Molecular Detection of Tick-Borne Pathogens in Dogs from Indigenous Communities, Amazon, Brazil.

Background: There are few reports of tick-borne pathogens infecting dogs living in indigenous communities of Brazil. Herein, we aimed to molecularly detect vector-borne pathogens in dogs from two indigenous communities in the Brazilian Amazon. Materials and Methods: We surveyed 327 dogs raised in Amazon region at 2 distinct indigenous ethnicities for the molecular detection of tick-borne pathogens (114 from Tapirapé and 213 from Karajá indigenous ethnicity). Whole blood samples were subjected to PCR and sequencing for Ehrlichia, Babesia, and Hepatozoon. Univariate and multivariate analyses were used to investigate the factors affecting the pathogen infection patterns in dogs. Results: Among the 327 blood samples, 40 were positive for Ehrlichia canis (12.2%), 2 for Anaplasma platys (0.61%), and 204 were positive for Hepatozoon canis (66.5%). Binary Logistic Regression showed association between E. canis infection and ethnicity (p = 0.010) and tick attachment (p = 0.041). Karajá dogs were 3.4 times (95% CI 1.3-8.5) more likely to be positive for E. canis than Tapirapé dogs. Dogs with ticks were 2.5 times more likely (95% CI 1.0-7.6) to be positive for E. canis than dogs without ticks. Conclusions: Our survey expands the knowledge regarding the presence of vector-borne pathogens in dogs from indigenous communities in the Amazon region.

Dogs naturally infected by Rangelia vitalii, Babesia canis vogeli, and Ehrlichia canis in São Paulo, Brazil / Cães naturalmente infectados por Rangelia vitalii, Babesia canis vogeli, e Ehrlichia canis em São Paulo, Brasil

Several agents can cause hemoparasitic diseases in dogs, and blood-sucking arthropods transmit these diseases. These agents can cause several clinical manifestations and, in some cases, can kill the host. Because these agents are essential in animal health, this study aims to detect the frequency of Ehrlichia canis, Rickettsia rickettsii, Anaplasma platys, and Rangelia vitalii by real-time PCR and Babesia vogeli in dogs in the southern region of the city of São Paulo, São Paulo. Of the 98 dog samples, 18 (18.4%) tested positive with real-time polymerase chain reaction for at least one studied agent. Of these 18 samples, 17 tested positive for a single agent (11.2% for B. canis vogeli, 1.02% for R. vitalii, and 5.1% for E. canis), and one showed co-infection with B. canis vogeli and R. vitalii. The results demonstrate the presence of hemoparasites in the studied animals, which can influence the quality and life expectancy of these animals. The Rangeliadetection warns small animal clinicians to include it as a differential diagnosis for hemoparasitosis.(AU)

As hemoparasitoses em cães podem ser causadas por diversos agentes, sendo essas doenças transmitidas por artrópodes hematófagos. Esses agentes podem causar diversas manifestações clínicas e, em alguns casos, podem matar o hospedeiro. Este estudo teve como objetivo detectar por PCR em tempo real a frequência de Ehrlichia canis, Rickettsia rickettsii, Anaplasma platys, Rangelia vitalii e Babesia canis vogeli em amostras de cães da zona sul da cidade de São Paulo, Brasil. Das 98 amostras de cães, 18 (18,4%) testaram positivo com reação em cadeia da polimerase em tempo real para pelo menos um agente estudado. Destas 18 amostras, 17 testaram positivo para um único agente (11,2% para B. canis vogeli, 1,02% para R. vitalii e 5,1% para E. canis), e uma apresentou coinfecção com B. canis vogeli e R. vitalii. Os resultados demonstram a presença de hemoparasitas nos animais estudados, o que pode influenciar a qualidade e a expectativa de vida desses animais. Além disso, é o primeiro relato da detecção de R. vitalli na zona sul de São Paulo e serve de alerta para os clínicos de pequenos animais incluírem esse agente como diagnóstico diferencial para as hemoparasitoses.(AU)

Toxoplasma gondii and Neospora caninum antibody seroprevalence and risk factors among dogs treated at Public Veterinary Hospitals in São Paulo, Brazil / Soroprevalência de anticorpos anti-Toxoplasma gondii e Neospora caninum em cães atendidos em Hospitais Públicos Veterinários em São Paulo, Brasil

