</strong>ntic salmon (Salmo sa<strong>la</strong>r L.) Fish & Shellfish<br />
Immunology 1993; 3: 170-90.<br />
[58] Baulny, MOD, Qu<strong>en</strong>tel, C, Fournier, V, Lamour, F and Gouvello, RL. Effect of<br />
long-term oral administration of β-glucan as an immunostimu<strong>la</strong>nt or an adjuvant<br />
on some non-specific parameters of the immune response of turbot Scophthalmus<br />
maximus. Diseases of Aquatic Organisms 1996; 26: 139-47.<br />
[59] Rodríguez, A, Cuesta, A, Esteban, MA and Meseguer, J. The effect of dietary<br />
administration of the fungu Mucor circinelloi<strong>de</strong>s on non-specific immune<br />
responses of gilthead seabream. Fish & Shellfish Immunology 2004; 16: 241-9.<br />
[60] Verschuere, L, Rombaut, G, Sorgeloos, P and Verstraete, W. Probiotic bacteria as<br />
biological control ag<strong>en</strong>ts. Aquaculture 2000; 64: 1092-2172.<br />
[61] Irianto, A and Austin, B. Use of <strong>de</strong>ad probiotic cells to control furunculosis in<br />
rainbow trout, Oncorhynchus mykiss (Walbaum). Journal of Fish Diseases 2003;<br />
26: 59:62.<br />
[62] Díaz-Rosales, P, Salinas, I, Rodríguez, A, Cuesta, A, Chabrillón, M, Balebona,<br />
MC, Moriñigo, MA, Esteban, MA and Meseguer, J. Gilthead seabream (Sparus<br />
aurata L.) innate immune response after dietary administration of heat-inactivated<br />
pot<strong>en</strong>tial probiotics. Fish & Shellfish Immunology 2006; 20: 482-92.<br />
[63] Nikoske<strong>la</strong>in<strong>en</strong>, S, Salmin<strong>en</strong>, S, Bylund, G and Ouwehand, AC. Characterization of<br />
the properties of human- and dairy- <strong>de</strong>rived probiotics for prev<strong>en</strong>tion of infectious<br />
diseases in fish. Applied and Environm<strong>en</strong>tal Microbiology 2001; 67: 2430-5.<br />
[64] Salinas, I, Cuesta, A, Esteban, MA and Meseguer, J. Dietary administration of<br />
Lactobacillus <strong>de</strong>lbrüeckii and Bacillus subtilis, single or combined, on gilthead<br />
seabream cellu<strong>la</strong>r innate immune responses. Fish & Shellfish Immunology 2005;<br />
19: 67-77.<br />
[65] Balcázar, JL, <strong>de</strong> B<strong><strong>la</strong>s</strong>, I, Ruíz-Zarzue<strong>la</strong>, I, Cunningham, D, V<strong>en</strong>drell, D and<br />
Múzquiz, JL. The role of probiotics in aquaculture. Veterinary Microbiology 2006;<br />
114: 173-84.<br />
19
[66] Salinas, I, Díaz-Rosales, P, Cuesta, A, Meseguer, J, Chabrillón, M, Moriñigo, MA<br />
and Esteban, MA. Effect of heat-inactivated fish and non-fish <strong>de</strong>rived probiotics<br />
on the innate immune parameters of a teleost fish (Sparus aurata L.). Veterinary<br />
Immunology &s Immunopathology 2006; 111: 279-89.<br />
20
Figure leg<strong>en</strong>ds<br />
Figure 1. Respiratory burst activity of sole phagocytes incubated with aqueous<br />
(a) or ethanolic (b) extracts of Porphyridium cru<strong>en</strong>tum (1, 2, 5, 10 mg ml -1 ) or β-glucan<br />
(c) in abs<strong>en</strong>ce ( ) or pres<strong>en</strong>ce ( ) of Photobacterium damse<strong>la</strong>e subsp. piscicida<br />
(2x10 6 cells per well). Results are expressed as stimu<strong>la</strong>tion in<strong>de</strong>x (mean ± SE; n=15)<br />
obtained by dividing each sample value by its mean control value (HBSS or P.<br />
damse<strong>la</strong>e subsp. piscicida cells). The symbol * <strong>de</strong>notes statistically significant<br />
differ<strong>en</strong>ces (P
Figure 1a<br />
3<br />
2.5<br />
Stimu<strong>la</strong>tion in<strong>de</strong>x<br />
2<br />
1.5<br />
1<br />
0.5<br />
0<br />
1 2 5 10 mg ml -1<br />
23
Figure 1b<br />
3<br />
2.5<br />
Stimu<strong>la</strong>tion in<strong>de</strong>x<br />
2<br />
1.5<br />
1<br />
0.5<br />
0<br />
1 2 5 10 mg ml -1<br />
25
Figure 1c<br />
Stimu<strong>la</strong>tion in<strong>de</strong>x<br />
3<br />
2.5<br />
2<br />
1. 5<br />
1<br />
0.5<br />
*<br />
*<br />
0<br />
1 2 5 10 mg ml -1<br />
27
Figure 2a<br />
Stimu<strong>la</strong>tion in<strong>de</strong>x<br />
3<br />
2.5<br />
2<br />
1. 5<br />
1<br />
0.5<br />
0<br />
2 weeks 3 weeks 4 weeks<br />
29
Figure 2b<br />
Stimu<strong>la</strong>tion in<strong>de</strong>x<br />
3<br />
2.5<br />
2<br />
1.5<br />
1<br />
0.5<br />
0<br />
*<br />
3 weeks 4 weeks<br />
31
A RTÍCULO 2.2.<br />
A RTICLE 2.2.
Effect of the extracellu<strong>la</strong>r polysaccharidic fraction from the red microalga<br />
Porphyridium cru<strong>en</strong>tum on the respiratory burst activity of sole (Solea s<strong>en</strong>egal<strong>en</strong>sis,<br />
Kaup 1858) phagocytes<br />
Patricia Díaz-Rosales a , Roberto T. Abda<strong>la</strong> b , Juan Decara b , Salvador Arijo a , Félix L.<br />
Figueroa b , M. Ángel Moriñigo a and M. Carm<strong>en</strong> Balebona a*<br />
a Departm<strong>en</strong>t of Microbiology, Faculty of Sci<strong>en</strong>ces. University of Má<strong>la</strong>ga. 29071<br />
Má<strong>la</strong>ga. Spain<br />
b Departm<strong>en</strong>t of Ecology and Geology, Group of Photobiology and Biotechnology of<br />
algae, Faculty of Sci<strong>en</strong>ces. University of Má<strong>la</strong>ga. 29071 Má<strong>la</strong>ga. Spain<br />
* Corresponding author. Tel. +34 952134233 ; fax: +34 952131889<br />
E-mail address: balebona@uma.es (M. Carm<strong>en</strong> Balebona)<br />
1
Abstract<br />
The pot<strong>en</strong>tial effect of extracellu<strong>la</strong>r polysacchari<strong>de</strong> fraction of the red microalga<br />
Porphyridium cru<strong>en</strong>tum on respiratory burst activity of sole phagocytes was evaluated.<br />
In vitro assays were carried out and no immunostimu<strong>la</strong>tory effects were obtained. In in<br />
vivo assays, fish were intraperitoneally injected with 500 μg of polysacchari<strong>de</strong> fraction<br />
and sampled after 24 h and 7 days. These fish were divi<strong>de</strong>d in two groups, immunized<br />
and not immunized with a bacterin against Photobacterium damse<strong>la</strong>e subsp. piscicida.<br />
The results obtained from in vivo assays did not show increase on respiratory burst, on<br />
the contrary, the only significant differ<strong>en</strong>ces were obtained from sole inocu<strong>la</strong>ted with<br />
polysaccharidic fraction or with bacterin, both of them showed a <strong>de</strong>crease on respiratory<br />
burst, 24 h post-inocu<strong>la</strong>tion.<br />
Keywords: Porphyridium cru<strong>en</strong>tum; Photobacterium damse<strong>la</strong>e subsp. piscicida;<br />
Polysacchari<strong>de</strong>s; Immunostimu<strong>la</strong>nts; Respiratory burst; Phagocytes; Sole (Solea<br />
s<strong>en</strong>egal<strong>en</strong>sis, Kaup 1858).<br />
2
1. Introduction<br />
Solea s<strong>en</strong>egal<strong>en</strong>sis (Kaup, 1858) is one of the new candidate species for<br />
aquaculture in the Portuguese and Spanish coasts [1]. One of the main limiting factors<br />
in its production is pseudotuberculosis, due to the bacterial pathog<strong>en</strong> Photobacterium<br />
damse<strong>la</strong>e subsp. piscicida [2-4]. There are several means of protection against this<br />
microorganism, including antibiotherapy, although the use of antibiotics leads to the<br />
appearance of resistant strains [5]. Vaccination is another option which has be<strong>en</strong><br />
<strong>de</strong>monstrated effective protection against pseudotuberculosis in sole [4].<br />
Since immunostimu<strong>la</strong>nts mainly facilitate the function of phagocytic cells and<br />
increase their bactericidal activities, it may be possible that immunostimu<strong>la</strong>nts<br />
administration to fish increase the respiratory burst activity of the phagocytes and<br />
<strong>en</strong>hance the protection against pseudotuberculosis. Several authors have reported that P.<br />
damse<strong>la</strong>e subsp. piscicida has the ability to survive as an intracellu<strong>la</strong>r pathog<strong>en</strong> [6-10].<br />
The search for new immunostimu<strong>la</strong>nt ag<strong>en</strong>ts is mainly due to the great <strong>de</strong>velopm<strong>en</strong>t of<br />
fish farming and the stress situations and diseases, which arise from int<strong>en</strong>sive culture<br />
[11].<br />
Several polysacchari<strong>de</strong>s, such as chitin, chitosan and β-glucans obtained from<br />
crustaceans and yeasts have be<strong>en</strong> used in fish and shellfish as immunostimu<strong>la</strong>nts [11-<br />
18]. Due to their cont<strong>en</strong>t in polysacchari<strong>de</strong>s, algae could be an alternative source to<br />
