- Email: [email protected]
Development of cathepsin-L cysteine proteinase based Dot-enzyme-linked immunosorbent assay for the diagnosis of Fasciola gigantica infection in buffaloes
Veterinary Parasitology 183 (2012) 382–385
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Development of cathepsin-L cysteine proteinase based Dot-enzyme-linked immunosorbent assay for the diagnosis of Fasciola gigantica infection in buffaloes Anju Varghese, O.K. Raina ∗ , Gaurav Nagar, Rajat Garg, P.S. Banerjee, B.R. Maharana, Justin D. Kollannur Division of Parasitology, Indian Veterinary Research Institute, Izatnagar 243122, India
a r t i c l e
i n f o
Article history: Received 23 December 2010 Received in revised form 12 April 2011 Accepted 15 July 2011 Keywords: Fasciola gigantica Buffalo Cathepsin-L cysteine proteinase Dot-ELISA Immunodiagnosis
a b s t r a c t Native cathepsin-L cysteine proteinase (28 kDa) was purified from the excretory secretory products of Fasciola gigantica and was used for sero-diagnosis of F. gigantica infection in buffaloes by Dot-enzyme-linked immunosorbent assay (Dot-ELISA). The test detected F. gigantica field infection in these animals with a sensitivity of ∼90%. No specific IgG antibody binding was displayed by sera obtained from 76 buffaloes considered to be Fasciola and other parasite-free by microscopic examination of faeces and necropsy examination of liver, rumen and intestine. Additionally, sera from 156 Fasciola-free buffaloes, yet infected with Gigantocotyle explanatum, Paramphistomum epiclitum, Gastrothylax spp., Strongyloides papillosus and hydatid cyst were all negative, indicating that F. gigantica cathepsin-L cysteine proteinase does not cross-react with these helminth parasites in natural infection of the host. The data indicated that cathepsin-L cysteine proteinase based Dot-ELISA reached ∼90% sensitivity and 100% specificity with relation to above parasites in the detection of bubaline fasciolosis. The present Dot-ELISA diagnostic assay is relevant to the field diagnosis of F. gigantica infection in buffaloes. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Tropical fasciolosis caused by Fasciola gigantica is a relentless constraint on the growth and productivity of cattle, buffaloes and sheep in the tropical countries (Spithill et al., 1997; Mehra et al., 1999; Yamasaki et al., 2002). Early prepatent diagnosis of fasciolosis in domestic ruminants is crucial for arresting its negative impact on productivity and sustained efforts towards developing specific serodiagnostic tests for early detection of fasciolosis in animals have shown a significantly high level of sensitivity with several antigenic preparations (Guobadia and Fagbemi, 1997; O’Neill et al., 1998; Cornelissen et al., 2001; Dixit
∗ Corresponding author. Tel.: +91 581 2315496; fax: +91 581 2302368. E-mail address: [email protected] (O.K. Raina). 0304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2011.07.032
et al., 2002, 2004; Yadav et al., 2005; Raina et al., 2006; Estuningsih et al., 2009; Anuracpreeda et al., 2011). Fasciola worms release substantial amounts of excretory–secretory products (ESPs) and cysteine proteinases are predominant antigens present in these ex vivo ESPs (Dalton and Heffernan, 1989; Smith et al., 1993; Jefferies et al., 2001; Morphew et al., 2007). The excretory–secretory products produced by Fasciola are key players in understanding the host–parasite interaction and offer targets for diagnosis, chemo- and immunotherapy (Morphew et al., 2007). Cathepsin-L cysteine proteinases have been exploited in the immunodiagnosis of Fasciola infection in human and animals and have given encouraging results for sensitive and specific diagnosis of the disease. The 28 and 26–27 kDa molecules of the excretory and secretory products were shown to essentially consist of cathepsin-L cysteine proteinases (Dixit et al., 2002, 2004) and when 28-kDa cysteine
A. Varghese et al. / Veterinary Parasitology 183 (2012) 382–385
proteinase was assayed in the prepatent diagnosis of fasciolosis, it detected F. gigantica experimental infection in bovine, bubaline and ovine hosts with 100% sensitivity (Dixit et al., 2002, 2004; Sriveny et al., 2006). This antigen was used in the ELISA format for the detection of bubaline fasciolosis and detected the infection with high sensitivity and specificity under field conditions (Raina et al., 2006). Present study aimed at developing a cysteine proteinase based Dot-ELISA in the immunodiagnosis of bubaline fasciolosis for field conditions. 2. Materials and methods 2.1. Parasite collection Fasciola gigantica flukes collected in physiological saline from the liver of buffaloes, slaughtered at a local abattoir, Bareilly, U.P., were brought to the laboratory and washed to remove host-origin material with normal saline. 