Performance of rapid tests for canine visceral leishmaniasis diagnosis in Argentina

To diagnose dogs infected by Leishmania infantum rK39 rapid diagnosis test is widely used in the Americas, while dual path platform (DPP) was recently adopted by Brazil. In this study we assessed the performance of rK39-RDT and DPP tests in recent urban transmission scenarios of Argentina. The sensitivity and specificity were evaluated with a sera panel and field samples, taken as true infected those from parasitological and/or PCR positive tests. Since none of these tests can be taken as a gold standard, the performance was also evaluated using Latent Class Analysis, a statistical modeling technique which allows to estimating sensitivity and specificity defining a latent class variable as the reference standard. The sensitivity of both tests in the panel was around 92% (symptomatic dogs 96%, asymptomatic 83%), while the sensitivity in field samples of rK39-RDT was 77%, and DPP 98% (mean in symptomatic dogs 89%, asymptomatic 82%). The specificity was similar for both tests and samples, around 98%. Therefore, these tests are acceptable for program dog population-based studies, as spatial stratification, focus intervention and follow up, and they could be used for individual screening and confirmation of clinical presumptive diagnosis in polysymptomatic dogs. The inability to discriminate between immunity and actual infectiousness suggest that a combination with other non-immunological based tests will be required for highly sensitive/specific diagnosis in order to targeting control measures in individual reservoirs from public health perspective, as for individual management from animal health perspective.

Para diagnosticar perros infectados por Leishmania infantum, en las Américas se utiliza ampliamente la prueba rápida rK39, mientras que DPP fue adoptado recientemente por Brasil. En este estudio se evaluó el desempeño de las pruebas rK39-RDT y DPP en escenarios de transmisión urbana reciente en Argentina. La sensibilidad y especificidad se evaluaron con un panel de sueros y muestras de campo, considerando muestras infectadas verdaderas aquellas con pruebas parasitológicas y/o de PCR positivas. Como ninguna de estas pruebas puede considerarse estándar de oro, el desempeño también se evaluó mediante análisis de clases latentes, una técnica de modelado estadístico que permite estimar sensibilidad y especificidad definiendo una variable de clase latente como estándar. La sensibilidad de ambas pruebas en el panel fue de alrededor del 92% (perros sintomáticos 96%, asintomáticos 83%), mientras que la sensibilidad en muestras de campo fue rK39-RDT: 77%, y DPP 98% (media en perros sintomáticos 89%, asintomáticos 82%). La especificidad fue similar para ambas pruebas y muestras, cerca de 98%. Por lo tanto, estas pruebas son aceptables para estudios programáticos caninos de base-poblacional, como estratificación espacial, intervención de foco y seguimiento, y podrían utilizarse para el tamizaje individual y la confirmación del diagnóstico clínico presuntivo en perros poli-sintomáticos. La incapacidad de discriminar entre inmunidad e infectividad real sugiere que se requerirá una combinación con otras pruebas, de base no inmunológica, para un diagnóstico suficientemente sensible/específico que permita definir las medidas de control en reservorios individuales, tanto para salud pública, como para la gestión individual en salud animal.

Transmission of Leishmania infantum from cats to dogs / Transmissão de Leishmania infantum de gatos para cães

Abstract Leishmania infantum infection in cats has been reported in several countries, including Brazil. However, the transmission of visceral leishmaniasis (VL) from cats to another host has not been proven yet. Therefore, the aim of this study was to verify the possibility of L. infantum transmission from cats to dogs. In order to verify the possibility of VL transmission from the cat to the dog, xenodiagnosis was carried out in a VL-positive cat, using 55 female Lutzomyia longipalpis. Five days later, 40 insects were dissected to verify Leishmania infection. The remaining 15 females were fed in a healthy dog. The potential infection of the dog was verified through clinical, serological, parasitological examinations, and PCR, at three, six, and twelve months post-infection. All 55 L. longipalpis females became visibly engorged. Leishmania promastigotes were detected in 27.5% of the dissected insects. Leishmania infection in the dog was confirmed upon first evaluation. DNA sequencing of the parasite isolated from the cat confirmed L. infantum infection and showed 99% similarity with the L. infantum DNA sequences from the dogs. Through this study, it was possible to confirm the L. infantum experimental transmission from a domestic cat to a domestic dog through its biological vector L. longipalpis.

Resumo A infecção por Leishmania infantum em gatos tem sido relatada em vários países, incluindo o Brasil. No entanto, a transmissão da leishmaniose visceral (LV) de gatos para outro hospedeiro ainda não foi comprovada. Portanto, o objetivo deste estudo foi verificar a possibilidade de transmissão de L. infantum de gatos para cães. Para verificar a possibilidade de transmissão da LV do gato para o cão, foi realizado xenodiagnóstico em um gato com LV, utilizando-se 55 fêmeas de Lutzomyia longipalpis. Cinco dias depois, 40 insetos foram dissecados para verificar a infecção por Leishmania. As 15 fêmeas restantes foram alimentadas em um cão saudável. A possível infecção no cão foi verificada por meio de exames clínicos, sorológicos, parasitológicos e PCR, três, seis e doze meses após a infecção. Todas as 55 fêmeas de L. longipalpis ficaram visivelmente ingurgitadas. Promastigotas de Leishmania foram detectadas em 27,5% dos insetos dissecados. A infecção por Leishmania no cão foi confirmada na primeira avaliação. O sequenciamento do DNA do parasito isolado do gato confirmou a infecção por L. infantum e apresentou 99% de similaridade com sequências de DNA de L. infantum de cães. Através deste estudo, foi possível confirmar a transmissão experimental de L. infantum de um gato doméstico para um cão doméstico através do seu vetor biológico L. longipalpis.

Epidemiological aspects of human and canine visceral leishmaniasis in State of Alagoas, Northeast, Brazil / Aspectos epidemiológicos da leishmaniose visceral canina e humana no estado de Alagoas, Nordeste, Brasil

Abstract Zoonotic visceral leishmaniasis (ZVL), caused by protozoans of the genus Leishmania, it is a worldwide of great importance disease. In the northeast region of Brazil, the state of Alagoas has an endemic status for ZVL. Thus, this work aimed to analyze the epidemiological situation of human and canine visceral leishmaniasis in Alagoas, Northeast, Brazil, from 2007 to 2013. We conducted a descriptive, observational, retrospective study using secondary data from the Notifiable Diseases Information System, the Center of Zoonosis Control of Maceió, and the Central Laboratory of Public Health of Alagoas. During the studied period, it was observed that the highest incidence of human visceral leishmaniasis was in 2011 and the lowest in 2013. On the other hand, canine visceral leishmaniasis had its highest incidence in 2007 and its lowest in 2012. Of the 55 municipalities in the State of Alagoas that showed human visceral leishmaniasis (HVL), São José da Tapera presented an average of 4.4 cases over the past five years, being classified as of intense transmission. Regarding canine visceral leishmaniasis, in the same studied period, 45,112 dogs were examined in the State, of which 4,466 were positive. It resulted, thus, in a 9.9% positivity rate. Conclusions: Our data are important because canine infection is an important risk factor for the human disease.

