Clinical, epidemiological and climatic factors related to the occurrence of cutaneous leishmaniasis in an endemic area in northeastern Brazil / Fatores clínicos, epidemiológicos e climáticos relacionados à ocorrência de leishmaniose cutânea em área endêmica no nordeste do Brasil

Abstract Cutaneous leishmaniasis (CL) is a neglected tropical disease with a wide distribution in the Americas. Brazil is an endemic country and present cases in all states. This study aimed to describe the occurrence, the underlying clinical and epidemiological factors, and the correlation of climatic variables with the frequency of reported CL cases in the municipality of Caxias, state of Maranhão, Brazil. This is a retrospective and descriptive epidemiological study based on data extracted from the Brazilian Information System of Diseases Notification, from 2007 to 2017. Maximum and minimum temperature, precipitation, and relative air humidity data were provided by the Brazilian National Institute of Meteorology. A total of 201 reported autochthonous CL cases were analyzed. The predominance of cases was observed in males (70.1%). The age range between 31 and 60 years old was the most affected, with 96 cases (47.9%). Of the total number of registered cases, 38.8% of the affected individuals were engaged in agriculture-related activities. The georeferenced distribution revealed the heterogeneity of disease occurrence, with cases concentrated in the Western and Southern regions of the municipality. An association was detected between relative air humidity (monthly mean) and the number of CL cases per month (p = 0.04). CL continues to be a concerning public health issue in Caxias. In this context, there is a pressing need to strengthen measures of prevention and control of the disease through the network of health services of the municipality, considering local and regional particularities.

Resumo A leishmaniose cutânea (CL) é uma doença tropical negligenciada, com ampla distribuição nas Américas. O Brasil é um país endêmico e apresenta casos em todos os estados. Este estudo teve como objetivo descrever a ocorrência, os fatores clínicos e epidemiológicos subjacentes e a correlação de variáveis climáticas com a frequência de casos de CL notificados no município de Caxias, estado do Maranhão, Brasil. Este é um estudo epidemiológico retrospectivo e descritivo, com base em dados extraídos da Notificação do Sistema Brasileiro de Informação de Doenças, de 2007 a 2017. Dados máximos e mínimos de temperatura, precipitação e umidade relativa do ar foram fornecidos pelo Instituto Nacional de Meteorologia. Foram analisados 201 casos de CL autóctones relatados. A predominância de casos foi observada no sexo masculino (70,1%). A faixa etária entre 31 e 60 anos foi a mais afetada, com 96 casos (47,9%). Do número total de casos registrados, 38,8% dos indivíduos afetados estavam envolvidos em atividades relacionadas à agricultura. A distribuição georreferenciada revelou a heterogeneidade da ocorrência da doença, com casos concentrados nas regiões oeste e sul do município. Foi detectada associação entre a umidade relativa do ar (média mensal) e o número de casos de CL por mês (p = 0,04). O CL continua sendo uma questão preocupante de saúde pública em Caxias. Nesse contexto, há uma necessidade premente de fortalecer medidas de prevenção e controle da doença por meio da rede de serviços de saúde do município, considerando as particularidades locais e regionais.

First molecular evidence of frogs as a food source for sand flies (Diptera: Phlebotominae) in Brazilian caves.