obtain polysacchari<strong>de</strong>s easily. Polysacchari<strong>de</strong>s from Spirulina p<strong>la</strong>t<strong>en</strong>sis and Chlorel<strong>la</strong><br />
have be<strong>en</strong> <strong>de</strong>monstrated their immunomodu<strong>la</strong>ting capacity in mammalian mo<strong>de</strong>ls [19-<br />
21]. However, few studies have be<strong>en</strong> focused on the immunostimu<strong>la</strong>nt properties of<br />
algal polysacchari<strong>de</strong>s in farmed fish [22, 23, 24]. The red microalga Porphyridium<br />
cru<strong>en</strong>tum excrete a sulphurized polysacchari<strong>de</strong>, commercially used in the industry as<br />
thick<strong>en</strong>er, stabilizer and emulsifier for its gelling properties [25-28]. This<br />
polysacchari<strong>de</strong> is an acidic heteropolymer composed of xylose, glucose, ga<strong>la</strong>ctose and<br />
sulphate esters [29]. In a previous study, Morris et al. [30] suggested a possible<br />
stimu<strong>la</strong>tion of the metabolic and functional action of phagocytic cells of Balb/c mice<br />
after an intraperitoneal administration of polysacchari<strong>de</strong>s iso<strong>la</strong>ted from cultures of P.<br />
cru<strong>en</strong>tum in stationary phase.<br />
3
The aim of this work is to evaluate the immunomodu<strong>la</strong>tory capacity of the<br />
extracellu<strong>la</strong>r polysaccharidic fraction iso<strong>la</strong>ted from P. cru<strong>en</strong>tum on respiratory burst<br />
activity of sole phagocytes against P. damse<strong>la</strong>e subsp. piscicida.<br />
2. Materials and Methods<br />
2.1. Microorganisms<br />
The virul<strong>en</strong>t strain of Photobacterium damse<strong>la</strong>e subsp. piscicida Lg h41/01 iso<strong>la</strong>ted<br />
from diseased sole [17] was selected to test the respiratory burst activity of phagocytes<br />
from S<strong>en</strong>egalese sole treated with and without polysaccharidic fraction of P. cru<strong>en</strong>tum.<br />
The bacterial strain was cultured on tryptic soy agar (Oxoid) supplem<strong>en</strong>ted with 1.5%<br />
NaCl (TSAs) for 24 h at 22 ºC. Bacterial susp<strong>en</strong>sions were obtained from one colony of<br />
the previous culture on TSAs, inocu<strong>la</strong>ted in culture in tryptic soy broth (Oxoid) ad<strong>de</strong>d<br />
with 1.5% NaCl (TSBs) and incubated at 22ºC for 24 h. Th<strong>en</strong>, the cultures were<br />
c<strong>en</strong>trifuged at 2000 xg for 20 min at 4ºC, and pellets resusp<strong>en</strong><strong>de</strong>d in L-15 medium at an<br />
optical <strong>de</strong>nsity (600 nm) equal to 1(10 8 cells ml -1 ).<br />
2.2. Iso<strong>la</strong>tion of the extracellu<strong>la</strong>r polysaccharidic fraction from Porphyridium cru<strong>en</strong>tum<br />
The red microalga P. cru<strong>en</strong>tum (S.F. Gray) Nägeli obtained from the collection<br />
of C<strong>en</strong>tro <strong>de</strong> Investigaciones Marinas <strong>de</strong> Cádiz, Cádiz, Spain was grown in<br />
Porphyridium medium [31] in batch culture at 25 ºC, with 12 h photoperiod for 7 days.<br />
Th<strong>en</strong>, the algal culture was c<strong>en</strong>trifuged at 10000 xg for 10 min.<br />
The polysaccharidic fraction was obtained by selective precipitation of the<br />
exocellu<strong>la</strong>r polysacchari<strong>de</strong>s from the culture supernatant with N-cetylpyridinium<br />
bromi<strong>de</strong> (Cetavlon) 2% (p/v), following the method <strong>de</strong>scribed by Morris et al. [30]. The<br />
pellet was redissolved with 4 M NaCl, and the polysacchari<strong>de</strong> was floccu<strong>la</strong>ted again<br />
with ethanol (96%), c<strong>en</strong>trifuged (10000 xg, 10 min), dialyzed against 2M NaCl and<br />
finally lyophilized. Dilutions from the lyophilised extract were prepared in HBSS to<br />
achieve conc<strong>en</strong>trations of 10 mg of lyophilised per ml, 5 mg ml -1 , 2 mg ml -1 and 1 mg<br />
ml -1 .<br />
4
On the other hand, β-1,3-glucan from Eugl<strong>en</strong>a gracilis (BioChemika Fluka,<br />
Sigma) was used as a positive control. T<strong>en</strong> mg of this compound were dissolved<br />
according to the manufacturer instructions, and diluted in HBSS to achieve<br />
conc<strong>en</strong>trations of 10 mg ml -1 , 5 mg ml -1 , 2 mg ml -1 and 1 mg ml -1 .<br />
2.3. Inocu<strong>la</strong>tion with the polysaccharidic fraction and immunization assay<br />
Specim<strong>en</strong>s of S<strong>en</strong>egalese sole of 50 g mean weight were randomly separated into<br />
groups of 20 fish each, and stocked into six 2500 l tanks with recircu<strong>la</strong>ting, aerated<br />
seawater at 22 ºC, 35‰ salinity and fed daily with a commercial pellet diet (Skreeting,<br />
Skreeting, Trouw España, Nutreco, Burgos, Spain).<br />
Two groups of 20 fish were intraperitoneally injected with a dose of 500 μg of the<br />
extracellu<strong>la</strong>r polysaccharidic fraction from P. cru<strong>en</strong>tum per fish. Phagocytes from two<br />
groups of sole were sampled at 2 and 8 days from algal extract administration. One<br />
group of them was intraperitoneally inocu<strong>la</strong>ted with bacterin of P. damse<strong>la</strong>e subsp.<br />
piscicida, 24 h after the administration by intraperitoneal injection of the<br />
polyssachari<strong>de</strong>. The formalin-killed aqueous vaccine was prepared according to the<br />
following <strong>de</strong>scription. Briefly, the selected strain of P. damse<strong>la</strong>e subsp. piscicida<br />
iso<strong>la</strong>ted from diseased sole in Spain was cultured on TSAs, and one colony was<br />
transferred to one tube containing 5 ml of TSBs for 18 h at 22 ºC. After incubation at 22<br />
ºC for 18 h, an aliquot of the culture, 50 μl, was inocu<strong>la</strong>ted in a f<strong><strong>la</strong>s</strong>k containing 50 ml<br />
TSBs and incubated at 22 ºC for 18 h with continuous shaking. Wh<strong>en</strong> the culture<br />
achieved O.D.600 of 1.2, corresponding to 6 x 10 8 bacteria ml -1 , the cells were killed by<br />
addition of formal<strong>de</strong>hy<strong>de</strong> (1% final conc<strong>en</strong>tration) and incubated overnight. Sterility<br />
trials were performed by spreading an aliquot of the vaccine preparation on TSAs p<strong>la</strong>tes<br />
and incubating for 2 days at 22 ºC. The vaccine was administered by intraperitoneal<br />
injection (0.1 ml per fish). The other two groups of fish were used as the controls and<br />
phosphate buffer saline (PBS, pH 7.2) was inocu<strong>la</strong>ted instead of the algal<br />
polysacchari<strong>de</strong>. One of these groups was vaccinated with a bacterin against P. damse<strong>la</strong>e<br />
subsp. piscicida as <strong>de</strong>scribed above.<br />
5
2.4. Iso<strong>la</strong>tion of head kidney phagocytes<br />
Five specim<strong>en</strong>s of soles of 200 g mean weight were sacrificed by an overdose of<br />
c<strong>la</strong>ve oil and head kidney phagocytes to test respiratory burst activity were iso<strong>la</strong>ted<br />
following the technique <strong>de</strong>scribed by Secombes [32]. Briefly, the kidney was removed<br />
aseptically and pushed through a 100 μm nylon mesh with Leibovitz medium (L-15)<br />
containing 2% foetal bovine serum (FBS, Sigma), 1% p<strong>en</strong>icillin-streptomycin (Sigma),<br />
0.1% (5 mg ml -1 ) g<strong>en</strong>tamicine sulfate (Sigma) (P/S/G) and 10 U heparine ml -1 . This cell<br />
susp<strong>en</strong>sion was <strong>la</strong>yered on a 30 to 51% Percoll (Amersham) gradi<strong>en</strong>t and c<strong>en</strong>trifuged at<br />
600 xg for 30 min, without brake. Th<strong>en</strong>, the bands separated at the interface were<br />
collected, c<strong>en</strong>trifuged for 15 min at 500 xg and resusp<strong>en</strong><strong>de</strong>d in L-15 medium<br />
supplem<strong>en</strong>ted with P/S/G. The viable cell conc<strong>en</strong>tration was <strong>de</strong>termined after staining<br />
with trypan blue and microscope counting. Aliquots of 100 μl containing 1x10 7 cells ml -<br />
1 in L-15 medium supplem<strong>en</strong>ted with P/S/G were ad<strong>de</strong>d to 96-well microtitre p<strong>la</strong>tes.<br />
After 3 h incubation at 22ºC, non-adher<strong>en</strong>t cells were removed and medium was<br />
substituted by L-15 and P/S/G supplem<strong>en</strong>ted with 2%FBS. Mono<strong>la</strong>yers were incubated<br />
overnight at 22ºC, before use.<br />
2.5. Respiratory burst activity<br />
The g<strong>en</strong>eration of intracellu<strong>la</strong>r superoxi<strong>de</strong> radicals by sole phagocytes, in<br />