Resumo Leishmaniose visceral zoonótica, causada por protozoários do gênero Leishmania, é uma doença importante no mundo. Na região nordeste do Brasil, do estado de Alagoas é endêmico para LVZ. Neste sentido, este trabalho teve como objetivo analisar a situação epidemiológica da leishmaniose visceral humana e canina em Alagoas, Nordeste, Brasil, no período de 2007 a 2013. Foi realizado um estudo descritivo, observacional, retrospectivo, usando-se secundário do Sistema de Informação de Agravos de Notificação (SINAN), Centro de Controle de Zoonoses de Maceió (CCZ) e Laboratório Central de Saúde Pública de Alagoas (LACEN/AL). Durante o período de estudo, observou-se que o ano de maior incidência de Leishmaniose visceral humana (LVH) foi o de 2011 e o de menor foi no ano de 2013. Já a LVC teve maior incidência em 2007 e menor em 2012. Dos 55 municípios do Estado de Alagoas que apresentaram LVH, São José da Tapera apresentou uma média de casos de 4,4 nos últimos cinco anos classificado como de transmissão intensa; No que diz respeito à leishmaniose visceral canina (LVC), no mesmo período de estudo, foram examinados 45.112 cães no Estado, dos quais 4.466 foram positivos. Resultou assim, em uma taxa de 9,9% de positividade. Nossos dados são importantes porque a infecção canina é um importante fator de risco para a doença humana.

La pérdida de la percepción al riesgo de zoonosis y la figura del perro comunitario / Loss of risk perception to zoonoses and the figure of community dog

Resumen Los perros son causantes de zoonosis y lesiones; en especial, si aceptamos como normales conductas peligrosas; perdiendo la percepción al riesgo a enfermar. El objetivo de este manuscrito fue demostrar, que la presencia de perros vagos y comunitarios, es un peligro para la salud y seguridad pública, a través de información epidemiológica que sustenta la medida de evitar su libre circulación. La agresividad de los perros regula sus relaciones sociales a través de sumisión o dominancia; características de difícil reconocimiento para la población humana. En Chile, 327.150 personas resultaron mordidas por perros, entre los años 2003-2012, con un costo de U$22,7 a U$177,9 millones; además de los U$3,13 millones por intervenir quirúrgicamente 767 sujetos humanos de hidatidosis durante el año 2012. Sin embargo, la zoonosis más temida es la rabia, dado el resultado mortal de ésta. No se debe tolerar la presencia de perros vagos y comunitarios en las calles, medida que debe tener un sustento técnico de salud y seguridad pública; debido a que la población ha perdido la capacidad de percibir el riesgo de contraer enfermedades zoonóticas, de altos costos para el sistema asistencial; sin considerar el desmedro económico al núcleo familiar y el daño psicológico a los afectados.

Dogs may cause zoonoses and damages; especially when we accept as normal some dangerous behavior, losing the perception of disease risk. The purpose of this review was to demonstrate that the presence of stray dogs as well as community dogs, are public health hazard. Through sustainable epidemiological information, we believe this measure could prevent it from spreading. Dog's aggressiveness regulates its social relationships through submission or dominance; those characteristics are difficult to be recognized by human population. In Chile, 327.150 people were bitten by dogs, between years 2003 and 2012 with a cost of USD$22.7 to USD$177,9 million in addition to the USD$3,13 million for the surgical intervention of 767 human subjects of Hydatidosis during 2012. However zoonosis more dangerous is Rabies with death results. As a conclusion we state that the presence of stray and community dogs in the streets cannot be tolerated. This measure must have a sustainable technical of Health and Public safety because population have lost its ability of recognize the risk of contracting zoonotic diseases, with high costs for the health care system, besides the economical decline and psychological damage to the affected individuals.

Human visceral leishmaniasis and relationship with vector and canine control measures / Leishmaniose visceral em humanos e relação com medidas de controle vetorial e canino

ABSTRACT OBJECTIVE Estimate the coverage of control measures of visceral leishmaniasis and relate them with the occurrence of human visceral leishmaniasis in endemic urban area. METHODS Cases of human and canine visceral leishmaniasis were considered as study population and evaluated by a serological survey conducted in Araçatuba, state São Paulo, from 2007 to 2015. The cases of human visceral leishmaniasis were geocoded by the address of the patients and the canine disease by the address of the dogs' owners. The coverage of serological survey, euthanasia, and insecticide spraying was calculated, as well as the canine seroprevalence and the incidence rates of human visceral leishmaniasis. The relationship between human visceral leishmaniasis and control measures was evaluated, as well as the seroprevalence by comparing maps and by linear regression. The relationship between the canine and the human disease was also evaluated by the Ripley's K function. RESULTS The incidence rates of human visceral leishmaniasis showed a period of decline (2007 to 2009) and a period of stability (2010 to 2015), a behavior similar to that of canine seroprevalence. In general, the coverage of control measures was low, and the non-association with the incidence of human visceral leishmaniasis can be a result of the period analyzed and of the small number of analyzed units (sectors of the Superintendence for the Control of Endemic Diseases). The distribution of human cases showed spatial dependence with the distribution of seropositive dogs from 2007 to 2009. CONCLUSIONS This study reaffirmed the relationship between the occurrence of the disease in humans and dogs, it verified a decrease in the rates of visceral leishmaniasis in Araçatuba over time, even at low coverage of control activities. However, further studies are needed to determine if factors beyond monitoring and control measures are involved in the reduction of incidences.

