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Polyclonal antibody based coproantigen detection immunoassay for diagnosis of Oesophagostomum columbianum infection in goats
Veterinary Parasitology 170 (2010) 262–267
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Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Polyclonal antibody based coproantigen detection immunoassay for diagnosis of Oesophagostomum columbianum infection in goats R. Jas a,*, J.D. Ghosh a, K. Das b a
Department of Veterinary Parasitology, Faculty of Veterinary and Animal Sciences, West Bengal University of Animal and Fishery Sciences, 37, Kshudiram Bose Sarani, Kolkata 700 037, India b Department of Veterinary Physiology, Faculty of Veterinary and Animal Sciences, West Bengal University of Animal and Fishery Sciences, 37, Kshudiram Bose Sarani, Kolkata 700 037, India
A R T I C L E I N F O
A B S T R A C T
Article history: Received 29 October 2009 Received in revised form 3 February 2010 Accepted 10 February 2010
A polyclonal antibody based coproantigen detection enzyme linked immunosorbent assay (cAg-ELISA) for diagnosis of experimental and natural Oesophagostomum columbianum infection in goats was developed and evaluated. Adult O. columbianum worms, collected from the caecum and colon of slaughtered goats, were triturated and cultured for obtaining infective third stage larvae (L3) and also used for preparation of excretory– secretory antigen (ESAg). Experimental goats were orally infected each with 600 L3/kg of the body weight. Filter sterilized faecal supernatant, i.e. the coproantigen (cAg) was harvested from the rectal faeces of all the infected goats, on alternate days from day-5 till day-31 after the infection. Hyperimmune serum (HIS) against ESAg of O. columbianum was raised in rabbits. Molecular and antigenic characterization of ES products of O. columbianum by HIS revealed that 50 and 39 kDa polypeptides were immuno-dominant. Coproantigen detection ELISA was standardized by using the cAg as coating antigen and its subsequent binding with the HIS against ESAg of O. columbianum. The sensitivity, specificity and accuracy of the standardized assay were determined by evaluating the assay on the faecal supernatant of 96 slaughtered goats taking into consideration their recorded parasitological status in respect of the abomasal and the intestinal parasites. The cAg-ELISA detected the prepatent oesophagostomosis on 20–24-day-post-infection with a sensitivity, specificity and accuracy of 88, 89.13 and 88.54%, respectively. The assay is relatively easy to perform and would serve as a reliable tool for detection of caprine nodular oesophagostomosis. ß 2010 Elsevier B.V. All rights reserved.
Keywords: Coproantigen ELISA Oesophagostomum columbianum Goat
1. Introduction Oesophagostomum columbianum, the cause of nodular enteritis or pimply gut, is one of the widely prevalent and pathogenic gastrointestinal nematodes causing considerable morbidity losses in small ruminants (Olivares et al., 2001) throughout the Asian sub-continent (Mohanta et al., 2007). Oesophagostomosis, like other helminth infections, is usually subclinical and chronic in nature. Major pathoge-
* Corresponding author. Tel.: +91 9748388238; fax: +91 3325571986. E-mail address: [email protected] (R. Jas). 0304-4017/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2010.02.013
nicity of this nematode parasite is caused due to the migratory or immature stages of the parasite. But the conventional method of diagnosis of caprine oesophagostomosis is usually based upon evaluation of clinical signs and microscopic faecal examination, which have their inherent limitations. Clinical signs usually become apparent only when the degree of infection is heavy and the eggs are passed in the faeces after the prepatent period of approximately 41 days (Soulsby, 1982) when the major damage is already done. In order to circumvent these limitations there is a pressing need for developing a reliable serological assay like enzyme linked immunosorbent assay (ELISA) to detect the infection, especially at the prepatent stage.
R. Jas et al. / Veterinary Parasitology 170 (2010) 262–267
Detection of specific serum antibodies against the parasite by ELISA is a rapid and simple method that allows processing of large number of samples at the same time. However such method detecting serum antibodies against parasites has yielded inconsistent results because of the complex nature of the parasite antigens (Charley et al., 1984) and the limited value of soluble extract as an antigen source (Parkhouse et al., 1987). Furthermore, detection of serum antibodies does not necessarily indicate the presence of a current infection. Assay for detection of parasitic antigens in the serum has recently emerged as the promising and reliable alternative to antibody detection assays. Consequently, detection of parasitic antigens in biological samples without involving any invasive procedures has, of late, attracted greater attention. Detection of parasitic antigens in host faeces, i.e. the coproantigens, therefore constitutes a novel method and holds considerable promise (Johnson et al., 1996; Deplazes et al., 1999; Lopera et al., 2003; Siavashi and Motamedi, 2006). Coproantigens possess potential diagnostic characteristics including stability in the gastrointestinal tract and under various temperatures (Deplazes et al., 1990). Such antigens are shed by both the mature and immature worms making their early diagnosis a plausible proposition. Moreover, their presence in detectable quantities and rapid disappearance after specific treatment make the antigen detection immunoassays a reliable alternative to the antibody detection assays (Deplazes et al., 1999). Hence a polyclonal antibody based coproantigen detection immunoassay was standardized for diagnosis of experimental and natural infection of O. columbianum in goat. 2. Materials and methods 2.1. Experimental animals Twenty Bengal goats (Capra hircus), 30–45-day-old, were procured from the university farm after coprological screening and maintained under helminth free conditions. All possible precautions were observed to preclude accidental parasitic infections. The goats were maintained on adequate amount of concentrate feed with provision for ad libitum clean drinking water. After attaining the age of 5 months (Alunda et al., 2003; Perez et al., 2003) they were used for the experimental establishment of O. columbianum infection. Adult New Zealand White rabbits (n = 6) were maintained in cages with provisions for recommended food and ad libitum water for raising hyperimmune serum (HIS) against excretory–secretory antigens (ESAg) of O. columbianum. 2.2. Experimental infections The caecum and colon of goats were dissected shortly after slaughter in the abattoir and the worms therein were recovered by a modified Baermann technique (Johnson et al., 2004). The adult female O. columbianum worms were microscopically separated out, triturated in small volume of distilled water and the triturates were then strained through a 300-mm sieve. The suspension so obtained was