Abstract Dogs can be infected by Toxoplasma gondii and Neospora caninum, for which they function, respectively, as intermediate, and definitive hosts. In the present study seroprevalence against T. gondii and N. caninum antibodies, were determined by indirect fluorescent antibody test (cut off of 16 and 50, respectively), in dogs that were treated at public veterinary hospitals in the metropolitan region of São Paulo and risk factors were identified. Out of the 1,194 samples 125 (10.5%; 95% CI: 8.8-12.3%) were positive for T. gondii and 9 (0.75%, 95% CI: 0.34-1.4%) for N. caninum. For T. gondii, statistical differences were observed between the proportions of positive dogs and different zones of the municipality (p = 0.025), and age (p = 0.02), higher among older dogs. The keepers were invited to answer an epidemiological questionnaire to analyze risk factors, and 471 (39.4%) agreed to be interviewed, and among their dogs 65 (13.8%) were T. gondii seropositive. Age group above 8 years (OR = 3.63; 95% CI: 1.08-12.23) was a risk factor and having a defined breed (OR = 0.49; 95% CI: 0.25-0.96) was a protective factor for T. gondii infection. Because of the low number of dogs positive for N. caninum, risk factors for this coccidium were not determined.

Resumo Cães podem ser infectados por Toxoplasma gondii e Neospora caninum, os quais funcionam, respectivamente, como hospedeiros intermediários e definitivos. Neste estudo, a soroprevalência contra anticorpos anti-T. gondii e N. caninum foi determinada pelo teste de imunofluorescência indireta (ponto de corte de 16 e 50, respectivamente), em cães atendidos em hospitais públicos veterinários da região metropolitana de São Paulo e fatores de risco foram identificados. Das 1.194 amostras, 125 (10,5%; IC 95%: 8,8-12,3%) foram positivas para T. gondii e 9 (0,75%, IC 95%: 0,34-1,4%) para N. caninum. Para T. gondii foram observadas diferenças entre as proporções de cães positivos e diferentes zonas do município (p = 0,025) e idade (p = 0,02), prevalência maior entre os mais velhos. Os tutores foram convidados a responder um questionário epidemiológico para análise de fatores de risco, e 471 (39,4%) concordaram em ser entrevistados, destes 65 cães (13,8%) eram soropositivos para T. gondii. Faixa etária acima de 8 anos (OR = 3,63; IC 95%: 1,08-12,23) foi fator de risco e raça definida (OR = 0,49; IC 95%: 0,25-0,96) foi fator de proteção para a infecção por T. gondii. Devido ao baixo número de positivos para anticorpos anti-N. caninum, fatores de risco para este coccídio não foram determinados.

Serosurvey of spotted fever group Rickettsia in equids from western Pará, Amazon, Brazil.

From a previous large epidemiological survey, we randomly selected 474 serum samples (463 horses and 11 mules) distributed among four municipalities of Pará state, Amazon region, Brazil, and from three types: farm animal, urban carthorse, and sport horse. Samples were tested by indirect fluorescence antibody test (IFAT ≥ 64) for antibodies reactive to spotted fever group (SFG) rickettsiae using Rickettsia rickettsii as crude antigens. From the 474 equids tested, 149 (31.4%) had ticks attached during sampling, belonging mostly to the species Dermacentor nitens. The overall seroprevalence for SFG rickettsiae was 31.4% (95% confidence interval: 27.3-35.9%) with 149 seropositive animals out of 474 screened. Notably, 77 equids (16.2%) had high endpoint titers ranging from 512 to 16,384, indicating that they had been exposed to SFG rickettsiae not long before sampling. Animal type affected rickettsial seroprevalence, with significantly higher values among farm horses when compared with urban and sport animals. Presence of dogs and tick infestation were negatively associated with equid seropositivity to R. rickettsii. This is the first report of SFG rickettsiae-reactive antibodies in equids from Pará state, Brazilian Amazon.

Ornithodoros cf. mimon infected with a spotted fever group Rickettsia in Brazil.

