Comparison of percutaneous vs oral infection of hamsters with the hookworm Ancylostoma ceylanicum: Parasite development, pathology and primary immune response.

BACKGROUND: Hundreds of millions of people in poor countries continue to suffer from disease caused by bloodfeeding hookworms. While mice and rats are not reliably permissive hosts for any human hookworm species, adult Golden Syrian hamsters are fully permissive for the human and animal pathogen Ancylostoma ceylanicum. Similar to humans, hamsters may be infected with A. ceylanicum third-stage larvae orally or percutaneously. Oral infection typically leads to consistent worm yields in hamsters but may not accurately reflect the clinical and immunological manifestations of human infection resulting from skin penetration. METHODOLOGY/PRINCIPAL FINDINGS: In this study we compared host responses following percutaneous infection to those utilizing an established oral infection protocol. Infected hamsters exhibited a dose-dependent pathology, with 1000 percutaneous larvae (L3) causing anemia and adult worm recovery comparable to that of 50 orally administered L3. A delayed arrival and maturity of worms in the intestine was observed, as was variation in measured cellular immune responses. A long-term study found that the decline in blood hemoglobin was more gradual and did not reach levels as low, with the nadir of disease coming later in percutaneously infected hamsters. Both groups exhibited moderate growth delay, an effect that was more persistent in the percutaneously infected group. Fecal egg output also peaked later and at lower levels in the percutaneously infected animals. In contrast to orally infected hamsters, antibody titers to larval antigens continued to increase throughout the course of the experiment in the percutaneous group. CONCLUSIONS/SIGNIFICANCE: These results demonstrate that the route of infection with A. ceylanicum impacts disease pathogenesis, as well as humoral and cellular immune responses in an experimental setting. These data further validate the utility of the Golden Syrian hamster as a model of both oral and percutaneous infection with human hookworms.

Epidemiology of hookworm infection in Kintampo North Municipality, Ghana: patterns of malaria coinfection, anemia, and albendazole treatment failure.

A cross-sectional pilot study of hookworm infection was carried out among 292 subjects from 62 households in Kintampo North, Ghana. The overall prevalence of hookworm infection was 45%, peaking in those 11-20 years old (58.5%). In children, risk factors for hookworm infection included coinfection with malaria and increased serum immunoglobulin G reactivity to hookworm secretory antigens. Risk factors for infection in adults included poor nutritional status, not using a latrine, not wearing shoes, and occupation (farming). Although albendazole therapy was associated with an overall egg reduction rate of 82%, 37 subjects (39%) remained infected. Among those who failed therapy, treatment was not associated with a significant reduction in egg excretion, and nearly one-third had higher counts on repeat examination. These data confirm a high prevalence of low-intensity hookworm infection in central Ghana and its association with poor nutritional status. The high rate of albendazole failure raises concern about emerging resistance.

Characterisation of a fatty acid and retinol binding protein orthologue from the hookworm Ancylostoma ceylanicum.

Hookworms, bloodfeeding intestinal nematodes, infect nearly one billion people in resource limited countries and are a leading cause of anaemia and malnutrition. Like other nematodes, hookworms lack the capacity to synthesise essential fatty acids de novo and therefore must acquire those from exogenous sources. The cDNA corresponding to a putative Ancylostoma ceylanicum fatty acid and retinol binding protein-1 (AceFAR-1) was amplified from adult hookworm mRNA. Studies using quantitative reverse transcriptase real-time PCR demonstrate that AceFAR-1 transcripts are most abundant in the earliest developmental stages of the parasite, and greater in females than males. Using in vitro assays, the recombinant AceFAR-1 (rAceFAR-1) was shown to bind individual fatty acids with equilibrium dissociation constants in the low micromolar range. The pattern of fatty acid uptake by live adult worms cultured ex vivo was similar to the in vitro binding profile of rAceFAR-1, raising the possibility that the native protein may be involved in acquisition of fatty acids by A. ceylanicum. Animals vaccinated orally with rAceFAR-1 and the mucosal adjuvant cholera toxin exhibited a statistically significant (40-47%) reduction in intestinal worm burden compared with controls immunized with antigen or adjuvant alone. Together, these data suggest a potential role for AceFAR-1 in hookworm biology, making it a potentially valuable target for drug and vaccine development.

Role for nitric oxide in hookworm-associated immune suppression.

