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)

Isolation, primary culture and morphological characterization of oenocytes from Aedes aegypti pupae.

Oenocytes are ectodermic cells that participate in a number of critical physiological roles such as detoxification and lipid storage and metabolism in insects. In light of the lack of information on oenocytes from Aedes aegypti and the potential role of these cells in the biology of this major yellow fever and dengue vector, we developed a protocol to purify and maintain Ae. aegypti pupa oenocytes in primary culture. Ae. aegypti oenocytes were cultured as clustered and as isolated ovoid cells with a smooth surface. Our results demonstrate that these cells remain viable in cell culture for at least two months. We also investigated their morphology in vivo and in vitro using light, confocal, scanning and transmission electron microscopes. This work is the first successful attempt in isolating and maintaining Ae. aegypti oenocytes in culture, and a significant step towards understanding the role of this cell type in this important disease vector. The purification and the development of primary cultures of insect oenocytes will allow future studies of their metabolism in producing and secreting compounds.

Crotalus durissus terrificus venom as a source of antitumoral agents

The basic knowledge on neoplasms is increasing quickly; however, few advances have been achieved in clinical therapy against tumors. For this reason, the development of alternative drugs is relevant in the attempt to improve prognosis and to increase patients' survival. Snake venoms are natural sources of bioactive substances with therapeutic potential. The objective of this work was to identify and characterize the antitumoral effect of Crotalus durissus terrificus venom (CV) and its polypeptide, crotoxin, on benign and malignant tumors, respectively, pituitary adenoma and glioblastoma. The results demonstrated that CV possess a powerful antitumoral effect on benign (pituitary adenoma) and malignant (glioblastoma multiforme) tumors with IC50 values of 0.96 ± 0.11 µg/mL and 2.15 ± 0.2 µg/mL, respectively. This antitumoral effect is cell-cycle-specific and dependent on extracellular calcium, an important factor for crotoxin phospholipase A2 activity. The CV antitumoral effect can be ascribed, at least partially, to the polypeptide crotoxin that also induced brain tumor cell death. In spite of the known CV nephrotoxicity and neurotoxicity, acute treatment with its antitumoral dose established in vitro was not found to be toxic to the analyzed animals. These results indicate the biotechnological potential of CV as a source of pharmaceutical templates for cancer therapy.

Primary culture of the region of the amebocyte-producing organ of the snail Biomphalaria glabrata, the intermediate host of Schistosoma mansoni.

Biomphalaria glabrata snails are major hosts for the digenetic trematoda Schistosoma mansoni, the causative agent of human schistosomiasis. The success or failure of the infection will be dependent on the mobilization of the molluskan internal defense system, where a major role will be played by circulating hemocytes produced by the APO (amebocyte-producing organ) of the snail. In this report, the primary culture of the APO region of B. glabrata was obtained for the first time, as well as a control culture of the ovotestis. Three different cell populations migrated easily from the explants in culture, with no need of any dispersion agent. The cells grew in suspension at an incubation temperature of 15 degrees C and the cultures were maintained viable for up to two weeks. Two of these cell populations obtained resembled cell types known to be present in the hemolymph of Biomphalaria. The availability of APO cells in culture may contribute to a better understanding of the internal defense in mollusks, in general, as well as the specific response of B. glabrata to S. mansoni infection.

Primary culture of the region of the amebocyte-producing organ of the snail Biomphalaria glabrata, the intermediate host of Schistosoma mansoni

Biomphalaria glabrata snails are major hosts for the digenetic trematoda Schistosoma mansoni, the causative agent of human schistosomiasis. The success or failure of the infection will be dependent on the mobilization of the molluskan internal defense system, where a major role will be played by circulating hemocytes produced by the APO (amebocyte-producing organ) of the snail. In this report, the primary culture of the APO region of B. glabrata was obtained for the first time, as well as a control culture of the ovotestis. Three different cell populations migrated easily from the explants in culture, with no need of any dispersion agent. The cells grew in suspension at an incubation temperature of 15°C and the cultures were maintained viable for up to two weeks. Two of these cell populations obtained resembled cell types known to be present in the hemolymph of Biomphalaria. The availability of APO cells in culture may contribute to a better understanding of the internal defense in mollusks, in general, as well as the specific response of B. glabrata to S. mansoni infection.

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.

Molecular cloning of a gamma-phospholipase A2 inhibitor from Lachesis muta muta (the bushmaster snake).