263
cultured in heat-sterilized goat faecal powder (Soulsby, 1982). The third stage infective larvae (L3) of O. columbianum was collected from the culture following the standard methods, washed twice in distilled water and stored at 4 8C till further use. Two of the 20 goats were used as donors for obtaining sufficient stock of O. columbianum L3. Prior to artificial infection the negative status of the goats for gastrointestinal parasites was ascertained by standard coprological examination on three alternate days. The goats, after overnight withdrawal of feed, were infected orally with 600 L3 of O. columbianum/kg of the body weight. When the infection became patent the faeces of the infected goats, collected in faeces collecting bags, was cultured (Soulsby, 1982), the L3 were harvested, cleared of the debris and concentrated by Baermannization (Anon., 1971) before storing at 4 8C for experimental infection of the goats. Twelve of the 18 goats were artificially infected with L3 of O. columbianum following the procedure followed for the donors keeping six goats as uninfected control. 2.3. Preparation of coproantigen Rectal samples of faeces from all the infected goats were collected on alternate days from 5- to 31-day-postinfection (DPI) and the coproantigen was extracted separately for each goat according to the method of Elayoubi and Craig (2004). Fresh faeces were mixed (1:2, w/v) with 0.15 M phosphate buffer saline (PBS, pH 7.2) containing 0.3% Tween-20. The mixture was thoroughly homogenized in a clean pestle and mortar and the homogenate was spun at 2000 g in 4 8C for 45 min. The supernatant was collected and filter-sterilized in 0.22mm-syringe filter (Sartorius, Germany). The sterilized filtrate, constituted the coproantigen (cAg), was stored at 20 8C for use in the assay. Reference negative cAg was extracted from the uninfected control goats following the same method as stated above. The protein content of cAg of the infected and the control goats was estimated (Lowry et al., 1951) which was 3.21 and 2.13 mg/ml, respectively. 2.4. Preparation of excretory–secretory antigen (ESAg) Live O. columbianum worms were collected in 0.15 M PBS (pH 7.2) by a modified Baermann technique (Johnson et al., 2004) and washed five times in the same buffer with the final two washings in 0.15 M PBS (pH 7.2) containing Penicillin (100 IU/100 ml), Streptomycin (1 mg/100 ml) and Nystatin (1 mg/100 ml). Subsequently, 300 of these worms in 20 ml of the same antibiotic–antimycotic added PBS in a petridish were incubated at 37 8C for 24 h in a Biological Oxidation Demand incubator. The spent medium, i.e. the buffer was then collected in a dialysis bag (Dialysis Membrane-70, HiMedia, India) and dialyzed against 0.15 M PBS (pH 7.2) for 24 h at 4 8C with constant stirring on a magnetic stirrer. The fluid in the bag was concentrated to one-sixth of its volume by keeping the membrane bag in dextrose crystals and it was collected as ESAg of O. columbianum (ESAg-Oc). The ESAg-Oc, with the protein concentration of 2.42 mg/ml as estimated by the
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method of Lowry et al. (1951), was preserved at using in the assay.
20 8C for
2.5. Raising of hyperimmune serum (HIS) against ESAg-Oc New Zealand White rabbits were immunized by subcutaneous injection at multiple sites of the antigen (antigenic protein = 0.5 mg) emulsified with equal volume of Freund’s complete adjuvant. Subsequently, three booster doses of the antigenic protein (i.e. 1.0, 1.5 and 2.0 mg), emulsified with Freund’s incomplete adjuvant, were similarly inoculated (Mayer and Walker, 1990) at 1week intervals. Blood from the hyperimmunized rabbits was collected after 7 days of the last booster; serum was separated and stored at 20 8C for using in the assay. 2.6. Molecular and antigenic characterization of ESAg-Oc Sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) in 10% gel (Laemmli, 1970) was used for separating the peptides in the ESAg-Oc. Broad range molecular weight marker (3.0–205 kDa, GeNei, India) was used. After completion of the run the separating gel containing the marker proteins and the proteins of the ESAg-Oc was stained by Coomassie brilliant blue. Gel Documentation System (BioRad, Japan) was used for determination of the number and the molecular weight (MW) of the polypeptides in the test protein mixture. Antigenic characterization of ESAg-Oc was done by Western blotting technique (Towbin et al., 1979) using the HIS raised in rabbits. After separating the polypeptides by SDS-PAGE the lane containing the separated marker proteins was cut out carefully, stained by Coomassie brilliant blue and was resolved in the destaining solution (methanol – 3 parts, glacial acetic acid – 1 part and distilled water – 6 parts). The separated polypeptides in the remaining portion of the gel were electrophoretically transferred (Mayer and Walker, 1990) overnight at 4 8C to a nitrocellulose membrane (NCM, Sigma, USA) at constant current (50 mA/gel) in a Transblot Chamber (GeNei, India). After transfer the NCM was washed five times with the washing buffer, i.e. 0.1 M PBS, pH 7.4 containing 0.05% Tween 20 (PBST) and blocked at 37 8C for 2 h with blocking buffer (BF, washing buffer containing 3% bovine serum albumin fraction V). After washing as above the NCM was incubated with the HIS diluted (1:30) in BF for 3 h at 37 8C. The NCM, after washing was incubated in Horse radish peroxidase (HRPO) labeled rabbit anti-goat IgG (Sigma, USA) conjugate, diluted (1:1000) in BF, at 37 8C for 1 h. After similar washing the immuno-reactive bands on the NCM were visualized by dipping it in freshly prepared diaminobenzidine (DAB system, GeNei, India) solution. On appearance of distinct bands the enzyme-substrate reaction on the NCM was stopped by washing in distilled water.
