Serological detection of Trichinella spiralis and Trichinella britovi in ...
Serological detection of Trichinella spiralis and Trichinella britovi in ...
Serological detection of Trichinella spiralis and Trichinella britovi in ...
- No tags were found...
You also want an ePaper? Increase the reach of your titles
YUMPU automatically turns print PDFs into web optimized ePapers that Google loves.
Artículo Orig<strong>in</strong>alRev. Ibero-Lat<strong>in</strong>oam. Parasitol. (2011); 70 (1): 49-57<strong>Serological</strong> <strong>detection</strong> <strong>of</strong> <strong>Trich<strong>in</strong>ella</strong> <strong>spiralis</strong> <strong>and</strong><strong>Trich<strong>in</strong>ella</strong> <strong>britovi</strong> <strong>in</strong> wild boar by ELISA us<strong>in</strong>gan excretor-secretor antigen <strong>and</strong> a crude antigenGAMITO-SANTOS J.A., BLANCO-CIUDAD J., SUÁREZ-LÓPEZ I., SERRANO-AGUILERA F.J.<strong>and</strong> PÉREZ-MARTÍN J.E.Parasitology, Animal Health Department, Veter<strong>in</strong>ary Faculty, 10071 Cáceres, Spa<strong>in</strong>.ABSTRACTThe enzyme-l<strong>in</strong>ked immunosorbent assay (ELISA) method is extensively used <strong>in</strong> epidemiologicalstudies <strong>in</strong> order to establish <strong>Trich<strong>in</strong>ella</strong> negligible areas. In this study, we exam<strong>in</strong>ed the sensitivity obta<strong>in</strong>edby the ELISA method us<strong>in</strong>g different antigens <strong>and</strong> the two <strong>Trich<strong>in</strong>ella</strong> species found <strong>in</strong> Spa<strong>in</strong>. We analyzedthe humoral evolution that is produced with the Excretor-Secretor (ES) <strong>and</strong> Crude Larval (CWE) antigens,<strong>in</strong> two species <strong>of</strong> <strong>Trich<strong>in</strong>ella</strong>. The results confirm greater precocity <strong>and</strong> persistence <strong>of</strong> antibodies when facedwith higher <strong>in</strong>fective doses <strong>and</strong> better <strong>detection</strong> <strong>of</strong> <strong>Trich<strong>in</strong>ella</strong> spp. when us<strong>in</strong>g the antigen homologous tothe <strong>in</strong>fected species. The sensitivity obta<strong>in</strong>ed with ELISA is at least equal to that <strong>of</strong> digestion <strong>and</strong> greater if<strong>Trich<strong>in</strong>ella</strong> <strong>britovi</strong> antigens are used. We have demonstrated a dist<strong>in</strong>ct k<strong>in</strong>etic pattern <strong>in</strong> the two species <strong>of</strong><strong>Trich<strong>in</strong>ella</strong> studied. Our results support the usefulness <strong>of</strong> this method <strong>in</strong> epidemiological surveillance <strong>and</strong>its optimization for the use as a serological test <strong>in</strong> the <strong>detection</strong> <strong>of</strong> this zoonosis.Key words: <strong>Trich<strong>in</strong>ella</strong>, ELISA, Excretor-Secretor antigen, Crude antigen, wild boar, Spa<strong>in</strong>.RESUMENEl método ELISA (enzyme-l<strong>in</strong>ked immunosorbent assay) es muy utilizado en estudios epidemiológicospara establecer áreas libres de <strong>Trich<strong>in</strong>ella</strong>. En este estudio, vemos la sensibilidad que se obtiene por el métodoELISA utiliz<strong>and</strong>o diferentes antígenos y las dos especies de <strong>Trich<strong>in</strong>ella</strong> presentes en España. Analizamos laevolución humoral que se produce ante los antígenos Excretor-Secretor (ES) y Bruto Larvario (CWE) y enlas dos especies de <strong>Trich<strong>in</strong>ella</strong>. Para esto, utilizamos 22 jabalíes encuadrados en cuatro grupos: <strong>in</strong>fectadoscon 200 larvas de <strong>Trich<strong>in</strong>ella</strong> <strong>spiralis</strong>/<strong>Trich<strong>in</strong>ella</strong> <strong>britovi</strong>, <strong>in</strong>fectados con 1.000 larvas de T. <strong>spiralis</strong>/T.