RESUMO OBJETIVO Estimar a cobertura das atividades de controle da leishmaniose visceral e relacioná-las com a ocorrência de leishmaniose visceral em humanos em área urbana endêmica. MÉTODOS Foram considerados como população de estudo os casos de leishmaniose visceral em humanos e em cães avaliados por inquérito sorológico censitário realizado em Araçatuba, SP, de 2007 a 2015. Os casos de leishmaniose visceral em humanos foram geocodificados pelo endereço de residência dos pacientes e, os cães, pelo endereço de residências dos respectivos tutores. Foram calculadas as coberturas do inquérito sorológico, da eutanásia e de borrifação de inseticida, as soroprevalências caninas e as taxas de incidência de leishmaniose visceral em humanos. A relação entre a leishmaniose visceral em humanos e as medidas de controle, bem como a soroprevalência foram avaliadas por comparação de mapas e por meio de regressão linear. A relação entre a doença canina e a humana também foi avaliada por meio da função K de Ripley . RESULTADOS As taxas de incidência de leishmaniose visceral em humanos apresentaram um período de declínio (2007 a 2009) e um período de estabilidade (2010 a 2015), comportamento semelhante ao das soroprevalências caninas. Em geral, a cobertura das medidas de controle foi baixa e a não associação com a incidência de leishmaniose visceral em humanos pode ser consequência do período analisado e do número pequeno de unidades analisadas (setores da Superintendência de Controle de Endemias). A distribuição dos casos humanos apresentou dependência espacial com a distribuição dos cães soropositivos de 2007 a 2009. CONCLUSÕES Este trabalho reafirmou a relação entre a ocorrência da doença no homem e no cão, verificou a diminuição das taxas de leishmaniose visceral em humanos e em cães em Araçatuba ao longo do tempo, mesmo em baixa cobertura das atividades de controle. Entretanto, novos estudos são necessários para averiguar se fatores além das atividades de vigilância e controle estariam envolvidos na diminuição das incidências.

Fatores ambientais associados à ocorrência de leishmaniose visceral canina em uma área de recente introdução da doença no Estado do Rio de Janeiro, Brasil / Factores ambientales asociados a la ocurrencia de leishmaniosis visceral canina en un área de reciente introducción de la enfermedad en el estado de Río de Janeiro, Brasil / Environmental factors associated with canine visceral leishmaniasis in an area with recent introduction of the disease in the State of Rio de Janeiro, Brazil

Resumo: Foi avaliada a associação entre características ambientais obtidas por sensoriamento remoto e a prevalência da leishmaniose visceral canina (LVC) no bairro do Jacaré, área de recente introdução da doença, no Município de Niterói, Estado do Rio de Janeiro, Brasil. Trata-se de um estudo seccional para avaliação da prevalência de LVC, definida por meio da positividade no teste imunocromatográfico rápido em dupla plataforma (dual path platform - DPP), confirmada com o ensaio imunoenzimático (EIE). Foram incluídos 97 cães com prevalência de LVC de 21,6%. Houve maior frequência de LVC em cães com a convivência com outro cão, gambá, mico e ouriço-terrestre, assim como com a história de remoção de outros cães com LVC do domicílio. Na análise multivariada, ajustada por sexo e idade do cão, cães residentes em áreas com maior cobertura de vegetação esparsa apresentaram prevalência da infecção por Leishmania infantum cinco vezes maior do que aqueles que residiam em áreas menos vegetadas (OR = 5,72; IC95%: 1,47-22,20). Por outro lado, áreas mais urbanizadas caracterizadas como comerciais ou residenciais carentes, identificadas pelo sensoriamento remoto como aquelas com alta densidade de estruturas cinza, estiveram associadas à menor ocorrência da LVC (OR = 0,09; IC95%: 0,01-0,92). A maior prevalência de infecção em cães convivendo com outros animais silvestres e em áreas com maior cobertura vegetal, associada com menor prevalência em áreas urbanizadas, indica um padrão rural de transmissão da LVC nessa área.

Resumen: Se evaluó la asociación entre las características ambientales obtenidas por teledetección y la prevalencia de la leishmaniosis visceral canina (LVC) en el barrio de Jacaré, área de reciente introducción de la enfermedad, en el municipio de Niteroi, Estado de Río de Janeiro, Brasil. Se trata de un estudio seccional para la evaluación de la prevalencia de LVC, definida mediante la positividad en el test inmunocromatográfico rápido en una plataforma de doble vía (dual path platform - DPP), confirmada con un ensayo imunoenzimático (EIE). Se incluyeron a 97 perros con una prevalencia de LVC de un 21,6%. Hubo una mayor frecuencia de LVC en perros que conviven con otros perros, zarigüeyas, monos y erizos terrestres, así como con el historial de retirada de otros perros con LVC del domicilio. En el análisis multivariado, ajustado por sexo y edad del perro, los perros residentes en áreas con una mayor cobertura de vegetación muy dispersa presentaron una prevalencia de infección por Leishmania infantum cinco veces mayor que aquellos que residían en áreas con menos vegetación (OR = 5,72; IC95%: 1,47-22,20). Por otro lado, las áreas más urbanizadas, caracterizadas como comerciales o residenciales con pocos recursos, identificadas mediante teledetección como aquellas con una alta densidad de estructuras en gris, estuvieron asociadas a una menor ocurrencia de la LVC (OR = 0,09; IC95%: 0,01-0,92). La mayor prevalencia de infección en perros, conviviendo con otros animales silvestres y en áreas con una mayor superficie vegetal, se asocia con una menor prevalencia en áreas urbanizadas, lo que indica un padrón rural de transmisión de la LVC en ese área.

Abstract: The study assessed the association between environmental characteristics obtained by remote sensing and prevalence of canine visceral leishmaniasis (CVL) in the neighborhood of Jacaré, an area with recent introduction of the disease in the municipality (county) of Niterói, Rio de Janeiro State, Brazil. This was a cross-sectional study to assess CVL prevalence, defined as a positive result in the dual path platform (DPP) rapid immunochromatographic assay, confirmed by immunoenzymatic assay (IEA). The study included 97 dogs, with 21.6% CVL prevalence. CVL prevalence was higher in dogs with contact with another dog, opossum, marmoset, or hedgehog, as well as history of culling of other dogs with CVL from the household. In the multivariate analysis, adjusted for the dog's sex and age, dogs in areas with sparse vegetation showed fivefold higher prevalence of Leishmania infantum infection compared to dogs in areas with less vegetation (OR = 5.72; 95%CI: 1.47-22.20). Meanwhile, less urbanized areas, characterized as commercial or low-income residential areas, identified by remote sensing as those with high density of gray structures, were associated with lower CVL prevalence (OR = 0.09; 95%CI: 0.01-0.92). The higher prevalence of infection in dogs living alongside wild animals and in areas with more vegetation and lower prevalence in more urbanized areas suggest a rural transmission pattern for CVL in this area.

