Establishment of a Tm-shift Method for Detection of Cat-Derived Hookworms.

Melting temperature shift (Tm-shift) is a new detection method that analyze the melting curve on real-time PCR thermocycler using SYBR Green I fluorescent dye. To establish a Tm-shift method for the detection of Ancylostoma ceylanicum and A. tubaeforme in cats, specific primers, with GC tail of unequal length attached to their 5 ́ end, were designed based on 2 SNP loci (ITS101 and ITS296) of the internal transcribed spacer 1 (ITS1) sequences. The standard curve of Tm-shift was established using the standard plasmids of A. ceylanicum (AceP) and A. tubaeforme (AtuP). The Tm-shift method stability, sensitivity, and accuracy were tested with reference to the standard curve, and clinical fecal samples were also examined. The results demonstrated that the 2 sets of primers based on the 2 SNPs could accurately distinguish between A. ceylanicum and A. tubaeforme. The coefficient of variation (CV) of Tm-values of AceP and AtuP was 0.07% and 0.06% in ITS101 and was 0.06% and 0.08% in ITS296, respectively. The minimum detectable DNA concentration was 5.22×10-6 and 5.28×10-6 ng/µl samples of AceP and AtuP, respectively. The accuracy of Tm-shift method reached 100% based on examination of 10 hookworm DNA samples with known species. In the clinical detection of hookworm in 69 stray cat fecal sample, the Tm-shift detection results were consistent with the microscopic examination and successfully differentiated between the 2-hookworm species. In conclusion, the developed method is a rapid, sensitive and accurate technique and can provide a promising tool for clinical detection and epidemiological investigation of cat-derived hookworms.

Ancylostoma ceylanicum Hookworm in the Solomon Islands.

Although hookworm is highly prevalent in the Solomon Islands, the species involved are unknown. We initiated this study in response to finding Ancylostoma ceylanicum hookworm in a peacekeeper in Australia who had returned from the Solomon Islands. Kato-Katz fecal surveys performed in 2013 and 2014 in 2 village groups in East Malaita, Solomon Islands, identified hookworm-positive samples. These specimens were tested by cytochrome oxidase 1 (cox-1) gene multiplex PCR and sequenced. Of 66 positive specimens, 54 (81.8%) contained only Necator americanus, 11 (16.7%) contained only A. ceylanicum, and 1 (1.5%) contained both species. A. duodenale was not found. Haplotype analysis of cox-1 sequences placed all human isolates (99% bootstrap support) of A. ceylanicum within the zoonotic clade rather than the human-specific clade. This study confirms that A. ceylanicum is endemic in the East Malaita region of this Pacific Island nation. The strain of the A. ceylanicum in this region can be shared among humans, dogs, and cats.

Hamsters vaccinated with Ace-mep-7 DNA vaccine produced protective immunity against Ancylostoma ceylanicum infection.

Hookworms are intestinal nematodes that infect up to 740 million people, mostly in tropical and subtropical regions. Adult worms suck blood from damaged vessels in the gut mucosa, digesting hemoglobin using aspartic-, cysteine- and metalloproteases. Targeting aspartic hemoglobinases using drugs or vaccines is therefore a promising approach to ancylostomiasis control. Based on homology to metalloproteases from other hookworm species, we cloned the Ancylostoma ceylanicum metalloprotease 7 cDNA (Ace-mep-7). The corresponding Ace-MEP-7 protein has a predicted molecular mass of 98.8 kDa. The homology to metallopeptidases from other hookworm species and its predicted transmembrane region support the hypothesis that Ace-MEP-7 may be involved in hemoglobin digestion in the hookworm gastrointestinal tract, especially that our analyses show expression of Ace-mep-7 in the adult stage of the parasite. Immunization of Syrian golden hamsters with Ace-mep-7 cDNA resulted in 50% (p < 0.01) intestinal worm burden reduction. Additionally 78% (p < 0.05) egg count reduction in both sexes was observed. These results suggest that immunization with Ace-mep-7 may contribute to reduction in egg count released into the environment during the A. ceylanicum infection.

Levels of Ancylostoma infections and phylogenetic analysis of cox 1 gene of A. ceylanicum in stray cat faecal samples from Guangzhou, China.

