Gastrointestinal nematodes infecting sheep in Limpopo province : Seasonal prevalence and anthelmintic resistance
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The annual cost associated with treatment of parasitic diseases in small ruminants is estimated to tens of billions of US dollars worldwide, from the sales of anthelmintic drugs by pharmaceutical companies, excluding production losses. In small ruminants, gastrointestinal nematodes (GINs) can result in anaemia due to the blood-sucking activities of some nematodes species which impact negatively on the profitability of the farm. Objectives of this study were to determine the seasonal occurrence of gastrointestinal nematodes (GINs) of sheep of of resource-poor farmers in Limpopo province of South Africa, risk factors associated with anthelmintic resistance (AR) and to assess the efficacy of most commonly used anthelmintics. Furthermore, to determine the phylogenetic position and genetic diversity of the most pathogenetic nematode species of sheep, Haemonchus contortus isolated from sheep in the Limpopo province. The study was conducted in five districts of the Limpopo province, namely Capricorn, Sekhukhune, Waterberg, Vhembe and Mopani for a period of 21 months. To determine the seasonal prevalence of GINs faecal samples were collected from 156 sheep in each district. They were analysed using the McMaster technique to determine faecal egg counts and faecal cultures were prepared for nematode identification. FAMACHA© was used to assess anaemia in study animals and monthly climate data were acquired from South African Weather Services (SAWS). A structured questionnaire with a combination of qualitative and quantitative, open-ended questions was administered to 77 sheep farmers in Limpopo province of South Africa to evaluate their knowledge on the use of anthelmintics. To determine anthelmintic resistance (AR) in GINs of sheep both in vivo and in vitro techniques were used. Forty sheep from flocks with high treatment frequencies from each of the five districts were divided into three treated groups and one untreated control group. Group 1 was treated subcutaneously with ivermectin (Ivomec®, Merial, 0.2 mg/kg bw), group 2 was orally dosed with levamisole (Tramisol Ultra®, Coopers and Intervet, 5 mg/kg bw) and the third group was orally dosed with albendazole (Valbazen®, Pfizer, 7.5 mg/kg bw). Group 4 represented the untreated control. Egg hatch assay (EHA) was used to determine AR against thiabendazole (TBZ) and micro-argar larval development test (MALDT) was used for both TBZ and levamisole (LEV). Phylogenetic position as well as genetic diversity of Haemonchus contortus isolated from naturally infected sheep in Limpopo Province, South Africa, in relation to the worldwide populations, was determined using the ITS2 gene region to amplify 259 bp DNA fragment using species-specific primers. Data were analysed using Statistical Analysis System (SAS). A high nematode prevalence ranging from 88 to 99% was recorded in all districts. During the cold dry season, prevalence decreased to a range between 75 and 83%. However, the observed decrease in egg per gram of faeces (EPG’s) during the cold dry season did not differ significantly (p ˃ 0.05) among the districts except for Mopani and Vhembe districts. Haemonchus contortus was the most dominant nematode species (70 – 93%) in all the districts followed by Trichostrongylus/Teladorsagia spp. (5-28%) and Oesophagostomum columbianum (˂5%) An increase in FAMACHA© scores was recorded when the FEC increased, resulting in a positive correlation (r = 0.959; p = ≤ 0.01). The most common risk factor associated with the occurrence of AR in all the five districts of was the use of anthelmintics without weighing the animals to determine the correct dosage band. Limited farming experience was also shown as one of the risks. Although 67.5% of farmers mentioned that they never dose their sheep, 32.5% used anthelmintics at varying times of the year. A strong correlation existed between faecal egg count reduction test (FECRT) and EHA as both tests confirmed the existence of AR for the tested anthelmintics in all the districts except for LEV in Sekhukhune. Haemonchus contortus was the most dominant resistant nematode speciesidentified. No polymorphism was observed within H. contortus isolates from Limpopo province. Phylogenetic analyses revealed four major lineages. Limpopo isolates shared common ancestry with reference sequences from Africa, as well as across the globe. The only isolates that did not cluster with the South African isolates were from the USA. Low levels of structure observed in the present study among our isolates and from elsewhere could imply a high level of gene flow. Seasonal pattern of GINs observed in this study have shown that climate change has not affected the seasonality of nematodes as the results compares with previous studies on nematode seasonality. Occurrence of AR and risk factors associated with AR Limpopo province suggest that there is a need to train rural resource-poor livestock farmers on proper use of anthelmintic treatment and to educate them on methods to prevent development of AR in their flocks. The findings of this study provide a basis for future studies in understanding and controlling the spread of anthelmintic resistance against H. contortus and other GINs in sheep in Limpopo province and South Africa at large and also for tracing changes in the population genetic structure of H. contortus in Limpopo.