Enhancement of experimental cutaneous leishmaniasis by Leishmania extract: identification of a disease-associated antibody specificity.

BACKGROUND: Both Leishmania braziliensis and Leishmania amazonensis induce cutaneous disease when injected in the skin of BALB/c mice. However, L. amazonensis may also visceralize in that strain of mice, infecting mainly the liver and spleen. In addition, whereas BALB/c mice die with a progressive cutaneous disease when infected by L. amazonensis, the infection by L. braziliensis is spontaneously cured. In a previous work, we have found that intravenous injections of L. amazonensis amastigote extract (LaE) potentiated a L. braziliensis infection in BALB/c mice, and that this infection-promoting activity could be inhibited by the addition of protease inhibitors to the extract. METHODS: In order to detect markers of disease evolution, in the present work we analyzed the specificity of the anti-L. amazonensis antibody response of L. braziliensis-infected BALB/c mice injected intravenously with saline or LaE, supplemented or not with protease inhibitors, by the Western blot technique. RESULTS: IgG1 antibodies recognizing an antigen with apparent molecular weight of 116 kDa were specifically detected in BALB/c mice that had been turned susceptible to L. braziliensis infection by injections of LaE. CONCLUSION: A Th2 immune response (IgG1 antibody-producing) against this 116 kDa antigen, therefore, could be associated with susceptibility to severe Leishmania infection.

Leishmania braziliensis and Leishmania amazonensis amastigote extracts differ in their enhancement effect on Leishmania infection when injected intradermally.

BACKGROUND: It has been reported that repeated intravenous injections of a relatively large amount of Leishmania amazonensis amastigote extract (LaE) in BALB/c mice exacerbates the infection of these mice by Leishmania braziliensis. The identification of the extract active principle(s) through physicochemical purification often involves dilution and losses of protein in the course of successive purification procedures. The large amount of the extract required to induce the phenomenon, therefore, hinders the carrying out of experiments aimed at identifying the active molecule(s) through extract purification. In the present work, a dose-response experiment was done to find out if smaller amounts of LaE than that necessary to be used by the intravenous route would reproduce the phenomenon when injected by the intradermal route. In addition, it was also investigated whether a Leishmania braziliensis amastigote extract (LbE) would exert the same effect and whether the effect would occur in C57BL/6 mice. RESULTS: It was found that a single injection of either LaE or LbE containing 5 µg of protein was capable of enhancing the infection in BALB/c but not in C57BL/6 mice. In addition, it was observed that the largest tested doses of LbE (containing 30 and 180 µg of protein) failed to enhance the infection by L. braziliensis, whereas all doses of LaE enhanced equally that infection. CONCLUSIONS: Those results indicate the possible existence in LbE, and not in LaE, of molecules that interfere with the extract infection-enhancing activity when it is injected in large amounts, and that the inoculation of Leishmania extracts through the intravenous and intradermal routes potentiate the infection by L. braziliensis through the same mechanism.

Temporal distribution of positive results of tests for detecting Leishmania infection in stray dogs of an endemic area of visceral leishmaniasis in the Brazilian tropics: a 13 years survey and association with human disease.

Human visceral leishmaniasis occurs in periodic waves in endemic areas of Brazil. In this study we followed the prevalence of human visceral leishmaniasis and of Leishmania infantum infection in stray dogs of an endemic area of visceral leishmaniasis at periods of time between 1997 and 2010. Prevalence of human visceral leishmaniasis had two peaks (40 cases) in 1997 and 2006 with sharp declines to 2 cases in 2001 and to 5 cases in 2008. Similar fluctuations were also observed in the occurrence of positive spleen culture and anti-Leishmania serology in dogs, although the proportion of dogs with active spleen parasitism remained relatively high even in the periods of low prevalence of human disease. These observations support the notion that stray dogs may constitute a renewable source of parasites, capable of sustaining the persistence of the infection in urban areas, even in periods of low transmission by phlebotomines.

