背景:在犬钩虫中已检测到对苯并咪唑的驱虫抗性,犬囊造孔。苯并咪唑抗性被认为最初是在灰狗中开发的,但在非灰狗宠物狗中也被检测到。这项研究的目的是验证基于探针的等位基因特异性实时PCR测试对β-微管蛋白同种型1基因的F167Y多态性,并确定地理分布。
方法:建立并验证了等位基因特异性实时PCR测试,以检测犬Ancylostomaβ-微管蛋白同种型1基因中167密码子的多态性。此外,实时PCR测试对Ancylostomaspp进行了验证。和海头鱼。验证了两种核酸提取方案,包括机械破坏粪便中的寄生虫结构。在钩虫确认的粪便样品中确定F167Y单核苷酸多态性(SNP)的频率。通过β-微管蛋白基因测序确认具有抗性167Y基因型的样品,并测定等位基因频率。
结果:Ancylostomaspp。和A.caninumF167Y等位基因特异性实时PCR测试在针对合成DNA进行测试时具有高度敏感性和特异性,加标样品,和特征寄生虫。使用优化的总核酸提取方案,发现511个中的54个(10.6%)含有苯并咪唑抗性等位基因。通过β-微管蛋白基因测序确认了所有55个含有具有抗性突变的钩虫的样品。大多数抗性钩虫(44抗性,183测试;24.4%)来自佛罗里达,来自加利福尼亚的五个(测试了103个,4.9%),三个来自爱达荷州(经过40次测试,7.5%),两个来自内华达州(22个测试,9.1%),和一个来自夏威夷的样本(13个测试,7.7%)。在14个不同的犬种中发现了抗性基因型,其中包括8个灰狗。等位基因频率测定显示抗性等位基因频率在1%至100%之间,其中58%高于50%。
结论:该数据强烈支持了在美国普通宠物狗群中存在的苯并咪唑抗性犬钩虫的最新发现。
Anthelmintic resistance to benzimidazole has been detected in the canine hookworm, Ancylostoma caninum. Benzimidazole resistance is believed to have developed originally in greyhounds, but has also been detected in non-greyhound pet dogs. The aim of this study was to validate a probe-based allele-specific real-time PCR tests for the F167Y polymorphism on the β-tubulin isotype-1 gene and to determine the geographic distribution.
Allele-specific real-time PCR tests were established and validated to detect the codon 167 polymorphism in the Ancylostoma caninum β-tubulin isotype-1gene. Additionally, real-time PCR tests were validated for Ancylostoma spp. and Uncinaria stenocephala. Two nucleic acid extraction protocols were validated including mechanical disruption of parasite structures in stool. The frequency of the F167Y single nucleotide polymorphism (SNP) was determined in hookworm confirmed stool samples. Samples with the resistant 167Y genotype were confirmed by β-tubulin gene sequencing and allele frequencies were determined.
The Ancylostoma spp. and A. caninum F167Y allele-specific real-time PCR tests were highly sensitive and specific when tested against synthetic DNA, spiked samples, and characterized parasites. Using an optimized total nucleic acid extraction protocol, 54 of 511 (10.6%) were found to contain the benzimidazole resistance allele. All 55 samples containing hookworms with the resistance mutation were confirmed by β-tubulin gene sequencing. The majority of resistant hookworms (44 resistant, 183 tested; 24.4%) originated from Florida, five from California (103 tested, 4.9%), three from Idaho (40 tested, 7.5%), two from Nevada (22 tested, 9.1%), and one sample from Hawaii (13 tested, 7.7%). Resistant genotypes were found in 14 different dog breeds including eight in Greyhounds. Allele-frequency determination revealed resistance allele frequencies between 1 and 100% with 58% above 50%.
This data strongly supports recent findings of benzimidazole resistant canine hookworms present throughout the general US pet dog population.