Several endogenous phospholipase A(2) inhibitors (PLIs) have been purified from the blood plasma of a number of snake species and are classified into three classes (alpha, beta and gamma) according to their structure and specificity. In the present study we have cloned transcripts of a protein homologous to CNF, a gammaPLI present in Crotalus durissus terrificus plasma, that is encoded in the liver of Lachesis muta muta (the bushmaster snake), a species evolutionarily related to Crotalus. The cDNA sequences code for two isoforms of a 200-residue protein including a 19-residue signal peptide followed by 181 amino acid residues in the mature form and a putative N-linked carbohydrate site. The deduced primary structures and some properties of those new proteins were compared to those of CNF. Multiple alignment was performed with the aminoacid sequences of all the gammaPLIs described so far and this used in the construction of a phylogenetic tree.

Toxicity of South American snake venoms measured by an in vitro cell culture assay.

Cytotoxicity of venoms from eight medically important South American Crotalidae snakes (Bothrops and Lachesis genera) was determined, based on a procedure originally described for the screening of cytotoxic agents in general. The assay, the conditions of which were adapted to snake venoms, determines the survival of viable cells in monolayer culture upon exposure to the toxic agent. Snake venom toxicity was expressed as the venom dose that killed 50% of the cells (CT(50)) under the assay conditions. Bothrops neuwieddi mattogrossensis (CT(50)=4.74+/-0.35 microg/ml) and Bothrops leucurus (CT(50)=4.95+/-0.51 microg/ml) were the most cytotoxic whereas Bothrops atrox (CT(50)=34.64+/-2.38 microg/ml) and Bothrops sp. (CT(50)=33.89+/-3.89 microg/ml) were the least cytotoxic venoms, respectively. The relationship between CT(50) and other biological activities of these snake venoms was evaluated.

Fingerprinting of cell lines by directed amplification of minisatellite-region DNA (DAMD)

The development of in vitro propagation of cells has been an extraordinary technical advance for several biological studies. The correct identification of the cell line used, however, is crucial, as a mistaken identity or the presence of another contaminating cell may lead to invalid and/or erroneous conclusions. We report here the application of a DNA fingerprinting procedure (directed amplification of minisatellite-region DNA), developed by Heath et al. [Nucleic Acids Research (1993) 21: 5782-5785], to the characterization of cell lines. Genomic DNA of cells in culture was extracted and amplified by PCR in the presence of VNTR core sequences, and the amplicons were separated by agarose gel electrophoresis. After image capture with a digital camera, the banding profiles obtained were analyzed using a software (AnaGel) specially developed for the storage and analysis of electrophoretic fingerprints. The fingerprints are useful for construction of a data base for identification of cell lines by comparison to reference profiles as well as comparison of similar lines from different sources and periodic follow-up of cells in culture

Fingerprinting of cell lines by directed amplification of minisatellite-region DNA (DAMD).

The development of in vitro propagation of cells has been an extraordinary technical advance for several biological studies. The correct identification of the cell line used, however, is crucial, as a mistaken identity or the presence of another contaminating cell may lead to invalid and/or erroneous conclusions. We report here the application of a DNA fingerprinting procedure (directed amplification of minisatellite-region DNA), developed by Heath et al. [Nucleic Acids Research (1993) 21: 5782-5785], to the characterization of cell lines. Genomic DNA of cells in culture was extracted and amplified by PCR in the presence of VNTR core sequences, and the amplicons were separated by agarose gel electrophoresis. After image capture with a digital camera, the banding profiles obtained were analyzed using a software (AnaGel) specially developed for the storage and analysis of electrophoretic fingerprints. The fingerprints are useful for construction of a data base for identification of cell lines by comparison to reference profiles as well as comparison of similar lines from different sources and periodic follow-up of cells in culture.

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.

Primary culture of venom gland cells from the South American rattlesnake (Crotalus durissus terrificus).

Primary cultures of venom gland cells from the South American rattlesnake (Crotalus durissus terrificus) were attempted. At first, six different cell types were obtained including potentially secreting epithelial-like cells. Nonepithelial cell cultures were later invaded by fibroblast-like cells. Cultures of epithelial-like gland cells were successfully maintained, after testing different culture conditions by varying the media, incubation temperature, use of dissociating agents and adhesion substrates. The best results were achieved using plates precoated with rattlesnake skin collagen and incubation in CMRL 1415 modified for snake gland cells plus 10% fetal calf serum at 30 degrees C. The presence of venom could be demonstrated in the supernatant of five out of six epithelial-like gland cell cultures tested by ELISA, in the very first passages. After the third passage, however, venom amounts dropped to undetectable values. A total of 23 venom gland cell lines were obtained and are kept frozen in the laboratory; among them, five epithelial-like gland cell lines with up to 12 passages, that were continuously cultured for more than 30 weeks. The methodology described here was successfully applied to C. d. terrificus kidney cells culturing, developed to be used as negative control.

