In vitro immunoregulatory role of recombinant Ancylostoma ceylanicum calreticulin.

Ancylostoma ceylanicum is a zoonotic soil-derived nematode that parasitizes the intestines of humans and animals (dogs and cats), leading to malnutrition and iron-deficiency anemia. Helminth parasites secrete calreticulin (CRT), which regulates or blocks the host's immune response. However, no data on A. ceylanicum calreticulin (Ace-CRT) are available. We investigated the biological function of recombinant Ace-CRT (rAce-CRT). rAce-CRT showed reliable antigenicity and stimulated the proliferation of mouse splenocytes and canine peripheral blood mononuclear cells. Quantitative reverse-transcription PCR assays revealed that rAce-CRT primarily promoted the expression of T helper 2 cytokines, particularly IL-13, in canine peripheral blood lymphocytes. rAce-CRT inhibited complement-mediated sheep erythrocyte hemolysis in vitro. Our findings indicate that Ace-CRT plays an immunomodulatory role and may be a promising candidate molecule for a hookworm vaccine.

Effect of Soil Temperature on Canine Soil-Transmitted Nematodes in Kandy District with the First Record of Hookworm, Ancylostoma tubaeforme from Sri Lanka.

PURPOSE: Soil-transmitted nematodes (STNs) are widespread in tropical and subtropical regions, particularly where the communities are socio-economically challenged. We investigated the effect of soil temperature on the prevalence and intensity of STN infection in free-roaming dogs. METHODS: Fresh faecal samples collected from free-roaming dogs in Digana and Pussellawa town areas in the Kandy District, Sri Lanka, were microscopically analysed for canine STNs. Soil temperature was measured at each sampling site. Highly prevalent canine hookworm Ancylostoma, was further studied using PCR and sequencing, followed by phylogenetic analysis. RESULTS: The soil temperature ranged between 28 and 31 °C (mean = 29.79 °C) and 18-21 °C (mean = 19.52 °C) in Digana and Pussellawa, respectively, showing a significant difference in the two sites (Students t-test t = 1.68, p < 0.0001). Of the total 44 dogs sampled, 41 (93.2%) were positive for STNs. During microscopic analysis, five nematodes: Ancylostoma spp., Capillaria sp., Strongyloides sp., Toxocara canis, and Trichuris sp., were identified. Ancylostoma species (93.2%) were the most prevalent, followed by Strongyloides sp. (22.7%) and Toxocara canis (15.9%). Infection prevalence of Strongyloides sp. was higher in Digana (40.9%) compared to that in Pussellawa (4.5%; Chi-square test, χ2 = 8.28, p = 0.004) and also the infection intensity from Digana (EPG = 8.02 ± 20.2) compared to that from Pussellawa (0.45 ± 2.1; Mann Whitney U test, p = 0.006). Amplicons (ITS1-5.8S-ITS2) of the expected size for A. caninum, and A. tubaeforme were produced. An A. caninum sequence reported here (OQ101719) illustrated the highest similarity of 99.2% to one of the local sequences (MZ707153) upon pairwise comparison. CONCLUSION: Digana, with a higher soil temperature than Pussellawa, had a significantly higher prevalence and infection intensity, particularly Strongyloides sp. This study also signifies the first molecular identification of hookworm species A. tubaeforme in Sri Lanka.

Characterisation of protective vaccine antigens from the thiol-containing components of excretory/secretory material of Ostertagia ostertagi.

