Detection of Wolbachia and Leishmania DNA in sand flies (Diptera: Psychodidae, Phlebotominae) from a focus of cutaneous leishmaniasis in Tabasco, Mexico.

Phlebotomine sand flies are the main vectors of Leishmania genus species worldwide; therefore, the detection of some reproductive parasites, such as Wolbachia, has been considered a possible strategy for biological control. In Mexico, leishmaniasis cases have been recorded in 25 states, yet only two sand fly species have been related to Wolbachia spp. Although the state of Tabasco has a high number of leishmaniasis cases, only few studies have been done on sand fly species. The aim of this study was to analyze the diversity of sand fly species and to detect Wolbachia spp. and/or Leishmania spp. in the captured specimens. Sand flies were collected at the locality of Huimango, Tabasco, Mexico, during October 2019, using nine light traps (CDC) and two Shannon traps per night. The specimens were identified and females were analyzed by PCR for the DNA detection for pathogens. A total of 193 sand fly specimens belonging to five species were morphologically identified. Pintomyia ovallesi was the most abundant species (76.84%), followed by Micropygomyia cayennensis (6.40%). Furthermore, first records of four sand fly species were established for the state of Tabasco, thereby increasing the species richness in the state from four to eight. We observed a natural infection rate of 9.7% (10/103) for Leishmania and 0.91% (1/103) for Wolbachia. The importance of conducting entomological surveys in endemic areas of leishmaniasis in Mexico is highlighted, to determine whether other sand fly species may be potential vectors of Leishmania spp., and if some Wolbachia strains could be relevant for the control of leishmaniasis.

Interaction between environmental and socioeconomic determinants for cutaneous leishmaniasis risk in Latin America.

OBJECTIVE: Determine and characterize potential risk areas for the occurrence of cutaneous leishmaniasis (CL) in Latin America (LA). METHOD: Ecological observational study with observation units defined by municipalities with CL transmission between 2014-2018. Environmental and socioeconomic variables available for at least 85% of the municipalities were used, combined in a single database, utilizing the R software. The principal component analysis methodology was combined with a hierarchical cluster analysis to group clusters of municipalities based on their similarity. The V-test was estimated to define the positive or negative association of the variables with the clusters and separation by natural breaks was used to determine which ones contributed the most to each cluster. Information on cases was also incorporated in the analyses to attribute CL risk for each cluster. RESULTS: This study included 4,951 municipalities with CL transmission (36.5% of the total in LA) and seven clusters were defined by their association with 18 environmental and socioeconomic variables. The historical risk of CL is positively associated with the Amazonian, Andean and Savannah clusters in a decreasingly manner; and negatively associated with the Forest evergreen, Forest/crop and Forest/populated clusters. The Agricultural cluster did not reveal any association with the CL cases. CONCLUSIONS: The study made it possible to identify and characterize the CL risk by clusters of municipalities and to recognize the epidemiological distribution pattern of transmission, which provides managers with better information for intersectoral interventions to control CL.

Interaction between environmental and socioeconomic determinants for cutaneous leishmaniasis risk in Latin America

[ABSTRACT]. Objective. Determine and characterize potential risk areas for the occurrence of cutaneous leishmaniasis (CL) in Latin America (LA).Method. Ecological observational study with observation units defined by municipalities with CL transmission between 2014-2018. Environmental and socioeconomic variables available for at least 85% of the municipal-ities were used, combined in a single database, utilizing the R software. The principal component analysis methodology was combined with a hierarchical cluster analysis to group clusters of municipalities based on their similarity. The V-test was estimated to define the positive or negative association of the variables with the clusters and separation by natural breaks was used to determine which ones contributed the most to each cluster. Information on cases was also incorporated in the analyses to attribute CL risk for each cluster.Results. This study included 4,951 municipalities with CL transmission (36.5% of the total in LA) and seven clusters were defined by their association with 18 environmental and socioeconomic variables. The histori-cal risk of CL is positively associated with the Amazonian, Andean and Savannah clusters in a decreasingly manner; and negatively associated with the Forest evergreen, Forest/crop and Forest/populated clusters. The Agricultural cluster did not reveal any association with the CL cases.Conclusions. The study made it possible to identify and characterize the CL risk by clusters of municipalities and to recognize the epidemiological distribution pattern of transmission, which provides managers with better information for intersectoral interventions to control CL.

[RESUMEN]. Objetivo. Determinar y caracterizar áreas de riesgo potencial de la ocurrencia de leishmaniasis cutánea (LC) en América Latina (AL).Método. Estudio observacional ecológico con unidades de observación definidas por municipios con transmisión de LC entre 2014-2018. Se utilizaron variables medioambientales y socioeconómicas disponibles para al menos 85% de los municipios, combinados en una sola base de datos, a través del software R. Se combinó la metodología de análisis de componentes principales con un análisis de conglomerados jerárquicos para la formación de conglomerados de municipios en función de su similitud. Se estimó el V-test para definir la asociación positiva o negativa de las variables con los conglomerados y separación por divisiones naturales para determinar cuáles contribuyeron más a cada conglomerado. Se incorporaron los casos para atribuir el riesgo de LC para cada conglomerado.Resultados. Se incluyeron en el estudio 4 951 municipios con transmisión de LC (36,5% del total en AL) y se definieron siete conglomerados por su asociación con 18 variables medioambientales y socioeconómicas. El riesgo histórico de LC se asocia de manera positiva y en forma decreciente con los conglomerados Amazónico, Andino y Sabana; y de manera negativa con los conglomerados Boscoso/perenne, Boscoso/cultivo y Boscoso/poblado. El conglomerado Agrícola no reveló ninguna asociación con los casos de LC.Conclusiones. El estudio permitió identificar y caracterizar el riesgo de LC por conglomerados de municipios y conocer el patrón propio epidemiológico de distribución de la transmisión, lo que proporciona a los gestores una mejor información para las intervenciones intersectoriales para el control de la LC.