against ESAg-Oc and the goat anti-rabbit IgG-HRPO conjugate (Sigma, USA) and the optimal test conditions were determined by chequer board dilution assay. Flat-bottom 96-well micro-ELISA plates (Nunc, Maxisorp) were coated with 100 ml of diluted (1:40) cAg-Oc (i.e. 6 mg of antigenic protein) in coating buffer (0.05 M carbonate–bicarbonate buffer, pH 9.6) and incubated overnight at 4 8C. After thorough washing with washing buffer (PBST) the wells were blocked by adding blocking buffer (2% bovine serum albumin in 0.05% PBST) and incubating the plate at 37 8C for 3 h. After thorough washing, 100 ml of heat-inactivated HIS (1:150) in blocking buffer was added in duplicate wells and the plate was incubated at 37 8C for 2 h. Goat anti-rabbit IgG-HRPO (100 ml, 1:1000 dilution) was added to the wells and the plate was incubated at 37 8C for 1 h. Freshly prepared solution of orthophenylene diamine dihydrochloride (OPD; Sigma, USA) in citrate phosphate buffer (pH 5.6) and H2O2 was then added after washing plates and incubated at 37 8C for 15 min. Thereafter the reaction was stopped by adding 50 ml of 2.5 M H2SO4 to all the wells. The optical density (OD) measured at 492 nm in the ELISA reader (Multiskan EX, ELISA Reader, Thermo, Japan). The mean OD492 nm value plus three times the standard deviation of the reference negative cAg was considered as the cut-off value for ELISA as per Johnson et al. (2004). 2.8. Faecal samples from natural source Rectal faeces and the respective abomasum and whole of the intestine of 96 goats slaughtered in the abattoir were collected following the standard procedures (Hansen and Perry, 1990). The parasitological status of the abomasum and the intestine was recorded separately for each goat. Coproantigens from the faeces was extracted and preserved as already described. 2.9. Evaluation of tests Coproantigens as extracted above were tested by the assay using the HIS prepared in the study and the assay was evaluated in respect of its sensitivity, specificity and accuracy as per Thrusfield (2003). 3. Results 3.1. Molecular and antigenic characterization of ESAg-Oc Out of 10 polypeptides in ESAg-Oc (MW ranged between 98 and 22.5 kDa) four polypeptides (52, 50, 39 and 22.5 kDa) were identified as the dominant ones by SDS-PAGE analysis (Fig. 1). Five of the 10 polypeptides (64, 50, 41, 39, 35.5 kDa) were immuno-reactive with ESAg-Oc in Western blot analysis (Fig. 2). There were two major immuno-reactive polypeptides having the MW of 50 and 39 kDa.
2.7. Coproantigen detection ELISA 3.2. Coproantigen detection ELISA The assay was performed following the methods used by Voller et al. (1976) and Singh et al. (1995), with some modifications. The optimal concentration of the ELISA reagents including dilution of the coating antigen, the HIS
The cut-off value for the standardized cAg-ELISA was 0.240. The earliest detection of cAg-Oc, in three goats only, by the assay was on 20 DPI, and on 24 DPI all the artificially
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Fig. 3. Follow-up coproantigen examination in 12 goats during experimentally induced infection with O. columbianum. The cut-off value was determined by calculating the mean OD492 nm value + 3 SD of faecal supernatant of negative control goats.
Fig. 1. Polypeptide profile of ESAg-Oc (L-1) as resolved by SDS-PAGE.
infected goats were positive in the assay (Fig. 3). The detection was much earlier than the patency of the infection, which in this study was 42 days. The ELISA was consistently detected the cAg in these goats during the subsequent period of observation till 31 DPI. 3.3. Sensitivity, specificity and accuracy of the assay The results of the standardized immunoassay performed on faecal supernatant of 96 slaughtered goats with known parasitological status are presented in Fig. 4. Seventy of the 96 gastrointestinal tracts were positive for different gastrointestinal (GI) parasites, while the remaining 26 were negative for any GI parasites. No monospecific infection with any parasite was recorded. In 50 of the 70 parasite-positive GI tracts O. columbianum (total worm burden, 9–191) along with other parasites viz., Haemonchus contortus, O. venulosum, Trichostrongylus sp., Bunostomum sp., Gaigeria sp., Moniezia expansa and
Trichuris ovis were recorded. While in the remaining 20 parasite-positive GI tracts mixed infections with a minimum of two of the above parasites excluding O. columbianum were recorded. In 44 of the faecal supernatants of these 50 O. columbianum positive goats the cAg was detected in the assay, while the remaining six were assay-negative. Mixed infection of O. columbianum (worm burden, 9–30) with Trichostrongylus, Moniezia expansa, Bunostomum sp. and T. ovis were recorded in these six false negative samples (total worm burden, 20–42). Out of 46 O. columbianum negative samples 41 were detected as negative by the cAg-ELISA, while five such goats were false positive in the assay. These five false positive goats had heavy worm burden of H. contortus (170–265) and T. ovis (143–239). The performance of the assay is summarized in Table 1. The sensitivity, specificity and accuracy of the cAg-ELISA in the detection of O. columbianum infection in goat were 88.00, 89.13 and 88.54%, respectively. 4. Discussion The immunological detection of soluble antigens of O. columbianum in the faeces, i.e. the coproantigen (cAg), of goats is a promising alternative to direct parasite detection
Fig. 2. Western blotting pattern of ESAg-Oc (L-1) against the homologous HIS.
