Fish-borne nematodiases in South America: neglected emerging diseases.

Fish-borne zoonotic nematodes may infect humans when fish or squid are ingested raw or inadequately cooked. Human infections may have serious consequences, including the unexpected deaths of infected people. This kind of disease is poorly known in general, and the characteristics of such infections in South American countries as a whole have never been assessed. In this paper the present status of fish-borne nematodiases in humans in South American countries is characterized. Potentially zoonotic nematode species are very common in both freshwater and marine fish in South America. Reports of human infections have only been found in some countries, and their incidence (especially with anisakids and Gnathostoma spp.) varies from country to country. Apparently they are more abundant in countries with strong traditions of eating raw fish, and are more frequent on the western coast of South America. So far fish-borne nematodes have been reported in Argentina, Brazil, Chile, Colombia, Ecuador and Peru. In recent years, cases of human infection have appeared in probably underestimated numbers. People need to be clearly informed about risky feeding habits, and physicians need to learn more about zoonotic diseases.

Serodiagnostic reliability of single-step enriched low-molecular weight proteins of Taenia solium metacestode of American and Asian isolates.

The low-molecular weight proteins (LMWPs) of Taenia solium metacestode (TsM) constituted pertinent serodiagnostic antigens for cysticercosis. We established a novel single-step purification of the LMWPs from TsM cyst fluid (CF). When the CF was precipitated with trichloroacetic acid/acetone mixture at the final concentrations of 5 and 50%, most LMWPs (ranging 7-38kDa) remained in the supernatant fraction. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analyses revealed that the LMWPs consisted mainly with the previously reported subunit proteins of the 120- and 150-kDa antigen complexes. Serum samples from neurocysticercosis (NC) and other helminthic infections, as well as those from healthy controls were tested by both immunoblotting and micro-ELISA. In 135 cases of active stage NC patients, 132 cases (97.7%) showed positive reactions. Serum samples from other helminthic diseases (n=125) and healthy controls (n=100) exhibited no positive reactions except for cystic echinococcosis, of which 12% (3/25 cases) exhibited low levels of cross-reactivity. The LMWPs from different geographical regions (Korea and Mexico) showed diagnostic sensitivity and specificity of 97.7% and 98.7% against active stage NC. Our single-step separation method for the LMWPs provided excellent performance with easy applicability and high reproducibility, which has a great benefit for preparation of potent antigen in endemic areas.

Evaluation of the antigenic similarities of adult-worm extracts from three Gnathostoma species, using sera from Mexican and Japanese patients with Gnathostoma infections.

The antigenic similarities of adult-worm extracts of Gnathostoma spinigerum, G. hispidum and G. doloresi, all of which are important food-borne parasites causing larva migrans in humans, were evaluated. The 40 sera used came from gnathostomiasis cases in Mexico, where G. binucleatum is endemic, or in Japan, where G. doloresi predominates. When used as the fixed antigens in microplate-ELISA, the adult-worm extracts from the three different species of Gnathostoma were found to have equal binding capacity to the Gnathostoma-specific IgG antibodies in the sera of the Mexican and Japanese patients. The correlation coefficients for the optical densities seen in the ELISA, between any two of the three Gnathostoma extracts, were all >0.900. The dose-response curves produced when four sera were tested, in the microplate-ELISA, against the three different Gnathostoma extracts were nearly identical, indicating that the antigens in each of the extracts had similar avidity. Furthermore, the results of competitive-inhibition ELISA indicated that the antigenic specificities of the three extracts were almost identical. An antigen of 40 kDa, which SDS-PAGE and immunoblot analysis revealed to be present in all three extracts, was recognized by the sera from the gnathostomiasis cases. When the sera were investigated by dot-blot ELISA, they also gave similar results whichever extract was used as the antigen source. It appears that, in the serodiagnosis of gnathostomiasis by microplate- or dot-ELISA, each of the three adult-worm extracts would be equally useful, regardless of the causative species of Gnathostoma.

The first human case in Mexico of conjunctivitis caused by the avian parasite, Philophthalmus lacrimosus.