response to in vitro contact with algal polysacchari<strong>de</strong>, was <strong>de</strong>termined by the reduction<br />
of nitro-blue tetrazolium (NBT) according to the technique <strong>de</strong>scribed by Secombes [32]<br />
and Boes<strong>en</strong> et al. [33]. Volumes of 20 μl containing 1, 2, 5 and 10 mg ml -1 of the<br />
lyophilized polysaccharidic fraction from P. cru<strong>en</strong>tum were ad<strong>de</strong>d to the wells<br />
containing phagocyte mono<strong>la</strong>yers obtained as <strong>de</strong>scribed above. The response of sole<br />
phagocytes to β-glucan from Eugl<strong>en</strong>a gracilis was also evaluated and 20 μl of serial<br />
dilutions containing 1, 2, 5 and 10 mg ml -1 to 12 wells with sole phagocyte mono<strong>la</strong>yers.<br />
Response of sole phagocytes to the infection with P. damse<strong>la</strong>e subsp. piscicida was<br />
<strong>de</strong>termined after inocu<strong>la</strong>tion of algal polysacchari<strong>de</strong> or β-glucan treated mono<strong>la</strong>yers<br />
with 20μl of bacterial susp<strong>en</strong>sions containing 10 8 bacteria ml -1 . Phorbol myristate<br />
acetate (PMA, Sigma) (1 μg ml -1 ) was used as a positive control to stimu<strong>la</strong>te the<br />
respiratory burst activity of sole phagocytes (data not shown). The specifity of the<br />
6
eaction was tested by adding superoxi<strong>de</strong> dismutase (SOD) (300 I.U. per well) to some<br />
wells containing PMA-stimu<strong>la</strong>ted phagocytes (data not shown).<br />
The effect of inocu<strong>la</strong>tion of polysaccharidic fraction to unvaccinated and<br />
vaccinated soles against P. damse<strong>la</strong>e subsp. piscicida on the production of superoxi<strong>de</strong><br />
anions by sole phagocytes was also <strong>de</strong>termined. In this case, phagocytes from control<br />
fish, fish inocu<strong>la</strong>ted with algal polysacchari<strong>de</strong>, and fish inocu<strong>la</strong>ted first with the algal<br />
polysacchari<strong>de</strong> and with the bacterin were iso<strong>la</strong>ted and mono<strong>la</strong>yers prepared. In all the<br />
cases, NBT (100 μl) dissolved at 1 mg ml -1 in HBSS was ad<strong>de</strong>d to the wells and<br />
phagocytes incubated at 22 ºC for 30 min. After incubation, cells were fixed in 70%<br />
methanol and the reduced formazan within phagocytes was solubilised by adding 120 μl<br />
2M KOH and 140 μl dimethyl sulfoxi<strong>de</strong> (DMSO, Sigma). Finally, absorbance was read<br />
at 630 nm in a multiscan spectrophotometer (UV-1601 Spectrophotometer, Whitakker<br />
Bioproducts).<br />
2.6. Statistical analysis<br />
Results are expressed as phagocyte stimu<strong>la</strong>tion in<strong>de</strong>x, calcu<strong>la</strong>ted by dividing each<br />
sample value by the mean control value. Values above 1 reflect an increase and un<strong>de</strong>r 1<br />
a <strong>de</strong>crease in each parameter. Data were statistically analysed by one-way analysis of<br />
variance (ANOVA) and Tukey’s comparison of means using SPSS for Windows.<br />
Differ<strong>en</strong>ces were consi<strong>de</strong>red statistically significant wh<strong>en</strong> P< 0.05.<br />
3. Results<br />
Results obtained in vitro after incubation of phagocytes with algal extracts are<br />
shown in Figure 1a. The data are expressed as a phagocytic stimu<strong>la</strong>tion in<strong>de</strong>x obtained<br />
by dividing superoxi<strong>de</strong> anion produced by phagocytes incubated with algal<br />
polysacchari<strong>de</strong> or with algal polysacchari<strong>de</strong> plus bacteria by the values of phagocytes<br />
incubated without the algal polysaccharidic fraction. Data obtained after incubation with<br />
β-glucan instead of algal polysacchari<strong>de</strong> are expressed in Figure 1b. Results obtained<br />
show that only at the commercial β-glucan significantly <strong>en</strong>hanced the respiratory burst<br />
activity of sole phagocytes at the highest conc<strong>en</strong>tration (10 mg ml -1 ), compared to non<br />
7
treated and algal extract treated phagocytes. Thus, the polysaccharidic fraction from P.<br />
cru<strong>en</strong>tum did not show ability to increase the respiratory burst activity of sole<br />
phagocytes, neither alone or in the pres<strong>en</strong>ce of P. damse<strong>la</strong>e subsp. piscicida, being<br />
stimu<strong>la</strong>tion in<strong>de</strong>x obtained 1.042 ± 0.069.<br />
Superoxi<strong>de</strong> anion production of phagocytes iso<strong>la</strong>ted from sole inocu<strong>la</strong>ted with<br />
P. cru<strong>en</strong>tum polysacchari<strong>de</strong> and P. damse<strong>la</strong>e subsp. piscicida bacterin are shown in<br />
Figure 2. Inocu<strong>la</strong>tion of the polysacchari<strong>de</strong> or the bacterin alone resulted in a significant<br />
<strong>de</strong>crease (P
Intraperitoneal inocu<strong>la</strong>tion of the polysaccharidic fraction of P. cru<strong>en</strong>tum to<br />
sole specim<strong>en</strong>s did not induce an increase of the respiratory burst activity. On the<br />
contrary, the respiratory burst activity <strong>de</strong>creased in fish inocu<strong>la</strong>ted with polysacchari<strong>de</strong><br />
fraction or with bacterin within 24 h post-inocu<strong>la</strong>tion. This <strong>de</strong>crease in respiratory burst<br />
activity may be due to a immunosupression caused by stress after handling [39, 40] as it<br />
is not observed after 7 days of the inocu<strong>la</strong>tion.<br />
In short, the results obtained in vivo indicate that the polysaccharidic fraction<br />
from P. cru<strong>en</strong>tum does not <strong>en</strong>hance the respiratory burst activity compared to non<br />
inocu<strong>la</strong>ted fish.<br />
Acknowledgem<strong>en</strong>ts<br />
Financial support was provi<strong>de</strong>d by the Ministry of Education and Sci<strong>en</strong>ce Spain<br />
(AGL2002-01488 and AGL 2005-02655) and Junta <strong>de</strong> Andalucía (RNM-295). The<br />
authors are grateful to Au<strong>la</strong> <strong>de</strong>l Mar of Má<strong>la</strong>ga (Spain) and PROMAN (Motril, Granada,<br />
Spain) for their help and participation in this study. P. Díaz-Rosales thanks Ministerio<br />
Español <strong>de</strong> Educación y Ci<strong>en</strong>cia for a F.P.U. scho<strong>la</strong>rship.<br />
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[30] Morris, HJ, Martínez, CE, Abda<strong>la</strong>, RT and Cobas, G. Evi<strong>de</strong>ncias preliminares <strong>de</strong> <strong>la</strong><br />
actividad inmunomodu<strong>la</strong>dora <strong>de</strong> <strong>la</strong> fracción polisacarídica <strong>de</strong> orig<strong>en</strong> marino PC-1.<br />
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Microalgal Biotechnology, Cambridge University Press; 1988, p. 122-35.<br />
[32] Secombes, CJ. Iso<strong>la</strong>tion of salmonid macrophages and analysis of their killing<br />
activity. In: Stol<strong>en</strong> JS, Fletcher DP, An<strong>de</strong>rson BS, Roberson, van Muiswinkel<br />
WB, editors. Techniques in Fish Immunology, Fair Hav<strong>en</strong>, NJ, SOS Publication;<br />
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[33] Boes<strong>en</strong>, HT, Lars<strong>en</strong>, MH, Lars<strong>en</strong>, LH and Ellis, AE. In vitro interactions betwe<strong>en</strong><br />
rainbow trout (Oncorhynchus mykiss) macrophages and Vibrio anguil<strong>la</strong>rum<br />
serogroup O2a. Fish & Shellfish Immunology 2001; 11: 415-31.<br />
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[35] Hou, WY and Ch<strong>en</strong>, JC. The immunostimu<strong>la</strong>tory effect of hot-water extract of<br />
Graci<strong>la</strong>ria t<strong>en</strong>uistipitata on the white shrimp Litop<strong>en</strong>aeus vannamei and its<br />
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127-38.<br />
[36] Santarém, M, Novoa, B and Figueras, A. Effects of β-glucans on the non-specific<br />
immune responses of turbot (Scophthalmus maximus L.). Fish & Shellfish<br />
Immunology 1997; 7: 429-37.<br />
[37] Cook, MT, Hayball, P, Hutchinson, W, Nowak, BF and Hayball, JD.<br />
Administration of a commercial immunostimu<strong>la</strong>nt preparation, EcoActiva, as<br />
feed supplem<strong>en</strong>t <strong>en</strong>hances macrophage respiratory burst and the growth rate of<br />
snapper (Pagrus auratus, Sparidae (Bloch and Schnei<strong>de</strong>r)) in winter. Fish &<br />
