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The human hookworm Necator americanus appears to have evolved a number of complementary strategies to overcome the host's haemostatic processes. These include the inhibition of blood coagulation, platelet aggregation and mediator release, and the secretion of fibrinogenolytic enzymes. These strategies presumably allow the parasite to establish the chronic infections so often documented in human populations.
The proteolytic activities present in adult Necator americanus excretory-secretory products have been assessed using biologically relevant, naturally occurring substrates (haemoglobin and fibrinogen) and a number of synthetic fluorogenic and chromogenic substrates. One broad peak of activity was observed against haemoglobin in the pH range 5 to 7, with maximum activity at pH 6.6, while fibrinogenolytic activity was shown to be greater at pH 3.5. Inhibition studies against haemoglobin, fibrinogen and synthetic substrates using a battery of appropriate protease inhibitors indicated the presence of a mixture of aspartyl, cysteinyl and serine proteases. Metal ion (Ca2+, Zn2+ and Fe2+) stimulation was demonstrated, with stimulation by Zn2+ being the most marked. These results are discussed in the context of recent developments in the field of parasite proteolytic enzymes, where they have been suggested as targets for immuno- and chemotherapy.
The course of a primary Necator americanus infection was studied in the lungs and small intestines of syngeneic mice. Following percutaneous infection no difference in initial larval establishment in the lungs was found between male BALB/c, NIH or B10.G mice. However, significant differences in the subsequent kinetics of infection were demonstrated between the BALB/c and NIH strains. Lung worm burdens declined more slowly in NIH mice than in BALB/c strain. Surprisingly, however, a greater proportion of larvae remaining in the lungs of BALB/c mice, 9 days p.i., were trapped than in NIH mice. Nevertheless, establishment in the small intestines of the BALB/c strain was consistently greater than in NIH mice. Host immunosuppression resulted in increased larval retention in the lungs of both the BALB/c and NIH strains as well as in the small intestines of BALB/c mice. Treatment with hydrocortisone acetate did not increase intestinal worm burdens in NIH mice. The data presented suggest that, in this complex, dynamic model system, designation of 'susceptible' and 'resistant' strains is inappropriate. The factors underlying the observed strain differences in resistance to infection are discussed.
Laboratory bred DSN hamsters were exposed to varying doses of infective larvae of Ancylostoma ceylanicum (orally) or Necator americanus (percutaneously) and were autopsied at times which corresponded to a period immediately before cessation of growth of worms or soon afterwards. A total of 829 (404 male and 425 female) A. ceylanicum and 1582 (781 male and 801 female) N. americanus were measured. At worm burdens of fewer than 100, the length of A. ceylanicum appeared to increase with infection intensity and no evidence was found that growth was retarded under crowded conditions. In an experiment comparing directly low (mean worm burden = 22) and heavy infections (mean worm burden = 180) significant negative associations between both weight and width, and worm burden were detected, but length again increased with worm burden. In contrast, 5 experiments with N. americanus indicated negative relationships between measures of worm size (length, width, wet and dry weight) and worm burden. It was concluded that N. americanus is subject to regulation by density-dependent processes within the host while A. ceylanicum is not sensitive to the same degree.
Neonatal hamsters were exposed to varying doses of Necator americanus larvae and changes in the stability of the resulting worm burdens were monitored over a period of 25 weeks. No change in worm burdens was evident for the first 5 weeks of infection, irrespective of the infection intensity, but the more heavily infected groups subsequently lost worms in a density-dependent manner. Male and female hamsters lost comparable proportions of their established parasite burdens indicating that there was no host sex-linked difference in this respect. By week 15 infections had stabilized and the residual worm burdens, usually a maximum of 30 worms survived for a considerably longer period of time. Initially the percentage of male worms varied from 45% to 50% but as infection progressed male worms comprised a significantly increasing proportion of the total worm burden. By week 25 the percentage of male worms was usually in excess of 60%. The growth of infected animals was not severely affected by N. americanus, even when heavy worm burdens established initially, but a significant effect was detected particularly in week 5, prior to worm loss, when the adult worms would have been feeding on intestinal tissues and causing blood loss for a period of about 2 weeks. The most severe depression in the packed cell volume was also recorded in week 5, indicating that anaemia had been initiated in infected hamsters. Whilst, the regulation of parasite burdens in weeks 5-10 post-infection may have resulted from host immunity, the persistence of the residual worm burdens, the marked density-dependent anaemia and the subtle effect on host weight, all reflected well-documented aspects of chronic human necatoriasis.