Previous vaccination trials have demonstrated that thiol proteins affinity purified from Ostertagia ostertagi excretory-secretory products (O. ostertagi ES-thiol) are protective against homologous challenge. Here we have shown that protection induced by this vaccine was consistent across four independent vaccine-challenge experiments. Protection is associated with reduced cumulative faecal egg counts across the duration of the trials, relative to control animals. To better understand the diversity of antigens in O. ostertagi ES-thiol we used high-resolution shotgun proteomics to identify 490 unique proteins in the vaccine preparation. The most numerous ES-thiol proteins, with 91 proteins identified, belong to the sperm-coating protein/Tpx/antigen 5/pathogenesis-related protein 1 (SCP/TAPS) family. This family includes previously identified O. ostertagi vaccine antigens O. ostertagi ASP-1 and ASP-2. The ES-thiol fraction also has numerous proteinases, representing three distinct classes, including: metallo-; aspartyl- and cysteine proteinases. In terms of number of family members, the M12 astacin-like metalloproteinases, with 33 proteins, are the most abundant proteinase family in O. ostertagi ES-thiol. The O. ostertagi ES-thiol proteome provides a comprehensive database of proteins present in this vaccine preparation and will guide future vaccine antigen discovery projects.

Hookworm infection as a model for deepen knowledge of iron metabolism and erythropoiesis in anemia.

Over the years, there has been progress in understanding the molecular aspects of iron metabolism and erythropoiesis. However, despite research conducted both in laboratories and living organisms, there are still unanswered questions due to the complex nature of these fields. In this study we investigated the effects of hookworm infection on iron metabolism and how the hosts response to anemia is affected using hamsters infected with Ancylostoma ceylanicum as a model. Our data revealed interesting relationships between infection-induced anemia, erythropoiesis, iron metabolism, and immune modulation, such that the elevated production of erythropoietin (EPO) in renal tissue indicated intensified erythropoiesis in response to anemia. Additionally, the increased expression of the erythroferrone (ERFE) gene in the spleen suggested its involvement in iron regulation and erythropoiesis. Gene expression patterns of genes related to iron metabolism varied in different tissues, indicating tissue-specific adaptations to hypoxia. The modulation of pro-inflammatory and anti-inflammatory cytokines highlighted the delicate balance between immune response and erythropoiesis. Data derived from the investigation of changes induced in iron metabolism and stress erythropoiesis following anemia aid in our understanding of mechanisms related to blood spoliation and anemia, which could potentially be extrapolated or compared to other types or causes of anemia. These findings also contribute to our understanding of the pathophysiology of erythropoiesis in the context of blood loss.

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.

Nationwide USA re-analysis of amplicon metabarcoding targeting ß-tubulin isoform-1 reveals absence of benzimidazole resistant SNPs in Ancylostoma braziliense, Ancylostoma tubaeforme and Uncinaria stenocephala.

Nationwide sampling by Venkatesan and colleagues (2023) described the resistance status of the canine hookworm, Ancylostoma caninum, to benzimidazoles across the USA via ß-tubulin isotype-1 amplicon metabarcoding. In this study, we aimed to use the existing public amplicon metabarcoding data and mine it for the presence of ß-tubulin isotype-1 sequences that belong to hookworm species other than A. caninum. Through bioinformatics analysis we assigned species to A. caninum, Ancylostoma braziliense, Ancylostoma tubaeforme and Uncinaria stenocephala. All non-A. caninum sequences contained only the benzimidazole susceptible residues of ß-tubulin isotype-1. Using two ß-tubulin isotype-1 metabarcoding sequence data (assay targeting 134 and 167 codons, and assay targeting 198 and 200 codons), 2.0% (6/307) and 2.9% (9/310) individual samples had hookworms other than A. caninum (A. braziliense n = 5, A. tubaeforme n = 4 and U. stenocephala n = 2), respectively. We identified one sample containing A. braziliense in each of the Northeastern region and Midwestern region, and in three samples from the Southern region. Presence of A. tubaeforme in dog faeces is considered as pseudoparasitism. There were no statistically significant regional differences for the distribution of each species, for either of the two assays independently or combined (χ2 tests, P > 0.05). Our work demonstrates the utility of the amplicon metabarcoding for the identification of species through antemortem assays, thus resolving the dilemma of assigning hookworm species based on either post-mortem or egg sizes for the identification of hookworms.

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.

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.

Immunity mediates host specificity in the human hookworm Ancylostoma ceylanicum.

