Altered larval activation response associated with multidrug resistance in the canine hookworm Ancylostoma caninum.

Parasitic gastrointestinal nematodes pose significant health risks to humans, livestock, and companion animals, and their control relies heavily on the use of anthelmintic drugs. Overuse of these drugs has led to the emergence of resistant nematode populations. Herein, a naturally occurring isolate (referred to as BCR) of the dog hookworm, Ancylostoma caninum, that is resistant to 3 major classes of anthelmintics is characterized. Various drug assays were used to determine the resistance of BCR to thiabendazole, ivermectin, moxidectin and pyrantel pamoate. When compared to a drug-susceptible isolate of A. caninum, BCR was shown to be significantly resistant to all 4 of the drugs tested. Multiple single nucleotide polymorphisms have been shown to impart benzimidazole resistance, including the F167Y mutation in the ß-tubulin isotype 1 gene, which was confirmed to be present in BCR through molecular analysis. The frequency of the resistant allele in BCR was 76.3% following its first passage in the lab, which represented an increase from approximately 50% in the founding hookworm population. A second, recently described mutation in codon 134 (Q134H) was also detected at lower frequency in the BCR population. Additionally, BCR exhibits an altered larval activation phenotype compared to the susceptible isolate, suggesting differences in the signalling pathways involved in the activation process which may be associated with resistance. Further characterization of this isolate will provide insights into the mechanisms of resistance to macrocyclic lactones and tetrahydropyrimidine anthelmintics.

Epidemiological and molecular updates on hookworm species in dogs from southern Italy.

BACKGROUND: The zoonotic hookworms Ancylostoma caninum and Uncinaria stenocephala are widespread soil-transmitted helminths in dogs in Europe. Given the veterinary and public health importance of hookworms in dogs and the recent changes in the molecular epidemiology of some species, there is a need to continuously monitor the epidemiological and molecular prevalence of these parasites also at the "local" level. The present study aimed to update the epidemiological scenario of hookworm infections in both owned and stray dogs in southern Italy and to discriminate between different hookworm species (A. caninum and U. stenocephala) through molecular analyses. For this purpose, a retrospective analysis was performed over 10 years (2011-2021), including a total of 7008 owned dogs and 5642 stray dogs referred to our laboratory for copromicroscopic examinations. Moreover, 72 faecal samples, from dogs naturally infected by hookworms, were used to discriminate between A. caninum and U. stenocephala using two PCR protocols. Prior to molecular analyses, a subsample of 40/72 positive faecal samples was used for morphometric investigations on hookworm eggs. RESULTS: The results of the ten-year retrospective analysis (2011-2021) showed an overall prevalence of hookworm infection of 9.16%, specifically 5.1% in owned dogs and 14.2% in stray dogs. Logistic regression showed a significant association between positivity to hookworms and the variable "puppies" both in stray (13.84%; OR = 2.4) and owned (7.07%; OR = 2.2) dogs. The results of molecular analyses showed that positivity was confirmed only in 21/72 samples, specifically, 6 samples using protocol A and 19 with protocol B. Sequencing revealed 15 samples positive to U. stenocephala and 6 to A. caninum. CONCLUSIONS: The findings of this study showed a high prevalence of hookworm infections in dogs in southern Italy, updating the epidemiological scenario of the last decade. Moreover, the results of the study revealed the first identification of hookworm species in dogs in Italy by molecular studies, highlighting that U. stenocephala is more prevalent than A. caninum.

Molecular variability of the Ancylostoma secreted Protein-2 (Aca-asp-2) gene from Ancylostoma caninum contributes to expand information on population genetic studies of hookworms.

