DEVELOPMENT OF MULTIPLEX POLYMERASE CHAIN REACTION FOR DIAGNOSIS OF AMPHISTOMOSIS AND SCHISTOSOMOSIS IN DAIRY CATTLE
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Date
2018
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COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR
Abstract
Amphistomosis and schistosomosis are two snail borne trematode
infections of dairy cattle in tropical and sub- tropical regions. During the present
study three species of amphistomes were identified namely Gastrothylax
crumenifer, Fischoederius cobboldi and Paramphistomum cervi and two species
of schistosome namely Schistosoma spindale and S. indicum from rumen and
mesentery respectively, collected from Municipal Corporation slaughter house,
Thrissur. Gradient PCR protocols were standardised with primers targeting cox 2
gene of mitochondrial DNA of amphistomes and schistosomes. An optimum
annealing temperature of 61.2°C amplified product size of 618 bp for amphistome
and 454 bp for schistosome in multiplex PCR. A minimum concentration of
0.13pg/ µl of amphistome DNA detected by PCR. Cross reaction of amphistome
primer was checked by using DNA isolated from common nematodes and
schsitosomes. Absence of cross reaction with strongyles and schistosomes
confirmed the specificity of mitochondrial primer for amphistomes. Out of 250
faecal samples collected from dairy cattle in and around Thrissur district, 28.4 per
cent was found positive for amphistome ova and 4.0 per cent for schistosome ova
with an overall parasitic infection of 36.4 per cent by conventional microscopical
examination. Ova positive samples were used for standardising multiplex coproPCR. Primer targeting cox 2 gene of mitochondrial DNA of amphistome yielded
618 bp and those targeting mitochondrial DNA of schistosome yielded 454 bp
products. Screening of 75 faecal samples from dairy cattle was done to ascertain
the sensitivity and specificity of multiplex copro- PCR. Mc Nemars test revealed
significant sensitivity of 70 per cent, specificity of 100 per cent, 100 per cent
positive predictive value, 62.5 per cent negative predictive value with 80 per cent
accuracy. The reliability of test checked by receiver operating characteristics
curve indicated good predictability of standardised multiplex copro-PCR.
Standardised multiplex copro-PCR could be used for mass screening of dairy
cattle and forms a valuable tool in epidemiological studies. Simultaneous
detection of the two fluke infection helps in adopting appropriate treatment
decisions and control strategies against trematode infections in dairy cattle.