Kerala Veterinary and Animal Sciences University, Wayanad

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DEVELOPMENT OF ELISA BASED DIAGNOSTICS FOR EARLY DETECTION OF COPROANTIGENS IN BOVINE AMPHISTOMOSIS
(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, 2016-12-30) H. SHAMEEM; K.DEVADA
Bovine amphistomosis is a relatively neglected snail borne trematode disease causing great economic loss to dairy farmers. Conventional ova detection often underestimates the true prevalence of the disease as many of the amphistomes are seasonally egg producing and the pathogenicity of the disease is due to immature flukes which do not lay eggs. The present study identified ten species of amphistomes viz. Fischoederius cobboldi, Gastrothylax crumenifer, F. elongatus, Carmyerius spatiosus, Paramphistomum epiclitum, P. cervi, Ceylonocotyle scoliocoelium, Cotylophoron cotylophorum, C. indicum and Explanatum explanatum from rumen collected from Municipal Corporation slaughter house, Thrissur. Fischoederius cobboldi was found to be the most predominant species. Protein profile of whole worm antigen of five species of amphistomes namely Fischoederius cobboldi, Gastrothylax crumenifer, F. elongatus, Carmyerius spatiosus, and Paramphistomum spp. and excretory-secretory antigens were analysed in one dimensional SDS-PAGE followed by western blotting with amphistome positive bovine sera. A 34 kDa polypeptide common to both whole worm antigen and excretory-secretory antigen was identified as immunogenic and useful for further diagnostic studies. Somatic antigens of G. crumenifer was partially purified by column chromatography and subjected to SDS-PAGE and immunoblotting. Cross reactivity of excretory-secretory antigen was ruled out by immunoblotting with schistosome and strongyle positive bovine sera. Excretory-secretory antigen was used to raise hyperimmune serum in guinea pigs and rabbits for use as detection antibodies. A coproantigen sandwich ELISA protocol was standardised using guinea pig and rabbit hyperimmune serum which could detect minimum 3 ng/µl of excretory-secretory antigen in dung. Out of 515 faecal samples collected from six agro-ecological zones of central Kerala, 362 (70%) were found to be positive by sandwich ELISA as against 165 (32%) by ova detection. Dot ELISA was also standardised as a rapid test for coproantigen detection in amphistomosis. Sensitivity and specificity of two tests were determined. Sandwich ELISA possessed a sensitivity of 90 per cent, specificity of 100 per cent while dot ELISA had a sensitivity of 76 per cent and specificity of 100 per cent. Statistical tests also depicted the reliability and high discriminating power of sandwich ELISA in early detection of amphistomosis.Statistical analysis showed no significant difference in the prevalence of amphistomes between the six agro-ecological zones of central Kerala but recorded highest infection in Palakkad plains (86%) followed by Central Midlands (80%). Molecular characterisation of four prevalent amphistomes namely F. cobboldi, G. crumenifer, F. elongatus, and Paramphistomum spp. was done by PCR targeting ITS-2+ rDNA sequences which yielded amplicons of 494 bp, 503 bp, 514 bp and 494 bp respectively. Nucelotide sequence analysis revealed eight base pair differences between pouched and unpouched species and also two base pair difference between G. crumenifer and Fischoederius spp. suggesting the utility of ITS-2+ region to be used as a species marker. In silico RE map analysis of four amphistomes identified unique recognition sites which could differentiate G. crumenifer from other three amphistomes. Phylogenetic analysis revealed clustering of Kerala isolates with Chennai, Shillong and Bareilly isolates.