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We conducted a cross-sectional study of zoonotic hookworm Ancylostoma ceylanicum in humans in Western Province, Papua New Guinea, confirmed by internal transcribed spacer sequencing. Overall hookworm prevalence was 54.9%; A. ceylanicum hookworms were present in 3.3% of specimens. One Health approaches are needed for hookworm control in Papua New Guinea.

Hookworms are widespread zoonotic parasites affecting humans and companion animals worldwide. We report molecular characterization of Ancylostoma ceylanicum and A. braziliense in a cat imported into Romania from Guadeloupe, an overseas administrative region of the French Republic in the Caribbean. This finding underscores the role of molecular diagnostics in detecting the transboundary introduction of exotic hookworms with significant zoonotic potential. Graphical Abstract

Background Gastrointestinal (GI) parasites of dogs, including helminths and protozoans, are of substantial relevance to veterinary medicine and public health. Nevertheless, epidemiological data are scarce worldwide, especially in remote locations. The emergence of novel technologies and diagnostic platforms facilitates comprehensive screening of multiple GI parasites. Our study aims to establish a baseline prevalence for GI parasites in dogs from Saipan, Northern Mariana Islands. Methods Fecal samples were collected from dogs ( n  = 420) from May to June 2023 during a spay-neuter campaign. Age, sex, ownership status, and residing location were recorded. Following genomic extraction, samples were screened using the KeyScreen™ GI Parasite PCR (Antech Diagnostics), a real-time PCR panel that detects 20 endoparasite infections, detects benzimidazole resistance in Ancylostoma caninum , and determines the zoonotic potential of Giardia duodenalis . If inconclusive results for Ancylostoma spp. were obtained, conventional PCR and Sanger sequencing were performed, targeting the ITS-1 region for species identification. Additionally, demographics (i.e. age, sex, ownership, and residing location) were evaluated as potential risk factors for each pathogen as the outcome with an initial univariate analysis, followed by multivariable logistic regression with backward stepwise selection. Results Overall, parasites were detected in 267/420 (63.5%; 95% CI: 58.7–68.1) canine samples. The most detected parasite genus was Ancylostoma spp. ( n  = 224; 53.3%), followed by G. duodenalis ( n  = 67; 15.9%), Trichuris ( n  = 39; 9.2%), Dipylidium ( n  = 25; 5.9%), Toxocara ( n  = 15; 3.5%), Cystoisospora ( n  = 10; 2.3%), and Cryptosporidium ( n  = 5; 1.1%). Assemblages with zoonotic potential of G. duodenalis and the SNPs 167Y and 134H in the isotype 1 Beta-tubulin gene associated with benzimidazole-resistance in A. caninum were not detected. Risk factors significantly associated with infection were age, district, and ownership with Trichuris ; age and ownership with Ancylostoma , Giardia , and Dipylidium ; and ownership with Toxocara and Cystoisospora . Hookworm-positive samples were further characterized to species level. Overall, Ancylostoma caninum and A. ceylanicum / A. duodenale were confirmed in 196 (46.7%) and 57 (13.5%) dogs, respectively. Further sequencing confirmed the presence of zoonotic A. ceylanicum in at least 21 samples, approximately 5% of the sampled dog population, distributed geographically across all districts. Conclusions To our knowledge, our study is the first to provide epidemiological data on canine gastrointestinal parasites in Saipan. The high prevalence of multiple parasites of One Health importance reinforces the need for surveillance and the implementation of prevention and control strategies island-wide, especially targeting A. ceylanicum , a zoonotic hookworm, that may establish patent infections in both companion animals and humans. Graphical Abstract

Hookworms are intestinal parasites that cause major public health problems, especially in developing countries. To differentiate eggs from different hookworm species, it is necessary to use molecular methodologies, since the eggs are morphologically similar. Here, we performed the molecular identification of single hookworm eggs from six Brazilian states. Of the 634 eggs individually analyzed, 98.1% (622/634) represented Necator americanus , and surprisingly, 1.9% (12/634 eggs from the same patient) represented Ancylostoma caninum . DNA analysis of the A. caninum -positive stool sample revealed no contamination with animal feces. This is the first report of the presence of A. caninum eggs in human feces, which may have a direct implication for the epidemiology of hookworm infection caused by this species. This suggests the need for special attention regarding prophylaxis, as different reservoirs, previously not described, may have great relevance for the spread of A. caninum .

