In a recent study published in the journal PLOS Pathogens, researchers in the United States of America (USA) and Canada found molecular evidence that the parasitic hookworms (Ancylostoma caninum) in pet dogs across the USA are becoming increasingly resistant to benzimidazole, a broad-spectrum anthelmintic drug used for infection control.
In addition, they observed that this drug resistance must have been occurring for many years. Accordingly, it was prevalent across the USA, explaining apparent hookworm treatment failures in recent years.
Most importantly, this study demonstrated that isotype-1 β-tubulin mutations governed benzimidazole resistance in A. caninum, a parasite closely related to hookworm species infecting humans.
Study: Molecular evidence of widespread benzimidazole drug resistance in Ancylostoma caninum from domestic dogs throughout the USA and discovery of a novel β-tubulin benzimidazole resistance mutation. Image Credit: Pressmaster / Shutterstock
Background
Severe A. caninum infections cause anemia and death in dogs, especially young puppies. In addition, this parasite is of public health concern due to its zoonotic potential and ability to cause infections in humans. Human infection is especially a cause for concern in tropical regions due to its cutaneous larval migrans.
A. caninum is now widespread in pet dogs worldwide, including in the United States of America (USA), and benzimidazoles are one of the most commonly used anthelmintic drugs for their treatment and control.
Previous studies have shown such resistance to anthelmintic drugs in the gastrointestinal nematodes of livestock. However, drug resistance in the gastrointestinal nematodes of domestic dogs has not previously been considered a significant issue.
About the study
The prevalence of A. caninum in kenneled greyhounds has been troublesome in the USA for the past few years, partly due to their crowded housing conditions and outdoor runs facilitating hookworm transmission. Furthermore, excessive and frequent use of anthelmintic drugs to control hookworm infections in greyhounds exerted a high drug selection pressure on A. caninum.
Accordingly, in vitro studies showed that all A. caninum samples from these dogs treated with benzimidazoles and macrocyclic lactones were resistant to these drugs. Previous studies investigating genotype-phenotype associations in greyhounds also suggested that benzimidazole resistance was due to Q134H(CAA>CAT) or F167Y(TTC>TAC) mutations.
In the present study, the researchers collected hundreds of A. caninum-positive dog fecal samples from four locations in the USA between February and December 2020. The samples were immediately refrigerated, stored, and shipped to the University of Georgia for analysis.
They categorized donor dogs into small, medium, and large breeds per the prespecified definitions of the American Kennel Club. The donor dogs belonged to different age groups, less than one year, one to three years, four to six years, and more than six years old.
First, the researchers identified a drug-resistance mutation in the genome of A. caninum, never previously identified in any parasite or fungal species, which occurs in codon 134 in the isotype-1 β-tubulin gene of A. caninum. Following the fecal floatation process, the researchers isolated A. caninum eggs, which they used to form individual and pooled egg samples for deoxyribonucleic acid (DNA) extraction.
Then, they amplified the 3,467 base pairs sequence of the isotype-1 β-tubulin gene retrieved from the genomic DNA preparations from eggs of two greyhound samples, BH45 and BH31, that contained the Q134H mutation at 44.4% and 84%, respectively, and from another A. caninum sample.
Further, the researchers sequenced the haplotype of the nearly full-length isotype-1 β-tubulin gene to determine if it had any additional non-synonymous mutations. In addition, they used AlphaFold2 to develop a structural model for the A. caninum isotype-1 β-tubulin gene.
Next, the team used the Caenorhabditis elegans beta-tubulin encoding ben-1 gene to edit the Q134H allele via clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) editing to demonstrate the functional significance of the Q134H mutation in hundreds of collected fecal samples.
Furthermore, the team used deep amplicon sequencing to identify and determine the frequency of single-nucleotide polymorphism (SNPs) at codons 134, 167, 198, and 200 of the isotype-1 β-tubulin gene. It provided estimates of mutation frequencies in each sample. Lastly, they performed two independent polymerase chain reactions (PCRs) on 50 randomly chosen samples to test the repeatability of the benzimidazole resistance SNP frequencies determined by amplicon sequencing.
Results
The researchers detected the presence of drug-resistant hookworms in over 50 percent of the dogs. In addition, screening of 685 hookworm-positive samples from pet dogs across several areas of the US using a state-of-the-art DNA sequencing technology revealed two benzimidazole resistance mutations in A. caninum.
"It means that many routine drug treatments are now ineffective and so we need to rethink how we control this parasite and use important antiparasitic drugs in companion animals in a more sustainable way." said Dr. John Gilleard, Ph.D., the University of Calgary Faculty of Veterinary Medicine (UCVM)
Previous studies had not detected the isotype-1 β-tubulin Q134H(CAA>CAT) mutation before. It is novel and was present at a high frequency, i.e., ≥90% in three A. caninum samples, BH25, DL13, and DL14. Notably, it was the only non-synonymous mutation on the same haplotype.
Intriguingly, another benzimidazole resistance mutation, F167Y(TTC>TAC) isotype-1 β-tubulin, was also present but at a much lower frequency of <6%. Protein-drug interaction modeling showed that an amino acid residue at codon 134 was in direct contact with the drug scaffold's constant region, disturbing which reduced its affinity to benzimidazole.
Deep amplicon sequencing revealed that these two mutations were highly prevalent, with prevalence rates for the F167Y and Q134H mutations being 49.7% and 31.2%, respectively, and overall median frequencies of 54% and 16.4%, respectively.
The infection intensities and occurrence of A. caninum in pet dogs were highest in the southern US regions and relatively lower in the northeastern and western regions, most likely due to varying ambient temperatures and humidity.
The western US regions, having the lowest prevalence of A. caninum infections, had a higher prevalence and frequency of both benzimidazole resistance mutations. Finally, CRISPR-Cas9 editing results showed that the Q134H mutation conferred a comparable benzimidazole resistance in vivo as canonical resistance mutations at codons 167, 198, and 200.
Conclusions
The current study found that benzimidazole resistance is geographically widespread in gastrointestinal parasitic hookworms infecting pet dogs. This poses a serious threat in the USA, where veterinarians commonly use anthelmintic drugs for treating A. caninum infections. Furthermore, since clinicians use similar drugs to treat cutaneous larval migrans in humans, this drug resistance could compromise their treatment.
The researchers are also investigating the hypothesis that resistance to other drugs, such as macrocyclic lactones, is similarly widespread in A. caninum of pet dogs, which would require mass phenotypic testing.
Nonetheless, the researchers advocated using next-generation DNA sequencing technologies for routine diagnosis of drug resistance for all kinds of drugs. Additionally, they emphasized the importance of pre-emptive surveillance to detect drug resistance at an early stage for the implementation of meaningful mitigation strategies.
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