Host cytokine production, lymphoproliferation, and antibody responses during the course of Ancylostoma ceylanicum infection in the Golden Syrian hamster

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.     

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 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.     

Altered host resistance to Trichinella spiralis infection following subchronic exposure to diethylstilbestrol

The effects of subchronic exposure to diethylstilbestrol on the host response to infection with Trichinella spiralis were examined in adult B6C3F1 mice. Expulsion of adult Trichinella from the small bowel, intestinal inflammation and delayed hypersensitivity responses to Trichinella antigens in artificially sensitized mice were investigated. Administration of 8 micrograms/g of diethylstilbestrol for five consecutive days beginning on days -5,0, +3 or +8 of infection inhibited adult worm expulsion and tissue reactions in the small intestine. Expulsion of adult parasites was also delayed in mice given 0.2 microgram/g of diethylstilbestrol for the first five days of infection. When the chemical was given during a primary infection, mice failed to expel a second infection as rapidly as untreated controls or previously infected mice exposed during a second infection. These findings indicate that diethylstilbestrol exposure altered the immune responses that mediate expulsion of adult worms from the gut, especially when exposure occurred during the inductive phase of host immunity. These results also suggest that use of diethyl-stilbestrol as a weight-gain promoter may lead to increased parasite burdens. While diethylstilbestrol-exposed mice retained adult worms longer than controls, no significant increase was found in numbers of encysted muscle-phase larvae, contrary to the usual findings in animals maintaining adult worm burdens for extended periods. The possibility that macrophages activated by diethylstilbestrol treatment had a role in limiting larvae encystment in the host musculature is discussed.     

Role for Activated Macrophages in Resistance Against Trichinella spiralis

To determine whether activated macrophages are important in resistance against the intestinal phase of nematode parasites, we studied Trichinella spiralis infections in mice with normal macrophages and in mice with macrophages activated by either chronic Toxoplasma gondii or acute Listeria monocytogenes infections. The peak T. spiralis adult worm burden in the intestines of normal C57BL/6 or Swiss Webster mice occurred from 6 to 14 days after infection. Subsequent expulsion of worms from the intestines occurred from 8 to 20 days after infection. C57BL/6 mice chronically infected with T. gondii and then challenged with T. spiralis larvae had significantly lower peak intestinal worm burdens (P < 0.05) than normal C57BL/6 mice similarly challenged. Swiss Webster mice infected 7 or 13 days earlier with L. monocytogenes and then challenged with T. spiralis larvae had significantly lower peak worm burdens (P < 0.01) than uninfected mice. The time of expulsion of adult worms was not affected by either infection. Swiss Webster mice infected 42 days earlier with L. monocytogenes (i.e., possessing lymphocytes sensitized to L. monocytogenes but not possessing activated macrophages) did not have a lower worm burden than uninfected mice. Serum factors (e.g., antibody) did not appear to play a role because normal mice injected with serum from L. monocytogenes-infected mice had worm burdens similar to those of mice injected with normal serum. The histopathology of intestines of mice infected with T. gondii or L. monocytogenes was the same as that of normal mice. When T. spiralis larvae were incubated with normal macrophages or macrophages from T. spiralis-infected mice in vitro for 24 h, the number of larvae with adherent T. spiralis macrophages was significantly (P < 0.005) greater than the number of larvae with adherent normal macrophages. These studies suggest a role for activated macrophages in resistance to T. spiralis.     

Ancylostoma Ceylanicum: I. Protein and Antigenic Composition of Adult and Larval Stages

The protein and antigenic composition of adult and larval stages of Ancylostoma ceylanicum, a human hookworm maintained in golden hamsters (Mesocricetus auratus), was studied employing immunochemical techniques. SDS-polyacrylamide gel electrophoresis revealed the presence of 47 and 43 protein bands in adult worms and infective larvae respectively in the molecular weight range of 10-170 kD. Crossed immunoelectrophoretic analysis, using immune rabbit sera, showed the presence of 32 antigenic peaks in adults and 19 in infective larval stage. Most of the antigens were common between adult and larval stage as evidenced by cross-line immunoelectrophoresis, although some stage specific antigens were also identified. These studies also demonstrate the complex nature of adult worms as compared to larvae.     