[RESUMO]. Objetivo. Determinar e caracterizar as áreas de risco de ocorrência de leishmaniose cutânea na América Latina.Método. Estudo observacional ecológico com unidades de observação definidas por municípios com transmissão de leishmaniose cutânea entre 2014 e 2018. Foram usadas as variáveis ambientais e socioeconômicas disponíveis em 85% ou mais dos municípios, reunidas em uma única base de dados com o uso do software R. A metodologia de análise de componentes principais foi combinada a uma análise de conglomerados com agrupamento hierárquico para formar conglomerados de municípios por semelhança. O teste V foi usado para estabelecer a associação (positiva ou negativa) das variáveis com os conglomerados e uma separação por divisões naturais foi usada para determinar as variáveis que mais contribuíram em cada conglomerado. Os casos foram incluídos para avaliar o risco de leishmaniose cutânea em cada conglomerado.Resultados. A amostra do estudo compreendeu 4.951 municípios com transmissão de leishmaniose cutânea (36,5% do total na América Latina). Foram definidos sete conglomerados por apresentarem associação com 18 variáveis ambientais e socioeconômicas. Foi observada associação positiva e decrescente do risco histórico de leishmaniose cutânea com os conglomerados Amazônico, Andino e Savana e negativa com os conglomerados Mata/perene, Mata/cultivo e Mata/povoado. O conglomerado Agrícola não demonstrou asso-ciação com casos de leishmaniose cutânea.Conclusões. Este estudo permitiu identificar e caracterizar o risco de leishmaniose cutânea por conglomerados de municípios e conhecer o padrão epidemiológico de distribuição da transmissão da doença, oferecendo às autoridades dados melhores para subsidiar as intervenções intersetoriais para o controle da leishmaniose cutânea.

[Interaction among environmental and socioeconomic determinants of risk for cutaneous leishmaniasis in Latin AmericaInteração entre os determinantes ambientais e socioeconômicos para o risco de leishmaniose cutânea na América Latina]. / Interacción entre los determinantes medioambientales y socioeconómicos para el riesgo para leishmaniasis cutánea en América Latina.

OBJECTIVE: Determine and characterize areas at potential risk for the occurrence of cutaneous leishmaniasis (CL) in Latin America. METHOD: Ecological observational study with observation units defined by municipalities with CL transmission during 2014-2018. Environmental and socioeconomic variables available for at least 85% of municipalities were combined in a single database, using R software. Principal component analysis was combined with hierarchical cluster analysis for the formation of clusters of municipalities according to their similarity. The V-test was used to define positive or negative association of variables with clusters and separation by natural divisions to determine which contributed more to each cluster. Cases were included to attribute CL risk for each cluster. RESULTS: The study included 4 951 municipalities with CL transmission (36.5% of municipalities in Latin America); seven clusters were defined by their association with 18 environmental and socioeconomic variables. Historical risk of CL is associated positively and in descending order with the Amazonian, Andean, and Savanna clusters; and negatively with the Forest/perennial, Forest/cultivated, and Forest/populated clusters. The Agricultural cluster showed no association with CL cases. CONCLUSIONS: The study made it possible to identify and characterize CL risk by clusters of municipalities and to understand the characteristic epidemiological distribution patterns of transmission, providing program managers with better information for intersectoral interventions to control CL.

OBJETIVO: Determinar e caracterizar as áreas de risco de ocorrência de leishmaniose cutânea na América Latina. MÉTODO: Estudo observacional ecológico com unidades de observação definidas por municípios com transmissão de leishmaniose cutânea entre 2014 e 2018. Foram usadas as variáveis ambientais e socioeconômicas disponíveis em 85% ou mais dos municípios, reunidas em uma única base de dados com o uso do software R. A metodologia de análise de componentes principais foi combinada a uma análise de conglomerados com agrupamento hierárquico para formar conglomerados de municípios por semelhança. O teste V foi usado para estabelecer a associação (positiva ou negativa) das variáveis com os conglomerados e uma separação por divisões naturais foi usada para determinar as variáveis que mais contribuíram em cada conglomerado. Os casos foram incluídos para avaliar o risco de leishmaniose cutânea em cada conglomerado. RESULTADOS: A amostra do estudo compreendeu 4.951 municípios com transmissão de leishmaniose cutânea (36,5% do total na América Latina). Foram definidos sete conglomerados por apresentarem associação com 18 variáveis ambientais e socioeconômicas. Foi observada associação positiva e decrescente do risco histórico de leishmaniose cutânea com os conglomerados Amazônico, Andino e Savana e negativa com os conglomerados Mata/perene, Mata/cultivo e Mata/povoado. O conglomerado Agrícola não demonstrou associação com casos de leishmaniose cutânea. CONCLUSÕES: Este estudo permitiu identificar e caracterizar o risco de leishmaniose cutânea por conglomerados de municípios e conhecer o padrão epidemiológico de distribuição da transmissão da doença, oferecendo às autoridades dados melhores para subsidiar as intervenções intersetoriais para o controle da leishmaniose cutânea.