In a combined field and laboratory study, the eggs and 3rd-stage infective larvae of the human hookworm Necator americanus were recovered from the creek waters of the Niger Delta, Nigeria. They were tested for viability and infectivity. Eggs recovered from fresh water creeks showed a viability or hatching index of between 17.5 and 23.7%. Eggs recovered from brackish water showed a viability index of 18.8-21.4% in 'normal' cultures, and a maximum index of 6.3% when cultured in brackish water. The differences in the viability indices of these eggs were not significant (P greater than 0.05). Infective 3rd-stage larvae were recovered from both fresh creek water (205-258 L3/litre of water), and brackish water (45 L3/litre of water). Larvae hatched from brackish water eggs that were cultured in the brackish water were 20% infective by 3 days, but lost all infective potential by 7 days post-hatching. Larvae from fresh water eggs, cultured in fresh water and 'normal' laboratory cultures reached 50% infectivity in 3-5 days, losing potential infectivity in 11-15 days post-hatching. The infectivity patterns of larvae recovered directly from fresh and brackish waters did not differ significantly (P greater than 0.05) from the infectivity patterns of larvae recovered from laboratory cultures of eggs from all the experimental sources. Although larvae recovered directly from fresh and brackish waters survived in these respective media under laboratory conditions, there was a significant difference (P less than 0.05) in their survival patterns. The survival and infectivity rates of field larvae were considerably reduced over time relative to control larvae.(ABSTRACT TRUNCATED AT 250 WORDS)
The blood and broncho-alveolar leucocyte (BAL) responses, to a primary Necator americanus infection, were studied in male BALB/c and NIH mice. Following percutaneous infection, a significant blood leucocytosis occurred in both the BALB/c and NIH strains. The peak response occurred, in both strains, on day 10 p.i. and this was reflected in the eosinophil response which peaked at the same time-point. The change in blood eosinophil numbers, as a result of infection, was found to be the greatest recorded for any cell type. In BALB/c mice, however, infection elicited a significantly more intense total leucocyte, lymphocyte and eosinophil response than in NIH mice. In contrast, the BAL response was more intense in the NIH strain. Peak BAL responses were observed between days 12 and 16 p.i., in both strains, and the change in broncho-alveolar eosinophil numbers, as a result of infection, was found to be the greatest recorded for any cell type. The relationship between the observed leucocyte responses and resistance to the migrating larvae of a primary infection is discussed.
Hookworms are gut-dwelling, blood-feeding nematodes that infect hundreds of millions of people, particularly in the tropics. As part of a program aiming to define novel drug targets and vaccine candidates for human parasitic nematodes, genes expressed in adults of the human hookworm Necator americanus were surveyed by the expressed sequence tag approach. In total 161 new hookworm genes were identified. For the majority of these, a function could be assigned by homology. The dataset includes proteases, protease inhibitors, a lipid binding protein, C-type lectins, an anti-bacterial factor, globins and other genes of interest from a drug or vaccine development viewpoint. Three different classes of small, secreted proteins were identified that may be involved in the host-parasite interaction, including potential potassium channel blocking peptides. One third of the genes were novel. These included highly expressed, secreted (glyco)proteins which may be part of the excretory-secretory products of these important pathogens. Of particular interest are a family of 9 genes with similarity to the immunomodulatory protein, neutrophil inhibitory factor, that may play a role in establishing an immunocompromised niche for this successful parasite.
Twenty-one to 58 individual Necator americanus were sampled from each of four villages in south-western China. Each nematode was sequenced for 588 bp of the mitochondrial cytochrome oxidase I gene. Allelic and nucleotide diversity varied two-fold among villages. Overall FST among populations was approximately 0.28, but this large value resulted from one low-diversity population that had a large genetic distance to the other three populations (F(ST) = 0.10 without that population). There was no correlation between geographical and genetic distance among sites. Thus, the genetic structure of this species in China may be characterized by variable effective sizes and uneven movement among sites. We discuss the implications of this genetic structure for vaccine development and the spread of drug resistance in human hookworms, and compare the genetic structure of hookworms with that of other nematodes.
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.
Calreticulin was recently identified as a hookworm (Necator americanus) allergen, implying secretion, and contact with cells of the immune system, or significant worm attrition in the tissues of the host. As human calreticulin has been shown to bind to and neutralize the haemolytic activity of the complement component C1q, and to be putatively involved in integrin-mediated intracellular signalling events in platelets, it was of interest to determine whether a calreticulin from a successful nematode parasite of humans, with known immune modulatory and antihaemostatic properties, exhibited a capacity to interfere with complement activation and to interact with integrin domains associated with cell signalling in platelets and other leucocytes. We can now report that recombinant calreticulin failed to demonstrate significant calcium binding capacity, which is a hallmark of calreticulins in general and may indicate inappropriate folding following expression in a prokaryote. Nevertheless, recombinant calreticulin retained sufficient molecular architecture to bind to, and inhibit the haemolytic capacity of, human C1q. Furthermore, recombinant calreticulin reacted in surface plasmon resonance analysis (SPR) with peptides corresponding to cytoplasmic signalling domains of the integrins alphaIIb and alpha5, in a calcium independent manner. SPR was also used to ratify the specificity of a polyclonal antibody to hookworm calreticulin, which was then used to assess the stage specificity of expression of the native molecule (in comparison with reverse transcriptase-polymerase chain reaction), to indicate its apparent secretion, and to purify native calreticulin from worm extracts by affinity chromatography. This development will allow the functional tests described above to be repeated for native calreticulin, to ascertain its role in the host-parasite relationship.