Hookworm infection is a major cause of anemia and malnutrition in resource-poor countries. Human and animal studies suggest that infection with these intestinal nematodes is associated with impaired cellular immunity, characterized by reduced lymphocyte proliferation in response to both parasite and heterologous antigens. We report here data from studies aimed at defining mechanisms through which hookworms modulate the host cellular immune response. Splenocytes and mesenteric lymph node (MLN) cells from hamsters infected with Ancylostoma ceylanicum showed minimal proliferation in response to mitogen at days 20 and 30 postinfection (p.i.), with partial recovery noted at day 70 p.i. The proliferative capacity of enriched splenocyte T-cell preparations from infected animals following stimulation with hookworm antigens was partially restored in the presence of antigen-presenting cells from uninfected hamsters. Analysis by fluorescence-activated cell sorting revealed that hookworm infection is associated with reduced percentages of both CD4(+) and surface immunoglobulin G-positive lymphocytes in the spleen and MLN cells. Splenocytes from infected hamsters also secreted more nitric oxide (NO) in culture than did those from naïve animals. Inhibition of NO secretion was associated with partial restoration of the proliferative capacity of splenocytes from infected animals in response to concanavalin A, suggesting a role for NO in mediating this effect. Together, these data demonstrate that hookworm infection is associated with impaired function of antigen-presenting cells and depletion of important lymphocyte subpopulations and also suggests a role for NO in parasite-induced immunosuppression.

A purified Bacillus thuringiensis crystal protein with therapeutic activity against the hookworm parasite Ancylostoma ceylanicum.

Crystal (Cry) proteins produced by the soil bacterium Bacillus thuringiensis (Bt) are harmless to vertebrates, but they are highly toxic to insects and nematodes. Their value in controlling insects that destroy crops and transmit human diseases is well established. Although it has recently been demonstrated that a few individual Bt Cry proteins, such as Cry5B, are toxic to a wide range of free-living nematodes, the potential activity of purified Cry proteins against parasitic nematodes remains largely unknown. We report here studies aimed at characterizing in vitro and in vivo anthelminthic activities of purified recombinant Cry5B against the hookworm parasite Ancylostoma ceylanicum, a bloodfeeding gastrointestinal nematode for which humans are permissive hosts. By using in vitro larval development assays, Cry5B was found to be highly toxic to early stage hookworm larvae. Exposure of adult A. ceylanicum to Cry5B was also associated with significant toxicity, including a substantial reduction in egg excretion by adult female worms. To demonstrate therapeutic efficacy in vivo, hamsters infected with A. ceylanicum were treated with three daily oral doses of purified Cry5B, the benzimidazole anthelminthic mebendazole, or buffer. Compared with control (buffer-treated) animals, infected hamsters that received Cry5B showed statistically significant improvements in growth and blood hemoglobin levels as well as reduced worm burdens that were comparable to the mebendazole-treated animals. These data demonstrate that Cry5B is highly active in vitro and in vivo against a globally significant nematode parasite and that Cry5B warrants further clinical development for human and veterinary use.

Dietary iron content mediates hookworm pathogenesis in vivo.

Hookworm infection is associated with growth delay and iron deficiency anemia in developing countries. A series of experiments were designed in order to test the hypothesis that host dietary iron restriction mediates susceptibility to hookworm infection using the hamster model of Ancylostoma ceylanicum. Animals were maintained on diets containing either 10 ppm iron (iron restricted) or 200 ppm iron (standard/high iron), followed by infection with A. ceylanicum third-stage larvae. Infected animals fed the standard diet exhibited statistically significant growth delay and reduced blood hemoglobin levels compared to uninfected controls on day 20 postinfection. In contrast, no statistically significant differences in weight or hemoglobin concentration were observed between infected and uninfected animals fed the iron-restricted diet. Moreover, iron-restricted animals were observed to have reduced intestinal worm burdens on day 10 and day 20 postinfection compared to those of animals maintained on the standard/high-iron diet. In a subsequent study, animals equilibrated on diets containing a range of iron levels (10 ppm, 40 ppm, 100 ppm, or 200 ppm) were infected with A. ceylanicum and followed for evidence of hookworm disease. Infected animals from the intermediate-dietary iron (40- and 100-ppm) groups exhibited greater weight loss and anemia than those in the low (10-ppm)- or high (200-ppm)-iron diet groups. Mortality was also significantly higher in the intermediate-dietary-iron groups. These data suggest that severe dietary iron restriction impairs hookworm development in vivo but that moderate iron restriction enhances host susceptibility to severe disease.

Detection of excretory/secretory coproantigens in experimental hookworm infection.

