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PubMed2003

Hookworms of feral cats in Florida.

Thirty feral cats (Felis catus) from Alachua county (northern Florida) and 30 from Palm Beach county (southern Florida) were examined for hookworms. Two species, Ancylostoma tubaeforme and Ancylostoma braziliense, were identified. Forty-five cats (75%) were infected with A. tubaeforme, with a mean intensity of 48 hookworms per cat. Twenty cats (33%) were infected with A. braziliense, with a mean intensity of 28 worms per cat. The prevalence of A. tubaeforme was greater than that of A. braziliense in Alachua (P=0.002) and Palm Beach (P=0.004) counties. The intensity of A. tubaeforme infections was higher in Palm Beach county than Alachua county (P=0.013). The intensities of A. tubaeforme and A. braziliense were positively correlated (increased together) in Palm Beach county (P=0.011). These hookworms have also been identified in bobcats (Felis rufus), gray foxes (Urocyon cinereoargenteus), and red foxes (Vulpes vulpes) in Florida. The prevalence of A. tubaeforme was significantly greater in feral cats than those reported in bobcats (P<0.001). The prevalence of A. braziliense was significantly greater in feral cats than in those reported in gray foxes (P=0.008). The hookworm that infects Florida panthers and bobcats, A. pluridentatum, was not found.

Ancylostoma↗

PubMed1993

Reinfection with hookworm after chemotherapy in Papua New Guinea.

Reinfection with hookworm (Necator americanus) following chemotherapy was studied over 2 years in a rural village in Madang Province, Papua New Guinea. The prevalence of hookworm infection had returned to pre-treatment levels after 2 years, and the geometric mean hookworm burden had returned to 58% of the pre-treatment value. The rate of acquisition of adult worms was independent of host age, and was estimated as a geometric mean of 2.9-3.3 worms/host/year (arithmetic mean 7.9-8.9 worms/host/year). There was significant predisposition to hookworm infection; the strength of this predisposition did not vary significantly between age or sex classes.

Age Factors↗

PubMed2003

Hookworm cathepsin D aspartic proteases: contributing roles in the host-specific degradation of serum proteins and skin macromolecules.

Cathepsin D aspartic proteases of hookworms were recently implicated in the host-specific digestion of haemoglobin by adult parasites. Ac-APR-1 from the dog hookworm, Ancylostoma caninum and Na-APR-1 from the human hookworm, Necator americanus, were shown to be expressed in the infective larval stage (L3) as well as adult worms. We now show that both proteases degraded skin macromolecules and serum proteins, some of which were cleaved more readily from permissive definitive hosts as opposed to non-permissive hosts. Na-APR-1 degraded human collagens more efficiently than did Ac-APR-1, and Ac-APR-1 degraded canine serum albumin more efficiently than did Na-APR-1. On the other hand, both enzymes degraded human serum proteins (albumin and fibrinogen) with approximately equal efficiency under the conditions of our assays in vitro. Molecular models of these 2 orthologous, aspartic proteases showed that, despite having active site clefts with identical primary sequences, residues in the S3 pocket adopted different conformations, likely accounting for different substrate preferences reported previously. Antisera raised to both proteases partially inhibited (16-26%) migration of hookworm L3 through hamster skin in vitro, further implying a connective tissue invasive role for these enzymes in addition to digestion of serum and erythrocyte proteins for nutrition.

Ancylostoma↗

PubMed1980

Homology between roundworm (Ascaris) and hookworm (N. americanus) antigens detected by human IgE antibodies.

Utilizing the techniques of radioallergosorbent test (RAST), inhibition of RAST and isoelectric focusing on polyacrylamide gels, antigenic cross-reactivity has been shown between extracts of Ascaris lumbricoides. Ascaris suum and Necator americanus (hookworm). Antigenic homology is extensive between the two Ascaris preparations. The antigens common to hookworm and Ascaris, while representing a minor component of Ascaris body fluid, are nonetheless potent stimulators of IgE antibody production in subjects infected only with hookworm larvae. These shared antigens appear to have a pI of less than 5.1. The hookworm specific antigens also have a pI of less than 5.1, whereas those specific to Ascaris have pI values in the ranges of 5.0 to 6.0 and 7.0 to 8.6. These latter antigens, i.e. those with pI between 7.0 and 8.6, apparently elicit an immune response only during an active infection with Ascaris.

