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.

Relationship between canine visceral leishmaniosis and the Leishmania (Leishmania) chagasi burden in dermal inflammatory foci.

The skin is the first point of contact with organisms of the genus Leishmania from sand fly vectors, and apparently normal skin of sick dogs harbours amastigote forms of Leishmania chagasi. In relation to canine visceral leishmaniosis (CVL), the ear skin was examined in 10 uninfected dogs (UDs) and in 31 dogs dogs naturally infected with L. chagasi. The infected animals consisted of 10 symptomless dogs (SLDs), 12 mildly affected dogs (MADs) and nine affected dogs (ADs). A higher parasite burden was demonstrated in ADs than in SLDs by anti-Leishmania immunohistochemistry (P<0.01), and by Leishman Donivan Unit (LDU) indices (P=0.0024) obtained from Giemsa-stained impression smears. Sections stained with haematoxylin and eosin demonstrated a higher intensity of inflammatory changes in ADs than in SLDs (P<0.05), and in the latter group flow cytometry demonstrated a correlation (P=0.05/r=0.7454) between the percentage of CD14(+) monocytes in peripheral blood and chronic dermal inflammation. Extracellular matrix assessment for reticular fibres by staining of sections with Masson trichrome and Gomori ammoniacal silver demonstrated a decrease in collagen type I and an increase in collagen type III as the clinical signs increased. The data on correlation between cellular phenotypes and histological changes seemed to reflect cellular activation and migration from peripheral blood to the skin, mediated by antigenic stimulation. The results suggested that chronic dermal inflammation and cutaneous parasitism were directly related to the severity of clinical disease.

Flow cytometric assay in peripheral blood of dogs--reference values for leukocytes from Brazilian beagles.

Use of domestic reference values in the flow cytometry analysis is known to improve its accuracy by integrating local variations as gender, race and age. Up to date application of flow cytometry in veterinary medicine has been limited to describe the percentual values just for peripheral lymphocytes subsets of blood. We now report establishment of reference values for a wide range of proportional and absolute numbers of peripheral blood leukocytes, including T cells subsets, B cells, monocytes and eosinophils, applicable to the healthy population of Beagles in Brazil and other regions with similar demographic characteristics. Normal reference values were also established to estimate the gender-related differences. This information will provide clinical aid in the evaluation of immunologic status as well as standard values for experimental animals of dogs from Brazil and other similar regions.

A randomized double-blind placebo-controlled trial to evaluate the immunogenicity of a candidate vaccine against American tegumentary leishmaniasis.

This study was aimed at evaluating the immunogenicity of a vaccine composed of killed Leishmania amazonensis promastigotes using several different protocols in a randomized, double-blind and controlled trial design in order to select one of them for further efficacy trials. One hundred and fourteen leishmanin skin test (LST)-negative healthy volunteers were allocated into eight groups that received either two or three deep intramuscular injections of vaccine at doses of 180, 360 and 540 microg or similar injections of placebo. Cell-mediated immune responses were evaluated before and after vaccination by means of LST as well as proliferative responses and cytokine production in Leishmania antigen-stimulated peripheral blood mononuclear cell cultures. The majority of the subjects who actually received vaccine converted to positive LST (89.5%). On the other hand, none of the subjects who received placebo converted to positive LST. Proliferative responses and production of interferon-gamma and interleukin-2 were significantly higher after vaccination than before vaccination in all groups, including those that received placebo. The dose of 360 microg provided the highest LST conversion rate (100%), as well as the greatest increase in interferon-gamma and interleukin-2 production after vaccination.

Impact of canine control on the epidemiology of canine and human visceral leishmaniasis in Brazil.