<strong>britovi</strong>, <strong>in</strong>fectados con 20.000 larvas de T. <strong>spiralis</strong>/T. <strong>britovi</strong> y grupo control. Los resultados concluyen unamayor precocidad y persistencia de anticuerpos ante dosis <strong>in</strong>fectivas más elevadas y una mejor detecciónde <strong>Trich<strong>in</strong>ella</strong> al utilizar el antígeno homólogo a la especie <strong>in</strong>fectante. La sensibilidad que se obtiene conReceived: 20 December 2010. Accepted: 15 February 2011.Correspond<strong>in</strong>g: J E Pérez MartínE-mail: juanerpm@gmail.comTelephone: 34-927257115, Fax: 34-92725711051
J. A. GAMITO SANTOS et al.el ELISA es como mínimo igual a la de la digestión y, mayor si se utiliza antígenos de T. <strong>britovi</strong>. Hemosevidenciado un diferente patrón c<strong>in</strong>ético en las dos especies de <strong>Trich<strong>in</strong>ella</strong> estudiadas. Nuestros resultadosavalan la utilidad de este método en vigilancia epidemiológica y su optimización para su utilización comotest serológico en la detección de esta zoonosis.Palabras clave: <strong>Trich<strong>in</strong>ella</strong>, ELISA, Antígeno Excretor-Secretor, Antígeno bruto larvario, jabalí,España.INTRODUCTIONImmunological diagnostic techniques are be<strong>in</strong>gused with greater frequency <strong>in</strong> the study <strong>of</strong> diseases.In the case <strong>of</strong> trich<strong>in</strong>ellosis, immunologicaldiagnosis is not used as the method <strong>of</strong> reference;accord<strong>in</strong>g to Regulation 2075/2005 <strong>of</strong> the EuropeanUnion, the <strong>of</strong>ficial method is magnetic artificialdigestion. The first studies were promis<strong>in</strong>g, serologicalmethods be<strong>in</strong>g found to be superior to directmethods (Van Knapen et al, 1980, 1981a,b, 1984).The high sensitivity detected surpasses that <strong>of</strong> otherserological techniques (Van Knapen et al, 1976).However, <strong>in</strong> spite <strong>of</strong> all this, follow<strong>in</strong>g deficienciesfound <strong>in</strong> terms <strong>of</strong> possible cross-reactivity, false negatives,etc., these authors arrive at the conclusion thatELISA should be accepted only <strong>in</strong> epidemiologicalstudies. The antigen <strong>of</strong> choice for the study <strong>of</strong> trich<strong>in</strong>ellosis<strong>in</strong> sw<strong>in</strong>e has been the excretor-secretor T. <strong>spiralis</strong>(Murrell et al, 1986; Van der Leek et al, 1992;Reiterová et al, 1999; Gamble 1996, 1998; Nöckleret al, 1995; Kapel et al, 1998; Ribicich et al, 2000).In terms <strong>of</strong> superiority <strong>of</strong> one antigen over another,there is no unanimity. Thus Gamble et al, (1983) statedthat it is better to use the ES antigen rather thanthe crude antigen, given that while with the latteralmost 9% <strong>of</strong> false positive animals were obta<strong>in</strong>ed,with the former type <strong>of</strong> antigen this does not occur.These results are also confirmed by other researcherssuch as Arriaga (1989a, b) or Bolás Fernández et al.