Advances in the control of Haemonchus contortus infection in sheep "Ovis aries" and comprehension of anthelminthic resistance to the monepantel

Abstract

Ovine nematodesioses added to anthelmintic resistance cause huge declines in productivity, especially increasing mortality and morbidity rates. Haemonchus contortus, Trichostrongylus spp., Oesophagostomum sp. and Cooperia sp. are the main species of nematodes that demonstrate resistance to the main anthelmintics. The present study aims to use deep amplicon sequencing methodology for the detection of polymorphisms in the exons of the mptl-1 gene; and validate these polymorphisms as molecular markers of resistance to monepantel in H. contortus. Young sheep from the "Embrapa Pecuária Sudeste" flock will be kept in stalls, and will be treated with 10% trichlorphone to remove the natural nematode infection. After 7 and 14 days of treatment, fecal samples will be collected to perform Egg count Per Gram of feces (EPG) and confirm the status of gastrointestinal nematode-free animals. To establish the infection, the animals will receive orally 4,000 H. contortus L3 larvae obtained by coproculture. Five animals will be infected with L3 larvae from each one of the following H. contortus isolates: Embrapa 2010 (sensitive isolate; Chagas et al., 2013), Botucatu (resistant isolate; Albuquerque et al., 2017) and 340, 344 and 345 (isolates selected by subdosing from Embrapa2010; Niciura et al., 2019a). Next, EPG will be followed weekly, and the feces will be collected for coproculture and L3 larvae recovery. About 1,000 L3 larvae recovered from the cocultures of each one of the five H. contortus isolates will be submitted to cuticle removal with sodium hypochlorite for DNA extraction (NemaBiome protocol) and PCR (WormBase ParaSite database for primer design). PCR products will be subjected to purification with magnetic beads, followed by Illumina P5/P7 adapter ligation and library preparation. The libraries will be sequenced on the MiSeq sequencer and with the addition of 25% Phix Control v3. The result of this study will enable the mapping and genotyping of mptl-1 gene for the identification of monepantel-specific molecular markers that can be used for detection and monitoring of H. contortus resistance, leading to the development of tools for the preventive diagnosis of resistance. (AU)