Eco-epidemiological study on sandflies and environmental aspects related to the transmission of leishmaniasis in a municipality of Minas Gerais, Brazil, 2015-2016 / Estudo eco-epidemiológico da fauna flebotomínica e dos aspectos ambientais relacionados à transmissão das leishmanioses em um município de Minas Gerais, Brasil, 2015 - 2016

Diante da escassez de dados sobre a topografia e a sintopia das vísceras abdominopélvicas do tamanduá-bandeira (Myrmecophage tridactyla - Linnaeus, 1758), o presente estudo teve como objetivo elucidar essas características e compará-las com as demais espécies animais, mormente as domésticas. Utilizaram-se três espécimes, dois machos e uma fêmea, provenientes de doação da Polícia Militar Ambiental de Franca ao Laboratório de Anatomia Veterinária da Universidade de Franca, após óbitos por atropelamentos. Os animais foram fixados e mantidos em solução aquosa de formaldeído a 10%, seguidos de dissecação convencional das cavidades abdominopélvicas para posterior inspeção direta e descrição topográfica das vísceras, visando a análises comparativas com outras espécies, cujo posicionamento e cujas particularidades já são bem estabelecidos na literatura. Observou-se que a maioria das vísceras dessas cavidades possuem localização e sintopia similares aos animais domésticos, exceto os rins e os testículos. Diante da metodologia estabelecida e dos resultados obtidos, admite-se que mais espécimes de tamanduás-bandeiras, de ambos os gêneros, devam ser avaliados e registrados cientificamente, visando à confirmação dos dados da atual pesquisa e à preconização anatômica da cavidade abdominopélvica, visto que variações anatômicas individuais são passíveis entre animais da mesma espécie.(AU)

Objetivou-se avaliar a fauna vetorial e os aspectos ambientais e climáticos relacionados à transmissão das leishmanioses. Foi realizado um estudo eco-epidemiológico prospectivo de coleta sistemática de flebotomíneos e inquérito censitário sorológico canino em áreas de um município do Brasil. Para determinar a taxa de prevalência de LVC, foram examinadas amostras de sangue de 1752 cães. Na avaliação entomológica, foram instaladas 24 armadilhas luminosas em 12 residências distribuídas, instaladas no ambiente de peridomicílio e intradomicílio durante 12 meses. Para análise dos aspectos climáticos, utilizou-se a correlação simples de Spearman e para análise espacial foram utilizadas a Lógica Fuzzy e a Função K. A taxa de prevalência em cães foi de 4,1% e 7,1%. No estudo entomológico, foram capturados 431 flebotomíneos. A maior parte (74%) dos espécimes foi capturada no peridomicílio. Em relação à infecção natural, 5,6 % das amostras analisadas por biologia molecular apresentaram positividade à infecção por Leishmania spp.. Em 100% das amostras positivas, encontrou-se infecção por Leishmania infantum. Na análise espacial uma Área apresentou maior concentração de pontos de sobreposição de alta densidade de Lutzomyia longipalpis e cães sororreagentes, indicando maior risco na ocorrência concomitante dos dois eventos. Os resultados mostram que a interface parasito-reservatório-vetor está ativa nas áreas estudadas.(AU)

Transmission of visceral leishmaniasis in dogs in a risk area of the metropolitan region of Belo Horizonte, Minas Gerais, Brazil / Transmissão da leishmaniose visceral em cães em área de risco da Região Metropolitana de Belo Horizonte, Minas Gerais, Brasil