2.2. Purification of cathepsin-L cysteine proteinase Cathepsin-L cysteine proteinases were purified from the F. gigantica regurgitant released in vitro in the excretory secretory products. Briefly, viable flukes were incubated in RPMI-1640 medium, pH 7.2 (Biological Industries, Israel) with one fluke/ml of the medium, supplemented with 0.2% sodium bicarbonate, 30 mM HEPES and 25 mg/l gentamycin. After 3–4 h incubation at 37 ◦ C, the culture medium with in vitro released parasite regurgitant, was centrifuged at 10,000 × g for 20 min and supernatant used for precipitation of the cysteine proteinase. Chilled ethanol was added to the culture supernatant, drop by drop, to a final concentration of 60% (v/v) and the suspension incubated at −20 ◦ C overnight. Proteins precipitating at 60% (v/v) ethanol concentration were pelleted at 6000 × g (20 min, 4 ◦ C) and discarded. Ethanol concentration in the supernatant was subsequently raised to 75% (v/v) and supernatant incubated overnight at −20 ◦ C. The precipitated proteins were centrifuged at 6000 × g (20 min, 4 ◦ C). The cysteine proteinases thus precipitated at 75% (v/v) ethanol concentration were rinsed in 70% ethanol, air dried and resuspended in PBS, pH 7.2. 2.3. Ion-exchange chromatography Three grams of DEAE Sephadex-A25 (Sigma Chemical Company, USA) were swollen in distilled water for 3–4 h and the resin was suspended in 10 bed volumes of 0.1 M NaOH. The resin was washed thoroughly with distilled water until its pH became neutral, re-suspended in 0.1 M HCl and washed as above. Thereafter, a 10 ml column was packed with the resin and equilibrated with 10–20 bed volumes of 100 mM, 50 mM, 20 mM and 10 mM sodium phosphate buffer (pH 8.0), respectively. CathepsinL cysteine proteinase earlier dialysed in 10 mM phosphate buffer (pH 8.0) was loaded onto the pre equilibrated column. Ion-exchanger bound cathepsin-L cysteine proteinase was eluted with a sodium chloride, non-linear gradient (0.1–0.5 M) in 10 mM phosphate buffer (pH 8.0). Purified protein was resolved on sodium dodecyl
383
sulphate-polyacrylamide gel electrophoresis and characterized as cathepsin-L cysteine proteinase (Yadav et al., 2005). 2.4. Dot-ELISA Dot-ELISA with soluble cathepsin-L cysteine proteinase was carried out with the antigen concentrations of 1 g, 500 ng, 200 ng and 100 ng, respectively, blotted to the nitrocellulose membrane (Biorad, California, USA). The antigen was reconstituted in PBS (pH 7.2) and dots of 1 l volume of the antigen were applied to the nitrocellulose strips. The antigen spots were air dried, blocked with 5% skimmed milk and washed with PBS-Tween-20 (wash buffer). Sera from F. gigantica infected buffaloes collected from a local abattoir and other areas were screened for anti-Fasciola antibodies at serial dilutions of 1:50–1:200. Sera collected from buffaloes infected with helminths other than Fasciola were also probed in the immunoassay and immuno-reactivity of the sera was probed with rabbit anti-bovine IgG–horse radish peroxidase (HRP) conjugate (Sigma Chemical Company, USA) at 1:2000 dilution. ELISA dots were developed with diaminobenzedine substrate (Sigma Chemical Company, USA) (DAB, 8 mg; PBS pH 7.2,10 ml; 8% nickle chloride, 50 l; hydrogen peroxide, 10 l, for a total 10 ml substrate volume). Sera positive for F. gigantica infection showed purple dots on the membrane while negative sera showed no dots. 2.5. Sensitivity and specificity Sera from buffaloes from F. gigantica non-endemic regions, mono-infected with helminths other than F. gigantica, were used to determine specificity of Dot-ELISA. The sensitivity was determined in F. gigantica infected buffaloes from F. gigantica endemic areas. The sensitivity ands specificity were calculated as, sensitivity = ELISA positive × 100/true positive and specificity = ELISA negative × 100/true negative. 3. Results Cathepsin-L cysteine proteinase (28 kDa) was purified to complete homogeneity following above purification protocol and the optimum concentration of the antigen for membrane coating was standardized to 200 ng/dot using chequer board titration. The results of the Dot-ELISA were read on the basis of colour development on nitrocellulose membrane, with positive reactions being read as purple dots and negative results with no dots (Fig. 1). Sensitivity of the Dot-ELISA was checked with the field sera collected from buffaloes at slaughter from F. gigantica endemic region of India. Fluke burden in the host was determined on necropsy as heavy (50 flukes and higher), moderate (11–50) and low (1–10). Out of 136 buffaloes, positive for F.gigantica infection by liver and faecal examination, 122 animals were positive by Dot-ELISA. The sera of the remaining 14 animals that were positive for adult flukes in the liver did not react with the antigen. The animals screened in this assay harboured either F. gigantica mono-infection or a mixed infection of F. gigantica with other helminths like
384
A. Varghese et al. / Veterinary Parasitology 183 (2012) 382–385
Fig. 1. Dot-ELISA showing reactivity of 11 animals infected with F. gigantica (3, 5, 6, 7, 8, 9, 13, 14, 15, 16 and 17) and 5 animals (2, 4, 10, 11 and 12) either negative for F. gigantica infection or infected with other helminths. Animals 1 and 18 (negative and positive controls).