Hookworm infection is a major public health problem in many regions of the world. Given the high levels of host morbidity and even mortality of the host caused by these infections, it is crucial to understand the genetic structure of hookworm populations. This understanding can provide insights into the ecology, transmission patterns, mechanisms of drug resistance, and the development of vaccines and immunotherapeutic strategies. Previously, we examined presumably neutral molecular markers, such as microsatellites and COI (Cytochrome C oxidase subunit 1) in Brazilian populations of Ancylostoma caninum. Here we analyze the molecular variability of a genomic fragment of the Aca-asp-2 (Ancylostoma secreted protein-2) gene from Ancylostoma caninum. This gene is a highly expressed and activated following the infection of the L3 larvae in the host. We obtained individuals of A. caninum from five different geographic locations in Brazil, sequenced and analyzed parts of the gene. The results revealed extensive polymorphism at this fragment, especially in the intronic region, indicating low selective pressure acting on these sequences. However, we also observed irregular distributions of nucleotides and polymorphisms in the coding region of this gene, resulting in the identification of 27 alleles. The data presented here contribute to expanding the understanding of population genetic studies of hookworms.

Epidemiological study of gastrointestinal helminths among dogs from Northeastern Nigeria: a potential public health concern.

Understanding the epidemiology of canine parasitic infections, gastrointestinal helminthic infections in particular, is crucial for designing an efficient control programs targeted at minimizing the risks of zoonotic transmission. A total of 470 dogs attending veterinary clinics in Northeastern Nigeria were screened for gastrointestinal helminths using standard parasitological techniques. Epidemiological risk factors associated with the canine parasitic infections were assessed using multivariable logistic regression analysis. In total, 77.9% (366/470) of dogs had harboured one or more species of the gastrointestinal helminths: Ancylostoma caninum (40.2%), Toxocara canis (35.1%) Trichuris vulpis (26.6%), Spirocerca lupi (5.7%), Taenia/Echinococcus (12.3%), Dipylidium caninum (10.9%) and Diphyllobothriid ova (2.8%). Female dogs (OR=2.1; p=0.02), local breeds (OR=2.3; p<0.01), younger dogs (OR=3.2; p<0.001), dogs adopted as security guards (OR=1.8; p<0.01) and poor body condition score (OR=3.1; p<0.001) were strongly associated with A. caninum infection. For T. canis and T. vulpis, younger dogs (OR=2.5; p=0.02 and OR=2.3; p<0.01) and poor body condition score (OR=4.2; p<0.001 and OR=2.2; p<0.01) were the strong predictors for infection, whereas female dogs (OR=2.8; p=0.02), younger dogs (OR=4.1; p<0.001) and poor body condition score (OR=4.0; p<0.001) were strongly associated with D. caninum infection in dogs. The results revealed high prevalence of gastrointestinal helminths-some of which have zoonotic potential-among dogs underlying huge public health risk.

Occurrence of Ancylostoma Caninum from a Gray Fox Urocyon Cinereoargenteus in Southeastern Mexico.

The hookworm Ancylostoma caninum is a common nematode of wild and domestic canids worldwide. In Mexico, there are few records of helminths in wild canids, especially in the southeastern region. The aim of the present study was to examine the helminths from a gray fox Urocyon cinereoargenteus in southeastern Mexico. A road-killed female gray fox found in Merida, Yucatan, Mexico, was examined for helminths. Only nematodes were found in the intestine of the gray fox and identified using morphological studies and molecular analysis of 28S rRNA gene fragments. The characteristics exhibited by the nematode specimens were in accordance with descriptions of A. caninum: e. g. oral opening with a pair of prominent chitinous plates bearing three pairs of ventral teeth, lateral rays with a common trunk, dorsal ray divided into two branches with each branch terminating in three digitations. BLAST analysis of the 28S sequence showed similarity and coverage values of 99.8 % and 100 %, respectively, with a sequence of A. caninum from the domestic dog Canis familiaris in Australia. The genetic distance between the Australian specimen and the Yucatan specimen of A. caninum was 0.1 %, that is, they were only different in a single nucleotide. The gray fox examined in this study was found close to a rural community where A. caninum has been recorded from domestic dogs, which could be the source of infection. Our study increases the distribution of this nematode parasitizing the gray fox in Mexico and provides the first nucleotide sequence of A. caninum from the gray fox.

Human density, economic level and frequency of canine helminths in Buenos Aires.