Fig. 4. Detection of O. columbianum coproantigens by cAg-ELISA in rectal faeces of 96 slaughtered goats. The cut-off value was determined by calculating the mean OD492 nm value + 3 SD of faecal supernatant of negative control goats.
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Table 1 Performance status of the coproantigen detection ELISA on field goat with known parasitological status on autopsy. Number of goat examined
Number of goat positive for O. columbianum plus other g. i. parasites on autopsy ELISA +ve
ELISA
96
44
6
ve
by microscopic faecal and indirect diagnosis by detection of the specific antibodies (Jenkins and Rickard, 1985; Gasser et al., 1988). This study provides sufficient indications that cAg-ELISA, with its considerably high performance values in respect of sensitivity and specificity and accuracy, could be developed as a reliable tool for diagnosis of nodular oesophagostomosis in goat. This assay vis-a`-vis the conventional immunoassays based on detection of serum antibodies, offers the distinct advantage of detecting the parasite antigens at the early prepatent stage. Moreover, this assay, unlike other immunoassays based on detection of parasite antigens and antibodies in the serum, precludes the involvement of invasive methods. Coproantigens with the desirable characteristics of stability in the gastrointestinal tract and under temperature variations (Deplazes et al., 1990) are the ideal diagnostic target. Besides, detectable quantities of these antigens are shed in the faeces by both the mature and immature worms of the gastrointestinal tract and these antigens rapidly disappear on elimination of the worms by medication (Deplazes et al., 1999). With these advantages cAg-ELISA would prove superior to all the currently available diagnostic methods. The assay, on evaluation of its performance on field goats with known parasitological status, proved to be 88% sensitive and only six of the 50 O. columbianum positive goats were false negative in the assay. It is apparent that concentration of helminth ESAg in the faeces of the infected host directly correlates with the presence and number of the parasites in the intestine (Deplazes et al., 1990, 1992, 1999). False negative result in this assay, which was encouragingly low, might therefore be attributed to the low worm burden (i.e. 9–30) with faecal excretion of presumably non-detectable quantity of coproantigens. This was further substantiated in this study which revealed that the assay on the goats with higher worm burden (130) recorded a much higher OD values than the cut-off value, which commensurates well with the earlier work on T. circumcincta in sheep (Johnson et al., 2004). This study was an attempt to standardize cAgELISA for reliable detection of O. columbianum infection in goats and the outcome has been quite promising, although the assay was not sensitive in detecting the infection with a low worm burden. Nevertheless, in practical terms an assay capable of identifying the animals carrying heavy worm burdens that warrants anthelmintic treatment may be useful to the livestock owners. The assay with 89.13 and 88.54% specificity and accuracy, respectively, has considerable promise as a reliable method for diagnosis and molecular epidemiological studies of caprine oesophagostomosis. Out of the 46 goats negative for O. columbianum only five showed false
Number of parasite negative goat
ELISA +ve
ELISA
00
26
ve
Number of goat positive for parasites other than Oesophagostomum sp. ELISA +ve
ELISA
05
15
ve
positive reaction in the assay. These five goats, although negative for O. columbianum, but had heavy burden of H. contortus and T. ovis. Therefore, cross-reaction between HIS against ESAg-Oc and the cAg of H. contortus and T. ovis could not be ruled out as the reason for such false positive results. The cAg-ELISA was able to detect the infection as early as on 20 DPI besides detecting the patent infection. Hence this assay would be useful in identifying the goats harbouring prepatent stages of the worms that could be taken care of by using suitable anthelmintics. Consequently, the economic losses caused due the prepatent infection, whatever the degree may be, can be minimized. 5. Conclusion Coproantigens with the desirable stability under the commonly used storage conditions including deep freezing (Deplazes et al., 1990) are a novel target for diagnosis of caprine oesophagostomosis using the ELISA. The assay is relatively easy to perform and its further simplification for field use appears to be feasible. However, the lowest quantitative level of infection that is detectable in the assay needs to be determined and the specificity of the assay under field conditions is to be improved. Once these shortcomings are suitably addressed by appropriate experimentations and by using species-specific monoclonal antibodies the cAg-ELISA could be refined and modified as a reliable tool for early detection of caprine nodular oesophagostomosis. Acknowledgements The authors thankfully acknowledge the financial assistance of the Indian Council of Agricultural Research, Government of India, New Delhi, in conducting this study under the research project entitled ‘‘All India Network Programme on Gastrointestinal Parasitism’’. References Alunda, J.M., Cabillan, F.A., Cuquerella, M., 2003. Immunization against ovine haemonchosis with three low molecular weight somatic antigens of adult Haemonchus contortus. J. Vet. Med. B 50, 70–74. Anon., 1971. Manual of Veterinary Parasitological Laboratory Techniques, Technical Bulletin No. 18. Her Majesty’s Stationery Office, Ministry of Agriculture, Fisheries and Food, London, UK. Charley, P.J., Luffau, G., Pery, P., 1984. Serum and abomasal antibody response of sheep to infection with Haemonchus contortus. Vet. Parasitol. 14, 129–141. Deplazes, P., Alther, P., Tanner, I., Thomson, R.C., Eckert, J., 1999. Echinococcus multilocularis coproantigen detection by enzyme linked immunosorbent assay in fox, dog and cat populations. J. Parasitol. 85 (1), 115–121.