Ancylostoma ceylanicum is a common zoonotic nematode. Cats act as natural reservoirs of the hookworm and are involved in transmitting infection to humans, thus posing a potential risk to public health. The prevalence of feline A. ceylanicum in Guangzhou (South China) was surveyed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). In total, 112 faecal samples were examined; 34.8% (39/112) and 43.8% (49/112) samples were positive with hookworms by microscopy and PCR method, respectively. Among them, 40.8% of samples harboured A. ceylanicum. Twelve positive A. ceylanicum samples were selected randomly and used for cox 1 sequence analysis. Sequencing results revealed that they had 97-99% similarity with A. ceylanicum cox 1 gene sequences deposited in GenBank. A phylogenetic tree showed that A. ceylanicum isolates were divided into two groups: one comprising four isolates from Guangzhou (South China), and the other comprising those from Malaysia, Cambodia and Guangzhou. In the latter group, all A. ceylanicum isolates from Guangzhou were clustered into a minor group again. The results indicate that the high prevalence of A. ceylanicum in stray cats in South China poses a potential risk of hookworm transmission from pet cats to humans, and that A. ceylanicum may be a species complex worldwide.

High prevalence of Ancylostoma ceylanicum hookworm infections in humans, Cambodia, 2012.

Ancylostoma ceylanicum, a hookworm of canids and felids in Asia, is becoming the second most common hookworm infecting humans. In 2012, we investigated the prevalence and infection dynamics of and risk factors for hookworm infections in humans and dogs in a rural Cambodian village. Over 57% of the population was infected with hookworms; of those, 52% harbored A. ceylanicum hookworms. The greatest intensities of A. ceylanicum eggs were in persons 21-30 years of age. Over 90% of dogs also harbored A. ceylanicum hookworms. Characterization of the cytochrome oxidase-1 gene divided isolates of A. ceylanicum hookworms into 2 groups, 1 containing isolates from humans only and the other a mix of isolates from humans and animals. We hypothesize that preventative chemotherapy in the absence of concurrent hygiene and animal health programs may be a factor leading to emergence of A. ceylanicum infections; thus, we advocate for a One Health approach to control this zoonosis.

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

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

Molecular detection of Ancylostoma duodenale, Ancylostoma ceylanicum, and Necator americanus in humans in northeastern and southern Thailand.

The 2 principal species of hookworms infecting humans are Necator americanus and Ancylostoma duodenale. Case studies on zoonotic hookworm infections with Ancylostoma ceylanicum and/or Ancylostoma caninum are known mainly from Asian countries. Of these 2 zoonotic species, only A. ceylanicum can develop to adulthood in humans. In the present study, we report a molecular-based survey of human hookworm infections present in southern and northeastern Thailand. Thirty larval hookworm samples were obtained from fecal agar plate cultures of 10 patients in northeastren Thailand and 20 in southern Thailand. Partial ITS1, 5.8S, and ITS2 regions of the ribosomal DNA genes were amplified using PCR. The amplicons were sequenced, aligned, and compared with other hookworm sequences in GenBank database. The results showed that, in Thailand, N. americanus is more prevalent than Ancylostoma spp. and is found in both study areas. Sporadic cases of A. ceylanicum and A. duodenale infection were seen in northeastern Thailand.

Cloning, sequencing and phylogenetic analysis of the small GTPase gene cdc-42 from Ancylostoma caninum.

CDC-42 is a member of the Rho GTPase subfamily that is involved in many signaling pathways, including mitosis, cell polarity, cell migration and cytoskeleton remodeling. Here, we present the first characterization of a full-length cDNA encoding the small GTPase cdc-42, designated as Accdc-42, isolated from the parasitic nematode Ancylostoma caninum. The encoded protein contains 191 amino acid residues with a predicted molecular weight of 21 kDa and displays a high level of identity with the Rho-family GTPase protein CDC-42. Phylogenetic analysis revealed that Accdc-42 was most closely related to Caenorhabditis briggsae cdc-42. Comparison with selected sequences from the free-living nematode Caenorhabditis elegans, Drosophila melanogaster, Xenopus laevis, Danio rerio, Mus musculus and human genomes showed that Accdc-42 is highly conserved. AcCDC-42 demonstrates the highest identity to CDC-42 from C. briggsae (94.2%), and it also exhibits 91.6% identity to CDC-42 from C. elegans and 91.1% from Brugia malayi. Additionally, the transcript of Accdc-42 was analyzed during the different developmental stages of the worm. Accdc-42 was expressed in the L1/L2 larvae, L3 larvae and female and male adults of A. caninum.