The aim of this study was to explore through cross-sectional study the variation in the prevalence of parasitic helminths in canine faeces collected from green spaces of Buenos Aires according to the human density (HD) and economic level (EL) in the surroundings. HD and EL were considered as independent variables with three categories each. Twenty public squares (one hectare of surface) were randomly selected for each existing combination of the two independent variables. Ten random samples of fresh canine faeces were obtained in each square and analysed for helminths by the sedimentation and flotation techniques. The prevalence for each of the species was analysed using generalized linear models (GLM). The prevalence was modelled with a binomial error distribution and a logit link function. Helminth eggs were detected in 45 out of the 200 (22.5%) faecal samples collected and in 18 of the 20 green spaces sampled. The species observed were Ancylostoma caninum (13% of samples), Trichuris vulpis (8%) and Toxocara canis (4.5%). The GLM indicated that the prevalence of A. caninum in the slum areas (very high HD and very low EL) was higher than that in the other areas studied. However, the HD seemed to contribute more than the EL to the variations in the prevalence of A. caninum in faecal samples. The GLM showed no differences in the prevalence of the other parasite species for the different levels of the independent variables.

Rare Case of Enteric Ancylostoma caninum Hookworm Infection, South Korea.

A 60-year-old man from South Korea underwent a colonoscopy. A juvenile female worm showing 3 pairs of teeth in the buccal cavity was recovered from the descending colon. Partial sequencing of the internal transcribed spacer region showed 100% identity with Ancylostoma caninum, the dog hookworm.

Nematicidal and ovicidal activity of Bacillus thuringiensis against the zoonotic nematode Ancylostoma caninum.

Ancylostoma caninum is a gastrointestinal parasite that affect dogs and humans, considered a worldwide public health problem. The control of these parasitosis is increasingly difficult due to the development of multi-drug resistance. Bacillus thuringiensis is an insecticidal, spore forming bacterium, often species specific. The strain GP526 of B. thuringiensis has toxic effect on the cestode Dipylidium caninum and the trematode Centrocestus formosanus, both of them zoonotic parasites. The high degrees of specificity and environment safe make B. thuringiensis suitable for use against pathogen parasites, especially those resistant to synthetic chemical insecticides. The objective of the current work was to evaluate the in vitro effect of B. thuringiensis on Ancylostoma caninum. Spore-crystal mixture of the strains was co-incubated with 120 adult nematodes (males, non-pregnant females and pregnant females) or with 4800 eggs. GP526 showed a nematicidal effect with an LT50 of 35.8 h and an LC50 of 60 µg/ml. It also showed an ovicidal effect with an LC50 of 94.9 µg/ml. Histological analyses showed detachment of the cuticle and of the uterus in adult females, and vacuolization with destruction of the eggs. The effects of GP526 strain were comparable to those of albendazole, allowing us to propose GP526 for A. caninum control, in both, the adult stage at a gastrointestinal level, and in egg stage in the environment. In addition, GP526 can be proposed as a potential broad spectrum antiparasitic drug.

Combined use of chemical and biological compounds to control hookworm.

The aim of this study was to evaluate the combined use of different chemical (albendazole, ivermectin, glycerine and Vaseline) and biological (Monacrosporium thaumasium) compounds in the control of Ancylostoma caninum. Infective larvae of A. caninum were obtained from coprocultures of positive faeces from naturally infected dogs. We used 1% ivermectin, 1% albendazole, 100% glycerine, 100% Vaseline and an isolate of the nematophagous fungus M. thaumasium (NF34), alone or in combinations. Next, an experimental test was set up with 16 groups in microtubes, with a 24-h interaction. The groups (G1 to G15) that contained any chemical or biological compound (NF34) and/or their combined use (chemical + biological) showed a difference in relation to the control group, except G5 - Vaseline 100% without combinations. It was concluded that, even on an experimental basis, the combined use of anthelmintic drugs with biological control was efficient; however, more studies must be carried out in order to elucidate the synergistic action between chemical and biological compounds to be used in the effective control of hookworms in the future.