Genus and species of phlebotomine sand flies have been recorded and described in caves in Brazil, but no study has provided the food source used by sand flies in these environments. Herein, we identified the blood source used by sand fly species in caves located at "Quadrilátero Ferrífero" (QF), Minas Gerais state. Specimens were manually collected near or on anurans inside ferruginous caves in Serra do Gandarela National Park and Serra do Rola Moça State Park. Males and females were placed in vials with 70% alcohol and stored at -10°C. Females engorged, after specific identification, had DNA extracted and followed for PCR amplification using specific primers. Sequencing was analyzed in the GenBank and Barcode of Life. A total of 198 specimens were collected (107 females and 91 males), all of them belonging to species Sciopemyia aff. microps (88.89%), Sciopemyia sordellii (10.61%), or Martinsmyia oliveirai (0.50%). When it comes to the females, 89 were S. aff. microps and 18 S. sordellii. Nineteen engorged females of S. aff. microps were analyzed and most of them (n=18) presented blood from Bokermannohyla martinsi and one contained blood from Scinax fuscovarius. The blood present in engorged females of S. sordellii (n=4) was from B. martinsi. Sciopemyia genus specimens are commonly found in collections carried out inside natural caves, but this was the first study to prove that females of this genus feed on cold-blooded animals in nature. HIGHLIGHTS: • Here we proved that sand flies feed in cold-blooded animals in in Brazilian caves. • Females of the Sciopemyia genus were for the first time found feeding in natural habitats. • Anurans of the family Hylidae were identified as source by molecular analyzes. • Insect bloodmeal identification can help assessing the fauna in several biomes. • This is the first record of S. aff. microps in caves of Brazil.

Clinical, epidemiological and climatic factors related to the occurrence of cutaneous leishmaniasis in an endemic area in northeastern Brazil.

Cutaneous leishmaniasis (CL) is a neglected tropical disease with a wide distribution in the Americas. Brazil is an endemic country and present cases in all states. This study aimed to describe the occurrence, the underlying clinical and epidemiological factors, and the correlation of climatic variables with the frequency of reported CL cases in the municipality of Caxias, state of Maranhão, Brazil. This is a retrospective and descriptive epidemiological study based on data extracted from the Brazilian Information System of Diseases Notification, from 2007 to 2017. Maximum and minimum temperature, precipitation, and relative air humidity data were provided by the Brazilian National Institute of Meteorology. A total of 201 reported autochthonous CL cases were analyzed. The predominance of cases was observed in males (70.1%). The age range between 31 and 60 years old was the most affected, with 96 cases (47.9%). Of the total number of registered cases, 38.8% of the affected individuals were engaged in agriculture-related activities. The georeferenced distribution revealed the heterogeneity of disease occurrence, with cases concentrated in the Western and Southern regions of the municipality. An association was detected between relative air humidity (monthly mean) and the number of CL cases per month (p = 0.04). CL continues to be a concerning public health issue in Caxias. In this context, there is a pressing need to strengthen measures of prevention and control of the disease through the network of health services of the municipality, considering local and regional particularities.

Evaluation of the presence of Leishmania infantum in bone stored in glycerol or sterilized by autoclave for cortical bone grafting / Avaliação da presença de Leishmania infantum em ossos armazenados em glycerol ou esterilizados em autoclave para enxertia óssea cortical

This study was performed to evaluate the effects of autoclaving and storage in 85% glycerol on cortical bone from dogs infected with Leishmania sp. We used 42 cadavers with leishmaniasis. The dogs were evaluated for the presence of Leishmania sp. in culture of bone marrow and by culturing cortical bone. From the infected animals, we harvested 42 diaphysis of the right femur for culture of cortical bone before and after autoclaving or storage in glycerol. There was no significant difference in growth of the parasite in culture of bone marrow or cortical bone. There was no growth of Leishmania sp. in culture of samples after autoclaving or storage in glycerol. Both treatments were effective in preventing the growth of the parasite in vitro, so it was considered viable for grafting.(AU)

Este estudo teve por objetivo avaliar os efeitos da esterilização em autoclave e do armazenamento em glicerol a 85% no osso cortical de cães infectados por Leishmania sp. Foram utilizados 42 cadáveres de cães com leishmaniose. Os cães foram avaliados para a presença de Leishmania sp. em cultura de medula óssea e de osso cortical. Foram coletadas 42 diáfises do fêmur direito para cultura do osso cortical antes e após a esterilização e o armazenamento em glicerol. Não houve diferença significativa no crescimento do parasito em cultura utilizando-se medula óssea ou osso cortical. Não houve crescimento de Leishmania sp. em cultura de amostras de osso cortical após a autoclavagem ou o armazenamento em glicerol. Ambos os tratamentos foram eficazes na prevenção do crescimento do parasita in vitro, de modo que foram considerados viáveis para enxerto.(AU)