This report describes the detection of hookworm excretory/secretory (ES) antigens in soluble hamster fecal extracts by an enzyme-linked immunosorbent assay (ELISA). A rabbit polyclonal IgG antibody against Ancylostoma ceylanicum ES was used to capture hookworm coproantigens that were then detected using pooled, high-titer, infected hamster serum. The ELISA was capable of detecting ES proteins over a range of 10 ng/mL to 10 mug/mL when the antigens were diluted in buffer or uninfected fecal extract, and ES could be detected in infected hamster feces at dilutions up to 1:256. Examination of the kinetics of coproantigen production demonstrated that detectable amounts of ES were produced as early as four days after A. ceylanicum infection, whereas fecal eggs were not observed until day 17. Moreover, fecal ES levels correlated well with intestinal worm burden and could be detected in wet or dry stool samples stored for 14 days over a temperature range of -80 degrees C to 37 degrees C. The fecal ELISA was then adapted to analyze the excretion of AceES-2, a novel immunogenic ES protein recently cloned from A. ceylanicum cDNA. AceES-2 was found to be excreted in feces with kinetics similar to that of whole ES. Examination of individual hookworm antigens by this method will provide new insights into the molecular host-parasite interaction and may form the basis for future diagnostic methods.

Purification and molecular cloning of and immunization with Ancylostoma ceylanicum excretory-secretory protein 2, an immunoreactive protein produced by adult hookworms.

Hookworms remain major agents of global morbidity, and vaccination against these bloodfeeding parasites may be an attractive complement to conventional control methods. Here we describe the cloning of Ancylostoma ceylanicum excretory-secretory protein 2 (AceES-2), a novel immunoreactive protein produced by adult worms. Native AceES-2 was purified from excretory-secretory (ES) products by reverse-phase high-pressure liquid chromatography, subjected to amino-terminal sequencing, and cloned from adult worm RNA by using reverse transcription-PCR. The translated AceES-2 cDNA predicts that the mature protein consists of 102 amino acids and has a molecular mass of 11.66 kDa. Western immunoblot and enzyme-linked immunosorbent assay analyses demonstrated that recombinant AceES-2 (rAceES-2) reacted strongly with antibodies from A. ceylanicum-infected hamsters. Immunization of hamsters with native ES products adsorbed to alum induced antibodies that recognized rAceES-2, while rAceES-2-alum vaccination resulted in antibodies that reacted with a single protein band in ES products that closely approximated the size predicted for the native molecule. Infected hamsters that were passively immunized with hyperimmune rabbit anti-rAceES-2 serum exhibited more rapid and complete recovery from anemia than controls that received normal serum. Oral immunization with rAceES-2 was associated with significantly reduced anemia upon challenge, an outcome similar to the outcome observed in hamsters that were orally vaccinated with soluble hookworm extract (the latter animals were also resistant to weight loss). These data suggest that AceES-2 plays an important role in the host-parasite interaction and that vaccination against this protein may represent a useful strategy for controlling hookworm anemia.

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.

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.

Molecular characterization of Ancylostoma inhibitors of coagulation factor Xa. Hookworm anticoagulant activity in vitro predicts parasite bloodfeeding in vivo.

Bloodfeeding hookworms, which currently infect over a billion people in the developing world, are a leading cause of gastrointestinal hemorrhage and iron deficiency anemia. The major anticoagulant inhibitor of coagulation factor Xa has been identified from the hookworm parasite Ancylostoma ceylanicum using reverse transcription PCR and 3'-rapid amplification of cDNA ends. This is the first anticoagulant cloned from a hookworm species for which humans are recognized permissive hosts. Despite approximately 50% amino acid similarity, A. ceylanicum anticoagulant peptide 1 (AceAP1) is both immunologically and mechanistically distinct from AcAP5, its homologue isolated from the dog hookworm Ancylostoma caninum. Studies using plasma clotting times and single stage chromogenic assays of factor Xa activity have demonstrated that the recombinant AceAP1 protein is substantially less potent than AcAP5 and that soluble whole worm protein extracts of adult A. ceylanicum possess less anticoagulant activity than extracts of A. caninum. These values correlate with previously reported differences in bloodfeeding capabilities between these two species of hookworm, suggesting that factor Xa inhibitory activity is predictive of hookworm bloodfeeding capabilities in vivo. These fundamental differences in the mechanism of action and immunoreactivity of the major anticoagulant virulence factors from related Ancylostoma hookworm species may have significant implications for human vaccine development.