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Background: Hookworms are blood-feeding nematodes that infect millions of people and animals worldwide. The most common species infecting dogs in Europe are representatives of the family Ancylostomatidae, which differ in invasiology, biology and morphological features. However, the differentiation of invasions of species such as Uncinaria stenocephala and Ancylostoma caninum based on a coproscopic examination is problematic. For this reason, it is recommended to use molecular diagnostics for this purpose. The authors of the article investigated the prevalence of U. stenocephala in dogs kept in various living conditions in Slovakia and developed a two-step morphology-molecular analysis-based strategy to identify the genus and the species of eggs and larvae of the Ancylostomatidae family in dogs., Conclusions: In our opinion, this work is very much needed as it shows how to effectively diagnose hookworm infestations. However, we do not agree with the information in the title of the article because such studies have already been carried out in Central Europe by other authors., (© 2023. The Author(s).)

Background: Diagnosis of soil-transmitted helminths (STHs) in developing countries is commonly based on microscopic detection of eggs in stool samples, using the Kato-Katz (KK) method, which has a poor sensitivity for detecting light intensity infections. We compared the performance of the KK method and real-time PCR in the framework of a randomized trial, which evaluated four novel treatments against Trichuris trichiura and concomitant STH infections., Results: Two stool samples obtained from 320 participants were examined at baseline and follow-up with quadruplicate KK and PCR analyses of one of the two samples using "bead-beating" for DNA extraction. At follow-up, 80 samples were negative according to both PCR and KK and 173 were positive with both methods for any of the STHs. Relative to PCR, the calculated sensitivity of KK at follow-up was 83.6%, 43.0% and 53.8% for T. trichiura, for hookworm and for Ascaris lumbricoides, respectively. The sensitivity of PCR compared with KK at this time point was 89.1% for T. trichiura, 72.7% for hookworm and 87.5% for A. lumbricoides. Cure rates (CRs) for T. trichiura and A. lumbricoides were slightly lower with the PCR method. For hookworm CRs with KK were mostly significantly lower, namely 36.7%, 91.1%, 72.2% and 77.8% for moxidectin, moxidectin in combination with tribendimidine, moxidectin in combination with albendazole and albendazole in combination with oxantel pamoate, respectively, whereas with PCR the CRs were 8.3%, 82.6%, 37.1% and 57.1%, respectively., Conclusions: In conclusion, a single real-time PCR is as sensitive as quadruplicate KK for T. trichiura and A. lumbricoides detection but more sensitive for hookworm, which has an influence on the estimated treatment efficacy. PCR method with DNA extraction using the "bead-beating protocol" should be further promoted in endemic areas and laboratories that can afford the needed equipment. The study is registered at ISRCTN (no. 20398469).

The canine hookworms Ancylostoma braziliense, Ancylostoma ceylanicum, Ancylostoma caninum and Uncinaria stenocephala are not only capable of producing morbidity and mortality in dogs but are also neglected tropical zoonoses. Each hookworm species differs considerably in its geographical distribution, life cycle, biology, pathogenic impacts on both canine and human hosts, zoonotic potential, and response to treatment with anthelminthics. Here we describe the development and validation of two Taq-Man based multiplex PCR assays capable of detecting and differentiating all four canine hookworm species in faeces of naturally infected dogs. The analytical sensitivity of both assays was assessed using 10-fold serial dilutions of synthetic gene block fragments containing individual sequence targets of each hookworm species. The sensitivity of the assays and ability to detect mixed species infections were compared to a conventional PCR-Restriction Fragment Length Polymorphism based-approach when applied to laboratory and field samples from endemic areas. The qPCRs detected at least one species of hookworms in 82.4% of PCR-RFLP-negative but microscopy-positive samples. The qPCRs detected an additional 68% mixed infections with different species of canine hookworms, and additional single species infection with A. caninum (47%), U. stenocephala (33%) and A. ceylanicum (0.02%) that were missed by PCR-RFLP. These multiplex qPCR assays will assist field based epidemiological surveillance studies towards an accurate and sensitive monitoring of canine hookworm infections in dogs, to inform their species-specific zoonotic risks to populations living in endemic areas, globally., Competing Interests: The authors have declared that no competing interests exist.

Public parks are leisure environments widely used by both, adults and children, often accompained by their pets. Soil contamination of these environments by enteric viruses and intestinal parasites occurs through these animals feces. The aim of this work was to detect Carnivore protoparvovirus 1 (CPV-1) and different species of Mastadenovirus in soils samples from a park located in a medium-sized city in Brazil and evaluate the presence of helminth eggs and larvae in 18 points of a public park soil samples, as well as feces found on this site during six months. Parasitological analyzes were conducted through flotation and sedimentation techniques, and polymerase chain reaction (PCR) was used for viral detection. Of the 216 soil and 16 feces samples, 49% (106/216) and 12% (2/16) were positivefor nematodes larvae, respectively, through sedimentation techniques. Toxocara spp eggs were found in one soil sample and one feces sample, Trichuris spp eggs were found in only one feces sample and Hookworms eggs were found in four soil samples. After reconstruction work in the streets near the park, 30% (64/216) of the samples were positive for Human Mastadenovirus C (HAdV-C), 1.4% (3/216) for HAdV-E and 0.4% (1/216) for Canine Mastadenovirus A (CAdV-A). The parasitic forms found in this study have demonstrated that the contamination of the park's soil pose a threat to human and animal health. This was the first study to report the presence of HAdVs and CAdVs in soil samples.

Hookworm infection is globally prevalent among dogs and cats representing a major public health risk. Although previous studies have surveyed canine and feline hookworms in Guangzhou city, the status of these infection needs to be further explored in other regions of South China. To investigate the prevalence and zoonotic risk of canine and feline hookworms in eight cities (Guangzhou, Foshan, Shenzhen, Huizhou, Zhongshan, Shaoguan, Shantou and Chaozhou) of Guangdong province, China, we developed specific PCR methods based on ITS sequence for identifying three common hookworm species. The results showed that the prevalence of hookworms from stray dogs and cats was 20.23% (142/702) and 15.26% (47/308), respectively. The established PCR methods could identify Ancylostoma ceylanicum, A. caninum and A. tubaeforme. The mixed infections of A. caninum and A. ceylanicum were detected in stray dogs of Guangzhou and Shaoguan, with the rate of 8.3% and 21.2%, respectively. Among the stray dogs in Foshan, the infection rate of A. ceylanicum was higher than that of A. caninum. The stray cats in four of five investigated cities were infected with A. ceylanicum. The different region, age and rearing environments had an impact on the hookworm infection rates of stray dogs and cats. In conclusion, the reported higher infection rate of A. ceylanicum than other hookworm species in stray dogs and cats poses a potential risk to public health., (Copyright © 2019. Published by Elsevier B.V.)

The presence and distribution of various species of canine hookworms in Africa are poorly known. The main objective of this study, therefore, was to identify the hookworm species present in canine faecal samples from Morogoro, Tanzania, using molecular techniques. Faecal samples from 160 local dogs were collected and hookworm positive samples processed to recover larvae for further molecular characterization. DNA was extracted from pools of larvae from individual samples (n = 66), which were analysed subsequently using two different molecular approaches, polymerase chain reaction-linked restriction fragment length polymorphism (PCR-RFLP) and species-specific PCR coupled with Sanger sequencing. The PCR-RFLP technique detected only the presence of the ubiquitous Ancylostoma caninum in the 66 samples. However, by species-specific PCR coupled with Sanger sequencing we identified ten samples with A. braziliense, two with Uncinaria stenocephala and five with A. ceylanicum. Thus, all four known species of canine hookworms were identified in Morogoro, Tanzania. To our knowledge this is the first report of the detection of the presence of U. stenocephala and A. ceylanicum in Africa using molecular techniques. In addition to their veterinary importance, canine hookworms have zoonotic potential and are of public health concern.