Serological tests fail to discriminate dogs with visceral leishmaniasis that transmit Leishmania infantum to the vector Lutzomyia longipalpis

Abstract INTRODUCTION The control of reservoirs for Leishmania infantum -induced zoonotic visceral leishmaniasis requires the identification of dogs posing a population risk. Here, we assessed the performance of several assays to identify Lutzomyia longipalpis infectious dogs. METHODS We evaluated 99 dogs that were positive for visceral leishmaniasis based on parasite identification. Serological analyses were performed using an enzyme-linked immunosorbent assay, immunofluorescence antibody tests in 1:40 and 1:80 dilutions, rapid dual path platform tests, immunochromatographic assay with a recombinant rK39 antigen, fast agglutination screening tests, and direct agglutination tests. We also performed PCR to analyze peripheral blood and xenodiagnosis. RESULTS Forty-six dogs infected at least one L. longipalpis specimen. Although the serological test sensitivities were above 85% for detecting L. longipalpis infectious dogs, none showed a satisfactory performance, as both specificity (0.06 to 13%) and the area under the receiver operating characteristic curve (45 to 53%) were low. The PCR results were also weak, with a sensitivity of 30%, specificity of 72%, and an area under the receiver operating characteristic curve of 51%. The infected L. longipalpis proportion was higher among asymptomatic dogs than symptomatic dogs. Among the symptomatic dogs, those with ulceration-free skin diseases were more infectious, with an odds ratio of 9.3 (confidence interval of 1.10 - 428.5). The larger the number of insects fed, the greater the detected infectiousness. CONCLUSIONS Our study supports the imperative to develop novel technologies for identifying the infectious dogs that transmit L. infantum for the benefit of public health.

Occurrence of Lutzomyia longipalpis andhuman and canine cases of visceral leishmaniasis and evaluation of their expansion in the Northwest region of the State of São Paulo, Brazil

Abstract: INTRODUCTION This paper aims to describe the dispersion of Lutzomyia longipalpis and the autochthonous occurrence of visceral leishmaniasis (VL) in the Northwest region of the State of São Paulo between 2007 and 2013 and to analyze their expansion. METHODS Information about the vector and associated cases was described using maps. The incidence, mortality, and lethality of human visceral leishmaniasis (HVL) were calculated. In municipalities in which more than one HVL case occurred, incidences were calculated according to census sector, and spatial and spatiotemporal clusters were identified. RESULTS The first case of HVL was reported in the municipality of Jales in 2007. By 2013, the vector and the disease had expanded from west to east, with the vector being detected in 29 municipalities. A total of 11 municipalities had cases of canine visceral leishmaniasis (CVL), and six had cases of HVL. Vector expansion occurred by vicinity with previously infested municipalities, and the expansion of VL was related to the major highways and the capital municipalities of the micro-regions in the study area. The highest incidence of HVL occurred in children between 0-4 years old, and the highest mortality and lethality occurred among persons aged 60 and older. The occurrence of HLV was more intense in the peripheral areas of municipalities with the disease. CONCLUSIONS The findings of this study may be useful for improving VL surveillance and control activities by slowing VL expansion and/or mitigating VL effects when they occur.

Assessment of experimental infection for dogs using Gallus gallus chorioallantoic membranes inoculated with Neospora caninum / Avaliação da infecção experimental de cães com membranas córion-alantóides de Gallus gallus inoculadas com Neospora caninum

The aim of this study was to evaluate parasitism kinetics and tissue lesions in the first week of infection by Neospora caninum in dogs fed Gallus gallus chorioallantoic membranes (CMs) previously infected in ovo. Five two-month-old pups were used. Each dog was given five CMs that were previously infected with N. caninum via the oral route. Four animals were euthanized in the first week of infection. All four dogs had their stools examined one week prior to and up to the day they were euthanized. The stools of the uneuthanized dog were collected for 30 days. After euthanasia, organ sections were utilized for histopathology, immunohistochemistry, indirect immunofluorescent tissue reactions, PCR and real-time PCR to detect parasites. Necropsy revealed that the small and large intestines, spleen, and lungs were affected. No oocysts or N. caninum DNA were identified in the stool samples. Real-time PCR was the most sensitive technique used to detect the protozoa in tissues, which were identified in 41% of the analyzed samples. Our results indicate that an experimental model using previously infected CMs appears to be a useful model for the study of the host-parasite relationship during the infection's acute phase.

O objetivo deste estudo foi avaliar a cinética de parasitismo e lesões teciduais, na primeira semana de infecção por Neospora caninum, em cães alimentados com membranas corioalantóicas (MCs) de Gallus gallus, previamente infectadas in ovo. Foram utilizados cinco filhotes de dois meses de idade. Cada cão recebeu cinco MCs previamente infectadas com N. caninum, por via oral. Quatro animais foram eutanasiados na primeira semana de infecção. Todos os quatro cães tiveram suas fezes examinadas uma semana antes e até o dia em que foram eutanasiados. O cão que não foi eutanasiado teve suas fezes colhidas durante 30 dias. Depois da eutanasia fragmentos de órgãos foram processados para histopatologia, imuno-histoquímica, reação de imunofluorescência indireta em tecidos, PCR e PCR em tempo real para detecção do parasito. A necropsia revelou que os intestinos delgado e grosso, baço e pulmões foram os órgãos afectados. Oocistos de N. caninum não foram identificados nas amostras de fezes. A PCR em tempo real foi a técnica mais sensível para detectar o protozoário nos tecidos, sendo identificados em 41% das amostras analisadas. Os nossos resultados indicam que o modelo experimental utilizando MCs evidenciou ser um bom modelo para estudar a relação parasito-hospedeiro durante a fase aguda da infecção.

Canine heartworm disease in Porto Velho: first record, distribution map and occurrence of positive mosquitoes / Dirofilariose canina em Porto Velho: primeiro registro, mapa de distribuicao e ocorrencia de mosquitos positivos

The aim of this study was to make the first report on canine heartworm disease in the state of Rondônia and confirm its transmission in this state. Blood samples were randomly collected from 727 dogs in the city of Porto Velho. The samples were analyzed to search for microfilariae and circulating antigens, using three different techniques: optical microscopy on thick blood smears stained with Giemsa; immunochromatography; and PCR. Mosquitoes were collected inside and outside the homes of all the cases of positive dogs and were tested using PCR to search for DNA of Dirofilaria immitis. Ninety-three blood samples out of 727 (12.8%) were positive according to the immunoassay technique and none according to the thick smear method. Among the 93 positive dogs, 89 (95.7%) were born in Porto Velho. No difference in the frequency of infection was observed between dogs raised indoors and in the yard. PCR on the mosquitoes resulted in only one positive pool. This result shows that the transmission of canine heartworm disease is occurring in the city of Porto Velho and that there is moderate prevalence among the dogs. The techniques of immunochromatography and PCR were more effective for detecting canine heartworm than thick blood smears. The confirmation of canine heartworm disease transmission in Porto Velho places this disease in the ranking for differential diagnosis of pulmonary nodules in humans in Rondônia.