Inhibition of nitric oxide production by aminoguanidine influences the number of Trichinella spiralis parasites in infected “low responders” (C57BL/6) and “high responders” (BALB/c) mice

The influence of nitric oxide (NO) on the development of adults and larvae Trichinella spiralis was examined in two strains of mice: C57BL/6 and BALB/c. The influence of aminoguanidine (AG)-inhibitor of inducible nitric oxide synthase (iNOS) administered in the first days after T. spiralis infection (1–5 dpi) on the number of adult parasites, as well as the influence of AG administered at the beginning of muscle phase of the T. spiralis infection (16–29 dpi) on the number of muscle larvae, was studied. In mice that were treated with AG from the 1st to the 5th day post infection (dpi), the numbers of adult T. spiralis were counted in intestines at 6, 9, 15, and 20 dpi. In this experiment, the impact of AG expressed as diminution of adult worms at 9, 15, and 20 dpi in BALB/c mice. The opposite effect of AG was demonstrated in C57BL/6 mice at 6 and 9 dpi. In mice in which AG was applied from the 16th to the 29th dpi T. spiralis larvae were counted at 30, 35, and 41 dpi. This experiment demonstrated that treating mice with AG at the beginning of muscle phase of the infection inhibits the reduction of muscle larvae number in mice of both strains.     

The relative importance of Ah versus H-2 genotype on Trichinella resistance following exposure to 3-methylcholanthrene

The relative influence of Ah vs H-2 genotype on the outcome of Trichinella spiralis (Tsp) infections of mice was examined following methylcholanthrene (MC) treatment. Female mice of four inbred strains were treated with MC and infected 24 h later with Tsp muscle larvae. The strains, with their respective major histocompatibility complex (MHC) haplotype, aryl hydrocarbon hydroxylase responsiveness (Ah phenotype) and level of susceptibility to Tsp infection, were: C3HeB/FeJ (C3), H-2k, Ahb, Tsp susceptible; C57BL/10.BR (B10.BR), H-2k, Ahb, Tsp susceptible; C57BL/10.Q (B10.Q), H-2q, Ahb, Tsp resistant; and AKR/J (AK), H-2k, Ahd, Tsp resistant. The proliferative response of splenic lymphocytes to crude Tsp L1 stage antigen was significantly depressed in all MC-treated groups, with the exception of the B10.BR strain. MC administered at 40 mg/kg impaired the ability of C3 and B10.Q mice to eliminate adult worms. At 80 mg/kg, C3 strain mice were also impaired, as well as AK strain mice. The fecundity of female worms recovered from B10 or AK strain mice was not significantly altered by MC treatment, although female worms from treated C3 mice exhibited increased fecundity on day 9 post infection. Muscle larvae burdens of MC-treated B10 and C3 mice were elevated, while those of AK strain mice were unaffected. These data suggest that with acute exposures to MC, the immunogenetic resistance or susceptibility of a given mouse strain may have a more pronounced effect on immune depression and the severity of Tsp infection than does the Ah phenotype.     

Resistance and susceptibility to filarial infection with Litomosoides sigmodontis are associated with early differences in parasite development and in localized immune reactions

In order to understand natural resistance to filariasis, we compared Litomosoides sigmodontis primary infection of C57BL/6 mice, which eliminate the worms before patency, and BALB/c mice, in which worms complete their development and produce microfilariae. Our analysis over the first month of infection monitoredmigration of the infective larvae from the lymph nodes to the pleural cavity, where the worms settle. Although immune responses from the mouse strains differed from the outset, the duration of lymphatic migration (4 days) and filarial recovery rates were similar, thus confirming that the proportion of larvae that develop in the host species upon infection is not influenced by host genetic variability. The majority of worms reached the adult stage in both mouse strains; however, worm growth and molting were retarded in resistant C57BL/6 mice. Surprisingly, the only immune responses detected at 60 h postinfection occurred in the susceptible mice and only upon stimulation of cells from lymph nodes draining the inoculation site with infective larva extract: massive production of interleukin-6 (IL-6) and IL-5 (the latter cytokine was previously suspected to have an effect on L. sigmodontis growth). However, between days 10 and 30 postinfection, extraordinarily high levels of type 1 and type 2 cytokines and expansion of pleural leukocyte infiltration were seen in the resistant C57BL/6 mice, explaining the destruction of worms later. Our results suggest that events early in the infection determine susceptibility or resistance to subsequent microfilarial production and a parasite strategy to use specific immune responses to its own benefit.     

