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This study investigated the co-infection dynamics of Schistosoma mansoni and Ancylostoma ceylanicum in an experimental model. Two experiments were conducted to investigate varying infection orders. In experimental group 1, subjects were initially infected with A. ceylanicum , followed by S. mansoni infection. In group 2, subjects were first infected with S. mansoni , followed by A. ceylanicum infection. The co-infected groups were compared to mono-infected groups for further analysis. Parameters assessed included weight, fecal egg elimination, blood cell counts, IgG response, histopathological analysis, and granuloma morphometry. Results indicated that the timing of infections influenced weight loss. Co-infected animals lost weight similarly to A. ceylanicum -infected animals when A. ceylanicum preceded S. mansoni. The co-infected groups did not experience worsened anemia, despite both parasites being hematophagous. No linear correlation was found between S. mansoni egg counts and anti-SEA IgG production. The response to the S. mansoni adult worm antigen was significantly higher in the S. mansoni mono-infected group compared to the group previously infected with hookworm. Co-infection with A. ceylanicum after S. mansoni infection also interfered with the host response. It caused a delay in the peak of S. mansoni egg elimination compared to the S. mansoni monoinfected group. Additionally, fewer eggs were retained in the liver compared to single infections. These findings help elucidate the interactions between different parasites and their hosts in the same endemic area, providing valuable information to enhance strategies for disease control and management.

Hookworms are bloodfeeding intestinal nematodes that are a major cause of anemia in resource-limited countries. Despite repeated exposure beginning in early childhood, humans retain lifelong susceptibility to infection without evidence of sterilizing immunity. In contrast, experimental infection of laboratory animals is typically characterized by varying degrees of resistance following primary infection, although the mechanisms underlying this phenomenon remain unknown. In this study, hamsters subjected to a single drug-terminated infection with 100 third stage hookworm larvae were confirmed to be resistant to pathological effects following a subsequent challenge. In a second experiment, hamsters infected twice-weekly with 10 third stage larvae (low inoculum) exhibited clinical and parasitological evidence of continued susceptibility, while those given 100 L3 (high inoculum) developed apparent resistance within 3 days following the initial exposure. The kinetics of parasite-specific IgA, IgM, and IgG antibody production varied by group, which suggests that the humoral immune response to hookworm infection is stimulated by the nature (frequency and intensity) of larval exposure. These results suggest that intermittent low-inoculum larval exposure, which is characterized by prolonged susceptibility to infection, may serve as a more representative model of human hookworm disease for studies of pathogenesis, as well as drug and vaccine development., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Hookworm infection is associated with anaemia and malnutrition in many resource-limited countries. Ancylostoma hookworms have previously been shown to modulate host cellular immune responses through multiple mechanisms, including reduced mitogen-mediated lymphocyte proliferation, impaired antigen presentation/processing, and relative reductions in CD4(+) T cells in the spleen and mesenteric lymph nodes. Syrian hamsters were depleted of CD4(+) for up to 9 days following intraperitoneal injection (200 microg) of a murine anti-mouse CD4 monoclonal IgG (clone GK1.5). CD4(+) T-cell-depleted hamsters infected with the hookworm Ancylostoma ceylanicum exhibited a threefold higher mean intestinal worm burden and more severe anaemia than animals that received isotype control IgG. In addition, depletion of CD4(+) T cells was associated with impaired cellular and humoral (serum and mucosal) immune responses to hookworm antigens. These data demonstrate an effector role for CD4(+) T cells in hookworm immunity and disease pathogenesis. Ultimately, these studies may yield important insights into the relationship between intestinal nematode infections and diseases that are associated with CD4(+) T-cell depletion, including HIV.

Exposure of encysted metacercariae of Clinostomum complanatum to UV light (254 nm) for 60 min reduced their development into adult worms in buff-backed herons (95.7% reduction in worm burden). Metacercariae that succeeded in developing into adult worms, showed low fecundity levels. Furthermore, 30% of eggs laid showed abnormal shape; however, all normal and abnormal eggs failed to hatch into miracidia. The effectiveness of UV-irradiated metacercariae as a vaccine was investigated. Compared to control unvaccinated herons, the vaccinated group showed a significantly high protection rate (73.8%) against challenge. In vitro, worm development after challenge showed decreased fecundity and increased egg abnormalities, where only 1.5% of all eggs produced hatched into miracidia. A passive haemagglutination test revealed increased antibody titres against soluble adult worm antigen in both vaccinated and vaccinated-challenged birds. It was concluded that vaccination of herons using encysted metacercariae UV-irradiated for 60 min can protect them against challenge infection.