Hookworm-Derived Metabolites Suppress Pathology in a Mouse Model of Colitis and Inhibit Secretion of Key Inflammatory Cytokines in Primary Human Leukocytes.

Iatrogenic hookworm therapy shows promise for treating disorders that result from a dysregulated immune system, including inflammatory bowel disease (IBD). Using a murine model of trinitrobenzenesulfonic acid-induced colitis and human peripheral blood mononuclear cells, we demonstrated that low-molecular-weight metabolites derived from both somatic extracts (LMWM-SE) and excretory-secretory products (LMWM-ESP) of the hookworm, Ancylostoma caninum, display anti-inflammatory properties. Administration to mice of LMWM-ESP as well as sequentially extracted fractions of LMWM-SE using both methanol (SE-MeOH) and hexane-dichloromethane-acetonitrile (SE-HDA) resulted in significant protection against T cell-mediated immunopathology, clinical signs of colitis, and impaired histological colon architecture. To assess bioactivity in human cells, we stimulated primary human leukocytes with lipopolysaccharide in the presence of hookworm extracts and showed that SE-HDA suppressed ex vivo production of inflammatory cytokines. Gas chromatography-mass spectrometry (MS) and liquid chromatography-MS analyses revealed the presence of 46 polar metabolites, 22 fatty acids, and five short-chain fatty acids (SCFAs) in the LMWM-SE fraction and 29 polar metabolites, 13 fatty acids, and six SCFAs in the LMWM-ESP fraction. Several of these small metabolites, notably the SCFAs, have been previously reported to have anti-inflammatory properties in various disease settings, including IBD. This is the first report showing that hookworms secrete small molecules with both ex vivo and in vivo anti-inflammatory bioactivity, and this warrants further exploration as a novel approach to the development of anti-inflammatory drugs inspired by coevolution of gut-dwelling hookworms with their vertebrate hosts.

Autochthonous Cutaneous Larva Migrans in France and Europe.

Cutaneous larva migrans is a dermatitis, typically acquired in warm tropical or sub-tropical countries, caused by migration of the larvae of nematodes (hookworm; mainly Ancylostoma braziliense and, occasionally, Ancylostoma caninum or Uncinaria stenocephala), which are parasitic on animals such as cats and dogs, into the patient's skin. The larvae penetrate the skin after contact with infected soil and cause a typical creeping eruption. Patients with cutaneous larva migrans seen in Europe have usually acquired the disease following a stay in a tropical or sub-tropical area. How-ever, some cases of cutaneous larval migrans are acquired in Europe. We report here 5 autochthonous cases in France and give an overview of European autochthonous cases.

Gene structure and expression patterns of Acdaf-1, a TGF-ß type I receptor in Ancylostoma caninum.

The components of the transforming growth factor ß (TGF-ß) signaling pathway in parasitic nematodes remain unknown. In this research, a type I receptor for TGF-ß was isolated from the hookworm Ancylostoma caninum. The new gene was designated as Acdaf-1, a Caenorhabditis elegans daf-1 homolog. The full-length cDNA of Acdaf-1 encodes a 595-amino-acid protein with an NH2-terminal signal peptide. This protein has a cytoplasm tail (209-595aa region) which corresponds to the type 1a membrane topology. Between amino acid position 295-500, the protein contains the ATP binding site, substrate binding sites, and PKC-kinase-like domain. Real-time RT-PCR showed that the transcript was expressed in three main stages of A. caninum. It reached the maximal level in the female adult worm stage with lower transcript level in the first and second larvae (L1/L2) and intermediate level in L3 stages as well as in the male worms. After serum activation, the activity of Acdaf-1 was decreased in L3 larvae. These data implied that Acdaf-1 might relate to the infection ability of the larvae. Immunolocalization revealed that AcDAF-1 was present in eggs, intestine, and epidermis cells of larvae (L1, L2, and L3 stages) with strong signal in primordium of the gonads in L3 and was abundant in epidermis, intestine, and ovary of adult worm. These results suggested that Acdaf-1 might be involved in the interaction of the parasite and host relationship and provide a potential target for parasite control.

