DEVELOPMENT OF COPRO-POLYMERASE CHAIN REACTION FOR DETECTION OF ECONOMICALLY IMPORTANT GASTROINTESTINAL STRONGYLES IN CATTLE
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Date
2018
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COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR
Abstract
The study was conducted for development of copro-PCR for detection of
common strongyle species in bovines viz., Haemonchus, Trichostrongylus and
Mecistocirrus spp. Three hundred and twenty faecal samples were randomly
collected from different areas of Thrissur and the occurrence of strongylosis was
studied by different diagnostic methods like floatation, sedimentation and
coproculture. Strongyles had an overall occurrence of 36.8 per cent. Concurrent
infections with amphistomes (3.4%) and coccidia (1.5%) were also obtained. The
strongyle larvae identified on coproculture were Haemonchus spp. (14.68%),
Mecistocirrus sp. (8.4%), Trichostrongylus spp. (3.4%), Bunostomum sp. (3.1%)
and Cooperia spp. (1.25%). Adult strongyle worms collected from the entrails of
cattle from slaughter houses revealed an overall occurrence of 31.88 per cent. The
species identified were Haemonchus spp. (8.6%), Trichostrongylus spp. (7.2%),
Mecistocirrus sp. (14.4%) and Cooperia spp. (1.4%). Hence the predominant
strongyles selected in this study were Haemonchus spp., Mecistocirrus sp. and
Trichostrongylus spp. Gradient PCR protocols were standardised with primers
targeting the ETS region in H. placei, ITS-2, 28S rRNA partial regions in T.
colubriformis and ITS-1, 5.8S, ITS-2 partial regions of rRNA in M. digitatus. The
optimum annealing temperature of 60o
C was chosen in PCR protocols. Multiplex
PCR was standardised for simultaneous detection of the DNA of predominant
adult strongyles. Copro-PCR was standardised using copro DNA as template and
adult worm DNA as positive control. Multiplex copro-PCR was standardised for
simultaneous detection of multiple strongyle infections in the faecal sample of
cattle. Ninty five faecal samples were randomly collected from Thrissur and
subjected to copro-PCR and multiplex copro-PCR. Detection limits of adult DNA
by PCR were determined. A minimum of 0.45 fg/µl of DNA was required for H.
placei, 0.34 pg/µl for T. colubriformis and 0.093 ng/µl for M. digitatus. The cross
reactivity of primers were checked with DNA isolated from the predominant
strongyle species as well as with common ruminant trematodes. No cross reaction
were noticed among strongyles and with amphistomes and schistosomes.
Sensitivity and specificity of multiplex copro-PCR by Mc Nemar test, using
coproculture as the standard were obtained as 66.7 and 98.2 per cent respectively.
The diagnosis of strongylosis mainly relies on routine coproscopy and
coproculture, which are time consuming and laborious. Development of a rapid
molecular diagnostic method for differentiating important species of
gastrointestinal strongyles infecting cattle will be a good diagnostic tool and will
also help to improve and extend the technology into epidemiological studies,
strategic control and prevention of strongylosis. However, multiplex copro-PCR
could be employed as a suitable test for simultaneous detection and speciation of
strongyle infection in cattle. This will be useful in strategic selective treatment
and also signifies the importance of further research on molecular identification of
gastrointestinal strongyles.