The effect of BCG-vaccine upon experimental visceral leishmaniasis in hamsters.

Stimulation with Bacillus Calmette-Guerin (BCG) vaccine has been reported to enhance resistance of mice against Leishmania donovani infection. Such infection is usually lethal in hamsters, thus providing a more stringent animal model to assess the effect of BCG upon visceral leishmaniasis. Animals receive two IP injections (2-8 X 10(7) BCG) pre or post IC challenge with 4 X 10(6) amastigotes. Controls received BCG alone (with no infection) or were untreated (NT). Pretreated animals exhibited significantly fewer (P less than 0.05) hepatic or splenic amastigotes than NT animals at days 7, 14, and 28 post challenge, but most BCG treated hamsters died earlier than NT. Post treated hamsters showed no significant reduction in parasite burdens, or in median time to death as compared to NT group. Hamsters which received BCG but were not infected appeared healthy during the study. The reason for increased susceptibility of BCG-treated hamsters to disease is not clear, but observed pathologic complications of L. donovani infected hamsters appear to be exacerbated by BCG stimulation.

Non-specific and specific stimulation of resistance against Leishmania donovani in C57BL/6 mice.

Stimulation by intravenous injections of glucan, a beta 1,3 polyglucose, provided a significant degree of resistance in mice against Leishmania donovani. The response of C57BL/6 animals was dose-dependent. A single glucan injection before or after infection induced significant resistance but to a lesser degree than two or three injections. Immunization by injections of formalin-killed promastigotes with glucan via the subcutaneous or intravenous routes provided greater resistance than glucan or dead parasites alone against subsequent infection with viable parasites. Subcutaneous immunization with promastigotes from cultures passaged 10 times in vitro and those from cultures maintained for 25 passages elicited a similar degree of resistance against infection and induced positive skin test responsiveness against leishmanial antigen prior to infection.

Immunogenicity of soluble and particulate antigens from Leishmania donovani: effect of glucan as an adjuvant.

The protective efficacy of glucan as an adjuvant with killed promastigotes of Leishmania donovani was compared with that of soluble or particulate fractions of the parasite. When these vaccine preparations were injected either intravenously or subcutaneously in CF-1 mice, glucan potentiated resistance against L. donovani infections as reflected by significant reductions in hepatic amastigote counts relative to infected control mice. The leishmanial antigens alone afforded no protection. Serum direct agglutination titers to leishmanial antigens were highest in all groups given the vaccine intravenously, whereas the delayed-type hypersensitivity response to the antigen was positive only in groups immunized subcutaneously with glucan as an adjuvant. Some index of protection and immune response against visceral infection with the parasite was seen in groups vaccinated with glucan and soluble antigens. However, the protection afforded by glucan and particulate antigens of L. donovani more closely paralleled the resistance of mice treated with glucan and unfractionated killed promastigotes. Further antigenic analysis of particulate fractions of L. donovani may optimize effective immunization when used with appropriate adjuvants, e.g., glucan.

Immunization of mice against Leishmania donovani by subcutaneous injections of dead promastigotes.

Mice immunized by a series of intravenous, intraperitoneal or subcutaneous injections of formalin-killed Leishmania donovani promastigotes with glucan, a beta 1, 3 polyglucose, exhibited a significant degree of resistance against subsequent infection with viable promastigotes. Intramuscular immunization was not effective. Immunization via subcutaneous injections of dead promastigotes simultaneously with glucan elicited protective resistance, positive skin test responsiveness before and after challenge and increased antipromastigote antibody levels. Injections of glucan alone induced a lesser degree of resistance against infection without significant skin test or humoral responsiveness. Injections of dead promastigotes alone elicited increased antibody levels but no skin test responsiveness or resistance against infection.

Protective effect of glucan against visceral leishmaniasis in hamsters.

The effect of pre- or posttreatment with glucan, a reticuloendothelial stimulant, on the course of Leishmania donovani infection was assessed in highly susceptible hamsters. Intravenous administration of glucan before or after L. donovani infection significantly suppressed proliferation of amastigote-stage parasites in liver and spleen. Glucan-activated peritoneal macrophages in vitro also significantly reduced multiplication of the intracellular parasite. Ultrastructural studies revealed a well-defined hepatic granulomatous response to glucan, with hypertrophic Kupffer cells and reduced numbers of intracellular parasites compared to the control group. In additional studies, groups of hamsters were immunized by intravenous injections of glucan with Formalin-killed promastigote-stage L. donovani cells and challenged 60 days after the last immunizing injection. This treatment regimen significantly prolonged the mean survival time of those hamsters which died after infection, relative to untreated control groups. Hamsters stimulated with the glucan-killed promastigote preparation also exhibited significant reductions in splenic amastigotes on days 10 and 21 postinfection compared with all other control groups, but on day 35, splenic amastigotes did not differ significantly from those of control animals. Our composite observations provide evidence for glucan-enhanced nonspecific resistance of hamsters to visceral leishmaniasis.

