RESISTA-Test©: refinement of the method for detection of anthelmintic resistance and validation in sheep flocks

Abstract

The increase in the occurrence of anthelmintic resistance of gastrointestinal nematodes in sheep has generated the need to develop new tests to detect resistance or improve existing ones. The Larval Development Test (LDT) can slow down the establishment of resistance and preserve the effectiveness of anthelmintics. A newly developed diagnostic test based on LDT, called RESISTA-Test© (Process FAPESP 2016 / 07132-8), uses 96-well plates and made possible a real savings of 52% of the costs of resistance diagnosis, compared to conventional LDT. The RESISTA-Test© was developed to evaluate the effectiveness of four chemical groups of anthelmintics used in Brazil (benzimidazoles, imidothiazoles, avermectin and amino-acetonitrile derivatives). He presented excellent results, comparable to the Faecal Egg Count Reduction Test (FECRT), having a good agreement between them. In this project, a refinement of the RESISTA-Test© is proposed: 1) to include another chemical group in the RESISTA-Test© by developing a protocol and validating the use of closantel; 2) improve the accuracy in the detection of resistance to levamisole and monepantel by performing the RESISTA-Test© and FECRT in 15 sheep herds; 3) validate the RESISTA-Test© for other genera of gastrointestinal nematodes; 4) elaborate a manual for the collection, packaging and sending of sheep faeces for the execution of the RESISTA-Test©. For the validation of closantel, four lambs will be used as faeces donors for the production of infective larvae (L3), two animals will be artificially infected with a Haemonchus contortus isolate susceptible to all anthelmintics and two other animals with a resistant isolate. Dilutions of anthelmintics will be performed as previously described in the RESISTA-Test©, while the dilutions to be produced for closantel will be standardized for Brazilian isolates. For the LDT, the faeces of the animals will be washed to recover the eggs with the sequential use of sieves and the test will be carried out following the methodology with the adaptations and validations performed for the RESISTA-test©. The FECRT will be carried out in 15 sheep flocks, which should have at least 42 animals (at least seven animals/treatment: albendazole, levamisole, ivermectin, monepantel, closantel and negative control). To improve the accuracy of the RESISTA-Test© for levamisole and monepantel, as well as for the validation for closantel and other parasitic genera, a comparative study of the results of the RESISTA-Test© (which detects in vitro resistance) and the TRCOF (which detects resistance in vivo). A manual for the RESISTA-Test© will also be developed, describing the process of collecting the sheep faeces, sending it to the laboratory and returning the report to the producers with the results of the most effective anthelmintic on the property. In addition, 30 H. contortus L3 from each property will be destined for DNA extraction and genotyping by molecular test to detect anthelmintic resistance. For this, the following polymorphisms will be evaluated: I) SNP F200Y in the isotype 1 of the beta-tubulin gene, associated with resistance to benzimidazois; II) 63 bp InDel in the acr-8 gene, associated with resistance to levamisole; and III) InDel of 6 bp 852_857del in exon 11 of the mptl-1 gene, associated with resistance to monepantel, after optimization of the electrophoresis methodology for small InDels. Thus, it is expected to provide tools to be used in the laboratory routine for monitoring anthelmintic resistance, reducing costs arising from the current situation of multidrug resistance, rationalizing the use of anthelmintics and delaying the appearance of resistance, minimizing the costs of activities production, making them more interesting to producers. (AU)