G. explanatum, P. epiclitum, Gastrothylax spp. and hydatid (Table 1). Results of the Dot-ELISA indicated a sensitivity of 89.7%. The specificity of the assay was determined with buffalo sera collected from F. gigantica-free areas of Uttar Pradesh and Rajasthan (North India). A total of 186 buffaloes, harbouring either mono or a mixed infection with G. explanatum (n = 67), P. epiclitum (n = 31), Gastrothylax spp. (n = 24) and hydatid cysts (n = 9) at their slaughter, were probed to determine the specificity of the assay. Four buffalo calves coprologically positive for S. papillosus, were also screened for the specificity of the test. The assay showed no cross-reaction with these helminth parasites, as no sero-reactivity was produced with these sera. Some buffalo sera (14/325) from F. gigantica endemic areas showed positive reactivity in Dot-ELISA, when the animals were otherwise negative for F. gigantica adult flukes on liver examination and for their eggs on faecal examination. 4. Discussion Cathepsin-L cysteine proteinases have been considered as valuable antigens for the immunodiagnosis of fasciolosis. Fagbemi and Guobadia (1995) showed that 28 kDa cysteine protease of F. gigantica ensured high sensitivity and specificity of the ELISA for ruminant fasciolosis. Immuno-diagnosis of both human and animal fasciolosis has been advocated using cysteine proteinases as specific antigens (O’Neill et al., 1998; Cordova et al., 1999; Carnevale et al., 2001; Sriveny et al., 2006). F. gigantica cathepsin-L cysteine proteinase-based synthetic peptides for immunodiagnosis of sheep fasciolosis were developed for discriminating between early and late, and low and high burden sheep infection with F. gigantica (Jezek et al., 2008). Coproantigen capture ELISA based on monoclonal antibodies to cathepsin-L showed sensitivity and
specificity of 95 and 91%, respectively and the test as a useful diagnostic method to determine patent F. gigantica infection in cattle (Estuningsih et al., 2009). Several monoclonal antibodies, developed against a recombinant F. gigantica cathepsin-B3 protein, when probed against antigens of G. explanatum, Eurytrema pancreaticum, P. cervi, Schistosoma spindale, S. mansoni, Haemonchus placei and Setaria labiato-papillosa did not show cross-reactivity, indicating their potential in diagnosis (Anuracpreeda et al., 2011). Likewise, the 28 kDa cathepsin-L cysteine proteinase of F. gigantica has shown diagnostic potential in goats with a high sensitivity and specificity (Gupta et al., 2011). In our previous studies on 28 kDa cysteine proteinase for the detection of experimental F. gigantica infection in sheep, cattle and buffaloes, exposed to varying levels of metacercarial infection, quite a high sensitivity of ELISA was shown by this antigen (Dixit et al., 2002, 2004; Yadav et al., 2005; Sriveny et al., 2006). F. gigantica experimental infection in these ruminants was detected 3–4 weeks after metacercarial challenge. The standard ELISA was validated in the field diagnosis of F. gigantica infection in buffaloes, where it showed over 90% sensitivity and no-cross reaction with P. epliclitum, G. explanatum, Gastrothylax spp., hydatid and S. papillosus (Raina et al., 2006). The same antigen was assayed in the present Dot-ELISA format for its utility in the field detection of F. gigantica infection in buffaloes that showed a sensitivity of ∼90%. In our earlier assay using plate-ELISA, it was observed that there was no direct correlation between the fluke burden and the antibody titre in the buffaloes infected with the parasite; hence fluke burden does not seem to influence the sensitivity of this assay, rather the antibody response of the individual host determines the sensitivity of the assay (Raina et al., 2006). The same results were observed in the present Dot-blot assay where 10% animals (14/136) positive for F. gigantica adult flukes gave false negative results. It could be because of low antibody response elicited by these animals
Table 1 Cysteine proteinase based Dot-ELISA in buffaloes infected with F. gigantica and other helminths. Dot-ELISA positive animals
Helminth spp.
Faecal/necropsy positive animals
F. gigantica G. explanatum + P. epiclitum + Gastrothylax spp. G. explanatum + P. epiclitum + Gastrothylax spp. + F. gigantica Hydatid Hydatid + F. gigantica Strogyloides papillosus Negative
35 140 94 12 7 4 33
31 0 84 0 7 0 0
Total
325
122
A. Varghese et al. / Veterinary Parasitology 183 (2012) 382–385
to F. gigantica infection, below the sensitivity level of the assay. Buffaloes suffer from infection with the common helminth parasites like G. explanatum, P. epiclitum, Gastrothylax spp., S. papillosus and cystic echinococcosis and the specificity of the present dot-assay was 100% with respect to these helminthic infections. Cathepsin-L antigen based Dot-ELISA detected F. gigantica infection with a high sensitivity and specificity, indicating relevance of the assay for the sero-diagnosis of F. gigantica infection in buffaloes in the field conditions. However, Dot-ELISA showed positive reactivity in 14/325 buffaloes from F. gigantica endemic areas, when the animals were otherwise negative for F. gigantica adult flukes on liver examination or for its eggs in faecal examination. This could be a result of infection of the host with either immature F. gigantica, missed on liver examination or due to the persistence of the antibodies from the previous exposure of the host or the animals had been treated with anthelmintics post-infection. The present assay validated for F. gigantica infection in buffalo host needs to be tested for its sensitivity and specificity in cattle and sheep as well, where prevalence of fasciolosis is quite high. Also, there are endemic pockets of schistosomosis in India, with ruminants suffering from S. indicum, S. spindale, and S. nasale infections. Dot-ELISA needs to be tested for its cross-reactivity with these parasites. Antibody based assays do not differentiate between current and the previous infection, due to the persistence of the antibodies for prolonged period of time after treatment, yet the high sensitivity and ease of application encourages use of antibody detection assays. Dot-ELISA does not require special equipment or reagent and is simple and cost-effective, therefore, useful for field screening of animals for fasciolosis. Acknowledgments The authors are thankful to the Director, Indian Veterinary Research Institute, for providing facilities for conduction of this research work. References Anuracpreeda, P., Songkoomkrong, S., Sethadavit, M., Chotwiwatthanakun, C., Tinikul, Y., Sobhon, P., 2011. Fasciola gigantica: production and characterization of a monoclonal antibody against recombinant cathepsin B3. Exp. Parasitol. 127, 340–345. Carnevale, S., Rodriguez, M.I., Guarnera, E.A., Carmona, C., Tanos, T., Angel, S.O., 2001. Immunodiagnosis of fasciolosis using recombinant procathepsin-L cysteine proteinase. Diag. Microbiol. Infect. Dis. 41, 43–49. Cordova, M., Reategui, L., Espinoza, J.R., 1999. Immunodiagnosis of human fasciolosis with Fasciola hepatica cysteine proteinases. Trans. R. Soc. Trop. Med. Hyg. 93, 54–57.