Ornithodoros mimon is an argasid tick primarily associated with bats that also infest other animals including birds, opossums and humans. In this paper, we report the finding of an argasid species resembling O. mimon, which similarly may be found in human dwellings and parasitize humans in Brazil. We also provide molecular evidence that this argasid tick species may carry a rickettsial organism, whose pathogenicity remains unknown. A total of 16 ticks (two females, two males and 12 nymphs) were collected in the bedroom and in the attic of a human house, where cases of "insect" bites have been recurrent. These ticks were identified morphologically and genetically as Ornithodoros cf. mimon. Upon PCR testing, four of these ticks (one female and three nymphs) were positive for human blood and for a bacterium closely related to "Candidatus Rickettsia paranaensis". In conclusion, we report for the first time in Brazil an argasid tick species morphologically and genetically related to O. mimon, which feeds on humans and carry a rickettsial organism belonging to the spotted fever group. Further studies are needed to formally assess the taxonomic status of this tick species and also to investigate the pathogenicity of its associated rickettsial organism.

Domestic dogs as amplifying hosts of Rickettsia rickettsii for Amblyomma aureolatum ticks.

Brazilian spotted fever (BSF) is an acute infectious disease caused by the bacterium Rickettsia rickettsii, which is transmitted by different tick species. Due to deleterious effects caused on ticks, the horizontal transmission of R. rickettsii through amplifying hosts is crucial for its maintenance in tick populations among BSF-endemic areas. The tick Amblyomma aureolatum is the main vector of R. rickettsii in the São Paulo metropolitan area; nevertheless, it is not known which vertebrate could act as an amplifying host for this tick species. Herein, we evaluated the potential of domestic dogs - primary hosts for A. aureolatum adults in BSF-endemic areas - to act as amplifying hosts. For this purpose, A. aureolatum non-infected adults were allowed to feed on two groups of dogs: the control group (G1), composed of one dog not exposed to R. rickettsii; and, the infected group (G2), composed of three dogs infected with R. rickettsii via tick parasitism. All G2-dogs became ill, seroconverted to R. rickettsii, and rickettsial DNA was detected in 87% of the engorged females that fed on them. Transovarial transmission rate was estimated to be 25% and infected larvae successfully transmitted R. rickettsii to guinea-pigs, confirming transovarial transmission and vector competence. No rickettsial DNA was detected in individual samples of eggs or larvae, which precluded the estimation of filial infection rate, but implies that it was low. Our results suggest that domestic dogs act as amplifying hosts of R. rickettsii for A. aureolatum ticks in BSF-endemic areas in Brazil.

Rickettsial infection in equids, opossums and ticks in the municipality of Monte Mor, state of São Paulo, Brazil.

The aim of this study was to investigate rickettsial infection in equids, opossums and ticks in the municipality of Monte Mor, a place where a Brazilian spotted fever case occurred in 2005. In addition, characteristics possibly associated with seropositivity in horses were analyzed. Serum samples from horses, mules and opossums (Didelphis albiventris) were subjected to indirect immunofluorescence assay (IFA) against Rickettsia rickettsii. The ticks collected from the animals were identified and Amblyomma sculptum ticks from the equids were tested using PCR for Rickettsia spp. Anti-R. rickettsii antibodies were detected in 22.6% (14/62) of the horses, none of the mules and 21.7% (5/23) of the opossums. Among the variables analyzed, only age > 12 years showed a statistically significant association with seropositivity among horses. All of the 166 A. sculptum ticks tested using PCR were negative. The results showed that rickettsiae of the spotted fever group was circulating in the municipality of Monte Mor when the samples were collected and indicate a need for surveillance of Brazilian spotted fever in this region.

Canine Visceral Leishmaniasis in São Paulo, Brazil, the Most Populous City of South America: Isolation, Molecular Diagnosis, and Phylogenetic Inferences.

Background and Objectives: Canine visceral leishmaniasis affects dogs, the main urban reservoirs, which favor the transmission and expansion of this zoonotic disease in areas with high anthropization process and human density. We investigated the occurence of Leishmania infatum based in molecular diagnosis, and phylogenetic analysis of isolates obtained from dogs in metropolitan region of São Paulo. Methods: A total of 201 dogs were tested by parasitological and molecular diagnosis. Phylogenetic analysis based sequences from SSUrDNA and gGAPDH genes were performed. Results: The parasitological diagnosis revealed 5% (10/201) of positivity, and the sequences obtained from seven isolates were clustered with L. infantum in phylogentic analysis based on SSUrDNA and gGAPDH genes. A total of 24.9% (50/201) of dogs were positive in molecular diagnosis based on cathepsin L-like marker. Interpretation and Conclusion: According to this study, it is necessary to implement a surveillance policy of visceral leishmaniasis, intensifying the actions of diagnosis, prevention, and control of this zoonosis.