Clinical, epidemiological and climatic factors related to the occurrence of cutaneous leishmaniasis in an endemic area in northeastern Brazil

Abstract Cutaneous leishmaniasis (CL) is a neglected tropical disease with a wide distribution in the Americas. Brazil is an endemic country and present cases in all states. This study aimed to describe the occurrence, the underlying clinical and epidemiological factors, and the correlation of climatic variables with the frequency of reported CL cases in the municipality of Caxias, state of Maranhão, Brazil. This is a retrospective and descriptive epidemiological study based on data extracted from the Brazilian Information System of Diseases Notification, from 2007 to 2017. Maximum and minimum temperature, precipitation, and relative air humidity data were provided by the Brazilian National Institute of Meteorology. A total of 201 reported autochthonous CL cases were analyzed. The predominance of cases was observed in males (70.1%). The age range between 31 and 60 years old was the most affected, with 96 cases (47.9%). Of the total number of registered cases, 38.8% of the affected individuals were engaged in agriculture-related activities. The georeferenced distribution revealed the heterogeneity of disease occurrence, with cases concentrated in the Western and Southern regions of the municipality. An association was detected between relative air humidity (monthly mean) and the number of CL cases per month (p = 0.04). CL continues to be a concerning public health issue in Caxias. In this context, there is a pressing need to strengthen measures of prevention and control of the disease through the network of health services of the municipality, considering local and regional particularities.

Resumo A leishmaniose cutânea (CL) é uma doença tropical negligenciada, com ampla distribuição nas Américas. O Brasil é um país endêmico e apresenta casos em todos os estados. Este estudo teve como objetivo descrever a ocorrência, os fatores clínicos e epidemiológicos subjacentes e a correlação de variáveis climáticas com a frequência de casos de CL notificados no município de Caxias, estado do Maranhão, Brasil. Este é um estudo epidemiológico retrospectivo e descritivo, com base em dados extraídos da Notificação do Sistema Brasileiro de Informação de Doenças, de 2007 a 2017. Dados máximos e mínimos de temperatura, precipitação e umidade relativa do ar foram fornecidos pelo Instituto Nacional de Meteorologia. Foram analisados 201 casos de CL autóctones relatados. A predominância de casos foi observada no sexo masculino (70,1%). A faixa etária entre 31 e 60 anos foi a mais afetada, com 96 casos (47,9%). Do número total de casos registrados, 38,8% dos indivíduos afetados estavam envolvidos em atividades relacionadas à agricultura. A distribuição georreferenciada revelou a heterogeneidade da ocorrência da doença, com casos concentrados nas regiões oeste e sul do município. Foi detectada associação entre a umidade relativa do ar (média mensal) e o número de casos de CL por mês (p = 0,04). O CL continua sendo uma questão preocupante de saúde pública em Caxias. Nesse contexto, há uma necessidade premente de fortalecer medidas de prevenção e controle da doença por meio da rede de serviços de saúde do município, considerando as particularidades locais e regionais.

Benchmark of Schemes for Multiscale Molecular Dynamics Simulations.

In multiscale molecular dynamics simulations the accuracy of detailed models is combined with the efficiency of a reduced representation. For several applications - namely those of sampling enhancement - it is desirable to combine fine-grained (FG) and coarse-grained (CG) approaches into a single hybrid approach with an adjustable mixing parameter. We present a benchmark of three algorithms that use a mixing of the two representation layers using a Lagrangian formalism. The three algorithms use three different approaches for keeping the particles at the FG level of representation together: 1) addition of forces, 2) mass scaling, and 3) temperature scaling. The benchmark is applied to liquid hexadecane and includes an evaluation of the average configurational entropy of the FG and CG subsystems. The temperature-scaling scheme achieved a 3-fold sampling speedup with little deviation of FG properties. The addition-of-forces scheme kept FG properties the best but provided little sampling speedup. The mass-scaling scheme yielded a 5-fold speedup but deviated the most from FG properties.

Parameters for Martini sterols and hopanoids based on a virtual-site description.