385
Cornelissen, J.B.W.J., Gassenbeek, C.P.H., Borgsteede, F.H.M., Holland, W.G., Harmsen, M.M., Bocrsma, W.J.A., 2001. Early immunodiagnosis of fasciolosis in ruminants using recombinant Fasciola hepatica cathepsin-L like protease. Int. J. Parasitol. 31, 728–737. Dalton, J.P., Heffernan, M., 1989. Thiol proteases released in vitro by Fasciola hepatica. Mol. Biochem. Parasitol. 35, 161–166. Dixit, A.K., Yadav, S.C., Sharma, R.L., 2002. The 28 kDa Fasciola gigantica cysteine proteinase in the diagnosis of prepatent ovine fasciolosis. Vet. Parasitol. 109, 233–247. Dixit, A.K., Yadav, S.C., Sharma, R.L., 2004. Experimental bubaline fasciolosis: kinetics of antibody response using 28 kDa Fasciola gigantica cysteine proteinase as antigen. Trop. Anim. Health Prod. 36, 49–54. Estuningsih, E., Spithill, T., Raadsma, H., Law, R., Adiwinata, G., Meeusen, E., Piedrafita, D., 2009. Development and application of a faecal antigen diagnostic sandwich ELISA for estimating prevalence of Fasciola gigantica in cattle in Central Java, Indonesia. J. Parasitol. 95, 450–455. Fagbemi, B.O., Guobadia, E.E., 1995. Immunodiagnosis of fasciolosis in ruminants using 28 kDa cysteine proteinase of Fasciola gigantica adult worms. Vet. Parasitol. 57, 309–318. Guobadia, E.E., Fagbemi, B.O., 1997. The isolation of Fasciola gigantica specific antigens and their use in the serodiagnosis of fasciolosis in sheep by the detection of circulating antigens. Vet. Parasitol. 68, 269–282. Gupta, A., Dixit, A.K., Dixit, P., Mahajan, C., Sharma, R.L., 2011. Evaluation of dipstick-ELISA using 28 kDa Fasciola gigantica cathepsin-L cysteine proteinase (FgCL3) for serodiagnosis of fasciolosis in naturally infected goats. Vet. Parasitol. 176, 165–169. Jefferies, J.R., Campbell, A.M., Van Rossum, A.J., Barrett, J., Brophy, P.M., 2001. Proteomic analysis of Fasciola hepatica excretory–secretory products. Proteomics 1, 1128–1132. Jezek, J., El Ridi, R., Salah, M., Wagih, A., Aziz, H.W., Tallima, H., El Shafie, M.H., Khalek, T.A., Ammou, F.F., Strongylis, C., Moussis, V., Tsikaris, V., 2008. Fasciola gigantica cathepsin L proteinase-based synthetic peptide for immunodiagnosis and prevention of sheep fasciolosis. Biopolymers 90, 349–357. Mehra, U.R., Dass, R.S., Verma, A.K., Sharma, R.L., Yadav, S.C., 1999. Effect of Fasciola gigantica infection on growth and nutrient utilization in buffalo calves. Vet. Rec. 145, 699–702. Morphew, R.M., Wright, H.A., LaCourse, E.J., Woods, D.J., Brophy, P.M., 2007. Comparative proteomics of excretory–secretory proteins released by the liver fluke Fasciola hepatica in sheep host bile and during in vitro culture ex-host. Mol. Cell. Proteomic. 6, 963–972. O’Neill, S.M., Parkinson, M., Strauss, W., Angles, R., Dalton, J.P., 1998. Immunodiagnosis of Fasciola hepatica infection (fasciolosis) in a human population inn the Bolivian Altiplano using purified cathepsin L cysteine proteinase. Am. J. Trop. Med. Hyg. 58, 417–423. Raina, O.K., Yadav, S.C., Sriveny, D., Gupta, S.C., 2006. Immunodiagnosis of bubaline fasciolosis with Fasciola gigantica cathepsin-L and recombinant cathepsin-L1-D proteases. Acta Trop. 98, 145–151. Smith, A., Dowd, A., McGonigle, S.M., Keegan, P., Brennan, G., Trudgget, A., Dalton, J.P., 1993. Purification of cathepsin L like proteinase secreted by adult Fasciola hepatica. Mol. Biochem. Parasitol. 62, 1–8. Spithill, T.W., Piedrafita, D., Smooker, P.M., 1997. Immunological approaches for the control of fasciolosis. Int. J. Parasitol. 27, 1221–1235. Sriveny, D., Raina, O.K., Yadav, S.C., Chandra, D., Jayraw, A.K., Singh, M., Velusamy, R., Singh, B.P., 2006. Cathepsin L cysteine proteinase in the diagnosis of bovine Fasciola gigantica infection. Vet. Parasitol. 135, 25–31. Yadav, S.C., Saini, M., Raina, O.K., Nambi, P.A., Jadav, K., Sriveny, D., 2005. Fasciola gigantica cathepsin-L cysteine proteinase in the detection of early experimental fasciolosis in ruminants. Parasitol. Res. 97, 527–534. Yamasaki, H., Mineki, R., Murayama, K., Ito, A., Aoki, T., 2002. Characterization and expression of the Fasciola gigantica cathepsin-L gene. Int. J. Parasitol. 32, 1031–1042.