Shellfish, 2003; 14: 333-45.<br />
[38] Couso, N, Castro, R, Magariños, B, Obach, A and Lamas, J. Effect of oral<br />
administration of glucans on the resistance of gilthead seabream to pasteurellosis.<br />
Aquaculture 2003; 219: 99-109.<br />
[39] Thompson, I, White, A, Fletcher, TC, Houlihan, DF and Secombes, CJ. The effect<br />
of stress on the immune response of At<strong>la</strong>ntic salmon (Salmo sa<strong>la</strong>r L.) fed diets<br />
containing differ<strong>en</strong>t amounts of vitamin C. Aquaculture 1993; 114: 1-18.<br />
[40] Pulsford, AL, Crampe, M, Langston, A and Glynn, PJ. Modu<strong>la</strong>tory effects of<br />
disease, stress, copper, TBT and vitamin E on the immune system of f<strong>la</strong>tfish. Fish<br />
& Shellfish Immunology 1995; 5: 631-43.<br />
13
Figure leg<strong>en</strong>ds<br />
Figure 1. Respiratory burst activity of sole phagocytes incubated with<br />
polysaccharidic fraction from Porphyridium cru<strong>en</strong>tum (a) or β-glucan (b) (1, 2, 5, 10<br />
mg ml -1 ) in abs<strong>en</strong>ce ( ) or pres<strong>en</strong>ce ( ) of Photobacterium damse<strong>la</strong>e subsp. piscicida<br />
(2x10 6 cells per well). Results are expressed as stimu<strong>la</strong>tion in<strong>de</strong>x (mean ± SE; n=15)<br />
obtained by dividing each sample value by its mean control value (HBSS or P.<br />
damse<strong>la</strong>e subsp. piscicida cells). Symbol * <strong>de</strong>notes statistically significant differ<strong>en</strong>ces<br />
(P
Figure 1a<br />
Stimu<strong>la</strong>tion in<strong>de</strong>x<br />
3<br />
2.5<br />
2<br />
1. 5<br />
1<br />
0.5<br />
0<br />
1 2 5 10 mg ml -1<br />
17
Figure 1b<br />
Stimu<strong>la</strong>tion in<strong>de</strong>x<br />
3<br />
2.5<br />
2<br />
1.5<br />
1<br />
0.5<br />
*<br />
*<br />
0<br />
1 2 5 10 mg ml -1<br />
19
Figure 2<br />
Stimu<strong>la</strong>tion in<strong>de</strong>x<br />
1.4<br />
1.2<br />
1.0<br />
0.8<br />
0.6<br />
0.4<br />
0.2<br />
0.0<br />
*<br />
P-24<br />
*<br />
P-7 B-24 B-7 PB-24 PB-7<br />
Treatm<strong>en</strong>ts<br />
21
A RTÍCULO 2.3.<br />
A RTICLE 2.3.
Effect of dietary administration of probiotics on respiratory burst activity of<br />
phagocytes and intestinal microbiota of S<strong>en</strong>egalese sole (Solea s<strong>en</strong>egal<strong>en</strong>sis, Kaup<br />
1858)<br />
P.Díaz-Rosales a , M. Chabrillón a , H. Smidt b , S. Arijo a , Juan M. León-Rubio a , Rosa M.<br />
Rico a , F. Javier A<strong>la</strong>rcón c , M. Ángel Sá<strong>en</strong>z <strong>de</strong> Rodrigáñez c , M.Carm<strong>en</strong> Balebona a and<br />
M.Ángel Moriñigo a<br />
a Departm<strong>en</strong>t of Microbiology, Faculty of Sci<strong>en</strong>ces, University of Má<strong>la</strong>ga. 29071<br />
Má<strong>la</strong>ga. Spain.<br />
b Laboratory of Microbiology, Agrotechnology and Food Sci<strong>en</strong>ces Group, Wag<strong>en</strong>ing<strong>en</strong><br />
University. 6703 CT Wag<strong>en</strong>ing<strong>en</strong>. The Nether<strong>la</strong>nds.<br />
c Departm<strong>en</strong>t of Applied Biology, University of Almería. 04120 Almería. Spain.<br />
* Corresponding author. Phone: +34 952 131 862; fax: +34 952 131 889<br />
E-mail address: morinigo@uma.es (M. Ángel Moriñigo)<br />
1
Abstract<br />
The effects of the dietary administration of two bacterial probiotic strains from<br />
the Alteromonadaceae family, Pdp11 and Pdp13, on S<strong>en</strong>egalese sole intestinal<br />
microbiota were studied. The fish were fed four differ<strong>en</strong>t diets: control (nonsupplem<strong>en</strong>ted),<br />
or diets supplem<strong>en</strong>ted with alginate alone, or as a carrier for Pdp11 (10 9<br />
cfu g -1 ) or Pdp13 (10 9 cfu g -1 ) for sixty days. The effect of the differ<strong>en</strong>t dietary<br />
treatm<strong>en</strong>ts was assessed by measuring respiratory burst activity, protection against<br />
experim<strong>en</strong>tal infection with Photobacterium damse<strong>la</strong>e subsp. piscicida and analysis of<br />
intestinal microbiota by 16S ribosomal RNA g<strong>en</strong>e-targeted PCR-DGGE (<strong>de</strong>naturing<br />
gradi<strong>en</strong>t gel electrophoresis). We also evaluated, to which ext<strong>en</strong>d and how this<br />
technique could be effectively applied to examine bacterial diversity and dynamics of<br />
the sole (Solea s<strong>en</strong>egal<strong>en</strong>sis) intestinal microbiota, as influ<strong>en</strong>ced by the diet. Bacterial<br />
ribotypic diversity was <strong>de</strong>termined using DGGE analysis of the V6 to V8 and V3<br />
regions of 16S rRNA g<strong>en</strong>es using two differ<strong>en</strong>t sets of primers for PCR amplification.<br />
To our knowledge, this is the first report re<strong>la</strong>ted to the effect of probiotics in the<br />
intestinal microbiota of sole, by cultivation-in<strong>de</strong>p<strong>en</strong><strong>de</strong>nt molecu<strong>la</strong>r ecological<br />
approaches.<br />
Keywords: Probiotics; Alteromonadaceae; Intestinal microbiota; DGGE; Sole (Solea<br />
s<strong>en</strong>egal<strong>en</strong>sis, Kaup 1858); Teleosts.<br />
2
1. Introduction<br />
S<strong>en</strong>egalese sole culture (Solea s<strong>en</strong>egal<strong>en</strong>sis, Kaup 1858) is nowadays a very<br />
promising industry in the Mediterranean countries [1]. One of the most serious<br />
problems concerning sole production is the exist<strong>en</strong>ce of infectious diseases,<br />
pseudotuberculosis caused by Photobacterium damse<strong>la</strong>e subsp. piscicida, being the<br />
main limiting factor [2].<br />
In fish farms, control of this bacterial pathog<strong>en</strong> is achieved by the<br />
administration of chemotherapeutic ag<strong>en</strong>ts. However, over the <strong><strong>la</strong>s</strong>t <strong>de</strong>ca<strong>de</strong>, drugresistant<br />
strains carrying a transferable R-p<strong><strong>la</strong>s</strong>mid have evolved, making treatm<strong>en</strong>t with<br />
antimicrobial chemotherapeutics less successful [3]. Therefore, immunoprophy<strong>la</strong>xis has<br />
become the best way to prev<strong>en</strong>t pseudotuberculosis [4].<br />
Throughout the <strong><strong>la</strong>s</strong>t 20 years, there has be<strong>en</strong> a variety of studies analyzing the<br />
effectiv<strong>en</strong>ess of immunization in prev<strong>en</strong>ting pseudotuberculosis [5]. A dival<strong>en</strong>t vaccine<br />
against V. harveyi and P. damse<strong>la</strong>e subsp. piscicida for sole has be<strong>en</strong> reported [6]. This,<br />
however, only provi<strong>de</strong>s protection for a short period. Therefore, searching for new<br />
prophy<strong>la</strong>ctic methods, such as immunostimu<strong>la</strong>nts or probiotics seems a very promising<br />
alternative.<br />
Probiotics have be<strong>en</strong> <strong>de</strong>fined as live microbial preparations that improve the<br />
health and well-being of the host [7-10]. The research on probiotics for aquatic animals<br />
is increasing with the <strong>de</strong>mand for <strong>en</strong>vironm<strong>en</strong>t-fri<strong>en</strong>dly aquaculture. Most of these<br />
studies have be<strong>en</strong> re<strong>la</strong>ted to chall<strong>en</strong>ge trials and suppression of pathog<strong>en</strong> growth by<br />
probiotic bacteria [11-21]. Rec<strong>en</strong>t works have be<strong>en</strong> focused on the immunological<br />
<strong>en</strong>hancem<strong>en</strong>t of fish <strong>de</strong>f<strong>en</strong>ce mechanisms by probiotic bacteria [22-27] and the effects<br />
that dietary administration of probiotics could exert on the intestinal microbiota of the<br />
host fish [28].<br />
As immunomodu<strong>la</strong>tors, probiotics may <strong>en</strong>hance phagocytic activity and increase<br />
the production of reactive oxyg<strong>en</strong> species by macrophages from species such as gilthead<br />
seabream [27, 29] and rainbow trout (Onchorynchus mykiss) [22]. The means of<br />
invasion and survival of P. damse<strong>la</strong>e subsp. piscicida insi<strong>de</strong> the host are still unknown,<br />
and while authors have reported the pres<strong>en</strong>ce of intact bacteria insi<strong>de</strong> fish cells,<br />
3
suggesting the ability of the bacterium to survive as an intracellu<strong>la</strong>r pathog<strong>en</strong> [30-33],<br />
others have observed that this pathog<strong>en</strong> is highly susceptible to oxidative radicals<br />
