Hookworm Ancylostoma Caninum Research Articles

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 β-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.

Updates on Emerging and Evolving Gastrointestinal Parasites in Dogs and Cats

Gastrointestinal parasites are a common veterinary concern and pet owner conversation. Globally, parasite ranges and abundances are evolving, and various clinical management challenges in dogs and cats have emerged. Some of these are novel, such as hookworm multi-drug resistance, and others have been veterinary challenges for years, like Giardia's zoonotic potential. For these examples and others, there has been increased awareness of the need for appropriate veterinary anthelmintic use and stewardship. This review provides an update on selected gastrointestinal parasites of One Health importance, highlights gastrointestinal parasite global management and prevention recommendations, and summarises recent research, along with potential risks associated with pet importation and travel. Suggestions on freely available online resources to assist with practical veterinary clinical management (screening, diagnosis, treatment, prevention) and pet owner communication, along with addressing One Health and antiparasitic stewardship concerns, are provided from a global perspective, for example The European Scientific Counsel for Companion Animal Parasites. Clinical cases are drawn from molecular diagnostic faecal surveillance of dogs and cats during 2022 and 2023, and include the following gastrointestinal parasites with zoonotic potential: hookworms (Ancylostoma caninum), tapeworms (Echinococcus multilocularis), roundworms (Baylisascaris procyonis) and Giardia.

Multiple anthelmintic drug resistance in hookworms (Ancylostoma caninum) in a Labrador breeding and training kennel in Georgia, USA.

To evaluate the efficacy of the 3 major classes of anthelmintics used for the treatment of hookworms in dogs in the US and an extralabel treatment with an FDA-approved product for use in cats in a Labrador kennel with a history of persistent hookworm infections. 22 dogs housed in a single kennel comprised of the following breeds: 19 Labrador Retrievers, 1 English Cocker Spaniel, 1 Chesapeake Bay Retriever, and 1 Boykin Spaniel. We performed a fecal egg count (FEC) reduction test using 22 dogs that were allocated randomly to 1 of 5 treatment groups: pyrantel pamoate (Pyrantel pamoate suspension), fenbendazole (Safe-Guard suspension 10%), milbemycin oxime (Interceptor), moxidectin plus imidacloprid (Advantage Multi), and emodepside plus praziquantel (Profender topical solution for cats). FEC was performed on samples collected on days 0 and 11. FEC reductions for the milbemycin oxime, moxidectin plus imidacloprid, and emodepside plus praziquantel groups were 43.9%, 57.4%, and 100%, respectively. The FEC increased following treatment for the pyrantel and fenbendazole groups. These data demonstrate that the Ancylostoma caninum infecting the dogs in this kennel are highly resistant to all major anthelmintic classes approved for use in dogs in the US but are susceptible to emodepside. This was the first report of multiple anthelmintic drug-resistant A caninum in a dog kennel that does not involve Greyhounds.

Anthelmintic Effects of Betel Nut (Areca catechu) Extracts on Roundworms (Toxocara canis) and Hookworms (Ancylostoma caninum) Isolated from Vietnamese Dogs

Anthelmintic Effects of Betel Nut (Areca catechu) Extracts on Roundworms (Toxocara canis) and Hookworms (Ancylostoma caninum) Isolated from Vietnamese Dogs

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

SummaryThe 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.

Efficacy evaluation of anthelmintic products against an infection with the canine hookworm (Ancylostoma caninum) isolate Worthy 4.1F3P in dogs