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Development of cathepsin-L cysteine proteinase based Dot-enzyme-linked immunosorbent assay for the diagnosis of Fasciola gigantica infection in buffaloes Anju Varghese, O.K. Raina ∗ , Gaurav Nagar, Rajat Garg, P.S. Banerjee, B.R. Maharana, Justin D. Kollannur Division of Parasitology, Indian Veterinary Research Institute, Izatnagar 243122, India
a r t i c l e
i n f o
Article history: Received 23 December 2010 Received in revised form 12 April 2011 Accepted 15 July 2011 Keywords: Fasciola gigantica Buffalo Cathepsin-L cysteine proteinase Dot-ELISA Immunodiagnosis
a b s t r a c t Native cathepsin-L cysteine proteinase (28 kDa) was purified from the excretory secretory products of Fasciola gigantica and was used for sero-diagnosis of F. gigantica infection in buffaloes by Dot-enzyme-linked immunosorbent assay (Dot-ELISA). The test detected F. gigantica field infection in these animals with a sensitivity of ∼90%. No specific IgG antibody binding was displayed by sera obtained from 76 buffaloes considered to be Fasciola and other parasite-free by microscopic examination of faeces and necropsy examination of liver, rumen and intestine. Additionally, sera from 156 Fasciola-free buffaloes, yet infected with Gigantocotyle explanatum, Paramphistomum epiclitum, Gastrothylax spp., Strongyloides papillosus and hydatid cyst were all negative, indicating that F. gigantica cathepsin-L cysteine proteinase does not cross-react with these helminth parasites in natural infection of the host. The data indicated that cathepsin-L cysteine proteinase based Dot-ELISA reached ∼90% sensitivity and 100% specificity with relation to above parasites in the detection of bubaline fasciolosis. The present Dot-ELISA diagnostic assay is relevant to the field diagnosis of F. gigantica infection in buffaloes. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Tropical fasciolosis caused by Fasciola gigantica is a relentless constraint on the growth and productivity of cattle, buffaloes and sheep in the tropical countries (Spithill et al., 1997; Mehra et al., 1999; Yamasaki et al., 2002). Early prepatent diagnosis of fasciolosis in domestic ruminants is crucial for arresting its negative impact on productivity and sustained efforts towards developing specific serodiagnostic tests for early detection of fasciolosis in animals have shown a significantly high level of sensitivity with several antigenic preparations (Guobadia and Fagbemi, 1997; O’Neill et al., 1998; Cornelissen et al., 2001; Dixit
∗ Corresponding author. Tel.: +91 581 2315496; fax: +91 581 2302368. E-mail address: [email protected] (O.K. Raina). 0304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2011.07.032
et al., 2002, 2004; Yadav et al., 2005; Raina et al., 2006; Estuningsih et al., 2009; Anuracpreeda et al., 2011). Fasciola worms release substantial amounts of excretory–secretory products (ESPs) and cysteine proteinases are predominant antigens present in these ex vivo ESPs (Dalton and Heffernan, 1989; Smith et al., 1993; Jefferies et al., 2001; Morphew et al., 2007). The excretory–secretory products produced by Fasciola are key players in understanding the host–parasite interaction and offer targets for diagnosis, chemo- and immunotherapy (Morphew et al., 2007). Cathepsin-L cysteine proteinases have been exploited in the immunodiagnosis of Fasciola infection in human and animals and have given encouraging results for sensitive and specific diagnosis of the disease. The 28 and 26–27 kDa molecules of the excretory and secretory products were shown to essentially consist of cathepsin-L cysteine proteinases (Dixit et al., 2002, 2004) and when 28-kDa cysteine
A. Varghese et al. / Veterinary Parasitology 183 (2012) 382–385
proteinase was assayed in the prepatent diagnosis of fasciolosis, it detected F. gigantica experimental infection in bovine, bubaline and ovine hosts with 100% sensitivity (Dixit et al., 2002, 2004; Sriveny et al., 2006). This antigen was used in the ELISA format for the detection of bubaline fasciolosis and detected the infection with high sensitivity and specificity under field conditions (Raina et al., 2006). Present study aimed at developing a cysteine proteinase based Dot-ELISA in the immunodiagnosis of bubaline fasciolosis for field conditions. 2. Materials and methods 2.1. Parasite collection Fasciola gigantica flukes collected in physiological saline from the liver of buffaloes, slaughtered at a local abattoir, Bareilly, U.P., were brought to the laboratory and washed to remove host-origin material with normal saline. 