Antigens↗

PubMed1999

A hookworm allergen which strongly resembles calreticulin.

Immmoglobulin E-rich plasma from patients from Papua New Guinea infected with Necator americanus has been used to probe an adult N. americanus cDNA library for the presence of hookworm allergens. Using this approach, one hookworm allergen has been identified as calreticulin, which was subsequently expressed in Escherichia coli. Little serological cross reactivity was seen between the recombinant calreticulins of this hookworm and its host. Prospective roles for hookworm calreticulin in the host-parasite relationship are discussed in depth.

Allergens↗

PubMed2000

The human hookworm pathogen Necator americanus induces apoptosis in T lymphocytes.

In the present study, we demonstrate that Jurkat T cells undergo apoptosis when cocultured with the human hook-worm Necator americanus. Pro-apoptotic activity was dose-dependent and readily detectable in hookworm secretions. This pro-apoptotic effect appears to be specific to cells of T lineage since the monocytic cell line, THP-1, the erythroleukaemic cell line, K562, and the basophil cell line, KU812, were unaffected. The induction of apoptosis in Jurkat T cells by the hookworm secretions did not involve cell activation or the Fas/Fas ligand interaction. In addition, the pro-apoptotic effect of the hookworm, or its secretions, was observed with activated human T cells but not with resting peripheral blood lymphocytes. These findings support the hypothesis that the hookworms' ability to recurrently infect humans is due to the parasite creating a site of 'immune privilege' around itself. This strategy promptly induces any reactive host leucocytes infiltrating the site of parasite colonization to undergo apoptosis, which reduces inflammation and renders the infection relatively asymptomatic.

Animals↗

PubMed2001

Vaccination with neutrophil inhibitory factor reduces the fecundity of the hookworm Ancylostoma ceylanicum.

Neutrophil inhibitory factor (NIF), a protein isolated from hookworms of the genus Ancylostoma, inhibits CD11b/18-dependent leucocyte function, binding to the I domain of CD11b. Historically, NIF was serendipitously isolated from whole worm extracts during a search for novel antihaemostatic agents, and little is known of its source or biological significance to the parasite. NIF has also been identified as a possible hookworm vaccine candidate. Ancylostoma ceylanicum recombinant NIF, expressed in its active form in Pichia pastoris, was purified and its functional activity confirmed using neutrophil adhesion assays and confirmatory immunoassay. Recombinant NIF was subsequently used in vaccination trials in the A. ceylanicum-hamster model system for human hookworm infection. Vaccinated and challenged animals were not protected in terms of worm burden or haematocrit values, despite the presence of high levels of specific antibody against NIF. However, adult worms resident in vaccinated animals showed a significant reduction in fecundity (85.8% by day 21 postinfection), indicating a degree of protection against subsequent transmission by vaccination. These data indicate that targeted vaccination with recombinant subunit material, derived from a known and effective immune suppressant secreted by the parasite, may offer partial protection against the transmission of hookworm infection. Furthermore, we can also report that a biological activity characteristic of NIF is detectable in the secretions of A. ceylanicum using two complementary bioassays. Complete neutralization of this secreted activity by vaccination in combination with other vaccine candidates may result in improved protection against A. ceylanicum infection.

Ancylostoma↗

PubMed1996

Cloning and characterization of Ancylostoma-secreted protein. A novel protein associated with the transition to parasitism by infective hookworm larvae.