Objective: The aim of this study was to determine the prevalence of soil transmitted helminthes among primary school children. School based cross-sectional study design was employed. A total of six hundred study subjects were selected by a multistage sampling method. Fresh stool specimens were collected using clean, dry and wide mouthed labeled stool cups. It was processed by Kato-Katz technique. The data were analyzed using SPSS version 20 and p-value < 0.05 was considered statistically significant., Result: The overall prevalence of soil transmitted helminthes was 57 (9.5%). Hookworm was the most prevalent helminthes species isolated (4.2%) followed by A. lumbricoide (3%). The prevalence of Taenia species, T. trichiura, H. nana and E. vermicularis were; 1.2%, 0.5%, 0.7% and 0.8% respectively. The prevalence of the Soil transmitted helminthes infection was low and all cases of Soil transmitted infections in this study were with low infection intensity. This might be due to the preventive chemotherapy given to the school children.

Hookworm infection persists focally in rural communities in Brazil. In this study, we analyze the mitochondrial nucleotide sequences obtained from hookworms infecting humans in order to characterize species composition and assess their genetic diversity and phylogenetic relationships. Field expeditions and cross-sectional surveys were carried out in three Brazilian municipalities from 2013 to 2017: Nossa Senhora de Nazaré (n = 605) and Teresina (n = 297), in the state of Piauí, and Russas (n = 213) in the State of Ceará. Parasitological methods were used to evaluate fecal samples. Hookworm-positive samples had a partial mtDNA cox1 amplified and sequenced. Maximum-likelihood and Bayesian analysis demonstrated two strongly-supported clades, including Group A, corresponding to Necator americanus, and Groups B and C, corresponding to Necator sp. Group A was divided into three main clusters: A1 grouped with Asian sequences, A2 grouped with African sequences, and A3 had only Asian sequences. Group B was closely related to Necator sp., showing a sequence similarity of 98%-99% with African samples circulating zoonotically among humans and non-human primates. Twenty three N. americanus haplotypes were identified. N. americanus Median-Joining network revealed three distinct groups, designated again as A1, A2, and A3. Group A1 presented a star-like shape, with one dominant haplotype. The molecular dating suggested that the two clades dividing N. americanus and Necator sp. began to diverge during the middle Pleistocene. The most recent common ancestor among N. americanus groups was dated to the late Pleistocene. Hookworms circulating in the studied communities are structured in well-defined subpopulations presenting both Asian and African genetic backgrounds. This reveals a double origin for hookworms in northeastern Brazil and opens up new possibilities in phylogeographic, evolutionary, and molecular epidemiological studies in regions where hookworms persists focally, despite control efforts. The presence of potentially zoonotic species and the specific identification of Necator sp. should be further investigated., (Copyright © 2018 Elsevier B.V. All rights reserved.)

To investigate the prevalence of canine and feline hookworms in South China, and to assess the risk of zoonotic hookworms to humans, one pair of primers (HRM-F/HRM-R) was designed to establish a high-resolution melting (HRM) method based on internal transcribed spacer 1 (ITS-1) rDNA for the detection of Ancylostoma ceylanicum, A. caninum and A. tubaeforme infection. The results showed that the HRM for the three hookworms produced different melting-curve profiles, where melting temperature (Tm) values were 84.50°C for A. ceylanicum, 82.25°C for A. caninum and 81.73°C for A. tubaeforme, respectively. The reproducibility of intra- and inter-assay melting curves was almost perfect. The lowest concentration detected was about 5.69 ×10-4 g/μl. The HRM detection results from 18 canine and feline hookworm samples were in complete accordance with their sequencing results. The HRM method was more sensitive than the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique in the detection of 98 clinical samples. It is concluded that the HRM method can differentiate between A. ceylanicum, A. caninum, A. tubaeforme and their mixed infections, which may provide important technical support for the zoonotic risk assessment and molecular epidemiological survey of canine and feline hookworms.

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.

There is a paucity of information on hookworm species in humans, domestic animals and wildlife in southern Africa. Our study aimed to identify hookworm species from stray dogs, humans, and selected wildlife from South Africa. A total of 356 faecal samples were screened for the presence of hookworm-like eggs and subsequently coproculture from the positive samples was carried out to obtain larvae. Hookworm-like eggs were detected in 23.03% (82/356) of samples. Of these samples, 78/296 were from dogs, 3/50 from humans and 1/10 from wildlife. DNA was then isolated from the larvae of 55 positive samples, which were subjected to polymerase chain reaction (PCR), polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequencing of the nuclear ribosomal internal transcribed spacer (ITS1) and 5.8S rRNA region. Presence of Ancylostoma caninum, A. braziliense and A. ceylanicum-like species was recorded in stray dogs and A. caninum was recorded in wildlife and humans, using PCR-RFLP. Although PCR-RFLP results pointed to the presence of A. ceylanicum, we did not get a sequence that matched with A. ceylanicum from GenBank. This may have been due to the low proportion of A. ceylanicum larvae in our samples. Twenty-two of the 27 positive amplicons from stray dogs matched with A. caninum, three with A. braziliense and two had mixed infections of A. braziliense and A. caninum. Sequences from a lion and three humans matched with A. caninum. This is the first confirmation of a patent A. caninum infection in humans as evidenced by the presence of eggs in faeces, with the subsequent larvae from coproculture being identified as A. caninum.

This study aimed to investigate the endoparasite fauna of wild European gray wolves, which are currently recolonizing Germany. In total, 69 fecal samples of wild wolves were collected in Lower Saxony, Germany, from 2013 to 2015, analyzed by the sedimentation-flotation and McMaster techniques and compared to previous results on captive European Gray wolves living in zoological gardens in Germany. In addition to coproscopy, taeniid-positive samples from wild as well as captive wolves were differentiated by amplification and sequencing of small subunit ribosomal RNA (SSU rRNA) and NADH dehydrogenase 1 (nad1) gene fragments. Missing Taenia krabbei SSU rRNA reference sequences were generated from two T. krabbei specimens. Overall, 60.87% (42/69) of wild wolve samples were microscopically positive for at least one of seven egg types. Capillaria/Eucoleus spp. showed the highest frequency (31.88% [22/69]), followed by Taeniidae (21.74% [15/69]), Ancylostomatidae (20.29% [14/69]), Alaria alata (15.94% [11/69]), Toxocara canis (13.04% [9/69]), and Toxascaris leonina and Trichuris vulpis (each 5.80% [4/69]). Amplification of SSU rRNA was successful for 7/15 Taeniidae-positive samples from wild and 20/39 samples from captive wolves, revealing T. hydatigena in two and 14 samples, respectively. Taenia krabbei was detected in two further samples of wild and three samples of captive wolves, while for the remaining samples, no differentiation between T. serialis/T. krabbei was possible. Echinococcus spp. were not detected. Sequence comparisons revealed that the SSU rRNA gene fragment was not suitable to differentiate between T. serialis and T. krabbei. Therefore, the use of this fragment alone cannot be recommended for species identification in future studies.

