Wilson Mayrink

Epidemiology of canine visceral leishmaniosis in the endemic area of Montes Claros Municipality, Minas Gerais State, Brazil

The Montes Claros City is located in an endemic area for visceral leishmaniosis in the Minas Gerais State, Brazil. With the implementation of a program for the control of visceral leishmaniosis in 1994, a sectional study was carried out to evaluate the infection by viscerotropic Leishmania in the population of dogs from Montes Claros, basically using indirect immunofluorescence antibody test (IFAT). Blood samples were collected on filter paper from 33,937 dogs, representing 96.1% of the canine local population. The prevalence for visceral leishmaniosis was found to be 9.7% in the municipality, being 9.9% in the urban area and 8.8% in the rural area. The annual incidence showed to be 64.3/1000 dogs. Prevalence of infection was not correlated with dogs age. The most affected breeds were: Boxer (24.6%) and Cocker (26.9%); Mongrel dogs had a prevalence of 7.8%. Short-hair animals had a prevalence of 11.9%, while long-furred animals had a prevalence of 8.9%. The isoenzymatic profile indicated that Leishmania (Leishmania) chagasi was the visceral leishmaniosis etiological agent in Montes Claros City, Minas Gerais State, Brazil. The main geographical areas for the parasite transmission were identified, and control measures were immediately started. The role of the dog as a reservoir for L. chagasi was confirmed. It was demonstrated that short-furred animals are at a higher risk of acquiring visceral leishmaniosis than the long-furred dogs.

Epidemiology of dermal leishmaniasis in the Rio Doce Valley, State of Minas Gerais, Brazil.

Dermal leishmaniasis is prevalent in the predominantly settled agricultural areas in the Rio Doce Valley in the eastern part of the State of Minas Gerais, Brazil. The disease has been recorded almost equally in both sexes. Cases have been confirmed in all age groups but youths aged ten to 14 years form the population segment at greatest risk to infection. Cases of single cutaneous lesions, multiple cutaneous lesions and muco-cutaneous lesions have been recorded in the area. Isolates of parasites include representatives of the Leishmania mexicana and L. braziliensis complexes and at least one parasite that does not fit into either category. Infections have not been detected in small mammals (mainly rodents) but about 3% of dogs are infected. The phlebotomine fauna includes no species (or close relatives of species) previously incriminated as vectors of mexicana and braziliensis infections in Brazil. In the complex and confusing epidemiological situation in the Rio Doce Valley it seems unwise to apply traditional specific names to Leishmania of the area.

A field trial of a vaccine against American dermal leishmaniasis

A field trial was carried out in the eastern part of the State of Minas Gerais (Brazil) of a vaccine containing killed promastigotes of five stocks of Leishmania. Tests with Montenegro antigen showed that a high proportion of the vaccinated persons became positive within three months, but circulating antibodies were not detected. A proportion of those vaccinated continued to give positive Montenegro reactions for up to three years. Lymphocyte sensitivity tests carried out, on a small sample, three years after vaccination were positive and gave no evidence of immunological depression. No cases of cutaneous or mucocutaneous leishmaniasis occurred in the trial area during the three years of observations.

Systemic and compartmentalized immune response in canine visceral leishmaniasis

Human visceral leishmaniasis (VL) and canine visceral leishmaniasis (CVL) are the most important emerging diseases with high prevalence in Latin American countries and are mainly caused by Leishmania (L.) chagasi (Syn=L. infantum). CVL has a great impact on Brazilian public health because domestic dogs are the most important VL peri-domicile reservoirs in both urban and peri-urban areas. Our findings highlight the complexity of cellular immunological events related to the natural infection from dogs by L. chagasi, additionally correlating major peripheral blood phenotypic markers with clinical status and tissues parasite density. Our main results demonstrated that lower frequency of circulating B cells and monocytes are important markers of severe CVL, whereas increased levels of CD8+ lymphocytes appear to be the major phenotypic feature of asymptomatic disease. Determination of the isotypes patterns during CVL demonstrated that asymptomatic dogs and those with low parasitism are associated with an increase of IgG1, while the symptomatic dogs and those with high parasitism are associated with an increase of IgG, IgG2, IgM, IgA and IgE immunoglobulins. Pioneer findings obtained by our group showed a correlation between clinical status of CVL with degree of tissue parasite density. This data demonstrated that asymptomatic dogs presented low parasitism while symptomatic dogs are associated with high parasite load in various tissues such as skin, bone marrow and spleen. We have also investigated the association between tissue parasitism and CVL clinical forms. Regardless of clinical status, skin and spleen are the major sites of high parasite density during ongoing CVL. Furthermore, we demonstrated that bone marrow and spleen parasite density are the most reliable parasitological markers to decode the clinical status of CVL. In this article, we have reviewed some aspects of the histopathological and immunological events occurring in natural and experimental L. chagasi/L. infantum infection, pointing out the main L. chagasi-parasitized tissue. We have discussed the importance of the association between parasite density, immunological/histopathological aspects and clinical status of the CVL, their current applications, challenges for the future and potential opportunities in CVL research.