The developmentally arrested third stage infective larva of hookworms resumes development upon entry into the definitive host. This transition to parasitism can be modeled in vitro by stimulating infective larvae with a low molecular weight ultrafiltrate of host serum together with methylated glutathione analogues. When stimulated to resume development in vitro, activated larvae of the hookworm Ancylostoma caninum released a 42-kDa protein, termed Ancylostoma-secreted protein (ASP). ASP was the major protein released by activated hookworm larvae. Degenerate oligonucleotide primers, based on a partial internal amino acid sequence of the protein, were used together with flanking vector sequence primers to amplify a fragment from a third stage larval cDNA library by polymerase chain reaction. The fragment was used as a probe to isolate a longer clone from the larval cDNA library. The full-length ASP cDNA was found to encode a 424-amino acid protein with homology to the antigen 5/antigen 3 family of proteins from hymenopteran venoms and a family of cysteine-rich secretory proteins. ASP was expressed in bacterial cells, and a polyclonal antiserum against purified recombinant ASP was produced. The antiserum, which was demonstrated to be specific for ASP, was used as a probe to measure the kinetics of ASP release by hookworm larvae. ASP is released within 30 min of stimulation, with the majority released by 4 h. Low levels of ASP were released continuously following activation, but only if the stimuli were present in the incubation medium. The compound 4,7-phenanthroline, previously shown to inhibit larval activation, also inhibited release of ASP. The specific, rapid release of ASP by activated infective larvae suggests that this molecule occupies a critical and central role in the transition from the external environment to parasitism.

Amino Acid Sequence↗

PubMed2000

A broad spectrum Kunitz type serine protease inhibitor secreted by the hookworm Ancylostoma ceylanicum.

Although blood-feeding hookworms infect over a billion people worldwide, little is known about the molecular mechanisms through which these parasitic nematodes cause gastrointestinal hemorrhage and iron deficiency anemia. A cDNA corresponding to a secreted Kunitz type serine protease inhibitor has been cloned from adult Ancylostoma ceylanicum hookworm RNA. The translated sequence of the A. ceylanicum Kunitz type inhibitor 1 (AceKI-1) cDNA predicts a 16-amino acid secretory signal sequence, followed by a 68-amino acid mature protein with a molecular mass of 7889 daltons. Recombinant protein (rAceKI-1) was purified from induced lysates of Escherichia coli transformed with the rAceKI-1/pET 28a plasmid, and in vitro studies demonstrate that rAceKI-1 is a tight binding inhibitor of the serine proteases chymotrypsin, pancreatic elastase, neutrophil elastase, and trypsin. AceKI-1 inhibitory activity is present in soluble protein extracts and excretory/secretory products of adult hookworms but not the infective third stage larvae. The native AceKI-1 inhibitor has been purified to homogeneity from soluble extracts of adult A. ceylanicum using size exclusion and reverse-phase high pressure liquid chromatography. As a potent inhibitor of mammalian intestinal proteases, AceKI-1 may play a role in parasite survival and the pathogenesis of hookworm anemia.

Amino Acid Sequence↗

PubMed1999

The hookworm platelet inhibitor: functional blockade of integrins GPIIb/IIIa (alphaIIbbeta3) and GPIa/IIa (alpha2beta1) inhibits platelet aggregation and adhesion in vitro.

Hookworms, aggressive, blood-feeding, intestinal nematodes, are currently a leading cause of iron deficiency anemia in the developing world. An inhibitor of platelet aggregation and adhesion has been partially purified and characterized from soluble protein extracts of adult Ancylostoma caninum hookworms. This protein, named the hookworm platelet inhibitor, has an estimated molecular mass of 15 kDa as determined by size-exclusion chromatography. In addition to blocking platelet aggregation in response to a variety of agonists, the partially purified inhibitor also prevents adhesion of resting platelets to immobilized fibrinogen and collagen. Inhibitory monoclonal antibodies were used to identify specific blockade of cell surface integrins GPIIb/IIIa (alphaIIbbeta3) and GPIa/IIa (alpha2beta1), the platelet receptors for fibrinogen and collagen, respectively. This broad-spectrum anti-platelet activity is also present in excretory and secretory products of adult worms, suggesting a biologic role for the hookworm platelet inhibitor in vivo.

Ancylostoma↗

PubMed2005

Crystallization and preliminary X-ray analysis of Na-ASP-1, a multi-domain pathogenesis-related-1 protein from the human hookworm parasite Necator americanus.