Hookworms of the genus Uncinaria parasitize pinniped pups in various locations worldwide. Four species have been described, two of which parasitize pinniped pups in the southern hemisphere: Uncinaria hamiltoni parasitizes Otaria flavescens and Arctocephalus australis from the South American coast, and Uncinaria sanguinis parasitizes Neophoca cinerea from the Australian coast. However, their geographical ranges and host specificity are unknown. Uncinaria spp. are morphologically similar, but molecular analyses have allowed the recognition of new species in the genus Uncinaria. We used nuclear genetic markers (internal transcribed spacer (ITS) and large subunit (LSU) rDNA) and a mitochondrial genetic marker (cytochrome c oxidase subunit I (COI)) to evaluate the phylogenetic relationships of Uncinaria spp. parasitizing A. australis and O. flavescens from South American coasts (Atlantic and Pacific coasts). We compared our sequences with published Uncinaria sequences. A Generalized Mixed Yule Coalescent (GMYC) analysis was also used to delimit species, and principal component analysis was used to compare morphometry among Uncinaria specimens. Parasites were sampled from A. australis from Peru (12°S), southern Chile (42°S), and the Uruguayan coast, and from O. flavescens from northern Chile (24°S) and the Uruguayan coast. Morphometric differences were observed between Uncinaria specimens from both South American coasts and between Uncinaria specimens from A. australis in Peru and southern Chile. Phylogenetic and GMYC analyses suggest that south-eastern Pacific otariid species harbour U. hamiltoni and an undescribed putative species of Uncinaria. However, more samples from A. australis and O. flavescens are necessary to understand the phylogenetic patterns of Uncinaria spp. across the South Pacific.

Appropriate diagnostic techniques are crucial to global soil-transmitted helminth (STH) control efforts. The recommended Kato-Katz method has low sensitivity in low-transmission settings. Quantitative polymerase chain reaction (qPCR) is a highly sensitive alternative diagnostic option. However, little is known about the variability in qPCR results, and there are few published comparisons between qPCR and other microscopy-based techniques such as sodium nitrate flotation (SNF). Using 865 stool samples collected from 571 individuals, we compared SNF and qPCR in terms of diagnostic sensitivity and infection intensity measurements. In addition, we conducted repeated examinations on a single Necator americanus -positive stool sample over a 6-month period. Results showed good diagnostic agreement between SNF and qPCR for Ascaris spp. (κ = 0.69, P < 0.001), and moderate agreement for hookworm (κ = 0.55, P < 0.001) and Trichuris spp. (κ = 0.50, P < 0.001). Quantitative polymerase chain reaction demonstrated higher sensitivity than SNF for Ascaris spp. (94.1% versus 68.1%) and hookworm (75.7% versus 66.9%) but not for Trichuris spp. (53.1% versus 81.3%), which had very low prevalence. Sodium nitrate flotation and qPCR infection intensity measurements were strongly correlated for Ascaris spp. (ρ = 0.82, P < 0.001) and moderately correlated for hookworm (ρ = 0.58, P < 0.001). Repeated examinations using qPCR showed that N. americanus cycle threshold values decreased significantly at 1 month and remained stable thereafter. Results confirm the high diagnostic sensitivity of qPCR for Ascaris spp. and hookworm, particularly for light-intensity infections, which is ideal for settings approaching transmission elimination. Results support the potential for qPCR to be used as a quantitative assay for STH. Further research is needed in settings where Trichuris trichiura is endemic.

Background: Hookworms (Necator americanus and Ancylostoma duodenale) remain a major public health problem worldwide. Infections with hookworms (e.g., A. caninum, A. ceylanicum and A. braziliense) are also prevalent in dogs, but the role of dogs as a reservoir for zoonotic hookworm infections in humans needs to be further explored., Methodology/principal Findings: As part of an open-label community based cluster-randomized trial in a tribal area in Tamil Nadu (India; 2013-2015), a total of 143 isolates of hookworm eggs from human stool were speciated based on a previously described PCR-RFLP methodology. The presence of hookworm DNA was confirmed in 119 of 143 human samples. N. americanus (100%) was the most prevalent species, followed by A. caninum (16.8%) and A. duodenale (8.4%). Because of the high prevalence of A. caninum in humans, dog samples were also collected to assess the prevalence of A. caninum in dogs. In 68 out of 77 canine stool samples the presence of hookworms was confirmed using PCR-RFLP. In dogs, both A. caninum (76.4%) and A. ceylanicum (27.9%) were identified. Additionally, to determine the contamination of soil with zoonotic hookworm larvae, topsoil was collected from defecating areas. Hookworm DNA was detected in 72 out of 78 soil samples that revealed presence of hookworm-like nematode larvae. In soil, different hookworm species were identified, with animal hookworms being more prevalent (A. ceylanicum: 60.2%, A. caninum: 29.4%, A. duodenale: 16.6%, N. americanus: 1.4%, A. braziliense: 1.4%)., Conclusions/significance: In our study we regularly detected the presence of A. caninum DNA in the stool of humans. Whether this is the result of infection is currently unknown but it does warrant a closer look at dogs as a potential reservoir.

To know the infection status of helminths in primary schoolchildren of southern parts of Vietnam, we performed an epidemiological study in Krong Pac district, Dak Lak Province, Vietnam. A total of 1,206 stool specimens were collected from ethnic Ede schoolchildren in 4 primary schools in 2015 and examined by the Kato-Katz technique. In addition, stool cultures were done by the Harada-Mori method to obtain hookworm larvae and then to clarify the species of hookworms infected. The results showed that the helminth infection rate was 25.0%, including 2.0% Ascaris lumbricoides, 0.33% Trichuris trichiura, and 22.8% hookworm infections. The average intensity of infection was 102.0 eggs per gram of feces (EPG) for Ascaris, 36.0 EPG for Trichuris, and 218.0 EPG for hookworms. ITS1 gene sequences of the hookworm larvae were identical with those of Necator americanus (100% homology) reported in GenBank. It has been confirmed in this study that the hookworm, N. americanus, is a dominant helminth species infected in primary schoolchildren of a southern part of Vietnam. Public health attention is needed for control of hookworm infections among schoolchildren in surveyed areas of Vietnam., Competing Interests: We have no conflict of interest related to this work.

Background: Intestinal parasitic infections are significant cause of morbidity and mortality in endemic countries. In Ethiopia, helminthiasis was the third leading cause of outpatient visits. Despite the health extension program was launched to address this problem, there is limited information on the burden of intestinal parasites after implementation of the program in our setting. Therefore, the aim of this study was to assess the intestinal helminthic infections among clients attending at Anbesame health center, South Gondar, Ethiopia., Methods: A cross sectional study was conducted at Anbesame health center from March to June 2015. A structured questionnaire was used to collect data from 464 study participants selected consecutively. Stool specimen collection, processing through formol-ether concentration technique and microscopic examination for presence of parasites were carried out. Data were entered, cleaned and analyzed using SPSS Version 20., Results: Among the total 464 study participants with median (±IQR) age of 25.0 (±21.75) years, 262 (56.5%) were females. Helminthic infection was found in 97 (20.9%) participants. Hookworm (68 [14.7%]) was the predominant parasite followed by S. mansoni (11 [2.4%]), E. vermicularis (9 [1.9%]) and S. stercoralis (5 [1.1%]). Patients with age group ≥15 years (AOR: 5.26; 95% CI: 2.05-13.46; P: 0.001) and walking barefoot (AOR: 2.20; 95% CI: 1.08-4.48; P: 0.031) were more vulnerable from the hookworm infections., Conclusions: There was a high burden of hookworm infections in our setting. Hence, regular shoes wearing, considering all age groups in the albendazole deworming as mass treatment and environmental hygiene are important interventions to reduce the burden of such neglected tropical disease.

