Authors
N Thundathil1; A Venkatesan1; E Redman1; A Morosetti1; PD Jimenez Castro2; R Kaplan2; J Gilleard1; 1 University of Calgary, United States; 2 University of Georgia, United States Discussion
The canine hookworm, Ancylostoma caninum, is a highly pathogenic and the most prevalent intestinal nematode of domestic dogs. Additionally, studies have shown that there was a 47% increase in the prevalence of A. caninum between 2012-2018. Management of infections is particularly problematic in greyhounds on breeding farms due to the unrestricted access these have to environments that are ideal for hookworm survival. Since greyhounds are exposed to this environment regularly, these are often subject to intense deworming protocols that present a high pressure for drug selection on hookworm populations on these farms and racing kennels. Our previous work revealed that A. caninum is resistant to multiple anthelmintic drug classes in kenneled greyhounds across the USA and that benzimidazole resistance is associated with mutations at codons 134 and 167 of the isotype-1 B-tubulin gene, the target of this drug class. Both these mutations are now widespread in A. caninum, not only from greyhounds, but also from pet dogs in the USA. Based on greyhound treatment history, we hypothesized that resistance originally emerged due to intense drug selection and housing conditions in kenneled greyhounds and subsequently spread to pet dogs via environmental contamination by retired greyhounds being rehomed across the USA. This project tested the specific hypothesis that there has been more intense selection at the A. caninum isotype-1 B-tubulin locus in greyhounds than in pet dogs. Deep amplicon sequencing data is already available for the isotype-1 B-tubulin locus, and we undertook additional deep amplicon sequencing of the cox-1 and nad-1 mitochondrial genes to use as neutral markers to assess the overall genetic diversity of the parasite populations. The A. caninum data was taken from 120 pet dog and 50 greyhound hookworm positive fecal samples, and primers were designed to target the 455bp and 394 bp regions of the cox-1 and nad-1 mitochondrial genes, respectively. Using the generated primer pairs, PCR amplicons were produced and sequenced at depth using the Illumina MiSeq platform. Finally, the DADA2 bioinformatics pipeline was used to generate a table of amplicon sequence variants that allowed for the comparison of genetic diversity between A. caninum in these two canine populations. We found that the isotype-1 B-tubulin locus has much lower expected heterozygosity and nucleotide diversity in A. caninum from kenneled greyhounds (Hd = 0.36, pi = 0.008) than from pet dogs (Hd = 0.89, pi = 0.017). Additionally, there was a greater departure from neutrality as measured by Tajima’s D for A. caninum from kenneled greyhounds (-2.70) than pet dogs (-1.85). In contrast, the cox-1 and nad-1 mitochondrial markers, had similar genetic diversity in A. caninum from kenneled greyhounds and pet dogs, respectively. Overall, these results indicate there has been more intense selection at the isotype-1 B-tubulin marker in A. caninum infecting greyhounds than pet dogs, which is consistent with the hypothesis of benzimidazole resistance in A. caninum in the USA originating in greyhound kennels. 
