University of Khartoum

Phenotypic and Molecular Identification of Albendazole Resistant Haemonchus contortus in Goats in South Darfur State, Sudan

Phenotypic and Molecular Identification of Albendazole Resistant Haemonchus contortus in Goats in South Darfur State, Sudan

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Title: Phenotypic and Molecular Identification of Albendazole Resistant Haemonchus contortus in Goats in South Darfur State, Sudan
Author: Adam, Khalid Mohammednour Mohammedsalih
Abstract: The present study aimed to evaluate albendazole resistance status of Haemonchus contortus in experimentally and naturally infected goats in different study areas of South Darfur State, Sudan, based on two hypotheses. The first indicated the change of albendazole efficacy is due to treatment failure as the result of under-dosing, while the second hypothesis indicated the actual development of resistance. Resistance to albendazole was evaluated using two different doses, 5 or 10 mg/kg body weight, and in different seasons during August 2014 – January 2017, based on phenotypic and molecular techniques. The obtained results were statistically analysed using NCSS and eggCounts package in R software. Four experimental infection trials were conducted during winter and summer using different H. contortus isolates that were derived from Nyala, Kass, Um Dafuq and Tulus. In each trial, 16 male goats, 3 – 6 months old, were infected individually with a single dose of 150 infective H. contortus larvae (L3)/kg body weight, then half were treated with 5 mg/kg. Also, resistance to albendazole was evaluated in goats naturally infected with gastrointestinal nematodes at Nyala and Beleil in autumn and winter. In these trials, 88 goats treated with 5 mg/kg, and 35 received 10 mg/kg dose. Assessing the resistance status at phenotypic level was depended on the faecal egg count reduction test (FECRT), the egg hatch test (EHT) and coproculture examination using samples collected on day 0, 8 and 14. Albendazole resistance is considered when the FECRT result is less than 95% and the effective concentration 50 (EC50) of the EHT is higher than 0.1 µg/ml thiabendazole, as recommended by the World Association for the advancement of Veterinary Parasitology. Molecular techniques were used to detect the presence of mutations at codons 167 (TTC/TAC), 198 (GAA/GCA) and 200 (TTC/TAC) in β-tubulin isotype 1 gene using 34 samples of H. contortus including L3 and adult worms. The techniques used were PCR, gene sequencing, gene cloning and pyrosequencing assays. The results of the FECRT as well as the EHT obtained from experimental and natural infection trials, and in different seasons, showed for the first time that albendazole resistant H. contortus are present in goats in South Darfur. With 5 mg/kg dose, the efficacy was 62, 71, 72 and 79% to H. contortus isolates from Nyala, Um Dafuq, Kass and Tulus, respectively. The EC50 of these trials was in a range of 0.11 – 0.18 µg/ml thiabendazole. In Nyala natural infection trials, the FECRT indicated percent reductions of 74% to 5 mg/kg and of 86% to 10 mg/kg dose. In Beleil, albendazole was 95% effective. The calculated EC50 ranged from 0.17 to 0.23 µg/ml thiabendazole in Nyala, while in Beleil was 0.06 µg/ml. No significant differences (P<0.05) detected when resistance evaluated in different seasons. The species-specific PCR detected H. contortus, Trichostrongylus colubriformis and Cooperia punctata in L3 samples collected from goats treated with 5 or 10 mg/kg body weight. The results of molecular tests showed no evidence for the presence of resistance-related alleles at codons 167 and 200. However, at codon 198 the sequencing results successfully showed the presence of two new candidates, i.e. replacement of wild-type glutamate with either leucine (E198L) (82%) or valine (E198V) (12%). These candidates have never been described before in H. contortus. It can be concluded that H. contortus has developed resistance to albendazole in South Darfur State. Additionally, this work elucidates the genetic information of albendazole resistance in H. contortus in Sudan with establishment of two new candidates, E198L and E198V. The study recommends the change of manufacturer package labels of albendazole to be used at 10 mg/kg body weight in goats in Sudan. The next steps of worm control in South Darfur will involve evaluation of target selective treatment, e.g. FAMACHA system, to decide which animals require treatment and identifying where there is resistance so that a change of anthelmintic from albendazole to, e.g. levamisole can be made.
URI: http://khartoumspace.uofk.edu/123456789/26693


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