Background: Differentiation of canine hookworm species is crucial from both a veterinary and public health standpoint. In Vietnam, three hookworm species, namely Ancylostoma caninum, Ancylostoma braziliense and Uncinaria stenocephala are reported to infect dogs. In light of the emerging distribution of A. ceylanicum in Asia, this study aims to re-evaluate the status of Ancylostoma in dogs in Vietnam., Methods: Faecal samples collected from 200 community dogs in Dak Lak province were subjected to faecal floatation for the detection of hookworm eggs. Hookworm-positive samples were subjected to a PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) assay targeting the internal transcribed spacer (ITS) region of rDNA for hookworm species identification. A subset of hookworm-positive samples was also subject to haplotype characterisation at the cytochrome oxidase-1 (COX-1) gene. Detailed morphological criteria were utilised in addition to molecular markers, to identify adult hookworms recovered from necropsied dogs., Results: Of 200 canine faecal samples, 111 (55.5 %) were positive for hookworm eggs on faecal flotation. Of these, 94/111 (84.7 %) were successfully amplified and assigned species status by PCR-RFLP targeting the ITS region. In total, 54.3 % (51/94) dogs harboured single infections with A. ceylanicum, 33.0 % (31/94) with A. caninum, and 12.7 % (12/94) harboured mixed infections with both A. ceylanicum and A. caninum. Adult worms recovered from necropsied dogs matched morphological description provided for A. ceylanicum, Looss (1911) for which the mediolateral and posteriolateral rays are parallel. Characterisation of the COX-1 gene placed all Vietnamese canine isolates of A. ceylanicum within the 'zoonotic' haplotype., Conclusion: Based on this information, it is apparent that the hookworms present in dogs in Vietnam are those of A. ceylanicum and not A. braziliense. Owing to the endemic nature of this significant zoonosis in dogs, the study strongly advocates for specific identification of this hookworm in human hookworm surveys.

The hookworms Uncinaria rauschi Olsen, 1968 and Uncinaria yukonensis ( Wolfgang, 1956 ) were formally described from grizzly ( Ursus arctos horribilis) and black bears ( Ursus americanus ) of North America. We analyzed the intestinal tracts of 4 grizzly and 9 black bears from Alberta and British Columbia, Canada and isolated Uncinaria specimens with anatomical traits never previously documented. We applied morphological and molecular techniques to investigate the taxonomy and phylogeny of these Uncinaria parasites. The morphological analysis supported polymorphism at the vulvar region for females of both U. rauschi and U. yukonensis. The hypothesis of morphological plasticity for U. rauschi and U. yukonensis was confirmed by genetic analysis of the internal transcribed spacers (ITS-1 and ITS-2) of the nuclear ribosomal DNA. Two distinct genotypes were identified, differing at 5 fixed sites for ITS-1 (432 base pairs [bp]) and 7 for ITS-2 (274 bp). Morphometric data for U. rauschi revealed host-related size differences: adult U. rauschi were significantly larger in black bears than in grizzly bears. Interpretation of these results, considering the historical biogeography of North American bears, suggests a relatively recent host-switching event of U. rauschi from black bears to grizzly bears which likely occurred after the end of the Wisconsin glaciation. Phylogenetic maximum parsimony (MP) and maximum likelihood (ML) analyses of the concatenated ITS-1 and ITS-2 datasets strongly supported monophyly of U. rauschi and U. yukonensis and their close relationship with Uncinaria stenocephala (Railliet, 1884), the latter a parasite primarily of canids and felids. Relationships among species within this group, although resolved by ML, were unsupported by MP and bootstrap resampling. The clade of U. rauschi, U. yukonensis, and U. stenocephala was recovered as sister to the clade represented by Uncinaria spp. from otariid pinnipeds. These results support the absence of strict host-parasite co-phylogeny for Uncinaria spp. and their carnivore hosts. Phylogenetic relationships among Uncinaria spp. provided a framework to develop the hypothesis of similar transmission patterns for the closely related U. rauschi, U. yukonensis, and U. stenocephala.

Canine hookworm infections are endemic worldwide, with zoonotic transmission representing a potentially significant public health concern. This study aimed to investigate hookworm infection and identify the prevalent species from stray and shelter dogs in Guangzhou city, southern China by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) based on internal transcribed spacer (ITS) sequences. From March 2011 to July 2012, fresh faecal samples from a total of 254 dogs were obtained from five locations, namely Conghua, Baiyun, Liwan, Haizhu and Panyu, in Guangzhou. These samples were screened for the presence of hookworm eggs using light microscopy, with an overall prevalence of 29.53% being recorded. The highest prevalence of 45.28% was found in suburban dogs from Conghua compared with lower values recorded in urban dogs in Haizhu (21.43%), Baiyun (18.97%), Panyu (18.18%) and Liwan (15%). The prevalence in stray dogs was significantly higher than that in shelter dogs. PCR-RFLP analysis showed that 57.33% were detected as single hookworm infections with Ancyclostoma caninum, and 22.67% as A. ceylanicum, while 20% were mixed infections. This suggests that high prevalences of both hookworm species in stray and shelter dogs in China pose a potential risk of transmission from pet dogs to humans.

The high natal site fidelity of endangered Australian sea lions (Neophoca cinerea) along the southern Australian coast suggests that their maternally transmitted parasitic species, such as hookworms, will have restricted potential for dispersal. If this is the case, we would expect to find a hookworm haplotype structure corresponding to that of the host mtDNA haplotype structure; that is, restricted among geographically separated colonies. In this study, we used a fragment of the cytochrome c oxidase I mitochondrial DNA (mtDNA) gene to investigate the diversity of hookworms (Uncinaria sanguinis) in N. cinerea to assess the importance of host distribution and ecology on the evolutionary history of the parasite. High haplotype (h=0.986) and nucleotide diversity (π=0.013) were seen, with 45 unique hookworm mtDNA haplotypes across N. cinerea colonies; with most of the variation (78%) arising from variability within hookworms from individual colonies. This is supported by the low genetic differentiation co-efficient (GST=0.007) and a high gene flow (Nm=35.25) indicating a high migration rate between the populations of hookworms. The haplotype network demonstrated no clear distribution and delineation of haplotypes according to geographical location. Our data rejects the vicariance hypothesis; that female host natal site fidelity and the transmammary route of infection restrict hookworm gene flow between N. cinerea populations and highlights the value of studies of parasite diversity and dispersal to challenge our understanding of parasite and host ecology., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: There is evidence to support that nutritional deficiency can reduce the body's immune function, thereby decreasing resistance to disease and increasing susceptibility to intestinal parasites., Methods: A cross-sectional survey was carried out on 693 school-aged children from 5 schistosomiasis-endemic villages in Northern Samar, the Philippines. Data on dietary intake, nutritional status, and intestinal parasitic infection were collected., Results: The prevalence of stunting, thinness, and wasting was 49.2%, 27.8%, and 59.7% of all children. The proportion of children infected with Schistosoma japonicum (15.6%, P = .03) and hookworm (22.0%, P = .05) were significantly lower among children who met the recommended energy and nutrient intake (RENI) for total calories. The percentage of children infected with Trichuris trichiura was highest among children who did not meet the RENI for energy (74.1%, P = .04), iron (73.4%, P = .01), thiamine (74.0%, P = .00), and riboflavin (73.3%, P = .01). Susceptibility to having 1 or more parasitic infections was significantly associated with poor intake of energy (P = .04), thiamine (P = .02), and riboflavin (P = .01).The proportion of stunted children was significantly higher among children who did not meet the RENI for energy (68.9%, P = .002), protein (54.0%, P = .004), or niacin (30.8%, P = .02) and for those infected with hookworm (31.8%, P = .0002). After adjusting for potential confounders, protein intake less than the RENI (odds ratio [OR], 1.48; 95% confidence interval [CI], 1.03-2.14), and hookworm infection (OR, 1.77; 95% CI, 1.22-2.55) were the major predictors of stunting., Conclusions: The results support the hypothesis that poor nutrient intake may increase susceptibility to parasitic diseases and together they negatively affect childhood nutritional status., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