Ancylostoma caninum anticoagulant peptide-5: immunolocalization and in vitro neutralization of a major hookworm anti-thrombotic

Hookworm infection is a major cause of gastrointestinal blood loss and iron deficiency anemia in the developing world. Recently two major anticoagulant serine protease inhibitors have been identified and cloned from adult Ancylostoma caninum hookworms. One of these, A. caninum anticoagulant peptide 5 (AcAP5), is a potent and specific inhibitor of human coagulation factor Xa. A polyclonal IgG has been purified from rabbits immunized with recombinant AcAP5 using affinity chromatography. Using immunohistochemistry, the polyclonal alpha-rAcAP5 IgG localized to the cephalic or amphidial glands, confirming previous biochemical studies that had identified this secretory gland as the primary source of anticoagulant activity in the adult worm. This polyclonal IgG also neutralized the inhibitory activity of recombinant and native AcAP using a single stage chromogenic assay of coagulation factor Xa activity. In addition, the polyclonal IgG also neutralized the anticoagulant activity of native and recombinant AcAP5 as measured by the activated partial thromboplastin time clotting assay. Importantly, this neutralizing activity is species specific, as the polyclonal IgG failed to neutralize the anticoagulant activity of A. ceylanicum. Taken together, these data suggest that the hookworm anticoagulant AcAP5 represents a viable target for future immunization strategies aimed at inhibiting the ability of the adult hookworm to feed on blood in vivo.     

Regulation of primary Strongyloides ratti infections in mice: a role for interleukin-5.

C57BL/6 mice genetically deficient in interleukin-5 (IL-5-/-) and normal C57BL/6 (IL-5+/+) mice were infected with larvae of a homogonic strain of the nematode Strongyloides ratti. In primary infections both male and female IL-5-/- mice released two to four times more eggs and larvae than IL-5+/+ mice. IL-5-/- mice harboured about 60% more intestinal worms, which were more fecund, than IL-5+/+ mice. The duration of the infection was similar in normal and IL-5-deficient mice. Both IL-5-/- and IL-5+/+ mice resisted a secondary infection. IL5-/- mice lost more weight during the infection than normal mice and took longer to regain their initial weight after expelling the worms. The number of eosinophils increased in the bone marrow, peritoneal cavity and small intestine of IL-5+/+ mice, but not IL-5-/- mice, following infection. No significant differences between infected IL-5+/+ and IL-5-/- mice in mast cells or other leucocytes were observed in the peritoneal cavity. Thus, IL-5 functions to protect the host in a primary infection of S. ratti by limiting the number and fecundity of worms establishing in the small intestine. This protection is correlated with elevated blood and tissue eosinophilia which occurs in normal mice but not in IL-5-/- mice.     

Anthelmintic activity of medicinal plants used in C?te d’Ivoire for treating parasitic diseases

Natural products play an important role in the discovery and development of new pharmaceuticals. In the present study, we assessed the anthelmintic properties of medicinal plants used in Cote d'Ivoire. Ethanolic extracts from 50 medicinal plants were tested in vitro against trematodes (Echinostoma caproni, Schistosoma mansoni) and nematodes (Ancylostoma ceylanicum, Heligmosomoides bakeri, Trichuris muris). Active extracts were evaluated for their cytotoxicity and followed up in vivo in mice harbouring adult S. mansoni, E. caproni and T. muris at single oral doses of 400 or 800 mg/kg. All extracts tested were active against at least one helminths species. Ten of the 65 extracts tested (15.4%) in vitro revealed activity against all helminths tested. Of 65 extracts tested in vitro at a concentration of 2 mg/ml, all caused death of schistosomula and 34.4% and 39.1% were lethal against adult S. mansoni and E. caproni 72 h post-incubation, respectively. The highest activity against A. ceylanicum in vitro was observed with Sclerocarya birrea at 2 mg/ml, which resulted in death of adult worms and inhibition of activity of third-stage larvae (L3). Of the extracts, 41.5% completely inhibited movement of H. bakeri L3 at minimal lethal concentration (MLC) values of 20-200 μg/ml 48 h post-incubation, and 15.4% paralysed adult H. bakeri at 200 μg/ml 72 h after incubation. Of the extracts, 19% resulted in death of adult T. muris at MLC values of 10-100 μg/ml. In vivo, none of the extracts tested revealed activity against E. caproni. Olax subscorpioidea achieved total and female worm burden reductions of 60% and 84%, respectively in S. mansoni-infected mice. Combretum mucronatum was the most active extracts in vivo against T. muris with a worm burden reduction of 85.3%. In conclusion, several of the medicinal plants used in C?te d'Ivoire are active against different helminths, hence might play a role in the treatment of helminthiases. Further studies are necessary to isolate the active components from these extracts.     