Human hookworm infection is a major cause of anemia and malnutrition in the developing world. In an effort to control hookworm infection, the Human Hookworm Vaccine Initiative has identified candidate vaccine antigens from the infective larval stage (L3) of the parasite, including a family of pathogenesis-related-1 (PR-1) proteins known as the ancylostoma-secreted proteins (ASPs). The functions of the ASPs are unknown. In addition, it is unclear why some ASPs have one while others have multiple PR-1 domains. There are no known structures of a multi-domain ASP and in an effort to remedy this situation, recombinant Na-ASP-1 has been expressed, purified and crystallized. Na-ASP-1 is a 406-amino-acid multi-domain ASP from the prevalent human hookworm parasite Necator americanus. Useful X-ray data to 2.2 A have been collected from a crystal that belongs to the monoclinic space group P2(1) with unit-cell parameters a = 67.7, b = 74.27, c = 84.60 A, beta = 112.12 degrees. An initial molecular-replacement solution has been obtained with one monomer in the asymmetric unit.

Animals↗

PubMed2005

The assessment of hookworm calreticulin as a potential vaccine for necatoriasis.

A vaccine against the human hookworm Necator americanus is urgently required to reduce hookworm-induced morbidity in endemic areas. In the present study, recombinant hookworm calreticulin, a nominated vaccine candidate, has been tested in mice. Mice given calreticulin had 43-49% fewer worms in their lungs, compared to non-vaccinated controls, following challenge infection with infective hookworm larvae. These levels of protection were achieved in the absence of adjuvant following intraperitoneal administration of three doses of 15 microg antigen. Antigen was also encapsulated in PLG microparticles. Encapsulated calreticulin elicited higher levels of anti-calreticulin IgG1 than free antigen but failed to induce protective immunity. The protection induced by free calreticulin was associated with low levels of serum IgE and moderate lung eosinophilia whilst administration of calreticulin-loaded microparticles was associated with high levels of serum IgE and higher lung eosinophil activity, suggesting that the classical Th2 phenotype may not always be associated with protective immunity, albeit in experimental necatoriasis.

Animals↗

PubMed1990

Metalloproteases of infective Ancylostoma hookworm larvae and their possible functions in tissue invasion and ecdysis.

To infect their hosts, hookworm larvae must exsheath and migrate through connective tissue. A modified in vitro skin chamber was used to show that the human hookworm Ancylostoma duodenale and the zoonotic canine hookworm Ancylostoma caninum penetrate epidermis, basement membrane, and dermis in similar ways. These similarities in tissue invasion properties reflect the observed biochemical similarities in parasite protease composition. The larvae of both species contain protease activity that is inhibited by o-phenanthroline; this identifies the proteases as metalloproteases. The enzyme activities exhibit an alkaline pH optimum between pH 9 and 10. During modified sodium dodecyl sulfate-polyacrylamide gel electrophoresis in which a protein substrate (either casein or gelatin) was used, the protease activities resolved into a major band at an Mr of 68,000 and a minor band at an Mr of 38,000. Proteases were released by living A. caninum larvae in vitro and degraded purified and radiolabeled casein to smaller peptides. Motile hookworm larvae were also incubated with purified and radiolabeled connective tissue macromolecules in vitro. Both Ancylostoma species degraded human fibronectin to a 60,000-Mr polypeptide intermediate, but could not degrade solubilized bovine elastin or human laminin. In contrast, the obligate skin-penetrating nematode Strongyloides stercoralis degraded all three substrates. This biochemical difference may explain some observed differences in invasiveness.

Ancylostoma↗

PubMed2006

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.

Ancylostoma↗

PubMed1983

Circulating antibodies against corneal epithelium and hookworm in patients with Mooren's ulcer from Sierra Leone.