Interacción entre los determinantes medioambientales y socioeconómicos para el riesgo de leishmaniasis cutánea en América Latina

[RESUMEN]. Objetivo. Determinar y caracterizar áreas de riesgo potencial de la ocurrencia de leishmaniasis cutánea (LC) en América Latina (AL). Método. Estudio observacional ecológico con unidades de observación definidas por municipios con transmisión de LC entre 2014-2018. Se utilizaron variables medioambientales y socioeconómicas disponibles para al menos 85% de los municipios, combinados en una sola base de datos, a través del software R. Se combinó la metodología de análisis de componentes principales con un análisis de conglomerados jerárquicos para la formación de conglomerados de municipios en función de su similitud. Se estimó el V-test para definir la asociación positiva o negativa de las variables con los conglomerados y separación por divisiones naturales para determinar cuáles contribuyeron más a cada conglomerado. Se incorporaron los casos para atribuir el riesgo de LC para cada conglomerado. Resultados. Se incluyeron en el estudio 4 951 municipios con transmisión de LC (36,5% del total en AL) y se definieron siete conglomerados por su asociación con 18 variables medioambientales y socioeconómicas. El riesgo histórico de LC se asocia de manera positiva y en forma decreciente con los conglomerados Amazónico, Andino y Sabana; y de manera negativa con los conglomerados Boscoso/perenne, Boscoso/cultivo y Boscoso/poblado. El conglomerado Agrícola no reveló ninguna asociación con los casos de LC. Conclusiones. El estudio permitió identificar y caracterizar el riesgo de LC por conglomerados de municipios y conocer el patrón propio epidemiológico de distribución de la transmisión, lo que proporciona a los gestores una mejor información para las intervenciones intersectoriales para el control de la LC.

[ABSTRACT]. Objective. Determine and characterize areas at potential risk for the occurrence of cutaneous leishmaniasis (CL) in Latin America. Method. Ecological observational study with observation units defined by municipalities with CL transmission during 2014-2018. Environmental and socioeconomic variables available for at least 85% of municipalities were combined in a single database, using R software. Principal component analysis was combined with hierarchical cluster analysis for the formation of clusters of municipalities according to their similarity. The V-test was used to define positive or negative association of variables with clusters and separation by natural divisions to determine which contributed more to each cluster. Cases were included to attribute CL risk for each cluster. Results. The study included 4 951 municipalities with CL transmission (36.5% of municipalities in Latin America); seven clusters were defined by their association with 18 environmental and socioeconomic variables. Historical risk of CL is associated positively and in descending order with the Amazonian, Andean, and Savanna clusters; and negatively with the Forest/perennial, Forest/cultivated, and Forest/populated clusters. The Agricultural cluster showed no association with CL cases. Conclusions. The study made it possible to identify and characterize CL risk by clusters of municipalities and to understand the characteristic epidemiological distribution patterns of transmission, providing program managers with better information for intersectoral interventions to control CL.

[RESUMO]. Objetivo. Determinar e caracterizar as áreas de risco de ocorrência de leishmaniose cutânea na América Latina. Método. Estudo observacional ecológico com unidades de observação definidas por municípios com transmissão de leishmaniose cutânea entre 2014 e 2018. Foram usadas as variáveis ambientais e socioeconômicas disponíveis em 85% ou mais dos municípios, reunidas em uma única base de dados com o uso do software R. A metodologia de análise de componentes principais foi combinada a uma análise de conglomerados com agrupamento hierárquico para formar conglomerados de municípios por semelhança. O teste V foi usado para estabelecer a associação (positiva ou negativa) das variáveis com os conglomerados e uma separação por divisões naturais foi usada para determinar as variáveis que mais contribuíram em cada conglomerado. Os casos foram incluídos para avaliar o risco de leishmaniose cutânea em cada conglomerado. Resultados. A amostra do estudo compreendeu 4.951 municípios com transmissão de leishmaniose cutânea (36,5% do total na América Latina). Foram definidos sete conglomerados por apresentarem associação com 18 variáveis ambientais e socioeconômicas. Foi observada associação positiva e decrescente do risco histórico de leishmaniose cutânea com os conglomerados Amazônico, Andino e Savana e negativa com os conglomerados Mata/perene, Mata/cultivo e Mata/povoado. O conglomerado Agrícola não demonstrou associação com casos de leishmaniose cutânea. Conclusões. Este estudo permitiu identificar e caracterizar o risco de leishmaniose cutânea por conglomerados de municípios e conhecer o padrão epidemiológico de distribuição da transmissão da doença, oferecendo às autoridades dados melhores para subsidiar as intervenções intersetoriais para o controle da leishmaniose cutânea.