O objetivo deste estudo foi de registrar pela primeira vez a dirofilariose canina no estado de Rondônia e confirmar sua transmissão neste estado. Amostras de sangue de 727 cães foram colhidas aleatoriamente na cidade de Porto Velho. As amostras foram analisadas em busca de microfilárias e antígenos circulantes usando três técnicas diferentes: microscopia ótica de gota espessa corada com Giemsa e imunocromatografia de fluxo lateral e PCR. Mosquitos foram colhidos no domicilio e peridomicílio de todos os casos de cães positivos, estes mosquitos foram testados pela PCR na detecção de DNA de Dirofilaria immitis. Noventa e três das 727 amostras de sangue foram positivas na técnica de imunoensaio (12,8%). Nenhuma amostra foi positiva na gota espessa. Entre os 93 cães positivos, 89 (95,7%) foram nascidos em Porto Velho. Nenhuma diferença na frequencia de infecção foi observada entre cães criados dentro da casa ou no quintal. O PCR dos mosquitos resultou em apenas um pool positivo. Este resultado mostra que a transmissão de dirofilariose canina está ocorrendo na cidade de Porto Velho e a frequência que ocorre nos cães é considerada moderada. As técnicas de imunocromatografia e PCR são mais eficazes na detecção de dirofilariose comparadas a gota espessa. A confirmação de transmissão de dirofilariose canina em Porto Velho, coloca esta doença no ranking de diagnóstico diferencial de nódulos pulmonares em seres humanos em Rondônia.

Leishmania infection and host-blood feeding preferences of phlebotomine sandflies and canine leishmaniasis in an endemic European area, the Algarve Region in Portugal

The Algarve Region (AR) in southern Portugal, which is an international tourist destination, has been considered an endemic region of zoonotic leishmaniasis caused by Leishmania infantum since the 1980s. In the present study, phlebotomine and canine surveys were conducted to identify sandfly blood meal sources and to update the occurrence of Leishmania infection in vectors and dogs. Four sandfly species were captured: Phlebotomus perniciosus, Phlebotomus ariasi, Phlebotomus sergenti and Sergentomyia minuta. In one P. perniciosus female, L. infantum DNA was detected. Blood meal tests showed that this species had no host preferences and was an opportunistic feeder. An overall canine leishmaniasis (CanL) seroprevalence of 16.06% was found; the seroprevalence was 3.88% in dogs housed in kennels and 40.63% in dogs that attended veterinary clinics. The simultaneous occurrence of dogs and P. perniciosus infected with L. infantum in the AR indicates that the region continues to be an endemic area for CanL. Our results reinforce the need for the systematic spatial distribution of phlebotomine populations and their Leishmania infection rates and the need to simultaneously perform pathogen monitoring in both invertebrate and vertebrate hosts to investigate the transmission, distribution and spreading of Leishmania infection.

Leishmaniasis visceral en la Argentina: Notificación y situación vectorial (2006-2012) / Visceral leishmaniasis in Argentina: Cases notification and distribution of vectors (2006-2012)

La leishmaniasis visceral (LV) en la Argentina se notifica obligatoriamente a través del Sistema Nacional de Vigilancia de la Salud (SNVS). El análisis de la notificación muestra un total de 103 casos distribuidos en cuatro provincias, siendo Misiones la de mayor casuística con un total de 80 casos en 15 localidades, desde mayo 2006 hasta julio de 2012. Los niños de 0 a 15 años de edad son el grupo con mayor incidencia. En el año 2004 fue registrado por primera vez el riesgo de LV urbana endémica en la provincia de Formosa tras el hallazgo del vector Lutzomyia longipalpis. En el año 2006 la provincia de Misiones registra casos de LV humana, LV canina, y la provincia de Corrientes en el verano 2008-2009. En Santiago del Estero los casos de LV humana y LV canina en el año 2008 estuvieron asociados posiblemente a vectores secundarios. En el año 2010 Lu. longipalpis fue hallado en Chajarí, Entre Ríos, mostrando su dispersión hacia el sur. Por ello, para conocer la distribución del riesgo, entre enero y abril del 2011 se realizó búsqueda sistemática del vector de LV en 13 localidades de las provincias de Entre Ríos, Santa Fe y Santiago del Estero, totalizando 275 trampas/noche. Se comprobó la colonización de Lu. longipalpis, en la localidad de Chajarí. En Santiago del Estero no se encontró Lu. longipalpis por lo que Migonemyia migonei sigue incriminada como vector putativo de LV en esta región.

Visceral leishmaniasis (VL) is a notifiable disease in Argentina. Every new case diagnosed must be reported to the National Health Surveillance System. In the period 2006-2012, a total of 103 cases have been notified in four provinces: Entre Rios, Santa Fe, Misiones and Santiago del Estero. Misiones has the largest amount of cases: a total of 80, detected in 15 towns from May 2006 to July 2012. The highest incidence is observed in children (less than 15 years of age). In 2004 the vector Lutzomyia longipalpis was detected for the first time in Formosa Province. Cases of human and canine VL, as well as the presence of the canine vector were reported in 2006 in the province of Misiones, and in 2008-2009 in the province of Corrientes. In Santiago del Estero cases of human and canine VL (2008) were possibly associated with secondary vectors. In 2010 Lu longipalpis was found in Entre Rios, showing its dispersal to the south. Between January and April 2011 a systematic search of the VL vector was carried out in 13 towns, in Entre Ríos, Santa Fe and Santiago del Estero Provinces (275 traps/night). The colonization of the vector Lu longipalpis was proved in Chajarí town (Entre Rios), but not in Santiago del Estero Province, where Migonemyia migonei appears as a putative LV vector.