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Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Polyclonal antibody based coproantigen detection immunoassay for diagnosis of Oesophagostomum columbianum infection in goats R. Jas a,*, J.D. Ghosh a, K. Das b a
Department of Veterinary Parasitology, Faculty of Veterinary and Animal Sciences, West Bengal University of Animal and Fishery Sciences, 37, Kshudiram Bose Sarani, Kolkata 700 037, India b Department of Veterinary Physiology, Faculty of Veterinary and Animal Sciences, West Bengal University of Animal and Fishery Sciences, 37, Kshudiram Bose Sarani, Kolkata 700 037, India
A R T I C L E I N F O
A B S T R A C T
Article history: Received 29 October 2009 Received in revised form 3 February 2010 Accepted 10 February 2010
A polyclonal antibody based coproantigen detection enzyme linked immunosorbent assay (cAg-ELISA) for diagnosis of experimental and natural Oesophagostomum columbianum infection in goats was developed and evaluated. Adult O. columbianum worms, collected from the caecum and colon of slaughtered goats, were triturated and cultured for obtaining infective third stage larvae (L3) and also used for preparation of excretory– secretory antigen (ESAg). Experimental goats were orally infected each with 600 L3/kg of the body weight. Filter sterilized faecal supernatant, i.e. the coproantigen (cAg) was harvested from the rectal faeces of all the infected goats, on alternate days from day-5 till day-31 after the infection. Hyperimmune serum (HIS) against ESAg of O. columbianum was raised in rabbits. Molecular and antigenic characterization of ES products of O. columbianum by HIS revealed that 50 and 39 kDa polypeptides were immuno-dominant. Coproantigen detection ELISA was standardized by using the cAg as coating antigen and its subsequent binding with the HIS against ESAg of O. columbianum. The sensitivity, specificity and accuracy of the standardized assay were determined by evaluating the assay on the faecal supernatant of 96 slaughtered goats taking into consideration their recorded parasitological status in respect of the abomasal and the intestinal parasites. The cAg-ELISA detected the prepatent oesophagostomosis on 20–24-day-post-infection with a sensitivity, specificity and accuracy of 88, 89.13 and 88.54%, respectively. The assay is relatively easy to perform and would serve as a reliable tool for detection of caprine nodular oesophagostomosis. ß 2010 Elsevier B.V. All rights reserved.
Keywords: Coproantigen ELISA Oesophagostomum columbianum Goat
1. Introduction Oesophagostomum columbianum, the cause of nodular enteritis or pimply gut, is one of the widely prevalent and pathogenic gastrointestinal nematodes causing considerable morbidity losses in small ruminants (Olivares et al., 2001) throughout the Asian sub-continent (Mohanta et al., 2007). Oesophagostomosis, like other helminth infections, is usually subclinical and chronic in nature. Major pathoge-
* Corresponding author. Tel.: +91 9748388238; fax: +91 3325571986. E-mail address: [email protected] (R. Jas). 0304-4017/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2010.02.013
nicity of this nematode parasite is caused due to the migratory or immature stages of the parasite. But the conventional method of diagnosis of caprine oesophagostomosis is usually based upon evaluation of clinical signs and microscopic faecal examination, which have their inherent limitations. Clinical signs usually become apparent only when the degree of infection is heavy and the eggs are passed in the faeces after the prepatent period of approximately 41 days (Soulsby, 1982) when the major damage is already done. In order to circumvent these limitations there is a pressing need for developing a reliable serological assay like enzyme linked immunosorbent assay (ELISA) to detect the infection, especially at the prepatent stage.
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Detection of specific serum antibodies against the parasite by ELISA is a rapid and simple method that allows processing of large number of samples at the same time. However such method detecting serum antibodies against parasites has yielded inconsistent results because of the complex nature of the parasite antigens (Charley et al., 1984) and the limited value of soluble extract as an antigen source (Parkhouse et al., 1987). Furthermore, detection of serum antibodies does not necessarily indicate the presence of a current infection. Assay for detection of parasitic antigens in the serum has recently emerged as the promising and reliable alternative to antibody detection assays. Consequently, detection of parasitic antigens in biological samples without involving any invasive procedures has, of late, attracted greater attention. Detection of parasitic antigens in host faeces, i.e. the coproantigens, therefore constitutes a novel method and holds considerable promise (Johnson et al., 1996; Deplazes et al., 1999; Lopera et al., 2003; Siavashi and Motamedi, 2006). Coproantigens possess potential diagnostic characteristics including stability in the gastrointestinal tract and under various temperatures (Deplazes et al., 1990). Such antigens are shed by both the mature and immature worms making their early diagnosis a plausible proposition. Moreover, their presence in detectable quantities and rapid disappearance after specific treatment make the antigen detection immunoassays a reliable alternative to the antibody detection assays (Deplazes et al., 1999). Hence a polyclonal antibody based coproantigen detection immunoassay was standardized for diagnosis of experimental and natural infection of O. columbianum in goat. 2. Materials and methods 2.1. Experimental animals Twenty Bengal goats (Capra hircus), 30–45-day-old, were procured from the university farm after coprological screening and maintained under helminth free conditions. All possible precautions were observed to preclude accidental parasitic infections. The goats were maintained on adequate amount of concentrate feed with provision for ad libitum clean drinking water. After attaining the age of 5 months (Alunda et al., 2003; Perez et al., 2003) they were used for the experimental establishment of O. columbianum infection. Adult New Zealand White rabbits (n = 6) were maintained in cages with provisions for recommended food and ad libitum water for raising hyperimmune serum (HIS) against excretory–secretory antigens (ESAg) of O. columbianum. 2.2. Experimental infections The caecum and colon of goats were dissected shortly after slaughter in the abattoir and the worms therein were recovered by a modified Baermann technique (Johnson et al., 2004). The adult female O. columbianum worms were microscopically separated out, triturated in small volume of distilled water and the triturates were then strained through a 300-mm sieve. The suspension so obtained was