Implications of domestic dogs in the epidemiology of Rickettsia parkeri strain Atlantic rainforest and Rangelia vitalii in Southeastern Brazil.

This study aimed to evaluate the occurrence of diseases transmitted by Amblyomma ovale in 61 dogs monitored for three years through collections of ticks and blood, interviews, telemetry and camera traps in three areas of Serra do Mar State Park, Brazil. Blood samples were used to investigate infection by Rangelia vitalii by real-time TaqMan PCR and Rickettsia parkeri by IIFA. The collected ticks were submitted to conventional PCR to investigate the presence of R. parkeri . These data were compared with the monitoring results and interviews with the owners. Dogs considered as companion presented a risk of infection by R. parkeri strain Mata Atlantica 5.4 times higher than those not considered as companion (p = 0.009). Dogs that had at least one A. ovale collected during the campaigns had a 10 times higher risk of infection by R. parkeri strain Mata Atlantica than those who did not (p = 0.009). One dog positive for R. vitalii by real-time TaqMan PCR was parasitized by A. ovale frequently during monitoring. Sequenced ompaA - positive DNA samples had 100% identity of R. parkeri strain Mata Atlantica clone As106. From the findings, it is urgent to control domestic dogs around rainforests to reduce zoonoses transmission.

Canine leptospirosis in asymptomatic populations from the Southwest Region of São Paulo State, Brazil / Leptospirose canina em uma população assintomática da região sudoeste do estado de São Paulo, Brasil

Leptospirosis is an infectious disease that can affect animals and humans. Distributed worldwide, the disease is more prevalent in tropical regions due to socioenvironmental characteristics. Dogs can serve as sentinels for environmental contamination due to their frequent contact with humans. This study investigated the frequency of occurrence of canine leptospirosis in asymptomatic populations from the Southwest Region of the State of São Paulo, Brazil. Thus, blood samples collected from 572 asymptomatic dogs from the cities of Apiaí, Cananeia, Itapeva, and Itu were tested with a microscopic agglutination test (MAT). A total of 40.5% of animals in Apiaí reacted to Leptospira spp., 42.6% in Itapeva, 7.1% in Cananeia, and 5.1% in Itu. The data from the present study demonstrate that at least one animal from the municipalities of Itapeva, Apiaí, and Cananeia had a titer equal to or higher than 800, indicating that Leptospira is circulating in these municipalities and that the teams working on castration campaigns need to be educated on the correct use of personal protective equipment, especially when mechanically emptying the bladder of these animals. This study also suggests that castration campaigns can strategically monitor zoonotic diseases and assist in establishing preventive strategies for human and animal health.(AU)

A leptospirose é uma enfermidade infectocontagiosa que pode acometer os animais e o homem. Nos países tropicais e em desenvolvimento ocorrem 70% dos casos humanos, com mortalidade variando entre 10 a 70%. Os cães podem se tornar portadores assintomáticos por um longo período, podendo transmitir a Leptospira para humanos. Devido ao intenso convívio com o ser humano, os cães podem servir como sentinelas da contaminação ambiental. Esse trabalho investigou a frequência de ocorrência da leptospirose canina em populações assintomáticas da região sudoeste do estado de São Paulo. Para isso foram examinadas pela técnica de soroaglutinação microscópica (MAT), amostras de sangue provenientes de 572 cães assintomáticos dos municípios de Apiaí, Cananeia, Itapeva e Itu por amostragem de conveniência, oriundos de campanhas de castração. Em Apiaí, foram encontrados 40,5% dos animais reagentes para Leptospira spp.; em Itapeva, 42,6%; em Cananeia, 7,7% e em Itu, 5,1%. Os dados encontrados demonstram que, pelo menos, um animal dos municípios de Itapeva, Apiaí e Cananeia apresentaram título igual ou maior que 800, indicando a circulação da bactéria nessas localidades e que a equipe envolvida nas campanhas de castração precisam ser alertadas sobre o correto uso de equipamento de proteção individual, principalmente no esvaziamento mecânico da bexiga antes do procedimento cirúrgico. O estudo também sugere que as campanhas de castração podem ser estratégicas no monitoramento de doenças zoonóticas e poderiam auxiliar no estabelecimento de ações preventivas para a saúde humana e animal.(AU)