Ancylostoma caninum is the most prevalent intestinal nematode of dogs, and has a zoonotic potential. Multiple-drug resistance (MDR) has been confirmed in a number of A. caninum isolates, including isolate Worthy 4.1F3P, against all anthelmintic drug classes approved for hookworm treatment in dogs in the United States (US). The cyclooctadepsipeptide emodepside is not registered to use in dogs in the US, but in a number of other countries/regions. The objective of this study was to evaluate the efficacy of emodepside + praziquantel, as well as three commercial products that are commonly used in the US for treatment of hookworms, against a suspected (subsequently confirmed) MDR A. caninum isolate Worthy 4.1F3P. 40 dogs infected on study day (SD) 0 with 300 third-stage larvae, were randomly allocated to one of five treatment groups with eight dogs each: pyrantel pamoate (Nemex®-2), fenbendazole (Panacur® C), milbemycin oxime (Interceptor®), emodepside + praziquantel tablets and non-treated control. Fecal egg counts (FEC) were performed on SDs 19, 20, 22, 27, 31 and 34. All treatments were administered as per label requirements on SD 24 to dogs in Groups 1 through 4. Two additional treatments were administered on SDs 25 and 26 to dogs in Group 2 as per label requirements. Dogs were necropsied on SD 34 and the digestive tract was removed/processed for worm recovery and enumeration. The geometric mean (GM) worm counts for the control group was 97.4, and for the pyrantel pamoate, fenbendazole, milbemycin oxime, and emodepside + praziquantel groups were 74.8, 72.0, 88.9, and 0.4, respectively. These yielded efficacies of 23.2%, 26.1%, and 8.8%, and 99.6%, respectively. These data support previous findings of the MDR status of Worthy 4.1F3P as treatments with pyrantel pamoate, fenbendazole and milbemycin oxime lacked efficacy. In sharp contrast, Worthy 4.1F3P was highly susceptible to treatment with emodepside + praziquantel.

Multiple drug resistance in the canine hookworm Ancylostoma caninum: an emerging threat?

BackgroundThe canine hookworm, Ancylostoma caninum is the most prevalent and important intestinal nematode parasite of dogs in the USA. Hookworms are typically well controlled by treatment with all commonly used anthelmintics that are approved for this use in dogs. However, in the past few years, cases of recurrent/persistent canine hookworm infections appear to have dramatically increased, suggesting that anthelmintic resistance (AR) may have evolved in this parasite. These cases are highly overrepresented by greyhounds, but multiple other breeds are also represented. The aim of this study was to characterize several of these suspected resistant isolates using in vitro, genetic and clinical testing to determine if these cases represent true anthelmintic resistance in A. caninum.MethodsFecal samples containing hookworm eggs from three cases of persistent hookworm infections; one from a greyhound, one from a miniature schnauzer and one from a hound-mix, were received by our laboratory. These were then used to establish infections in laboratory dogs and to perform egg hatch assays (EHA) and larval development assays (LDA) for detecting resistance to benzimidazoles and macrocyclic lactones, respectively. Additional EHA and LDA were performed on eggs recovered from the laboratory-induced infections. Fecal egg count reduction tests were performed to detect resistance to pyrantel. Deep amplicon sequencing assays were developed to measure the frequency of non-synonymous single nucleotide polymorphisms (SNP) at codons 167, 198 and 200 of the A. caninum isotype-1 β-tubulin gene.ResultsResistance ratios for the three A. caninum isolates tested ranged from 6.0 to > 100 and 5.5 to 69.8 for the EHA and LDA, respectively. Following treatment with pyrantel, reduction in faecal egg counts was negative or 0%. Deep amplicon sequencing of the isotype-1 β-tubulin gene identified a high frequency of resistance-associated SNPs at codon 167 in all three resistant isolates and in two additional clinical cases.ConclusionsThese data conclusively demonstrate multiple anthelmintic resistance in multiple independent isolates of A. caninum, strongly suggesting that this is an emerging problem in the USA. Furthermore, evidence suggest that these resistant hookworms originate from racing greyhound farms and kennels, though additional research is needed to confirm this.