Interacción entre los determinantes medioambientales y socioeconómicos para el riesgo para leishmaniasis cutánea en América Latina / Interaction among environmental and socioeconomic determinants of risk for cutaneous leishmaniasis in Latin America / Interação entre os determinantes ambientais e socioeconômicos para o risco de leishmaniose cutânea na América Latina

RESUMEN Objetivo. Determinar y caracterizar áreas de riesgo potencial de la ocurrencia de leishmaniasis cutánea (LC) en América Latina (AL). Método. Estudio observacional ecológico con unidades de observación definidas por municipios con transmisión de LC entre 2014-2018. Se utilizaron variables medioambientales y socioeconómicas disponibles para al menos 85% de los municipios, combinados en una sola base de datos, a través del software R. Se combinó la metodología de análisis de componentes principales con un análisis de conglomerados jerárquicos para la formación de conglomerados de municipios en función de su similitud. Se estimó el V-test para definir la asociación positiva o negativa de las variables con los conglomerados y separación por divisiones naturales para determinar cuáles contribuyeron más a cada conglomerado. Se incorporaron los casos para atribuir el riesgo de LC para cada conglomerado. Resultados. Se incluyeron en el estudio 4 951 municipios con transmisión de LC (36,5% del total en AL) y se definieron siete conglomerados por su asociación con 18 variables medioambientales y socioeconómicas. El riesgo histórico de LC se asocia de manera positiva y en forma decreciente con los conglomerados Amazónico, Andino y Sabana; y de manera negativa con los conglomerados Boscoso/perenne, Boscoso/cultivo y Boscoso/poblado. El conglomerado Agrícola no reveló ninguna asociación con los casos de LC. Conclusiones. El estudio permitió identificar y caracterizar el riesgo de LC por conglomerados de municipios y conocer el patrón propio epidemiológico de distribución de la transmisión, lo que proporciona a los gestores una mejor información para las intervenciones intersectoriales para el control de la LC.

ABSTRACT Objective. Determine and characterize areas at potential risk for the occurrence of cutaneous leishmaniasis (CL) in Latin America. Method. Ecological observational study with observation units defined by municipalities with CL transmission during 2014-2018. Environmental and socioeconomic variables available for at least 85% of municipalities were combined in a single database, using R software. Principal component analysis was combined with hierarchical cluster analysis for the formation of clusters of municipalities according to their similarity. The V-test was used to define positive or negative association of variables with clusters and separation by natural divisions to determine which contributed more to each cluster. Cases were included to attribute CL risk for each cluster. Results. The study included 4 951 municipalities with CL transmission (36.5% of municipalities in Latin America); seven clusters were defined by their association with 18 environmental and socioeconomic variables. Historical risk of CL is associated positively and in descending order with the Amazonian, Andean, and Savanna clusters; and negatively with the Forest/perennial, Forest/cultivated, and Forest/populated clusters. The Agricultural cluster showed no association with CL cases. Conclusions. The study made it possible to identify and characterize CL risk by clusters of municipalities and to understand the characteristic epidemiological distribution patterns of transmission, providing program managers with better information for intersectoral interventions to control CL.

RESUMO Objetivo. Determinar e caracterizar as áreas de risco de ocorrência de leishmaniose cutânea na América Latina. Método. Estudo observacional ecológico com unidades de observação definidas por municípios com transmissão de leishmaniose cutânea entre 2014 e 2018. Foram usadas as variáveis ambientais e socioeconômicas disponíveis em 85% ou mais dos municípios, reunidas em uma única base de dados com o uso do software R. A metodologia de análise de componentes principais foi combinada a uma análise de conglomerados com agrupamento hierárquico para formar conglomerados de municípios por semelhança. O teste V foi usado para estabelecer a associação (positiva ou negativa) das variáveis com os conglomerados e uma separação por divisões naturais foi usada para determinar as variáveis que mais contribuíram em cada conglomerado. Os casos foram incluídos para avaliar o risco de leishmaniose cutânea em cada conglomerado. Resultados. A amostra do estudo compreendeu 4.951 municípios com transmissão de leishmaniose cutânea (36,5% do total na América Latina). Foram definidos sete conglomerados por apresentarem associação com 18 variáveis ambientais e socioeconômicas. Foi observada associação positiva e decrescente do risco histórico de leishmaniose cutânea com os conglomerados Amazônico, Andino e Savana e negativa com os conglomerados Mata/perene, Mata/cultivo e Mata/povoado. O conglomerado Agrícola não demonstrou associação com casos de leishmaniose cutânea. Conclusões. Este estudo permitiu identificar e caracterizar o risco de leishmaniose cutânea por conglomerados de municípios e conhecer o padrão epidemiológico de distribuição da transmissão da doença, oferecendo às autoridades dados melhores para subsidiar as intervenções intersetoriais para o controle da leishmaniose cutânea.

Premature deaths by visceral leishmaniasis in Brazil investigated through a cohort study: A challenging opportunity?

BACKGROUND: Visceral Leishmaniasis (VL) is the most severe form of leishmaniasis because it can lead to death. In the Americas, 96% of cases are in Brazil, and despite efforts, the fatality rate has increased in the past years. We analyzed deaths associated to VL in Brazil and investigated the factors that could influence on the timeliness of fatal outcome with emphasis on time (tStoD). METHODOLOGY: The registered deaths by VL were sourced from the Brazilian National Notification System from 2007-2014. Through a retrospective cohort study, univariate and multivariable Cox proportional hazards model analysis were performed and investigated the factors that could influence the time (tStoD). These factors were analyzed through survival models. RESULTS: Out of the 1,589 reported deaths, the median for onset of the symptoms and the case notification date (tStoN) is 25 days (10-61), and for date of case notification and death (tNotD) is 9 days (4-17). The time (tStoN) to event investigation for HIV non-infected individuals was 1.4 (1.16-1.68) greater than the HIV positive group. At the same time peri-urban and urban area were 0.83 (0.47-1.44) and 1.33 (1.16-1.52), respectively. The explorations revealed apparent differences between the time to event investigation (both for tStoN and tNotD) and the age at the onset of the symptoms. According to the tStoN the rate of notification is 1.73 times greater in patients under 5 years old at the onset of the clinical symptoms compared to older patients. CONCLUSION: VL patients under 5 years old were diagnosed earlier and had shorter survival. It could mean that in younger population, although properly diagnosed, the fatality pattern might be related to the severity of the disease. Main host characteristics were evaluated, and age and co-infections seem to have an impact in the disease progression.