Brazil is the only country endemic for zoonotic visceral leishmaniasis (ZVL) that regularly conducts epidemiologic and prophylactic control programs that involve the treatment of human cases, insect vector control, and the removal of seropositive infected dogs. This report reviews 60 studies reporting data on the efficacy of these recommended control tools and concludes that in Brazil 1) eradication of the disease in Minas Gerais was achieved by the concomitant use of the three control methods, 2) although seropositivity by an immunofluorescent assay is not completely related to infectiousness, the removal of seropositive dogs leads to a significant reduction of canine and human incidence, 3) improvement of the sensitivity of the diagnostic tool used for canine control should optimize the efficacy of control, and 4) although difficult and expensive, the public health dog control campaigns performed in Brazil reduced the incidence of ZVL and should be maintained since treatment of dogs is an unrealistic intervention, both because of its prohibitive cost and relatively poor effectiveness.

Immunochemotherapy in American cutaneous leishmaniasis: immunological aspects before and after treatment.

In this study, we evaluated the immune response of patients suffering from cutaneous leishmaniasis treated with two distinct protocols. One group was treated with conventional chemotherapy using pentavalent antimonium salts and the other with immunochemotherapy where a vaccine against cutaneous leishmaniasis was combined with the antimonium salt. Our results show that, although no differences were observed in the necessary time for complete healing of the lesions between the two treatments, peripheral blood mononuclear cells from patients treated by chemotherapy showed smaller lymphoproliferative responses at the end of the treatment than those from patients in the immunochemotherapy group. Furthermore, IFN-gamma production was also different between the two groups. While cells from patients in the chemotherapy group produced more IFN-gamma at the end of treatment, a significant decrease in this cytokine production was associated with healing in the immunochemotherapy group. In addition, IL-10 production was also less intense in this latter group. Finally, an increase in CD8+ -IFN-gamma producing cells was detected in the chemotherapy group. Together these results point to an alternative treatment protocol where healing can be induced with a decreased production of a potentially toxic cytokine.

Immunochemotherapy in American cutaneous leishmaniasis: immunological aspects before and after treatment

In this study, we evaluated the immune response of patients suffering from cutaneous leishmaniasis treated with two distinct protocols. One group was treated with conventional chemotherapy using pentavalent antimonium salts and the other with immunochemotherapy where a vaccine against cutaneous leishmaniasis was combined with the antimonium salt. Our results show that, although no differences were observed in the necessary time for complete healing of the lesions between the two treatments, peripheral blood mononuclear cells from patients treated by chemotherapy showed smaller lymphoproliferative responses at the end of the treatment than those from patients in the immunochemotherapy group. Furthermore, IFN-gamma production was also different between the two groups. While cells from patients in the chemotherapy group produced more IFN-gamma at the end of treatment, a significant decrease in this cytokine production was associated with healing in the immunochemotherapy group. In addition, IL-10 production was also less intense in this latter group. Finally, an increase in CD8+ -IFN-gamma producing cells was detected in the chemotherapy group. Together these results point to an alternative treatment protocol where healing can be induced with a decreased production of a potentially toxic cytokine

Simple form of clinical sample preservation and Leishmania DNA extraction from human lesions for diagnosis of American cutaneous leishmaniasis via polymerase chain reaction.

The diagnosis value of polymerase chain reaction (PCR) was assessed in patients from an area endemic for American cutaneous leishmaniasis (ACL) in Brazil. Different forms of clinical sample preservation and DNA extraction for PCR were tested. The 4 preservation forms of the skin biopsies from patients suspected to have ACL were as follows: imprinted on filter paper (FP); imprinted on nitrocellulose paper (NP); frozen at -20 degrees C (FB); or immersed in 70% ethanol (EB). The DNA was extracted by elution from FP and NP and by enzyme digestion from FB and EB. Clinical examinations and parasitological or immunological tests confirmed the cases of ACL. Of 164 patients suspected to have ACL, 133 patients (81.1%) were confirmed. The PCR was positive in 76.8% of the suspected cases and in 90.2% of the confirmed cases. Polymerase chain reaction alone showed nearly the same positivity of the parasitological and immunological tests together; positivity varied 73.3-82.2%, according to the means by which the samples were preserved or the way the DNA was extracted. This variation was not significantly different (P > 0.05). Therefore, we recommend that clinical samples from patients with ACL should be collected and preserved on FP and the DNA further extracted by elution. The samples can be mailed to reference laboratories for the definitive diagnosis of ACL. This alternative is simple, inexpensive, and adequate for field conditions in developing countries.