The effect of the intestinal worms and migrating L1 larvae of Trichinella spiralis on the production of antiparasitic IgE antibodies

The effect of the adult worms and migrating L1 larvae of Trichinella spiralis on the production of specific IgE antibodies was determined in BCF1 mice. To achieve this, we combined the effect of two anthelminthics: thiabendazole, to produce chemosterilization of adult females, and napthalophos, to expel adult worms from the intestine of infected mice on the desired day. Our results demonstrate that when the natural route of infection is used the production of IgE antibodies is not dependent on the infection dose or the number of migrating L1 larvae, and that both intestinal worms and migrating L1 larvae contribute to the production of reaginic antibodies. In addition to this, an extended period of antigenic stimulation (10–12 days) is required for the induction of a detectable, specific IgE response by adult worms. Finally, our results seem to indicate that although the effects of adult worms and migratory L1 larvae on the IgE production are not additive, the presence of adult worms in the intestine of mice may stimulate a secondary exposure to common antigens released by the migrating L1 larvae.     

漳州市芗城区浦南村人群钩虫病调查

目的了解芗城区浦南村钩虫人群感染及流行情况。方法采用改良加藤氏厚涂片法检查钩虫卵;对部分重度感染者进行驱虫导泻治疗,并收集虫体进行虫种鉴定;对部分环境类型土壤的钩蚴进行分离培养与鉴定。结果检查4812人,钩虫感染者1601人,感染率为33.27%。感染程度以轻度感染为主;女性感染率（18.37%）高于男性（14.90%）（χ2=7.74,P〈0.05）;人群分布以〈15岁年龄段的感染率最低（9.31%）,55～年龄段的老年人最高（47.43%）,呈年龄增长而升高趋势（χ2=300.00,P〈0.01）;文化程度以高中以上者感染率最低（8.06%）,与文盲半文盲（31.89%）、小学（37.83%）、初中（31.35%）文化程度者比较差异有统计学意义（χ2=7.74,P〈0.05）。驱虫导泻20人,收集钩虫成虫326条,均鉴定为美洲钩虫;分离培养土壤20份,分离阳性钩蚴12份,均鉴定为美洲钩蚴。结论芗城区浦南村为严重的钩虫流行区,人群钩虫感染率高,且为单纯的美洲钩虫;因此,加强健康教育,开展防治措施具有重要意义。     

Hepatic microsomal cytochrome P450 system during experimental hookworm infection

Experimental infection of golden hamsters with the hookworm, Ancylostoma ceylanicum, caused a profound decline in the hepatic microsomal cytochrome P450 content. Concomitant decrease was also noticed in aminopyrine N-demethylase and benzo[a]pyrene hydroxylase activities. However, aniline hydroxylase activity was only marginally elevated during the infection. Microsomal markers, viz., cytochrome b5, NADH-cytochrome-c reductase, and glucose-6-phosphatase, were not significantly altered. Hepatic tissue exhibited an accumulation of lipids, especially phospholipids, triglycerides, and cholesterol, resulting in fatty necrosis around the central vein region. Isolated hepatic microsomes showed a decrease in phosphatidylcholine content. Impairment in hepatic mixed function oxidase (MFO) activities was further confirmed by prolongation in hexobarbital sleeping time and zoxazolamine-induced paralysis. The hepatic MFO system of A. ceylanicum-infected hamsters responded qualitatively and quantitatively in a manner similar to that of control hamsters, upon stimulation with selective chemical inducers like phenobarbitone and 3-methylcholanthrene. Kinetic and in vitro substrate binding studies revealed that for aminopyrine the substrate affinity and the maximum enzyme activity (Vmax) were decreased, while for aniline the binding affinity was decreased and the binding capacity was enhanced. Results indicate specific/selective impairment of the hepatic microsomal cytochrome P450 system during hookworm infection and may have many practical implications in toxicology and pharmacology.     