Hookworms are bloodfeeding nematodes that reside in the intestinal mucosa. These parasites secrete proteins that induce robust systemic immune responses in humans and experimental animals. By contrast, mucosal immune responses in and around the site of attachment are not described as well. This paper presents data from studies aimed at examining hookworm-specific mucosal antibody responses in a hamster model of Ancylostoma ceylanicum infection. Intestinal flush prepared from infected hamsters was analysed by ELISA and shown to be enriched in IgA-specific for A. ceylanicum excretory-secretory (ES) products. Evaluation of mucosal IgA responses by immunoblot demonstrated that infected hamsters recognized a broad range of ES proteins. Hamsters repeatedly exposed to drug-terminated infections were shown to have enhanced serum IgG and mucosal IgA responses, as well as a high level of protection from challenge infection. Parasite-specific IgA was also detected in the faeces of hamsters undergoing a primary infection, and increasing faecal IgA responses were coincident with significant reductions in intestinal worm burdens and faecal ES output over time. Together these results suggest that secretory IgA may act in concert with other components of the mucosal and systemic immune response to promote protective immunity against hookworm infection and/or disease.

Background: The effect of concurrent (active) and treated hookworm infections on the immunogenicity of vaccination with the recombinant fusion protein Ay-Ancylostoma-secreted protein 2 was analyzed in the Golden Syrian hamster., Methods: Hamsters were infected with the hookworm Ancylostoma ceylanicum and vaccinated with the recombinant protein, with Quil A used as adjuvant. As controls, hookworm-infected hamsters were treated with the anthelmintic drug pyrantel pamoate before vaccination. Naive hamsters (i.e., those with neither previous hookworm infections nor treatment) were also vaccinated., Results: The proliferation capacities of carboxyfluorescein diacetate succinimidyl ester-positive lymphocytes from the hookworm-infected vaccinated group were reduced by 50% relative to the capacities of lymphocytes from uninfected or treated vaccinated hamsters; capacities were comparable to the rates observed in lymphocytes from the hamsters vaccinated with the adjuvant alone. Immunoglobulin G1 antibody responses were also reduced in the actively infected, untreated hamsters, and interferon- gamma and interleukin-4 cytokine mRNAs were down-regulated. Conversely, interleukin-10 and tumor necrosis factor- alpha mRNAs were up-regulated in those hamsters., Conclusions: These results suggest that hookworm infections have an immunomodulatory effect by impairing the immune response to an exogenous antigen during infection. The hookworm-associated immunodepression may have important implications for design of clinical trials of human vaccines and vaccination strategies.

The Syrian Golden hamster (Mesocricetus auratus) has been used to model infections with the hookworm Ancylostoma ceylanicum. New molecular immunological reagents to measure cellular immune responses in hamsters were developed and used to determine the impact of A. ceylanicum hookworm infection on host cytokine responses and lymphoproliferation. Initial larval infection with 100 third-stage A. ceylanicum larvae resulted in predominant Th1 responses (upregulation of proinflammatory cytokines) that lasted for the duration of larval migration and continued up to 14 days postinfection (prepatency). Subsequently, development of larvae into egg-laying adult hookworms (patency) coincided with a switch to Th2 predominant responses (interleukin-4 [IL-4]) as well as a marked increase in IL-10 production. This switch also concurred with reduced host lymphoproliferative responses to hookworm antigens. The findings demonstrate a similarity in immune responses between hamsters and humans infected with hookworms, suggesting that hamsters will be a useful animal model species for examining host immunity to human hookworm infections.

The serum anti-Ancylostoma duodenale immunoglobulin (Ig) G4 antibody response to fraction III of the partially purified excretory secretory antigen of adult worm (Ad III ESA) was studied. The work included 60 patients with A. duodenale infection (GI), 40 patients with other parasitic infections (GII) and 30 apparently healthy parasite-free controls (GIII). Level of serum specific IgG4 was measured by indirect enzyme linked immunosorbent assay and compared with serum specific IgG, IgG 1, 2 & 3 subclass antibodies. Patients of GI had gastro-intestinal manifestations and symptoms suggestive of anaemia, and by investigations they had anaemia in 31.7% & eosinophilia in 100%. Measuring the intensity of A. duodenale infection, quantified as fecal egg counts, in patients of GI revealed that 60%, 30% & 10% had light, moderate, and heavy infections, respectively. The serum anti-Ad III ESA IgG & IgG 1-4 subclass antibodies were significantly elevated (P < 0.001) in patients of GI compared with GIII. Serum specific IgG4 was expressed in 100% of patients of GI at a significantly highly elevated level than IgGI (P < 0.01), IgG2 & IgG3 (P < 0.001). Specific IgG1 was expressed in 88.3% of patients of GI at a significantly elevated level (P < 0.001) than IgG2 & IgG3 which were expressed in 31.7% & 38.3%, respectively and elevated to a moderate extent. Serum specific IgG4 showed a 1.0, 1.1, 3.1 & 2.6-fold increase in detection rate of positive cases than IgG, IgG1, IgG2 & IgG3, respectively. The highest ability to differentiate between infected and healthy subjects was by serum specific IgG4 recording a discrimination coefficient of 9.4, while IgG, IgG1, IgG2 & IgG3 recorded 5.2, 6.3, 3.2 & 3.4, respectively. Serum specific IgG4 showed a significant positive correlation (r = 0.41, P < 0.001) with the intensity of A. duodenale infection, as was demonstrated by IgG & IgG3 (P < 0.01 & P < 0.05, respectively). Detection of serum anti-Ad III ESA IgG4 antibody recorded a 100% sensitivity that was significantly higher (P < 0.001) than IgG1, IgG2 & IgG3, but insignificantly different (P > 0.05) from IgG. Finally, serum specific IgG4 recorded a 100% specificity that was significantly higher than IgG, IgG2, IgG3 (P < 0.01) & IgG1 (P < 0.05). They showed cross-reactions with ascariasis, lymphatic filariasis and strongyloidiasis. The results are discussed.