2.2. Purification of cathepsin-L cysteine proteinase Cathepsin-L cysteine proteinases were purified from the F. gigantica regurgitant released in vitro in the excretory secretory products. Briefly, viable flukes were incubated in RPMI-1640 medium, pH 7.2 (Biological Industries, Israel) with one fluke/ml of the medium, supplemented with 0.2% sodium bicarbonate, 30 mM HEPES and 25 mg/l gentamycin. After 3–4 h incubation at 37 ◦ C, the culture medium with in vitro released parasite regurgitant, was centrifuged at 10,000 × g for 20 min and supernatant used for precipitation of the cysteine proteinase. Chilled ethanol was added to the culture supernatant, drop by drop, to a final concentration of 60% (v/v) and the suspension incubated at −20 ◦ C overnight. Proteins precipitating at 60% (v/v) ethanol concentration were pelleted at 6000 × g (20 min, 4 ◦ C) and discarded. Ethanol concentration in the supernatant was subsequently raised to 75% (v/v) and supernatant incubated overnight at −20 ◦ C. The precipitated proteins were centrifuged at 6000 × g (20 min, 4 ◦ C). The cysteine proteinases thus precipitated at 75% (v/v) ethanol concentration were rinsed in 70% ethanol, air dried and resuspended in PBS, pH 7.2. 2.3. Ion-exchange chromatography Three grams of DEAE Sephadex-A25 (Sigma Chemical Company, USA) were swollen in distilled water for 3–4 h and the resin was suspended in 10 bed volumes of 0.1 M NaOH. The resin was washed thoroughly with distilled water until its pH became neutral, re-suspended in 0.1 M HCl and washed as above. Thereafter, a 10 ml column was packed with the resin and equilibrated with 10–20 bed volumes of 100 mM, 50 mM, 20 mM and 10 mM sodium phosphate buffer (pH 8.0), respectively. CathepsinL cysteine proteinase earlier dialysed in 10 mM phosphate buffer (pH 8.0) was loaded onto the pre equilibrated column. Ion-exchanger bound cathepsin-L cysteine proteinase was eluted with a sodium chloride, non-linear gradient (0.1–0.5 M) in 10 mM phosphate buffer (pH 8.0). Purified protein was resolved on sodium dodecyl
383
sulphate-polyacrylamide gel electrophoresis and characterized as cathepsin-L cysteine proteinase (Yadav et al., 2005). 2.4. Dot-ELISA Dot-ELISA with soluble cathepsin-L cysteine proteinase was carried out with the antigen concentrations of 1 g, 500 ng, 200 ng and 100 ng, respectively, blotted to the nitrocellulose membrane (Biorad, California, USA). The antigen was reconstituted in PBS (pH 7.2) and dots of 1 l volume of the antigen were applied to the nitrocellulose strips. The antigen spots were air dried, blocked with 5% skimmed milk and washed with PBS-Tween-20 (wash buffer). Sera from F. gigantica infected buffaloes collected from a local abattoir and other areas were screened for anti-Fasciola antibodies at serial dilutions of 1:50–1:200. Sera collected from buffaloes infected with helminths other than Fasciola were also probed in the immunoassay and immuno-reactivity of the sera was probed with rabbit anti-bovine IgG–horse radish peroxidase (HRP) conjugate (Sigma Chemical Company, USA) at 1:2000 dilution. ELISA dots were developed with diaminobenzedine substrate (Sigma Chemical Company, USA) (DAB, 8 mg; PBS pH 7.2,10 ml; 8% nickle chloride, 50 l; hydrogen peroxide, 10 l, for a total 10 ml substrate volume). Sera positive for F. gigantica infection showed purple dots on the membrane while negative sera showed no dots. 2.5. Sensitivity and specificity Sera from buffaloes from F. gigantica non-endemic regions, mono-infected with helminths other than F. gigantica, were used to determine specificity of Dot-ELISA. The sensitivity was determined in F. gigantica infected buffaloes from F. gigantica endemic areas. The sensitivity ands specificity were calculated as, sensitivity = ELISA positive × 100/true positive and specificity = ELISA negative × 100/true negative. 3. Results Cathepsin-L cysteine proteinase (28 kDa) was purified to complete homogeneity following above purification protocol and the optimum concentration of the antigen for membrane coating was standardized to 200 ng/dot using chequer board titration. The results of the Dot-ELISA were read on the basis of colour development on nitrocellulose membrane, with positive reactions being read as purple dots and negative results with no dots (Fig. 1). Sensitivity of the Dot-ELISA was checked with the field sera collected from buffaloes at slaughter from F. gigantica endemic region of India. Fluke burden in the host was determined on necropsy as heavy (50 flukes and higher), moderate (11–50) and low (1–10). Out of 136 buffaloes, positive for F.gigantica infection by liver and faecal examination, 122 animals were positive by Dot-ELISA. The sera of the remaining 14 animals that were positive for adult flukes in the liver did not react with the antigen. The animals screened in this assay harboured either F. gigantica mono-infection or a mixed infection of F. gigantica with other helminths like
384
A. Varghese et al. / Veterinary Parasitology 183 (2012) 382–385
Fig. 1. Dot-ELISA showing reactivity of 11 animals infected with F. gigantica (3, 5, 6, 7, 8, 9, 13, 14, 15, 16 and 17) and 5 animals (2, 4, 10, 11 and 12) either negative for F. gigantica infection or infected with other helminths. Animals 1 and 18 (negative and positive controls).