Babassu aqueous extract (BAE) as an adjuvant for T helper (Th)1-dependent immune responses in mice of a Th2 immune response-prone strain.

BACKGROUND: The aqueous extract of a Brazilian palm-tree fruit - the babassu - (BAE) exerts a clear immunostimulative activity in vivo. In the present work, the possibility that BAE can promote Th1 immune responses in mice of a Th2 immune response-prone strain - the BALB/c was investigated. BAE itself, and preparations consisting of Leishmania amazonensis promastigote extract (LE), adsorbed or not to Al(OH)3, and in the presence or not of BAE, were used as immunogens. LE and Al(OH)3 have been shown to preferentially elicit Th2 immune responses. RESULTS: The addition of BAE to LE-containing immunogenic preparations, adsorbed or not to Al(OH)3, clearly promoted the in vitro production of interferon γ (IFN-γ), a major Th1-dependent cytokine, and not of interleukin (IL-)4 (a Th2-dependent cytokine), by LE-stimulated splenocytes of immunized BALB/c mice. It also promoted the in vivo formation of IgG2a anti-LE antibodies. However, immunization with LE by itself led to an increased production of IL-4 by LE-stimulated splenocytes, and this production, albeit not enhanced, was not reduced by the addition of BAE to the immunogen. On the other hand, the IL-4 production by LE-stimulated splenocytes was significantly lower in mice immunized with a preparation containing Al(OH)3-adsorbed LE and BAE than in mice immunized with the control preparation of Al(OH)3-adsorbed LE without BAE. Moreover, an increased production of IFN-γ, and not of IL-4, was observed in the culture supernatants of splenocytes, from BAE-immunized mice, which were in vitro stimulated with BAE or which received no specific in vitro stimulus. No differences in IL-10 (an immunoregulatory cytokine) levels in the supernatants of splenocytes from mice that were injected with BAE, in relation to splenocytes from control mice, were observed. The spontaneous ex vivo production of NO by splenocytes of mice that had been injected with BAE was significantly higher than the production of NO by splenocytes of control mice. CONCLUSIONS: Based on the results described above, BAE, or biologically active molecules purified from it, should be further investigated as a possible adjuvant, in association or not with aluminium compounds, for the preferential induction of Th1-dependent immune responses against different antigens in distinct murine strains and animal species.

Enhancement of experimental cutaneous leishmaniasis by Leishmania molecules is dependent on interleukin-4, serine protease/esterase activity, and parasite and host genetic backgrounds.

Most inbred strains of mice, like the BALB/c strain, are susceptible to Leishmania amazonensis infections and resistant to Leishmania braziliensis infections. This parasite-related difference could result from the activity of an L. amazonensis-specific virulence factor. In agreement with this hypothesis, it is shown here that the intravenous injection of BALB/c mice with L. amazonensis amastigote extract (LaE) but not the L. braziliensis extract confers susceptibility to L. braziliensis infection. This effect was associated with high circulating levels of IgG1 anti-L. amazonensis antibodies and with an increase in interleukin-4 (IL-4) production and a decrease in gamma interferon production by draining lymph node cells. Moreover, the effect was absent in IL-4-knockout mice. The biological activity in the LaE was not mediated by amphiphilic molecules and was inhibited by pretreatment of the extract with irreversible serine protease inhibitors. These findings indicate that the LaE contains a virulence-related factor that (i) enhances the Leishmania infection by promoting Th2-type immune responses, (ii) is not one of the immunomodulatory Leishmania molecules described so far, and (iii) is either a serine protease or has an effect that depends on that protease activity. In addition to being Leishmania species specific, the infection-enhancing activity was also shown to depend on the host genetic makeup, as LaE injections did not affect the susceptibility of C57BL/6 mice to L. braziliensis infection. The identification of Leishmania molecules with infection-enhancing activity could be important for the development of a vaccine, since the up- or downmodulation of the immune response against a virulence factor could well contribute to controlling the infection.