(1993). On the other h<strong>and</strong>, L<strong>in</strong>d et al, (1991) arriveat the conclusion that the crude antigen is more sensitive,given that earlier sero-conversions are obta<strong>in</strong>edthan with other more purified antigens, po<strong>in</strong>t<strong>in</strong>g outthat with the latter the sensitivity <strong>of</strong> the techniquecould be reduced.We carried out an ELISA us<strong>in</strong>g Excretor-Secretor antigen <strong>and</strong> crude Antigen <strong>of</strong> the twospecies <strong>of</strong> <strong>Trich<strong>in</strong>ella</strong> found <strong>in</strong> Spa<strong>in</strong>: T. <strong>spiralis</strong><strong>and</strong> T. <strong>britovi</strong>. Our study focused on wild boar, animportant species that can act as a source <strong>of</strong> bothwild <strong>and</strong> domestic trich<strong>in</strong>ellosis <strong>and</strong> for which thereare few previous studies.MATERIALS AND METHODSTest animals <strong>and</strong> blood recovery22 wild boars from various estates from theregion <strong>of</strong> Cáceres (Spa<strong>in</strong>), <strong>of</strong> both sexes, aged between2 <strong>and</strong> 4 months at the beg<strong>in</strong>n<strong>in</strong>g <strong>of</strong> the experiment,weigh<strong>in</strong>g between 3.95 <strong>and</strong> 22 kg, wereused <strong>in</strong> the experiment. They were captured accord<strong>in</strong>gto authorized methods with the required <strong>of</strong>ficialpermits.For the duration <strong>of</strong> the experiment the animalswere held <strong>in</strong> the facilities designed for <strong>in</strong>fectious<strong>and</strong> contagious diseases <strong>in</strong> the Cl<strong>in</strong>ical Hospital <strong>of</strong>the Veter<strong>in</strong>ary Faculty <strong>of</strong> Cáceres, the University <strong>of</strong>Extremadura (Spa<strong>in</strong>).The two isolates used were obta<strong>in</strong>ed from wildboars shot <strong>in</strong> hunts that took place <strong>in</strong> the AutonomousRegion <strong>of</strong> Extremadura (Spa<strong>in</strong>). For T. <strong>spiralis</strong>, (T1)the isolate ISS-512, from a wild boar shot <strong>in</strong> 1997<strong>in</strong> Deleitosa (Cáceres, Spa<strong>in</strong>) was used, <strong>and</strong> for T.<strong>britovi</strong> (T3), the isolate ISS-308, from a wild boarshot <strong>in</strong> 1993 <strong>in</strong> Carrascalejo (Cáceres, Spa<strong>in</strong>). Bothisolates were ma<strong>in</strong>ta<strong>in</strong>ed by periodic passage <strong>in</strong>Swiss NBC mice. The <strong>in</strong>fectious dose was appliedto the wild boars through a gastric tube. The dosesadm<strong>in</strong>istered <strong>and</strong> the division <strong>in</strong>to groups are shown<strong>in</strong> Table 1.Blood samples were collected by jugular venipuncture.Wild boars were anesthetized us<strong>in</strong>gblowgun. We use Zoletil 100® whose compositionper milliliter is: Teletam<strong>in</strong>a 50 milligram <strong>and</strong> Zolacepam50 milligram. We <strong>in</strong>ject 10 milligram per kilogram<strong>of</strong> weight. From each wild boar blood wastaken on days -5, 0, 5, 10, 15, 20, 25, 30, 40, 50, 60,75, 90, 105 <strong>and</strong> 125 d.pi (days post <strong>in</strong>fection). Serawere isolated by centrifugation <strong>and</strong> frozen at -60º Cuntil used.52Rev. Ibero-Lat<strong>in</strong>oam. Parasitol. (2011); 70 (1): 49-57
DETECTION OF TRICHINELLA SPP. IN WILD BOAR BY ELISATable 1. Distribution <strong>in</strong> groups <strong>and</strong> dosesGroups Wild boar Species Dose1 1, 2 & 3 T. <strong>spiralis</strong> 2002 4, 5 & 6 T. <strong>spiralis</strong> 1,0003 7, 8 & 9 T. <strong>spiralis</strong> 20,000Control A, B, C & D ******* *******4 13, 14 & 15 T. <strong>britovi</strong> 2005 16, 17 & 18 T. <strong>britovi</strong> 1,0006 19, 20 & 21 T. <strong>britovi</strong> 20,000Larval recovery <strong>and</strong> count<strong>in</strong>gThe numbers <strong>of</strong> muscle larvae were determ<strong>in</strong>ed<strong>in</strong> the diaphragm. Muscles were exam<strong>in</strong>ed by theartificial digestion procedure for the presence <strong>of</strong> T.