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.

Cognitive and Microbiome Impacts of Experimental Ancylostoma ceylanicum Hookworm Infections in Hamsters.

Hookworms are one of the most prevalent and important parasites, infecting ~500 million people worldwide. Hookworm disease is among the leading causes of iron-deficiency anemia in the developing world and is associated with significant growth stunting and malnutrition. In humans, hookworms appear to impair memory and other forms of cognition, although definitive data are hard to come by. Here we study the impact of a human hookworm parasite, Ancylostoma ceylanicum, on cognition in hamsters in a controlled laboratory setting. We developed tests that measure long-term memory in hamsters. We find that hookworm-infected hamsters were fully capable of detecting a novel object. However, hookworm-infected hamsters were impaired in detecting a displaced object. Defects could be discerned at even at low levels of infection, whereas at higher levels of infection, hamsters were statistically unable to distinguish between displaced and non-displaced objects. These spatial memory deficiencies could not be attributed to defects in infected hamster mobility or to lack of interest. We also found that hookworm infection resulted in reproducible reductions in diversity and changes in specific taxanomic groups in the hamster gut microbiome. These data demonstrate that human hookworm infection in a laboratory mammal results in a specific, rapid, acute, and measurable deficit in spatial memory, and we speculate that gut alterations could play some role in these cognitive deficits. Our findings highlight the importance of hookworm elimination and suggest that finer tuned spatial memory studies be carried out in humans.

Multiparasitism and intensity of helminth infections in relation to symptoms and nutritional status among children: a cross-sectional study in southern Lao People's Democratic Republic.

The occurrence and spatial distribution of intestinal helminth infection in children is fairly well understood. However, knowledge on how helminth infections govern intestinal morbidity is scarce. We conducted a cross-sectional study to assess and quantify the relationship between single and multiple species helminth infection with clinical and self-reported morbidity indicators and nutritional status in Champasack province, southern Lao People's Democratic Republic (Lao PDR). A random sample of 1313 children, aged 6 months to 12 years, from villages in nine rural districts were enrolled and examined for helminth infection using duplicate Kato-Katz thick smears. Morbidity was assessed by self-reported symptoms, coupled with clinical examination and appraisal of nutritional status and anaemia. Bivariate and multivariate logistic regression was employed to study associations between helminth infection and morbidity indicators and anaemia. We found considerable morbidity among the surveyed children, including hepatomegaly (13.7%), pale conjunctiva (13.2%) and abdominal pain (10.4%). Anaemia was recorded in 60.4% of the children, whilst signs of stunting and low body mass index (BMI) were observed in 49.8% and 33.3% of the surveyed children, respectively. Hookworm and Opisthorchis viverrini were the predominant helminth species with prevalences of 51.0% and 43.3%, respectively. The prevalence of Schistosoma mekongi in the surveyed children was 5.6%. Multiple species helminth infections were recorded in 40.4% of the study cohort. Morbidity was associated with specific helminth species infection (e.g. S. mekongi with hepatomegaly; adjusted odds ratio (aOR): 9.49, 95% confidence interval (CI): 2.07-43.51) and multiparasitism (e.g. two or more helminth species with abdominal pain; aOR: 2.40, 95% CI: 1.46-3.93). Anaemia was associated with hookworm infection (aOR: 1.64, 95% CI: 1.16-2.34) and multiparasitism (aOR: 1.64, 95% CI: 1.18-2.29). Low BMI was associated with O. viverrini infection (aOR: 1.68, 95% CI: 1.14-2.49) and multiparasitism (aOR: 1.42, 95% CI: 1.01-2.00). The multiple strong associations reported here between helminth infections (single or multiple species) and intestinal morbidity among children in rural parts of southern Lao PDR call for concerted efforts to control helminth infections, which in turn might improve children's health and development.

Ancilostomíase / Ancylostomiasis

A ancilostomíase é uma doença parasitária causada pelos helmintos Ancylostoma duodenale e Necator americanus, com importante.

Molecular characterization of Ancylostoma ceylanicum Kunitz-type serine protease inhibitor: evidence for a role in hookworm-associated growth delay.