Rickettsial infection in equids, opossums and ticks in the municipality of Monte Mor, state of São Paulo, Brazil / Infecção por Rickettsia spp. em equídeos, gambás e carrapatos do município de Monte Mor, estado de São Paulo, Brasil

Abstract The aim of this study was to investigate rickettsial infection in equids, opossums and ticks in the municipality of Monte Mor, a place where a Brazilian spotted fever case occurred in 2005. In addition, characteristics possibly associated with seropositivity in horses were analyzed. Serum samples from horses, mules and opossums (Didelphis albiventris) were subjected to indirect immunofluorescence assay (IFA) against Rickettsia rickettsii. The ticks collected from the animals were identified and Amblyomma sculptum ticks from the equids were tested using PCR for Rickettsia spp. Anti-R. rickettsii antibodies were detected in 22.6% (14/62) of the horses, none of the mules and 21.7% (5/23) of the opossums. Among the variables analyzed, only age > 12 years showed a statistically significant association with seropositivity among horses. All of the 166 A. sculptum ticks tested using PCR were negative. The results showed that rickettsiae of the spotted fever group was circulating in the municipality of Monte Mor when the samples were collected and indicate a need for surveillance of Brazilian spotted fever in this region.

Resumo Este trabalho objetivou pesquisar a infecção por Rickettsia spp. em equídeos, gambás e carrapatos, do município de Monte Mor, local que teve um caso de febre maculosa brasileira, em 2005. Além disso, características possivelmente associadas com a soropositividade nos equinos foram analisadas. Soros de equinos, muares e gambás Didelphis albiventris foram submetidos à reação de imunofluorescência indireta (RIFI) contra Rickettsia rickettsii. Os carrapatos coletados dos animais foram identificados e os carrapatos Amblyomma sculptum dos equídeos foram testados pela PCR para Rickettsia spp. Anticorpos anti-R. rickettsii foram detectados em 22,6% (14/62) equinos, zero muares e 21,7% (5/23) gambás. Entre as variáveis analisadas, apenas a idade maior que 12 anos mostrou associação estatisticamente significante com a soropositividade em equinos. De 166 carrapatos A. sculptum testados pela PCR, todos foram negativos. Os resultados mostram que riquétsias do grupo da febre maculosa estavam circulando no município de Monte Mor, quando as amostras foram coletadas, e apontam para a necessidade de vigilância para a febre maculosa brasileira nessa região.

Implications of domestic dogs in the epidemiology of Rickettsia parkeri strain Atlantic rainforest and Rangelia vitalii in Southeastern Brazil / Implicações dos cães domésticos na epidemiologia da Rickettsia parkeri cepa Mata Atlântica e da Rangelia vitalii no Sudeste do Brasil

Abstract This study aimed to evaluate the occurrence of diseases transmitted by Amblyomma ovale in 61 dogs monitored for three years through collections of ticks and blood, interviews, telemetry and camera traps in three areas of Serra do Mar State Park, Brazil. Blood samples were used to investigate infection by Rangelia vitalii by real-time TaqMan PCR and Rickettsia parkeri by IIFA. The collected ticks were submitted to conventional PCR to investigate the presence of R. parkeri . These data were compared with the monitoring results and interviews with the owners. Dogs considered as companion presented a risk of infection by R. parkeri strain Mata Atlantica 5.4 times higher than those not considered as companion (p = 0.009). Dogs that had at least one A. ovale collected during the campaigns had a 10 times higher risk of infection by R. parkeri strain Mata Atlantica than those who did not (p = 0.009). One dog positive for R. vitalii by real-time TaqMan PCR was parasitized by A. ovale frequently during monitoring. Sequenced ompaA - positive DNA samples had 100% identity of R. parkeri strain Mata Atlantica clone As106. From the findings, it is urgent to control domestic dogs around rainforests to reduce zoonoses transmission.

