DEVELOPMENT OF ELISA BASED DIAGNOSTICS FOR EARLY DETECTION OF COPROANTIGENS IN BOVINE AMPHISTOMOSIS
Loading...
![Thumbnail Image](assets/images/Item.jpg)
Date
2016-12-30
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR
Abstract
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.
Description
Submitted in partial fulfilment of the requirement for the degree of
Doctor of Philosophy in Veterinary Parasitology