Species of Philophthalmus parasitize primarily the eyes of wild and domestic birds. A variety of mammals, including humans, occasionally serve as the intermediate as well as the definitive hosts for this parasite, although human cases are extremely rare. Here, we report a case of human conjunctivitis caused by an infection with Philophthalmus sp. in Mexico. The patient was a 31-yr-old male who visited an ophthalmologist in Los Mochis, Sinaloa, Mexico, because of a foreign-body sensation in his left eye for 2 mo. A small live parasite was found in the connective tissue of the bulbar conjunctiva and was removed surgically under local anesthesia under ophthalmoscopic observation. The parasite was identified morphologically as Philophthalmus lacrimosus Braun, 1902. This is the first case of human philophthalmosis in Mexico and, to our knowledge, the first human case of P. lacrimosus infection in the world.

Goblet cell hyperplasia in the airway of Nippostrongylus brasiliensis-infected rats.

BACKGROUND: In rats, the intestinal parasite Nippostrongylus brasiliensis is recognized as a strong inducer of intestinal goblet cell hyperplasia. Although this parasite migrates through the airways during the course of its infection, airway goblet cell response remains unknown. OBJECTIVE: This study was designed to examine airway goblet cell response during the course of N. brasiliensis infection in rats and to characterize these goblet cells. METHODS: Airway goblet cells were stained with Alcian blue and periodic acid-Schiff. To characterize the goblet cells, mebendazole treatment, lectin histochemistry, and RNA blot analysis using probes for rat MUC2 and trefoil peptides were examined. RESULTS: Airway and small intestinal goblet cell hyperplasia were observed at days 14 and 21 after infection but not at day 7. In rats treated with mebendazole, goblet cell hyperplasia was not present in the small intestine, but was observed in the lung on day 14. These results indicate that airway goblet cell hyperplasia may be induced by local pulmonary factors. By lectin histochemistry, the stainability of airway goblet cells at day 21 was similar to that of small intestine goblet cells even though rat MUC2 and trefoil peptide mRNA were not detected in the lung. CONCLUSIONS: Airway goblet cell hyperplasia observed at days 14 and 21 after N. brasiliensis infection may be induced by local factors. Airway goblet cells have characteristics that differ from those of the small intestine.

Short report: gnathostomiasis in Mexico.

Gnathostomiasis is an important food-borne parasitic zoonosis that is endemic mainly in Asian countries where some people prefer to eat raw freshwater fish. In North America, the first recorded case of gnathostomiasis was in Mexico in 1970, and the numbers of gnathostomiasis patients in Mexico seems to be increasing dramatically with time. However, the epidemiology of this disease in Mexico has never been described in detail. Here we review the current status of gnathostomiasis in Mexico.

Clinical manifestations and immunodiagnosis of gnathostomiasis in Culiacan, Mexico.

Gnathostomiasis was first described in Mexico in 1970, and endemic areas have been spreading in six states of this country. In Culiacan, Sinaloa, 300 cases of cutaneous larva migrans were recorded between January 1992 and December 1995. In addition, a Gnathostoma larva was surgically removed from the eye of one patient. Cutaneous lesions were observed mainly on the face, neck, arms, and legs. About 70% of the patients showed eosinophilia. A skin biopsy was carried out on 35 patients and the parasite was identified in histopathologic sections of 12 of these patients. In four patients, the larva migrated out spontaneously from the skin. An enzyme-linked immunosorbent assay using a crude somatic extract of adult Gnathostoma doloresi worms showed that 93% of the patients were seropositive, confirming the reliability of clinical diagnosis. A total of 14 advanced third-stage Gnathostoma larvae were found in four species of ichthyophagous birds captured on dams and dikes near the city of Culiacan. Scanning electron micrographs of human and bird larvae showed that they were morphologically indistinguishable from G. spinigerm. We conclude that the life cycle of Gnathostoma has been established in Sinaloa, and has become a serious public health issue for residents.

Development of basophils in Mongolian gerbils: formation of basophilic cell clusters in the bone marrow after Nippostrongylus brasiliensis infection.