Molecular identification of zoonotic hookworms in dogs from four counties of Kenya.

All canine hookworms are known to be zoonotic, causing infections ranging from transient skin irritations to prolonged 'creeping eruptions', eosinophilic enteritis and even patent intestinal infections. There is little information on canine hookworm species and their public health significance in sub-Saharan Africa. This study determined the prevalence and species of hookworms in dogs from different climatic zones of Kenya. Dog faecal samples were collected from the environment, and hookworm eggs were isolated by zinc chloride flotation and subjected to DNA extraction. Polymerase chain reaction (PCR) assays targeting the internal transcribed spacer (ITS) 1 and 2, 5.8S and 28S ribosomal RNA of Ancylostoma spp. and Uncinaria stenocephala were performed, and hookworm species were identified by PCR-restriction fragment length polymorphism (RFLP) or DNA sequencing. Hookworm eggs were detected by microscopy in 490/1621 (30.23%, 95% CI 28.01-32.54) faecal samples. Estimates of faecal prevalence were high in counties receiving higher rainfall (Narok 46.80%, Meru 44.88%) and low in those with a more arid climate (Isiolo 19.73%, Turkana 11.83%). In a subset of 70 faecal samples, Ancylostoma caninum (n = 59) was the most common species, followed by A. braziliense (n = 10) and A. cf. duodenale (n = 1). This study reports for the first time the detection of A. cf. duodenale in dog faeces and zoonotic hookworm species in Kenyan dogs. These findings emphasize the need for control measures such as enforcing laws for restraining stray dogs, regular deworming of dogs, and public health awareness programmes aimed at informing communities on outdoor use of footwear.

Revisiting the Ancylostoma Caninum Secretome Provides New Information on Hookworm-Host Interactions.

Hookworm infection is a major tropical parasitic disease affecting almost 500 million people worldwide. These soil-transmitted helminths can survive for many years in the intestine of the host, where they feed on blood, causing iron deficiency anemia and other complications. These parasites release a variety of molecules known as excretory/secretory products (ESPs) that are involved in many different biological processes that govern parasite survival. Using a combination of separation techniques such as SDS-PAGE and OFFGEL electrophoresis, in combination with state-of-the-art mass spectrometry we have reanalyzed the dog hookworm, Ancylostoma caninum, ESPs (AcESP). We identified 315 proteins present in the AcESP, compared with just 105 identified in previous studies. The most highly represented family of proteins is the SCP/TAPs (110 of the 315 proteins), and the most abundant constituents of AcESP are homologues of the tissue inhibitors of metalloproteases (TIMP) family. Interestingly, we identified new homologs of well-known vaccine candidates and immunomodulatory proteins. This study provides novel information about the proteins secreted by A. caninum, and constitutes a comprehensive dataset to study the proteins involved in host-hookworm interactions.

Mucosal tolerance of the hookworm Ancylostoma caninum in the gut of naturally infected wild dogs.

Ancylostoma caninum is a very pathogenic hookworm that locates in the small intestine of the dog and other canid species. The mucosal response of wild dogs naturally infected with A. caninum was investigated in this study. In spite of diffuse infiltrations of the mucosa with CD3+ , CD4+ , CD8+ , CD11c+ , CD21+ or MHC class II antigen cells, no focal infiltrations with any of these cell phenotypes were observed around the buccal capsule or the body of the feeding worms. Very few or no apoptotic cells could be detected around the worms fixed into the mucosa but they were detected on the tip of villi and in the superficial layer of cellular debris and proteinaceous exudate that covers the mucosa. Muc5AC, a mucine associated with expulsion of gut worms (Trichuris muris) was expressed extremely weakly or was not expressed at all in the intestine of the wild dogs infected with A. caninum. Our data show that individual specimens of A. caninum can reside for some time in the mucosa of the gut of dogs undetected and most likely unaffected by the effectors of the local immune response.

Comparison of different molecular protocols for the detection of Uncinaria stenocephala infection in dogs.