The mitochondrial genome of the dog hookworm Ancylostoma caninum (Nematoda, Ancylostomatidae) from Southwest China

The dog hookworm Ancylostoma caninum (Nematoda, Ancylostomatidae) is a blood-feeding intestinal parasitic nematode and can cause ancylostomiasis in humans. In this study, the complete mitochondrial genome of this anthropozoonotic hookworm was sequenced through Illumina deep sequencing technology. The whole genome was 13,721 bp in length and encoded 36 genes including 12 protein-coding genes, 22 transfer RNAs, and 2 ribosomal RNAs. Phylogeny revealed that A. caninum grouped with species from Ancylostomatinae and separated from species of Bunostominae in the family Ancylostomatidae. Amongst the subfamily Ancylostomatinae, three dog-originated A. caninum, regardless of isolate origins, clustered together and were more closely related to the cat hookworm A. tubaeforme and the human hookworm A. duodenale than to the dog/cat hookworm A. ceylanicum and the sea lion hookworm Uncinaria sanguinis. Taken together, the cumulative mitochondrial DNA data provides insights into phylogenetic studies among Ancylostomatidae nematodes.

Isolation and characterization of a naturally occurring multidrug-resistant strain of the canine hookworm, Ancylostoma caninum

Soil-transmitted nematodes infect over a billion people and place several billion more at risk of infection. Hookworm disease is the most significant of these soil-transmitted nematodes, with over 500 million people infected. Hookworm infection can result in debilitating and sometimes fatal iron-deficiency anemia, which is particularly devastating in children and pregnant women. Currently, hookworms and other soil-transmitted nematodes are controlled by administration of a single dose of a benzimidazole to targeted populations in endemic areas. While effective, people are quickly re-infected, necessitating frequent treatment. Widespread exposure to anthelmintic drugs can place significant selective pressure on parasitic nematodes to generate resistance, which has severely compromised benzimidazole anthelmintics for control of livestock nematodes in many areas of the world. Here we report, to our knowledge, the first naturally occurring multidrug-resistant strain of the canine hookworm Ancylostoma caninum. We reveal that this isolate is resistant to fenbendazole at the clinical dosage of 50 mg/kg for 3 days. Our data shows that this strain harbors a fixed, single base pair mutation at amino acid 167 of the β-tubulin isotype 1 gene, and by using CRISPR/Cas9 we demonstrate that introduction of this mutation into the corresponding amino acid in the orthologous β-tubulin gene of Caenorhabditis elegans confers a similar level of resistance to thiabendazole. We also show that the isolate is resistant to the macrocyclic lactone anthelmintic ivermectin. Understanding the mechanism of anthelmintic resistance is important for rational design of control strategies to maintain the usefulness of current drugs, and to monitor the emergence of resistance. The isolate we describe represents the first multidrug-resistant strain of A. caninum reported, and our data reveal a resistance marker that can emerge naturally in response to heavy anthelminthic treatment.

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

The components of the transforming growth factor β (TGF-β) signaling pathway in parasitic nematodes remain unknown. In this research, a type I receptor for TGF-β 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.

Development of new microsatellites for the hookworm Ancylostoma caninum and analysis of genetic diversity in Brazilian populations

Considering the great efforts towards formulating a vaccine against hookworms, and the concerns about the spread of drug resistance through hookworm populations, it is justified to study the molecular diversity and population genetic structure of these nematodes. This work had the aim to develop microsatellite markers to investigate the genetic structure and the molecular diversity of Brazilian populations of Ancylostoma caninum. Seven microsatellites markers were successfully used to characterize five Brazilian populations. These findings may contribute to a better comprehension of the ecology, patterns of transmission, drug resistances and development of immunotherapeutic strategies in hookworms.

An approach to reduce false viability assessment of hookworm eggs with vital stains

The effectiveness of vital stains to assess the viability of hookworm eggs depends on the permeability of eggshells. The eggshells may not be permeable immediately after inactivation, and this can lead to over-estimation of viable eggs in a sample. In this study, heat-inactivated eggs of hookworm (Ancylostoma caninum) were used to evaluate the time required for eggshells to become permeable to three vital stains (eosin Y, methyl red, and methylene blue). The results of this study showed that heat-inactivated eggs incubated for 1h at room temperature (24±1°C) contain 32–53% non-viable eggs or permeable eggshells. The numbers of non-viable eggs increased to 88–94% after 6h of incubation. The accurate viability assessment was achieved after 12h of incubation at room temperature. Vital stains did not play a significant (P>0.05) role in reducing the false viability. This study provides information that can aid in the accurate detection of viable hookworm eggs from environmental samples comprised of mixed populations that are viable and non-viable with permeable or impermeable eggshells.