Protection of C57BL/10 mice by vaccination with association of purified proteins from Leishmania (Leishmania) amazonensis.

In the past few years, induction of protective immunity to cutaneous leishmaniasis has been attempted by many researchers using a variety of antigenic preparations, such as living promastigotes or promastigote extracts, partially purified, or defined proteins. In this study, eleven proteins from Leishmania (Leishmania) amazonensis (LLa) with estimated molecular mass ranging from 97 to 13.5kDa were isolated by polyacrylamide gel electrophoresis and electro-elution. The proteins were associated as vaccine in different preparations with gp63 and BCG (Bacilli Calmette-Guérin). The antigenicity of these vaccines was measured by their ability to induce the production of IFN-gamma by lymphocyte from subjects vaccinated with Leishvacinregister mark or target. The immunogenicity was evaluated in vaccinated mice. C57BL/10 mice were vaccinated with three doses of each vaccine consisting of 30 microg of each protein at 15 days interval. One hundred microg of live BCG was only used in the first dose. Seven days after the last dose, they received a first challenge infection with 10(5) infective promastigotes and four months later, a second challenge was done. Two months after the second challenge, 42.86% of protection was obtained in the group of mice vaccinated with association of proteins of gp63+46+22kDa, gp63+13.5+25+42kDa, gp63+46+42kDa, gp63+66kDa, and gp63+97kDa; 57. 14% of protection was demonstrated with gp63+46+97+13.5kDa, gp63+46+97kDa, gp63+46+33kDa, and 71.43% protection for gp63 plus all proteins. The vaccine of gp63+46+40kDa that did not protect the mice, despite the good specific stimulation of lymphocytes (LSI = 7. 60) and 10.77UI/ml of IFN-gamma production. When crude extract of L. (L.) amazonensis was used with BCG a 57.14% of protection was found after the first challenge and 28.57% after the second, the same result was observed for gp63. The data obtained with the vaccines can suggest that the future vaccine probably have to contain, except the 40kDa, a cocktail of proteins that would protect mice against cutaneous leishmaniasis.

Short report: evaluation of the potency and stability of a candidate vaccine against American cutaneous leishmaniasis.

Availability of a safe, immunogenic, and affordable vaccine would represent the best strategy for control of cutaneous leishmaniasis (CL). Stability in field conditions is a essential property for any candidate vaccine. The stability and immunogenicity of three different preparations (thimerosal-preserved, autoclaved, and lyophilized) of a killed Leishmania amazonensis vaccine were assessed using fresh products and after 12 months of storage at 4 degrees C. Autoclaving was associated with a time-dependent decrease in the immunogenicity of the vaccine, as measured by the leishmanin skin test and production of interferon-gamma. These findings are of importance in the decision of which preparation of candidate killed CL vaccines should move to phase III trials.

The fucose-mannose ligand-ELISA in the diagnosis and prognosis of canine visceral leishmaniasis in Brazil.

The fucose-mannose ligand (FML)-ELISA assay showed a sensitivity of 100% and a specificity of 100% in diagnosis of canine visceral leishmaniasis (CVL) (kala-azar) in sera from naturally infected dogs from São Gonçalo do Amaranto, Rio Grande de Norte, Brazil. The overall prevalence of antibodies to Leishmania in the endemic area was 23% (79 of 343). Seroreactivity detected by a Leishmania chagasi immunofluorescent (IF) assay was much lower (2.9%) and similar to the percentage of dogs with kala-azar symptoms (2.6%). Twenty-one of 21 asymptomatic, FML-seropositive animals died of kala-azar in a period ranging from 0 to 6 months after diagnosis. The predictive value was 100% for the FML-ELISA, 43% for an L. mexicana ELISA, and 24% for the L. mexicana and L. chagasi IF assays, respectively. In experimentally infected dogs, all assays detected seropositivity between 90 and 120 days after infection. Since the current strategy for control of CVL is based on detection and destruction of infected dogs, the highly predictive, sensitive, and specific FML-ELISA represents a useful tool for field control of the disease.

