Serological detection of Trichinella spiralis and Trichinella britovi in ...

Artículo OriginalRev. Ibero-Latinoam. Parasitol. (2011); 70 (1): 49-57Serological detection of Trichinella spiralis andTrichinella britovi in wild boar by ELISA usingan excretor-secretor antigen and a crude antigenGAMITO-SANTOS J.A., BLANCO-CIUDAD J., SUÁREZ-LÓPEZ I., SERRANO-AGUILERA F.J.and PÉREZ-MARTÍN J.E.Parasitology, Animal Health Department, Veterinary Faculty, 10071 Cáceres, Spain.ABSTRACTThe enzyme-linked immunosorbent assay (ELISA) method is extensively used in epidemiologicalstudies in order to establish Trichinella negligible areas. In this study, we examined the sensitivity obtainedby the ELISA method using different antigens and the two Trichinella species found in Spain. We analyzedthe humoral evolution that is produced with the Excretor-Secretor (ES) and Crude Larval (CWE) antigens,in two species of Trichinella. The results confirm greater precocity and persistence of antibodies when facedwith higher infective doses and better detection of Trichinella spp. when using the antigen homologous tothe infected species. The sensitivity obtained with ELISA is at least equal to that of digestion and greater ifTrichinella britovi antigens are used. We have demonstrated a distinct kinetic pattern in the two species ofTrichinella studied. Our results support the usefulness of this method in epidemiological surveillance andits optimization for the use as a serological test in the detection of this zoonosis.Key words: Trichinella, ELISA, Excretor-Secretor antigen, Crude antigen, wild boar, Spain.RESUMENEl método ELISA (enzyme-linked immunosorbent assay) es muy utilizado en estudios epidemiológicospara establecer áreas libres de Trichinella. En este estudio, vemos la sensibilidad que se obtiene por el métodoELISA utilizando diferentes antígenos y las dos especies de Trichinella presentes en España. Analizamos laevolución humoral que se produce ante los antígenos Excretor-Secretor (ES) y Bruto Larvario (CWE) y enlas dos especies de Trichinella. Para esto, utilizamos 22 jabalíes encuadrados en cuatro grupos: infectadoscon 200 larvas de Trichinella spiralis/Trichinella britovi, infectados con 1.000 larvas de T. spiralis/T.britovi, infectados con 20.000 larvas de T. spiralis/T. britovi y grupo control. Los resultados concluyen unamayor precocidad y persistencia de anticuerpos ante dosis infectivas más elevadas y una mejor detecciónde Trichinella al utilizar el antígeno homólogo a la especie infectante. La sensibilidad que se obtiene conReceived: 20 December 2010. Accepted: 15 February 2011.Corresponding: 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. britovi. Hemosevidenciado un diferente patrón cinético en las dos especies de Trichinella 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: Trichinella, ELISA, Antígeno Excretor-Secretor, Antígeno bruto larvario, jabalí,España.INTRODUCTIONImmunological diagnostic techniques are beingused with greater frequency in the study of diseases.In the case of trichinellosis, immunologicaldiagnosis is not used as the method of reference;according to Regulation 2075/2005 of the EuropeanUnion, the official method is magnetic artificialdigestion. The first studies were promising, serologicalmethods being found to be superior to directmethods (Van Knapen et al, 1980, 1981a,b, 1984).The high sensitivity detected surpasses that of otherserological techniques (Van Knapen et al, 1976).However, in spite of all this, following deficienciesfound in terms of possible cross-reactivity, false negatives,etc., these authors arrive at the conclusion thatELISA should be accepted only in epidemiologicalstudies. The antigen of choice for the study of trichinellosisin swine has been the excretor-secretor T. spiralis(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 of superiority of 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% of false positive animals were obtained,with the former type of 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 hand, Lind et al, (1991) arriveat the conclusion that the crude antigen is more sensitive,given that earlier