Resumo A ocorrência de doenças transmitidas por Amblyomma ovale em 61 cães monitorados por três anos através de coletas de carrapatos, sangue, entrevistas, telemetria e armadilhas fotográficas foi avaliada em três áreas do Parque Estadual da Serra do Mar - SP. Amostras de sangue foram utilizadas para investigação de Rangelia vitalii através de PCR TaqMan em tempo real e Rickettsia parkeri através da RIFI. Carrapatos coletados foram submetidos à PCR convencional para investigação de R. parkeri . Estes dados foram comparados considerando os resultados do monitoramento e entrevistas. Cães de companhia apresentaram risco de infecção pela R. parkeri cepa Mata Atlântica 5,4 vezes maior que os não considerados como de companhia (p = 0,009). Cães que tiveram pelo menos um A. ovale coletado apresentaram risco de infecção por R. parkeri cepa Mata Atlântica 10 vezes maior do que aqueles que não tiveram (p = 0,009). Um cão positivo para R. vitalii através de PCR TaqMan em tempo real foi parasitado por A. ovale durante o monitoramento. Amostras positivas para o gene ompaA possuíam 100% de identidade do clone As106 de R. parkeri cepa de Mata Atlântica. Assim, é urgente o controle de cães na Mata Atlântica para redução dos riscos de zoonoses.

Survey of protozoan vector-borne diseases in dogs from Atlantic Rainforest fragment around Billings Dam, São Paulo, Brazil / Levantamento de protozoários transmitidos por vetores em cães de um fragmento de Mata Atlântica nos arredores da Barragem Billings, São Paulo, Brasil

ABSTRACT: Vector-borne diseases are currently one of the biggest public health concerns worldwide. Dogs, being the closest companion animals to humans, are considered the main reservoir of some of these diseases in the urban environment. Therefore, the study of the disease behavior in dogs can help to understand the disease affecting human health. Serological and molecular diagnoses of Babesia vogeli, Rangelia vitalli, Leishmania infantum, and other trypanosomatids, were performed by immunochromatographic and PCR assays, respectively, on dogs in a dog shelter located in an Atlantic Forest fragment near the Billings Dam, São Bernardo do Campo, São Paulo-Brazil. Our molecular diagnostic results showed a high prevalence of Babesia vogeli, at 20.9% (17/81). No other protozoan was detected in any of the tests. Determining the prevalence of major vector-borne diseases is essential to establish preventive and control measures for zoonotic diseases in animals kept in shelters, in order to minimize the impact of vector-borne diseases on animal health.

RESUMO: As doenças transmitidas por vetores são atualmente um dos maiores problemas de saúde pública. Os cães, sendo os animais de companhia mais próximos dos seres humanos, são considerados os principais reservatórios de algumas dessas doenças no ambiente urbano, e o estudo de seu comportamento em cães ajuda a entender a doença como um todo na saúde humana. Diagnósticos sorológicos e moleculares de Babesia vogeli, Rangelia vitalli, Leishmania infantum e outros tripanossomatídeos, em um abrigo para cães localizado em um fragmento da Mata Atlântica próximo à Barragem Billings, São Bernardo do Campo, São Paulo, Brasil. Foram realizadas sorologias e diagnósticos moleculares, no ensaio de PCR foram utilizados marcadores moleculares de oligonucleotídeos específicos para alguns protozoários de importância na saúde animal, como Babesia vogeli, Rangelia vitalli, Leishmania infantum e outros tripanossomatídeos. Nossos resultados de diagnóstico molecular mostraram uma alta prevalência de 20,9% (17/81) de Babesia vogeli. Nenhum outro protozoário foi detectado em nenhum dos testes. A determinação da prevalência das principais doenças transmitidas por vetores é essencial para estabelecer medidas preventivas e de controle de doenças zoonóticas em animais mantidos em abrigos. Essas medidas devem ser propostas para minimizar o impacto de doenças transmitidas por vetores na saúde animal.

Serological survey of Rickettsia in equids from Vale do Paraíba, São Paulo, Brazil, and their tick identification and molecular investigation of Rickettsia / Levantamento sorológico de Rickettsias em equinos no Vale do Paraíba, São Paulo, Brasil. Identificação e pesquisa molecular de Rickettsias em carrapatos