Development of basophilic leukocytes was studied in the Mongolian gerbil, Meriones unguiculatus, after infection with the nematode Nippostrongylus brasiliensis. After infection, peripheral blood basophilia developed and peaked at 2 weeks. In bone marrow sections, numbers of alcian blue+/safranine- basophilic cells were increased. These cells did not bind berberine sulfate and were clearly distinguishable from the bone marrow-resident mast cells, safranine+ and berberine sulfate+. Alcian blue+/safranine- cells were identified by electron microscopy as basophilic myelocytes in various stages of maturation. In the early period of infection, these cells had round-to-oval granules with a homogenous electron-dense matrix, a well-developed Golgi apparatus and rough endoplasmic reticulum, and a nonsegmented nucleus. By enzyme cytochemical analysis, intense peroxidase activity was demonstrated in all of the specific granules as well as in the rough endoplasmic reticulum and Golgi apparatus. Two weeks after infection, the number of bone marrow basophilic cells further increased, forming distinct clusters or islands composed of up to 100 cells each. On electron micrographs, the basophilic cells in these clusters appeared to be late-stage basophilic myelocytes, ie, having an increased number of granules, a less-conspicuous Golgi apparatus and rough endoplasmic reticulum, a horseshoe-shaped-to-lobulated nucleus, and reduced peroxidase activity. Eosinophils and mast cells were rarely found in the basophilic cell clusters. Four weeks after infection, the clusters had disappeared. These results show that gerbil basophilic myelocytes tend to form cell clusters in the bone marrow during their active proliferation. The comparative paucity of other cell lineages in basophilic cell clusters suggests that basophilia is generated from differentiation/proliferation of precommitted basophil progenitors independently from cells of other lineages.

Long-term survival of 'damaged' Nippostrongylus brasiliensis adult worms in the testosterone-treated Indian soft-furred rat, Millardia meltada.

When testosterone-treated female Millardia meltada were infected with Nippostrongylus brasiliensis, adult worms persisted for over seven weeks. The kinetics of faecal egg counts showed a biphasic pattern having a transient decline at around two weeks post infection (p.i.). Thus the status of N. brasiliensis adult worms surviving in the small intestines of testosterone-treated M. meltada was examined. The fecundity and maturity of eggs in the uteri of female adult worms were examined at one, two, three and seven weeks p.i. Both the fecundity and maturity of eggs transiently decreased at two and three weeks p.i. and then completely recovered by seven weeks. Adoptive transfer of N. brasiliensis adult worms into naive recipients can discriminate the status of worms. Those obtained from the stable phase of a primary infection ('normal' worm) can establish and survive in the recipients, whereas those obtained at the time of expulsion ('damaged' worm) are rapidly expelled. Therefore, 300 each of N. brasiliensis adult worms collected from the testosterone-treated female M. meltada at one, two and seven weeks p.i. were transferred intraduodenally into normal rats to determine their status. Those collected at one week p.i. persisted for eight days, indicating that they were still 'normal'. In contrast, worms collected at two and seven weeks p.i. were expelled within four days, indicating that they had already been 'damaged'. Moreover, when the 'damaged' worms obtained from rats were intraduodenally transferred into testosterone-treated female M. meltada, they were not expelled, suggesting that testosterone-treatment affected the final expulsive step, but not the damaging process, of the mucosal defence of M. meltada against N. brasiliensis adult worms.

Reduced amount of intestinal mucus by treatment with anti-CD4 antibody interferes with the spontaneous cure of Nippostrongylus brasiliensis-infection in mice.