Flow Cytometric Determination of Cellular Sources and Frequencies of Key Cytokine-Producing Lymphocytes Directed against Recombinant LACK and Soluble Leishmania Antigen in Human Cutaneous Leishmaniasis

ABSTRACT Leishmaniasis, caused by infection with the protozoan parasiteLeishmania, affects millions of individuals worldwide, causing serious morbidity and mortality. This study directly determined the frequency of cells producing key immunoregulatory cytokines in response to the recombinant antigen Leishmania homolog of receptors for activated kinase C (LACK) and soluble leishmania antigen (SLA), and it determined relative contributions of these antigens to the overall cytokine profile in individuals infected for the first time with Leishmania braziliensis. All individuals presented with the cutaneous clinical form of leishmaniasis and were analyzed for proliferative responses to LACK antigen and SLA, frequency of lymphocyte subpopulations (analyzed ex vivo), and antigen-induced (LACK and SLA) cytokine production at the single-cell level (determined by flow cytometry). The following were determined. (i) The Th1-type response previously seen in patients with cutaneous leishmaniasis is due to gamma interferon (IFN-γ) production by several different sources, listed in order of contribution: CD4+ T lymphocytes, CD4−, CD8− lymphocytes, and CD8+ T lymphocytes. (ii) SLA induced a higher frequency of lymphocytes producing IFN-γ and tumor necrosis factor alpha (TNF-α) than did LACK. (iii) LACK induced an activation of monocyte populations as reflected by an increased percentage of CD14-positive cells. (iv) Neither SLA nor LACK induced detectable frequencies of cells producing interleukin-4 (IL-4) or IL-5. These data demonstrated a multifaceted immune response to SLA in human leishmaniasis involving Th1 CD4+ T lymphocytes (IFN-γ+ and IL-10−/IL-4−), Tc1 CD8+ T cells (IFN-γ+, and IL-10−/IL-4−), and a high frequency of TNF-α-producing lymphocytes. Moreover, it was determined that the recombinant antigen LACK acts as a weak inducer of Th1-type lymphocyte responses compared to SLA.

An experimental vaccine against American dermal leishmaniasis: experience in the State of Espírito Santo, Brazil

A vaccine prepared from killed and sonicated promastigotes of five Brazilian strains of Leishmania was used during an epidemic of American dermal leishmaniasis that occurred in Viana county, State of Espírito Santo, Brazil. Initially, all of the participants in the vaccination programme had negative reactions to Montenegro antigen. Forty days after the last dose of vaccine had been given, 87.6% of the 216 vaccinated individuals had become Montenegro-positive whereas the 266 unvaccinated persons remained Montenegro-negative. The study area had an unstable population and details are given about the human population changes that occurred during the two-year study period. Taking into account population movements, 1.5% of those vaccinated and 6.4% of the unvaccinated group developed dermal leishmanial lesions by the end of the first year. At the end of the second year, 1.7% of those vaccinated and 8.9% of the unvaccinated group had become infected. The difference in infection rates of the two groups is statistically significant at both the end of the first and second years of observation. Diagnosis of the disease(s) was based on the clinical appearance of lesions combined with parasitological and/or immunological evidence and subsequent responses to treatment. The experience gained in Viana also provided information about the storage and administration of the experimental vaccine which have been used in mounting a randomized clinical trial.

