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

Abstract

Rhipicephalus annulatus field populations collected from small cattle farms in Beni-Suef province in Egypt were evaluated for deltamethrin resistance by toxicological in vitro bioassays (adult immersion test-AIT and larval packet test-LPT). Moreover, a quantitative PCR high resolution melting (PCR-HRM) technique was used 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 (populations El-Wasta - A, and El-Hakamna - C) or resistant (populations El-Wasta - B, El-Hakamna - D, EL-Halabia - E, and Kom-abokhalad - F). Based on LPT findings, the phenotypic resistant populations were found to have a resistance ratio between 6.5 – 10.8. The PCR-HRM genotyping of the ticks showed variable melting curves among the populations in domain II of the Na-channel gene. Analysis of the curves showed the presence of wild type, mutant homozygous, and mutant heterozygous tick individuals. By sequencing the PCR amplified fragments, the C190A mutation was the only detected nucleotide polymorphism of domain II among the phenotypically resistant populations, which was present in 39.5 % (34/86) of the ticks tested. On the other hand, the phenotypically susceptible populations A and C did not show C190A mutant homozygous (RR) individuals. Meanwhile, in domain III all of the examined populations revealed melting curves like the wild type. Furthermore, the sequence analysis of these populations 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 efficacy of chemical compounds used by farmers to control ticks on cattle should be considered as part of animal health programs to manage the emerging resistance to acaricides in R. annulatus populations.