SisLeish: A multi-country standardized information system to monitor the status of Leishmaniasis in the Americas.

BACKGROUND: In the Americas, leishmaniasis is endemic in 18 countries, and from 2001 through 2015, 17 countries reported 843,931 cases of cutaneous and mucocutaneous leishmaniasis, and 12 countries reported 52,176 cases of visceral leishmaniasis. A Regional Information System (SisLeish) was created in order to provide knowledge of the distribution and tendency of this disease to analyze and monitor the leishmaniasis status. This article analyses the performance and progress of SisLeish from 2012-2015. METHODOLOGY: The performance of SisLeish was evaluated by country adhesion, data completeness and delay in entering the data, and also by the SWOT technique. Furthermore, we outlined the structure and modus operandi of the system and indicators utilized. RESULTS: In 2012, only 18% of the countries entered the data in SisLeish before the deadline, where 66.7% and 50% of the countries with autochthonous CL/ML and VL reported their cases to the system, respectively. Whereas in 2015, 59% of the countries reached the deadline, where 94.4% and 58.3% of the countries reported their CL/ML and VL data, respectively. Regarding data completeness, there was great progress for different variables since its launch, such as gender, which had an approximately 100% improvement from 2012 to 2015. The SWOT analysis of SisLeish showed 12 strengths, 11 opportunities, seven weaknesses and six threats. CONCLUSIONS: From 2012-2015 there has been an improvement in the adhesion, quality and data completeness, showing the effort of the majority of the countries to enhance their national database. The SWOT analysis demonstrated that strengths and opportunities exceed weaknesses and threats; however, it highlighted the system frailties and challenges that need to be addressed. Furthermore, it has stimulated several National Programs to advance their surveillance system. Therefore, SisLeish has become an essential tool to prioritize areas, assist in decision-making processes, and to guide surveillance and control actions.

Infection of mice with oocysts of Toxoplasma gondii by oral route showed differences of virulence from Brazilian RFLP genotypes BrI and BrIII.

South American strains of Toxoplasma gondii present higher genetic diversity than classical European strains. We compared the virulence of two non-archetypal Brazilian genotypes of T. gondii to mice. Oocysts of four isolates, two genotype BrI (TgCatBr71 and TgShBr11) and two BrIII (TgCatBr74 and TgCatBr60) were obtained from cats fed experimentally infected mice. After sporulation, 5.0×10(1) and 1.0×10(2) oocysts were orally administrated to Swiss albine mice in Experiments #1 and #2, respectively (4-10 mice/group). Humoral response from dead and surviving mice was analyzed on days 9 to 35 post-infection. Microscopic observations of lungs and brains were performed for tachyzoites and cysts visualization in fresh preparations. Negative results were tested by PCR. Virulence after infection with oocysts is dose dependent for genotype BrIII isolates, but not for BrI. Differences in mortality were observed among isolates from genotype BrIII on Experiment #1. Intra-genotype phenotypic variation related to the parasite stage of infection was demonstrated and this characteristic should be further studied and may influence future work regarding the role of virulence amid hosts.

Molecular phylogeny of Toxoplasmatinae: comparison between inferences based on mitochondrial and apicoplast genetic sequences.

Phylogenies within Toxoplasmatinae have been widely investigated with different molecular markers. Here, we studied molecular phylogenies of the Toxoplasmatinae subfamily based on apicoplast and mitochondrial genes. Partial sequences of apicoplast genes coding for caseinolytic protease (clpC) and beta subunit of RNA polymerase (rpoB), and mitochondrial gene coding for cytochrome B (cytB) were analyzed. Laboratory-adapted strains of the closely related parasites Sarcocystis falcatula and Sarcocystis neurona were investigated, along with Neospora caninum, Neospora hughesi, Toxoplasma gondii (strains RH, CTG and PTG), Besnoitia akodoni, Hammondia hammondiand two genetically divergent lineages of Hammondia heydorni. The molecular analysis based on organellar genes did not clearly differentiate between N. caninum and N. hughesi, but the two lineages of H. heydorni were confirmed. Slight differences between the strains of S. falcatula and S. neurona were encountered in all markers. In conclusion, congruent phylogenies were inferred from the three different genes and they might be used for screening undescribed sarcocystid parasites in order to ascertain their phylogenetic relationships with organisms of the family Sarcocystidae. The evolutionary studies based on organelar genes confirm that the genus Hammondia is paraphyletic. The primers used for amplification of clpC and rpoB were able to amplify genetic sequences of organisms of the genus Sarcocystisand organisms of the subfamily Toxoplasmatinae as well.