This study investigates the identity of hookworms parasitising the Australian sea lion, Neophoca cinerea (Péron), from three colonies in South Australia, Australia. The Australian sea lion is at risk of extinction because its population is small and genetically fragmented. Using morphological and molecular techniques, we describe a single novel species, Uncinaria sanguinis sp. n. (Nematoda: Ancylostomatidae). The new species is most similar to hookworms also parasitic in otariid hosts, Uncinaria lucasi Stiles, 1901 and Uncinaria hamiltoni Baylis, 1933. Comparative morphometrics offered limited utility for distinguishing between species within this genus whilst morphological features and differences in nuclear ribosomal DNA sequences delineated U. sanguinis sp. n. from named congeners. Male specimens of U. sanguinis sp. n. differ from U. lucasi and U. hamiltoni by relatively shorter anterolateral and externodorsal rays, respectively, and from other congeners by the relative lengths and angulations of bursal rays, and in the shape of the spicules. Female specimens of U. sanguinis sp. n. are differentiated from Uncinaria spp. parasitic in terrestrial mammals by differences in vulval anatomy and the larger size of their eggs, although are morphologically indistinguishable from U. lucasi and U. hamiltoni. Molecular techniques clearly delimited U. sanguinis sp. n. as a distinct novel species. Obtaining baseline data on the parasites of wildlife hosts is important for the investigation of disease and the effective implementation and monitoring of conservation management.

Bunostomum trigonocephalum and Bunostomum phlebotomum are blood-feeding hookworms of sheep and cattle, causing considerable economic losses to the live stock industries. Studying genetic variability within and among hookworm populations is critical to addressing epidemiological and ecological questions. Mitochondrial (mt) DNA is known to provide useful markers for investigations of population genetics of hookworms, but mt genome sequence data are scant. In the present study, the complete mitochondrial DNA (mtDNA) sequences of the sheep and goat hookworm B. trigonocephalum were determined for the first time, and the mt genome of B. phlebotomum from yak in China was also sequenced for comparative analyses of their gene contents and genome organizations. The lengths of mt DNA sequences of B. trigonocephalum sheep isolate, B. trigonocephalum goat isolate and B. phlebotomum China yak isolate were 13,764bp, 13,771bp and 13,803bp in size, respectively. The identity of the mt genomes was 99.7% between B. trigonocephalum sheep isolate and B. trigonocephalum goat isolate. The identity of B. phlebotomum China yak isolate mt genomes was 85.3% with B. trigonocephalum sheep isolate, and 85.2% with B. trigonocephalum goat isolate. All the mt genes of the two hookworms were transcribed in the same direction and gene arrangements were consistent with those of the GA3 type, including 12 protein-coding genes, 2 rRNA genes and 22 tRNA genes, but lacking ATP synthetase subunit 8 gene. The mt genomes of B. trigonocephalum and B. phlebotomum were similar to prefer bases A and T, the contents of A+T are 76.5% (sheep isolate), 76.4% (goat isolate) and 76.9% (China yak isolate), respectively. Phylogenetic relationships reconstructed using concatenated amino acid sequences of 12 protein-coding genes with three methods (maximum likelihood, Bayesian inference and neighbor joining) revealed that the B. trigonocephalum and B. phlebotomum represent distinct but closely-related species. These data provide novel and useful genetic markers for studying the systematics, and population genetics of the two ruminant hookworms., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Hookworms of the genus Uncinaria have been widely reported from juvenile pinnipeds, however investigations of their systematics has been limited, with only two species described, Uncinaria lucasi from northern fur seals (Callorhinus ursinus) and Uncinaria hamiltoni from South American sea lions (Otaria flavescens). Hookworms were sampled from these hosts and seven additional species including Steller sea lions (Eumetopias jubatus), California sea lions (Zalophus californianus), South American fur seals (Arctocephalus australis), Australian fur seals (Arctocephalus pusillus), New Zealand sea lions (Phocarctos hookeri), southern elephant seals (Mirounga leonina), and the Mediterranean monk seal (Monachus monachus). One hundred and thirteen individual hookworms, including an outgroup species, were sequenced for four genes representing two loci (nuclear ribosomal DNA and mitochondrial DNA). Phylogenetic analyses of these sequences recovered seven independent evolutionary lineages or species, including the described species and five undescribed species. The molecular evidence shows that U. lucasi parasitises both C. ursinus and E. jubatus, whereas U. hamiltoni parasitises O. flavescens and A. australis. The five undescribed hookworm species were each associated with single host species (Z. californianus, A. pusillus, P. hookeri, M. leonina and M. monachus). For parasites of otarids, patterns of Uncinaria host-sharing and phylogenetic relationships had a strong biogeographic component with separate clades of parasites from northern versus southern hemisphere hosts. Comparison of phylogenies for these hookworms and their hosts suggests that the association of U. lucasi with northern fur seals results from a host-switch from Steller sea lions. Morphometric data for U. lucasi shows marked host-associated size differences for both sexes, with U. lucasi individuals from E. jubatus significantly larger. This result suggests that adult growth of U. lucasi is reduced within the host species representing the more recent host-parasite association. Intraspecific host-induced size differences are inconsistent with the exclusive use of morphometrics to delimit and diagnose species of Uncinaria from pinnipeds., (Copyright © 2013 Australian Society for Parasitology Inc. All rights reserved.)

The present study examined sequence variability in four mitochondrial genes, namely cytochrome c oxidase subunit (cox1), cytochrome b (cytb) and NADH dehydrogenase subunits 1 and 5 (nad1 and nad5), among Bunostomum trigonocephalum isolates from four different geographic regions in China. Ten B. trigonocephalum samples were collected from each of the four provinces (Heilongjiang, Jilin, Shaanxi and Yunnan), China. A part of the cox1 (pcox1), cytb (pcytb), nad1 and nad5 genes (pnad1 and pnad5) were amplified separately from individual hookworms by polymerase chain reaction (PCR) and were subjected to direct sequencing in order to define sequence variations and their phylogenetic relationships. The intra-specific sequence variations within B. trigonocephalum were 0-1.9% for pcox1, 0-2.0% for pcytb, 0-1.6% for pnad1 and 0-1.7% for pnad5. The A+T contents of the sequences were 69.6-70.4% (pcox1), 71.9-72.7 (pcytb), 70.4-71.1% (pnad1) and 72.0-72.6% (pnad5). However, the inter-specific sequence differences among members of the family Ancylostomatidae were significantly higher, being 12.1-14.2% for pcox1, 13.7-16.0 for cytb, 17.6-19.4 for nad1 and 16.0-21.6 for nad5. Phylogenetic analyses based on the combined partial sequences of cox1, cytb, nad1 and nad5 using three inference methods, namely Bayesian inference (Bayes), maximum likelihood (ML) and maximum parsimony (MP), revealed that all the B. trigonocephalum samples form monophyletic groups, but samples from the same geographical origin did not always cluster together, suggesting that there was no obvious geographical distinction within B. trigonocephalum based on sequences of the four mtDNA genes. These results demonstrated the existence of low-level intra-specific variation in mitochondrial DNA (mtDNA) sequences among B. trigonocephalum isolates from different geographic regions.