Sterols play an essential role in modulating bilayer structure and dynamics. Coarse-grained molecular dynamics parameters for cholesterol and related molecules are available for the Martini force field and have been successfully used in multiple lipid bilayer studies. In this work, we focus on the use of virtual sites as a means of increasing the stability of cholesterol and cholesterol-like structures. We improve and extend the Martini parameterization of sterols in four different ways: 1-the cholesterol parameters were adapted to make use of virtual interaction sites, which markedly improves numerical stability; 2-cholesterol parameters were also modified to address reported shortcomings in reproducing correct lipid phase behavior in mixed membranes; 3-parameters for ergosterol were created and adapted from cholesterols; and 4-parameters for the hopanoid class of bacterial polycyclic molecules were created, namely, for hopane, diploptene, bacteriohopanetetrol, and for their polycyclic base structure.

Detection of Leishmania infantum in the smegma of infected dogs / Detecção de Leishmania infantum no esmegma de cães infectados

Considering the venereal transmission of visceral leishmaniasis from dogs to bitches, the aim of this study was to verify if the penile surface and smegma from infected dogs can be the source of parasites in bitches. Twelve Leishmania infantum infected dogs had semen and smegma samples collected for submission to PCR identification of the DNA of the parasite. Semen (41.7 percent) and smegma (50.0 percent) have similar positive incidence (P>0.05; Fisher's exact test), with 58.3 percent of the dogs positive for semen and/or smegma samples. The proportion of positivity for both semen and smegma was 33.3 percent, but 8.3 percent was positive only for semen, and 16.7 percent only for smegma, revealing a moderate agreement between tests (K=0.5; Kappa index). It was concluded that Leishmania infantum is present in the smegma of contaminated dogs and it can be a source of parasites for the semen and the bitch...

Tendo em vista a transmissão venérea da leishmaniose visceral do cão para a cadela, o objetivo deste estudo foi verificar se a superfície peniana e o esmegma de cães infectados poderiam ser a fonte de parasitas para a fêmea. Amostras de sêmen e esmegma de 12 cães infectados com Leishmania infantum foram submetidas à identificação do DNA do parasita por PCR. As incidências de positividade no sêmen (41,7 por cento) e no esmegma (50,0 por cento) foram semelhantes (P>0,05; teste exato de Fisher), sendo 58,3 por cento dos cães positivos para sêmen e/ou esmegma. A positividade para sêmen e esmegma juntos ocorreu em 33,3 por cento, mas em 8,3 por cento dos casos apenas no sêmen, e em 16,7 por cento apenas no esmegma, o que revela uma concordância moderada entre os testes (K=0,5; índice Kappa). Conclui-se que a Leishmania infantum está presente no esmegma de cães contaminados, podendo ser a fonte de parasitas para o sêmen e a cadela...

Leishmania infantum: Lipophosphoglycan intraspecific variation and interaction with vertebrate and invertebrate hosts.

Interspecies variations in lipophosphoglycan (LPG) have been the focus of intense study over the years due its role in specificity during sand fly-Leishmania interaction. This cell surface glycoconjugate is highly polymorphic among species with variations in sugars that branch off the conserved Gal(ß1,4)Man(α1)-PO(4) backbone of repeat units. However, the degree of intraspecies polymorphism in LPG of Leishmania infantum (syn. Leishmania chagasi) is not known. In this study, intraspecific variation in the repeat units of LPG was evaluated in 16 strains of L. infantum from Brazil, France, Algeria and Tunisia. The structural polymorphism in the L. infantum LPG repeat units was relatively slight and consisted of three types: type I does not have side chains; type II has one ß-glucose residue that branches off the disaccharide-phosphate repeat units and type III has up to three glucose residues (oligo-glucosylated). The significance of these modifications was investigated during in vivo interaction of L. infantum with Lutzomyia longipalpis, and in vitro interaction of the parasites and respective LPGs with murine macrophages. There were no consequential differences in the parasite densities in sand fly midguts infected with Leishmania strains exhibiting type I, II and III LPGs. However, higher nitric oxide production was observed in macrophages exposed to glucosylated type II LPG.

