Molecular diagnosis of benzimidazole resistance in Haemonchus contortus in sheep from different geographic regions of North India

• Main Authors: S. Chandra, A. Prasad, M. Sankar, N. Yadav, S. Dalal
• Format: Article
• Language: English
• Published: Veterinary World 2014-05-01
• Series: Veterinary World
• Subjects: β-tubulin; allele specific PCR; benzimidazole resistance; Haemonchus contortus
• Online Access: http://www.veterinaryworld.org/Vol.7/May-2014/13.pdf

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 -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 -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.