[Visceral leishmaniasis: a study on phlebotomine sand flies and canine infection in Montes Claros, State of Minas Gerais]. / Leishmaniose visceral: estudo de flebotomíneos e infecção canina em Montes Claros, Minas Gerais.

Visceral leishmaniasis in Brazil was initially associated with rural areas. However, due to several environmental modifications such as deforestation, urbanization and intense migratory processes, there has been an expansion of endemic areas, leading to urbanization of the disease, mainly in the central and northeastern regions of Brazil. In the municipality of Montes Claros, located in the north of the state of Minas Gerais, an epidemiological survey on VL was carried out. A canine serological inquiry was carried out in 2002 and an entomological survey, using luminous CDC traps, was performed from September 2002 to August 2003. Canine VL prevalence showed an average infection rate of approximately 5%. An estimated 16 species comprised the phlebotomine sand fly fauna, based on a total of 1043 specimens. The predominant species was Lutzomyia longipalpis with a rate of 74%, suggesting its participation in the transmission of VL in the municipality of Montes Claros.

Standardization of a rapid immunochromatographic test with the recombinant antigens K39 and K26 for the diagnosis of canine visceral leishmaniasis.

The serological diagnosis of canine visceral leishmaniasis (CVL) remains problematic because there areno reliable commercially available tests. Most laboratories use domestically prepared tests such as the enzyme-linked immunosorbent assay (ELISA) or the indirect immunofluorescent antibody test (IFAT). We evaluated rapid immunochromatographic (RICH) test kits for the diagnosis of CVL. The tests were assembled with either Leishmania chagasi recombinant antigens K39 or K26 and with either gold-labelled Staphylococcus aureus protein A or Streptococcus pyogenes protein G. Fifty sera from dogs with CVL, 14 sera from dogs with Chagas disease, and 50 sera from normal healthy dogs were tested. The results show that the RICH test using recombinant antigen K39 has a sensitivity of 96% and 100% specificity for the diagnosis of CVL. No significant differences were observed in the tests assembled with either protein A or protein G. The RICH tests using recombinant antigen K26 were equally specific but less sensitive than those using K39. However, the 2 antigens complemented each other and increased the overall sensitivity of the test. Because of its simplicity and performance the RICH test is a quick and reliable alternative for the diagnosis of CVL either in conventional laboratories or for remote areas where laboratories are not readily accessible for conventional assays.

Identification and purification of immunogenic proteins from nonliving promastigote polyvalent Leishmania vaccine (Leishvacin ).

Immunogenic proteins from nonliving promastigote polyvalent Leishmania vaccine against American tegumentary leishmaniasis (Leishvacin ), produced by Biobr s (Biochemistry of Brazil ), Montes Claros, State of Minas Gerais, Brazil, were identified and purified by polyacrylamide electrophoresis gel and electroelution. C57BL/10 mice were vaccinated with proteins with estimated molecular weights of 42, 46, 63, 66, 73, 87, 97, and 160kDa in three doses of 30 g of each protein at 15-day intervals combined with 250 microg of Corynebacterium parvum followed by a challenge infection with 10(5) infective promastigotes from Leishmania (Leishmania) amazonensis. The ability of these proteins to induce immune response and protection was analyzed. No statistical difference was observed in the level of IFN-gamma induced by proteins in vaccinated groups in comparison with control groups. Six months after challenge infection, protection levels of 28.57; 42.86; 57.14; 42.86; 42.86, 57.14; 42.86 and 57.14% were demonstrated for each purified protein.

Evaluation of an immunochemotherapeutic protocol constituted of N-methyl meglumine antimoniate (Glucantime) and the recombinant Leish-110f + MPL-SE vaccine to treat canine visceral leishmaniasis.

The evaluation of the efficacy of an immunochemotherapy protocol to treat symptomatic dogs naturally infected with Leishmania chagasi was studied. This clinical trial had the purpose to test the combination of N-methyl meglumine antimoniate (Glucantime and the second generation recombinant vaccine Leish-110f plus the adjuvant MPL-SE to treat the canine leishmaniasis (CanL). Thirty symptomatic naturally infected mongrel dogs were divided into five groups. Animals received standard treatment with Glucantime or treatment with Glucantime Leish-110f + MPL-SEas immunochemotherapy protocol. Additional groups received Leish-110f + MPL-SE only, MPL-SE only, or placebo. Evaluation of haematological, biochemical (renal and hepatic function) and plasmatic proteins, immunological (humoral and cellular immune response) and the parasitological test revealed improvement of the clinical parameters and parasitological cure in dogs in both chemotherapy alone and immunochemotherapy cohorts. However, the immunotherapy and immunochemotherapy cohorts had reduced number of deaths, higher survival probability, and specific cellular reactivity to leishmanial antigens, in comparison with chemotherapy cohort only and control groups (adjuvant alone and placebo). These results support the notion of using well-characterized recombinant vaccine as an adjunct to improve the current chemotherapy of CanL.

Immunogenicity in dogs of three recombinant antigens (TSA, LeIF and LmSTI1) potential vaccine candidates for canine visceral leishmaniasis.

Control of canine visceral leishmaniasis (VL) remains a difficult and serious problem mostly because there is no reliable and effective vaccine available to prevent this disease. A mixture of three recombinant leishmanial antigens (TSA, LeIF and LmSTI1) encoded by three genes highly conserved in the Leishmania genus have been shown to induce excellent protection against infection in both murine and simian models of cutaneous leishmaniasis. A human clinical trial with these antigens is currently underway. Because of the high degree of conservation, these antigens might be useful vaccine candidates for VL as well. In the present study, using the dog model of the visceral disease, we evaluated the immunogenicity of these three antigens formulated with two different adjuvants, MPL-SE and AdjuPrime. The results were compared with a whole parasite vaccine formulated with BCG as the adjuvant. In order to investigate if sensitization with the recombinant antigens would result in recognition of the corresponding native parasite antigens upon infection, the animals were exposed for four weeks after the termination of the immunization protocol with the recombinant antigens to a low number of L. chagasi promastigotes, an etiological agent of VL. Immune response was evaluated by quantitative ELISA in the animal sera before and after exposure to the viable parasites. Both antigen specific IgG1 and IgG2 antibody levels were measured. Immunization of dogs with the recombinant antigens formulated in either MPL-SE or AdjuPrime resulted in high antibody levels particularly to LmSTI1. In addition, this immunization although to low levels, resulted in the development of antibody response to the whole parasite lysate. Importantly, experimental exposure with low numbers of culture forms of L. chagasi promastigotes caused a clear boost in the immune response to both the recombinant antigens and the corresponding native molecules. The boost response was predominantly of the IgG2 isotype in animals primed with the recombinant antigens plus MPL-SE. In contrast, animals primed with the recombinant antigens formulated in AdjuPrime as well as animals vaccinated with crude antigen preparation responded with mixed IgG1/IgG2 isotypes. These results point to the possible use of this antigen cocktail formulated with the adjuvant MPL-SE in efficacy field trials against canine VL.