Inflammatory response, parasite load and AgNOR expression in ear skin of symptomatic and asymptomatic Leishmania (Leishmania) chagasi infected dogs

The skin has an important role in the transmission of visceral leishmaniasis (VL) as the infection pathway in dogs. To better characterize the inflammatory response of intact skin in VL, sixty infected dogs (30 symptomatic and 30 asymptomatic) and six non-infected controls were studied. Diagnosis of visceral leishmaniasis was confirmed by RIFI and ELISA; direct visualization of the parasite in bone marrow aspirate; imprints of popliteal lymph nodes, spleen, liver and skin; culture in NNN-phase liquid Schneider's medium; and PCR (performed only in the ear skin). Amastigote forms of the parasite in intact skin were found only in symptomatic dogs. Inflammatory infiltrates were observed in all groups, varying from intense and/or moderate in symptomatic to discrete and/or negligible in asymptomatic and control animals. Parasite load was associated with the intensity of the inflammatory response and with clinical manifestations in canine visceral leishmaniasis. AgNOr as active transcription markers were expressed in inflammatory cells and within apoptotic bodies in all groups, including controls, with no statistical difference. Therefore, cell activation and transcription do occur in both symptomatic and asymptomatic canine visceral leishmaniasis and may result in more necrosis and inflammation or in apoptosis and less symptoms, depending on the parasite load.

Phlebotomine sand flies (Diptera: Psychodidae) and Leishmania infection in Gafanhoto Park, Divinópolis, Brazil.

The potential of Gafanhoto Park as an American cutaneous leishmaniasis (ACL) focus was evaluated by examination of sand fly vectors of the Leishmania parasite. This forest remnant is located in a periurban area of Divin6polis, Brazil, where autochthonous cases of ACL have been reported. Sand fly populations were monitored over a 2-yr period (2006-2008) by using light traps (HP and Shannon). During systematic collections with HP traps, 824 specimens in total (342 males and 482 females) of 21 species were captured. Most prevalent species were as follows: Brumptomyia brumpti (Larrouse), Lutzomyia aragaoi (Costa Lima), Lutzomyia lutziana (Costa Lima), Lutzomyia sordellii (Shannon & Del Ponte), and Lutzomyia whitmani (Antunes & Coutinho). Using Shannon traps, 257 specimens representing 15 species were collected (159 females and 98 males), with a high prevalence of L. whitmani and Lutzomyia neivai (Pinto), both vectors of Leishmania braziliensis (Vianna). To ascertain the level of natural infection, a sample of females captured in Shannon traps was assayed for the presence of Leishmania by using polymerase chain reaction-restriction fragment length polymorphism, where 39% of insects were positive. The most infected species was L. whitmani (29 sand flies; 18.2%), followed by L. neivai (21; 13.2%), Lutzomyia christenseni (Young & Duncan) (five; 3.1%), Lutzomyia pessoai (Coutinho & Barreto) (three; 1.9%), L. aragaoi (one; 0.6%), Lutzomyia fischeri (Pinto) (one; 0.6%), Lutzomyia lenti (Mangabeira) (one; 0.6%), L. lutziana (one; 0.6%), and Lutzomyia monticula (Costa Lima) (one; 0.6%). The finding of potential and incriminated vectors naturally infected with Leishmania reinforces the need of epidemiologic surveillance in the area.

Humoral immunological profile and parasitological statuses of Leishmune vaccinated and visceral leishmaniasis infected dogs from an endemic area.

Dogs represent the major reservoir of Leishmaniao chagasi and vaccination against the canine disease is a potential control strategy. However, seroconversion occurs post-vaccine and hence, there is need to discriminate between the former group and naturally infected dogs. The present study represents a comparison of the humoral immunological profiles of both groups using Leishmania soluble antigen (LSA) and fucose-mannose ligand (FML). For both categories, ear skin samples were evaluated immunohistochemically and through PCR, that was also performed in blood specimens, as well as their ability to infect Lutzomyia (Lutzomyia) longipalpis. All these tests showed negative results for the vaccinated dogs. Differences between groups were observed regarding IgG, IgG2 and IgE absorbances as determined by FML-ELISA, and for IgG1 and IgE absorbances as measured by LSA-ELISA, showing that Leishmune-immunised animals and VL naturally infected dogs present different immunological profiles, even though these differences cannot be used to distinguish between these two groups of dogs.