Epidemiology of intestinal helminth parasites in stray dogs from markets in south-eastern Nigeria.

A survey of gastrointestinal helminth parasites of stray dogs (Canis familiaris) was conducted at Obollo-Afor and Ekwulobia markets, in Enugu and Anambra States, south-eastern Nigeria, respectively, to determine the patterns of infection among dogs in different parts of south-eastern Nigeria. Faecal samples collected, using long forceps, from every dog encountered in the markets between June 2007 and December 2008 were analysed by the Kato-Katz technique. Out of 413 dogs examined in both markets, 217 (52.6%) were infected with at least one of five parasites (Toxocara spp., Dipylidium caninum, Ancylostoma caninum, Taenia spp. and Trichuris vulpis). Overall faecal egg intensity of infection was 49.9 ± 58.7 eggs/g (epg). The prevalence of infection was comparable between the markets and between the male and female dogs, but varied significantly (P < 0.05) by age, decreasing from 78.9% in pups to 36.0% in adult dogs. The mean intensity pattern was similar to that of prevalence, decreasing from 86.7 ± 63.0 epg in pups to 22.1 ± 34.4 in adults. The most important individual parasite infection was Ancylostoma spp. (39.2%; 30.0 ± 41.2 epg) while T. vulpis was the least important (1.9%; 0.7 ± 5.4 epg). Generally, prevalence and intensity patterns of each parasite were also comparable between the markets and between sexes, but significantly (P < 0.05) age-dependent. The implications of these findings to public health in Nigeria and other endemic countries are discussed in relation to options for cost-effective control design and implementation.

Insights into hookworm prevalence in Asia: a systematic review and meta-analysis.

INTRODUCTION: Hookworm infections are neglected tropical diseases of humans and animals worldwide. A meta-analysis and systematic review was designed to evaluate the prevalence of hookworm infection in animal and human hosts in Asia until July 2018. METHODS: The available online articles of five English databases (PubMed, Scopus, Science Direct, Web of Science and Google Scholar) were explored. RESULTS: The most parasitized carnivores were jackal (48%, CI: 4 to 91%), followed by dog (41%, CI: 29 to 53%), cat (26%, CI: 14 to 38%) and the red fox (19%, CI: 13 to 24%). The weighted prevalence of Ancylostoma braziliensis, A. caninum, A. ceylanicum, A. tubaeforme and Uncinaria stenocephala isolated from different canids were found to be 27% (CI: 21 to 33%), 23% (CI: 7.0 to 53%), 24% (CI: 12 to 35%), 44% (CI: 37 to 51%) and 37% (CI: 18 to 55%), respectively. In total, 98 records were obtained for human hookworms from 3209 760 examined individuals and the calculated weighted prevalence in this population was 19% (CI: 17 to 20%). CONCLUSIONS: These findings highlight a desirable ecological milieu for parasite survival and transmission in such territories, which implicates revisiting control programs and public health infrastructures in those areas.

Egg genotyping reveals the possibility of patent Ancylostoma caninum infection in human intestine.

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

The mitochondrial genomes of Ancylostoma caninum and Bunostomum phlebotomum--two hookworms of animal health and zoonotic importance.