sero-conversions are obtainedthan with other more purified antigens, pointing outthat with the latter the sensitivity of the techniquecould be reduced.We carried out an ELISA using Excretor-Secretor antigen and crude Antigen of the twospecies of Trichinella found in Spain: T. spiralisand T. britovi. Our study focused on wild boar, animportant species that can act as a source of bothwild and domestic trichinellosis and for which thereare few previous studies.MATERIALS AND METHODSTest animals and blood recovery22 wild boars from various estates from theregion of Cáceres (Spain), of both sexes, aged between2 and 4 months at the beginning of the experiment,weighing between 3.95 and 22 kg, wereused in the experiment. They were captured accordingto authorized methods with the required officialpermits.For the duration of the experiment the animalswere held in the facilities designed for infectiousand contagious diseases in the Clinical Hospital ofthe Veterinary Faculty of Cáceres, the University ofExtremadura (Spain).The two isolates used were obtained from wildboars shot in hunts that took place in the AutonomousRegion of Extremadura (Spain). For T. spiralis, (T1)the isolate ISS-512, from a wild boar shot in 1997in Deleitosa (Cáceres, Spain) was used, and for T.britovi (T3), the isolate ISS-308, from a wild boarshot in 1993 in Carrascalejo (Cáceres, Spain). Bothisolates were maintained by periodic passage inSwiss NBC mice. The infectious dose was appliedto the wild boars through a gastric tube. The dosesadministered and the division into groups are shownin Table 1.Blood samples were collected by jugular venipuncture.Wild boars were anesthetized usingblowgun. We use Zoletil 100® whose compositionper milliliter is: Teletamina 50 milligram and Zolacepam50 milligram. We inject 10 milligram per kilogramof weight. From each wild boar blood wastaken on days -5, 0, 5, 10, 15, 20, 25, 30, 40, 50, 60,75, 90, 105 and 125 d.pi (days post infection). Serawere isolated by centrifugation and frozen at -60º Cuntil used.52Rev. Ibero-Latinoam. Parasitol. (2011); 70 (1): 49-57

DETECTION OF TRICHINELLA SPP. IN WILD BOAR BY ELISATable 1. Distribution in groups and dosesGroups Wild boar Species Dose1 1, 2 & 3 T. spiralis 2002 4, 5 & 6 T. spiralis 1,0003 7, 8 & 9 T. spiralis 20,000Control A, B, C & D ******* *******4 13, 14 & 15 T. britovi 2005 16, 17 & 18 T. britovi 1,0006 19, 20 & 21 T. britovi 20,000Larval recovery and countingThe numbers of muscle larvae were determinedin the diaphragm. Muscles were examined by theartificial digestion procedure for the presence of T.spiralis or T. britovi. We digested approximately25g of muscular tissue, with the exception of lowinfection loads for which the tissue digested wasincreased. The larvae were isolated and the muscularload was calculated in larvae per gram (LPG).Preparation of antigens• Preparation of crude muscle larva antigen.We followed the guidelines proposed by theCEE workgroup on trichinellosis (Ruitenberg etal, 1976), with slight modifications. The larvaeobtained following artificial digestion werecleaned of tissue residues with 10 washes in PBS(Phosphate Buffer Saline) to which one gramof streptomycin sulphate and 1,000,000 I.U. ofpenicillin G sodium per liter of PBS was added.For approximately 30 seconds, sedimentationof the larvae was carried out using gentlecentrifugation, aspirating the supernatant witha vacuum pump. They were then homogenizedin PBS with ultrasound at 4º C for one hour.With this, the soluble fraction of the antigenwas obtained through centrifugation at 2,000gdiminishing the insoluble part. In order todetermine protein concentration, a modifiedversion of the Bradford method (CoomassiePlus Protein Assay, of Pierce), was carried out,according to commercial instructions.