More than 470 million people globally are infected with the hookworms Ancylostoma ceylanicum and Necator americanus , resulting in an annual loss of 2.1 to 4 million disability-adjusted-life-years. Current infection management approaches are limited by modest drug efficacy, the costs associated with frequent mass drug administration campaigns, and the risk of reinfection and burgeoning drug resistance. Subunit vaccines based on proteins excreted and secreted (ES) by hookworms that reduce worm numbers and associated disease burden are a promising management strategy to overcome these limitations. However, studies on the ES proteomes of hookworms have mainly described proteins from the adult life stage which may preclude the opportunity to target the infective larva. Here, we employed high resolution mass spectrometry to identify 103 and 57 ES proteins from the infective third larvae stage (L3) as well as 106 and 512 ES proteins from the adult N . americanus and A . ceylanicum respectively. Comparisons between these developmental stages identified 91 and 41 proteins uniquely expressed in the L3 ES products of N . americanus and A . ceylanicum , respectively. We characterized these proteins based on functional annotation, KEGG pathway analysis, InterProScan signature and gene ontology. We also performed reciprocal BLAST analysis to identify orthologs across species for both the L3 and adult stages and identified five orthologous proteins in both life stages and 15 proteins that could be detected only in the L3 stage of both species. Last, we performed a three-way reciprocal BLAST on the L3 proteomes from both hookworm species together with a previously reported L3 proteome from the rodent hookworm Nippostrongylus brasiliensis , and identified eight L3 proteins that could be readily deployed for testing using well established rodent models. This novel characterization of L3 proteins and taxonomic conservation across hookworm species provides a raft of potential candidates for vaccine discovery for prevention of hookworm infection and disease.

Background Dogs and cats can harbor hookworms, which may contribute to zoonotic infections. This study investigates hookworm infections in carnivores from the Caspian Sea littoral region of northern Iran, focusing on molecular and morphological identification. Methods A cross-sectional study was conducted between September 2015 and October 2024, involving 172 road-killed carnivores including 78 stray dogs ( Canis familiaris ), 62 golden jackals ( Canis aureus ), and 32 stray cats ( Felis catus ), all collected from the northern Iranian provinces of Guilan and Mazandaran. Results Overall, 46 (26.74%) animals tested positive for hookworms. Among these, Ancylostoma caninum was found in dogs and golden jackals, Ancylostoma tubaeforme in cats, and Uncinaria stenocephala in dogs and golden jackals. Morphological analyses confirmed key differences between A. caninum and A. tubaeforme , including size, esophageal features, and bursal structure. Molecular identification was supported by 18S, ITS1-5.8S-ITS2 rDNA, 28S, and mitochondrial cytochrome c oxidase 1 (COX1) gene sequences, demonstrating high similarity with previously identified isolates in GenBank. Phylogenetic analysis of the ITS and COX1 sequences revealed distinct clades for each species, with A. caninum and A. tubaeforme clustering together in the Ancylostoma spp. group. Conclusion These results provide important insights into hookworm diversity and highlight the zoonotic risks posed by these parasites.

Background The 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 . Methods Fecal 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. Results Resistance 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. Conclusions These 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.