Brazilian spotted fever is a serious and lethal illness for humans and is caused by the Rickettsia rickettsii bacteria. In the state of São Paulo/SP (Brazil), the etiological agent of this disease is transmitted by the Amblyomma sculptum tick. It was already shown that horses infected with this bacteria produce a strong immune response and could be important sentinels for the detection of the disease in a proper region. The present investigation performed a serological survey in horses from five farms of Vale do Paraíba, São Paulo state, Brazil, searching for antibodies against, Rickettsia rickettsii, Rickettsia parkeri, Rickettsia amblyommatis, Rickettsia rhipicephali, and Rickettsia bellii. In each farm, ticks were also collected that were taxonomically identified and examined by real-time PCR for Rickettsia spp DNA. Blood samples were collected from 206 horses, and 334 ticks were picked up from these animals from January to December 2017. Eighty ticks were A. sculptum and 254 Dermacentor nitens. Of the blood samples, 7.3% seroconverted to Rickettsia spp. Of these, 0.97% had a positive serological response to R. bellii. None of the 80 A. sculptum ticks were positive through real-time PCR for Rickettsia spp. Although there was no detection of ticks infected by Rickettsia spp in five farms of Paraíba Valley, the horses presented serological positive reactions against this agent. Thus, further large studies should be conducted in the area targeting hosts and vectors to generate data for control measures of the transmission of Brazilian spotted fever(AU)

A febre maculosa brasileira é uma doença grave e letal para seres humanos causada pela bactéria Rickettsia rickettsii. No estado de São Paulo, SP, Brasil, o agente etiológico desta enfermidade é transmitido pelo carrapato Amblyomma sculptum. Conforme descrito na literatura científica, os cavalos infectados com esta bactéria produzem uma forte resposta imune e podem ser importantes sentinelas para a detecção da doença. A presente investigação realizou um levantamento sorológico em cavalos de cinco fazendas do Vale do Paraíba, São Paulo, Brasil, à procura de anticorpos contra Rickettsia rickettsii, Rickettsia parkeri, Rickettsia amblyommatis, Rickettsia rhipicephali e Rickettsia bellii. Em cada fazenda, também foram coletados carrapatos identificados taxonomicamente e examinados por PCR em tempo real para o DNA de Rickettsia spp. Foram coletadas amostras de sangue de 206 cavalos e coletados 334 carrapatos desses animais entre os meses de janeiro e dezembro de 2017. Oitenta carrapatos foram identificados como A. sculptum e 254 Dermacentor nitens. Das amostras de sangue, 7,3% soroconverteram para Rickettsia spp., sendo que, 0,97% apresentaram soropositividade homóloga para R. bellii. Nenhum dos 80 carrapatos de A. sculptum foi positivo com o emprego de PCR em tempo real para Rickettsia spp. Embora não tenham sido detectados carrapatos infectados por Rickettsia spp em cinco fazendas do Vale do Paraíba, os animais apresentaram reações sorológicas positivas para este agente. Assim, outros estudos abrangentes deverão ser realizados na área investigando hospedeiros e vetores, gerando dados para medidas de controle da transmissão da febre maculosa brasileira.(AU)

Brazilian spotted fever serological survey among equids at the Guarapiranga Dam area in the city of São Paulo, Brazil / Investigação sorológica da febre maculosa brasileira entre equídeos na área da represa de Guarapiranga, na cidade de São Paulo, Brasil

In the Guarapiranga dam region located in the metropolitan area of São Paulo, human cases have been reported of Brazilian spotted fever (BSF), a tick-borne disease caused by the bacterium Rickettsia rickettsii. In this area, R. rickettsii is known to be transmitted to humans by Amblyomma aureolatum, a typical dog tick that is not associated with horses. In other BSF-endemic areas, R. rickettsii transmission is associated with Amblyomma sculptum, a tick species that typically infest capybaras and horses. The Guarapiranga Dam bears abundant populations of capybaras and horses. However, since nothing is known about a possible cycle of transmission of R. rickettsii by A. sculptum in this area, this study evaluated such transmission by performing a serosurvey of horses living in the Guarapiranga Dam region. A total of 206 equids living in the margins of the Guarapiranga Dam were serologically tested for antibodies reactive to five Rickettsia species, four of the spotted fever group (R. rickettsii, R. parkeri, R. amblyommatis, R. rhipicephali) and one basal group species, R. bellii. Overall, 171 (83%) equids reacted positively to at least one Rickettsia species. A total of 160 (78%), 123 (60%), 80 (39%), 72 (35%), and 71 (34%), equid sera reacted to R. bellii, R. rickettsii, R. parkeri, R. rhipicephali, and R. amblyommatis, respectively, with endpoint titers ranging from 64 to 1024 for R. bellii, and 64 to 512 for the remaining four Rickettsia species. Endpoint titers to R. bellii (median: 256) was significantly higher (P<0.05) than the endpoint titers to the other four Rickettsia species, for which the median values varied from 64 to 128. A total of 65 (32%) equid sera showed endpoint titers to R. bellii at least 4-fold higher than those to any of the other four antigens, indicating that they have been exposed to R. bellii or a very closely related species. Our results provide serological evidence that the sampled equids were not frequently exposed to R. rickettsii-infected ticks. Since horses are a highly suitable sentinel for R. rickettsii transmission by A. sculptum, we conclude that this tick species has no epidemiological role in the transmission of R. rickettsii in the BSF-endemic area of the Guarapiranga Dam in the metropolitan area of São Paulo.(AU)