• Preparation of E/S antigen.The obtaining of larvae 1, and the post washwith sterile antibiotic PBS were carried out asdescribed for crude larva antigen. They werethen transferred to a RPMI 1640 Mediumsupplemented with L-Glutamine and with 20mM HEPES, to which penicillin (500 I.U./ ml)and streptomycin (500 I.U./ml) were addedin the ratio of 5,000 larvae 1/ml. The culturewas incubated in a heater at 37º C with 90%humidity and 10% CO 2for 48 hours. At 24and 48 hours, the viability of the larvae waschecked through observation of the culturein an inverted microscope, those displayingmore than 5% dead larvae being discarded.The medium was then centrifuged at 1,000g,obtaining and filtering the supernatant througha sterile membrane of 0.20 µm pore diameter.The protein content was assessed.ELISA testAn indirect double-antibody micro-ELISA(enzyme-linked immunosorbent assay) method wasused in order to detect circulating antibodies fromthe serum of the wild boars.Micro-ELISA 96-well flat bottomed polystyreneplates (Corning Incorporated), of 300 µl capacity,were used. The protocol used was based on thestandardization carried out by Pérez Martín etal, (1994). In each well, 100 µl of antigen at aconcentration of 3µg/ml was placed, dissolved ina 0.1 M carbonate buffer pH 9.6. They were thenincubated with forced air at 37 ºC for 4 hours.Following the incubation, washing was carriedout with a solution of 0.01 M PBS pH 7.2-7.3 with0.05% Tween-20 (PBS-Tw20), with the help of anautomatic washer (AM60 Multi-Reagent Washer,Dinex Technologies), performing three washes of30 seconds. Following the first wash, 5% bovineserum albumin (BSA) was added (200 µl) in PBSpH 7.2, the plates being incubated at 37 ºC for30 minutes. A further wash was then carried out.Rev. Ibero-Latinoam. Parasitol. (2011); 70 (1): 49-5753

J. A. GAMITO SANTOS et al.Once the plates were coated, we filled each wellwith 100µl of prediluted serum. This predilutionwas obtained by diluting the serum in PBS-Tw20 to 1/1000. We incubated the serum in thewells for 45 minutes at 37 ºC and then washedit. Following this step we added 100 µl ofimmuno-conjugate (anti-pig IgG conjugate withperoxidase) per well, diluted to 1/25000 in PBS-Tw20. It was incubated again for 45 minutes at37 º C, being washed three more times. Finally,100µl of substrate was added per well (0.01%3,3´, 5,5´- Tetramethylbenzidine dihydrochloridein a 0.1 M pH 5 citrate buffer, also adding 3µl/ml ofH 2O 2of vol. 10) and incubated at 37ºC in darknessfor approximately 8-10 minutes. The reaction wasstopped by applying 50 µl of H 2SO 43N per well.We carried out a measurement of the plates usingthe spectrophotometer (MRX Microplate Reader,Dinex Technologies), at a wavelength (λ) of 450nm.The percentage of reactivity was expressed inaccordance with Pérez Martín et al, (1994) according%reactivity = [ ODsample -ODnegative control] x 100ODpositivecontrol - ODnegativecontrolto the formula:Diagnostic limits used in the studyThe diagnostic limits used in this study wereobtained from the analysis of 1,000 wild boarsshot in various hunts in the Autonomous Region ofExtremadura (Spain).From statistical data from the Junta deExtremadura on animals shot in previous years, wecalculated an average of 11,902 wild boars huntedper season, this being used as a reference for thecalculation of the representative sample.90% and 99% confidence limits are obtainedwith the help of the average and the standarddeviation of the wild boars studied. Thus:• 90% is the Average of the Reactivity percentage+1,645 x Standard Deviation (DS). This isthe limit up to which the serum is considerednegative (cut-off 1).• 99% is the Average of the Reactivity percentage+3 x Standard Deviation (DS). This is the limitfrom which the serum is considered positive(cut-off 2).• Between both limits, the wild boars areTable 2. Confidence limits99% confidencelimit(cut-off 2 value)considered doubtful.Diagnostic limits used for ELISA are shown inTable 2.RESULTS% Reactivity90% confidencelimit(cut-off 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 obtained are shown in Table 3.All the wild boars of the experiment wereparasitized with larvae 1 Trichinella spp., with theexception of number 17, infected with T. britovi,for which no larva was detected using artificialdigestion.Wild boar number 19 showed a negative resultin digestion, although a humoral reaction wasobserved. On carrying out trichinelloscopy, weobserved calcified cysts which, due to infectioneffects, would