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cultured in heat-sterilized goat faecal powder (Soulsby, 1982). The third stage infective larvae (L3) of O. columbianum was collected from the culture following the standard methods, washed twice in distilled water and stored at 4 8C till further use. Two of the 20 goats were used as donors for obtaining sufficient stock of O. columbianum L3. Prior to artificial infection the negative status of the goats for gastrointestinal parasites was ascertained by standard coprological examination on three alternate days. The goats, after overnight withdrawal of feed, were infected orally with 600 L3 of O. columbianum/kg of the body weight. When the infection became patent the faeces of the infected goats, collected in faeces collecting bags, was cultured (Soulsby, 1982), the L3 were harvested, cleared of the debris and concentrated by Baermannization (Anon., 1971) before storing at 4 8C for experimental infection of the goats. Twelve of the 18 goats were artificially infected with L3 of O. columbianum following the procedure followed for the donors keeping six goats as uninfected control. 2.3. Preparation of coproantigen Rectal samples of faeces from all the infected goats were collected on alternate days from 5- to 31-day-postinfection (DPI) and the coproantigen was extracted separately for each goat according to the method of Elayoubi and Craig (2004). Fresh faeces were mixed (1:2, w/v) with 0.15 M phosphate buffer saline (PBS, pH 7.2) containing 0.3% Tween-20. The mixture was thoroughly homogenized in a clean pestle and mortar and the homogenate was spun at 2000 g in 4 8C for 45 min. The supernatant was collected and filter-sterilized in 0.22mm-syringe filter (Sartorius, Germany). The sterilized filtrate, constituted the coproantigen (cAg), was stored at 20 8C for use in the assay. Reference negative cAg was extracted from the uninfected control goats following the same method as stated above. The protein content of cAg of the infected and the control goats was estimated (Lowry et al., 1951) which was 3.21 and 2.13 mg/ml, respectively. 2.4. Preparation of excretory–secretory antigen (ESAg) Live O. columbianum worms were collected in 0.15 M PBS (pH 7.2) by a modified Baermann technique (Johnson et al., 2004) and washed five times in the same buffer with the final two washings in 0.15 M PBS (pH 7.2) containing Penicillin (100 IU/100 ml), Streptomycin (1 mg/100 ml) and Nystatin (1 mg/100 ml). Subsequently, 300 of these worms in 20 ml of the same antibiotic–antimycotic added PBS in a petridish were incubated at 37 8C for 24 h in a Biological Oxidation Demand incubator. The spent medium, i.e. the buffer was then collected in a dialysis bag (Dialysis Membrane-70, HiMedia, India) and dialyzed against 0.15 M PBS (pH 7.2) for 24 h at 4 8C with constant stirring on a magnetic stirrer. The fluid in the bag was concentrated to one-sixth of its volume by keeping the membrane bag in dextrose crystals and it was collected as ESAg of O. columbianum (ESAg-Oc). The ESAg-Oc, with the protein concentration of 2.42 mg/ml as estimated by the
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method of Lowry et al. (1951), was preserved at using in the assay.
20 8C for
2.5. Raising of hyperimmune serum (HIS) against ESAg-Oc New Zealand White rabbits were immunized by subcutaneous injection at multiple sites of the antigen (antigenic protein = 0.5 mg) emulsified with equal volume of Freund’s complete adjuvant. Subsequently, three booster doses of the antigenic protein (i.e. 1.0, 1.5 and 2.0 mg), emulsified with Freund’s incomplete adjuvant, were similarly inoculated (Mayer and Walker, 1990) at 1week intervals. Blood from the hyperimmunized rabbits was collected after 7 days of the last booster; serum was separated and stored at 20 8C for using in the assay. 2.6. Molecular and antigenic characterization of ESAg-Oc Sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) in 10% gel (Laemmli, 1970) was used for separating the peptides in the ESAg-Oc. Broad range molecular weight marker (3.0–205 kDa, GeNei, India) was used. After completion of the run the separating gel containing the marker proteins and the proteins of the ESAg-Oc was stained by Coomassie brilliant blue. Gel Documentation System (BioRad, Japan) was used for determination of the number and the molecular weight (MW) of the polypeptides in the test protein mixture. Antigenic characterization of ESAg-Oc was done by Western blotting technique (Towbin et al., 1979) using the HIS raised in rabbits. After separating the polypeptides by SDS-PAGE the lane containing the separated marker proteins was cut out carefully, stained by Coomassie brilliant blue and was resolved in the destaining solution (methanol – 3 parts, glacial acetic acid – 1 part and distilled water – 6 parts). The separated polypeptides in the remaining portion of the gel were electrophoretically transferred (Mayer and Walker, 1990) overnight at 4 8C to a nitrocellulose membrane (NCM, Sigma, USA) at constant current (50 mA/gel) in a Transblot Chamber (GeNei, India). After transfer the NCM was washed five times with the washing buffer, i.e. 0.1 M PBS, pH 7.4 containing 0.05% Tween 20 (PBST) and blocked at 37 8C for 2 h with blocking buffer (BF, washing buffer containing 3% bovine serum albumin fraction V). After washing as above the NCM was incubated with the HIS diluted (1:30) in BF for 3 h at 37 8C. The NCM, after washing was incubated in Horse radish peroxidase (HRPO) labeled rabbit anti-goat IgG (Sigma, USA) conjugate, diluted (1:1000) in BF, at 37 8C for 1 h. After similar washing the immuno-reactive bands on the NCM were visualized by dipping it in freshly prepared diaminobenzidine (DAB system, GeNei, India) solution. On appearance of distinct bands the enzyme-substrate reaction on the NCM was stopped by washing in distilled water.