g<strong>en</strong>erated during the macrophage respiratory burst [34, 35]. For this reason, the<br />
stimu<strong>la</strong>tion of the respiratory burst activity of S<strong>en</strong>egalese sole phagocytes could<br />
facilitate a more effective <strong>de</strong>struction of P. damse<strong>la</strong>e subsp. piscicida.<br />
H<strong>en</strong>ce, the first objective of the pres<strong>en</strong>t study was to evaluate the possible<br />
immunostimu<strong>la</strong>tory effect of dietary administration of two probiotics [36-38] on<br />
respiratory burst activity of sole phagocytes. In addition, possible changes in the<br />
intestinal microbiota were evaluated by <strong>de</strong>termining the g<strong>en</strong>etic diversity of gut<br />
microbiota by DGGE (<strong>de</strong>naturing gradi<strong>en</strong>t gel electrophoresis) [39]. Several authors<br />
have reported on the composition of intestinal microbiota of differ<strong>en</strong>t farmed fish [40-<br />
44], although none of them have studied sole microbiota. On the other hand, the<br />
possible effect of probiotics on fish intestinal microbiota is still <strong>la</strong>rgely unknown [28].<br />
Thus, this is the first report on sole gut microbiota and the possible changes in response<br />
to probiotic treatm<strong>en</strong>t.<br />
2. Materials and Methods<br />
2.1. Microorganisms<br />
Two bacteria strains iso<strong>la</strong>ted from gilthead seabream skin, Pdp11 and Pdp13,<br />
belonging to Alteromonadaceae family, Shewanel<strong>la</strong> g<strong>en</strong>us, were selected for this study<br />
because their in vitro characteristics suggested they could be consi<strong>de</strong>red as pot<strong>en</strong>tial fish<br />
probiotics [36-38].<br />
These strains were grown in tubes containing 5 ml of trypticase soy broth (Oxoid<br />
Ltd., Basingstoke, UK) supplem<strong>en</strong>ted with 1.5 % NaCl (TSBs) for 18 h at 22 ºC, with<br />
continuous shaking. Appropriate dilutions of the culture were spread onto p<strong>la</strong>tes of<br />
trypticase soy agar (Oxoid) supplem<strong>en</strong>ted with 1.5 % NaCl (TSAs). The number of<br />
culturable bacteria was <strong>de</strong>termined by p<strong>la</strong>te counting on TSAs. Bacteria were recovered<br />
from the p<strong>la</strong>tes, lyophilized and the conc<strong>en</strong>tration of lyophilized bacteria was adjusted<br />
per gram of pellet diet.<br />
4
2.2. Fish and experim<strong>en</strong>tal <strong>de</strong>sign<br />
S<strong>en</strong>egalese sole specim<strong>en</strong>s (Solea s<strong>en</strong>egal<strong>en</strong>sis, Kaup, 1838) of 15-30 g weight,<br />
were kept in four 300 l seawater tanks (75 fish per tank), 36 ‰ salinity, at 22 ºC and fed<br />
with a commercial pellet diet (Gemma 0.5, Skreeting, Trouw España, Nutreco, Burgos,<br />
Spain). Specim<strong>en</strong>s were sacrificed by overdose of c<strong>la</strong>ve oil.<br />
Experim<strong>en</strong>tal diets were prepared in the <strong>la</strong>boratory from the commercial pellet<br />
diet. The diet preparation was carried out with alginate (0.5% kg -1 ) and 50 mM calcium<br />
chlori<strong>de</strong> (0.4% kg -1 ) to facilitate the lyophilized bacteria incorporation to the pellet.<br />
Alginate and Ca Cl 2 were sprayed into the feed slowly, mixing with the required amount<br />
of lyophilized bacteria (10 9 cfu g -1 ).<br />
Thus, fish in each tank received one of four differ<strong>en</strong>t diets: a commercial diet<br />
supplem<strong>en</strong>ted with Pdp11 (10 9 cfu g -1 ) (diet A); the same diet supplem<strong>en</strong>ted with Pdp13<br />
(10 9 cfu g -1 ) (diet B); a diet consisting of the commercial diet (control without alginate)<br />
(diet C); and, finally, the fourth group of fish received a diet supplem<strong>en</strong>ted with alginate<br />
and calcium chlori<strong>de</strong> (control with alginate) (diet D). Fish were fed at a rate of 15 g dry<br />
diet Kg -1 biomass (1.5 %) per day for 60 days. The biomass of the fish in each aquarium<br />
was measured at the beginning of the experim<strong>en</strong>t and daily ratio was adjusted<br />
accordingly. No mortality was observed during the experim<strong>en</strong>t.<br />
Sampling was carried out at the <strong>en</strong>d of the feeding trial, day 60, for the chall<strong>en</strong>ge<br />
test and study of intestinal microbiota. Samples for measurem<strong>en</strong>t of the respiratory burst<br />
activity were tak<strong>en</strong> at the middle of the trial and at the <strong>en</strong>d, at day 30 and 60,<br />
respectively.<br />
2.3. Iso<strong>la</strong>tion of head kidney phagocytes<br />
The influ<strong>en</strong>ce of the treatm<strong>en</strong>t on respiratory burst activity was tested in<br />
phagocytes iso<strong>la</strong>ted from the kidney of soles following the technique <strong>de</strong>scribed by<br />
Secombes [45]. Briefly, the kidney was removed aseptically and pushed through a 100<br />
μm nylon mesh with Leibovitz medium (L-15) containing 2% foetal calf serum (FCS,<br />
Sigma), 1% p<strong>en</strong>icillin-streptomycin (Sigma), 0.1 % (5 mg ml -1 ) g<strong>en</strong>tamicine (Sigma)<br />
(P/S/G) and 10 U heparine ml -1 . This cell susp<strong>en</strong>sion was <strong>la</strong>yered on a 30 to 51%<br />
Percoll (Amersham) gradi<strong>en</strong>t and c<strong>en</strong>trifuged at 600 × g for 30 min. Th<strong>en</strong>, the bands<br />
5
separated at the interface were collected, c<strong>en</strong>trifuged for 15 min at 500 × g and<br />
resusp<strong>en</strong><strong>de</strong>d in L-15 medium supplem<strong>en</strong>ted with P/S/G. The viable cell conc<strong>en</strong>tration<br />
was <strong>de</strong>termined after staining with trypan blue and microscope counting. Aliquots of<br />
100 μl containing 1x10 7 cells ml -1 in L-15 medium supplem<strong>en</strong>ted with P/S/G were<br />
ad<strong>de</strong>d to 96-well microtitre p<strong>la</strong>tes. After 3 h incubation at 22 ºC, non-adher<strong>en</strong>t cells<br />
were removed and medium was substituted by L-15 and P/S/G supplem<strong>en</strong>ted with<br />
2%FCS. Mono<strong>la</strong>yers were incubated overnight at 22 ºC.<br />
2.4. Respiratory burst activity<br />
Fish of the four experim<strong>en</strong>tal groups, fed with diets A, B, C or D, were assayed<br />
for respiratory burst activity.<br />
The g<strong>en</strong>eration of intracellu<strong>la</strong>r superoxi<strong>de</strong> radicals by sole phagocytes was<br />
<strong>de</strong>termined by the reduction of nitro-blue tetrazolium (NBT) according to the technique<br />
<strong>de</strong>scribed by Secombes [45] and Boes<strong>en</strong> et al. [46]. Phagocyte mono<strong>la</strong>yers were washed<br />
with L-15 medium and HBSS (Hank´s Ba<strong>la</strong>nced Salt Solution) to remove any trace of<br />
antibiotic. Th<strong>en</strong>, NBT (100 μl) dissolved at 1 mg ml -1 in HBSS was ad<strong>de</strong>d to the wells<br />
and the phagocytes incubated at 22 ºC for 30 min. Wells containing phagocytes were<br />
infected with P. damse<strong>la</strong>e subsp. piscicida (10 8 bacterias ml -1 ) and used to <strong>de</strong>termine the<br />
response of the phagocytes to the fish pathog<strong>en</strong>. As a positive control phorbol myristate<br />
acetate (PMA, Sigma) (1 μg ml -1 ), an activating ag<strong>en</strong>t of the respiratory burst, was used<br />
to stimu<strong>la</strong>te the respiratory burst of non-infected phagocytes. The specificity of the<br />
reaction was tested by adding superoxi<strong>de</strong> dismutase (SOD) (300 I.U. per well) to some<br />
wells containing PMA-stimu<strong>la</strong>ted phagocytes (data not shown).<br />
After incubation, cells were fixed in 70% methanol and reduced formazan within<br />
phagocytes was solubilised by adding 120 μl 2M KOH and 140 μl dimethyl sulfoxi<strong>de</strong><br />
(DMSO, Sigma). Finally, absorbance was read at 630 nm in a multiscan<br />
spectrophotometer (UV-1601 Spectrophotometer, Whitakker Bioproducts).<br />
6
2.5. Chall<strong>en</strong>ge with Photobacterium damse<strong>la</strong>e subsp. piscicida<br />
Groups of 10 fish (15-30 g weight) were chall<strong>en</strong>ged at the <strong>en</strong>d of the feeding trial,<br />
after 60 days of beginning. For the chall<strong>en</strong>ge, three groups of fish were assayed, fed<br />
with diets A, B and C.<br />
Soles were intraperitoneally injected with a dose of P. damse<strong>la</strong>e subsp. piscicida<br />