Studies on the specificity of CNF, a phospholipase A2 inhibitor isolated from the blood plasma of the South American rattlesnake (Crotalus durissus terrificus). I. Interaction with PLA2 from Lachesis muta muta snake venom.

A phospholipase A2 inhibitor has been previously purified and cloned from the blood plasma of the South American rattlesnake, Crotalus durissus terrificus. This inhibitor, named CNF for Crotalus neutralizing factor, interacts with crotoxin, the main neurotoxin from C. d. terrificus venom, abolishing its phospholipase A2 activity. Crotoxin is a heterodimer of an acidic subunit (CA) and a basic phospholipase A2 (CB). CNF acts by forming a stable non-toxic complex with CB, replacing CA in the toxic CA-CB of crotoxin. In the present investigation, we have shown that CNF has a broader specificity. It is able to inhibit the PLA2 activity of the whole venom from the bushmaster snake (Lachesis muta muta), a species evolutionary related to Crotalus. Inhibition experiments have been carried out with four PLA2 active components isolated from L. m. muta venom, one basic and three acidic ones. CNF inhibition is not restricted to the basic PLA2, but extended to the three acidic forms as well.

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.

A phospholipase A2 inhibitor from the plasma of the South American rattlesnake (Crotalus durissus terrificus). Protein structure, genomic structure, and mechanism of action.

The lethal toxicity of the South American rattlesnake (Crotalus durissus terrificus) venom can be attributed mainly to the presence of a pre-synaptic neurotoxin, crotoxin, with phospholipase A2 activity. Crotoxin is a heterodimer of an acidic protein (CA) and a basic phospholipase A2 (CB). An anti-toxic protein of subunit molecular mass 23.6 kDa that neutralizes both lethal and PLA2 activity of crotalid venom and crotoxin has been previously purified from the plasma of this snake (Fortes-Dias, C., Fonseca, B. C. B., Kochva, E., and Diniz, C. R. (1991) Toxicon 29, 997-1008). The protein has been named CNF for Crotalus neutralizing factor. In the present study, we have shown that CNF exists as an oligomeric aggregate of (CNF)n, where n = 6-8, and when it interacts with crotoxin, it replaces the acidic protein CA of crotoxin to form a stable near stoichiometric complex of CNF.CB. The CNF.CB complex no longer exhibits PLA2 activity and is inert in vivo. Thus, the exchange reaction between CA.CB of crotoxin and CNF to form CNF.CB and free CA is reminiscent of the interaction of crotoxin with its target receptor at the neuromuscular transmission site in the presynaptic cells. A cDNA encoding CNF has been isolated from a liver cDNA library using an appropriate nucleotide probe. The nucleotide sequence codes for a 19-residue signal peptide, followed by a 181-residue protein of which 16 are half-cystines. Calculated molecular mass is 20.06 kDa, and there is a putative N-linked carbohydrate site at Asn157.

C-reactive proteins, limunectin, lipopolysaccharide-binding protein, and coagulin. Molecules with lectin and agglutinin activities from Limulus polyphemus.

In 1964, Levin and Bang discovered that gram-negative bacterial endotoxin could rapidly induce gelation of Limulus amebocyte lysate. This observation has led to the development of the most sensitive and specific method for the detection of bacterial endotoxin in pharmaceuticals and drugs intended for human use. Over 10 years ago, Bang injected endotoxin into young horseshoe crabs and observed a time and dose-dependent coagulation of the whole hemolymph. Limunectin, LEBP-PI, and Limulus CRP are found together with coagulin as part of the hemolymph clot at the time of endotoxin-induced exocytosis of amebocytes. In this manner, these molecules with agglutinin/lectin activities could work in concert to assist in the recognition and eventual removal of invading microorganisms from the circulating system. Although the mechanism of endotoxin-induced clot formation is to a large extent understood, the mechanism of clot dissolution and removal in the Limulus hemolymph remains to be clarified.

Agglutination activity of Limulus polyphemus coagulogen following limited proteolysis.