This study presents morphological and molecular data on hookworms from the Australian fur seal Arctocephalus pusillus doriferus (Schreber) currently identified in Australian waters as Uncinaria hamiltoni Baylis, 1933. Additional specimens from the Australian sea lion Neophoca cinerea (Péron) and the New Zealand fur seal Arctocephalus forsteri (Lesson) from Australia, and the Southern elephant seal Mirounga leonina (Linnaeus) from Antarctica, were included. Using the internal transcribed spacer (ITS), hookworms from A. p. doriferus, N. cinerea and A. forsteri were found to be genetically similar but distinct from Uncinaria spp. found in M. leonina from Antarctica, as well as from Zalophus californianus (Lesson) and Callorhinus ursinus (Linnaeus) from California. Few morphological differences were detected between these taxa.

In the present study, samples representing Bunostomum trigonocephalum and Bunostomum phlebotomum from sheep and cattle in Heilongjiang Province, China, were characterized and grouped genetically by the first (ITS-1) and second (ITS-2) internal transcribed spacers (ITS) of nuclear ribosomal DNA (rDNA). The rDNA region including the ITS-1, 5.8S, ITS-2, and flanking 18S and 28S rDNA sequences was amplified by polymerase chain reaction (PCR), then sequenced and compared with that of other members of the hookworms available in GenBank™, and phylogenetic relationships between them were reconstructed using the Maximum-Parsimony method. The ITS-1, 5.8S, and ITS-2 sequences of the sheep hookworm were 381, 153, and 231bp in length, respectively, and the corresponding sequences of the cattle hookworm were 392, 153, and 240bp in length. The identity of ITS sequences of B. trigonocephalum and B. phlebotomum from sheep and cattle was 87.4%. A PCR-linked restriction fragment length polymorphism (PCR-RFLP) assay using restriction endonuclease Nde I was established for the unequivocal differentiation of the two hookworm species. Phylogenetic analyses based on the ITS sequences revealed that B. trigonocephalum and B. phlebotomum were closely related, but they represent two different species., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The peritoneal cavity (PNC) and intestine of northern fur seal (Callorhinus ursinus) pups and California sea lion (Zalophus californianus) pups that died in late July and early August, 2003, on San Miguel Island, California, were examined for hookworms. Prevalence and morphometric studies were done with the hookworms in addition to molecular characterization. Based on this and previous molecular studies, hookworms from fur seals are designated as Uncinaria lucasi and the species from sea lions as Uncinaria species A. Adult hookworms were found in the PNC of 35 of 57 (61.4%) fur seal pups and of 13 of 104 (12.5%) sea lion pups. The number of hookworms located in the PNC ranged from 1 to 33 (median = 3) for the infected fur seal pups and 1 to 16 (median = 2) for the infected sea lion pups. In addition to the PNC, intestines of 43 fur seal and 32 sea lion pups were examined. All of these pups were positive for adult hookworms. The worms were counted from all but one of the sea lion pups. Numbers of these parasites in the intestine varied from 3 to 2,344 (median = 931) for the fur seal pups and 39 to 2,766 (median = 643) for the sea lion pups. Sea lion pups with peritoneal infections had higher intensity infections in the intestines than did pups without peritoneal infections, lending some support for the hypothesis that peritoneal infections result from high-intensity infections of adult worms. There was no difference in intestinal infection intensities between fur seal pups with and without peritoneal infections. Female adult hookworms in the intestines of both host species were significantly larger than males, and sea lion hookworms were larger than those in fur seals. Worms in the intestine also were larger than worms found in the PNC. Gene sequencing and (RFLP) analysis of (PCR) amplified (ITS) ribosomal DNA were used to diagnose the species of 172 hookworms recovered from the PNC and intestine of 18 C. ursinus and seven Z. californianus hosts. These molecular data revealed that U. lucasi (hookworm of C. ursinus) and Uncinaria species A (of Z. californianus) infrequently mature in the intestine of the opposite host species in California rookeries. However, there is no support from molecular data for the hypothesis that cross-infection with "the wrong" Uncinaria species is a contributing factor in these cases of host peritonitis. The major significance of this research is the unusual finding of adult hookworms in the PNC of so many dead pups. No obvious explanation for this occurrence could be determined. Further research, like in the present study, should help understand and monitor the apparent ever changing role of hookworm disease in the health of northern fur seal and California sea lion pups on SMI.

Background: An enhanced understanding of the hookworm genome and its resident mobile genetic elements should facilitate understanding of the genome evolution, genome organization, possibly host-parasite co-evolution and horizontal gene transfer, and from a practical perspective, development of transposon-based transgenesis for hookworms and other parasitic nematodes., Methodology/principal Findings: A novel mariner-like element (MLE) was characterized from the genome of the dog hookworm, Ancylostoma caninum, and termed bandit. The consensus sequence of the bandit transposon was 1,285 base pairs (bp) in length. The new transposon was flanked by perfect terminal inverted repeats of 32 nucleotides in length with a common target site duplication TA, and it encoded an open reading frame (ORF) of 342 deduced amino acid residues. Phylogenetic comparisons confirmed that the ORF encoded a mariner-like transposase, which included conserved catalytic domains, and that the bandit transposon belonged to the cecropia subfamily of MLEs. The phylogenetic analysis also indicated that the Hsmar1 transposon from humans was the closest known relative of bandit, and that bandit and Hsmar1 constituted a clade discrete from the Tc1 subfamily of MLEs from the nematode Caenorhabditis elegans. Moreover, homology models based on the crystal structure of Mos1 from Drosophila mauritiana revealed closer identity in active site residues of the catalytic domain including Ser281, Lys289 and Asp293 between bandit and Hsmar1 than between Mos1 and either bandit or Hsmar1. The entire bandit ORF was amplified from genomic DNA and a fragment of the bandit ORF was amplified from RNA, indicating that this transposon is actively transcribed in hookworms., Conclusions/significance: A mariner-like transposon termed bandit has colonized the genome of the hookworm A. caninum. Although MLEs exhibit a broad host range, and are identified in other nematodes, the closest phylogenetic relative of bandit is the Hsmar1 element of humans. This surprising finding suggests that bandit was transferred horizontally between hookworm parasites and their mammalian hosts.

Arthrostoma miyazakiense (Nematoda: Ancylostomatidae) is a hookworm species reported from the small intestines of raccoon dogs (Nyctereutes procyonoides) in Japan. Five Korean raccoon dogs (N. procyonoides koreensis) caught from 2002 to 2005 in Jeollanam-do (Province), a southeastern area of South Korea, contained helminth eggs belonging to 4 genera (roundworm, hookworm, whipworm, and Capillaria spp.) and cysts of Giardia sp. in their feces. Necropsy findings of 1 raccoon dog revealed a large number of adult hookworms in the duodenum. These hookworms were identified as Arthrostoma miyazakiense based on the 10 articulated plates observed in the buccal capsule and the presence of right-sided prevulval papillae. Eggs of A. miyazakiense were 60-65 x 35-40 micrometer (av. 62.5 x 35 micrometer), and were morphologically indistinguishable from those of Ancylostoma caninum. The eggs were cultured to infective 2nd stage larvae via charcoal culture, and 100 infective larvae were used to experimentally infect each of 3 mixed-bred puppies. All puppies harbored hookworm eggs in their feces on the 12th day after infection. This is the first report thus far concerning A. miyazakiense infections in raccoon dogs in Korea, and the first such report outside of Japan.