A recombinant fusion protein derived from dog hookworm inhibits autoantibody-induced dermal-epidermal separation exvivo.

The proteins secreted by parasitic nematodes are evolutionarily optimized molecules with unique capabilities of suppressing the immune response of the host organism. Neutrophil inhibitory factor (NIF), which is secreted by the dog hookworm Ancylostoma caninum, binds to the β2 integrin CD11b/CD18, which is expressed on human neutrophils, eosinophils, monocytes and macrophages and inhibits neutrophil-dependent lung injury and neutrophil invasion of ischaemic brain tissue. Neutrophils are key players in the pathogenesis of subepidermal autoimmune blistering diseases (sAIBDs), and their pathogenic activities are crucially dependent on β2 integrin functionality. Based on the template of single-stranded, dimerizing antibody derivatives, which are already used in cancer treatment, we designed a novel biologic, NIF-IGHE-CH4, comprising NIF and the dimerizing but otherwise inert constant heavy subdomain 4 (CH4) of human IgE (IGHE). This molecule was evaluated in a variety of invitro assays, demonstrating its ability to inhibit pathogenically relevant neutrophil functions such as migration, adhesion and spreading, and release of reactive oxygen species. Finally, we confirmed that NIF-IGHE-CH4 inhibits blister formation in an exvivo assay of sAIBD. These results suggest that NIF-IGHE-CH4 is a novel potential anti-inflammatory drug for the treatment of neutrophil-mediated diseases such as sAIBDs. This study promotes the drugs from bugs concept and encourages further research and development focused on turning parasite proteins into useful anti-inflammatory biologics.

Rapid concentration and sensitive detection of hookworm ova from wastewater matrices using a real-time PCR method

The risk of human hookworm infections from land application of wastewater matrices could be high in regions with high hookworm prevalence. A rapid, sensitive and specific hookworm detection method from wastewater matrices is required in order to assess human health risks. Currently available methods used to identify hookworm ova to the species level are time consuming and lack accuracy. In this study, a real-time PCR method was developed for the rapid, sensitive and specific detection of canine hookworm (Ancylostoma caninum) ova from wastewater matrices. A. caninum was chosen because of its morphological similarity to the human hookworm (Ancylostoma duodenale and Necator americanus). The newly developed PCR method has high detection sensitivity with the ability to detect less than one A. caninum ova from 1 L of secondary treated wastewater at the mean threshold cycle (CT) values ranging from 30.1 to 34.3. The method is also able to detect four A. caninum ova from 1 L of raw wastewater and from ∼4 g of treated sludge with mean CT values ranging from 35.6 to 39.8 and 39.8 to 39.9, respectively. The better detection sensitivity obtained for secondary treated wastewater compared to raw wastewater and sludge samples could be attributed to sample turbidity. The proposed method appears to be rapid, sensitive and specific compared to traditional methods and has potential to aid in the public health risk assessment associated with land application of wastewater matrices. Furthermore, the method can be adapted to detect other helminth ova of interest from wastewater matrices.

Expression profile of heat shock response factors during hookworm larval activation and parasitic development