against ESAg-Oc and the goat anti-rabbit IgG-HRPO conjugate (Sigma, USA) and the optimal test conditions were determined by chequer board dilution assay. Flat-bottom 96-well micro-ELISA plates (Nunc, Maxisorp) were coated with 100 ml of diluted (1:40) cAg-Oc (i.e. 6 mg of antigenic protein) in coating buffer (0.05 M carbonate–bicarbonate buffer, pH 9.6) and incubated overnight at 4 8C. After thorough washing with washing buffer (PBST) the wells were blocked by adding blocking buffer (2% bovine serum albumin in 0.05% PBST) and incubating the plate at 37 8C for 3 h. After thorough washing, 100 ml of heat-inactivated HIS (1:150) in blocking buffer was added in duplicate wells and the plate was incubated at 37 8C for 2 h. Goat anti-rabbit IgG-HRPO (100 ml, 1:1000 dilution) was added to the wells and the plate was incubated at 37 8C for 1 h. Freshly prepared solution of orthophenylene diamine dihydrochloride (OPD; Sigma, USA) in citrate phosphate buffer (pH 5.6) and H2O2 was then added after washing plates and incubated at 37 8C for 15 min. Thereafter the reaction was stopped by adding 50 ml of 2.5 M H2SO4 to all the wells. The optical density (OD) measured at 492 nm in the ELISA reader (Multiskan EX, ELISA Reader, Thermo, Japan). The mean OD492 nm value plus three times the standard deviation of the reference negative cAg was considered as the cut-off value for ELISA as per Johnson et al. (2004). 2.8. Faecal samples from natural source Rectal faeces and the respective abomasum and whole of the intestine of 96 goats slaughtered in the abattoir were collected following the standard procedures (Hansen and Perry, 1990). The parasitological status of the abomasum and the intestine was recorded separately for each goat. Coproantigens from the faeces was extracted and preserved as already described. 2.9. Evaluation of tests Coproantigens as extracted above were tested by the assay using the HIS prepared in the study and the assay was evaluated in respect of its sensitivity, specificity and accuracy as per Thrusfield (2003). 3. Results 3.1. Molecular and antigenic characterization of ESAg-Oc Out of 10 polypeptides in ESAg-Oc (MW ranged between 98 and 22.5 kDa) four polypeptides (52, 50, 39 and 22.5 kDa) were identified as the dominant ones by SDS-PAGE analysis (Fig. 1). Five of the 10 polypeptides (64, 50, 41, 39, 35.5 kDa) were immuno-reactive with ESAg-Oc in Western blot analysis (Fig. 2). There were two major immuno-reactive polypeptides having the MW of 50 and 39 kDa.
2.7. Coproantigen detection ELISA 3.2. Coproantigen detection ELISA The assay was performed following the methods used by Voller et al. (1976) and Singh et al. (1995), with some modifications. The optimal concentration of the ELISA reagents including dilution of the coating antigen, the HIS
The cut-off value for the standardized cAg-ELISA was 0.240. The earliest detection of cAg-Oc, in three goats only, by the assay was on 20 DPI, and on 24 DPI all the artificially
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Fig. 3. Follow-up coproantigen examination in 12 goats during experimentally induced infection with O. columbianum. The cut-off value was determined by calculating the mean OD492 nm value + 3 SD of faecal supernatant of negative control goats.
Fig. 1. Polypeptide profile of ESAg-Oc (L-1) as resolved by SDS-PAGE.
infected goats were positive in the assay (Fig. 3). The detection was much earlier than the patency of the infection, which in this study was 42 days. The ELISA was consistently detected the cAg in these goats during the subsequent period of observation till 31 DPI. 3.3. Sensitivity, specificity and accuracy of the assay The results of the standardized immunoassay performed on faecal supernatant of 96 slaughtered goats with known parasitological status are presented in Fig. 4. Seventy of the 96 gastrointestinal tracts were positive for different gastrointestinal (GI) parasites, while the remaining 26 were negative for any GI parasites. No monospecific infection with any parasite was recorded. In 50 of the 70 parasite-positive GI tracts O. columbianum (total worm burden, 9–191) along with other parasites viz., Haemonchus contortus, O. venulosum, Trichostrongylus sp., Bunostomum sp., Gaigeria sp., Moniezia expansa and
Trichuris ovis were recorded. While in the remaining 20 parasite-positive GI tracts mixed infections with a minimum of two of the above parasites excluding O. columbianum were recorded. In 44 of the faecal supernatants of these 50 O. columbianum positive goats the cAg was detected in the assay, while the remaining six were assay-negative. Mixed infection of O. columbianum (worm burden, 9–30) with Trichostrongylus, Moniezia expansa, Bunostomum sp. and T. ovis were recorded in these six false negative samples (total worm burden, 20–42). Out of 46 O. columbianum negative samples 41 were detected as negative by the cAg-ELISA, while five such goats were false positive in the assay. These five false positive goats had heavy worm burden of H. contortus (170–265) and T. ovis (143–239). The performance of the assay is summarized in Table 1. The sensitivity, specificity and accuracy of the cAg-ELISA in the detection of O. columbianum infection in goat were 88.00, 89.13 and 88.54%, respectively. 4. Discussion The immunological detection of soluble antigens of O. columbianum in the faeces, i.e. the coproantigen (cAg), of goats is a promising alternative to direct parasite detection
Fig. 2. Western blotting pattern of ESAg-Oc (L-1) against the homologous HIS.