The purpose of this paper is to disseminate, more widely, certain historically important aspects of research by O. Wilford Olsen on hookworms (Uncinaria lucasi) in northern fur seals (Callorhinus ursinus) and Steller sea lions (Eumatopias jubatus) in 1951 on St. Paul Island, Alaska. Highly meaningful facets of the research were recorded only in a report with limited availability by Olsen in 1952. Portions of his research presented here include measurements of hookworm free-living infective third-stage larvae (L3) and adults, photographs of bursae, and the conclusion that the species of hookworms (U. lucasi) is probably the same in both pinniped hosts. This information is especially important because very little has been published on taxonomy of hookworms in E. jubatus.

Thirty-one South American sea lion pups (Otaria flavescens) found dead in Punta León, Argentina, during the summer of 2002, were examined for hookworms (Uncinaria hamiltoni). Parasite parameters were analyzed in 2 locations of the rookery, i.e., a traditional, well-structured breeding area and an expanding area with juveniles and a lax social structure. Prevalence of hookworms was 50% in both localities, and no difference was observed in prevalence between pup sexes (P > 0.05). Hookworms were concentrated in the small intestine. Transmammary transmission is assumed because only adult hookworms were found in the pups. The mean intensity of hookworms per pup was 135; the mean intensity in females (92.78) was significantly different (P < 0.05) from that of males (230.25). No difference (P > 0.05) in intensity was found between the 2 breeding areas, although prevalence was higher in the traditional breeding area than in the other area. Location was the only factor affecting hookworm prevalence (P log-linear model: 0.9552; chi2: 1.5629). No apparent trend between body condition and intensity of hookworms was observed.

Canine parasitic zoonoses pose a continuing public health problem, especially in developing countries and communities that are socioeconomically disadvantaged. Our study combined the use of conventional and molecular epidemiological tools to determine the role of dogs in transmission of gastrointestinal (GI) parasites such as hookworms, Giardiaand Ascarisin a parasite endemic tea-growing community in northeast India. A highly sensitive and specific PCR-RFLP was developed to detect and differentiate the zoonotic species of canine hookworm eggs directly from faeces. This allowed epidemiological screening of canine hookworm species in this community to be conducted with ease and accuracy. Seventy two percent of dogs were found to harbour A. caninum, 60% A. braziliense and 37% harboured mixed infections with both hookworms. No A. ceylanicum was detected in the dog population. The zoonotic potential of canine Giardiawas also investigated by characterising Giardia duodenalisrecovered from humans and dogs living in the same locality and households, at three different loci. Phylogenetic and epidemiological analysis provided compelling evidence to support the zoonotic transmission of canine Giardia. Molecular tools were also used to identify the species of Ascarisegg present in over 30% of dog faecal samples. The results demonstrated the role of dogs as a significant disseminator and environmental contaminator of Ascaris lumbricoidesin communities where promiscuous defecation practices exist. Our study demonstrated the usefulness of combining conventional and molecular parasitological and epidemiological tools to help solve unresolved relationships with regards to parasitic zoonoses.

Uncinaria spp. were recovered from the milk of California sea lions ( Zalophus californianus) collected from the: (1) teats of a cow just after parturition (one parasitic third-stage larva, L(3)), (2) stomach of her nursing pup (two L(3)), and (3) stomach of a dead pup about 2 days old (one L(3), one headless, probably L(3), and four L(4)) on San Miguel Island, California in May 2001. This, in addition to earlier research, indicates transmammary transmission of hookworms in this host. Uncinaria spp. were found in dead northern fur seals ( Callorhinus ursinus) in the: (1) intestines of 2 of 75 pups (either one or two adult specimens in each infected pup) and (2) ventral abdominal blubber of 3 of 78 subadult males (one to seven L(3) in each infected seal) on St. Paul Island (SPI), Alaska in July and August 2001. These findings verify the low current prevalence of Uncinariaspp. in fur seals on SPI. Rectal fecal samples taken from 50 live Steller sea lion ( Eumetopias jubatus) pups, about 1 month old, on Rogue Reef in Curry County, Oregon in July 2001, were all negative for the eggs of Uncinaria spp. The apparent zero infection rate in these pups is possibly because the rocky terrain of this rookery is not suitable for hookworm transmission.

Ancylostomatoidea or hookworms recorded in Australia are reviewed and the attempt is made to provide the biogeographical background to their occurrence. The poor representation of this nematode superfamily is probably a reflection of the fact that they are primarily parasites of Carnivora, Artiodactyla, Insectivora, Rodentia, Edentata, Proboscoidea and primates, eutherian mammals which are either absent from the Australian fauna or which have only recently reached the continent. The principal genera of hookworms recorded to date from Australia are Ancylostoma, Bunostomum, Necator and Uncinaria. The majority of the ancylostomatoid fauna is represented by introduced species of man and domestic animals. Native or endemic species of hookworms are restricted to members of the genus Uncinaria with two species occurring in rodents and pinnipeds. Only a single endemic species of hookworm is known, U. hydromyidis, which is found in the small intestine of a rat. Significant problems remain in understanding the systematics, epidemiology and evolutionary relationships of the Australian ancylostomatoid fauna.

California sea lions (Zalophus californianus) and northern fur seals (Callorhinus ursinus) are each believed to host distinct hookworm species (Uncinaria spp.). However, a recent morphometric analysis suggested that a single species parasitizes multiple pinniped hosts, and that the observed differences are host-induced. To explore the systematics of these hookworms and test these competing hypotheses, we obtained nucleotide sequences of nuclear ribosomal DNA (D2/D3 28S, D18/D19 28S, and internal transcribed spacer [ITS] regions) from 20 individual hookworms parasitizing California sea lion and northern fur seal pups where their breeding grounds are sympatric. Five individuals from an allopatric population of California sea lions were also sampled for ITS-1 and D18/D19 28S sequences. The 28S D2/D3 sequences showed no diagnostic differences among hookworms sampled from individual sea lions and fur seals, whereas the 28S D18/D19 sequences had one derived (apomorphic) character demarcating hookworms from northern fur seals. ITS sequences were variable for 7 characters, with 4 derived (apomorphic) states in ITS-1 demarcating hookworms from California sea lions. Multivariate analysis of morphometric data also revealed significant differences between nematodes representing these 2 host-associated lineages. These results indicate that these hookworms represent 2 species that are not distributed indiscriminately between these host species, but instead exhibit host fidelity, evolving independently with each respective host species. This evolutionary approach to analyzing sequence data for species delimitation is contrasted with similarity-based methods that have been applied to numerous diagnostic studies of nematode parasites.

