Detection of single nucleotide polymorphism in the para-sodium channel gene of Rhipicephalus annulatus populations from Egypt resistant to deltamethrin

Authors: ARAFA, WALEED - Beni Suef University - Egypt; KLAFKE, GUILHERME - Centro De Pesquisas Rene' Rachou; Tidwell, Jason; Perez De Leon, Adalberto - Beto; ESTEVE-GASSENT, MARIA - Texas A&M University

Submitted to: Ticks and Tick Borne Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/1/2020
Publication Date: 6/3/2020
Citation: Arafa, W.M., Klafke, G.M., Tidwell, J.P., Perez De Leon, A.A., Esteve-Gassent, M. 2020. Detection of single nucleotide polymorphism in the para-sodium channel gene of Rhipicephalus annulatus populations from Egypt resistant to deltamethrin. Ticks and Tick Borne Diseases. https://doi.org/10.1016/j.ttbdis.2020.101488.
DOI: https://doi.org/10.1016/j.ttbdis.2020.101488

Interpretive Summary: Rhipicephalus annulatus is the scientific name of the most common tick found infesting cattle in Egypt. This tick species was eradicated from the United States, but still threatens the livestock industry of this country because it has an invasive biology and remains established in Mexico. R. annulatus is a pest that affects cattle health and production directly because it feeds on blood and indirectly due to its biological role as vector of disease-causing agents, or pathogens. Important R. annulatus-borne pathogens include those that cause bovine babesiosis and anaplasmosis. Pyrethroids comprise a class of synthetic pesticides used to treat tick infestations in cattle that are also known as acaricides. Deltamethrin is a pyrethroid commonly used in Egypt as an acaricide to try to control R. annulatus. Field reports of treatment failure with veterinary products containing deltamethrin prompted this investigation to determine if R. annulatus were resistant to this acaricide. Laboratory tests were conducted using R. annulatus collected from several regions in Egypt. This was complemented with the adaptation of a novel molecular assay to determine if changes known to be associated with deltamethrin resistance could be detected in the gene coding for components of the sodium channel, which is the target for pyrethroids in the tick nervous system. Detection of the single point mutation designated C190A represents the first time that this genetic change associated with deltamethrin resistance is reported in R. annulatus from Egypt. Detection of this mutation with the novel molecular assay known as high resolution melting technique was confirmed in the tests were ticks were shown to be resistant to deltamethrin. Screening for acaricidal activity of chemical compounds used by farmers in Egypt should be considered as part of animal health programs to manage the emerging resistance to deltamethrin and potentially other acaricides in R. annulatus populations.

Technical Abstract: Rhipicephalus annulatus field isolates from Egypt were evaluated for deltamethrin resistance by toxicological in vitro bioassays (Adult Immersion Test: AIT, and Larval Packet Test: LPT) and a quantitative PCR high resolution melting technique to detect nucleotide substitutions in the voltage-gated sodium channel (Na-channel) gene. By the in vitro bioassays, the examined ticks were phenotypically categorized as deltamethrin susceptible (isolates El-Wasta - A, and El-Hakamna - C) or resistant (isolates El-Wasta - B, El-Hakamna - D, EL-Halabia - E, and Kom-abokhalad - F). The quantitative PCR high resolution melting (PCR-HRM) genotyping of the R. annulatus samples showed variable melting curves among the isolates in domain II of the Na-channel gene. Analysis of the curves showed the presence of wild type, mutant homozygous, and mutant heterozygous individuals. By sequencing the PCR amplified fragments, the C190A mutation was confirmed in the phenotypically resistant populations. On the other hand, the phenotypically susceptible isolates A and C did not show nucleotide mutations in domain II. Meanwhile, in domain III all the examined isolates revealed melting curves like the wild type. Furthermore, the sequence analysis of these isolates confirmed the absence of SNPs in domain III. The C190A single point mutation was detected for the first time in domain II of the Na-channel gene of deltamethrin-resistant R. annulatus in Egypt using PCR-HRM. Screening for acaricidal activity of chemical compounds used by farmers should be considered as part of animal health programs to manage the emerging resistance to acaricides in R. annulatus populations.