Molecular phylogeny of Toxoplasmatinae: comparison between inferences based on mitochondrial and apicoplast genetic sequences / Filogenia molecular de Toxoplasmatinae: comparação entre inferências baseadas em sequências genéticas mitocondriais e de apicoplasto

Abstract Phylogenies within Toxoplasmatinae have been widely investigated with different molecular markers. Here, we studied molecular phylogenies of the Toxoplasmatinae subfamily based on apicoplast and mitochondrial genes. Partial sequences of apicoplast genes coding for caseinolytic protease (clpC) and beta subunit of RNA polymerase (rpoB), and mitochondrial gene coding for cytochrome B (cytB) were analyzed. Laboratory-adapted strains of the closely related parasites Sarcocystis falcatula and Sarcocystis neurona were investigated, along with Neospora caninum, Neospora hughesi, Toxoplasma gondii (strains RH, CTG and PTG), Besnoitia akodoni, Hammondia hammondiand two genetically divergent lineages of Hammondia heydorni. The molecular analysis based on organellar genes did not clearly differentiate between N. caninum and N. hughesi, but the two lineages of H. heydorni were confirmed. Slight differences between the strains of S. falcatula and S. neurona were encountered in all markers. In conclusion, congruent phylogenies were inferred from the three different genes and they might be used for screening undescribed sarcocystid parasites in order to ascertain their phylogenetic relationships with organisms of the family Sarcocystidae. The evolutionary studies based on organelar genes confirm that the genusHammondia is paraphyletic. The primers used for amplification of clpC and rpoB were able to amplify genetic sequences of organisms of the genus Sarcocystisand organisms of the subfamily Toxoplasmatinae as well.

Resumo A filogenia da subfamília Toxoplasmatinae tem sido amplamente investigada com diversos marcadores moleculares. Neste estudo, a filogenia molecular da subfamília Toxoplasmatinae foi analisada através de genes de apicoplasto e mitocondriais. Foram analisadas sequências parciais de genes de apicoplasto codificadores da protease caseinolítica (clpC), e da subunidade beta da RNA polimerase (rpoB) e de gene mitocondrial codificador de citocromo B (cytB). Foram investigadas cepas adaptadas em laboratório de Sarcocystis neurona eSarcocystis falcatula, parasitos estreitamente relacionados, além de Neospora caninum, Neospora hughesi, Toxoplasma gondii (cepas RH, CTG e PTG),Besnoitia akodoni, Hammondia hammondi e duas linhagens geneticamente divergentes de Hammondia heydorni. A análise molecular, baseada em genes de organelas, não diferenciou claramenteN. caninum de N. hughesi, porém foi possível confirmar as duas linhagens de H. heydorni. Foram encontradas pequenas diferenças entre as cepas adaptadas em laboratório deS. falcatula e S. neurona em todos os marcadores moleculares avaliados. Concluindo, filogenias congruentes foram reconstruídas com os três diferentes genes que podem ser úteis em triagem de parasitos sarcocistídeos não identificados, para identificar sua relação com organismos da família Sarcocystidae. Os estudos evolutivos com genes organelares confirmam que o gênero Hammondia é parafilético. Osprimers utilizados para amplificação declpC e rpoB foram capazes de amplificar sequências genéticas de organismos do gênero Sarcocystis e da subfamília Toxoplasmatinae.

Diversity of Sarcocystis spp shed by opossums in Brazil inferred with phylogenetic analysis of DNA coding ITS1, cytochrome B, and surface antigens.

Although few species of Sarcocystis are known to use marsupials of the genus Didelphis as definitive host, an extensive diversity of alleles of surface antigen genes (sag2, sag3, and sag4) has been described in samples of didelphid opossums in Brazil. In this work, we studied 25 samples of Sarcocystis derived from gastrointestinal tract of opossums of the genus Didelphis by accessing the variability of sag2, sag3, sag4, gene encoding cytochrome b (cytB) and first internal transcribed spacer (ITS1). Reference samples of Sarcocystis neurona (SN138) and Sarcocystis falcatula (SF1) maintained in cell culture were also analyzed. We found four allele variants of cytB, seven allele variants of ITS1, 10 allele variants of sag2, 13 allele variants of sag3, and 6 allele variants of sag4. None of the sporocyst-derived sequences obtained from Brazilian opossums revealed 100% identity to SN138 at cytB gene, nor to SN138 or SF1 at ITS1 locus. In addition, none of the sag alleles were found identical to either SF1 or SN138 homologous sequences, and a high number of new sag allele types were found other than those previously described in Brazil. Out of ten sag2 alleles, four are novel, while eight out of 13 sag3 alleles are novel and one out of six sag4 alleles is novel. Further studies are needed to clarify if such a vast repertoire of allele variants of Sarcocystis is the consequence of re-assortments driven by sexual exchange, in order to form individuals with highly diverse characteristics, such as pathogenicity, host spectrum, among others or if it only represents allele variants of different species with different biological traits.

A review of Sarcocystis spp. shed by opossums (Didelphis spp.) in Brazil / Revisão sobre Sarcocystis spp. excretados por gambás (Didelphis spp.) no Brasil

South American opossums are the definitive hosts of Sarcocystis neurona, Sarcocystis falcatula, Sarcocystis speeri and Sarcocystis lindsayi. The sporocysts of these species of Sarcocystis are morphologically similar and methods like infectivity and pathogenicity for intermediate hosts (immunodeficient mice and psittacine birds) and molecular tools are used for identification. Opossums are synanthropic wild animals, and widely distributed in Brazilian territory. Previous studies have shown high environmental contamination with S. neurona sporocysts in several Brazilian regions. This paper reviews information on Sarcocystis spp. shed by various opossum species and its occurrence in Brazil.(AU)

Os gambás Sul-americanos são os hospedeiros definitivos de Sarcocystis falcatula, Sarcocystis neurona, Sarcocystis speeri e Sarcocystis lindsayi. Estas espécies de Sarcocystis são morfologicamente similares, mas podem ser distinguidas por sua patogenicidade e infectividade em hospedeiros intermediários (aves e camundongos imunodeficientes) e técnicas moleculares. Os gambás são animais silvestres e sinantrópicos e amplamente distribuídos no território nacional. Estudos anteriores demonstraram uma alta contaminação ambiental com esporocistos de S. neurona em diversas regiões brasileiras. Este artigo revisa informações sobre Sarcocystis spp. excretados por gambás e sua ocorrência no Brasil.(AU)

Serosurvey for tick-borne diseases in dogs from the Eastern Amazon, Brazil.