Respiratory allergy to Blomia tropicalis: immune response in four syngeneic mouse strains and assessment of a low allergen-dose, short-term experimental model.

BACKGROUND: The dust mite Blomia tropicalis is an important source of aeroallergens in tropical areas. Although a mouse model for B. tropicalis extract (BtE)-induced asthma has been described, no study comparing different mouse strains in this asthma model has been reported. The relevance and reproducibility of experimental animal models of allergy depends on the genetic background of the animal, the molecular composition of the allergen and the experimental protocol. OBJECTIVES: This work had two objectives. The first was to study the anti-B. tropicalis allergic responses in different mouse strains using a short-term model of respiratory allergy to BtE. This study included the comparison of the allergic responses elicited by BtE with those elicited by ovalbumin in mice of the strain that responded better to BtE sensitization. The second objective was to investigate whether the best responder mouse strain could be used in an experimental model of allergy employing relatively low BtE doses. METHODS: Groups of mice of four different syngeneic strains were sensitized subcutaneously with 100 microg of BtE on days 0 and 7 and challenged four times intranasally, at days 8, 10, 12, and 14, with 10 microg of BtE. A/J mice, that were the best responders to BtE sensitization, were used to compare the B. tropicalis-specific asthma experimental model with the conventional experimental model of ovalbumin (OVA)-specific asthma. A/J mice were also sensitized with a lower dose of BtE. RESULTS: Mice of all strains had lung inflammatory-cell infiltration and increased levels of anti-BtE IgE antibodies, but these responses were significantly more intense in A/J mice than in CBA/J, BALB/c or C57BL/6J mice. Immunization of A/J mice with BtE induced a more intense airway eosinophil influx, higher levels of total IgE, similar airway hyperreactivity to methacholine but less intense mucous production, and lower levels of specific IgE, IgG1 and IgG2 antibodies than sensitization with OVA. Finally, immunization with a relatively low BtE dose (10 microg per subcutaneous injection per mouse) was able to sensitize A/J mice, which were the best responders to high-dose BtE immunization, for the development of allergy-associated immune and lung inflammatory responses. CONCLUSIONS: The described short-term model of BtE-induced allergic lung disease is reproducible in different syngeneic mouse strains, and mice of the A/J strain was the most responsive to it. In addition, it was shown that OVA and BtE induce quantitatively different immune responses in A/J mice and that the experimental model can be set up with low amounts of BtE.

Comparison between indirect enzyme-linked immunosorbent assays for Anaplasma marginale antibodies with recombinant major surface protein 5 and initial body antigens.

Indirect enzyme-linked immunosorbent assays (ELISAs) based on recombinant major surface protein 5 (rMSP5) and initial body (IB) antigens from a Brazilian isolate of Anaplasma marginale were developed to detect antibodies against this rickettsia in cattle. Both tests showed the same sensitivity (98.2%) and specificities (100% for rMSP5 and 93.8% for IB ELISA) which did not differ statistically. No cross-reactions were detected with Babesia bigemina antibodies, but 5 (rMSP5 ELISA) to 15% (IB ELISA) of cross-reactions were detected with B. bovis antibodies. However, such difference was not statistically significant. Prevalences of seropositive crossbred beef cattle raised extensively in Miranda county, state of Mato Grosso do Sul, Brazil, were 78.1% by rMSP5 ELISA and 79.7% by IB ELISA. In the analysis of sera from dairy calves naturally-infected with A. marginale, the dynamics of antibody production was very similar between both tests, with maternal antibodies reaching the lowest levels at 15-30 days, followed by an increase in the mean optical densities in both ELISAs, suggesting the development of active immunity against A. marginale. Results showed that all calves were seropositive by one-year old, characterizing a situation of enzootic stability. The similar performances of the ELISAs suggest that both tests can be used in epidemiological surveys for detection of antibodies to A. marginale in cattle.