Anti-hookworm antibody serologic responses were measured in residents of an Anhui provincial Chinese village where Ancylostoma duodenale is the predominant hookworm. Antibody responses were measured against either soluble infective third-stage larval (L3) or adult antigens. Immunoglobulins of the IgG class, especially IgG4 correlated with both the prevalence and intensity of A. duodenale hookworm infections. In contrast, there was an inverse correlation with IgM, but no correlation with IgA or IgE. Circulating IgG4 antibody responses might serve as a surrogate marker for active A. duodenale hookworm infection.

Vaccination of mice with either third-stage Ancylostoma caninum infective hookworm larvae (L3) or alum-precipitated recombinant Ancylostoma secreted protein 1 from A. caninum (Ac-ASP-1) results in protection against hookworm challenge infections. Vaccine protection is manifested by reductions in lung hookworm burdens at 48 h postchallenge. Mice actively immunized 4 times with Ac-ASP-1 also exhibited reductions in hookworm burden in the muscles. Hookworm burden reductions from Ac-ASP-1 immunization were associated with elevations in all immunoglobulin subclasses, with the greatest rise observed in host IgG1 and IgG2b. The addition of a fourth immunization resulted in even higher levels of IgG and IgE. In contrast, L3-vaccinated mice exhibited marked elevations in IgG1 and IgM, including anti-Ac-ASP-1 IgM antibody. Passive immunization with pooled sera from recombinant Ac-ASP-1-vaccinated mice also resulted in lung hookworm burden reductions. It is hypothesized that recombinant Ac-ASP-1 vaccinations elicit antibody that interferes with parasite larval migration.

Correlation between the degree of protection and induced serum antibody response in hamsters immunized with somatic and excretory-secretory (ES) antigens of adult. A. ceylanicum was investigated. Hamsters were immunized with non purified and Sephadex G-200 fractions (F1, F2 and F3) of somatic and ES antigens. The degree of protection was assessed in terms of percent worm reduction compared to controls against challenge infection. Induced humoral antibody response was determined by ELISA. Both somatic and ES antigens had shown moderate to significant protection but the latter was found more immunogenic as highest level of protection (67.02%) was achieved by these antigens. Humoral antibody was found highly elevated in animals immunized with protective doses of somatic and ES antigens. The maximum serum antibody titer i.e. 1:3200 was noticed in animals immunized with high protective dose (64.59% protection) of fraction F1 of ES antigens. Antibody titer correspond to the degree of protection and a positive correlation between induced humoral antibodies and protection level was established.

Most patients with proven or suspected enteric infection with the common hookworm of dogs, Ancylostoma caninum, produce immunoglobulin G (IgG) and IgE antibodies to an immunodominant excretory/secretory antigen (Ac68) of the parasite. These antibodies were detected in both enzyme-linked immunosorbent assay (ELISA) and Western blots; the Western blot to detect IgG antibodies to Ac68 was the most specific and sensitive. The subclasses of IgG of the antibody response to the parasite were analyzed using Western blots with anti-IgG subclass-specific monoclonal antibodies as marker systems in an attempt to further improve the specificity of the assay. Eight patients with confirmed enteric infections with A. caninum (positive controls) were tested; six had antibodies in all IgG subclasses against Ac68. Twenty sera from patients with suspected enteric infection with A. caninum (manifested as eosinophilic enteritis or unexplained abdominal pain with peripheral eosinophilia) were tested; 16 had total IgG antibodies to Ac68, while IgG1, IgG2, IgG3, and IgG4 responses were found in 11, 10, 9, and 12 of these sera, respectively. Small numbers of sera from groups of patients infected with other helminths and from healthy blood donors had various combinations of IgG, IgG1, IgG2, and IgG3 antibodies to Ac68, but none of these sera had IgG4 antibodies to Ac68. Sera from all nine patients with human hookworm infection had IgG, IgG1, IgG2, and IgG3 antibodies to Ac68 and eight of the nine were also positive for IgG4 antibodies. These results indicate the Western blot to detect IgG4 antibodies to Ac68 is the most reliable immunodiagnostic test yet described for enteric infection with A. caninum, although this test does not discriminate between infections with human and canine hookworms.