Mechanism of spontaneous cure was studied in mice infected with mouse-nonadaptive Nippostrongylus brasiliensis. Adult BALB/c mice were cured spontaneously of infection with this strain of N. brasiliensis by Day 7 post-infection. Expulsion of intestinal worms was delayed dose-dependently by a treatment with anti-CD4 antibody. However, the treatment had no significant effect on larval recovery from the lungs. Treatment of mice with anti-IL-5 antibody suppressed intestinal tissue eosinophilia induced by the infection, but did not affect intestinal worm recovery. Antigen specific IgE antibody was not detected in the sera obtained from Days 5 to 15. Therefore, IL-5 and specific IgE antibody are probably not important in the spontaneous cure. Treatment of mice with anti-CD4 antibody had no significant effect on number of intestinal goblet cells or on expression of terminal sugars of goblet cell mucins. However, histological and quantitative analyses revealed that significantly less intestinal mucus was released in anti-CD4 antibody treated mice than in control mice. These results suggest that CD4+ lymphocytes control the amount of intestinal mucus and consequently the reduced mucus interferes with the spontaneous cure. Quantity of mucus released in the intestinal lumen may have an essential role in the spontaneous cure of N. brasiliensis-infection of mice.

The Indian soft-furred rat, Millardia meltada, a new host for Nippostrongylus brasiliensis, showing androgen-dependent sex difference in intestinal mucosal defence.

Susceptibility of the Indian soft-furred rat, Millardia meltada, to infection with the intestinal helminth, Nippostrongylus brasiliensis, was examined. After subcutaneous infection with 1500 infective larvae (L3), daily faecal egg output (EPG) of both male and female animals reached a peak at 1 week post-infection (p.i.) with the same magnitude (about 20,000 epg faeces) and then rapidly decreased below detection level at around 2 weeks p.i. In male animals, however, after a transient cessation at 2 weeks p.i., parasite eggs reappeared in faeces 3 weeks afterwards, though the counts were far lower than the first peak. This phenomenon was rarely seen in female animals. High susceptibility of M. meltada to N. brasiliensis was confirmed by worm burdens. About 70% of the initial dose of larvae became adult worms in the intestine of male and female hosts. As suggested by the decline in egg counts, the majority of adult worms were expelled by 2 weeks p.i. The residual worm burden at 2 and 4 weeks p.i. was significantly higher in male than female animals. When orchidectomized males were infected with N. brasiliensis, the magnitude of residual worm burden was significantly reduced. On the other hand, ovariectomy did not affect the number of residual worms in females.

Inhibitory effects of concurrently present 'normal' Nippostrongylus brasiliensis worms on expulsion of 'damaged' worms and associated goblet cell changes in rats.

Nippostrongylus brasiliensis worms obtained from the stable phase of a primary infection ('normal' worms) are able to establish and survive in naïve recipient rats into which they have been adoptively transferred, whereas those obtained at the time of expulsion ('damaged' worms) are rapidly expelled. Expulsion is associated with quantitative/qualitative goblet cell changes. Thus, N. brasiliensis adult worms seem to stimulate the expulsion process only after being 'damaged' by the host's immunity, whereas it is possible that 'normal' worms may inhibit the expulsion process. To see whether 'normal' worms have such inhibitory effects, the fate of concurrently implanted 'normal' and 'damaged' worms was examined with reference to goblet cell changes in the intestine. By using either male or female worms as 'normal' or 'damaged' populations, it was possible to determine the origin of worms remaining in the intestine. The results show that not only 'normal' worms but also 'damaged' worms, which would normally be expelled, remained in the intestines of the recipients on Day five. Goblet cell hyperplasia and alteration of terminal sugars of mucins, which was induced within five days after implantation of 'damaged' worms, were completely inhibited by the concurrent presence of 'normal' worms. The inhibitory effects of 'normal' worms were destroyed when they were killed by heating before implantation. Possible mechanisms leading to expulsion of N. brasiliensis are discussed.

Reconstitution by bone marrow grafting of the defective protective capacity at the migratory phase but not at the intestinal phase of Nippostrongylus brasiliensis infection in W/Wv mice.