Canine ehrlichiosis and babesiosis are the most prevalent tick-borne diseases in Brazilian dogs. Few studies have focused attention in surveying tick-borne diseases in the Brazilian Amazon region. A total of 129 blood samples were collected from dogs living in the Brazilian eastern Amazon. Seventy-two samples from dogs from rural areas of 19 municipalities and 57 samples from urban stray dogs from Santarém municipality were collected. Serum samples were submitted to Indirect Immunofluorescence Assay (IFA) with antigens of Babesia canis vogeli, Ehrlichia canis, and six Rickettsia species. The frequency of dogs containing anti-B. canis vogeli, anti-E. canis, and anti-Rickettsia spp. antibodies was 42.6%, 16.2%, and 31.7%, respectively. Anti-B. canis vogeli antibodies were detected in 59.6% of the urban dogs, and in 29.1% of the rural dogs (P < 0.05). For E. canis, seroprevalence was similar among urban (15.7%) and rural (16.6%) dogs. For Rickettsia spp., rural dogs presented significantly higher (P < 0.05) prevalence (40.3%) than urban animals (21.1%). This first study on tick-borne pathogens in dogs from the Brazilian eastern Amazon indicates that dogs are exposed to several agents, such as Babesia organisms, mostly in the urban area; Spotted Fever group Rickettsia organisms, mostly in the rural area; and Ehrlichia organisms, in dogs from both areas studied.

Serosurvey for tick-borne diseases in dogs from the Eastern Amazon, Brazil / Pesquisa sorológica por doenças transmitidas por carrapatos em cães da Amazônia oriental, Brasil

Canine ehrlichiosis and babesiosis are the most prevalent tick-borne diseases in Brazilian dogs. Few studies have focused attention in surveying tick-borne diseases in the Brazilian Amazon region. A total of 129 blood samples were collected from dogs living in the Brazilian eastern Amazon. Seventy-two samples from dogs from rural areas of 19 municipalities and 57 samples from urban stray dogs from Santarém municipality were collected. Serum samples were submitted to Indirect Immunofluorescence Assay (IFA) with antigens of Babesia canis vogeli, Ehrlichia canis, and six Rickettsia species. The frequency of dogs containing anti-B. canis vogeli, anti-E. canis, and anti-Rickettsia spp. antibodies was 42.6%, 16.2%, and 31.7%, respectively. Anti-B. canis vogeli antibodies were detected in 59.6% of the urban dogs, and in 29.1% of the rural dogs (P < 0.05). For E. canis, seroprevalence was similar among urban (15.7%) and rural (16.6%) dogs. For Rickettsia spp., rural dogs presented significantly higher (P < 0.05) prevalence (40.3%) than urban animals (21.1%). This first study on tick-borne pathogens in dogs from the Brazilian eastern Amazon indicates that dogs are exposed to several agents, such as Babesia organisms, mostly in the urban area; Spotted Fever group Rickettsia organisms, mostly in the rural area; and Ehrlichia organisms, in dogs from both areas studied.

Ehrliquiose canina e babesiose canina são as doenças parasitárias transmitidas por carrapatos de maior prevalência em cães do Brasil. Poucos estudos pesquisaram doenças transmitidas por carrapatos na região da Amazônia brasileira. Um total de 129 amostras de sangue foram colhidas de cães da Amazônia oriental brasileira. Setenta e dois cães eram de áreas rurais de 19 municípios do Estado do Pará, e 57 amostras foram colhidas de cães errantes vadios da área urbana do município de Santarém-PA. As amostras de soro foram submetidas ao ensaio de imunofluorescência indireta, com antígenos de Babesia canis vogeli, Ehrlichia canis, e seis espécies de Rickettsia. A frequência de cães com anticorpos anti-B. canis vogeli, anti-E. canis, e anti-Rickettsia spp. foi de 42,6%, 16,2% e 31,7%, respectivamente. Anticorpos anti-B. canis vogeli foram detectados em 59,6% dos cães urbanos, e em 29,1% dos cães rurais (P < 0.05). Para E. canis, a soroprevalência foi parecida entre os cães urbanos (15,7%) e rurais (16,6%). Para Rickettsia spp., cães rurais apresentaram prevalência (P < 0.05) significativamente maior (40,3%) do que os cães urbanos (21,1%). Esse primeiro estudo sobre agentes transmitidos por carrapatos entre cães da Amazônia oriental brasileira indica que estes animais estão expostos a vários agentes. Estes incluem Babesia principalmente na área urbana, Riquétsias do grupo da Febre Maculosa principalmente nas áreas rurais, e Erliquia em cães de ambas as áreas, rural e urbana.

Evaluation of a rapid immunochromatographic dipstick test for detection of antibodies to Trypanosoma cruzi in dogs experimentally infected with isolates obtained from opossums (Didelphis virginiana), armadillos (Dasypus novemcinctus), and dogs (Canis familiaris) from the United States.