Biotic factors and occurrence of Lutzomyia longipalpis in endemic area of visceral leishmaniasis, Mato Grosso do Sul, Brazil

The relationships between environmental exposure to risk agents and health conditions have been studied with the aid of remote sensing imagery, a tool particularly useful in the study of vegetation cover. This study aims to evaluate the influence of environmental variables on the spatial distribution of the abundance of Lutzomyia longipalpis and the reported canine and human visceral leishmaniasis (VL) cases at an urban area of Campo Grande, state of Mato Grosso do Sul. The sandfly captures were performed in 13 residences that were selected by raffle considering four residences or collection station for buffer. These buffers were generated from the central house with about 50, 100 and 200 m from it in an endemic area of VL. The abundance of sandflies and human and canine cases were georreferenced using the GIS software PCI Geomatica. The normalized difference vegetation index (NDVI) and percentage of land covered by vegetation were the environmental variables extracted from a remote sensing IKONOS-2 image. The average NDVI was considered as the complexity of habitat and the standard deviation as the heterogeneity of habitat. One thousand three hundred sixty-seven specimens were collected during the catch. We found a significant positive linear correlation between the abundance of sandflies and the percentage of vegetation cover and average NDVI. However, there was no significant association between habitat heterogeneity and the abundance of these flies.

The distribution pattern of Lutzomyia longipalpis (Diptera: Psychodidae) in the peridomiciles of a sector with canine and human visceral leishmaniasis transmission in the municipality of Dracena, São Paulo, Brazil

The specimen distribution pattern of a species can be used to characterise a population of interest and also provides area-specific guidance for pest management and control. In the municipality of Dracena, in the state of São Paulo, we analysed 5,889 Lutzomyia longipalpis specimens collected from the peridomiciles of 14 houses in a sector where American visceral leishmaniasis (AVL) is transmitted to humans and dogs. The goal was to analyse the dispersion and a theoretical fitting of the species occurrence probability. From January-December 2005, samples were collected once per week using CDC light traps that operated for 12-h periods. Each collection was considered a sub-sample and was evaluated monthly. The standardised Morisita index was used as a measure of dispersion. Adherence tests were performed for the log-series distribution. The number of traps was used to adjust the octave plots. The quantity of Lu. longipalpis in the sector was highly aggregated for each month of the year, adhering to a log-series distribution for 11 of the 12 months analysed. A sex-stratified analysis demonstrated a pattern of aggregated dispersion adjusted for each month of the year. The classes and frequencies of the traps in octaves can be employed as indicators for entomological surveillance and AVL control.

Giardiasis as zoonosis: between proof of principle and paradigm in the Northwestern region of São Paulo State, Brazil

OBJECTIVE: In order to evaluate the potential zoonotic transmission of Giardia duodenalis, isolates from humans and dogs in the Northwestern region of the São Paulo State, Brazil were characterized based on the β-giardin gene. METHODS: The samples were analyzed by sequencing of the Nested-PCR products. RESULTS: The A1 and A2 subgenotypes were detected in human and dogs. Cysts of assemblage B, C and D have not been found in any isolates studied. CONCLUSIONS: These results are consistent with the view that giardiasis in the largest endemic region of the Brazil should not be seen as a single entity.

Diagnóstico de Dirofilaria immitis en el municipio Sucre, estado Sucre, Venezuela / Diagnosis of Dirofilaria immitis in the municipality of Sucre, Sucre state, Venezuela

La dirofilariasis es una patología parasitaria, causada por los nematodos Dirofilaria immitis y D. repens, principalmente. Mientras que D. immitis presenta distribución mundial, D. repens se extiende por el viejo mundo. Ambas especies afectan a caninos y felinos domésticos y salvajes, así como a humanos, por lo que es un problema de salud pública mundial. En este estudio se determinó la prevalencia de D. immitis, en caninos del municipio Sucre, estado Sucre, Venezuela. Se tomaron muestras sanguíneas a 138 caninos domésticos, mayores de seis meses, sin distinción de raza ni sexo, previo consentimiento informado por sus propietarios. El diagnóstico parasitológico de las microfilarias circulantes se realizó por examen directo y método de concentración Knott modificado; las características morfológicas fueron observadas en extendidos teñidos con Giemsa. Se utilizó un estuche comercial, para la captura de antígenos de excreción-secreción de D. immitis. De los 138 caninos estudiados, 12 casos positivos (8,7%) fueron detectados parasitológicamente, mientras que por el estuche comercial se detectaron antígenos en 20 caninos (14,5%), lo que demuestra que existe una dirofilariasis oculta en el 5,8% de los casos evaluados. Uno de los perros microfilarémicos, no presentó antígenos circulantes. La prevalencia de dirofilariasis en el municipio Sucre, estado Sucre fue de 15,2%. Este reporte de dirofilariasis canina en el municipio Sucre, representa un importante aporte en el levantamiento epidemiológico de la enfermedad. Futuros estudios permitirán evaluar el riesgo de infección de las personas que conviven con los perros con dirofilariasis.

Heartworm is a parasitic disease, caused by nematodes Dirofilaria immitis and D. repens, mainly. While D. immitis presents worldwide distribution, D. repens extends the old world. Both species affect canines and felines domestic and wild, as well as human, so it is a global public health problem. In this study we determined the prevalence of D. immitis in dogs in the municipality of Sucre, Sucre state, Venezuela. Blood samples were taken for 138 domestic dogs, over six months, regardless of race or sex, prior informed consent by their owners. The parasitological diagnosis of circulating microfilariae was done by direct examination and concentration method modified Knott, the morphological characteristics were observed in Giemsa-stained smears. We used a commercial kit, for the capture of excretory-secretory antigens of D. immitis. Of the 138 dogs studied, 12 cases (8.7%) were detected parasitologically, whereas the commercial kit antigens were detected in 20 dogs (14.5%), demonstrating that there is an occult infections in 5.8% of the cases. One of the dogs with microfilariae in blood, did not submit antigens circulating. The prevalence of heartworm in the municipality of Sucre, Sucre state was 15.2%. This report of canine heartworm in the municipality of Sucre, represents an important contribution in an epidemiologic survey of the disease. Future studies will assess the risk of infection for people living with dogs with heartworm disease.