<strong>spiralis</strong> or T. <strong>britovi</strong>. We digested approximately25g <strong>of</strong> muscular tissue, with the exception <strong>of</strong> low<strong>in</strong>fection loads for which the tissue digested was<strong>in</strong>creased. The larvae were isolated <strong>and</strong> the muscularload was calculated <strong>in</strong> larvae per gram (LPG).Preparation <strong>of</strong> antigens• Preparation <strong>of</strong> crude muscle larva antigen.We followed the guidel<strong>in</strong>es proposed by theCEE workgroup on trich<strong>in</strong>ellosis (Ruitenberg etal, 1976), with slight modifications. The larvaeobta<strong>in</strong>ed follow<strong>in</strong>g artificial digestion werecleaned <strong>of</strong> tissue residues with 10 washes <strong>in</strong> PBS(Phosphate Buffer Sal<strong>in</strong>e) to which one gram<strong>of</strong> streptomyc<strong>in</strong> sulphate <strong>and</strong> 1,000,000 I.U. <strong>of</strong>penicill<strong>in</strong> G sodium per liter <strong>of</strong> PBS was added.For approximately 30 seconds, sedimentation<strong>of</strong> the larvae was carried out us<strong>in</strong>g gentlecentrifugation, aspirat<strong>in</strong>g the supernatant witha vacuum pump. They were then homogenized<strong>in</strong> PBS with ultrasound at 4º C for one hour.With this, the soluble fraction <strong>of</strong> the antigenwas obta<strong>in</strong>ed through centrifugation at 2,000gdim<strong>in</strong>ish<strong>in</strong>g the <strong>in</strong>soluble part. In order todeterm<strong>in</strong>e prote<strong>in</strong> concentration, a modifiedversion <strong>of</strong> the Bradford method (CoomassiePlus Prote<strong>in</strong> Assay, <strong>of</strong> Pierce), was carried out,accord<strong>in</strong>g to commercial <strong>in</strong>structions.• Preparation <strong>of</strong> E/S antigen.The obta<strong>in</strong><strong>in</strong>g <strong>of</strong> larvae 1, <strong>and</strong> the post washwith sterile antibiotic PBS were carried out asdescribed for crude larva antigen. They werethen transferred to a RPMI 1640 Mediumsupplemented with L-Glutam<strong>in</strong>e <strong>and</strong> with 20mM HEPES, to which penicill<strong>in</strong> (500 I.U./ ml)<strong>and</strong> streptomyc<strong>in</strong> (500 I.U./ml) were added<strong>in</strong> the ratio <strong>of</strong> 5,000 larvae 1/ml. The culturewas <strong>in</strong>cubated <strong>in</strong> a heater at 37º C with 90%humidity <strong>and</strong> 10% CO 2for 48 hours. At 24<strong>and</strong> 48 hours, the viability <strong>of</strong> the larvae waschecked through observation <strong>of</strong> the culture<strong>in</strong> an <strong>in</strong>verted microscope, those display<strong>in</strong>gmore than 5% dead larvae be<strong>in</strong>g discarded.The medium was then centrifuged at 1,000g,obta<strong>in</strong><strong>in</strong>g <strong>and</strong> filter<strong>in</strong>g the supernatant througha sterile membrane <strong>of</strong> 0.20 µm pore diameter.The prote<strong>in</strong> content was assessed.ELISA testAn <strong>in</strong>direct double-antibody micro-ELISA(enzyme-l<strong>in</strong>ked immunosorbent assay) method wasused <strong>in</strong> order to detect circulat<strong>in</strong>g antibodies fromthe serum <strong>of</strong> the wild boars.Micro-ELISA 96-well flat bottomed polystyreneplates (Corn<strong>in</strong>g Incorporated), <strong>of</strong> 300 µl capacity,were used. The protocol used was based on thest<strong>and</strong>ardization carried out by Pérez Martín etal, (1994). In each well, 100 µl <strong>of</strong> antigen at aconcentration <strong>of</strong> 3µg/ml was placed, dissolved <strong>in</strong>a 0.1 M carbonate buffer pH 9.6. They were then<strong>in</strong>cubated with forced air at 37 ºC for 4 hours.Follow<strong>in</strong>g the <strong>in</strong>cubation, wash<strong>in</strong>g was carriedout with a solution <strong>of</strong> 0.01 