When organisms are exposed to an increase in temperature, they undergo a heat shock response (HSR) regulated by the transcription factor heat shock factor 1 (HSF-1). The heat shock response includes the rapid changes in gene expression initiated by binding of HSF-1 to response elements in the promoters of heat shock genes. Heat shock proteins function as molecular chaperones to protect proteins during periods of elevated temperature and other stress. During infection, hookworm infective third stage larvae (L3) undergo a temperature shift from ambient to host temperature. This increased temperature is required for the resumption of feeding and activation of L3, but whether this increase initiates a heat shock response is unknown. To investigate the role of the heat shock in hookworm L3 activation and parasitic development, we identified and characterized the expression profile of several components of the heat shock response in the hookworm Ancylostoma caninum. We cloned DNAs encoding an hsp70 family member (Aca-hsp-1) and an hsp90 family member (Aca-daf-21). Exposure to a heat shock of 42°C for one hour caused significant up-regulation of both genes, which slowly returned to near baseline levels following one hour attenuation at 22°C. Neither gene was up-regulated in response to host temperature (37°C). Conversely, levels of hsf-1 remained unchanged during heat shock, but increased in response to incubation at 37°C. During activation, both hsp-1 and daf-21 are down regulated early, although daf-21 levels increase significantly in non-activated control larvae after 12h, and slightly in activated larvae by 24h incubation. The heat shock response modulators celastrol and KNK437 were tested for their effects on gene expression during heat shock and activation. Pre-incubation with celastrol, an HSP90 inhibitor that promotes heat shock gene expression, slightly up-regulated expression of both hsp-1 and daf-21 during heat shock. KNK437, an inhibitor of heat shock protein expression, slightly down regulated both genes under similar conditions. Both modulators inhibited activation-associated feeding, but neither had an effect on hsp-1 levels in activated L3 at 16h. Both celastrol and KNK437 prevent the up-regulation of daf-21 and hsf-1 seen in non-activated control larvae during activation, and significantly down regulated expression of the HSF-1 negative regulator Aca-hsb-1 in activated larvae. Expression levels of heat shock response factors were examined in developing Ancylostoma ceylanicum larvae recovered from infected hosts and found to differ significantly from the expression profile of activated L3, suggesting that feeding during in vitro activation is regulated differently than parasitic development. Our results indicate that a classical heat shock response is not induced at host temperature and is suppressed during larval recovery and parasitic development in the host, but a partial heat shock response is induced after extended incubation at host temperature in the absence of a developmental signal, possibly to protect against heat stress.

Infectious disease and red wolf conservation: assessment of disease occurrence and associated risks.

Infectious diseases pose a significant threat to global biodiversity and may contribute to extinction. As such, establishing baseline disease prevalence in vulnerable species where disease could affect persistence is important to conservation. We assessed potential disease threats to endangered red wolves (Canis rufus) by evaluating regional (southeastern United States) disease occurrences in mammals and parasite prevalence in red wolves and sympatric coyotes (Canis latrans) in North Carolina. Common viral pathogens in the southeast region, such as canine distemper and canine parvovirus, and numerous widespread endoparasites could pose a threat to the red wolf population. The most prevalent parasites in red wolves and sympatric coyotes were heartworm (Dirofilaria immitis), hookworm (Ancylostoma caninum), and Ehrlichia spp.; several red wolves and coyotes were also positive for bacteria causing Lyme disease (Borrelia burgdorferi). Coyotes had a more species-rich parasite community than red wolves, suggesting they could harbor more parasites and act as a disease reservoir. Species identity and sex did not significantly affect parasite loads, but young canids were less likely to have heartworm and more likely to have high levels of endoparasites. Continued disease monitoring is important for red wolf recovery because low levels of genetic variability may compromise the wolves’ abilities to combat novel pathogens from closely related species, such as domestic dogs and coyotes.

Differences in the faecal microbiome of non-diarrhoeic clinically healthy dogs and cats associated with Giardia duodenalis infection: impact of hookworms and coccidia