Hepatic granulomas in canine visceral leishmaniasis and clinical status

The histopathological description of intralobular hepatic granulomas in animals with a defined clinical status (asymptomatic, oligosymptomatic and symptomatic animals) was reported. Seventy-one mongrel dogs naturally infected with Leishmania chagasi were obtained from two Brazilian endemic areas: João Pessoa, PB and Belo Horizonte, MG. The hepatic parasite load was determined and compared to granuloma formation. Liver fragments from all infected animals showed remarkable leishmaniotic granulomatous inflammatory reaction. Granulomas with variable size were constituted by macrophages (parasitized or not with amastigotes of L. chagasi), some epithelioid cells, small numbers of lymphocytes, plasma cells, and rare neutrophils. Asymptomatic dogs had higher numbers of granulomas than oligosymptomatic and symptomatic animals from both geographical regions. However, the average diametric size of granulomas was very heterogeneous in all groups, independently of the geographic region (P>0.05). Parasite tissue load did not show any difference among liver fragments of all animals, especially when considering the defined clinical status and/or their geographic origin

Descreve-se a formação de granulomas hepáticos na leishmaniose canina em animais com classificação clínica definida - assintomáticos, oligossintomáticos e sintomáticos. Setenta e um animais, sem raça definida e naturalmente infectados com Leishmania chagasi, foram obtidos de duas regiões endêmicas brasileiras: João Pessoa, PB e Belo Horizonte, MG. A carga parasitária tecidual foi determinada mediante emprego do Leishmania Donovani Units (LDU) e comparada com a formação de granulomas hepáticos. Fragmentos de fígado de todos os animais infectados mostraram reação granulomatosa notadamente leishmaniótica. Granulomas de variáveis tamanhos eram constituídos por macrófagos, parasitados ou não com formas amastigotas de L. chagasi, algumas células epitelióides, pequeno número de linfócitos e plasmócitos, e raros neutrófilos. Cães assintomáticos apresentaram maior número de granulomas do que os animais oligossintomáticos e sintomáticos, em ambas as regiões geográficas. As médias dos diâmetros foram heterogêneas em todos os grupos, independente da região geográfica (P>0,05). Quanto ao parasitismo (LDU), não houve diferença entre as amostras de fígado, especialmente quando se consideraram a classificação clínica e a região geográfica

Comparison of duplex PCR and microscopic techniques for the identification of Babesia bigemina and Babesia bovis in engorged female ticks of Boophilus microplus.

A single-step duplex polymerase chain reaction (PCR) technique and traditional microscopic examination of haemolymph smears were used to detect Babesia bigemina and/or Babesia bovis infection in engorged female ticks of Boophilus microplus recovered from calves raised in an endemic area of the State of Minas Gerais, Brazil. In the PCR amplification of tick-derived DNA, pairs of oligonucleotide primers specific for a 278-bp sequence from B. bigemina and for a 350-bp sequence from B. bovis were used conjointly. The microscopic examination of haemolymph revealed that 16.7% of the engorged ticks were infected with Babesia spp., although no significant differences (rho > 0.05) were found in the infection rate of ticks collected from calves of different age groups. PCR analysis showed that 77.8% of the engorged ticks whose haemolymph contained sporokinetes were infected with B. bigemina, 7.8% with B. bovis and 14.4% with both protozoan species. However, the PCR assay further revealed that, amongst the engorged female ticks whose haemolymph was apparently negative for the presence of sporokinetes, 15.6% were infected with B. bigemina, 2.2% with B. bovis and 10.0% with both species. The duplex PCR method is thus more efficient and sensitive than the microscopic assay and also permits facile identification of the protozoa species present in engorged female ticks.