M PBS pH 7.2-7.3 with0.05% Tween-20 (PBS-Tw20), with the help <strong>of</strong> anautomatic washer (AM60 Multi-Reagent Washer,D<strong>in</strong>ex Technologies), perform<strong>in</strong>g three washes <strong>of</strong>30 seconds. Follow<strong>in</strong>g the first wash, 5% bov<strong>in</strong>eserum album<strong>in</strong> (BSA) was added (200 µl) <strong>in</strong> PBSpH 7.2, the plates be<strong>in</strong>g <strong>in</strong>cubated at 37 ºC for30 m<strong>in</strong>utes. A further wash was then carried out.Rev. Ibero-Lat<strong>in</strong>oam. Parasitol. (2011); 70 (1): 49-5753
J. A. GAMITO SANTOS et al.Once the plates were coated, we filled each wellwith 100µl <strong>of</strong> prediluted serum. This predilutionwas obta<strong>in</strong>ed by dilut<strong>in</strong>g the serum <strong>in</strong> PBS-Tw20 to 1/1000. We <strong>in</strong>cubated the serum <strong>in</strong> thewells for 45 m<strong>in</strong>utes at 37 ºC <strong>and</strong> then washedit. Follow<strong>in</strong>g this step we added 100 µl <strong>of</strong>immuno-conjugate (anti-pig IgG conjugate withperoxidase) per well, diluted to 1/25000 <strong>in</strong> PBS-Tw20. It was <strong>in</strong>cubated aga<strong>in</strong> for 45 m<strong>in</strong>utes at37 º C, be<strong>in</strong>g washed three more times. F<strong>in</strong>ally,100µl <strong>of</strong> substrate was added per well (0.01%3,3´, 5,5´- Tetramethylbenzid<strong>in</strong>e dihydrochloride<strong>in</strong> a 0.1 M pH 5 citrate buffer, also add<strong>in</strong>g 3µl/ml <strong>of</strong>H 2O 2<strong>of</strong> vol. 10) <strong>and</strong> <strong>in</strong>cubated at 37ºC <strong>in</strong> darknessfor approximately 8-10 m<strong>in</strong>utes. The reaction wasstopped by apply<strong>in</strong>g 50 µl <strong>of</strong> H 2SO 43N per well.We carried out a measurement <strong>of</strong> the plates us<strong>in</strong>gthe spectrophotometer (MRX Microplate Reader,D<strong>in</strong>ex Technologies), at a wavelength (λ) <strong>of</strong> 450nm.The percentage <strong>of</strong> reactivity was expressed <strong>in</strong>accordance with Pérez Martín et al, (1994) accord<strong>in</strong>g%reactivity = [ ODsample -ODnegative control] x 100ODpositivecontrol - ODnegativecontrolto the formula:Diagnostic limits used <strong>in</strong> the studyThe diagnostic limits used <strong>in</strong> this study wereobta<strong>in</strong>ed from the analysis <strong>of</strong> 1,000 wild boarsshot <strong>in</strong> various hunts <strong>in</strong> the Autonomous Region <strong>of</strong>Extremadura (Spa<strong>in</strong>).From statistical data from the Junta deExtremadura on animals shot <strong>in</strong> previous years, wecalculated an average <strong>of</strong> 11,902 wild boars huntedper season, this be<strong>in</strong>g used as a reference for thecalculation <strong>of</strong> the representative sample.90% <strong>and</strong> 99% confidence limits are obta<strong>in</strong>edwith the help <strong>of</strong> the average <strong>and</strong> the st<strong>and</strong>arddeviation <strong>of</strong> the wild boars studied. Thus:• 90% is the Average <strong>of</strong> the Reactivity percentage+1,645 x St<strong>and</strong>ard Deviation (DS). This isthe limit up to which the serum is considerednegative (cut-<strong>of</strong>f 1).• 99% is the Average <strong>of</strong> the Reactivity percentage+3 x St<strong>and</strong>ard Deviation (DS). This is the limitfrom which the serum is considered positive(cut-<strong>of</strong>f 2).