Glucan as an adjuvant for a murine Babesia microti immunization trial.

A vaccination protocol against murine Babesia microti infection, using glucan, a beta-1,3-glucopyranose derivative of yeast cell walls, and glutaraldehyde-fixed infected erythrocytes was evaluated. BALB/c mice were immunized intravenously four times at 2-day intervals with 2 X 10(8) fixed infected erythrocytes with and without glucan (0.45 mg). The mice were challenged 2 weeks after the last immunization with 10(8) viable infected erythrocytes. Peak parasitemia was significantly reduced (8.9 +/- 1.0%; P less than 0.001) in glucan-immunized mice as compared with nonimmunized controls (41.2 +/- 1.4%), glucan-treated controls (31.7 +/- 2.5%; P less than 0.05), or mice which received fixed infected erythrocytes without glucan (21.0 +/- 1.2%; P less than 0.01). Humoral and cellular immunity to B. microti was evaluated before challenge by measuring antibody titers and splenocyte blastogenic responses to B. microti antigens. The in vitro cellular response was inversely correlated with mean peak parasitemia (P less than 0.05). These observations demonstrate that glucan is an effective adjuvant in enhancing immunity to murine babesiosis.

Effects of malaria (Plasmodium berghei) on the maternal-fetal relationship in mice.

Plasmodium berghei infection was more severe in pregnant than in nonpregnant mice. Infection initiated on gestation day 7 resulted in rapidly increasing parasitemia and deaths of all pregnant mice within 12 days, while some nonpregnant mice survived until day 21 postinfection. When mice were infected on gestation day 12 or 14, a proportion of mice died before parturition; but some animals survived to deliver living pups. Reduced birthweights and increased spleen weight to body weight ratios were seen in pups from infected mice as compared with pups from uninfected animals. Histopathological abnormalities of placentae from infected animals included degeneration of the normal labyrinthine architecture and thickening of the trophoblast separating maternal and fetal blood vessels.

Trichomonas vaginalis: alternative pathway activation of complement.

Serum from adult human donors lysed Trichomonas vaginalis. The lytic effect was eliminated by heat-inactivation of serum (56 degrees C, 30 min). No serum donor exhibited significantly increased antibody against the parasite as measured by indirect immunofluorescence. Treatment of serum with ethylene glycol-bis (B-aminoethyl ether)-N, N-tetraacetic acid to eliminate classical pathway complement activation did not prevent C3 leads to C3i conversion in serum incubated with T. vaginalis. Release of complement products during alternative pathway activation may contribute to pathogenesis of trichomonal vaginitis.

Immunization against Leishmania donovani: glucan as an adjuvant with killed promastigotes.

Mice were immunized by a series of intravenous injections of formalin-killed Leishmania donovani promastigotes alone and combined with glucan, a beta 1,3 polyglucose derivative of baker's yeast. In three separate experiments animals were challenged with viable parasites on day 21, 40 or 80 after immunization. Mice which received dead parasites and glucan exhibited resistance against challenge up to 80 days after immunization. Animals which had been injected with glucan alone exhibited a lesser degree of resistance but injections of killed promastigotes alone conferred no measurable resistance against infection.

Glucan-enhanced immunogenicity of killed erythrocyte stages of Plasmodium berghei.

Intravenous injections of glucan simultaneously with Formalin-killed erythrocytic stages of Plasmodium berghei elicited a greater degree of resistance in mice against subsequent infection with viable parasites than injections of killed erythrocytic stages alone. In two experiments with P. berghei strain NK 65, 100% of mice immunized with the glucan-dead parasite preparation survived challenge, whereas only 28.6% of mice receiving dead parasites alone survived. In the third experiment, using P. berghei strain NYU-2, the same proportion of mice survived after immunization with glucan and dead parasites as with dead parasites alone (i.e., 10 of 11 in each group), but mice immunized with the glucan-dead parasite preparation experienced parasitemias of significantly less intensity and shorter duration than mice which received only dead parasites before infection. Inoculation of glucan alone or with normal erythrocytes conferred no protection against challenge.