After a primary infection by subcutaneous inoculation with the infective larvae (L3) of Nippostrongylus brasiliensis, the intestinal worm burden was higher and expulsion was slower in W/Wv mice than in +/+ mice. When the course of infection was segregated into the migratory and intestinal phases, protection during the migratory phase examined by the larval recovery from the lungs and that during the intestinal phase measured by worm burden after intraduodenal implantation with adult worms were both defective in W/Wv mice. The higher susceptibility of W/Wv mice during the migratory phase was normalized by bone marrow reconstitution. On the other hand, higher susceptibility of W/Wv mice during the intestinal phase, which was measured by worm burden 24 h after intraduodenal implantation of the larvae recovered from the lungs of rats, was not normalized by bone marrow grafting. Furthermore, slower expulsion seen in W/Wv mice after intraduodenal implantation with adult worms was not hastened by bone marrow reconstitution. These results indicate that the protective mechanisms against N. brasiliensis operating during the migratory phase and those during the intestinal phase were different in terms of bone marrow dependency and that non-myeloid cells utilizing c-kit ligand/receptor system to express their functions are involved in the mucosal defence against N. brasiliensis.

Goblet cell mucins as the selective barrier for the intestinal helminths: T-cell-independent alteration of goblet cell mucins by immunologically 'damaged' Nippostrongylus brasiliensis worms and its significance on the challenge infection with homologous and heterologous parasites.

The aim of this study was to examine the role of T cells on the alteration of terminal sugars of goblet cell mucins in the small intestinal mucosa of parasitized rats and to clarify the biological significance of the altered mucins in the mucosal defence against intestinal helminths. For this purpose, Nippostrongylus brasiliensis adult worms obtained from donor rats at 7 ('normal' worms) or 13 days ('damaged' worms) post-infection were implanted intraduodenally into euthymic and hypothymic (rnu/rnu) rats. Expulsion of implanted normal worms and associated goblet cell changes were extremely delayed in hypothymic recipients compared with euthymic recipients. In contrast, intraduodenally implanted damaged worms were expelled by day 5 regardless of the strains. Around the time of expulsion of implanted damaged worms, euthymic recipients showed both goblet cell hyperplasia and alteration of mucins, whereas hypothymic rats showed only the latter. Dexamethasone treatment completely abolished goblet cell changes of both strains of recipients. To clarify the importance of the constitutional changes of goblet cell mucins in mucosal defence, euthymic rats were primed by implantation of damaged worms to induce goblet cell changes, and then 3 or 5 days later they were challenged by implantation with normal worms. The results show that when goblet cell changes were induced by priming with damaged worms, recipient rats could completely prevent the establishment of normal worms. When hypothymic rats were primed and challenged in the same manner, a similar but slightly less preventive effect was observed. Such a protective effect of altered mucins seems to be selective because priming of euthymic rats with damaged N. brasiliensis did not affect the establishment of Strongyloides venezuelensis. These results suggest that: (1) once N. brasiliensis adult worms are 'damaged' by the host's T-cell-dependent immune mechanisms, they can induce alteration of sugar residues of goblet cell mucins via host-mediated, T-cell-independent processes; (2) the expression of such altered mucins is highly effective not only in causing expulsion of established damaged worms but also in preventing establishment of normal worms; and (3) the preventive effect of altered mucins is selective against parasite species.

Persistent infection of Strongyloides venezuelensis and normal expulsion of Nippostrongylus brasiliensis in Mongolian gerbils, Meriones unguiculatus, with reference to the cellular responses in the intestinal mucosa.

The kinetics of daily faecal egg count, worm burdens, and intestinal cellular responses were examined in Mongolian gerbils after infection with either Strongyloides venezuelensis or Nippostrongylus brasiliensis alone, or concurrently with both parasites. The results show that, both in individual and concurrent infections, S. venezuelensis infection persisted for over 10 weeks and elicited a gradual increase in number of mast cells in the jejunal mucosa. On the other hand, N. brasiliensis worms were expelled by 3 weeks in association with goblet cell hyperplasia. These results suggest that effector/regulator cells involved in worm expulsion are different and highly selective depending on the genus of intestinal helminths.

Immune-mediated alteration of the terminal sugars of goblet cell mucins in the small intestine of Nippostrongylus brasiliensis-infected rats.