(Lg h41/01 ) of 5 x 10 4 cfu g -1 from a bacterial susp<strong>en</strong>sion in PBS (phosphate buffer saline)<br />
adjusted to 5 x 10 8 cfu ml -1 . As control, the same number of fish was inocu<strong>la</strong>ted with 0.1<br />
ml PBS. Inocu<strong>la</strong>ted fish were followed daily for 10 days, and all mortalities were<br />
recor<strong>de</strong>d, consi<strong>de</strong>ring only those that could be linked to the bacterial chall<strong>en</strong>ge by reiso<strong>la</strong>tion<br />
in pure culture from internal organs of <strong>de</strong>ad fish.<br />
2.6. Analysis of the sole intestinal microbiota<br />
2.6.1. Sample collection<br />
Three groups of fish were analyzed for intestinal microbiota composition, i.e. fish<br />
fed with diets A, B and C.<br />
Per treatm<strong>en</strong>t group, three specim<strong>en</strong>s of S<strong>en</strong>egalese sole were sacrificed at the <strong>en</strong>d<br />
of the feeding trial (60 days), and whole intestines were collected and stored at -80 ºC<br />
until further analysis.<br />
2.6.2. DNA iso<strong>la</strong>tion from intestinal cont<strong>en</strong>t and probiotic cultures<br />
The gut cont<strong>en</strong>ts were homog<strong>en</strong>ized in 4 ml PBS (phosphate buffer saline) pH<br />
7.2, and a 1 ml aliquot was c<strong>en</strong>trifuged at 1000 × g for 5 min. The supernatant was pretreated<br />
by <strong>en</strong>zymatic digestion with 40 μl proteinase K (20 mg/ml) and 50 μl do<strong>de</strong>cyl<br />
sulphate (SDS) 10% and incubated at 65 ºC for 30 min. Subsequ<strong>en</strong>tly, DNA extraction<br />
from the susp<strong>en</strong>sion was performed with the Fast DNA Spin kit for soil (Qbiog<strong>en</strong>e, Inc.,<br />
Carlsbad, CA) according to manufacturer’s instructions. Agarose gel (1.5% [wt/vol])<br />
electrophoresis in the pres<strong>en</strong>ce of ethidium bromi<strong>de</strong> was used to check visually for<br />
DNA quality and yield.<br />
Pure cultures of probiotic strains were grown until expon<strong>en</strong>tial phase in TSBs,<br />
and th<strong>en</strong> c<strong>en</strong>trifuged at 2500 × g for 15 min. Pellets were washed with PBS and<br />
c<strong>en</strong>trifuged again. DNA was extracted from the resulting pellet resusp<strong>en</strong><strong>de</strong>d in 500 μl<br />
PBS with the Fast DNA Spin kit (Qbiog<strong>en</strong>e, Inc., Carlsbad, CA).<br />
7
2.6.3. PCR amplification<br />
Two differ<strong>en</strong>t sets of primers were tested in this study to select those that yiel<strong>de</strong>d<br />
the best results to compare DGGE patterns based on their resolution and observed<br />
diversity (Table 1). Primer set Bact-0968-GC-F / Bact-1401-R was used to amplify the<br />
V6 to V8 regions of the 16S rRNA g<strong>en</strong>e [47, 48] and PRBA-338-GC-F and PRUN-518-<br />
R amplified the V3 hypervariable region [44]. PCR was performed using the Taq DNA<br />
polymerase kit from Life Technologies (Gaithersburg, Md). PCR mixtures (50 μl)<br />
contained 0.5 μl Taq polymerase (1.25 U), 20 mM Tris-HCl (pH 8.5), 50 mM KCl, 3<br />
mM MgCl 2 , 200 μM of each <strong>de</strong>oxynucleosi<strong>de</strong> triphosphate, 5 pmol of the primers, 1 μl<br />
of DNA temp<strong>la</strong>te, and UV-sterilized water. The samples were amplified in a T1<br />
thermocycler (Whatman Biometra, Götting<strong>en</strong>, Germany), and the cycling conditions for<br />
each pair of primers are listed in Table 1. Aliquots (5 μl) were analyzed by<br />
electrophoresis on 1.5% (wt/vol) agarose gels containing ethidium bromi<strong>de</strong> to check for<br />
product size and quantity.<br />
2.6.4. DGGE analysis<br />
The amplicons obtained from the intestinal lum<strong>en</strong>-extracted DNA and the<br />
probiotic strains were separated by DGGE according to the specifications of Muyzer et<br />
al. [39] using a Dco<strong>de</strong> TM system (Bio-Rad Laboratories, Hercules, CA).<br />
Electrophoresis was performed in an 8% polyacry<strong>la</strong>mi<strong>de</strong> gel (37.5:1 acry<strong>la</strong>mi<strong>de</strong>bisacry<strong>la</strong>mi<strong>de</strong>;<br />
dim<strong>en</strong>sions, 200 by 200 by 1 mm) using a 30 to 55% <strong>de</strong>naturing gradi<strong>en</strong>t<br />
for separation of PCR products. The gels contained a 30 to 55% gradi<strong>en</strong>t of urea and<br />
formami<strong>de</strong> increasing in the direction of the electrophoresis. A 100% <strong>de</strong>naturing<br />
solution contained 7 M urea and 40% (vol/vol) <strong>de</strong>ionized formami<strong>de</strong>. PCR samples<br />
were applied to gels in aliquots of 13 μl per <strong>la</strong>ne. The gels were electrophoresed for 16<br />
h at 85 V in 0.5 X TAE (20 mM Tris acetate [pH 7.4], 10 mM sodium acetate, 0.5 mM<br />
Na 2 -EDTA) [50] buffer at a constant temperature of 60 ºC and subsequ<strong>en</strong>tly stained<br />
with AgNO 3 [51].<br />
Gel image processing and comparative analysis of banding patterns was done<br />
with Bionumerics version 4.0 (Applied Maths BVBA, Sint-Mart<strong>en</strong>s-Latem, Belgium).<br />
Simi<strong>la</strong>rity betwe<strong>en</strong> DGGE profiles was <strong>de</strong>termined by calcu<strong>la</strong>ting simi<strong>la</strong>rity indices of<br />
8
the <strong>de</strong>nsitometric curves of the profiles compared using the Pearson product-mom<strong>en</strong>t<br />
corre<strong>la</strong>tion [52, 53].<br />
Clustering of DGGE patterns was achieved by construction of <strong>de</strong>ndrograms using<br />
UPGMA (Unweighted Pair Groups Method using Arithmetic Averages).<br />
3. Results<br />
3.1. Respiratory burst activity<br />
Respiratory burst activity of phagocytes iso<strong>la</strong>ted from assayed soles was<br />
expressed as stimu<strong>la</strong>tion in<strong>de</strong>x, which was calcu<strong>la</strong>ted as the ratio betwe<strong>en</strong> the<br />
absorbance obtained from phagocytes from fish fed with the differ<strong>en</strong>t experim<strong>en</strong>tal diets<br />
and absorbance from fish fed with the control diet, without any supplem<strong>en</strong>tation.<br />
The respiratory burst activity of phagocytes was measured at days 30 and 60.<br />
Only the phagocytes from sole specim<strong>en</strong>s fed with the diet supplem<strong>en</strong>ted with probiotic<br />
strain Pdp11 showed a significant increase (P
3.3. Intestinal microbiota analysis<br />
In or<strong>de</strong>r to compare DGGE patterns of the intestinal microbiota of soles receiving<br />
the differ<strong>en</strong>t diets assayed, two sets of primers, targeting differ<strong>en</strong>t variable regions of<br />
the 16S rRNA g<strong>en</strong>e, were used to select the optimal set with respect to diversity and<br />
resolution. DGGE profiles were compared using Pearson’s simi<strong>la</strong>rity coeffici<strong>en</strong>t<br />
analysis, a test that consi<strong>de</strong>rs quantitative values of a <strong>de</strong>nsitometric curve, repres<strong>en</strong>ting<br />
bands and their int<strong>en</strong>sities.<br />
Profiles were compared within (Table 2) and betwe<strong>en</strong> experim<strong>en</strong>tal groups (Table<br />
3). The results obtained show that the V6-V8 targeting primers Bact-0968-GC-F and<br />
Bact-1401-R <strong>de</strong>tected higher simi<strong>la</strong>rity in DGGE patterns among fish from the same<br />
group (intragroup) in two of the three groups assayed, control group, without any<br />
supplem<strong>en</strong>tation, and fish fed with Pdp11 strain (Table 2). On the other hand, the same<br />
set of primers (Bact-0968-GC-F and Bact-1401-R) <strong>de</strong>tected lower perc<strong>en</strong>tages and more<br />
pronounced differ<strong>en</strong>ces with respect to intergroup simi<strong>la</strong>rities (Table 3). There is only<br />
one statistically significant differ<strong>en</strong>ce (P
most effici<strong>en</strong>t probiotics for aquaculture, as a consequ<strong>en</strong>ce of the specificity of aquatic<br />
microbiota [8]. H<strong>en</strong>ce, we assessed the probiotic pot<strong>en</strong>tial of bacteria of the g<strong>en</strong>us<br />
Shewanel<strong>la</strong>, naturally pres<strong>en</strong>t on seabream skin [36]. Moreover, the selection of the<br />
probiotic Pdp11 was based on its adhesion to intestinal mucus, its resistance to intestinal<br />
mucus and bile [37, 38] and its ability to increase several immunological parameters in<br />
gilthead seabream [27, 29].<br />
Despite of their affiliation within the same g<strong>en</strong>us, Shewanel<strong>la</strong>, and same origin of<br />