• Between both limits, the wild boars areTable 2. Confidence limits99% confidencelimit(cut-<strong>of</strong>f 2 value)considered doubtful.Diagnostic limits used for ELISA are shown <strong>in</strong>Table 2.RESULTS% Reactivity90% confidencelimit(cut-<strong>of</strong>f 1 value)ES T1 Antigen 47.41% 29.71%ES T3 Antigen 51.16% 30.77%Crude T1 Antigen 37.97% 22.92%Crude T3 Antigen 45.48% 25.82%Artificial digestionThe results obta<strong>in</strong>ed are shown <strong>in</strong> Table 3.All the wild boars <strong>of</strong> the experiment wereparasitized with larvae 1 <strong>Trich<strong>in</strong>ella</strong> spp., with theexception <strong>of</strong> number 17, <strong>in</strong>fected with T. <strong>britovi</strong>,for which no larva was detected us<strong>in</strong>g artificialdigestion.Wild boar number 19 showed a negative result<strong>in</strong> digestion, although a humoral reaction wasobserved. On carry<strong>in</strong>g out trich<strong>in</strong>elloscopy, weobserved calcified cysts which, due to <strong>in</strong>fectioneffects, would lead to this animal be<strong>in</strong>g consideredpositive.ELISAThe results <strong>of</strong> the humoral evolution are shown<strong>in</strong> Figures 1, 2, 3 <strong>and</strong> 4.The four wild boars <strong>in</strong>cluded <strong>in</strong> the controlgroup were free from <strong>Trich<strong>in</strong>ella</strong> follow<strong>in</strong>gartificial digestion <strong>of</strong> more than 500g <strong>of</strong> muscletissue, <strong>and</strong> were shown to be negative for the fourantigens used by the ELISA technique throughoutthe experiment.In groups 3 <strong>and</strong> 6, higher <strong>in</strong>fective doses wereapplied (20,000 larvae 1) <strong>and</strong> we have obta<strong>in</strong>edhigher <strong>in</strong>fective loads. In groups 3 <strong>and</strong> 6 serologicalresponse <strong>in</strong>crease from 15 d.p.i. <strong>and</strong> the globalreactivity was significantly higher than <strong>in</strong> other<strong>in</strong>fected groups. In groups 2, 3 <strong>and</strong> 6, we obta<strong>in</strong> thehighest persistence <strong>of</strong> high reactivity, up until the54Rev. Ibero-Lat<strong>in</strong>oam. Parasitol. (2011); 70 (1): 49-57
DETECTION OF TRICHINELLA SPP. IN WILD BOAR BY ELISATable 3. Artificial digestion resultsGroups <strong>and</strong> doses Wild boar l.p.g. a1 1 0.28T. <strong>spiralis</strong> 2 0.15(200 L1) 3 0.322 4 4.49T. <strong>spiralis</strong> 5 91.16(1,000 L1) 6 134.513 7 1276.19T. <strong>spiralis</strong> 8 206.77(20,000 L1) 9 1673.064 13 0.12T. <strong>britovi</strong> 14 0.04*(200 L1) 15 0.45 16 0.48**T. <strong>britovi</strong> 17 0(1,000 L1) 18 0.26 19 0T. <strong>britovi</strong> 20 203.28(20,000 L1) 21*** 196.35* The larvae were detected only <strong>in</strong> abdom<strong>in</strong>al muscles(follow<strong>in</strong>g st<strong>and</strong>ard procedure, on non-<strong>detection</strong> <strong>of</strong>larvae <strong>in</strong> the diaphragm, we analyzed the rest <strong>of</strong> themuscles us<strong>in</strong>g artificial digestion).** The larvae were detected only <strong>in</strong> <strong>in</strong>ter-costal muscles.*** Died at 30 days PI. Thus, it has not been <strong>in</strong>cluded<strong>in</strong> humoral evolution graphics.al.p.g., larvae per gram muscle tissueFigure 1. Antibody level <strong>of</strong> groups <strong>of</strong> wild boarsmeasured by ELISA employ<strong>in</strong>g crude antigen from T.<strong>spiralis</strong>.Figure 2. Antibody level <strong>of</strong> groups <strong>of</strong> wild boars measuredby ELISA employ<strong>in</strong>g crude antigen from T. <strong>britovi</strong>.Figure 3. Antibody level <strong>of</strong> groups <strong>of</strong> wild boars measuredby ELISA employ<strong>in</strong>g ES antigen from T. <strong>spiralis</strong>.Figure 4. Antibody level <strong>of</strong> groups <strong>of</strong> wild boars measuredby ELISA employ<strong>in</strong>g ES antigen from T. <strong>britovi</strong>.Rev. Ibero-Lat<strong>in</strong>oam. Parasitol. (2011); 70 (1): 49-5755