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.

Histopathology of human American cutaneous leishmaniasis before and after treatment.

Chemical therapy for the treatment of leishmaniasis is still inadequate, and a number of drugs and therapeutic programs are being tested. Besides treatment, the ultimate goal is an effective cure, and histopathological analyses of the lesion cicatrices constitute an important measure of treatment success, or otherwise, in this respect. In this paper, we describe histopathological patterns in cases of American cutaneous leishmaniasis in 32 patients from the municipality of Caratinga, Minas Gerais, Brazil, before and after treatment with the following therapeutic methods: 1) leishvacin + glucantime; 2) leishvacin + BCG associated with glucantime; 3) glucantime; 4) leishvacin + BCG. Lesion fragments were collected from all patients by biopsy prior to, and approximately 30 days after, each treatment which resulted in a clinical diagnosis of cure. Following the analysis of slides, the preparations were described from a histopathological point of view and grouped taking into account the prevalence or significance of the characteristic elements. This process resulted in the following classification: 1. exudative reaction (ER); 2. exudative giant cell reaction (EGCR); 3. exudative productive reaction (EPR); 4. exudative productive giant cell reaction (EPGCR); 5. exudative productive necrotic reaction (EPNR); 6. necrotic exudative reaction (NER); 7. productive exudative reaction (PER), 8. productive giant cell reaction (PGCR); 9. productive exudative giant cell reaction (PEGCR); 10. productive exudative giant cell granulomatous reaction (PEGCGR); 11. productive reaction (PR) and 12. productive cicatricial (cure) reaction (PCR). After this analysis, it was noted that clinical cure did not always coincide with histopathological cure.

Histopathology of human American cutaneous leishmaniasis before and after treatment

Chemical therapy for the treatment of leishmaniasis is still inadequate, and a number of drugs and therapeutic programs are being tested. Besides treatment, the ultimate goal is an effective cure, and histopathological analyses of the lesion cicatrices constitute an important measure of treatment success, or otherwise, in this respect. In this paper, we describe histopathological patterns in cases of American cutaneous leishmaniasis in 32 patients from the municipality of Caratinga, Minas Gerais, Brazil, before and after treatment with the following therapeutic methods: 1) leishvacin + glucantime; 2) leishvacin + BCG associated with glucantime; 3) glucantime; 4) leishvacin + BCG. Lesion fragments were collected from all patients by biopsy prior to, and approximately 30 days after, each treatment which resulted in a clinical diagnosis of cure. Following the analysis of slides, the preparations were described from a histopathological point of view and grouped taking into account the prevalence or significance of the characteristic elements. This process resulted in the following classification: 1. exudative reaction (ER); 2. exudative giant cell reaction (EGCR); 3. exudative productive reaction (EPR); 4. exudative productive giant cell reaction (EPGCR); 5. exudative productive necrotic reaction (EPNR); 6. necrotic exudative reaction (NER); 7. productive exudative reaction (PER), 8. productive giant cell reaction (PGCR); 9. productive exudative giant cell reaction (PEGCR); 10. productive exudative giant cell granulomatous reaction (PEGCGR); 11. productive reaction (PR) and 12. productive cicatricial (cure) reaction (PCR). After this analysis, it was noted that clinical cure did not always coincide with histopathological cure.