Invading infective third-stage larvae (L3) of parasitic nematodes execute a series of programmed developmental events in response to a host-specific signal encountered during infection. One of these early events is the release of excretory/secretory products. Using an in vitro feeding assay that mimics these early events of infection, a protein released by in vitro activated larvae of the hookworm Ancylostoma caninum was identified. This protein, Ac-ASP-2, was partially sequenced, and the cDNA encoding it isolated by PCR and screening of an A. caninum L3 cDNA library. The Ac-asp-2 cDNA encodes a protein of 219 amino acids that is related to a previously identified protein, Ac-ASP-1, from hookworms. Both molecules are members of an evolutionarily diverse family of molecules that include the venom allergens of the Hymenoptera, and the testes specific proteins/sperm-coating glycoproteins of mammals. Homologues are present in nearly all nematodes tested, as demonstrated by PCR-hybridization and database searching. The Ac-asp-2 mRNA is synthesized in all life history stages, but the gene product is released only by L3 activated to feed in vitro. The wide distribution of the Ac-asp-2 in nematodes and its release in response to host specific signals suggests that Ac-ASP-2 serves an important function in nematode physiology and development, and possibly in the infective process of parasitic species.

To overcome limitations in the morphological identification of different developmental stages of hookworms to species, we have established a polymerase chain reaction-linked single strand conformation polymorphism technique (PCR-SSCP) utilizing the internal transcribed spacers (ITS) of ribosomal (r)DNA. These spacers were specifically chosen because they provide reliable species markers for strongylid nematodes. ITS spacers were amplified by PCR from DNA derived from individual parasites of seven species of hookworm, then denatured and subjected to electrophoresis in a mutation detection enhancement (MDE) (non-denaturing) gel matrix. PCR SSCP analysis showed that the single-strand ITS patterns produced allowed the unequivocal identification of all species. The method also allowed the direct display of sequence variation within some species where multiple individual worms were examined. These findings demonstrate the usefulness of the SSCP approach for hookworm identification, the detection of population variation and the direct display of sequence variation in rDNA.

Polymerase chain reaction-linked restriction fragment length polymorphism (PCR-RFLP) analysis of ribosomal (r) DNA was conducted on Uncinaria stenocephala, Ancylostoma caninum, A. tubaeforme and A. ceylanicum. The rDNA region spanning the first and second internal transcribed spacers (ITS1 and ITS2) plus the 5.8S (ITS+) gene was amplified by PCR from each of the species, digested separately with 9 restriction endonucleases and the fragments separated by agarose gel electrophoresis. Characteristic PCR-RFLP patterns were produced for each morphologically defined species using some of the endonucleases. The present study demonstrated that the ITS+ provides genetic markers for the delineation of each species examined and suggests that this region of rDNA will be useful for the identification of other hookworms from a range of hosts. The results are likely to have important implications for studying the genetic structure of hookworm populations, the systematics and the epidemiology of hookworm infections.

Within the past 2 years, progress has been made in the identification, isolation, and cDNA cloning of several macromolecules from hookworms. While the predicted amino acid sequences of some cDNAs resemble those from other nematodes, such as Caenorhabditis elegans, other cDNAs are unique to hookworms. Studies are under way to evaluate the function of these recombinant hookworm polypeptides with respect to the biology of hookworms in experimental animal models. The recombinant polypeptides are also under evaluation as vaccine targets and as natural products used in the treatment of human cardiovascular and autoimmune diseases.

Estimation of the prevalence of hookworm infection is usually based on finding eggs of the parasite in the stool. Eggs of the different hookworm species are difficult to distinguish and can be confused with those of hookworm-like species. Proper identification can be made after hatching the eggs and cultivating larvae to the infective stage. We have used the Harada-Mori method to study the stools of Bushnegroes in Suriname and have identified the emerged larvae. Of the 804 individuals examined, 53.6% had positive fecal cultures. The identification of emerged larvae revealed that 75% of the positive cultures contained Necator americanus and Strongyloides stercoralis was detected in 19.7%. Oesophagostomum and Ternidens species were also found. Ancylostoma duodenale larvae were not found. Six percent of fecal cultures in which larvae emerged contained only hookworm-like larvae hatching from eggs that resembled hookworm eggs. The diagnosis of hookworm infection, based solely on the microscopic detection of eggs in the stool, must be considered with special caution.

The surface structures of adult Arthrostoma longespiculum were studied with the aid of the scanning electron microscope. In the mouth opening, a pair of ventral cutting plates was present; each was semilunar, thick and rounded at the inner edge. Cervical papillae were paired, small and sharp. The vulvar papilla was a single, round, wart-like projection adjacent to the vulva.

Thirteen species of trichostronglyloid nematodes have so far been recorded from wild birds and mammals in Israel and surrounding territories. Three species were found in birds: Amidostomum fulicae (Rudolphi, 1819) in Fulica atra L., 1758, A. acutum (Lundahl, 1848) in Anas crecca L., 1758 AND Amidostomum sp. in Ceryle rudis L., 1758. Ten species, 3 of which are new, were found in small mammals: Trichostrongylus colubriformis (Giles, 1892) in Hystrix indica Kerr, 1792; Tenorastrongylus josephi n. sp. in Mus musculus L., 1758; Nippostrongylus brasiliensis (Travassos, 1914) in Rattus norvegicus Berk, 1796 and Rattus rattus L., 1758; Nippostrongylus witenbergi Greenberg, 1972, in Nesokia indica Gray et Hardw., 1832; Heligmonina nevoi n. sp. in Spalax ehrenbergi, Nehring, 1898; Boreostrongylus seurati (Travassos et Darriba, 1929) in Gerbillus allenbyi Thomas, 1918, G. pyramidum Geoffrey, 1825, G. (Dipodillus) dasyurus, Meriones sacramenti Thomas, 1922 and M. tristrami Thomas, 1892; Boreostrongylus minutus (Dujardin, 1845) in Microtus guentheri Danford et Alsen, 1880; Heligmosomoides polygyrus polygyrus (Dujardin, 1845) in Apodemus mystacinus Danf. et Alst., 1877 and A. sylvaticus L., 1758; Suncinema witenbergi n. sp. in Crocidura russula Herm., 1780. Ecologic and zoogeographic relationships are discussed.

Necator miyazakiensis Nagayosi 1955, a hookworm from the raccoon-like dog, Nyctereutes procyonoides, in Miyazaki Prefecture, Kyushu, Japan, is redescribed and placed in the genus Arthrostoma Cameron 1927, and compared with the 6 other species of the genus. Arthrostoma miyazakiense comb. n. is found in raccoon-like dogs not only in Miyazaki Prefecture, but also in Kyoto and Hokkaido Prefectures. This is the first record of a species of Arthrostoma in Japan.

Uncinaria (Uncinaria) maya n. sp. (Nematoda: Ancylostomatidae) and Molineus springsmithi yayeyamanus n. subsp. (Nematoda: Molineidae) are described from the Iriomote cat, Prionailurus iriomotensis, on Iriomote Island, Okinawa, Japan. Uncinaria (U.) maya resembles Uncinaria (Uncinaria) felidis Maplestone, 1939, from Prionailurus bengalensis of India but is distinguished in that the body is much smaller, the ventral rays are set closely with the lateral rays, and the externolateral ray is much shorter than other laterals. Molineus springsmithi yayeyamanus differs from Molineus springsmithi springsmithi Inglis and Ogden, 1965, from Prionailurus bengalensis horsfieldi of East Nepal in that the body is much longer, whereas the esophagus is somewhat shorter and the spicules are divided more distally. Presence of the closely related nematodes in both the Iriomote cat and P. bengalensis suggests a close evolutionary relationship of the 2 hosts.

The species is closely related to U. bauchoti parasite of malagasian Tenrecoidea (sub-genus Megadeirides). The two species may be considered as a link with the primitive sub-family Globocephalinae. The genus Uncinaria itself is considered as an archaic group, which could be at the origin of Uncinariinae-Ancylostomatinae.