Epidemiology and molecular determination of benzimidazole resistance in Haemonchus contortus (Trichostrongylidae: Nematoda) from naturally infected Merino sheep in Lesotho

Abstract

Small ruminants, especially Merino sheep, play an important role in the socio-economic and livelihoods of rural, resource-poor communities in Lesotho. Diseases and poor livestock husbandry practices threaten the small ruminant industry in low-income countries. The present study was designed to investigate the epidemiology, genetic characterization, and detection of anthelmintic resistance in Haemonchus contortus in communally grazed Merino sheep in the four ecological zones of Lesotho. Chapter 1 of the thesis encapsulates the general background and epidemiology of gastrointestinal nematodes (GIN) in small ruminants. Objectives and hypotheses of the study are presented in this chapter. GINs cause substantial economic losses in the small ruminant industry and constitute a limiting factor worldwide. There is, however, little knowledge on the occurrence of GINs, their distribution, intensity, and development of anthelmintic resistance (AR) in small ruminants. Chapter 2 discusses an overview of H. contortus epidemiology in small ruminants. It further delves into GIN species identification, molecular characterization, and molecular detection of anthelmintic resistance. Chapter 3, presents the anthropogenic barriers and drivers associated with GIN control among smallholder Merino sheep farms in the four ecological zones of Lesotho. A questionnaire survey was designed to assess the activities in livestock management associated with GIN control in four ecological zones (Highlands, Foothills, Lowlands and Senqu River Valley) of Lesotho. Two hundred and twelve (212) respondents were interviewed across the four ecological zones. The study revealed a low literacy rate among Merino sheep farmers across Lesotho with male farmers (77.8%) dominating the industry. Highlands had significantly more animals per farm and predominantly practiced transhumance (93.6%). The respondents used commercial anthelmintic drugs enormously (98%) while herbal and indigenous remedies use was at 57.3%. Chapter 4, presents a stratified random and cross-sectional study investigating the prevalence, abundance, and geographical distribution of gastrointestinal parasites (GIPs) in Merino sheep. Six hundrend and two (602) Merino sheep from 20 ollection sites were vii sampled for faecal matter. The McMaster method of the faecal egg count was used to determine the identity, diversity, and intensity of GIPs from Merino sheep from four ecological zones of Lesotho. Coprocultures were prepared, and third-stage larvae were identified by morphological and morphometric methods under the microscope. The three GIPs identified in the present study comprised, strongyles, Eimeria spp., and Monezia spp., recording prevalence rates of 64%, 18%, and 1.3%, respectively. The Highlands had the highest strongyle burdens (1170.97 ± 113.134) that were statistically significant (p < 0.05) in comparison with other ecological zones. Third-stage larvae from coproculture were subjected to a morphometric species identification protocol and were exclusively identified as H. contortus. Chapter 5, provides a molecular confirmatory test for presumptive morphometric GIN identification owing to the close morphological resemblance between the larvae GINs of ruminants, to give a more conclusive identity. PCR protocol that amplified the Second Internal Transcribed Spacer gene (ITS-2) of the ribosomal DNA was run using universal NC1 and NC2 primer sets. Gel electrophoresis of the ITS-2 resulted in about 350 bp gene segment and the Sanger sequencing protocol confirmed the third-stage larvae identity as 100% H. contortus. The present initiative provides the first molecular characterization of H. contortus in Lesotho. The phylogenetic tree analysis clustered 17 out of 20 sequences into one clade. The median haplotype analysis produced four haplotypes out of 20 ITS-2 sequences. The mean haplotype and nucleotide diversity values of the ITS-2 gene were 0.7684 ± 0.0689 and 0.00519, respectively. The molecular variation had distribution within topographic populations at 94.35 % while 5.65% was between populations (FST=0.05651, p > 0.05). The ITS-2 gene demonstrated a genetic structure with the most diversity within the population but less differentiation between topographic sub-populations. Chapter 6, presents the molecular genotyping of the beta-tubulin isotype-1 gene to determine defined point mutations associated with benzimidazole resistance of H. contortus in the four ecological zones of Lesotho. Immoderate administration of anthelmintic drugs has been reported to exacerbate the development of anthelmintic resistance (AR) in GINs in small ruminants, adding an unforeseen financial burden to the industry. Genotyping the beta- tubulin isotype-1 partial gene revealed the presence of a benzimidazole resistance-associated mutation (TAC) in codon 200. Three different types of alleles associated with AR in H. viii contortus were identified: homozygous susceptible (SS = 51.7%), heterozygous (SR = 32.6%), and homozygous resistant (RR = 15.7%). The present study presents the first molecular evidence of AR of H. contortus from Merino sheep in Lesotho. The phylogenetic tree analysis of the beta-tubulin isotype-1 partial gene clustered all sequences into one clade, depicting a close evolutionary relationship among all seven sequences. The partial gene exhibited haplotype and nucleotide diversity of 1.000 ± 0.076 and 0.01891, respectively. The result depicted absolute haplotypic differentiation among all seven sequences. Chapter 7, entails general discussions on the interconnections of different factors from the respective project's sub-sections. The overwhelming intensity and vast distribution of H. contortus in the study suggest it is the predominant cause of helminthiasis in Merino sheep in Lesotho. The present study suggests that extensive use of anthelmintic drugs in Merino sheep is a probable cause of AR development in H. contortus. Genetic characterization is the basis for molecular epidemiology and molecular tracer studies that inform on prevention initiatives and resistance detection. Timely and periodic parasite surveys on AR are essential for strategic management, prevention, and control of GINs