BACKGROUND: Hookworms are blood-feeding nematodes that parasitize the small intestines of many mammals, including humans and cattle. These nematodes are of major socioeconomic importance and cause disease, mainly as a consequence of anaemia (particularly in children or young animals), resulting in impaired development and sometimes deaths. Studying genetic variability within and among hookworm populations is central to addressing epidemiological and ecological questions, thus assisting in the control of hookworm disease. Mitochondrial (mt) genes are known to provide useful population markers for hookworms, but mt genome sequence data are scant. RESULTS: The present study characterizes the complete mt genomes of two species of hookworm, Ancylostoma caninum (from dogs) and Bunostomum phlebotomum (from cattle), each sequenced (by 454 technology or primer-walking), following long-PCR amplification from genomic DNA (approximately 20-40 ng) isolated from individual adult worms. These mt genomes were 13717 bp and 13790 bp in size, respectively, and each contained 12 protein coding, 22 transfer RNA and 2 ribosomal RNA genes, typical for other secernentean nematodes. In addition, phylogenetic analysis (by Bayesian inference and maximum likelihood) of concatenated mt protein sequence data sets for 12 nematodes (including Ancylostoma caninum and Bunostomum phlebotomum), representing the Ascaridida, Spirurida and Strongylida, was conducted. The analysis yielded maximum statistical support for the formation of monophyletic clades for each recognized nematode order assessed, except for the Rhabditida. CONCLUSION: The mt genomes characterized herein represent a rich source of population genetic markers for epidemiological and ecological studies. The strong statistical support for the construction of phylogenetic clades and consistency between the two different tree-building methods employed indicate the value of using whole mt genome data sets for systematic studies of nematodes. The grouping of the Spirurida and Ascaridida to the exclusion of the Strongylida was not supported in the present analysis, a finding which conflicts with the current evolutionary hypothesis for the Nematoda based on nuclear ribosomal gene data.

Male-enriched transcription of genes encoding ASPs and Kunitz-type protease inhibitors in Ancylostoma species.

Various transcripts coding for proteins considered to be central to parasite-host interactions were identified previously as male-enriched in the hookworm Ancylostoma braziliense. Among these genes were an ASP-5-like homologue and a Kunitz-type protease inhibitor. The present study extends this previous work to investigate similar molecules in other hookworms (Ancylostomatidae). Specifically, partial cDNA sequences encoding three different ASP molecules and two different Kunitz-type protease inhibitors were isolated, and the differential transcription between adult male and female worms was compared by conventional and quantitative reverse transcription (RT)-PCR for three species, A. braziliense, Ancylostoma caninum and Ancylostoma ceylanicum. In accordance with previous findings, male-enriched transcription was observed for all molecules explored. Based on this information, it is hypothesized that adult males are responsible for producing proteins essential to the survival of hookworms inside the host and for supporting developmental and reproductive processes in female worms.

A New Species of Ancylostoma (Nemata: Strongylida: Ancylostomatidae) from Two Species of Ctenomys in Lowland Bolivia.

From the small intestines of both Ctenomys boliviensis and Ctenomys steinbachi collected from August 1984 through June 1990 from the eastern lowlands of the Department of Santa Cruz, Bolivia a total of 36 specimens of Ancylostoma were recovered. Morphological investigation and comparisons with known species described and reported from mammals in the Neotropical Region show that this is an undescribed species, herein described as new. These nematans were collected from individuals of C. steinbachi collected from near a locality called Caranda (northwest of Santa Cruz de la Sierra) and from C. boliviensis from near Santa Rosa de la Roca (northeast of Santa Cruz de la Sierra) and from cajuchis collected from 3 km west of Estación El Pailón, 30 km east of Santa Cruz de la Sierra. The new species of Ancylostoma differs from all other species of Ancylostoma known from the Neotropical Region in the presence of paired sub-terminal papillae on the dorsal ray of males.

Prevalence survey of gastrointestinal and respiratory parasites of shelter cats in northeastern Georgia, USA.