Background Hookworms belonging to the genus Ancylostoma (Dubini, 1843) cause ancylostomiasis, a disease of considerable concern in humans and domestic and wild animals. Molecular and epidemiological data support evidence for the zoonotic potential among species of Ancylostoma where transmission to humans is facilitated by rapid urbanization and increased human-wildlife interactions. It is important to assess and describe these potential zoonotic parasite species in wildlife, especially in hosts that have physiological similarities to humans and share their habitat. Moreover, defining species diversity within parasite groups that can circulate among free-ranging host species and humans also provides a pathway to understanding the distribution of infection and disease. In this study, we describe a previously unrecognized species of hookworm in the genus Ancylostoma in the giant panda, including criteria for morphological and molecular characterization. Methods The hookworm specimens were obtained from a wild giant panda that died in the Fengtongzai Natural Reserve in Sichuan Province of China in November 2013. They were microscopically examined and then genetically analyzed by sequencing the nuclear internal transcribed spacer (ITS, ITS1-5.8S-ITS2) and mitochondrial cytochrome c oxidase subunit 1 ( cox 1) genes in two representative specimens (one female and one male, FTZ1 and FTZ2, respectively). Results Ancylostoma ailuropodae n. sp. is proposed for these hookworms. Morphologically the hookworm specimens differ from other congeneric species primarily based on the structure of the buccal capsule in males and females, characterized by 2 pairs of ventrolateral and 2 pairs of dorsolateral teeth; males differ in the structure and shape of the copulatory bursa, where the dorsal ray possesses 2 digitations. Pairwise nuclear and mitochondrial DNA comparisons, genetic distance analysis, and phylogenetic data strongly indicate that A. ailuropodae from giant pandas is a separate species which shared a most recent common ancestor with A. ceylanicum Looss, 1911 in the genus Ancylostoma (family Ancylostomatidae). Conclusion Ancylostoma ailuropodae n. sp. is the fourth species of hookworm described from the Ursidae and the fifteenth species assigned to the genus Ancylostoma. A sister-species association with A. ceylanicum and phylogenetic distinctiveness from the monophyletic Uncinaria Frölich, 1789 among ursids and other carnivorans indicate a history of host colonization in the evolutionary radiation among ancylostomatid hookworms. Further, phylogenetic relationships among bears and a history of ecological and geographical isolation for giant pandas may be consistent with two independent events of host colonization in the diversification of Ancylostoma among ursid hosts. A history for host colonization within this assemblage and the relationship for A. ailuropodae n. sp. demonstrate the potential of this species as a zoonotic parasite and as a possible threat to human health. The cumulative morphological, molecular and phylogenetic data presented for A. ailuropodae n. sp. provides a better understanding of the taxonomy, diagnostics and evolutionary biology of the hookworms.

Ancylostoma caninum is an important zoonotic gastrointestinal nematode of dogs worldwide and a close relative of human hookworms. We recently reported that racing greyhound dogs in the USA are infected with A . caninum that are commonly resistant to multiple anthelmintics. Benzimidazole resistance in A . caninum in greyhounds was associated with a high frequency of the canonical F167Y(T T C>T A C) isotype-1 β-tubulin mutation. In this work, we show that benzimidazole resistance is remarkably widespread in A . caninum from domestic dogs across the USA. First, we identified and showed the functional significance of a novel benzimidazole isotype-1 β-tubulin resistance mutation, Q134H(CA A >CA T ). Several benzimidazole resistant A . caninum isolates from greyhounds with a low frequency of the F167Y(T T C>T A C) mutation had a high frequency of a Q134H(CA A >CA T ) mutation not previously reported from any eukaryotic pathogen in the field. Structural modeling predicted that the Q134 residue is directly involved in benzimidazole drug binding and that the 134H substitution would significantly reduce binding affinity. Introduction of the Q134H substitution into the C . elegans β-tubulin gene ben-1 , by CRISPR-Cas9 editing, conferred similar levels of resistance as a ben-1 null allele. Deep amplicon sequencing on A . caninum eggs from 685 hookworm positive pet dog fecal samples revealed that both mutations were widespread across the USA, with prevalences of 49.7% (overall mean frequency 54.0%) and 31.1% (overall mean frequency 16.4%) for F167Y(T T C>T A C) and Q134H(CA A >CA T ), respectively. Canonical codon 198 and 200 benzimidazole resistance mutations were absent. The F167Y(T T C>T A C) mutation had a significantly higher prevalence and frequency in Western USA than in other regions, which we hypothesize is due to differences in refugia. This work has important implications for companion animal parasite control and the potential emergence of drug resistance in human hookworms.

Biologics, protein- and peptide-based drugs derived from living organisms or cell lines have emerged as effective therapies across a broad range of indications. However, parenteral administration and the need for frequent dosing increase costs and limit compliance, creating an urgent need for innovative platforms capable of continuously, safely, and efficiently delivering sustained biologics in situ within the host. Here, we describe a bioengineered hookworm platform to manufacture and deliver biologic therapeutics in vivo. As proof of concept, we engineer the Ancylostoma ceylanicum secretome by inserting a human single-chain variable fragment antibody (s16-HuScFv) into its genome. Transgene expression does not perturb surrounding gene expression, and heritable transgenesis is confirmed. The s16-HuScFv transgene product is secreted into host circulation and partially neutralizes tetrodotoxin. Given the availability of controlled human infections, our disease-agnostic bioengineered hookworm platform offers a next-generation approach to address a suite of chronic human diseases, and with a single-dose administration, could potentially produce and deliver biologic medicines within the human host for years. Biologics and protein-based drugs often require frequent, costly injections. Here, authors have engineered a hookworm to continuously produce and secrete a therapeutic human antibody directly into a host, demonstrating potential as a single-dose platform for long-term continuous delivery of therapeutics.