How effective is dog culling in controlling zoonotic visceral leishmaniasis? a critical evaluation of the science, politics and ethics behind this public health policy / Quanto é efetivo o abate de cães para o controle do calazar zoonótico? uma avaliação crítica da ciência, política e ética por trás desta política de saúde pública

INTRODUCTION: Zoonotic kala-azar, a lethal disease caused by protozoa of the genus Leishmania is considered out of control in parts of the world, particularly in Brazil, where transmission has spread to cities throughout most of the territory and mortality presents an increasing trend. Although a highly debatable measure, the Brazilian government regularly culls seropositive dogs to control the disease. Since control is failing, critical analysis concerning the actions focused on the canine reservoir was conducted. METHODS: In a review of the literature, a historical perspective focusing mainly on comparisons between the successful Chinese and Soviet strategies and the Brazilian approach is presented. In addition, analyses of the principal studies regarding the role of dogs as risk factors to humans and of the main intervention studies regarding the efficacy of the dog killing strategy were undertaken. Brazilian political reaction to a recently published systematic review that concluded that the dog culling program lacked efficiency and its effect on public policy were also reviewed. RESULTS: No firm evidence of the risk conferred by the presence of dogs to humans was verified; on the contrary, a lack of scientific support for the policy of killing dogs was confirmed. A bias for distorting scientific data towards maintaining the policy of culling animals was observed. CONCLUSIONS: Since there is no evidence that dog culling diminishes visceral leishmaniasis transmission, it should be abandoned as a control measure. Ethical considerations have been raised regarding distorting scientific results and the killing of animals despite minimal or absent scientific evidence.

INTRODUÇÃO: O calazar zoonótico, uma doença fatal causada por protozoários do gênero Leishmania, é considerada fora de controle, particularmente no Brasil, onde se urbaniza e a letalidade aumenta. Apesar de ser uma medida muito controversa, o governo brasileiro abate cães soropositivos regularmente para controlar a doença. Assim, diante da falha do controle, foi efetuada uma análise crítica das ações para o controle do reservatório canino. MÉTODOS: Em uma revisão da literatura, foi feita uma abordagem histórica focalizada principalmente na comparação das bem sucedidas tentativas chinesas e soviéticas de controlar a doença. Também foi efetuada uma análise dos principais estudos acerca do papel de cães como fatores de risco para humanos e dos principais ensaios de intervenção acerca da eliminação destes animais. A reação política do Brasil a uma revisão sistemática recentemente publicada que concluiu pela ineficácia do programa de eliminação de cães e os seus efeitos nas políticas públicas são revisadas. RESULTADOS: Não foram encontradas evidências firmes do risco conferido por cães para os seres humanos. Além disto, foi confirmada a falta de apoio científico à política de eliminação de cães. Foi notada uma tendência para distorção dos dados científicos para o suporte da política de eliminação dos animais. CONCLUSÕES: Uma vez que não existem evidências de que o abate de cães diminui a transmissão de leishmaniose visceral, este programa deve ser abandonado como estratégia de controle. São levantadas as implicações éticas acerca da distorção da ciência e sobre a eliminação de animais na ausência de mínima ou nenhuma evidência científica.

Distribución de Lutzomyia longipalpis en la Mesopotamia Argentina, 2010 / Distribution of Lutzomyia longipalpis in the Argentine Mesopotamia, 2010

El primer caso autóctono de leishmaniasis visceral (LV) en la Argentina se notificó en el año 2006 en Posadas, Misiones, y en el verano 2008-2009 se comprobó la dispersión del vector de LV, Lutzomyia longipalpis y casos de LV canina en la provincia de Corrientes. Para conocer la distribución del riesgo, entre febrero y marzo del 2010 se realizó la búsqueda sistemática del vector de LV en 18 localidades de las provincias de Entre Ríos, Corrientes y en la ciudad de Puerto Iguazú, Misiones, totalizando 313 trampas/noche. Se comprobó la presencia de Lu. longipalpis, por primera vez, en las localidades de Chajarí (Entre Ríos), Alvear, La Cruz, Curuzú Cuatiá y Bella Vista (Corrientes), y en Puerto Iguazú (Misiones). En Santo Tomé y Monte Caseros (Corrientes) se volvió a registrar la presencia del vector, y se obtuvieron las trampas con más ejemplares, 830 y 126 Lu. longipalpis trampa/sitio/noche respectivamente. Los resultados muestran que el vector de la LV urbana, continúa dispersándose en el territorio argentino. Simultáneamente, la propagación del parásito, y los consecuentes casos de LV humana se asocian al aumento de reservorios, perros infectados con o sin clínica, debidos al tránsito humano.

The first case of visceral leishmaniasis (VL) in Argentina was reported in 2006 in Posadas, Misiones. During the summer 2008-2009 Lutzomyia longipalpis, the VL vector, and canine VL cases were already spread along the province of Corrientes. In order to know the distribution of VL risk, systematic captures of the vector were performed between February and March 2010, in 18 areas of the provinces of Entre Ríos and Corrientes, and the city of Puerto Iguazú, Misiones, with a total of 313 traps/night. We confirmed the presence of Lu. longipalpis, for the first time in Chajarí (Entre Ríos), Alvear, La Cruz, Curuzú Cuatiá and Bella Vista (Corrientes), and Puerto Iguazú (Misiones). In Santo Tome and Monte Caseros (Corrientes), where the vector had been previously reported, traps with more samples were obtained with 830 and 126 Lu. Longipalpis trap/site/night respectively. These results show that the vector of urban VL continues spreading in the Argentine territory. Simultaneously, the spread of the parasite and the resulting human VL cases are associated with the dispersion of reservoirs, infected dogs, with or without clinical symptoms or signs, due to human transit.

Evidência de transmissão de leishmaniose visceral por Lutzomyia cruzi no município de Jaciara, Estado de Mato Grosso, Brasil / Evidence of transmission of visceral leishmaniasis by Lutzomyia cruzi in the municipality of Jaciara, State of Mato Grosso, Brazil

INTRODUÇÃO: O município de Jaciara foi classificado em 2003, como área de transmissão de leishmaniose visceral em situação de surto. O trabalho objetivou determinar evidência de transmissão de Leishmania (Leishmania) infantum chagasi por Lutzomyia cruzi no município de Jaciara, Estado de Mato Grosso, Brasil. MÉTODOS: O município situa-se a 127km da capital Cuiabá e é um importante ponto de atração para os praticantes de eco-turismo. Fêmeas de Lutzomyia cruzi, capturadas com armadilha de CDC, foram dissecadas para confirmação da espécie e armazenadas a -20ºC em pools de 10 indivíduos para extração de DNA, PCR genérico, RFLP específico e eletroforese em gel de poliacrilamida. RESULTADOS: O levantamento entomológico demonstrou a ocorrência abundante de Lutzomyia cruzi e ausência de Lutzomyia longipalpis, principal vetora da Leishmania (Leishmania) infantum chagasi. Uma das três amostras analisadas apresentou banda característica de DNA de Leishmania (120pb) em PCR genérico. Para confirmação da espécie de Leishmania, na RFLP utilizaram-se controles positivos de Leishmania (Leishmania) amazonensis, Leishmania (Viannia) braziliensis e Leishmania (Leishmania) infantum chagasi digeridas com enzima de restrição HaeIII. Constatou-se um padrão de bandas semelhante à Leishmania (Leishmania) infantum chagasi em uma amostra, confirmando a detecção de infecção natural de Leishmania (Leishmania) infantum chagasi em Lutzomyia cruzi. CONCLUSÕES: A ocorrência de casos humanos e cães positivos, a presença da Lutzomyia cruzi e a ausência de Lutzomyia longipalpis, bem como a detecção de infecção natural por Leishmania (Leishmania) infantum chagasi, evidenciam a participação de Lutzomyia cruzi na transmissão da leishmaniose visceral em Jaciara, Estado de Mato Grosso, Brasil.