The goal of this study was to assess the prevalence of gastrointestinal and respiratory parasites of shelter cats from northeast Georgia, thus promoting a more targeted approach in parasite diagnosis and treatment. Fecal samples of cats kept in a shelter located in Lavonia, northeastern Georgia, USA, were processed for the presence of parasites using double centrifugation sugar flotation (n = 103) and Baermann techniques (n = 98). Flotation revealed eggs of Toxocara cati (17.5%), Ancylostoma sp. (11.7%), Taeniidae (3.9%), Spirometra mansonoides (2.9%), Mesocestoides sp. (1%), Dipylidium caninum (1%), and Eucoleus aerophilus (1%), and oocysts of Cystoisospora felis (16.5%), and Cystoisospora rivolta (8.7%). Baermann diagnosed Aelurostrongylus abstrusus larvae in 5 cats (5.1%), while fecal flotation alone identified only 2 of these infections. Taeniidae eggs were identified to species-level by PCR and sequencing targeting the cytochrome oxidase c subunit 1 (cox1) of the mitochondrial DNA. All isolates belong to Hydatigera taeniaeformis sensu stricto, which is the first unequivocal report of the species in North America. Overall, 45.6% of the cats were infected with at least one parasite. This prevalence of infection is much higher than what is generally reported in client owned animals, highlighting the importance of using appropriate fecal diagnostic techniques to detect gastrointestinal and respiratory parasites on newly adopted cats. Correct diagnosis may direct appropriate treatment and control strategies, which would mitigate the risk of infection of other animals in household, and human exposure to zoonotic parasites.

Mitochondrial DNA variation of the dog hookworm Ancylostoma caninum in Brazilian populations.

The mitochondrial cytochrome oxidase I gene was partially sequenced for 164 Ancylostoma caninum individuals, originating from five different localities in Brazil, with the aim of describing the genetic diversity and genetic structure of Brazilian hookworm populations. Allelic and nucleotide diversity were moderate (overall h=0.88 and pi=0.016) and were similar among cities. There was moderate genetic differentiation among the populations sampled (approximately Phi(ST)=0.12) and a weak but nonsignificant correlation between geographical and genetic distance. This genetic structure was similar to that observed among populations of the human hookworm, Necator americanus, but distinct from that typically found in trichostrongylid nematode parasites of livestock. Thus, a pattern of different genetic structures among different groups of nematodes is emerging. We also observed a few individuals that had a highly divergent mtDNA sequence (almost 7% sequence divergence from the other sequences). These results in combination with data from other studies suggest that A. caninum populations worldwide consist of a mix of previously differentiated populations, or perhaps even cryptic species. This study contributes to the knowledge of genetic structure and diversity of hookworms, which in turn will be useful in developing methods for their control.

[Differentiating the morphology of infective larvae between two human hookworms].

The morphological differentiation of the infective larvae between human Ancylostoma duodenale and Necator americanus is of great significance for the epidemiological survey of hookworm diseases and human parasitology teaching. Understanding of features of the oral spear and transverse lines on the tunica vaginalis is able to accurately differentiate the infective larvae between these two human hookworms.

Diversity and prevalence of gastrointestinal parasites in two wild Galago species in Gabon.

In this study, we characterize the diversity and estimated infection levels of gastrointestinal parasites circulating in two galago species, Galago demidoff and G. thomasi in two sites situated in the Southeastern forests of Gabon. Our study reveals that eleven parasites including nine helminthes (Ascaris spp., Ankylostoma spp., Dicrocoelium spp., Gongylonema spp., Oesophagostomum spp., Lemuricola spp., Strongyloides spp. Trichostrongylus spp. and Trichuris spp.) and two protozoans (Balantidium spp. and Entamoeba spp.) may infect Galago spp. with high infection rates. The results show that: a very similar parasite spectrum is found in both host species; all the taxa identified were previously observed in other Primate species and/or Man. They also show that age, gender and forest type may influence infection rates and/or parasite diversity found in a particular host and/or geographic area.

Ancylostoma ailuropodae n. sp. (Nematoda: Ancylostomatidae), a new hookworm parasite isolated from wild giant pandas in Southwest China.