lead to this animal being consideredpositive.ELISAThe results of the humoral evolution are shownin Figures 1, 2, 3 and 4.The four wild boars included in the controlgroup were free from Trichinella followingartificial digestion of more than 500g of muscletissue, and were shown to be negative for the fourantigens used by the ELISA technique throughoutthe experiment.In groups 3 and 6, higher infective doses wereapplied (20,000 larvae 1) and we have obtainedhigher infective loads. In groups 3 and 6 serologicalresponse increase from 15 d.p.i. and the globalreactivity was significantly higher than in otherinfected groups. In groups 2, 3 and 6, we obtain thehighest persistence of high reactivity, up until the54Rev. Ibero-Latinoam. Parasitol. (2011); 70 (1): 49-57

DETECTION OF TRICHINELLA SPP. IN WILD BOAR BY ELISATable 3. Artificial digestion resultsGroups and doses Wild boar l.p.g. a1 1 0.28T. spiralis 2 0.15(200 L1) 3 0.322 4 4.49T. spiralis 5 91.16(1,000 L1) 6 134.513 7 1276.19T. spiralis 8 206.77(20,000 L1) 9 1673.064 13 0.12T. britovi 14 0.04*(200 L1) 15 0.45 16 0.48**T. britovi 17 0(1,000 L1) 18 0.26 19 0T. britovi 20 203.28(20,000 L1) 21*** 196.35* The larvae were detected only in abdominal muscles(following standard procedure, on non-detection oflarvae in the diaphragm, we analyzed the rest of themuscles using artificial digestion).** The larvae were detected only in inter-costal muscles.*** Died at 30 days PI. Thus, it has not been includedin humoral evolution graphics.al.p.g., larvae per gram muscle tissueFigure 1. Antibody level of groups of wild boarsmeasured by ELISA employing crude antigen from T.spiralis.Figure 2. Antibody level of groups of wild boars measuredby ELISA employing crude antigen from T. britovi.Figure 3. Antibody level of groups of wild boars measuredby ELISA employing ES antigen from T. spiralis.Figure 4. Antibody level of groups of wild boars measuredby ELISA employing ES antigen from T. britovi.Rev. Ibero-Latinoam. Parasitol. (2011); 70 (1): 49-5755

J. A. GAMITO SANTOS et al.Table 4. Sensitivity found depending of 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 infection; ES, Excretor Secretor; T1, T. spiralis; T3, T. britovi.end of the experiment. Other groups reach levels ofdoubtful with some of the antigens used.We observed greater precocity and persistenceof antibodies using antigens homologous to theinfected species. This can be seen in figure 3 and4 where we observed greater precocity and higherpersistence of antibodies in groups of wild boars infectedwith T. britovi (groups 4, 5 and 6) as opposedto the ES T3 Antigen (Figure 4). This precocityand persistence is lower if we compare these samegroups as opposed to the ES T1 Antigen (Figure 3).The sensitivity found at 40 and 125 d.pi for eachantigen is displayed in Table 4.DISCUSSIONThe results obtained show a greater susceptibilityof wild boar to T. spiralis. This finding in wild boaris in accordance with results published by Kapel in2001 and Gamito-Santos et al, (2009).The immunoenzymatic diagnostic techniquesare more sensitive than artificial digestion, coincidingwith studies undertaken in porcine livestock,such as that of Van Knapen et al. (1980, 1981a,b,1984), Madden and Murrell (1990) or that of Nöckleret al, (2000). Thus, with ES T1 Antigen at 40d.pi, and ES T1 Antigens and crude T1 Antigen,both at 125 d.pi, the same sensitivity as digestionis obtained. The rest surpass digestion in terms ofsensitivity.Assessing both antigens, in our study ES T1Antigen is less sensitive than the crude T1 Antigenat 40 d.pi, contradicting studies by Gamble et al, in1983 and corroborating other studies in pigs suchas that of Lind et al, (1991), Bolás Fernández etal, (1993) or Pozio et al, (2003). ES T3 Antigenand crude T3 Antigen have the same sensitivity at40 d.pi as at 125 d.pi. We agree with Serrano et al,(1992) in a study of pigs.We have demonstrated a tendency for serologicalresponse to be more intense and earlier in wildboars infected with 20,000 larvae 1 (groups 3 and6) in comparison to those infected with 200 larvae 156Rev. Ibero-Latinoam. Parasitol. (2011); 70 (1): 49-57