both microorganisms assayed in this study, their effect on the respiratory burst activity<br />
was completely differ<strong>en</strong>t, as only Pdp11 strain produced statistically significant increase<br />
in superoxi<strong>de</strong> anion production by sole kidney phagocytes. This could suggest that the<br />
immunostimu<strong>la</strong>tion of the respiratory burst of sole phagocytes is strain-<strong>de</strong>p<strong>en</strong><strong>de</strong>nt rather<br />
than a g<strong>en</strong>eral characteristic of a bacterial species. This strain-<strong>de</strong>p<strong>en</strong><strong>de</strong>nce has be<strong>en</strong><br />
<strong>de</strong>scribed for other characteristics such as the mucus adhesion of certain iso<strong>la</strong>tes of<br />
<strong>la</strong>ctic acid bacteria [56] and fish iso<strong>la</strong>tes [37, 38]. In a previous work it was reported<br />
that Pdp11 strain did not induce an increase on the respiratory burst activity of<br />
phagocytes from gilthead seabream wh<strong>en</strong> the microorganism was administered heatinactivated<br />
[27]. These results are in agreem<strong>en</strong>t with Panigrahi et al. [24], who indicated<br />
that probiotic viability could probably influ<strong>en</strong>ce the induced immune responses. It was<br />
found that viable <strong>la</strong>ctic acid bacteria were more effici<strong>en</strong>t in <strong>en</strong>hancing certain aspects of<br />
immune response in rainbow trout (Oncorhynchus mykiss) compared to non-viable heatkilled<br />
cells, although the respiratory burst activity in this case, was not affected.<br />
Although several authors reported the induction of respiratory burst activity by<br />
probiotics [23, 57-59]. The fact that the pot<strong>en</strong>tial probiotic strain Pdp13 did not increase<br />
superoxi<strong>de</strong> anion production does not rule out that the possibility to induce a significant<br />
change in other immunological parameters. This is supported by the fact that we<br />
observed increased survival after the chall<strong>en</strong>ge with the pathog<strong>en</strong>ic bacteria P. damse<strong>la</strong>e<br />
subsp. piscicida in fish fed with diet supplem<strong>en</strong>ted with Pdp13 strain, compared to<br />
control fish (fed with normal diet) and fish fed with the diet supplem<strong>en</strong>ted with Pdp11.<br />
Although the <strong>de</strong>gree of protection achieved with Pdp13 was not very high (i.e. 20%<br />
survival), the reduced and retar<strong>de</strong>d mortality might provi<strong>de</strong> additional time that could be<br />
useful to prev<strong>en</strong>t the infection by using other differ<strong>en</strong>t treatm<strong>en</strong>ts.<br />
11
Several authors have reported a stimu<strong>la</strong>tory effect of differ<strong>en</strong>t probiotics on<br />
differ<strong>en</strong>t immunological parameters, such as phagocytic [22, 24, 26, 27, 58],<br />
complem<strong>en</strong>t [24, 27], lysozyme [22, 24] or cytotoxic [26, 27] activities. Moreover it has<br />
be<strong>en</strong> <strong>de</strong>scribed that probiotic treatm<strong>en</strong>t resulted in an increase of immunoglobulin levels<br />
in serum [23, 59]. H<strong>en</strong>ce, Pdp13 strain can be consi<strong>de</strong>red as probiotic although further<br />
studies should be carried out, evaluating additional immunological aspects.<br />
On the other hand, the probiotic bacteria have to be administered at an optimal<br />
dose that may <strong>de</strong>p<strong>en</strong>d on the size and species of experim<strong>en</strong>tal fish and used probiotic<br />
strain [23]. This might exp<strong>la</strong>in differ<strong>en</strong>t effects of both probiotic strains observed in this<br />
study wh<strong>en</strong> applied to sole, and differ<strong>en</strong>ces of Pdp11 obtained for sole (this study) and<br />
gilthead seabream [27].<br />
Besi<strong>de</strong>s the effect of the probiotics on the immune response of sole, the other<br />
objective of this work was to assess changes in bacterial community structure of the sole<br />
intestine, as influ<strong>en</strong>ced by dietary treatm<strong>en</strong>t with probiotics. The <strong>de</strong>velopm<strong>en</strong>t of<br />
molecu<strong>la</strong>r methods for studying microbial communities has rec<strong>en</strong>tly also resulted in<br />
numerous works focusing on fish microbiota [20, 40-44, 60]. Nevertheless, no study has<br />
be<strong>en</strong> reported evaluating possible shifts in fish intestinal microbiota in response to<br />
probiotics administration. In the pres<strong>en</strong>t study, PCR-DGGE has be<strong>en</strong> applied to the<br />
analysis of fragm<strong>en</strong>ts <strong>de</strong>rived from differ<strong>en</strong>t hypervariable regions of 16S rRNA g<strong>en</strong>es.<br />
The set of primers selected are specific for all bacteria (V6-V8 region), and were<br />
previously used by several authors for the analysis of dietary effects on intestinal<br />
microbiota [47-49]. The results obtained in this study showed very simple patterns.<br />
Muyzer et al. [39] <strong>de</strong>scribed that communities with a few dominant species will produce<br />
simpler patterns and that less abundant species may not be a<strong>de</strong>quately repres<strong>en</strong>ted in the<br />
community pattern. It could be possible that some of these bands may not repres<strong>en</strong>t<br />
individual species, but rather groups which have the same re<strong>la</strong>tive G+C cont<strong>en</strong>t within<br />
the 16S V6-V8 region and have comigrated [61, 62]. These limitations may account in<br />
part for the <strong>de</strong>creased band number in the pres<strong>en</strong>t study and may also have influ<strong>en</strong>ced<br />
the appar<strong>en</strong>t diversity and simi<strong>la</strong>rity values [63]. The use of universal primers (e.g.<br />
those targeting the V3 or V6-V8 region of the 16S rRNA g<strong>en</strong>e) allows any bacterial<br />
community to be analyzed, although in case of an ecosystem with a re<strong>la</strong>tively high<br />
12
acterial diversity only the dominant microbiota (i.e. popu<strong>la</strong>tions with re<strong>la</strong>tive<br />
abundances of > 1%) will be visualized on the DGGE gels [64].<br />
Simi<strong>la</strong>r to the situation <strong>de</strong>scribed by several authors in homeotherms [64, 65],<br />
each individual fish showed a unique DGGE pattern. As expected, the fish intestinal<br />
microbiota fluctuates over time and is more <strong>de</strong>p<strong>en</strong><strong>de</strong>nt on the <strong>en</strong>vironm<strong>en</strong>t than<br />
homeothers [8].<br />
In our study, the only significant change in the intestinal microbiota could be<br />
observed in intergroup comparisons after comparing fish from the control group with<br />
fish fed with strain Pdp13. This might partly be exp<strong>la</strong>ined by the fact that closely re<strong>la</strong>ted<br />
popu<strong>la</strong>tions were already installed in the tissue, as corresponding bands were also<br />
observed in control groups. This is in accordance with the i<strong>de</strong>a that the microbiota of<br />
fish intestine consists of bacteria that are also pres<strong>en</strong>t in the surrounding water, but<br />
which are able to persist and multiply in the <strong>en</strong>vironm<strong>en</strong>t provi<strong>de</strong>d by intestinal tract<br />
[66, 67]. Therefore, the gastrointestinal microbiota of fish and shellfish are peculiarly<br />
<strong>de</strong>p<strong>en</strong><strong>de</strong>nt on the external <strong>en</strong>vironm<strong>en</strong>t, Vibrio being one of the most abundant g<strong>en</strong>era in<br />
marine fish [55]. The probiotics were iso<strong>la</strong>ted from the skin of another farmed fish,<br />
gilthead seabream. It can be expected that simi<strong>la</strong>r microbiota colonize both hosts.<br />
Further studies using direct sequ<strong>en</strong>cing of excised bands and 16S rRNA g<strong>en</strong>e clone<br />
library analysis need to be carried out to confirm that these bands correspond to<br />
popu<strong>la</strong>tions i<strong>de</strong>ntical to Pdp11 or Pdp13 strains.<br />
Although probiotics did not produce dramatic changes in the intestinal microbiota<br />
<strong>de</strong>tected by DGGE analysis, this does not imply that they do not exert any effect.<br />
According to Ouwehand et al. [68], it is not necessary that a probiotic induces changes<br />