Histopathological and immunohistochemical study of type 3 complement receptors (CD11b/CD18) in livers and spleens of asymptomatic and symptomatic dogs naturally infected with Leishmania (Leishmania) chagasi.

Leishmania promastigotes interact with macrophages through the association of multiple membrane surface receptors. Macrophage complement receptor CR3 (CD11b/CD18 or Mac-1) has been implicated in the interaction of both human and murine macrophages with serum-opsonized promastigotes. The aim of this study was to determine CR3 expression in the livers and spleens of dogs naturally infected with Leishmania (Leishmania) chagasi. CR3 expression in liver was higher in asymptomatic than in symptomatic animals. Moreover, the hepatic parasitism load determined by immunocytochemical analysis was lower in parallel with higher numbers of granulomas. In contrast, in spleens, CR3 expression was higher in symptomatic animals than in asymptomatic ones. However, the tissue parasite load was greater in spleens of symptomatic dogs. There was a strict correlation between the parasite load and cellular CR3 expression in the spleens of dogs naturally infected with L. chagasi. CR3 macrophage integrins could be essential receptors for Leishmania survival. Considering that the symptomatic animals showed higher parasite loads and higher CD11b/CD18 expression in their spleens, we can conclude that these splenic cells (monocyte-macrophages) might serve to perpetuate intracellular infection.

Characterization of Toxoplasma gondii from domestic animals from Minas Gerais, Brazil.

In an attempt to isolate and characterize Toxoplasma gondii from the State of Minas Gerais, Brazil, musculature samples from 72 pigs, 25 dogs, 28 free-range chickens and 50 chickens produced in industrialized farms were collected. Antibodies to T. gondii have not been detected in pigs, but were found in nine (40.9 %) out of 22 dogs, and in 15 (53.6 %) of 28 free range chickens. T. gondii was not isolated from pigs and industrialized chickens, but from eight dogs and 11 free range chickens. In order to determine T. gondii virulence, female BALB/c mice were inoculated with 10(3), 10(2), 10(1) and 10(0) tachyzoites of the 19 isolates. The strains RH (virulent) and ME49 (non-virulent) were used as references. Isolates were divided into three groups according to the virulence phenotype: five isolates were classified into virulent in mice, one into non-virulent and 13 into intermediate virulent. Nested-PCR of T. gondii SAG2 locus amplified DNA from 21 out of 22 DNA samples directly extracted from heart of free range chickens. These samples were genotyped through a PCR-RFLP assay. Seventeen (80.9 %) were classified into type I; one (4.8 %) into type III and three (14.3 %) into type I or II.

Leishmania amazonensis: chemotaxic and osmotaxic responses in promastigotes and their probable role in development in the phlebotomine gut.

Taxic responses may play a role in development of Leishmania in their phlebotomine sand fly vectors. They are possibly responsible for movement of the parasites towards the anterior regions of the gut, from where they would be transmitted to the vertebrate host. A methodology capable to distinguish chemotaxic from osmotaxic responses was described and used to characterise taxic responses in Leishmania promastigotes. These were able to respond to chemotaxic as well as to osmotaxic stimuli. Like bacteria, promastigotes were capable to undergo "adaptation," a phenomenon by which they stop responding to a continuos stimulus. A model capable to explain how a relatively small number of different receptors works to perceive gradients in chemotaxic responses was proposed. According to this model, these receptors possess low specificity and a wide range of affinities varying from high to low. A low specificity makes the same receptor able to bind to a large number of different but structurally related molecules and; a wide range of affinities (considering a population of receptors), implies that the number of receptors "occupied" by attractant molecules along a gradient would go growing step by step.