The present study aims to assess the performance of different molecular targets using various matrices of samples for the detection of Uncinaria stenocephala (US) in hookworm infected dogs. To this end, the DNA extraction was performed on the following matrices of samples: (i) larvae of US obtained from experimentally infected dogs with US with different larvae counts per microliter (µl); (ii) pure US eggs suspension in distilled water with different egg counts per µl; (iii) spiked dog fecal samples with different US eggs per gram (EPG) of feces; (iv) feces from dogs naturally infected with hookworm eggs; (v) fecal suspension with hookworm eggs recovered from the FLOTAC apparatus. All the samples were tested with four different PCR protocols targeting specific regions for the detection of both hookworms US and AC as follows: Protocol A (ITS1, 5.8 S, ITS2) and Protocol B (18 S) for the detection of both species, Protocol C (ITS1) for the detection of AC and Protocol D (ITS1) for the detection of US. The best results were obtained with DNA extracted from US larvae matrix obtained from experimentally infected dogs, showing a detection limit of 3.5 larvae/ml for the protocols A, B and D. A moderate correlation was found between the FLOTAC technique and PCR protocols B and D with respect to fecal samples from dogs naturally infected with hookworms. Indeed, PCR protocols B (18 S) and D (ITS1) gave the best results for feces and fecal suspension from naturally infected dogs. However, all the PCR protocols used showed lower sensitivity than FLOTAC technique. Perhaps, isolating US eggs in advance could help to obtain better quality and quantity of DNA, avoiding some notable factors such as inhibitors present in faecal samples. However, a further study is needed to evaluate and standardise a protocol for the recovery of parasitic elements, that could be applied prior to DNA extraction. Therefore, this could lead to a better amplification of US eggs DNA. In conclusion, our results showed that the type of sample (sample-matrix) used for the DNA extraction samples is crucial, as this affects the diagnostic sensitivity of the technique.

Retrospective analysis of canine fecal flotation and coproantigen immunoassay hookworm positive results in Greyhounds and other dog breeds.

Recent studies demonstrated that Greyhounds are commonly infected with Ancylostoma caninum and these infections have been shown to be resistant to anthelmintics. This study evaluated samples submitted to a commercial reference laboratory (IDEXX Laboratories) for canine fecal flotation zinc sulfate centrifugation and coproantigen immunoassay between January 1, 2019, and July 30, 2023 for evidence that Greyhounds were more often positive for Ancylostoma spp. (hookworms) compared to other breeds. The purpose of the study was to determine if Greyhounds were more likely to be hookworm-positive compared to other breeds, if Greyhounds on preventives with efficacy against hookworm infections are more likely to test positive than other breeds, if their infections take longer to resolve, to estimate how long this takes and to assess whether the proportion of hookworm positive tests for all breeds is increasing over time. Records of 25,440,055 fecal results were obtained representing 17,671,724 unique dogs. Of these, 49,795 (∼0.3%) were Greyhounds. The overall odds ratio (OR) of 15.3 (p < 0.001) suggests that Greyhounds are at significantly higher risk than other breeds for hookworm positive float findings, and the OR of 14.3 (p < 0.001) suggests significantly higher risk for hookworm antigen positive results. The median time to negative testing event from the Turnbull distribution estimate was in the interval of 1-2 days for other breeds and 71-72 days for Greyhounds. These results provide evidence that anthelmintic resistant A. caninum strains may be having population-level impacts on the frequency and duration of infections in Greyhounds. The findings have broader health implications beyond Greyhounds as MADR A. caninum strains could spread to other breeds and even pet owners.

CANINE HOOKWORM PRESENCE IN TONGATAPU, TONGA.

Dogs are common in Tonga, where they exist in a close relationship with humans as pets and guard dogs and often are free-roaming due to limited property fencing and the need to scavenge for food. Little is known about the health status of these dogs and the type and level of pathogens carried by them. In this study, blood and fecal samples were analyzed to identify the presence of endoparasites and determine packed cell volume (PCV) values for dogs in Tonga. The current data deficit in this area made it necessary to develop a methodology and collect preliminary data to assist with future research in Tonga. Fecal analysis revealed hookworm in 73% of dogs, primarily those 4-12 mo old. Hematologic evaluation revealed low to borderline low PCV in 87% of dogs. The zoonotic nature of some of the pathogens identified poses a public health risk, with a particular risk for children.