BACKGROUND: Hookworms belonging to the genus Ancylostoma (Dubini, 1843) cause ancylostomiasis, a disease of considerable concern in humans and domestic and wild animals. Molecular and epidemiological data support evidence for the zoonotic potential among species of Ancylostoma where transmission to humans is facilitated by rapid urbanization and increased human-wildlife interactions. It is important to assess and describe these potential zoonotic parasite species in wildlife, especially in hosts that have physiological similarities to humans and share their habitat. Moreover, defining species diversity within parasite groups that can circulate among free-ranging host species and humans also provides a pathway to understanding the distribution of infection and disease. In this study, we describe a previously unrecognized species of hookworm in the genus Ancylostoma in the giant panda, including criteria for morphological and molecular characterization. METHODS: The hookworm specimens were obtained from a wild giant panda that died in the Fengtongzai Natural Reserve in Sichuan Province of China in November 2013. They were microscopically examined and then genetically analyzed by sequencing the nuclear internal transcribed spacer (ITS, ITS1-5.8S-ITS2) and mitochondrial cytochrome c oxidase subunit 1 (cox1) genes in two representative specimens (one female and one male, FTZ1 and FTZ2, respectively). RESULTS: Ancylostoma ailuropodae n. sp. is proposed for these hookworms. Morphologically the hookworm specimens differ from other congeneric species primarily based on the structure of the buccal capsule in males and females, characterized by 2 pairs of ventrolateral and 2 pairs of dorsolateral teeth; males differ in the structure and shape of the copulatory bursa, where the dorsal ray possesses 2 digitations. Pairwise nuclear and mitochondrial DNA comparisons, genetic distance analysis, and phylogenetic data strongly indicate that A. ailuropodae from giant pandas is a separate species which shared a most recent common ancestor with A. ceylanicum Looss, 1911 in the genus Ancylostoma (family Ancylostomatidae). CONCLUSION: Ancylostoma ailuropodae n. sp. is the fourth species of hookworm described from the Ursidae and the fifteenth species assigned to the genus Ancylostoma. A sister-species association with A. ceylanicum and phylogenetic distinctiveness from the monophyletic Uncinaria Frölich, 1789 among ursids and other carnivorans indicate a history of host colonization in the evolutionary radiation among ancylostomatid hookworms. Further, phylogenetic relationships among bears and a history of ecological and geographical isolation for giant pandas may be consistent with two independent events of host colonization in the diversification of Ancylostoma among ursid hosts. A history for host colonization within this assemblage and the relationship for A. ailuropodae n. sp. demonstrate the potential of this species as a zoonotic parasite and as a possible threat to human health. The cumulative morphological, molecular and phylogenetic data presented for A. ailuropodae n. sp. provides a better understanding of the taxonomy, diagnostics and evolutionary biology of the hookworms.

A novel, species-specific, real-time PCR assay for the detection of the emerging zoonotic parasite Ancylostoma ceylanicum in human stool.

BACKGROUND: Molecular-based surveys have indicated that Ancylostoma ceylanicum, a zoonotic hookworm, is likely the second most prevalent hookworm species infecting humans in Asia. Most current PCR-based diagnostic options for the detection of Ancylostoma species target the Internal Transcribed Spacer (ITS) regions of the ribosomal gene cluster. These regions possess a considerable degree of conservation among the species of this genus and this conservation can lead to the misidentification of infecting species or require additional labor for accurate species-level determination. We have developed a novel, real-time PCR-based assay for the sensitive and species-specific detection of A. ceylanicum that targets a non-coding, highly repetitive genomic DNA element. Comparative testing of this PCR assay with an assay that targets ITS sequences was conducted on field-collected samples from Argentina and Timor-Leste to provide further evidence of the sensitivity and species-specificity of this assay. METHODS/PRINCIPAL FINDINGS: A previously described platform for the design of primers/probe targeting non-coding highly repetitive regions was used for the development of this novel assay. The assay's limits of detection (sensitivity) and cross-reactivity with other soil-transmitted helminth species (specificity) were assessed with real-time PCR experiments. The assay was successfully used to identify infections caused by A. ceylanicum that were previously only identified to the genus level as Ancylostoma spp. when analyzed using other published primer-probe pairings. Further proof of sensitive, species-specific detection was provided using a published, semi-nested restriction fragment length polymorphism-PCR assay that differentiates between Ancylostoma species. CONCLUSIONS/SIGNIFICANCE: Due to the close proximity of people and domestic/wild animals in many regions of the world, the potential for zoonotic infections is substantial. Sensitive tools enabling the screening for different soil-transmitted helminth infections are essential to the success of mass deworming efforts and facilitate the appropriate interpretation of data. This study describes a novel, species-specific, real-time PCR-based assay for the detection of A. ceylanicum that will help to address the need for such tools in integrated STH deworming programs. TRIAL REGISTRATION: ANZCTR.org.au ACTRN12614000680662.