Evaluation of enzyme-linked immunosorbent assay using crude Leishmania and recombinant antigens as a diagnostic marker for canine visceral leishmaniasis

The performances of ELISA assays with different antigen preparations, such as Leishmania amazonensis or L. chagasi lysates and the recombinant antigens rK-39 and rK-26, were compared using sera or eluates from dried blood collected on filter paper to detect anti-Leishmania antibodies in dogs from a visceral leishmaniasis-endemic area in Brazil. Of 115 IFAT-reactive dogs at 1:40 titre, 106 (92.2%) were positive in parasitological exams (skin and/or spleen). These animals were compared to healthy animals (n = 25), negative for IFAT at a titre of 1:40 and parasitological exams. The sensitivities of crude and recombinant antigens were similar and remarkably high for both sera and eluates (97-100%). Specificity was higher than 96% for sera and eluates for different antigens, except for L. chagasi antigen using eluates (88%). Concordance values among the tests were higher either for sera or eluates (J = 0.95-1.00). High concordances were observed between sera and eluates tested with different antigens (kappa = 0.93-0.97). Crude and recombinant antigens identified different clinical phases of canine leishmaniasis. These results show that eluates could be used in canine surveys to identify L. chagasi infection. Recombinant antigens added little when compared to crude antigen in identifying positive dogs. Cross-reactivity with other diseases whose distribution often overlaps VL-endemic areas is a limitation of crude antigen use however.

Immunochemotherapy for cutaneous leishmaniasis: a controlled trial using killed Leishmania (Leishmania) amazonensis vaccine plus antimonial

Background Leishmaniasis is endemic in 88 countries in the world, and 350 million individuals are at risk of acquiring the disease. Treatment for American cutaneous leishmaniasis (ACL) is long, expensive, and associated with important side‐effects.

Phenotypic features of circulating leucocytes as immunological markers for clinical status and bone marrow parasite density in dogs naturally infected by Leishmania chagasi

Canine visceral leishmaniasis (CVL) manifests itself as a broad clinical spectrum ranging from asymptomatic infection to patent severe disease. Despite relevant findings suggesting changes on lymphocytes subsets regarding the CVL clinical forms, it still remains to be elucidated whether a distinct phenotypic profile would be correlated with degree of tissue parasite density. Herein, we have assessed the correlation between the clinical status as well as the impact of bone marrow parasite density on the phenotypic profile of peripheral blood leucocytes in 40 Brazilian dogs naturally infected by Leishmania chagasi. Our major findings describe the lower frequency of B cells and monocytes as the most important markers of severe CVL. Our main statistically significant findings reveal that the CD8+ T cell subset reflects most accurately both the clinical status and the overall bone marrow parasite density, as increased levels of CD8+ lymphocytes appeared as the major phenotypic feature of asymptomatic disease and dogs bearing a low parasite load. Moreover, enhanced major histocompatibility complex (MHC)‐II density as well as a higher CD45RB/CD45RA expression index seems to represent a key element to control disease morbidity. The association between clinical status, bone marrow parasitism and CD8+ T cells re‐emphasizes the role of the T cell‐mediated immune response in the resistance mechanisms during ongoing CVL. Higher levels of circulating T lymphocytes (both CD4+ and CD8+ T cells) and lower MHC‐II expression by peripheral blood lymphocytes seem to be the key for the effective immunological response, a hallmark of asymptomatic CVL.

Evaluation of the stability and immunogenicity of autoclaved and nonautoclaved preparations of a vaccine against American tegumentary leishmaniasis

This study was designed to evaluate the immunogenicity of autoclaved and nonautoclaved preparations of a vaccine composed of whole antigens from killed promastigotes of Leishmania amazonensis. Leishmanin skin-test (LST)-negative volunteers were immunized with either autoclaved or nonautoclaved vaccine preparations (32 and 36 subjects, respectively) that had been maintained at 4 degrees C for one year before the onset of this trial. Immunological tests were performed two days before and 40 days after vaccination. The LST conversion rates induced by the autoclaved and nonautoclaved vaccines were significantly different: 59% and 83%, respectively. Leishmania antigen-stimulated proliferative responses of peripheral blood mononuclear cells (PBMC) were significantly higher after vaccination than before vaccination in both groups. The CD8+ subset was predominant over the CD4+ subset among the leishmania-reactive cells after vaccination in both groups. The production of IFN-gamma by the leishmania antigen-stimulated PBMC was significantly higher after vaccination than before vaccination in the group receiving the nonautoclaved vaccine but not in the autoclaved vaccine group. IL-2 was found both before and after vaccination with no differences between its levels in these time points in either group. IL-4 was not detected for either group during the study period.