G. explanatum, P. epiclitum, Gastrothylax spp. and hydatid (Table 1). Results of the Dot-ELISA indicated a sensitivity of 89.7%. The specificity of the assay was determined with buffalo sera collected from F. gigantica-free areas of Uttar Pradesh and Rajasthan (North India). A total of 186 buffaloes, harbouring either mono or a mixed infection with G. explanatum (n = 67), P. epiclitum (n = 31), Gastrothylax spp. (n = 24) and hydatid cysts (n = 9) at their slaughter, were probed to determine the specificity of the assay. Four buffalo calves coprologically positive for S. papillosus, were also screened for the specificity of the test. The assay showed no cross-reaction with these helminth parasites, as no sero-reactivity was produced with these sera. Some buffalo sera (14/325) from F. gigantica endemic areas showed positive reactivity in Dot-ELISA, when the animals were otherwise negative for F. gigantica adult flukes on liver examination and for their eggs on faecal examination. 4. Discussion Cathepsin-L cysteine proteinases have been considered as valuable antigens for the immunodiagnosis of fasciolosis. Fagbemi and Guobadia (1995) showed that 28 kDa cysteine protease of F. gigantica ensured high sensitivity and specificity of the ELISA for ruminant fasciolosis. Immuno-diagnosis of both human and animal fasciolosis has been advocated using cysteine proteinases as specific antigens (O’Neill et al., 1998; Cordova et al., 1999; Carnevale et al., 2001; Sriveny et al., 2006). F. gigantica cathepsin-L cysteine proteinase-based synthetic peptides for immunodiagnosis of sheep fasciolosis were developed for discriminating between early and late, and low and high burden sheep infection with F. gigantica (Jezek et al., 2008). Coproantigen capture ELISA based on monoclonal antibodies to cathepsin-L showed sensitivity and
specificity of 95 and 91%, respectively and the test as a useful diagnostic method to determine patent F. gigantica infection in cattle (Estuningsih et al., 2009). Several monoclonal antibodies, developed against a recombinant F. gigantica cathepsin-B3 protein, when probed against antigens of G. explanatum, Eurytrema pancreaticum, P. cervi, Schistosoma spindale, S. mansoni, Haemonchus placei and Setaria labiato-papillosa did not show cross-reactivity, indicating their potential in diagnosis (Anuracpreeda et al., 2011). Likewise, the 28 kDa cathepsin-L cysteine proteinase of F. gigantica has shown diagnostic potential in goats with a high sensitivity and specificity (Gupta et al., 2011). In our previous studies on 28 kDa cysteine proteinase for the detection of experimental F. gigantica infection in sheep, cattle and buffaloes, exposed to varying levels of metacercarial infection, quite a high sensitivity of ELISA was shown by this antigen (Dixit et al., 2002, 2004; Yadav et al., 2005; Sriveny et al., 2006). F. gigantica experimental infection in these ruminants was detected 3–4 weeks after metacercarial challenge. The standard ELISA was validated in the field diagnosis of F. gigantica infection in buffaloes, where it showed over 90% sensitivity and no-cross reaction with P. epliclitum, G. explanatum, Gastrothylax spp., hydatid and S. papillosus (Raina et al., 2006). The same antigen was assayed in the present Dot-ELISA format for its utility in the field detection of F. gigantica infection in buffaloes that showed a sensitivity of ∼90%. In our earlier assay using plate-ELISA, it was observed that there was no direct correlation between the fluke burden and the antibody titre in the buffaloes infected with the parasite; hence fluke burden does not seem to influence the sensitivity of this assay, rather the antibody response of the individual host determines the sensitivity of the assay (Raina et al., 2006). The same results were observed in the present Dot-blot assay where 10% animals (14/136) positive for F. gigantica adult flukes gave false negative results. It could be because of low antibody response elicited by these animals
Table 1 Cysteine proteinase based Dot-ELISA in buffaloes infected with F. gigantica and other helminths. Dot-ELISA positive animals
Helminth spp.
Faecal/necropsy positive animals
F. gigantica G. explanatum + P. epiclitum + Gastrothylax spp. G. explanatum + P. epiclitum + Gastrothylax spp. + F. gigantica Hydatid Hydatid + F. gigantica Strogyloides papillosus Negative
35 140 94 12 7 4 33
31 0 84 0 7 0 0
Total
325
122
A. Varghese et al. / Veterinary Parasitology 183 (2012) 382–385
to F. gigantica infection, below the sensitivity level of the assay. Buffaloes suffer from infection with the common helminth parasites like G. explanatum, P. epiclitum, Gastrothylax spp., S. papillosus and cystic echinococcosis and the specificity of the present dot-assay was 100% with respect to these helminthic infections. Cathepsin-L antigen based Dot-ELISA detected F. gigantica infection with a high sensitivity and specificity, indicating relevance of the assay for the sero-diagnosis of F. gigantica infection in buffaloes in the field conditions. However, Dot-ELISA showed positive reactivity in 14/325 buffaloes from F. gigantica endemic areas, when the animals were otherwise negative for F. gigantica adult flukes on liver examination or for its eggs in faecal examination. This could be a result of infection of the host with either immature F. gigantica, missed on liver examination or due to the persistence of the antibodies from the previous exposure of the host or the animals had been treated with anthelmintics post-infection. The present assay validated for F. gigantica infection in buffalo host needs to be tested for its sensitivity and specificity in cattle and sheep as well, where prevalence of fasciolosis is quite high. Also, there are endemic pockets of schistosomosis in India, with ruminants suffering from S. indicum, S. spindale, and S. nasale infections. Dot-ELISA needs to be tested for its cross-reactivity with these parasites. Antibody based assays do not differentiate between current and the previous infection, due to the persistence of the antibodies for prolonged period of time after treatment, yet the high sensitivity and ease of application encourages use of antibody detection assays. Dot-ELISA does not require special equipment or reagent and is simple and cost-effective, therefore, useful for field screening of animals for fasciolosis. Acknowledgments The authors are thankful to the Director, Indian Veterinary Research Institute, for providing facilities for conduction of this research work. References Anuracpreeda, P., Songkoomkrong, S., Sethadavit, M., Chotwiwatthanakun, C., Tinikul, Y., Sobhon, P., 2011. Fasciola gigantica: production and characterization of a monoclonal antibody against recombinant cathepsin B3. Exp. Parasitol. 127, 340–345. Carnevale, S., Rodriguez, M.I., Guarnera, E.A., Carmona, C., Tanos, T., Angel, S.O., 2001. Immunodiagnosis of fasciolosis using recombinant procathepsin-L cysteine proteinase. Diag. Microbiol. Infect. Dis. 41, 43–49. Cordova, M., Reategui, L., Espinoza, J.R., 1999. Immunodiagnosis of human fasciolosis with Fasciola hepatica cysteine proteinases. Trans. R. Soc. Trop. Med. Hyg. 93, 54–57.