Hookworm infection is a major cause of iron deficiency anemia and malnutrition in developing countries. The Ancylostoma ceylanicum Kunitz-type inhibitor (AceKI) is a 7.9-kDa broad-spectrum inhibitor of trypsin, chymotrypsin, and pancreatic elastase that has previously been isolated from adult hookworms. Site-directed mutagenesis of the predicted P1 inhibitory reactive site amino acid confirmed the role of Met(26) in mediating inhibition of the three target serine proteases. By using reverse transcription-PCR, it was demonstrated that the level of AceKI gene expression increased following activation of third-stage larvae with serum and that the highest level of expression was reached in the adult stage of the parasite. Immunohistochemistry studies performed with polyclonal immunoglobulin G raised against recombinant AceKI showed that the inhibitor localized to the subcuticle of the adult hookworm, suggesting that it has a potential in vivo role in neutralizing intestinal proteases at the surface of the parasite. Immunization with recombinant AceKI was shown to confer partial protection against hookworm-associated growth delay without a measurable effect on anemia. Taken together, the data suggest that AceKI plays a role in the pathogenesis of hookworm-associated malnutrition and growth delay, perhaps through inhibition of nutrient absorption in infected hosts.

The second messenger cyclic GMP mediates activation in Ancylostoma caninum infective larvae.

The developmentally arrested infective larva (L(3)) of hookworms encounters a host-specific signal during infection that initiates previously suspended developmental pathways. Activated L(3) express a parasitic gene set that encodes proteins involved in moulting, growth and development to the adult stage. Early events in this activation to parasitism can be investigated using an in vitro larval feeding assay. When Ancylostoma caninum L(3) are exposed to a host-like stimulus, they resume feeding and release molecules involved in infection. The dauer larva of the free-living nematode Caenorhabditis elegans is a developmentally arrested stage analogous to the hookworm L(3). Recovery from the dauer stage has been proposed as a model for the transition to parasitism in hookworm. Dauer formation and recovery involve several tightly regulated pathways, including a cyclic GMP mediated signalling pathway. To determine if hookworm L(3) activation uses a similar pathway, 8-bromo-cyclic GMP, a membrane permeant analogue of cyclic GMP, was tested for its ability to stimulate feeding. Populations of L(3) incubated with 0.5 mM 8-bromo-cyclic GMP began feeding, and reached maximum feeding at 3.5-5.0 mM. Unlike the serum stimulus, which triggers feeding after a short exposure, 8-bromo-cyclic GMP must be present throughout the entire incubation. Both serum stimulated and 8-bromo-cyclic GMP stimulated L(3) secreted Ancylostoma secreted protein 1, indicating that the stimuli activate the same pathway. Serum and 8-bromo-cyclic GMP stimulated feeding was inhibited by atropine, a muscarinic receptor antagonist. However, only serum stimulated feeding was inhibited by 4,7-phenanthroline, a non-chelating isomer of the metalloprotease inhibitor 1,10-phenantholine. The results indicate that cyclic GMP mediates activation in hookworm larvae, and that a muscarinic receptor is involved in activation. This also suggests that hookworm activation and dauer recovery share similar signalling pathways, and that C. elegans dauer recovery can be used as a model for the transition to parasitism in hookworms.

Oral transfer of adult Ancylostoma ceylanicum hookworms into permissive and nonpermissive host species.

Syrian hamsters become anemic and exhibit delayed growth following oral infection with third-stage Ancylostoma ceylanicum hookworm larvae. Here we describe experiments designed to determine the feasibility of adult worm transfer (AWT) between hosts, a technique that would facilitate the specific study of bloodfeeding hookworms in vivo without prior exposure of the host to larva-specific antigens, permit the ex vivo manipulation of adult parasites prior to reimplantation, and also allow for cross-species transfer of worms. Weanling hamsters given an oral AWT of 40 or 60 mixed-sex A. ceylanicum worms rapidly developed anemia; in the higher-dose group, hemoglobin levels declined from prechallenge levels by 44% within 4 days following AWT. Long-term survival of transferred worms was demonstrated by recovery of parasites from the intestines 42 days after AWT. AWT hamsters acquired humoral immune responses against soluble adult hookworm extracts and excretory-secretory products that were comparable in magnitude to those of animals given a typical infection with larvae. In AWT experiments employing the nonpermissive murine model, C57BL/6 mice given adult worms rapidly became anemic and lost weight in a manner similar to AWT hamsters. Infection of additional mouse strains demonstrated that while C57BL/10 and CD-1 mice also developed anemia following AWT, BALB/c mice were resistant. The technique of AWT to mice may further our understanding of hookworm pathogenesis by allowing the study of adult hookworm infections in a species with well-characterized genetics and an abundance of available reagents.

Experimental human infection with the dog hookworm, Ancylostoma caninum.