Trapping and Immobilization of Nippostrongylus brasiliensis Larvae at the Site of Inoculation in Primary Infections of Interleukin-5 Transgenic Mice

Interleukin-5 (IL-5) transgenic mice are highly resistant to primary infections with the intestinal nematode Nippostrongylus brasiliensis; few parasites are found in the intestines of infected animals, and egg production is minimal. While adult worms may be damaged in the intestine, larval migration, development, and viability may also be impaired in other tissues. This study addresses the migration of N. brasiliensis larvae through the skin and lungs and associated cellular responses in primary infections of IL-5 transgenic mice. Although some larvae may have been trapped and killed in the lungs of IL-5 transgenic mice, most apparently failed to reach this site. Two or more hours after infection of IL-5 transgenic mice, eosinophils were a major component of the cellular infiltrate at the subcutaneous site of injection, and localized eosinophil degranulation was extensive. Seventy-five to ninety-five percent of the larvae injected into subcutaneous air pouches in IL-5 transgenic mice were retained there for at least 24 h. In contrast, in nontransgenic mice, less than 20% of larvae could be recovered from the skin 2 or more h postinjection, and eosinophil activity was modest at all times. The data strongly suggest that eosinophils can restrict the movement of N. brasiliensis larvae in the first few hours of a primary infection and that this has profound effects on later stages of parasite development. Preexisting eosinophilia, due either to allergy or to infection with tissue-invasive helminth species, may therefore confer some degree of immediate and nonspecific resistance in primary infections with parasitic worms.     

Protective immunity againstAngiostrongylus cantonensis in rats following sensitizing infections

Three strains (ACI, August and Wistar) of rats previously sensitized by oral infection with intact third-stage larvae ofAngiostrongylus cantonensis developed significant protective immunity to challenge infections 6 weeks later. The degree of the immunity was highest in the August strain of rats, followed by Wistar and ACI rats. Protective immunity appears to affect both third-stage larvae and fourth- and/or fifth-stage worms. ACI rats showed poor antibody responses, especially in the IgE fraction. When 24-day-old young adult worms were transferred from the brain of donor rats into the peritoneal cavity of sensitized rodents, peritoneal eosinophils predominantly adhered to the worm surfaces in vivo.     

Tegumental phosphodiesterase SmNPP-5 is a virulence factor for schistosomes

The intravascular trematode Schistosoma mansoni is a causative agent of schistosomiasis, a disease that constitutes a major health problem globally. In this study we cloned and characterized the schistosome tegumental phosphodiesterase SmNPP-5 and evaluated its role in parasite virulence. SmNPP-5 is a 52.5-kDa protein whose gene is rapidly turned on in the intravascular parasitic life stages, following invasion of the definitive host. Highest expression is found in mated adult males. As revealed by immunofluorescence analysis, SmNPP-5 protein is found prominently in the dorsal surface of the tegument of males. Localization by immuno-electron microscopy illustrates a unique pattern of immunogold-labeled SmNPP-5 within the tegument; some immunogold particles are scattered throughout the tissue, but many are clustered in tight arrays. To determine the importance of the protein for the parasites, RNA interference (RNAi) was employed to knock down expression of the SmNPP-5-encoding gene in schistosomula and adult worms. Both quantitative real-time PCR (qRT-PCR) and Western blotting confirmed successful and robust gene suppression. In addition, the suppression and the ectolocalization of this enzyme in live parasites were evident because of a significantly impaired ability of the suppressed parasites to hydrolyze exogenously added phosphodiesterase substrate p-nitrophenyl 5'-dTMP (p-Nph-5'-TMP). The effects of suppressing expression of the SmNPP-5 gene in vivo were tested by injecting parasites into mice. It was found that, unlike controls, parasites whose SmNPP-5 gene was demonstrably suppressed at the time of host infection were greatly impaired in their ability to establish infection. These results demonstrate that SmNPP-5 is a virulence factor for schistosomes.