385
Cornelissen, J.B.W.J., Gassenbeek, C.P.H., Borgsteede, F.H.M., Holland, W.G., Harmsen, M.M., Bocrsma, W.J.A., 2001. Early immunodiagnosis of fasciolosis in ruminants using recombinant Fasciola hepatica cathepsin-L like protease. Int. J. Parasitol. 31, 728–737. Dalton, J.P., Heffernan, M., 1989. Thiol proteases released in vitro by Fasciola hepatica. Mol. Biochem. Parasitol. 35, 161–166. Dixit, A.K., Yadav, S.C., Sharma, R.L., 2002. The 28 kDa Fasciola gigantica cysteine proteinase in the diagnosis of prepatent ovine fasciolosis. Vet. Parasitol. 109, 233–247. Dixit, A.K., Yadav, S.C., Sharma, R.L., 2004. Experimental bubaline fasciolosis: kinetics of antibody response using 28 kDa Fasciola gigantica cysteine proteinase as antigen. Trop. Anim. Health Prod. 36, 49–54. Estuningsih, E., Spithill, T., Raadsma, H., Law, R., Adiwinata, G., Meeusen, E., Piedrafita, D., 2009. Development and application of a faecal antigen diagnostic sandwich ELISA for estimating prevalence of Fasciola gigantica in cattle in Central Java, Indonesia. J. Parasitol. 95, 450–455. Fagbemi, B.O., Guobadia, E.E., 1995. Immunodiagnosis of fasciolosis in ruminants using 28 kDa cysteine proteinase of Fasciola gigantica adult worms. Vet. Parasitol. 57, 309–318. Guobadia, E.E., Fagbemi, B.O., 1997. The isolation of Fasciola gigantica specific antigens and their use in the serodiagnosis of fasciolosis in sheep by the detection of circulating antigens. Vet. Parasitol. 68, 269–282. Gupta, A., Dixit, A.K., Dixit, P., Mahajan, C., Sharma, R.L., 2011. Evaluation of dipstick-ELISA using 28 kDa Fasciola gigantica cathepsin-L cysteine proteinase (FgCL3) for serodiagnosis of fasciolosis in naturally infected goats. Vet. Parasitol. 176, 165–169. Jefferies, J.R., Campbell, A.M., Van Rossum, A.J., Barrett, J., Brophy, P.M., 2001. Proteomic analysis of Fasciola hepatica excretory–secretory products. Proteomics 1, 1128–1132. Jezek, J., El Ridi, R., Salah, M., Wagih, A., Aziz, H.W., Tallima, H., El Shafie, M.H., Khalek, T.A., Ammou, F.F., Strongylis, C., Moussis, V., Tsikaris, V., 2008. Fasciola gigantica cathepsin L proteinase-based synthetic peptide for immunodiagnosis and prevention of sheep fasciolosis. Biopolymers 90, 349–357. Mehra, U.R., Dass, R.S., Verma, A.K., Sharma, R.L., Yadav, S.C., 1999. Effect of Fasciola gigantica infection on growth and nutrient utilization in buffalo calves. Vet. Rec. 145, 699–702. Morphew, R.M., Wright, H.A., LaCourse, E.J., Woods, D.J., Brophy, P.M., 2007. Comparative proteomics of excretory–secretory proteins released by the liver fluke Fasciola hepatica in sheep host bile and during in vitro culture ex-host. Mol. Cell. Proteomic. 6, 963–972. O’Neill, S.M., Parkinson, M., Strauss, W., Angles, R., Dalton, J.P., 1998. Immunodiagnosis of Fasciola hepatica infection (fasciolosis) in a human population inn the Bolivian Altiplano using purified cathepsin L cysteine proteinase. Am. J. Trop. Med. Hyg. 58, 417–423. Raina, O.K., Yadav, S.C., Sriveny, D., Gupta, S.C., 2006. Immunodiagnosis of bubaline fasciolosis with Fasciola gigantica cathepsin-L and recombinant cathepsin-L1-D proteases. Acta Trop. 98, 145–151. Smith, A., Dowd, A., McGonigle, S.M., Keegan, P., Brennan, G., Trudgget, A., Dalton, J.P., 1993. Purification of cathepsin L like proteinase secreted by adult Fasciola hepatica. Mol. Biochem. Parasitol. 62, 1–8. Spithill, T.W., Piedrafita, D., Smooker, P.M., 1997. Immunological approaches for the control of fasciolosis. Int. J. Parasitol. 27, 1221–1235. Sriveny, D., Raina, O.K., Yadav, S.C., Chandra, D., Jayraw, A.K., Singh, M., Velusamy, R., Singh, B.P., 2006. Cathepsin L cysteine proteinase in the diagnosis of bovine Fasciola gigantica infection. Vet. Parasitol. 135, 25–31. Yadav, S.C., Saini, M., Raina, O.K., Nambi, P.A., Jadav, K., Sriveny, D., 2005. Fasciola gigantica cathepsin-L cysteine proteinase in the detection of early experimental fasciolosis in ruminants. Parasitol. Res. 97, 527–534. Yamasaki, H., Mineki, R., Murayama, K., Ito, A., Aoki, T., 2002. Characterization and expression of the Fasciola gigantica cathepsin-L gene. Int. J. Parasitol. 32, 1031–1042.