Dogs are reservoir hosts for Trypanosoma cruzi , the causative agent of American trypanosomiasis. A rapid immunochromatographic dipstick test (ICT) is available commercially for canine serological testing. The ICT was developed with the use of sera from South American dogs, but it is not routinely used in the United States. We evaluated the utility of the ICT in detecting anti-T. cruzi antibodies in dogs from the United States. Dogs (N  =  64) were experimentally infected with United States' isolates of T. cruzi from an opossum (Didelphis virginiana), an armadillo (Dasypus novemcinctus), and a domestic dog (Canis familiaris), and were tested after experimental infection. Sera from uninfected United States dogs (n  =  79; hemaculture negative) were used as negative controls. In a blind study, sera were tested by the ICT and compared to the indirect immunofluorescent antibody test with the use of Brazil-strain epimastigotes as antigen. The sensitivity of the ICT was 91% and the specificity was 98% in dogs experimentally infected with United States isolates. Our study indicates that the ICT could be a useful screening tool for serological surveillance of canine T. cruzi exposure in the United States.

Prevalence of antibodies to Trypanosoma Cruzi, Leishmania Infantum, Encephalitozoon Cuniculi, Sarcocystis Neurona, and Neosporacanium in Capybara, Hidrochoerus Hydrochaeris, From São Paulo State, Brazil / Prevalência de anticorpos para Trypanosoma cruzi, Leishmania infantum,

Pouco se sabe sobre a importância da capivara, Hydrochoerus hydrochaeris, como reservatórios de parasitas de zoonóticas ou

importância veterinária. Sera de 63 capivaras, de 6 municípios do Estado de Sa ~ o Paulo, Brasil, foram examinados para detecção de anticorpos

Trypanosoma cruzi, Leishmania infantum, Encephalitozoon cuniculi, Sarcocystis neurona e Neospora caninum utilizando uma indireta

teste de imunofluorescência. Cinco (8%) dos 63 capivaras tinham anticorpos para T. cruzi epimastigotas. Nenhuma das amostras de

capivara reagiram positivamente com promastigotas L. infantum ou com esporos de E. cuniculi. Dois (3%) das amostras de soro foram positivas

para anticorpos para S. neurona merozoítos, e 2 (3%) das amostras de soro foram positivas para anticorpos para N. caninum taquizoítos. A

amostra de soro a partir de 1 capivara foi positivo para anticorpos contra ambos T. cruzi e N. caninum. Nenhum dos restantes 62 amostras reagiram

com mais de um parasita.

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Occurrence of antibodies anti-Neospora caninum, anti-Toxoplasma gondii, and anti-Leishmania chagasi in serum of dogs from Pará State, Amazon, Brazil.

A cross-sectional study was conducted to determine the occurrence of anti-Toxoplasma gondii, anti-Neospora caninum, and anti- Leishmania chagasi antibodies in dogs of the state of Pará, Brazil. For this purpose, 129 blood samples were collected from dogs of different ages and gender. Samples of 72 dogs were collected from 39 rural properties from 19 municipalities, and 57 samples were from stray dogs, collected after captivity by the Center of Zoonosis Control from the municipality of Santarém. The sera were analyzed for anti-T. gondii and anti-N. caninum antibodies by indirect fluorescent antibody tests with cutoff values of 1:16 and 1:50, respectively. For the presence of L. chagasi antibodies, enzyme-linked immunosorbent assay was used and positive results were confirmed by immunochromatographic method using the recombinant antigen K39. Of the total of 129 dogs, 90 (69.8%) were positive for T. gondii, 16 (12.4%) for N. caninum, and 30 (23.3%) for L. chagasi. Antibodies for all three parasites were found simultaneously in seven dogs (5.4%), mostly in urban dogs (six of seven). No association was observed related to gender and location (urban or rural) of dogs and occurrence of N. caninum and T. gondii antibodies although, regarding L. chagasi, higher prevalence was found in females (P < 0.02) and in dogs from urban location (P < 0.001). From the 39 farms, in 30 (76.9%) at least one dog was positive for T. gondii or N. caninum or both. Higher occurrence of Leishmania antibodies was observed in N. caninum-negative dogs (P < 0.05).

Prevalence of antibodies to Trypanosoma cruzi, Leishmania infantum, Encephalitozoon cuniculi, Sarcocystis neurona, and Neospora caninum in Capybara, Hydrochoerus hydrochaeris, from São Paulo State, Brazil.

Little is known about the importance of capybara, Hydrochoerus hydrochaeris, as reservoirs for parasites of zoonotic or veterinary importance. Sera from 63 capybaras, from 6 counties in the state of São Paulo, Brazil, were examined for antibodies to Trypanosoma cruzi, Leishmania infantum, Encephalitozoon cuniculi, Sarcocystis neurona, and Neospora caninum using an indirect immunofluorescent antibody test. Five (8%) of the 63 capybaras had antibodies to T. cruzi epimastigotes. None of the samples from capybara reacted positively with L. infantum promastigotes or with spores of E. cuniculi . Two (3%) of the serum samples were positive for antibodies to S. neurona merozoites, and 2 (3%) of the serum samples were positive for antibodies to N. caninum tachyzoites. A serum sample from 1 capybara was positive for antibodies to both T. cruzi and N. caninum. None of the remaining 62 samples reacted with more than 1 parasite.