INTRODUCTION: The municipality of Jaciara was classified in 2003 as a transmission area for visceral leishmaniasis in outbreak situations. This study aimed to establish evidence of transmission of Leishmania (Leishmania) infantum chagasi by Lutzomyia cruzi in the municipality of Jaciara, State of Mato Grosso, Brazil. METHODS: This municipality is located 127 km from the state capital (Cuiabá) and is an important center for ecotourism practitioners. Females of Lutzomyia cruzi, captured using CDC traps, were dissected to confirm the species and stored at -20ºC in pools of 10 individuals for DNA extraction, generic PCR, specific RFLP and electrophoresis on polyacrylamide gel. RESULTS: The entomological survey showed abundant occurrence of Lutzomyia cruzi and absence of Lutzomyia longipalpis (the main vector for Leishmania (Leishmania) infantum chagasi). One of the three samples showed a characteristic DNA band from Leishmania (120 bp) in generic PCR. To confirm the Leishmania species via RFLP, positive controls for Leishmania (Leishmania) amazonensis, Leishmania (Viannia) braziliensis and Leishmania (Leishmania) infantum chagasi digested with restriction enzyme HaeIII were used. A pattern of bands similar to Leishmania (Leishmania) infantum chagasi was found in one sample, thus confirming the detection of natural infection with Leishmania (Leishmania) infantum chagasi in Lutzomyia cruzi. CONCLUSIONS: The occurrences of positive cases in humans and dogs, the presence of Lutzomyia cruzi and the absence of Lutzomyia longipalpis together with the detection of natural infection with Leishmania (Leishmania) infantum chagasi, indicate that Lutzomyia cruzi participates in visceral leishmaniasis transmission in Jaciara, State of Mato Grosso, Brazil.

Canine visceral leishmaniasis due to Leishmania (L.) infantum chagasi in Amazonian Brazil: comparison of the parasite density from the skin, lymph node and visceral tissues between symptomatic and asymptomatic, seropositive dogs / Leishmaniose visceral canina causada por Leishmania (L.) infantum chagasi na Amazônia brasileira: comparação da densidade parasitária da pele, linfonodo e vísceras entre cães soropositivos, sintomáticos e assintomáticos

Canine visceral leishmaniasis (CVL) is recognizable by characteristic signs of disease and is highly lethal. The infection, however, may be quite inapparent in some seropositive dogs, and this has raised the polemic question as to whether or not such animals can be a source of infection for Lutzomyia longipalpis, the vector of American visceral leishmaniasis (AVL). In this study we have examined 51 dogs with acute CVL from an AVL area in Pará State, northern Brazil, and compared the parasite density, amastigotes of Leishmania (L.) infantum chagasi, in the skin, lymph node and viscera of symptomatic with that of nine asymptomatic but seropositive dogs (IFAT-IgG). Post-mortem biopsy fragments of these tissues were processed by immunohistochemistry, using a polyclonal antibody against Leishmania sp. The X² and Mann Whitney tests were used to evaluate the means of infected macrophage density (p < 0.05). There was no difference (p > 0.05) in the skin (10.7/mm² x 15.5/mm²) and lymph node (6.3/mm² x 8.3/mm²), between asymptomatic and symptomatic dogs, respectively. It was higher (p < 0.05), however, in the viscera of symptomatic (5.3/mm²) than it was in asymptomatic (1.4/mm²) dogs. These results strongly suggest that asymptomatic or symptomatic L. (L.) i. chagasi-infected dogs can serve as a source of infection, principally considering the highest (p < 0.05) parasite density from skin (10.7/mm² x 15.5/mm²), the place where the vetor L. longipalpis takes its blood meal, compared with those from lymph node (6.3/mm² x 8.3/mm²) and viscera (1.4/mm²x 5.3/mm²).

A leishmaniose visceral canina (LVC) é reconhecida pelas características clínicas da doença e é altamente letal. A infecção, entretanto, pode ser totalmente assintomática em alguns cães soropositivos, o que tem levantado questão polêmica sobre a possibilidade desses animais, serem ou não uma fonte importante da infecção para o flebotomíneo, Lutzomyia longipalpis, o principal vetor da leishmaniose visceral americana (LVA). Neste estudo foram examinados 51 cães com LVC aguda, provenientes de área endêmica de LVA no Estado do Pará, Brasil, e a carga parasitária, formas amastigotas de Leishmania (L.) infantum chagasi, na pele, linfonodo poplíteo e vísceras (fígado e baço) foi comparada com a de nove cães assintomáticos soropositivos (IFAT-IgG). Fragmentos de biópsia desses tecidos obtidos post-mortem foram processados para análise através de imunohistoquímica, usando um anticorpo policlonal contra Leishmania sp. Os testes do Qui-quadrado (X²) e Mann Whitney foram usados para avaliar as médias da densidade de macrófagos infectados (p < 0,05). Os resultados mostraram que não houve diferença (p > 0,05) na densidade de macrófagos infectados da pele (10,7/mm² x 15,5/mm²) e do linfonodo (6,3/mm² x 8,3/mm²) entre cães assintomáticos e sintomáticos. Entretanto, a densidade de macrófagos infectados da víscera de cães sintomáticos (5,3/mm²) foi maior (p < 0,05) que a de cães assintomáticos (1,4/mm²). Estes resultados sugerem, fortemente, que cães naturalmente infectados por L. (L.) i. chagasi, assintomáticos ou sintomáticos, podem servir como fonte de infecção, principalmente, considerando-se que a densidade de macrófagos infectados da pele (10,7/mm² x 15,5/mm²), local onde o flebotomíneo vetor Lu. longipalpis realiza a hematofagia, foi maior (p < 0,05) que as do linfonodo (6,3/mm² x 8.3/mm²) e vísceras (1,4/mm²x 5,3/mm²).