Hookworm infection affects millions globally, leading to chronic conditions like malnutrition and anaemia. Among the hookworm species, Ancylostoma ceylanicum stands out as a generalist, capable of infecting various hosts, including humans, cats, dogs and hamsters. Surprisingly, it cannot establish in mice, despite their close phylogenetic relationship to hamsters. The present study investigated the development of A. ceylanicum in immunodeficient NSG mice to determine the contribution of the immune system to host restriction. The infections became patent on day 19 post-infection (PI) and exhibited elevated egg production which lasted for at least 160 days PI. Infective A. ceylanicum larvae reared from eggs released by infected NSG mice were infectious to hamsters and capable of reproduction, indicating that the adults in the NSG mice were producing viable offspring. In contrast, A. ceylanicum showed limited development in outbred Swiss Webster mice. Furthermore, the closely related canine hookworm Ancylostoma caninum was unable to infect and develop in NSG mice, indicating that different mechanisms may determine host specificity even in closely related species. This is the first report of any hookworm species completing its life cycle in a mouse and implicate the immune system in determining host specificity in A. ceylanicum.

Occurrence of zoonotic enteric parasites in fecal samples from dogs in shelters, parks, squares and public roads, and the dog guardians' perception of zoonoses as for the risk to public health in the city of Guarapuava, Paraná, Brazil.

The aim of this study was to determine the occurrence of zoonotic enteroparasites in the feces of dogs from public shelters, squares, parks, and public roads in the city of Guarapuava, Paraná, Brazil, and to evaluate the perception of dog guardians regarding zoonoses and their risk to public health. Fecal samples were collected, coproparasitological examinations were performed to detect zoonotic enteroparasites, and polymerase chain reaction (PCR) was used to identify Giardia spp. and Cryptosporidium spp. Questionnaires were given to guardians who walked their dogs in parks, squares, and public roads, as to assess their perception of zoonoses. A total of 333 samples were collected, of these 75, 123, and 135 of them were from public shelters, squares and parks, and public roads, respectively. One or more parasites were identified in 166 (50 %) samples, of which 58/75 (77 %) were from public shelters, 50/123 (41 %) from squares and parks, and 58/135 (43 %) from public roads. The parasites detected included Ancylostoma spp., Giardia spp., Trichuris spp., Toxocara spp., and Cystoisospora spp., with Ancylostoma spp. having the highest occurrence. PCR was performed on 161 samples for convenience due to financial limitations, because only a portion of the study was funded by the municipal government, of which 15.6 % were positive for Giardia spp., and all were negative for Cryptosporidium spp. In total, 246 guardians were interviewed, of which 36 % said they did not collect their animals' feces during walks, 20 % did not use anti-helminthics on their dogs, and 23 % did not know which diseases could be transmitted by feces. Therefore, we conclude that there is a high infection rate of parasites with zoonotic potential in public places, showing the need to raise awareness among guardians about the diseases transmitted by dog feces, correct vermifugation and the importance of collecting feces in public places.

New insight into genetic diversity of zoonotic-potential Ancylostoma ceylanicum in stray cats living in Bangkok, Thailand, based on deep amplicon sequencing.

AIMS: This study aimed to characterize feline hookworms from stray cats living in Bangkok. METHODS AND RESULTS: A total of 56 hookworm-positive faecal samples were identified for hookworm species by using PCR targeting the ITS1, 5.8S, and ITS2 fragment and qPCR targeting ITS2. Of 56 samples, 96.4% (54/56) were identified as Ancylostoma ceylanicum and 1.8% (1/56) as Ancylostoma caninum. With qPCR, 89.3% (50/56) were identified as single A. ceylanicum infection and 5.4% (3/56) as coinfection of A. ceylanicum and A. caninum. For genetic characterization of A. ceylanicum, 10 samples were pooled, and the partial COI gene was amplified, followed by deep amplicon sequencing. Five pooled samples were analysed, and 99.73% were identified with A. ceylanicum sequences, which were allocated into 19 haplotypes (AC01-AC19). Genetic diversity findings for A. ceylanicum in Asia revealed that three of eight haplotypes considered of zoonotic significance occurred in humans, dogs, and cats, including haplotypes H01, H20, and H21. The predominant haplotype in this study, AC01, was clustered with H01-a zoonotic haplotype. CONCLUSIONS: The diversity obtained by deep amplicon sequencing supported that the A. ceylanicum community had high genetic variation. Deep amplicon sequencing was a useful method to determine source, zoonotic potential, and host-parasite relationship.