Pharmacokinetic and parasitological evaluation of the bone marrow of dogs with visceral leishmaniasis submitted to multiple dose treatment with liposome-encapsulated meglumine antimoniate

The aim of the present study was to evaluate the impact of a multiple dose regimen of a liposomal formulation of meglumine antimoniate (LMA) on the pharmacokinetics of antimony in the bone marrow of dogs with visceral leishmaniasis and on the ability of LMA to eliminate parasites from this tissue. Dogs naturally infected with Leishmania chagasi received 4 intravenous doses of either LMA (6.5 mg antimony/kg body weight, N = 9), or empty liposomes (at the same lipid dose as LMA, N = 9) at 4-day intervals. A third group of animals was untreated (N = 8). Before each administration and at different times after treatment, bone marrow was obtained and analyzed for antimony level (LMA group) by electrothermal atomic absorption spectrometry, and for the presence of Leishmania parasites (all groups). There was a significant increase of antimony concentration from 0.76 æg/kg wet organ (4 days after the first dose) to 2.07 æg/kg (4 days after the fourth dose) and a half-life of 4 days for antimony elimination from the bone marrow. Treatment with LMA significantly reduced the number of dogs positive for parasites (with at least one amastigote per 1000 host cells) compared to controls (positive dogs 30 days after treatment: 0 of 9 in the LMA group, 3 of 9 in the group treated with empty liposomes and 3 of 8 in the untreated group). However, complete elimination of parasites was not achieved. In conclusion, the present study showed that multiple dose treatment with LMA was effective in improving antimony levels in the bone marrow of dogs with visceral leishmaniasis and in reducing the number of positive animals, even though it was not sufficient to achieve complete elimination of parasites.

Pharmacokinetic and parasitological evaluation of the bone marrow of dogs with visceral leishmaniasis submitted to multiple dose treatment with liposome-encapsulated meglumine antimoniate.

The aim of the present study was to evaluate the impact of a multiple dose regimen of a liposomal formulation of meglumine antimoniate (LMA) on the pharmacokinetics of antimony in the bone marrow of dogs with visceral leishmaniasis and on the ability of LMA to eliminate parasites from this tissue. Dogs naturally infected with Leishmania chagasi received 4 intravenous doses of either LMA (6.5 mg antimony/kg body weight, N = 9), or empty liposomes (at the same lipid dose as LMA, N = 9) at 4-day intervals. A third group of animals was untreated (N = 8). Before each administration and at different times after treatment, bone marrow was obtained and analyzed for antimony level (LMA group) by electrothermal atomic absorption spectrometry, and for the presence of Leishmania parasites (all groups). There was a significant increase of antimony concentration from 0.76 microg/kg wet organ (4 days after the first dose) to 2.07 microg/kg (4 days after the fourth dose) and a half-life of 4 days for antimony elimination from the bone marrow. Treatment with LMA significantly reduced the number of dogs positive for parasites (with at least one amastigote per 1000 host cells) compared to controls (positive dogs 30 days after treatment: 0 of 9 in the LMA group, 3 of 9 in the group treated with empty liposomes and 3 of 8 in the untreated group). However, complete elimination of parasites was not achieved. In conclusion, the present study showed that multiple dose treatment with LMA was effective in improving antimony levels in the bone marrow of dogs with visceral leishmaniasis and in reducing the number of positive animals, even though it was not sufficient to achieve complete elimination of parasites.

Germ-free mice produce high levels of interferon-gamma in response to infection with Leishmania major but fail to heal lesions.

In order to investigate the importance of the host microbiota on differentiation of T cell subsets in response to infection, Swiss/NIH germ-free mice and conventional (microbiota-bearing) mice were infected with Leishmania major, and lesion development, parasite loads, and cytokine production were assessed. Germ-free mice failed to heal lesions and presented a higher number of parasites at the site of infection than their conventional counterparts. In addition, histopathological analysis indicated a higher density of parasitized macrophages in lesions from germ-free mice than in conventional mice. The initial production of interleukin (IL)-12 and interferon-gamma (IFN-gamma) in germ-free mice was comparable to the conventional controls. Also, germ-free mice produced elevated levels of IFN-gamma and lower levels of IL-4 throughout the course of infection, suggesting the development of a Th1 response. Macrophages from germ-free mice exposed to IFN-gamma and infected with amastigotes in vitro were not as efficient at killing parasites as macrophages from conventional animals. These observations indicate that the microbiota is not essential for the development of Th1 immune responses, but seems to be important for macrophage activation.