J. A. GAMITO SANTOS et al.Table 4. Sensitivity found depend<strong>in</strong>g <strong>of</strong> the techniqueTechnique Wild Boar SensitivityDigestion method(5 gr.)1 2 3 4 5 6 7 8 9 13 14 15 16 18 19 20+ - + + + + + + + - - + + - - + 68.75 %ES T1 Antigen - + - - + + + + + - + - + + + + 68.75 %40 d.piES T1 Antigen - + - + + + + + + - D - - D D D 68.75 %125 d.piES T3 Antigen - + D + + + + + + - + + + + + + 87.5 %40 DPIES T3 Antigen - + D + + + + + + D + D D D + + 93.75 %125 d.piCrude T1 Antigen- + - + + + + + + - + + + + + + 81.25 %40 d.piCrude T1 - + - + + + + + + D - - - + + D 68.75 %Antigen 125d.piCrude T3 Antigen- + D + + + + + + - + + + + + + 87.5 %40 d.piCrude T3 - + D + + + + + + + D D D + + + 93.75 %Antigen 125d.piD, doubtful; d.pi, days post <strong>in</strong>fection; ES, Excretor Secretor; T1, T. <strong>spiralis</strong>; T3, T. <strong>britovi</strong>.end <strong>of</strong> the experiment. Other groups reach levels <strong>of</strong>doubtful with some <strong>of</strong> the antigens used.We observed greater precocity <strong>and</strong> persistence<strong>of</strong> antibodies us<strong>in</strong>g antigens homologous to the<strong>in</strong>fected species. This can be seen <strong>in</strong> figure 3 <strong>and</strong>4 where we observed greater precocity <strong>and</strong> higherpersistence <strong>of</strong> antibodies <strong>in</strong> groups <strong>of</strong> wild boars <strong>in</strong>fectedwith T. <strong>britovi</strong> (groups 4, 5 <strong>and</strong> 6) as opposedto the ES T3 Antigen (Figure 4). This precocity<strong>and</strong> persistence is lower if we compare these samegroups as opposed to the ES T1 Antigen (Figure 3).The sensitivity found at 40 <strong>and</strong> 125 d.pi for eachantigen is displayed <strong>in</strong> Table 4.DISCUSSIONThe results obta<strong>in</strong>ed show a greater susceptibility<strong>of</strong> wild boar to T. <strong>spiralis</strong>. This f<strong>in</strong>d<strong>in</strong>g <strong>in</strong> wild boaris <strong>in</strong> accordance with results published by Kapel <strong>in</strong>2001 <strong>and</strong> Gamito-Santos et al, (2009).The immunoenzymatic diagnostic techniquesare more sensitive than artificial digestion, co<strong>in</strong>cid<strong>in</strong>gwith studies undertaken <strong>in</strong> porc<strong>in</strong>e livestock,such as that <strong>of</strong> Van Knapen et al. (1980, 1981a,b,1984), Madden <strong>and</strong> Murrell (1990) or that <strong>of</strong> Nöckleret al, (2000). Thus, with ES T1 Antigen at 40d.pi, <strong>and</strong> ES T1 Antigens <strong>and</strong> crude T1 Antigen,both at 125 d.pi, the same sensitivity as digestionis obta<strong>in</strong>ed. The rest surpass digestion <strong>in</strong> terms <strong>of</strong>sensitivity.Assess<strong>in</strong>g both antigens, <strong>in</strong> our study ES T1Antigen is less sensitive than the crude T1 Antigenat 40 d.pi, contradict<strong>in</strong>g studies by Gamble et al, <strong>in</strong>1983 <strong>and</strong> corroborat<strong>in</strong>g other studies <strong>in</strong> pigs suchas that <strong>of</strong> L<strong>in</strong>d et al, (1991), Bolás Fernández etal, (1993) or Pozio et al, (2003). ES T3 Antigen<strong>and</strong> crude T3 Antigen have the same sensitivity at40 d.pi as at 125 d.pi. We agree with Serrano et al,(1992) <strong>in</strong> a study <strong>of</strong> pigs.We have demonstrated a tendency for serologicalresponse to be more <strong>in</strong>tense <strong>and</strong> earlier <strong>in</strong> wildboars <strong>in</strong>fected with 20,000 larvae 1 (groups 3 <strong>and</strong>6) <strong>in</strong> comparison to those <strong>in</strong>fected with 200 larvae 156Rev. Ibero-Lat<strong>in</strong>oam. Parasitol. (2011); 70 (1): 49-57