OBJECTIVE: To investigate possible routes for human infection by the dog hookworm (Ancylostoma caninum). DESIGN, SETTING AND PARTICIPANT: Relatively small numbers of infective larvae were administered orally and percutaneously to an informed healthy volunteer (J K L) under medical supervision, at intervals between May 1998 and May 1999. MAIN OUTCOME MEASURES: Symptoms; weekly blood eosinophil counts; faecal microscopy. RESULTS: A marked blood eosinophilia followed a single oral exposure to 100 infective larvae, while faecal examination remained negative. Eosinophil counts then declined gradually, although a rapid, spontaneous rise several months later, at the beginning of spring, possibly indicated reactivation of dormant larvae. Blood eosinophil numbers did not rise significantly after percutaneous infection with 200 larvae. A subsequent, smaller, oral inoculum of 20 larvae provoked an eosinophil response similar to that of the first experiment. CONCLUSIONS: Our findings suggest that, following ingestion, some infective larvae of A. caninum develop directly into adult worms in the human gut (as they do in dogs). While the percutaneous route might be the most common means of human exposure to canine hookworm larvae, leading generally to subclinical infection, oral infection may be more likely to provoke symptomatic eosinophilic enteritis.

Parasitose intestinal: profilaxia e tratamento dessa esquecida realidade brasileira / Intestinal parasite: prophylaxis and treatment of that forgotten brazilian reality

Com o evoluir da Medicina, inúmeras técnicas, métodos de diagnóstico e tratamento foram surgindo para as novas doenças constantemente descobertas. E, nessa realidade, com freqüência deparamo-nos com médicos capazes de tratar doenças complexas, com tecnologias modernas. Porém muitos se esquecem de considerar doenças básicas da comunidade, como é o caso das parasitoses intestinais, uma realidade brasileira que, embora prevalecente nas camadas socioeconomicamente menos favorecidas, afeta todos os níveis sociais. A finalidade deste artigo é promover uma revisão bibliográfica sobre o tema e abordar as principais parasitoses, dando subsídio ao diagnóstico, e, principalmente, frisar a conscientização da necessidade das medidas preventivas

Ancylostoma ceylanicum infection in female hamsters: an observation on altered reproductive function.

Reproductive performances of female hamsters were investigated during Ancylostoma ceylanicum (hookworm) infection. Animals having the highest levels of infection (34.96 +/- 1.11 worms) showed degenerative changes in the reproductive system. Ovaries of infected animals contained a few primary or secondary follicles. On cocaging with males of proven fertility, only 7-8% (80% in controls) of the infected females mated but did not conceive as evidenced by the absence of corpora lutea or implantation sites on day 10 postcoitum. Animals with low worm burdens (5.94 +/- 0.65 worms), however, showed almost normal fertility. The uterine weight bioassay and compensatory ovarian hypertrophy suggest strong suppression of pituitary gonadotrophin contents in infected females. Resorptive effects on the pregnancy outcome of infected female hamsters were also recorded.

The response of captive rodents to the migration of Ancylostoma caninum larvae.

The distribution of the larvae of Ancylostoma caninum in tissues of captive wild rodents (Rattus rattus) and the leucocytic and behavioural responses of these rodents were studied after experimental oral infection. There was a wide distribution of larvae in tissues with a preponderance of the larvae in the skeletal muscles of the anterior part of the body in older infection. The rats responded by an elevation of total leucocytes and eosinophils in blood, alternation of locomotory activity and behavioural dominance that may have a correlation with predation and epidemiology of A. caninum in a sylvatic setting.

Ancylostoma ceylanicum: immunization with soluble worm extract and responses to challenge infection of dogs.

When dogs were immunized with soluble extract of adult Ancylostoma ceylanicum antigen, they were partially resistant to challenge infection in this model of human hookworm infection. Two immunizing doses, each of 1 mg protein suspended in Freund's complete adjuvant, were administered to one group of animals 1 and 3 weeks prior to infection with 5000 larvae. When compared with control dogs given the same infective dose, fecal egg excretion and intestinal adult worm burden in the immunized animals were reduced by 59 and 74%, respectively. Infection had no significant effect on hemoglobin concentrations, mean red cell volumes, total white cell counts, platelet levels, or spontaneous and phytohemagglutinin-induced lymphocyte transformations in both control and immunized animals. Both groups developed an eosinophilia, and lymphocytes from the immunized dogs responded transiently to stimulation with both larval and adult worm antigens. Specific IgM antibodies were transitory in both groups of dogs following infection. IgG antibodies developed significantly 2 weeks after infection in the immunized group; however, they did not appear until 4 weeks after infection in the control group. Both groups developed IgA antibodies 1 week after infection. They were maintained in the control dogs, in contrast to the levels in immunized animals which subsided rapidly 4 weeks after infection. Therefore, when animals are injected with soluble adult worm antigen prior to infection, specific protective immunity is acquired.