Genetic analysis of emergence and spread of benzimidazole resistance genesin blood sucking Haemonchus placei in bovine

Abstract

Gastrointestinal parasitic (GI) nematodes are among the most economically important parasites to livestock costing the North American cattle industry alone more than $2 billion per year. The GI parasitic nematode species H. placei and H. contortus are amongst the most common and highly pathogenic parasite species of ruminants worldwide. Although these parasites have strong host preferences, they can infect multiple host species and co-infections have been reported. H. contortus is most commonly found in sheep and goats, whereas the closely related nematode H. placei is most common in cattle and buffalo. The two parasite species are phylogenetically close and experimental co-transplantation of adult parasites can result in hybridization between the two species to produce F1 progeny which, although sterile for inter-F1 mating, can propagate when backcrossed against the parental species. Hybridization between the species is an interesting phenomenon for the transfer of genetic material. The transfer of the resistance mutations from the H. contortus (high resistant) to H. placei (low resistant) is possible due to the process of hybridization. Hence, the interspecies hybridization is of particular interest due to its potential to provide a mechanism of drug resistance. Samples of Haemonchus spp. were collected from each abattoir of each district from 40 animals (cattle and buffaloes) over the year. A total of 240 (n=180 cattle, n=60 buffalo) worms were subjected for the collection of samples from 6 districts of Punjab over the year. Twenty worms per population on an average were subjected for the collection of samples from each abattoir. Age and sex of the animal were recorded at the time of collection of samples. Samples were preserved in 70% ethanol and transferred to lab for further processing. Genetic analysis was accomplished in a set of studies. Through Pyrosequencing of the rDNA, ITS-2 and P24, SNPs were investigated the extent to which co-infections between H. placei and H. contortus species occured in cattle and buffalo hosts in Pakistan. Interspecies hybridization and/or introgression of BZ-conferring mutations between the H. placei and H. contortus in the populations is analysed through 4 different validated genetic markers. (i) Sequencing of the rDNA ITS-2 region. (ii) PCR amplification and sequencing of the isotype-1 β-tubulin (iii) Microsatellite analysis of hybrid worms. Phylogenetic network analysis of hybrid β-tubulin isotype-1 sequences also used for identification of resistance alleles arisen independently in different parasite populations, as opposed to being introduced by migration, determined the relative importance of quarantine drenching versus other aspects of ‘on-farm’ anthelmintic use. We then undertook a phylogenetic analysis of resistance and susceptible alleles in these populations to investigate the different models of the origins and spread of BZ resistance mutations. Two statistical tests (Sign Test and Wilcoxon Test) were used for the population bottlenecking effect. According to the mod shift analysis, all populations had a normal L-shaped distribution. The overall results suggested that population studies did not diverge from genetic drift equilibrium. To prevent co-infection/ hybridization, stop co-grazing of small and large ruminants. For the control of spread of drug resistance, stop animal movement from one (resistant) to other (susceptible) area. By the use of deep amplicon sequencing technique, diagnose early stage of resistance to prevent the emergence and spread of drug resistance by using alternative drug groups.