1. A 14 kDa protein with cell agglutination properties has been purified from endotoxin-activated L. polyphemus amebocyte lysate. Amino terminal sequence analysis indicates that this protein corresponds to a proteolytically cleaved product (coagulin) of coagulogen. 2. Similar cell agglutination activity can be generated, in vitro, by proteolytic cleavage of the coagulogen with either trypsin, endogenous protease or an alpha 2M/enzyme complex isolated from amebocytes. 3. Studies with [125I]-labeled coagulogen showed that only coagulin, not the intact coagulogen, binds to rabbit erythrocytes and formalin-fixed amebocytes. 4. The cell agglutination activity of coagulin towards erythrocytes was not inhibited by various sugars tested, and was not Ca(2+)-dependent. 5. These findings suggest that coagulogen and coagulin are reminiscent of their mammalian counterparts, fibrinogen and fibrin, in their clotting and relative adhesive properties.

[The efficacy of the bothropic-crotalic antivenom in the neutralization of the main Bothrops jararacussu venom effects]. / A eficácia do antiveneno botrópico-crotálico na neutralização das principais atividades do veneno de Bothrops jararacussu.

Myonecrosis is one of the effects of Bothrops jararacussu venom, from which a myotoxin was isolated showing structural homology to phospholipase A2 (PLA2), but without enzymatic activity. Such myotoxic activity is also present in the Crotalus durissus terrifucus venom, and is attributed to crotoxin and to PLA2 (crotoxin B), the basic component of the crotoxin complex. The Bothrops jararacussu venom showed three proteins with immunologic identity to PLA2 from crotoxin. The bothropic (AB) and the bothropic/crotalic (AB/C) anti-venoms, two commercial polyvalent anti-venoms produced at Instituto Butantan, were compared in order to assess their capacity for neutralization of the lethal, hemorrhagic, coagulant and myotoxic activities of Bothrops jararacussu venom. Both anti-venoms showed the same level of hemorrhagic activity neutralization. However, AB/C was about three times more efficient than AB in neutralizing the myotoxic activity, and two times more potent for neutralization of lethality and coagulant activity of Bothrops jararacussu venom. These data suggest that the use of AB/C could be of value in the treatment of patients bitten by snakes of this species.

Immunization of horses with Crotalus durissus Terrificus (South American Rattlesnake) venom: a comparision of four different procedures

A comparative study was carried out on horses immunized with Crotalus durissus terrificus venom using four different inoculation procedures, which included the use Freund's adjuvant, A1(OH)3 and liposomes as adjuvants. The antibody titer was assessed by enzyme linked immunosorbent assay (ELISA) and the neutralizing potency by the neutralizing median effective dose (ED50). The inoculation schedule used in horses to obtain antivenom serum consisted of scinjections of a 7.5 mg venom starting dose in 5.0ml sterile saline emulsified with an equal volume sterile saline at 2-dayintervals. This immunization procedure, based in low doses of antigen (37.5mg/horse) emulsified with Freund's adjuvant, proceduced a more protective andsustained immune response whencompared with other procedures using A1(OH)3 and 5.0 mg/horse in liposome) or high (870.0 mg/horse in A1(OH)3 and 20.0 mg/horse in liposome) antigen doses. The ED50 values evaluated at the end of the procedure were 15.4 *l serum/20 gmouse when antigen was emulsified with Freund's adjuvant; 21.7 * serum/20 g mouse when 870,0 mg antigen/horse was emulsified with A1(OH)3 and 30.0 *l serum/20 g mouse when 50.0 mg antigen/hors was emulsified with a1(OH)3.When antigen was emulsified with liposome, the immune serum was ineffective against the lethal effects of C.d terrificus venom. The inoculation schedule used in horses to obtain hyperimmune serum consisted of reimmunization with sc booster injections of 7.5 mg venom in 5.0 ml sterile saline emulsified with an equal volume of Freund's incomplete adjuvant. One week later, 2.5 mg venom in 12.0 ml sterile saline was inoculated at 2-day intervals.This reimmunization schedule,based on low doses of antigen (15.0 mg/horse) emulsified with Freund's incomplete adjuvant or with saline, produced a protective andsustained immune response, regardless of the initial immunization procedure. The ED50 evaluatedfor each of the animals five days after the reimmunization period was never more than 20 * serum/20 g mouse. The liposome inoculation method employed a membrane-stabilized reverse phase evaporation preparation of sphingomyelin/cholesterol 2.5/1 (w/w) liposomes. This procedure permits incorporation of 1.0 mg protein per mg of phospholipid. This liposome inoculation method , which stimulates a rapid, sustained and protective immune response in mice and rabbits inoculated with both C.d. collineatus and C.d. terrificus, was not effective when horses were immunized with C.