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Research
(Published
online: 20-05-2014)
13. Molecular diagnosis of benzimidazole
resistance in Haemonchus contortus in sheep from different
geographic regions of North India - S. Chandra, A.
Prasad, M. Sankar, N. Yadav and S. Dalal
Veterinary World, 7(5): 337-341
doi:
10.14202/vetworld.2014.337-341
S.
Chandra:
Division of Veterinary Parasitology, Indian Veterinary Indian
Veterinary Research Institute, Izatnagar - 243 122, Uttar Pradesh,
India
A.
Prasad:
Division of Veterinary Parasitology, Indian Veterinary Indian
Veterinary Research Institute, Izatnagar - 243 122, Uttar Pradesh,
India
M.
Sankar:
Tropical Animal Health and Production, Indian Veterinary Research
Institute, Mukteswar, Uttarakhand, India
N.
Yadav:
Division of Veterinary Parasitology, Indian Veterinary Indian
Veterinary Research Institute, Izatnagar - 243 122, Uttar Pradesh,
India
S.
Dalal:
Division of Veterinary Parasitology, Indian Veterinary Indian
Veterinary Research Institute, Izatnagar - 243 122, Uttar Pradesh,
India
Received: 06-03-2014, Revised: 09-04-2014, Accepted: 16-04-2014,
Published online: 20-05-2014
Corresponding author:
A.
Prasad, email: drarvindivri@rediffmail.com
Abstract
Aim: To monitor the
benzimidazole resistance in Haemonchus contortus in sheep from
different agro climatic zones of India reared in extensive or
intensive managemental conditions.
Materials and Methods: Faecal materials were collected from
sheep from organized as well as private farms located in
Rohilkhand region, Bundelkhand region, and Eastern Utter Pradesh.
Larvae were obtained by coproculture and identified as L3 of
Haemonchus contortus. Exsheathment of larvae was done by
incubating them in 180 µl of sodium hypochlorite for 5-20 minutes.
Genomic DNA was isolated from single larvae and amplification of β-tubulin
of Haemonchus contortus was done by primary and nested PCR.
PCR-RFLP was done with RsaI enzyme for species identification.
Genotyping was done by Allele specific PCR using Nested PCR
product as template and resulting fragments were separated by gel
electrophoresis in a 1.5% agarose gel in TAE buffer.
Results: Primary PCR size of β-tubulin gene of H. contortus
was 840 bp, whereas nested PCR amplified 774 bp size product.
Species was confirmed as H. contortus by PCR-RFLP which shows
three fragments at 440 bp, 190 bp, and 150 bp. From Rohilkhand
region out of 30 larvae, 19 (63%) were homozygous resistant (RR),
6 (20%) were homozygous susceptible (SS), and 5 (17%) were
heterozygous (RS). From Bundelkhand 34 (85%) were RR, 4 (10%) were
SS, and 2 (5%) were RS out of 40 larvae screened. From Eastern UP
(Jaunpur and Varanasi) 30 larvae each from Jaunpur and Varanasi
were genotyped;larvae from Jaunpur revealed 53% RR, 27% SS, and
20% RS whereas larvae from Varanasi genotyped showed 57% RR, 17%
SS, and 26% RS.
Conclusion: Due to indiscriminate use of Benzimidazole
group of drugs in organized and private farms, resistance to this
group of drugs is increasing at the field level.
Keywords: β-tubulin, allele specific PCR, benzimidazole
resistance, Haemonchus contortus.
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