Visceral leishmaniasis (VL) has spread rapidly across cities in the metropolitan region of Belo Horizonte. The aim of this study was to investigate VL dynamics in a prospective cohort study of dogs in Juatuba, between 2010 and 2011, to confirm the incidence of Leishmania infantum, and to assess possible risk factors associated with infection. An observational and prospective closed cohort study was performed using serology testing in dogs, randomly selected from the whole municipality. All seronegative dogs, or dogs with inconclusive results were monitored using indirect immunofluorescence (IIF) at 6-month intervals. The dog's owners completed a semi-structured questionnaire to assess possible causal factors of seroconversion, and the responses were assessed using logistic regression. The canine incidence coefficient was 206/1,000 dogs per year (CI: 178-238), and a cluster was identified in an area with a high concentration of seropositive dogs, but a low overall canine population. Large dogs were identified as a risk factor and the following variables were identified as protection factors: dogs aged over 4 years, daily peridomicile cleaning, and better socioeconomic conditions. VL is spreading over a large area in Juatuba in a short period of time.(AU)

A leishmaniose visceral (LV) expandiu-se de forma rápida e extensa pelos municípios da Região Metropolitana de Belo Horizonte. Objetivou-se estudar a dinâmica da LV em uma coorte prospectiva de cães em Juatuba, entre 2010 e 2011, para verificar a incidência e fatores de risco associados à infecção por Leishmania infantum. Foi feito um estudo observacional e prospectivo de coorte fechada por meio de análise sorológica em cães selecionados aleatoriamente em todo o município, com acompanhamento semestral dos resultados soronegativos e indeterminados na imunofluorescência indireta (IFI). Usou-se questionário semiestruturado junto aos proprietários de cães para avaliação da soroconversão e dos fatores determinantes a essa, por meio da regressão logística. O coeficiente de incidência canina foi de 206/1000 cães.ano (IC: 178 - 238), e foi identificado cluster em área com elevada concentração de cães soropositivos, mas com baixa densidade populacional canina. A variável cão de porte grande foi identificada como fator de risco, e as variáveis idade do cão superior a quatro anos, limpeza diária do peridomicílio e melhores condições socioeconômicas como fatores de proteção. A infecção por LV está ocorrendo em curto período de tempo e com ampla distribuição em Juatuba.(AU)

Standardization of a molecular method for epidemiologic identification of Leishmania strains.

Molecular studies of the evolutionary relationships among Leishmania species suggest the presence of high genetic variation within this genus, which has a direct effect on public health in many countries. The coexistence of species in a particular region can result in different leishmaniasis clinical forms and treatment responses. We aimed to standardize the kinetoplast DNA (kDNA) enterobacterial repetitive intergenic consensus (ERIC) sequence polymerase chain reaction (PCR) method for molecular epidemiological identification of Leishmania strains, and estimate existing inter-strain genomic differences and kDNA signatures using this technique. ERIC-PCR of genomic DNA revealed genetic polymorphisms between species, although some strains shared many DNA fragments. Leishmania guyanensis, L. amazonensis, and L. braziliensis clustered together in a dendrogram with similarities ranging from 42.0 to 61.0%, whereas L. chagasi grouped with these three species with a similarity of 28.0%. After amplification of kDNA, 780-bp bands were extracted from an agarose gel and purified for analysis of its genetic signature. kDNA ERIC-PCR electrophoretic patterns consisted of 100- to 600- bp fragments. Using these profiles, L. braziliensis and L. guyanensis grouped with a similarity of 26.0%, and L. amazonensis and L. chagasi clustered based on a similarity of 100%. The electrophoretic profiles and dendrograms showed that, for epidemiological identification by ERIC-PCR, genomic DNA had greater discriminatory power than kDNA did. More strains need to be analyzed to validate the kDNA ERIC-PCR method. The genomes of these strains should be sequenced for better epidemiological identification of Leishmania species.

First record of Ceratium furcoides (Dinophyta), an invasive species, in a temporary high-altitude lake in the Iron Quadrangle (MG, Southeast Brazil).

