Rapid detection of fasciolosis in ruminants by Coproantigen ELISA and Immunoblotting techniques

Abstract

Background: Fasciolosis is infection of small and large ruminants causing major economic losses and health issues to the livestock population. For the sake of successful treatment of the infection early diagnosis is the only choice for the farmers. Traditional methods like egg count method involved in the detection of fasciolosis are not viable because these methods require expertise as trematode eggs are not easily distinguished. Therefore, current study is a step towards application and development of rapid and accurate diagnostic method. Objective: To detect the antigenic proteins in somatic (SA) and excretory secretory (ES) products of Fasciola by using western blotting and development of an indirect ELISA against Fasciola copro-antigens for accurate detection of fasciolosis. Methodology: Adult worms were collected from the liver of cattle and buffaloes which were brought to the slaughterhouse of Peshawar for the purpose of slaughtering. More than 200 slaughtered animals were examined for Fasciola helminths collection. Adult helminths (n=150) were collected and subjected to phosphate buffer saline (PBS-pH-7.2) to clear contamination. Faecal samples from positive (n=33) and negative (n=20) animals were collected. The excretory/secretory antigen was extracted from live helminths kept in O.OIM PBS (1 helminth / 5 ml) for 24 hr and somatic antigen was collected from fluke homogenate. The protein concentration was determined by the Bradford method. The polyclonal antibodies were produced by inm1Unization of rabbits with somatic and excretory/secretory antigens. SDS-PAGE and western blot was performed to find the antigenic proteins. Copro-antigen ELISA was developed, and its sensitivity and specificity was calculated. Kappa value of developed ELISA was calculated to check the diagnostic performance of test. Results: The Fasciola ES Western blotting results with rabbit anti-Fasciola polyclonal IgG antisera recognized a minimum of 4 bands with maximal response against polypeptides of 70 kDa, 98 kDa, and 110 kDa and one above 250 kDa. In case of somatic antigen, the rabbit antiFasciola polyclonal IgG antiserum analysed a minimum of 6 protein bands with maximal reaction with polypeptides of 16 kDa, 35 kDa, and 100 kDa, 103 kDa, 250 kDa and one above 250 kDa. To evaluate the suitability of the ELISA for the detection of Fasciola antigens in faecal sample copro-antigen ELISA is performed. The sensitivity and specificity of diagnostic test with ES-polyclonal antibodies were 100% (95% CI: 89.42%- 100.00%) and 76.19% (95% CI: 52.83.30%-91.78%), respectively. The results of Kappa value revealed that the strength VI of agreement is almost substantial. The result with somatic antigen polyc1onal antibodies showed the specificity and sensitivity of diagnostic test were 100% (95% CI: 89.42%- 100.00%) and 90.00% (95% CI: 68.30%- 98.77%), respectively. However, Kappa value of test revealed that the strength of agreement is perfect. Conclusion: The result showed immunoblotting and copro-antigen ELISA was suitable rapid and accurate diagnostic methods to detect fasciolosis in ruminants. The present study recommended using purified fractions of somatic and excretory secretory antigens for development of rapid diagnostic tests.