DEVELOPMENT OF COPRO-POLYMERASE CHAIN REACTION FOR DETECTION OF ECONOMICALLY IMPORTANT GASTROINTESTINAL STRONGYLES IN GOATS
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Date
2021-11-30
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COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR
Abstract
The proposed study was undertaken to develop copro-PCR for detection of
economically important gastrointestinal strongyles in caprines. Strongylosis is an
economically important nematode infection that adversely affects the goat
population of small scale farmers in India. Adult strongyle worms were collected
from 76 visceral organs of goats and its overall prevalence was estimated to be
43.42 per cent. Haemonchus contortus was found to be the most prevalent (61.5 per
cent) followed by T. colubriformis (44 per cent) and O. columbianum (24 per cent).
Hence, these strongyles were selected, as they were predominant species which
cause production loss. The protocol for performing gradient PCR was standardised
using adult worms with the designed primers targeting partial 5.8S and ITS-2 region
of H. contortus, partial ITS-1 for O. columbianum and partial ITS-2 region was
selected for amplifying T. colubriformis. The optimum annealing temperature was
selected as 60.7oC and multiplex PCR was standardised using adult worm DNA
samples. The minimum detection limit of adult H. contortus, O. columbianum and
T. colubriformis DNA were 4.7 ag/µl, 0.31 fg/µl and 0.079 ng/µl respectively. No
cross reactions were noticed among these strongyles. The sequences of these
strongyles were accessed in GenBank with accession numbers. Copro-PCR was
standardised to detect strongyle infections of economically important strongyles in
the faecal sample with their adult DNA as positive control. Multiplex copro-PCR
was standardised to detect multiple infections in faecal samples. A total of 257
faecal samples were collected randomly from various places in and around Thrissur.
Various diagnostic procedures were conducted to study the occurrence of
strongylosis viz., direct examination, sedimentation, floatation and coproculture. The
overall prevalence of strongylosis was 70.42 per cent. Concurrent infections with
Strongyloides spp. was observed in 47 (19.66 per cent), Moniezia spp. in 23 (9.62
per cent), coccidia in nine (3.76 per cent) and Trichuris spp. in four (1.67 per cent)
samples. Coproculture was found to be the most specific conventional method for
species identification of strongyles which could detect 69.92 per cent of infection. In
coproculture also Haemonchus contortus was the predominant species (77.65 per
cent), followed by T. colubriformis (17.8 per cent), O. columbianum (7.82 per cent),
Bunostomum trigonocephalum (5.02 per cent) and Cooperia spp. (1.11 per cent).
Fifty faecal samples were subjected to copro-PCR and multiplex copro-PCR.
Sensitivity and specificity of multiplex copro-PCR with coproculture were assessed
with McNemar test and it was found to be 76.9 and 90.9 per cent respectively. The
area under receiver operating characteristic curve (ROC) showed 83.9 per cent
which was found to be a good diagnostic predictability. Hence, it was found to be a
rapid diagnostic tool for simultaneous detection of economically important
strongyles in goats and can be employed in epidemiological studies. The inclusion
of other primers of strongyles prevalent in that area could be effective for their
strategic control.
Description
Thesis Submitted in the partial fulfillment of the requirement for the degree of Master of Veterinary Science in Veterinary Parasitology