Dinoflagellates of the genus Ceratium are generally marine organisms, but rare occurrences in freshwater have been observed in Brazil. In this paper we are recording for the first time the presence of Ceratium furcoides, an invasive species, in a shallow, natural intermittent pool formed at a high-altitude at the southern end of the Iron Quadrangle, an iron-mining district of Minas Gerais State (Southeast Brazil). Samples were collected in October and November of 2010 (rainy period). The population density of this organism observed in Lagoa Seca ("Dry Pool") was very low, at most 4 ind L-1. Mountain lakes are extremely vulnerable to atmospheric deposition of organisms, making them valuable witnesses both of the many forms of impact arising from human activities and of the extended global connections that facilitate the dispersion and introduction of new species over great distances. Studies on the population dynamics of C. furcoides in natural tropical systems are still rare and very recent to the brazilian scenario and hence the monitoring of its dynamics and the potential impact on aquatic communities of its becoming established are essential to an understanding of the process of bioinvasion by this species.

Genetic variability in geographical populations of Lutzomyia whitmani elucidated by RAPD-PCR.

Unequivocal identification of phlebotomine sand flies is of crucial importance in epidemiological studies of leishmaniasis, because certain species may act as vectors, depending on behavior and physiology. For Lutzomyia whitmani, a major vector of American human cutaneous leishmaniasis in Brazil, an increasing number of studies have suggested the existence of a species complex. In the present work, we evaluated the genetic variability of L. whitmani populations from four Brazilian foci of that disease: Corte de Pedra, Ilhéus, Martinho Campos, and Serra de Baturité. Computational analysis of 85 characters, generated by RAPD-polymerase chain reaction, demonstrated high intrapopulational variability. Those characters led to sex discrimination in three of the populations, with the exception of Martinho Campos individuals, in which sex distinction was not complete. One and two interpopulational phenograms were obtained for females and males, respectively. A higher similarity was observed among the specimens from Ilhéus, Corte de Pedra, and Serra de Baturité, whereas the Martinho Campos population remained external to that cluster. These results, which are in partial accordance with a previous morphometric survey of L. whitmani from the same regions, provide additional evidence to support the existence of at least two spatial clusters of biogeographical populations of L. whitmani in Brazil.

Epidemiological studies of an outbreak of cutaneous leishmaniasis in the Rio Jequitinhonha Valley, Minas Gerais, Brazil.

We detected an outbreak of American cutaneous leishmaniasis in the Jequitinhonha River Valley, Minas Gerais, Brazil. Clinical and epidemiological aspects were studied for a period of two years. Data include results of physical examinations, Montenegro skin test and serology. In total 72 of the 299 individuals evaluated presented active lesions. Only one case out of these 72 patients showed the mucosal form of the disease. The precarious sanitary conditions, low educational level and low income found in the population studied demonstrated that, as with the other parasitic diseases, cutaneous leishmaniasis occurs with greater frequency in needy populations. A canine serological survey detected 20.3% (30/148) of dogs reactive to the Leishmania antigen. Lutzomyia intermedia was the predominant phlebotomine species and the majority of the specimens (84.9%) were captured in the peridomicile. Four samples from human and three from canine cases were isolated and characterised by PCR and isoenzymes as being Leishmania (Viannia) braziliensis. The peridomiciliary nature of the disease is discussed.

O sistema imune inato intestinal em pacientes com estrongiloidíase / The innate intestinal immune system in patients with strongyloidiasis

Foram estudados fragmentos da mucosa duodenal obtidos de nove pacientes apresentando sintomas leves de estrogiloidiase, nove com sintomas moderados, sete com sintomas graves e sete indivíduos aparentemente normais. A muramidase(lisozima) foi imunocitoquimicamente demonstrada em cortes contracorados pela técnica do PAS. Havia aparente aumento progressivo na secreçao de muramidase pela célula de Paneth aaacompanhado o agravamento dos sintomas, näo obstante o fato de que a sua populaçao permanecesse constante. Decréscimo progressivo no número de células caliciformes foi observado enquanto, concomitantemente, haavia aumento na populaçao de células intermediarias. Esses resultados foram interpretados como a indicaçao da participaçao do sistema imune inato intestinal no estabelecimento da interaçao Strongyloides stercoralis/hospedeiro, através do aumento da secreçao da enzima mucolítica muramidase

Microbial diversity during maturation and natural processing of coffee cherries of Coffea arabica in Brazil.