Human eosinophilic enteritis (EE) may result from hypersensitivity to the excretory/secretory (ES) antigens of adult Ancylostoma caninum. The origin of several antigens were identified by probing adult A. caninum with mouse monoclonal antibodies (MoAbs), sera from mice vaccinated with ES antigens and sera from human EE patients. Six MoAbs (AC/ES 1-6) were produced against ES antigens, two being IgG3 and four IgM. Western blots demonstrated four different antigen specificities: MoAb AC/ES 1 bound strongly to an ES product at about 30 kDa; AC/ES 2 recognized a broad band ranging from 50-200 kDa; AC/ES 3, AC/ES 5 and AC/ES 6 reacted at about 68 kDa, and AC/ES 4 at about 97 kDa. Sections of formalin-fixed, paraffin embedded adult A. caninum were then incubated with these MoAbs and immunostained by the peroxidase-anti-peroxidase (PAP) technique. The target epitope of MoAb AC/ES 1 was found mainly in the oesophageal, amphidial and excretory glands; AC/ES 2 reacted weakly with many structures in the sections; AC/ES 3, AC/ES 5 and AC/ES 6 were specific for excretory glands only, and AC/ES 4 bound to amphidial glands. Sera from immunized mice reacted with all three (especially the excretory) glands and the cuticle. In an indirect assay, worm sections probes with three human EE patient sera demonstrated maximal staining in the amphidial glands. Our findings confirm that ES products of A. caninum include immunogenic glandular secretions which may be involved in the pathogenesis of human EE.

The relation between the level of serum immunoglobulins (IgG, IgM, IgA and IgE) percentage of T-lymphocyte and egg counts was studied in dogs infected with Ancylostoma duodenale. The serum IgG levels were significantly high and a marked significance was noticed also in the levels of IgM and IgE. An inverse relationship between the concentration of IgG and egg count and a direct association between the percentage of T-lymphocyte and IgG level was proved.

To evaluate the role of canine hookworms in human eosinophilic enteritis (EE) in north-eastern Australia, we tested human sera in an enzyme-linked immunosorbent assay (ELISA) which incorporated antigens of adult Ancylostoma caninum. Sera from the following groups were examined: 10 patients with EE (unexplained recurrent abdominal pain and related symptoms, with peripheral eosinophilia) from Townsville and Brisbane; 2 persons known to be infected with A. caninum and 20 presumed unexposed healthy controls; 20 patients with other gastrointestinal diseases; 20 with other identified parasitic infections; and 20 with atopic conditions. High levels of specific immunoglobulin (Ig) G and IgE antibodies were found in patients with EE but not other gastrointestinal disease. Excretory-secretory (ES) products were more discriminating than somatic antigens in the ELISA and the IgG/ES-ELISA was the most specific; occasional cross-reactions could be explained on epidemiological or parasitological grounds. The IgM-ELISA was neither specific nor sensitive. We conclude that canine ancylostomiasis is a major cause of human EE in north-eastern Australia, and the pathogenesis is based probably on hypersensitivity to antigens secreted by the parasite.

Sera of patients with either hookworm infections or ascariasis, who are from areas nonendemic for Schistosoma mansoni, contain antibody reactivity against extracts of S. mansoni schistosomula. Such patients with hookworm, but not those with Ascaris lumbricoides, also express considerable antibody activity against intact schistosomula and schistosomular membrane preparations. Sera from patients with schistosomiasis mansoni also contain these activities. Because these three helminthic infections often occur in the same areas, and concomitantly in patients, anti-schistosomular reactivity should not be ascribed only to exposure to schistosomes. The demonstration and definition of these surface-specific cross-reactivities point out the problems of serodiagnosis, and the potential of interactive influences in the common occurrence of multiple helminthic infections in humans.

The immune response of hamsters to a chronic hookworm infection has been investigated. Ancylostoma ceylanicum caused long term infections in hamsters which were associated with prominent changes in secondary lymphoid organs. The mesenteric lymph nodes and spleens increased rapidly in size stabilizing at approximately 3-4 times the weight in control animals by weeks 3-7. Cells from both the mesenteric lymph node and spleen, after an initial period of increased blast cell activity, became less reactive in the latter stages of infection. Serum antibody responses were marked, commencing in weeks 3-4 and increasing in intensity throughout the 10 week period of measurement. The results are discussed in relation to their contribution to the understanding of human hookworm infection.