Fig. 4. Detection of O. columbianum coproantigens by cAg-ELISA in rectal faeces of 96 slaughtered goats. The cut-off value was determined by calculating the mean OD492 nm value + 3 SD of faecal supernatant of negative control goats.
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Table 1 Performance status of the coproantigen detection ELISA on field goat with known parasitological status on autopsy. Number of goat examined
Number of goat positive for O. columbianum plus other g. i. parasites on autopsy ELISA +ve
ELISA
96
44
6
ve
by microscopic faecal and indirect diagnosis by detection of the specific antibodies (Jenkins and Rickard, 1985; Gasser et al., 1988). This study provides sufficient indications that cAg-ELISA, with its considerably high performance values in respect of sensitivity and specificity and accuracy, could be developed as a reliable tool for diagnosis of nodular oesophagostomosis in goat. This assay vis-a`-vis the conventional immunoassays based on detection of serum antibodies, offers the distinct advantage of detecting the parasite antigens at the early prepatent stage. Moreover, this assay, unlike other immunoassays based on detection of parasite antigens and antibodies in the serum, precludes the involvement of invasive methods. Coproantigens with the desirable characteristics of stability in the gastrointestinal tract and under temperature variations (Deplazes et al., 1990) are the ideal diagnostic target. Besides, detectable quantities of these antigens are shed in the faeces by both the mature and immature worms of the gastrointestinal tract and these antigens rapidly disappear on elimination of the worms by medication (Deplazes et al., 1999). With these advantages cAg-ELISA would prove superior to all the currently available diagnostic methods. The assay, on evaluation of its performance on field goats with known parasitological status, proved to be 88% sensitive and only six of the 50 O. columbianum positive goats were false negative in the assay. It is apparent that concentration of helminth ESAg in the faeces of the infected host directly correlates with the presence and number of the parasites in the intestine (Deplazes et al., 1990, 1992, 1999). False negative result in this assay, which was encouragingly low, might therefore be attributed to the low worm burden (i.e. 9–30) with faecal excretion of presumably non-detectable quantity of coproantigens. This was further substantiated in this study which revealed that the assay on the goats with higher worm burden (130) recorded a much higher OD values than the cut-off value, which commensurates well with the earlier work on T. circumcincta in sheep (Johnson et al., 2004). This study was an attempt to standardize cAgELISA for reliable detection of O. columbianum infection in goats and the outcome has been quite promising, although the assay was not sensitive in detecting the infection with a low worm burden. Nevertheless, in practical terms an assay capable of identifying the animals carrying heavy worm burdens that warrants anthelmintic treatment may be useful to the livestock owners. The assay with 89.13 and 88.54% specificity and accuracy, respectively, has considerable promise as a reliable method for diagnosis and molecular epidemiological studies of caprine oesophagostomosis. Out of the 46 goats negative for O. columbianum only five showed false
Number of parasite negative goat
ELISA +ve
ELISA
00
26
ve
Number of goat positive for parasites other than Oesophagostomum sp. ELISA +ve
ELISA
05
15
ve
positive reaction in the assay. These five goats, although negative for O. columbianum, but had heavy burden of H. contortus and T. ovis. Therefore, cross-reaction between HIS against ESAg-Oc and the cAg of H. contortus and T. ovis could not be ruled out as the reason for such false positive results. The cAg-ELISA was able to detect the infection as early as on 20 DPI besides detecting the patent infection. Hence this assay would be useful in identifying the goats harbouring prepatent stages of the worms that could be taken care of by using suitable anthelmintics. Consequently, the economic losses caused due the prepatent infection, whatever the degree may be, can be minimized. 5. Conclusion Coproantigens with the desirable stability under the commonly used storage conditions including deep freezing (Deplazes et al., 1990) are a novel target for diagnosis of caprine oesophagostomosis using the ELISA. The assay is relatively easy to perform and its further simplification for field use appears to be feasible. However, the lowest quantitative level of infection that is detectable in the assay needs to be determined and the specificity of the assay under field conditions is to be improved. Once these shortcomings are suitably addressed by appropriate experimentations and by using species-specific monoclonal antibodies the cAg-ELISA could be refined and modified as a reliable tool for early detection of caprine nodular oesophagostomosis. Acknowledgements The authors thankfully acknowledge the financial assistance of the Indian Council of Agricultural Research, Government of India, New Delhi, in conducting this study under the research project entitled ‘‘All India Network Programme on Gastrointestinal Parasitism’’. References Alunda, J.M., Cabillan, F.A., Cuquerella, M., 2003. Immunization against ovine haemonchosis with three low molecular weight somatic antigens of adult Haemonchus contortus. J. Vet. Med. B 50, 70–74. Anon., 1971. Manual of Veterinary Parasitological Laboratory Techniques, Technical Bulletin No. 18. Her Majesty’s Stationery Office, Ministry of Agriculture, Fisheries and Food, London, UK. Charley, P.J., Luffau, G., Pery, P., 1984. Serum and abomasal antibody response of sheep to infection with Haemonchus contortus. Vet. Parasitol. 14, 129–141. Deplazes, P., Alther, P., Tanner, I., Thomson, R.C., Eckert, J., 1999. Echinococcus multilocularis coproantigen detection by enzyme linked immunosorbent assay in fox, dog and cat populations. J. Parasitol. 85 (1), 115–121.
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