Cutaneous larva migrans: A One Health Perspective on Familial Infection Among Tourists Returning from Southeast Asia.

Cutaneous larva migrans (CLM) is a dermatosis caused by accidental infestation with animal hookworms and is widely distributed in tropical and subtropical regions. Humans become infected when their skin comes into contact with soil contaminated with dog faeces. The filariform larvae penetrate and burrow into human skin, causing a condition known as "creeping eruption". We describe a case, well-documented by photos, of CLM infection in a family of three who returned from Thailand.

RNA interference-mediated hookworm control by gut-dwelling lactic acid bacteria.

Background: The complex life cycle of geohelminth parasites make it difficult to manage repeated infections that occur in endemic areas, that requires the development of new therapeutic strategies. Human hookworm Ancylostoma sp. and Necator sp. are a serious problem in many areas of the world where the old age anti-helminthics are ineffective. To address this, we have tried a non-traditional therapeutic approach for a sustainable solution to manage parasite infections. Aims and Objectives: In this study, we attempted to develop a new type of therapy using value-added probiotics-producing antiparasitic RNA interference (RNAi) molecules against a vital hookworm (Ancylostoma sp.) enzyme, astacin-like metalloprotease Ac-MTP-1. A new gut delivery RNAi vector was designed to produce double-stranded RNA (dsRNA) against the target to be delivered by feeding with a probiotic Lactococcus lactis that when administered in endemic areas can potentially be used to control the spread of infection by interrupting the life cycle of hookworm. Results: The engineered probiotics colonizing the gut, when consumed by the parasite released the dsRNA that which knocked down the target by RNAi interfering with their moulting and tissue migration. The initial penetration of the larvae into the gut lining was reduced by 70%, followed by a reduction of up to 50% in migration to the critical organs. The damage caused to the liver (30%-40%) and the kidneys (50%-60%) by the hookworm in mouse models as quantitated by enzymes released in the blood was totally reversed when the worms were pre fed with the engineered L. lactis before the parasite challenge. Conclusions: We believe that this is the first demonstrated knockdown of a target gene in hookworms using probiotics genetically engineered to stimulate RNAi (RNAi food), administered through the oral route. This novel method of parasite control when extended to other hookworms, A. duodenalis and A. ceylanicum can augment the efficacy of the existing anthelminthics if combined with them.

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.

Prevalence of intestinal parasites in street dogs (Canis lupus familiaris) with highlights on zoonosis in Lalitpur, Nepal.

BACKGROUND: The presence of intestinal parasites influences the growth and well-being of canids. Additionally, infected dogs and their faeces with considerably higher eggs/oocysts released per gram (epg/opg) of zoonotic parasites contribute to parasitic spillover to humans, domestic animals and sympatric wildlife. OBJECTIVES: The current study aimed to reveal the prevalence of intestinal parasites (protozoa and helminths) and to list the zoonotically significant parasites in free-roaming street dogs in Lalitpur Metropolitan City, Nepal. METHODS: Fresh faecal samples (n = 332) were collected from feral dogs of varying ages and sexes and transported to the research laboratory. The copro microscopic examination was carried out via direct wet mount, formalin ethyl acetate sedimentation, saturated salt flotation, acid-fast staining and sporulation techniques. RESULTS: Coproscopy revealed an overall 95.7% (318/332) prevalence rate with 23 diverse species of intestinal parasites (10 protozoa and 13 helminths). Among them, 5 protozoa and 11 helminths possessed zoonotic potential, and their overall prevalence was 92.5%. Helminth's overall prevalence was double that of the protozoa (87.7% vs. 43%). Polyparasitism was dominant over mono parasitism (79.5% vs. 16.3%), and co-infection of up to seven species of parasites at a time was recorded. CONCLUSIONS: Urban street dogs harboured a higher prevalence of intestinal parasites that varied with age and sex. Since most of the reported parasites are zoonotic, dog density and parasitic richness indicate a greater spillover risk to humans and domestic animals. Furthermore, this study also provides appropriate 'baseline' data for assessing effective control measures against parasitic infestations among street dogs and controlling their transmission to humans.