A quimioterapia para a leishmaniose não é satisfatória e existem hoje, várias drogas e esquemas terapêuticos em teste. Além do tratamento ideal, busca-se um critério de cura efetivo, onde a análise da histopatologia da cicatriz poderá ser de grande valia. Este trabalho propõe caracterizar o padrão histopatológico de casos humanos de leishmaniose tegumentar americana, em 32 pacientes do município de Caratinga-MG, antes e após o tratamento com os seguintes métodos terapêuticos: 1) leishvacin + glucantime; 2) leishvacin + BCG associado ao glucantime; 3) glucantime; 4) leishvacin + BCG. Foram colhidos fragmentos das lesões de todos os pacientes, através de biópsias, antes e após o tratamento, com diagnóstico de cura. Após análise das lâminas, as preparações foram descritas, do ponto de vista histopatológico, e agrupadas levando em conta a prevalência e a significância do elemento característico. Tal processo resultou na classificação: 1. reação exsudativa; 2. reação exsudativa giganto-celular; 3. reação exsudativa produtiva; 4. reação exsudativa produtiva giganto-celular; 5. reação exsudativa produtiva necrótica; 6. reação necrótica exsudativa; 7. reação produtiva exsudativa; 8. reação produtiva giganto-celular; 9. reação produtiva exsudativa giganto-celular; 10. reação produtiva exsudativa giganto-celular granulomatosa; 11. reação produtiva e 12. reação produtiva cicatricial (cura histopatológica). Observamos após tal análise, que nem sempre a cura clínica coincide com a cura histopatológica.

Leishmaniose visceral canina: estudo imunocitoquímico dos antígenos do MHC classe II no fígado e órgäos linfóides / Canine visceral leishmaniasis: immunocytochemical study of the MHC class II antigens in liver and lymphoid organs

O objetivo deste trabalho foi o de estudar a expressäo imunocitoquímica dos antígenos do MHC classe II no fígado e órgäos linfóides (baço, linfonodos e placas de Peyer) de cäes, sem raça definida, experimentalmente e naturalmente infectados com Leishmania chagasi. Cortes em criostato de fígado, baço, linfonodos e placas de Peyer foram corados pela técnica imunocitoquímica peroxidase anti-peroxidase (PAP). A marcaçäo imunocitoquímica para os antígenos do MHC classe II revelou a mesma topografia em todos os órgäos examinados de todos os animais infectados. Entretanto, a expressäo dos antígenos do MHC classe II foi menos intensa nos linfonodos cervicais e abdominais dos cäes naturalmente infectados, indicando que L. chagasi é capaz de inibir a expressäo dos antígenos do MHC classe II

Histopathology and immunocytochemical study of type 3 and type 4 complement receptors in the liver and spleen of dogs naturally and experimentally infected with Leishmania (Leishmania) chagasi.

The objective of this study was to compare the histopathological changes and expression of CR3 and CR4 in the liver and spleen of dogs naturally and experimentally infected with L. chagasi. The basic histopathological lesions observed mainly in naturally infected dogs were: epithelioid hepatic granulomas, hyperplasia and hypertrophy of Kupffer cells, Malpigui follicles and mononucleated cells of the red pulp of the spleen. Sections from the liver and spleen by immunocytochemistry technique showed the presence of CD11b, c/CD 18 antigens in the control and infected animals and no qualitative or quantitative differences in the liver. Nevertheless, CD18 was always increased in the spleen of naturally and experimentally infected dogs. These results indicate that there is a difference in the activation of CD18 in both experimental and natural cases of canine visceral leishmaniasis that should play an important role in the immunological response to Leishmania chagasi infection.

Assessment of immunity induced in mice by glycoproteins derived from different strains and species of Leishmania.

A comparative study was undertaken on the immunogenic properties of 63kDa glycoproteins obtained from five different strains/species of Leishmania and assessed in C57BL/10 mice. The humoral immune response was assessed by ELISA against the five different antigens of the immunized animals. The cellular immune response was derived from Leishmania. The response was found to be species-specific in all of determined by means of the cytokine profiles secreted by the spleen cells of immunized animals. The presence of gamma-IFN and IL-2, and the absence of IL-4 in the supernatants of cells stimulated by L. amazonensis antigen established that the cellular response is of Th1 type. The five glycoproteins tested were equally effective in protecting C57BL/10 mice against challenge by L. amazonensis. About 50% of the immunized animals were protected for six months.