DETECTION AND MOLECULAR CHARACTERIZATION OF BENZIMIDAZOLE RESISTANCE IN GASTROINTESTINAL NEMATODES OF GOATS
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Date
2017-06-23
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COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR
Abstract
The study was conducted with the objectives of assessing the status of
benzimidazole resistance in gastrointestinal (GI) nematodes of goats in Kerala,
detection of single nucleotide polymorphisms (SNPs) associated with
benzimidazole resistance in the β-tubulin gene of predominant GI nematode
species and to evaluate the efficacy of the egg hatch assay, larval development
assay and PCR-RFLP in detection of benzimidazole resistance. Microscopical
examination of 520 faecal samples collected from goats from 10 organized farms
and 16 small holder farmers’ flocks in eight agro-ecological zones of Kerala
revealed an overall prevalence of 81.5 + 5.54 per cent strongyles in goats. There
was significant difference between the prevalence of strongylosis in organized
farms (91.19 + 4.33 %) and small holder farmers’ flocks (65.34 + 10.27 %). The
mean faecal egg counts (FECs) also differed significantly between organized
farms and small holder farmers’ flocks. The prevalent genera of strongyles
identified on coproculture were Haemonchus spp., Trichostrongylus spp. and
Oesophagostomum spp. Faecal egg count reduction test (FECRT) done for
screening the benzimidazole resistance status in 10 organized farms and 16 small
holder farmers’ flocks identified benzimidazole resistance in all the organized
farms. Among the small holder farmers’ flocks, 43.75 per cent were found to be
resistant to benzimidazoles. Susceptibility was identified in 37.5 per cent of the
small holder farmers’ flocks while resistance was suspected in 18.75 per cent of
the flocks. Statistical analysis revealed significant association between the
resistance status and farm type. Haemonchus spp. was found to be the most
predominant GI nematode in post-treatment faecal cultures indicating that it is the
major species responsible for benzimidazole resistance. Resistance status was
found to be significantly correlated with the frequency of deworming in flocks.
Molecular genotyping by PCR-RFLP revealed E198A polymorphism in isotype 1
β-tubulin gene in Haemonchus spp. with an overall frequency of 0.516 for the
resistant allele (r). The overall prevalence of homozygous resistant genotype (rr)
at codon 198 in Haemonchus spp. was 25.6 per cent. No polymorphism was
identified at codons 167 and 200 in Haemonchus spp. in this study. In
Trichostrongylus spp., F200Y polymorphism was identified in isotype 1 β-tubulin
gene with an overall gene frequency of 0.337 for the resistant allele and with 28
per cent of the larvae genotyped being homozygous resistant (rr). Susceptible
genotype was identified at codons 167 and 198. All the Oesophagostomum spp.
larvae genotyped were found to be of the susceptible genotype at codons 198 and
200. In vitro detection of benzimidazole resistance was done by egg hatch assay
(EHA) and larval development assay (LDA). Correlation of the results of FECRT,
EHA, LDA and PCR-RFLP revealed significant correlation (p < 0.05) between
FECR per cent, ED50 in EHA, Pdd (proportion of larvae hatching at the
discriminating dose of 0.02 µg/ml) in LDA and the percentage of homozygous
resistant (rr) genotype in PCR-RFLP. There was significant correlation between
ED50 and Hdd (hatching ratio of strongyle eggs at the discriminating dose of 0.1
µg/ml) values in egg hatch assay. Pdd values were found to be significantly
correlated with other resistance parameters indicating that it is a better criterion
for resistance detection than LD50 in LDA. To predict the genotypic resistance
using phenotypic resistance indicators, regression equations were derived with rr
genotype per cent as dependent variable and FECR per cent, ED50 and Pdd as the
independent variables.
Description
Thesis Submitted in partial fulfilment of the requirement for the degree of Doctor of Philosophy in Veterinary Parasitology.