Activation of the alternative complement pathway by Naegleria fowleri.

Naegleria fowleri amoebae were lysed by adult fresh human serum, and their multiplication was inhibited in culture medium supplemented with 10% fresh human serum. Heat inactivation (56 degrees C, 30 min) of serum abrogated these lytic and inhibitory effects. Absorption of human serum with amoebae failed to reduce immunoglobulin levels, and no specific antibody was detected in untreated or treated sera by counterimmunoelectrophoresis. Conversion of C3 and C3i occurred after incubation of n. fowleri with serum which had been treated with ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid, indicating activation of complement via the alternative pathway.

The human materno-foetal relationship in malaria: I. Identification of pigment and parasites in the placenta.

To facilitate investigations of the consequences of malarial infection during human pregnancy, several methods for the recognition of pigment and parasites in the placenta were evaluated. Pigment was visualized in infected blood smears and placental tissue using both white light and modified fluorescence microscopy. However, the characteristic pigment dots observed with fluorescent light were also apparent in unstained cryostat and deparaffinized placental sections, and following reaction with immunohistological reagents. Intact parasites were recognized immunohistologically in placental sections and blood smears using rabbit antisera to Plasmodium falciparum and P. berghei. Using these procedures, numerous erythrocytes containing parasites associated with small pigment dots were seen in intervillous spaces in heavily infected placentae. In these organs, larger irregular pigment aggregates were present within maternal cells which were shown to be monocytes by esterase staining. Pigment was also observed in the cytoplasm of the trophoblast and not infrequently in the mesenchymal stroma, but no intact parasites were observed within chorionic villi. These simple and sensitive methods thus confirm placental localization of parasites and pigment. Furthermore, the finding of pigment in all Gambian placentae examined, of which seven were thought initially to be uninfected, indicates that malaria may complicate pregnancy more frequently than hiterto anticipated.

Naegleria fowleri in chick embryos. Effects of embryo age and incubation temperature, and the infectivity of embryo-derived amebae for mice.

Chick embryos were infected with Naegleria fowleri which was initially isolated from an ultimately fatal human case. Following inoculation of equivalent numbers of amebae on the chorioallantoic membrane, younger embryos died earlier than older embryos infected at the same time. Incubation of infected embryos at 32 degrees C prolonged survival only slightly in comparison with those at 37 degrees C. N. fowleri maintained for more than 25 serial passages in chick embryos retained infectivity for mice and the ability to convert to the biflagellate form in vitro.

Primary amebic meningoencephalitis.

Primary amebic meningoencephalitis a rapidly fatal CNS infection caused by the free-living ameba Naegleria fowleri. The disease is acquired by swimming in fresh water and is being recognized with increasing frequency. Results of early diagnosis and treatment with amphotericin and other drugs suggest therapeutic optimism. Epidemiological surveys have shown the organism to be commonly present in fresh-water lakes in the warmer parts of the world. Prompt recognition and treatment is vital.

Interference of human spermatozoal motility by trichomonas vaginalis.

Human spermatozoa were exposed to concentrations of live Trichomonas vaginalis varying from 10(4) to 10(7) organisms per ml. A striking decrease in spermatozoal motility ensued. The possible role of trichomoniasis in human reproductive failure is considered.

PIP: Trichomonas vaginalis is a flagellated parasite often found as an occult resident of the genital tract of sexually active women. A pure culture of T. vaginalis was isolated from urine and maintained at 37 degrees C by in vitro passage in Diamond's broth. Fresh semen specimens were incubated in .2 ml of serial dilutions of T. vaginalis culture. Spermatozoal motility was observed immediately on mixing and again after 2, 4, and 6 hours of incubation. Marked lessening of spermatozoal activity was observed but no agglutination. The motion of T. vaginalis continued normally. In control samples containing saline or Diamond's broth, only minor retardation of spermatozoal activity was noted during 6 hours of incubation. Filtered Diamond's broth, which prior to filtration had supported a dense concentration of protozoa, when added to equal volumes of fresh human ejaculate caused a similar retardation of spermatozoal activity. It was speculated that some metabolic metabolites of T. vaginalis decrease spermatozoal activity. T. vaginalis should be included among other factors that lead to infertility. In subfertile couples eradication of T. vaginalis may enhance fertility.