The magnitude and diversity of the microbial population associated with dry (natural) processing of coffee (Coffea arabica) has been assessed during a 2-year period on 15 different farms in the Sul de Minas region of Brazil. Peptone water-washed samples were taken of maturing cherries on trees (cherries, raisins and dried cherries) and from ground fermentations. The microbial load varied from 3 x 10(4) to 2.2 x 10(9) cfu/cherry with a median value of 1.6 x 10(7) cfu/cherry. The microbial load increased after heavy rainfall on cherries that were drying on the ground. At all stages, bacteria were usually the most abundant group, followed by filamentous fungi and finally yeasts. Counts of bacteria, yeasts and fungi varied considerably between farms and at different stages of maturation and processing and no consistent pattern could be seen. Yeasts showed an increase during the fermentation process. Median counts were not significantly different for fungi, yeasts and bacteria between the 2 years although Gram-negative bacteria dominated in the wet year and Gram-positive bacteria dominated in the dry year. Of a total of 754 isolates, 626 were identified to at least genus level comprising 44 genera and 64 different species. The 164 isolates of Gram-negative bacteria included 17 genera and 26 species, the most common of which were members of the genera Aeromonas, Pseudomonas, Enterobacter and Serratia. Of 191 isolates of Gram-positive bacteria, 23 were spore-forming and included six Bacillus species, and 118 were non-spore-formers of which over half were Cellulomonas with lesser numbers of Arthrobacter, Microbacterium, Brochothrix, Dermabacter and Lactobacillus. Of the 107 yeast isolates, 90 were identified into 12 genera and 24 different species and almost all were fermentative. The most common genera, in decreasing frequency, were Pichia, Candida, Arxula and Saccharomycopsis. There were many rarely described yeasts including Pichia lynferdii and Arxula adeninivorans. Almost all 292 fungal isolates were identified to genus level and 52 were identified to species level. Cladosporium, Fusarium and Penicillium each comprised about one third of the isolates and were found on all farms. Only 3% of the isolates were Aspergillus. Beauvaria, Monilia, Rhizoctonia and Arthrobotrys species were also occasionally found. The microbial flora is much more varied and complex than found in wet fermentations. The genera and species identified include members known to have all types of pectinase and cellulase activities.

Description of Lutzomyia bianchigalatiae n. sp. A sand fly within the subgenus Pintomyia Costa Lima 1932 (Diptera; Psychodidae).

A new sand fly species is described based on males collected in Bananal, municipality of Mariana and the female from Sabará city, State of Minas Gerais, Brazil. Taxonomic remarks, geographic distribution and the description of new species are presented. The name Lutzomyia (Pintomyia) bianchigalatiae is in honour of Dr Eunice Aparecida Bianchi Galati, friend and researcher at the Faculdade de Saúde Pública, Universidade de São Paulo.

Morphometric and phenetic studies of five geographical populations of Lutzomyia whitmani (Diptera: Psychodidae) in Brazil.

A morphometric survey examined adult specimens of Lutzomyia whitmani (Antunes & Coutinho) captured at 5 municipalities in southeastern and northeastern Brazil to compare the populations. The localities were Ilhéus (Bahia), Martinho Campos (Minas Gerais), Corte de Pedra (Bahia), Baturité (Ceará), and Amaraji (Pernambuco): all are known foci of American cutaneous leishmaniasis. Fifteen males and 15 females from each population were analyzed morphometrically for 42 and 37 characters, respectively. Statistical data alone were insufficient to discriminate among the 5 populations. Further analysis generated phenograms that indicated there were 2 spatial clusters: the 1st was composed of specimens from Ilhéus (Bahia) and Baturité (Ceará) and the 2nd of specimens from Martinho Campos (Minas Gerais), Corte de Pedra (Bahia), and Amaraji (Pernambuco). Although insufficient to define the taxonomic status of the populations studied, the results delineated the existence of biogeographical structuring within L. whitmani. Complementary studies on the susceptibility to Leishmania braziliensis infection in the 5 populations are in progress to clarify the relationship between the 2 biogeographical clusters and American cutaneous leishmaniasis transmission in those Brazilian regions.

