Comparison of real-time PCR and the Kato-Katz method for the diagnosis of soil-transmitted helminthiasis and assessment of cure in a randomized controlled trial.

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).

High-throughput multiplex qPCRs for the surveillance of zoonotic species of canine hookworms.

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.

Soil contamination of a public park by human and canine mastadenovirus, as well as hookworms and Toxocara spp eggs.

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.

Prevalence and potential zoonotic risk of hookworms from stray dogs and cats in Guangdong, China.

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.

Prevalence, infection intensity and associated factors of soil transmitted helminths among primary school children in Gurage zone, South Central Ethiopia: a cross-sectional study design.

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.

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

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

Molecular identification of hookworm isolates from stray dogs, humans and selected wildlife from South Africa.

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.

Helminth infections of wild European gray wolves (Canis lupus Linnaeus, 1758) in Lower Saxony, Germany, and comparison to captive wolves.

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.

Molecular identification of zoonotic hookworm species in dog faeces from Tanzania.

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.

Molecular differentiation of three canine and feline hookworms in South China through HRM analysis.

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.

Mitochondrial DNA reveals species composition and phylogenetic relationships of hookworms in northeastern Brazil.

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.

Morphometrical and molecular evidence suggests cryptic diversity among hookworms (Nematoda: Uncinaria) that parasitize pinnipeds from the south-eastern Pacific coasts.

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.

Quantitative Polymerase Chain Reaction for Diagnosis of Soil-Transmitted Helminth Infections: A Comparison with a Flotation-Based Technique and an Investigation of Variability in DNA Detection.

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.

Prevalence of Soil-Transmitted Helminths and Molecular Clarification of Hookworm Species in Ethnic Ede Primary Schoolchildren in Dak Lak Province, Southern Vietnam.

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.

Molecular Identification of Hookworm Isolates in Humans, Dogs and Soil in a Tribal Area in Tamil Nadu, India.

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.

Helminthiasis: Hookworm Infection Remains a Public Health Problem in Dera District, South Gondar, Ethiopia.

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.

Re-evaluation of the species of hookworms infecting dogs in Central Vietnam.

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.

Morphological variability and molecular identification of Uncinaria spp. (Nematoda: Ancylostomatidae) from grizzly and black bears: new species or phenotypic plasticity?

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.

Molecular identification of hookworms in stray and shelter dogs from Guangzhou city, China using ITS sequences.

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.

Unexpected absence of genetic separation of a highly diverse population of hookworms from geographically isolated hosts.

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.