A relationship between the occurrence of Mooren's ulcer and hookworm infections was suggested by Kuriakose in 1963. Sixteen patients with clinical diagnosis of Mooren's ulcer and 15 local controls from Sierra Leone were tested with respect to serum immunoglobulin levels, circulating antibody to hookworm, circulating antibodies to corneal epithelium, stool smears, and eosinophil and lymphocyte levels. Both patients and healthy controls had circulating antibodies to corneal epithelium and to hookworm. In the controls the titres of hookworm antibodies were significantly lower than in the patients, though in both groups most people had intestinal parasite infestations as detected by the stool smear. Further investigation failed to demonstrate any other significant immune alteration in the patients as compared with local controls.

Adolescent↗

PubMed1997

Failure of mebendazole in treatment of human hookworm infections in the southern region of Mali.

Preliminary studies indicated that single-dose (500 mg) mebendazole gave disappointing results in the treatment of hookworm infections (Necator americanus) in Mali. A placebo-controlled, randomized trial conducted with the participation of 103 infected subjects (background hookworm prevalence > 50%) confirmed that mebendazole (Vermox) did not reduce parasite burdens significantly, as assessed through fecal egg counts. In contrast, a group of subjects treated with pyrantel (Combantrin) experienced a significant reduction in fecal worm egg counts (overall, both sexes combined showed a 75% reduction). Male subjects carried significantly more intense infections compared with females, but there was no gender difference in response to treatment. A standard egg hatch assay showed that N. americanus from our subjects in Mali was more resistant to benzimidazoles compared with a laboratory-maintained strain that had not been exposed to anthelmintics in more than 100 generations (50% effective dose = 0.12 and 0.07 microg/ml of thiabendazole, respectively), suggesting that, among other possibilities, the development of resistance to the benzimidazoles by N. americanus may have contributed to the drug failure. Whatever the underlying explanation, our results indicate that single-dose treatment with mebendazole is an ineffective treatment for hookworm infections and despite its relatively cheap cost and wide availability, mebendazole should not be considered a drug of choice in the mass treatment of hookworm infections in this region of Mali.

Adolescent↗

PubMed1999

Epidemiology of human ancylostomiasis among rural villagers in Nanlin County (Zhongzhou village), Anhui Province, China: age-associated prevalence, intensity and hookworm species identification.

Hookworm infection has traditionally been highly endemic to Anhui Province, China. Zhongzhou village in southwestern Anhui was identified as an endemic focus of infection caused by the hookworm Ancylostoma duodenale. This species was found to predominate over Necator americanus in ratios ranging from 35:1 based on the recovery of third-stage infective larvae (L,) to 21:1 based on the recovery of adult hookworms after anthelmintic chemotherapy. The overall prevalence of Ancylostoma infection in Zhongzhou was 33.2% with a greater prevalence among males. Unlike the age-associated prevalence patterns for N. americanus in Hainan and other southern Chinese provinces that show increasing prevalence with age and the highest prevalence among the elderly, the age-associated prevalence for A. duodenale in Zhongzhou exhibited a peak in middle-aged adults with subsequent decline. The age-associated intensity pattern exhibited a similar trend although the most of the hookworm infections were light or moderate infections as defined by quantitative egg counts.

Adolescent↗

PubMed2000

Successful eradication of Ascaris lumbricoides and hookworm infection after three repeated doses of albendazole.

Three repeated orally doses of albendazole 400 mg in 6 weekly intervals were evaluated in Thai hill-tribe students who had at least one kind of soil-transmitted helminths (i.e. Ascaris lumbricoides, hookworm and Trichuris trichiura). Stool examination and parasite egg count were performed using Beaver's standard direct smear method and Kato-Katz's cellophane thick smear method prior to treatment and then 1 month after the first, second and third dose of drug administrations. A single dose of albendazole was very effective against A. lumbricoides and hookworm infections, with cure rates of 98.68 per cent and 92.16 per cent, respectively. The second and third dosages eradicated A. lumbricoides and hookworm infections, respectively. Conversely, the first to third cure rates for T. trichiura infection were relatively low, being 37.76-58.16 per cent. Three repeated doses of albendazole proved to be beneficial in eradication of A. lumbricoides and hookworm infections, and decreased the prevalence of T. trichiura infected cases. For eradication of T. trichiura infection, further regimen and period of drug administration is required.

Albendazole↗