EPR spectrometry was used to investigate the effect of excretory/secretory product from Necator americanus on superoxide radical anions generated by xanthine/xanthine oxidase as a measure of excretory/secretory product superoxide dismutase activity. Using 1,1',5,5'-dimethylpyrollidine-N-oxide (DMPO) as a superoxide spin-trapping agent a 12-line EPR spectrum characteristic of the DMPO-OOH adduct was observed to decrease in the presence of excretory/secretory product. Superoxide dismutase activity was proportional to excretory/secretory protein concentration, was inhibited with cyanide treatment and was progressively destroyed with increasing time of heat denaturation of excretory/secretory product. Using a purpose-built chamber the superoxide dismutase activity of excretory/secretory product from live worms in culture was shown to accumulate with time to a maximum at 4 h. The electron paramagnetic resonance spectrum obtained for the frozen excretory/secretory product of N. americanus recorded at 77 K is typical of Cu(II) in a protein matrix. The results are consistent with the presence of an active Cu/Zn superoxide dismutase in excretory/secretory product from N. americanus and demonstrate a method for the unequivocal determination of the fate of superoxide anions in the presence of live worms.
We report a case of the endoscopic discovery and removal of a specimen of Necator americanus hooked into the gastric mucosa of a black woman from Zaire. We extracted the parasite by means of biopsy forceps. Signs of previous bites were seen in the antrum. This exceptional localization might be due to a jejuno-duodeno-gastric reflux.
The clearance of hookworm (Necator americanus) ova by a single dose of tetrachlorethylene (T.C.E.) was compared with that produced by single and multiple dose regimes of bephenium hydroxynaphthoate and pyrantel pamoate. Single doses of bephenium and pyrantel were inferior to T.C.E. Three daily doses of bephenium or pyrantel produced effects comparable with a single dose of T.C.E. Because of the low cost and lack of side effects, T.C.E. remains the drug of choice in the treatment of N. americanus in infections; the only disadvantage of T.C.E. is its unnoticed deterioration under tropical conditions.
A longitudinal study was carried out in Owerri, capital of Imo State, Nigeria, to assess the pattern of Necator americanus infection during a 27-month period. The mean annual prevalence fell from 59.0% in 1978, to 55.5% in 1979 and 51.1% in 1980. The egg count (apparent intensity of infection) fell in a similar pattern. Maximum and minimum conversion rates were 55.2 and 13.7%, respectively, and conversion rates fell by approximately 20% between 1979 and 1980. The highest reversion rate was 55.0% between 1979 and 1980. The population density of available third-stage larvae fluctuated seasonally and this was significant in determining the pattern of infection.
Adult Necator americanus infection in laboratory hamsters (the hamster-hookworm model) was examined as an anthelminthic screening system. Three reference anthelminthics--pyrantel (PYTL), mebendazole (MBZ) and ivermectin (IVRN)--were used to assess the sensitivity of adult N. americanus and also to investigate the value of the hamster-hookworm model for predicting clinical results. Serial drug dosages were used, and the ED50 was determined from the resulting cure rates. In addition, percentage worm reductions were calculated by reference to the worm burdens in control groups. The results showed that the hamster-hookworm model was able to differentiate anthelminthics on their efficacy. Absolute activity (100% worm reduction) followed treatment with 8 mg kg-1 MBZ, 38-40 mg kg-1 PYTL and 18 mg kg-1 IVRN. Based on ED50 data of PYTL and MBZ, adult N. americanus appeared to be two to five times more sensitive than pre-adult stages. However, with IVRN the reverse appeared true. MBZ appeared to be most active and PYTL least active in terms of curing infected animals, but there were no obvious differences between the rates of worm reductions following single or multiple doses of anthelminthics. It is considered that the hamster-hookworm model will prove of value in identifying and characterizing possible new anthelminthics.
When filariform larvae of Necator americanus, enclosed within the second sheath, were cultured in various semi-defined media, the best larval development was achieved in Waymouth's MB 752/1 with 20% foetal calf serum (FCS). In this medium, most of the filariform larvae cast off the second sheath and developed into third-stage larvae with provisional buccal capsules and some larvae developed further; 26% were fourth stage after 49 days of culture. Of several supplements tested, only FCS supported such development and no further development occurred if the medium was further supplemented with human red blood cells, liver extract, intestinal extract or tryptose phosphate broth. Larval development stopped at or before early third stage in Eagle's Basal Medium, F-12, NCTC 135 and L-15, even when these media were supplemented with FCS.
The population dynamic parameters of Necator americanus infections were estimated from longitudinal intensity and prevalence data from a village in south-eastern China. The theoretical predictions of a deterministic model incorporating these parameters were then compared with observed, horizontal, age-prevalence data from an infected population (N = 565), and an estimate of the basic reproduction rate (R0 = 3.7) of N. americanus was obtained. The findings indicate that populations of N. americanus might be easier to control by mass-treatment chemotherapy (eradication requiring only 63% of the population to be treated every year for 4 years) than those of Ascaris lumbricoides and Trichuris trichiura.