Late relapse of cutaneous larva migrans.

Cutaneous larva migrans is a common disease in the tropics and among travelers. The itchy, serpentigious rash often appears within days to weeks after transmission. There are only few reported cases of late relapses. Here, we present a case of a relapse of cutaneous larva migrans more than one year after exposure.

Comparative study of a broad qPCR panel and centrifugal flotation for detection of gastrointestinal parasites in fecal samples from dogs and cats in the United States.

BACKGROUND: For decades, zinc sulfate centrifugal fecal flotation microscopy (ZCF) has been the mainstay technique for gastrointestinal (GI) parasite screening at veterinary clinics and laboratories. Elsewhere, PCR has replaced microscopy because of generally increased sensitivity and detection capabilities; however, until recently it has been unavailable commercially. Therefore, the primary aim of this study was to compare the performance of real-time PCR (qPCR) and ZCF for fecal parasite screening. Secondary aims included further characterization of markers for hookworm treatment resistance and Giardia spp. assemblages with zoonotic potential and qPCR optimization. METHODS: A convenience sampling of 931 canine/feline fecal samples submitted to a veterinary reference laboratory for routine ZCF from the Northeast US (11/2022) was subsequently evaluated by a broad qPCR panel following retention release. Detection frequency and agreement (kappa statistics) were evaluated between ZCF and qPCR for seven GI parasites [hookworm/(Ancylostoma spp.), roundworm/(Toxocara spp.), whipworm/(Trichuris spp.), Giardia duodenalis, Cystoisospora spp., Toxoplasma gondii, and Tritrichomonas blagburni] and detections per sample. Total detection frequencies were compared using a paired t-test; positive sample and co-infection frequencies were compared using Pearson's chi-squared test (p ≤ 0.05 significant) and qPCR frequency for hookworm benzimidazole (BZ) resistance (F167Y) and zoonotic Giardia spp. assemblage markers calculated. Confirmatory testing, characterization, and qPCR optimization were carried out with Sanger sequencing. RESULTS: qPCR detected a significantly higher overall parasite frequency (n = 679) compared to ZCF (n = 437) [p = < 0.0001, t = 14.38, degrees-of-freedom (df) = 930] and 2.6 × the co-infections [qPCR (n = 172) vs. ZCF (n = 66)], which was also significant (p = < 0.0001, X2 = 279.49; df = 1). While overall agreement of parasite detection was substantial [kappa = 0.74; (0.69-0.78], ZCF-undetected parasites reduced agreement for individual and co-infected samples. qPCR detected markers for Ancylostoma caninum BZ resistance (n = 5, 16.1%) and Giardia with zoonotic potential (n = 22, 9.1%) as well as two parasites undetected by ZCF (T. gondii/T. blagburni). Sanger sequencing detected novel roundworm species, and qPCR optimization provided detection beyond ZCF. CONCLUSIONS: These results demonstrate the statistically significant detection frequency advantage offered by qPCR compared to routine ZCF for both single and co-infections. While overall agreement was excellent, this rapid, commercially available qPCR panel offers benefits beyond ZCF with detection of markers for Giardia assemblages with zoonotic potential and hookworm (A. caninum) BZ resistance.

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.

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.