Abstract
Small ruminants greatly influence the economic sustainability of rural community of a country. However, parasitic diseases, especially gastrointestinal (GI) nematodes are the major constraint on profitable small ruminant production worldwide, including Bangladesh, and one of the major problems is the emergence of anthelmintic resistance (AR) to commonly used anthelmintics. The present study was conducted in seven topographic zones of Bangladesh and also six organized sheep/ goat farms of Rajshahi, Dhaka, Sylhet, Jhenaidah and Mymensingh to investigate the level of GI nematodes infection and its associated risk factors, status of anthelmintic resistance, and explore genetic diversity of Haemonchus contortus populations of small ruminants during August- 2015 to July - 2018. Of the 2,570 animals examined, 1,566 (60.9%) were infected with one or more species of GI nematodes. The identified eggs were strongyles (55.3%), Strongyloides spp. (18.2%), and Trichuris spp. (2.4%). Haemonchus spp., Oesophagostomum spp., Trichostrongylus spp. and Bunostomum spp. were identified in strongyles group by coproculture. According to univariate analysis, species, age, breed, body condition, farming system, farm type, housing, socio-economic status, knowledge about GI nematodes and education level of farmers and season were significantly associated with GI nematode infections. However, by a final logistic regression model, poor body conditioned animals, backyard rearing, housing on muddy floor, poor socio-economic status and low education background of farmers were identified as the risk factors of GI nematode infections in small ruminants. Fecal egg count reduction test (FECRT) revealed that GI nematodes were resistant against albendazole, levamisole and ivermectin in studied farms. Coproculture revealed that Haemonchus spp., Oesophagostomum spp. and Trichostrongylus spp. were the resistant parasites. Albendazole resistance was also confirmed by the egg hatch assay in all the farms except the farm in Mymensingh. To further validate benzimidazole (BZ) resistance, allele specific PCR (AS-PCR) of H. contortus were performed which unmasked three genotype such as homozygous resistant (rr), heterozygous (rS) and homozygous susceptible (SS). The genotype frequencies were homozygous resistant (rr, 6%), heterozygous (rS, 59%) and homozygous susceptible (SS, 35%) among different topographic zones of Bangladesh. The allelic frequency of the mutation conferring resistance (r) ranged from 25% to 47% in zones. The genotype frequencies (rr, rS and SS) and allelic frequencies (r and s) varied significantly (p<0.05) in different zones of Bangladesh. To explore genetic variation within and among H. contortus populations, internal transcribed spacer-2 (ITS2) of nuclear ribosomal DNA and the mitochondrial nicotinamide dehydrogenase subunit 4 (nad4) genes were amplified and sequenced for 95 and 85 worms, respectively. Genetic analysis defined 19 distinct ITS2 genotypes and 77 unique nad4 haplotypes among the H. contortus isolates. The nucleotide diversities were 0.0098 and 0.025 for ITS2 and nad4 gene, respectively. Phylogenetic analysis (neighbour joining, maximum likelihood and maximum parsimony) of 77 haplotypes indicated the existence of two populations without marked specification of host and locations within H. contortus populations. By population genetic analysis, 93.67% of genetic variance was partitioned within the population. Very low genetic differentiation but high gene flow was observed within H. contortus populations in Bangladesh. In conclusion, the present study showed that GI nematodes are highly prevalent and AR is an emerging concern in Bangladesh. A high degree of gene flow was observed within and among H. contortus populations. To the best of my knowledge, this is the first comprehensive epidemiological investigation of GI nematodes and molecular characterization of H. contortus isolates of small ruminants in Bangladesh. These epidemiological findings would help formulate sustainable control strategy against GI nematode infections, and the obtained molecular data of H. contortus creating a basis for further epidemiological investigations, using more discriminative markers for tracing possible changes in the population structure of H. contortus.