Benzimidazole F167Y polymorphism in the canine hookworm, Ancylostoma caninum: Widespread geographic, seasonal, age, and breed distribution in United States and Canada dogs.

Surveillance data for Ancylostoma spp. and the A. caninum benzimidazole treatment resistance associated F167Y polymorphism using molecular diagnostics was obtained in a large population of dogs from the United States and Canada. Real-time PCR (qPCR) for Ancylostoma spp. and allele-specific qPCR detecting a single nucleotide polymorphism (SNP) F167Y was used in 262,872 canine stool samples collected between March and December of 2022. Ancylostoma spp. was found at an overall prevalence of 2.5% (6538/262,872), with the highest prevalence in the Southern US, 4.4% (4490/103,095), and the lowest prevalence in Canada 0.6% (101/15,829). The A. caninum F167Y polymorphism was found with the highest prevalence (13.4%, n = 46/343) in the Western US and the lowest in Canada at 4.1% (4/97). The F167Y polymorphism was detected every month over the 10-month collection period. Seasonal distribution showed a peak in June for both Ancylostoma spp. (3.08%, 547/17,775) and A. caninum F167Y (12.25%, 67/547). However, the A. caninum F167Y polymorphism prevalence was highest in September (13.9%, 119/856). Age analysis indicates a higher prevalence of both hookworm infections and occurrence of resistant isolates in puppies. The breeds with the highest F167Y polymorphism prevalence in Ancylostoma spp. detected samples were poodles (28.9%), followed by Bernese Mountain dogs (25%), Cocker spaniels (23.1%), and greyhounds (22.4%). Our data set describes widespread geographic distribution of the A. caninum benzimidazole resistance associated F167Y polymorphism in the United States and Canada, with no clear seasonality compared to the Ancylostoma spp. prevalence patterns. The F167 polymorphism was present in all geographic areas with detected hookworms, including Canada. Our study highlights that the F167Y polymorphism is represented in many dog breeds, including greyhounds.

Emergence of canine hookworm treatment resistance: Novel detection of Ancylostoma caninum anthelmintic resistance markers by fecal PCR in 11 dogs from Canada.

OBJECTIVE: To describe dogs with detected Ancylostoma caninum anthelmintic treatment resistance markers in Canada. ANIMALS: 11 client-owned dogs with fecal quantitative PCR (qPCR) assay detected A caninum with benzimidazole (BZ) resistance genotypic markers. METHODS: Signalment, presenting concern, duration of clinical signs, fecal testing, treatment, and outcomes were obtained. Where available, follow-up data were collected via telephone or email with the primary veterinarian. RESULTS: Ancylostoma spp was detected from 184/32,205 dog fecal samples by reference laboratory qPCR surveillance, between May 15, 2022, and April 26, 2023. 11 of these 184 samples had A caninum with genetic BZ F167Y resistance marker detection. 4 dogs had not traveled outside Canada, 6 had been imported from the US, and the travel history was unclear in 1 dog. 7 of the dogs had gastro-intestinal signs (diarrhea or soft stool) on initial presentation. Clinical improvement was reported in 6 of these dogs (resolution of diarrhea and soft stool), with 1 dog lost to follow-up. All 11 dogs received anthelmintic treatment (varied drugs and duration). CLINICAL RELEVANCE: Identification of genetic markers of BZ resistance raises concerns about the potential animal and human impacts of resistant hookworms. 4 dogs lacked an origin from or travel history to the US, indicating true emergence and/or novel spread within Canada, not just importation from an area where resistance has been reported. Fecal surveillance was performed with a qPCR test incorporating treatment (BZ) resistance markers. There is a need to raise clinician awareness around treatment-resistant hookworm in dogs and the capability of fecal surveillance for genotypic and phenotypic resistance.