Random amplified polymorphic DNA (RAPD) analysis of Lutzomyia longipalpis laboratory populations.

The phlebotomine sand fly Lutzomyia longipalpis has been incriminated as a vector of American visceral leishmaniasis, caused by Leishmania chagasi. However, some evidence has been accumulated suggesting that it may exist in nature not as a single but as a species complex. Our goal was to compare four laboratory reference populations of L. longipalpis from distinct geographic regions at the molecular level by RAPD-PCR. We screened genomic DNA for polymorphic sites by PCR amplification with decamer single primers of arbitrary nucleotide sequences. One primer distinguished one population (Marajó Island, Pará State, Brazil) from the other three (Lapinha Cave, Minas Gerais State, Brazil; Melgar, Tolima Department, Colombia and Liberia, Guanacaste Province, Costa Rica). The population-specific and the conserved RAPD-PCR amplified fragments were cloned and shown to differ only in number of internal repeats.

Production and regeneration of protoplasts from the mycorrhizal fungus Suillus granulatus.

Experiments were performed with the mycorrhizal fungus Suillus granulatus to define the parameters for production and regeneration of protoplasts. Protoplasts were released at frequencies between 1 and 3×10(7)/ml from mycelium 3 to 7 days old. The best osmotic stabilizer for protoplast release was MgSO4 (0.7 M). To optimize protoplast release and regeneration an enzyme (Novozym 234) concentration 1.7 mg/ml was chosen, with a digestion time of 1 to 2 h. Regenerated colonies formed mycorrhizae within 60 days after inoculation in Pinus caribaea var. hondurensis seedlings.

Epidemiological aspects of American Cutaneous Leishmaniasis in a periurban area of the metropolitan region of Belo Horizonte, Minas Gerais, Brazil

In order to characterize the epidemiology of American Cutaneous Leishmaniasis (ACL) in a periurban area of the municipality of Sabará in the metropolitan region of Belo Horizonte (MRBH), an area until then considered free of the disease, a cross sectional survey was undertaken in 1990. The survey of the population consisted of 1119 interviews and 881 clinical examinations using Montenegro's skin test (MST). A low prevalence (3.7%) of positive MST was encountered. The disease had been occuring in the area for about 20 years in the form of sporadic cases. The predominant species of sandfly both in domestic areas and nearby areas of secondary vegetation was Lutzomyia whitmani. A canine survey of delayed hypersensitivity to the antigen P10,000 identified only one dog with a positive reaction out of 113 examined. The transmission of ACL in MRBH was confirmed. The occurrence of the disease in women, children and individuals with no contact with forest areas as well as the presence of potential vector species in the domiciliar environment, suggests the transmission of the disease in this environmewnt

Epidemiological aspects of American cutaneous leishmaniasis in a periurban area of the metropolitan region of Belo Horizonte, Minas Gerais, Brazil.

In order to characterize the epidemiology of American Cutaneous Leishmaniasis (ACL) in a periurban area of the municipality of Sabará in the metropolitan region of Belo Horizonte (MRBH), an area until then considered free of the disease, a cross sectional survey was undertaken in 1990. The survey of the population consisted of 1119 interviews and 881 clinical examinations using Montenegro's skin test (MST). A low prevalence (3.7%) of positive MST was encountered. The disease had been occurring in the area for about 20 years in the form of sporadic cases. The predominant species of sandfly both in domestic areas and nearby areas of secondary vegetation was Lutzomyia whitmani. A canine survey of delayed hypersensitivity to the antigen P10,000 identified only one dog with a positive reaction out of 113 examined. The transmission of ACL in MRBH was confirmed. The occurrence of the disease in women, children and individuals with no contact with forest areas as well as the presence of potential vector species in the domiciliar environment, suggests the transmission of the disease in this environment.