The protozoan parasite Giardia duodenalis causes a waterborne diarrhoeal disease in animals and humans, yet many Giardia-infected hosts remain asymptomatic. Mixed parasite infections are common in both animals and humans with unknown consequences for Giardia or other parasites. We compared the composition and diversity of bacterial communities from 40 dogs, including free-roaming dogs, and 21 surrendered cats from Australia. The dog cohort included 17 (42.5%) dogs positive for Giardia and 13 (32.5%) dogs positive for dog hookworm (Ancylostoma caninum). The cat samples included eight positive for Giardia and eight positive for Cystoisospora. The V4 region of 16S rRNA was sequenced at an average of 36,383 high quality sequences (>200bp) per sample using the Ion Torrent PGM™ platform. In dogs we found significant (P<0.05, AnoSim) difference between the Giardia-positive and -negative groups when evaluating bacterial genera. No such difference was demonstrated between Ancylostoma-positive and -negative dogs. However, there was a modest but not significant separation of the Giardia-negative and -positive dogs (P=0.09, UniFrac) using principal coordinate analysis. Removal of dogs with hookworms further separated Giardia-positive and -negative groupings (P=0.06, UniFrac). In cats, the presence of Giardia was not associated with a significant difference based on bacterial genera (P>0.05, AnoSim). Cystoisospora-positive cats, however, exhibited significantly different profiles from Cystoisospora-negative cats (P=0.02, AnoSim) and UniFrac showed significant separation of Cystoisospora-positive and -negative samples (P<0.01). The results suggest that in clinically heathy dogs and cats, helminths and protozoa are associated with different microbiomes and possibly variable gut microbiota functions. Understanding the association of parasites and microbiomes has important consequences for the administration of antiparasitic drugs in animals and humans.

First identification of the F200Y SNP in the β-tubulin gene linked to benzimidazole resistance in Ancylostoma caninum

Single nucleotide polymorphisms (SNPs) in the β-tubulin isotype 1 gene in codons 167, 198 and 200 have been associated with benzimidazole resistance in some nematodes, although no polymorphisms in this gene have been described in the hookworm Ancylostoma caninum to date. This study aimed to screen for SNPs in the β-tubulin isotype 1 gene at codons 198 and 200 associated with benzimidazole resistance in A. caninum worms recovered from naturally infected dogs from two Brazilian states (Minas Gerais and Piauí). To perform this analysis, a molecular technique based on the amplification refractory mutation system (ARMS-PCR) was standardized. A total of 110 individual worms recovered from 9 dogs in Piauí and 124 individual worms recovered from 10 dogs in Minas Gerais were analyzed using this methodology. The presence of a SNP at position 200 was observed at a low frequency (0.8%), which was only detected in the worm population from Minas Gerais State. A total of 75 randomly selected samples were sequenced to validate this technique, and the presence of the SNP was confirmed. Furthermore, SNP at position 198 were evaluated by sequencing, and none were detected in this region. This is the first report of the presence of a SNP in the β-tubulin isotype 1 gene associated with benzimidazole resistance in an A. caninum population.

Kv1.3 channel-blocking immunomodulatory peptides from parasitic worms: implications for autoimmune diseases.

The voltage-gated potassium (Kv) 1.3 channel is widely regarded as a therapeutic target for immunomodulation in autoimmune diseases. ShK-186, a selective inhibitor of Kv1.3 channels, ameliorates autoimmune diseases in rodent models, and human phase 1 trials of this agent in healthy volunteers have been completed. In this study, we identified and characterized a large family of Stichodactyla helianthus toxin (ShK)-related peptides in parasitic worms. Based on phylogenetic analysis, 2 worm peptides were selected for study: AcK1, a 51-residue peptide expressed in the anterior secretory glands of the dog-infecting hookworm Ancylostoma caninum and the human-infecting hookworm Ancylostoma ceylanicum, and BmK1, the C-terminal domain of a metalloprotease from the filarial worm Brugia malayi. These peptides in solution adopt helical structures closely resembling that of ShK. At doses in the nanomolar-micromolar range, they block native Kv1.3 in human T cells and cloned Kv1.3 stably expressed in L929 mouse fibroblasts. They preferentially suppress the proliferation of rat CCR7(-) effector memory T cells without affecting naive and central memory subsets and inhibit the delayed-type hypersensitivity (DTH) response caused by skin-homing effector memory T cells in rats. Further, they suppress IFNγ production by human T lymphocytes. ShK-related peptides in parasitic worms may contribute to the potential beneficial effects of probiotic parasitic worm therapy in human autoimmune diseases.