Alteration of terminal sugars of goblet cell mucins in the small intestinal mucosa was examined by lectin histochemistry in rats infected with Nippostrongylus brasiliensis. To see whether alteration of the nature of mucins was regulated by the local immune system, adult worms were implanted intraduodenally into recipient rats of various immune status. When 7-day-old ('normal') adult worms were implanted into naive euthymic rats, about 60% remained until day 7 and then were expelled. The number of goblet cells transiently decreased on day 5 and then progressively increased from day 9 onwards. In parallel with the hyperplasia, mucins in and released from goblet cells strongly expressed terminal N-acetyl-D-galactosamine which was specifically recognized by Helix pomatia agglutinin (HPA). When 13-day-old ('damaged') adult worms were implanted into naive euthymic rats, they were rapidly expelled by day 9 in association with hastened goblet cell hyperplasia and the alteration of terminal sugars of the mucins. Hastened worm expulsion, goblet cell hyperplasia and alteration of terminal sugars of the mucins were also observed when 'normal' worms were implanted into immune recipients. On the other hand, after implantation of 'normal' worms into hypothymic (rnu/rnu) rats, goblet cell hyperplasia or the alteration of terminal sugars of the mucins was almost completely absent. These results suggest that alteration of sugar residues of goblet cell mucins, especially the strong expression of terminal N-acetyl-D-galactosamine, is regulated by the host's immune system and seems to be important in the expulsion of N. brasiliensis.

Induction of the expulsion of Strongyloides ratti and retention of Nippostrongylus brasiliensis in athymic nude mice by repetitive administration of recombinant interleukin-3.

A repetitive administration of recombinant interleukin-3 (rIL-3), which can induce the expulsion of Strongyloides ratti in athymic nude mice, did not affect the expulsion of Nippostrongylus brasiliensis. Nude mice infected with N. brasiliensis were injected i.p. with a total of 6.8 x 10(5) U rIL-3 or medium twice a day from Day 5 to Day 11 post-infection. The kinetics of expulsion estimated by egg excretion in faeces up to Day 20 post-infection and adult worm burden on Day 21 was not affected by the IL-3 administration. A similar administration with a higher dose (total 10.6 x 10(5) U) of rIL-3 did not alter the adult worm burden on Day 13. The number of intestinal mucosal mast cells on Day 13 was markedly increased by the treatment, although the number of intestinal goblet cells was comparable between the treated and control mice. When nude mice were infected concurrently with N. brasiliensis and S. ratti and then injected repeatedly with rIL-3 (total 2.2 x 10(5) U) from Day 5 to Day 11, adult worms of S. ratti were expelled from the small intestine by Day 13; however adult worms of N. brasiliensis were retained. Again in the concurrent infection, the number of intestinal mucosal mast cells was significantly increased but that of intestinal goblet cells was not altered by the rIL-3 administration. These results indicate that the expulsion of S. ratti is dependent on IL-3 whereas that of N. brasiliensis is less dependent on IL-3.

Regulation of eosinophilia in rats infected with Nippostrongylus brasiliensis. I. Eosinophil chemotactic factor produced spontaneously by mesenteric lymph node cells of infected rats.

The possible mechanism of eosinophilia was studied in rats undergoing primary infection with Nippostrongylus brasiliensis (Nb). In vivo studies showed that the kinetics of intestinal tissue eosinophilia was not directly related to those of the intestinal worm burden. Furthermore, Nb worm extract has no or only very weak in vitro eosinophil chemotactic activity, suggesting that parasite-derived eosinophil chemotactic factor (ECF) is, if at all, not a major regulator for intestinal tissue eosinophilia in this Nb rat system. On the other hand, when mesenteric lymph node (MLN) cells obtained various days after infection were cultured, potent ECF activity was detected in the cell-free supernatant from the cultures of MLN cells 15-20 days after infection, at which time marked intestinal tissue eosinophilia was observed in vivo. Production of ECF by MLN cells from Nb-infected rats seems to be spontaneous, since these cultures were performed without adding worm antigen. ECF-producing activity of day-20 MLN cells was suppressed by adding various metabolic inhibitors such as cycloheximide, mitomycin C, or puromycin. After Sephadex G-75 gel filtration, ECF activity produced by day-20 MLN cells was associated with two different molecules.