in intestinal microbiota or so-called colonization to exert an effect locally or during<br />
transi<strong>en</strong>t passage through the gastrointestinal system. In fact, the variation of the<br />
microbiota in fish is substantial and fluctuates strongly on a daily basis [17, 20, 24].<br />
Therefore, stabilisation of the protective intestinal microbiota may be a means of<br />
improving the natural resistance to pot<strong>en</strong>tial pathog<strong>en</strong>s.<br />
13
Acknowledgem<strong>en</strong>ts<br />
The authors thank PREDOMAR (Carboneras, Almería, Spain) fishery for help<br />
and participation in this study and the financial support by the Ministerio Español <strong>de</strong><br />
Ci<strong>en</strong>cia y Tecnología (AGL 2005-07454-CO2-O2). P. Díaz-Rosales wishes to thank the<br />
Ministerio Español <strong>de</strong> Educación y Ci<strong>en</strong>cia for a F.P.U. scho<strong>la</strong>rship.<br />
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and host-specific communities of active bacteria. Applied & Environm<strong>en</strong>tal<br />
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[65] Simpson, JM, McCrack<strong>en</strong>, VJ, Gaskins, HR and Mackie, RI. D<strong>en</strong>aturing gradi<strong>en</strong>t<br />
gel electrophoresis analysis of 16S ribosomal DNA amplicons to monitor changes<br />
in fecal bacterial popu<strong>la</strong>tions of weaning pigs after introduction of Lactobacillus<br />
reuteri strain MM53. Applied & Environm<strong>en</strong>tal Microbiology 2000; 66: 4705-14.<br />
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effects. Antoine van Leeuw<strong>en</strong>hoek 2002; 82: 279-89.<br />
21
Figures leg<strong>en</strong>ds<br />
Table 1. Primers and PCR conditions used in this study.<br />
Figure 1a. Respiratory burst activity of kidney phagocytes from sole specim<strong>en</strong>s<br />
fed diets containing alginate alone, or in combination with 10 9 cfu g -1 Pdp11 or 10 9 cfu<br />
g -1 Pdp13 for 30 days ( ) or 60 days ( ). Results are expressed as stimu<strong>la</strong>tion in<strong>de</strong>x<br />
obtained by dividing each sample value by the mean control value. Symbol * <strong>de</strong>notes<br />
statistically significant differ<strong>en</strong>ces (P
Table 1<br />
Primer Sequ<strong>en</strong>ce (5’-3’) PCR Refer<strong>en</strong>ce<br />
Bact-0968-GC-F CGC CCG GGG CGC GCC 94 ºC for 2 min and 35 [47-49]<br />
CCG GGC GGG GCG cycles of 95 ºC for 30 s,<br />
GGG<br />
56 ºC for 40 s, 72 ºC<br />
GCA CGG GGG GAA CGC<br />
GAA GAA CCT TAC<br />
for 1 min, and 72 ºC for<br />
5 min (final ext<strong>en</strong>sion)<br />
Bact-1401-R CGG TGT GTA CAA GAC<br />
CC<br />
PRBA-338-GC-F CGC CCG CCG CGC GCG 92 ºC for 2 min and 30 [44]<br />
GCG GGC GGG GCG GGG<br />
GCA CGG GGG GAC TCC<br />
cycles of 92 ºC for 1<br />
min, 55 ºC for 30 s, 72<br />
TAC GGG AGG CAG ºC for 1 min, and 72 ºC<br />
CAG<br />
for 6 min (final<br />
ext<strong>en</strong>sion)<br />
PRUN-518-R ATT ACC GCG GCT GCT<br />
GG<br />
* Primer with a 40-bp GC c<strong>la</strong>mp at the 5’<strong>en</strong>d.<br />
25
Figure 1a<br />
2.5<br />
2<br />
*<br />
Stimu<strong>la</strong>tion in<strong>de</strong>x<br />
1. 5<br />
1<br />
0.5<br />
0<br />
Alginate Pdp11 Pdp13<br />
27
Figure 1b<br />
Stimu<strong>la</strong>tion in<strong>de</strong>x<br />
2.5<br />
2<br />
1.5<br />
1<br />
0.5<br />
*<br />
0<br />
Alginate Pdp11 Pdp13<br />
29
Figure 2<br />
Cumu<strong>la</strong>tive mortality (%)<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
Control<br />
Pdp11<br />
Pdp13<br />
0 1 2 3 4<br />
Days after chall<strong>en</strong>ge<br />
31
Table 2<br />
Primer Control Pdp11 Pdp13<br />
Bact-0968-GC-F & 72.85±7 83.01±4.22 65.81±4.92<br />
Bact-1401-R<br />
PRBA-338-GC-F &<br />
PRUN-518-R<br />
71.4±4.67 71.33±7.19 73.11±5<br />
33
Table 3<br />
Primer Bact-0968-GC-F &<br />
Bact-1401-R<br />
PRBA-338-GC-F &<br />
PRUN-518-R<br />
Control-Pdp11 57.08±2.91 68.95±4.52<br />
Control-Pdp13 41.54±2.88 70.21±3.82<br />
Pdp11-Pdp13 65.08±1.87 79.78±4.18<br />
35
Figure 3<br />
Pearson corre<strong>la</strong>tion [0.0%-100.0%]<br />
PREDOMARSalvador<br />
PREDOMARSalvador<br />
40<br />
50<br />
60<br />
70<br />
80<br />
90<br />
100<br />
Pdp11 . Pdp11 (I)<br />
Pdp11 . Pdp11 (I)<br />
Pdp11 . Pdp11(I)<br />
Pdp13 . Pdp13 (I)<br />
Pdp13 . Pdp13 (I)<br />
Pdp13 . Pdp13 (I)<br />
Control . Control (I)<br />
Control . Control (I)<br />
Control . Control (I)<br />
Pure control . Pure Pdp13 culture Pdp1<br />
Pure culture . Pure Pdp11 culture Pdp1<br />
37
Agra<strong>de</strong>cimi<strong>en</strong>tos / Acknowledgem<strong>en</strong>ts<br />
En primer lugar, un agra<strong>de</strong>cimi<strong>en</strong>to muy especial a Miguel Ángel Moriñigo<br />
y a Mª Carm<strong>en</strong> Balebona, mis directores. Agra<strong>de</strong>ceros el apoyo y <strong>la</strong> confianza<br />
que <strong>de</strong>positasteis <strong>en</strong> mí, espero no haberos <strong>de</strong>fraudado. Gracias por <strong>en</strong>señarme,<br />
por haber hecho fácil y gratificante este camino, y por transmitirme <strong>la</strong> ilusión por<br />
<strong>la</strong> investigación. Ha sido un honor ser vuestra discípu<strong>la</strong>.<br />
Gracias a Eduardo Martínez por transmitir sus conocimi<strong>en</strong>tos y por su<br />
apoyo.<br />
Mi agra<strong>de</strong>cimi<strong>en</strong>to al director <strong>de</strong>l Departam<strong>en</strong>to, Antonio <strong>de</strong> Vic<strong>en</strong>te, a<br />
Juan José Borrego, Dolores Castro, Alejandro Pérez, Mª Carm<strong>en</strong> Alonso,<br />
Francisco Cazor<strong>la</strong> y Esther García.<br />
Gracias a Roberto Abda<strong>la</strong> y Félix López, <strong>de</strong>l Departam<strong>en</strong>to <strong>de</strong> Ecología,<br />
por esta gratificante y <strong>en</strong>riquecedora co<strong>la</strong>boración inter<strong>de</strong>partam<strong>en</strong>tal.<br />
Muchas gracias a Salvador Arijo y Mariana Chabrillón: esta Tesis también<br />
es vuestra. Durante estos años vosotros habéis sido mi ejemplo a seguir. Gracias<br />
por todo lo que me habéis <strong>en</strong>señado. Muchas gracias a Rosa Mª Rico, por tu<br />
sonrisa y <strong>la</strong> alegría que transmites. Gracias a los que comi<strong>en</strong>zan ahora, <strong>en</strong><br />
especial a Silvana Tapia.<br />
Gracias a Daniel <strong>de</strong>l Pino, Paco Olea, Eva Arrebo<strong>la</strong>, Diego Romero, Ir<strong>en</strong>e<br />
Cano y Pedro Fierro. Gracias a María Múñoz y Carm<strong>en</strong> Vi<strong>la</strong>.<br />
Agra<strong>de</strong>cer a <strong>la</strong> piscifactoría PROMAN (Promotora Alpujarreña <strong>de</strong><br />
Negocios, S.L., Motril, Granada, España), especialm<strong>en</strong>te a Víctor Fernán<strong>de</strong>z,<br />
Director Técnico, y al Au<strong>la</strong> <strong>de</strong>l Mar (Má<strong>la</strong>ga, España) su inestimable<br />
co<strong>la</strong>boración.<br />
Thank you Dr. Secombes, Chris, to afford me the opportunity to work at<br />
your <strong>la</strong>boratory in Aber<strong>de</strong><strong>en</strong>. I am very grateful that you have trusted me again. I<br />
hope you will not be disappointed by me. Thank you Dr. Jun Zou.<br />
José Meseguer y Mª Ángeles Esteban, gracias por <strong>de</strong>jarme trabajar con<br />
vosotros, por aceptarme como un miembro más. Muchas gracias a Alberto<br />
Cuesta, Alejandro Rodríguez y, <strong>en</strong> especial, a Ir<strong>en</strong>e Salinas.<br />
Thank you Dr. Smidt, Hauke, to allow me working at your <strong>la</strong>boratory.<br />
Danke Schön. Dr. Edwin Zoet<strong>en</strong>dal, dank u wel. Mariana Chabrillón, agra<strong>de</strong>certe
que perdieras parte <strong>de</strong> tu tiempo <strong>en</strong> <strong>en</strong>señarme y todo lo que me has ayudado;<br />
creo que esto no podría haber salido sin ti.<br />
A mis amigos que, a pesar <strong>de</strong> los años transcurridos, sigu<strong>en</strong> estando ahí,<br />
apoyándome <strong>en</strong> cada mom<strong>en</strong>to. Muchas gracias.<br />
Dar <strong><strong>la</strong>s</strong> gracias a mis padres por tantas horas <strong>de</strong> <strong>de</strong>dicación.<br />
Y, sobre todo, a mi hermano Raúl. Raúl, sin ti esta Tesis no habría visto <strong>la</strong><br />
luz; gracias por apoyarme siempre, por escucharme y por soportarme. Gracias<br />
por tu ayuda <strong>en</strong> todo el proceso <strong>de</strong> e<strong>la</strong>boración, esta Tesis está completa gracias a<br />
tus conocimi<strong>en</strong>tos filológicos. Gracias.