Na região da represa de Guarapiranga localizada na área metropolitana de São Paulo, têm sido relatados casos humanos de Febre Maculosa Brasileira (FMB), uma doença transmitida por carrapatos causada pela bactéria Rickettsia rickettsii. Nesta área de estudo, R. rickettsii é conhecida por ser transmitida aos seres humanos pelo Amblyomma aureolatum, um carrapato de cão que não está associado a cavalos. Em outras áreas endêmicas da FMB, a transmissão de R. rickettsii está associada ao Amblyomma sculptum, uma espécie de carrapato que normalmente infesta capivaras e cavalos. A represa de Guarapiranga possui populações abundantes de capivaras e cavalos; no entanto, como nada se sabe sobre um possível ciclo de transmissão de R. rickettsii por A. sculptum nessa área, este estudo avaliou essa transmissão realizando um levantamento sorológico em cavalos que vivem na região da represa de Guarapiranga. Um total de 206 equídeos que vivem nas margens da represa de Guarapiranga foram testados sorologicamente para cinco espécies de Rickettsia, sendo quatro do grupo da FMB (R. rickettsii, R. parkeri, R. amblyommatis, R. rhipicephali) e um do grupo basal (R. bellii). No geral, 171 (83%) equídeos reagiram positivamente a pelo menos uma espécie de Rickettsia. Um total de 160 (78%), 123 (60%), 80 (39%), 72 (35%) e 71 (34%), reagiram a R. bellii, R. rickettsii, R. parkeri, R. rhipicephali e R. amblyommatis, respectivamente, com títulos finais variando de 64 a 1024 para R. bellii e 64 a 512 para as quatro espécies restantes de Rickettsia. Os títulos finais para R. bellii (mediana: 256) foram significativamente maiores (P <0,05) do que os títulos para as outras quatro espécies de Rickettsia, para os quais os valores medianos variaram de 64 a 128. Um total de 65 (32%) equideos, os soros mostraram títulos finais para R. bellii pelo menos quatro vezes maior que os de qualquer um dos outros quatro antígenos, indicando que eles foram expostos a R. bellii ou a uma espécie muito próxima. Os resultados obtidos fornecem evidências sorológicas de que os equídeos amostrados não eram frequentemente expostos a carrapatos infectados por R. rickettsii. Como os cavalos são um sentinela altamente adequado para a transmissão de R. rickettsiipor A. sculptum, a conclusão obtida foi que essa espécie de carrapato não tem papel epidemiológico na transmissão da bactéria na área endêmica de FMB da represa de Guarapiranga na região metropolitana de São Paulo.(AU)

Rangelia vitalii infection in a dog from São Paulo city, Brazil: case report / Infecção por Rangelia vitalli em cão da zona norte de São Paulo/SP: relato de caso

Canine rangeliosis is an extravascular hemolytic disease caused by the protozoan Rangelia vitalii, which is transmitted by ticks of the species Amblyomma aureolatum. The most common clinical signs are apathy, hyperthermia and spontaneous bleeding. Anemia and thrombocytopenia are the most common hematological findings. This work reports a clinical case of canine Rangeliosis treated at a private veterinary hospital, in São Paulo city in 2017. A dog was treated at a veterinary hospital in the north of São Paulo, with progressive weight loss, apathy and tail injury. Anemia and thrombocytopenia were observed on the hemogram. Rangelia vitalii DNA was detected in animal blood by real-time PCR (qPCR). In addition to the supportive treatment, doxycycline and subcutaneous imidocarb applications were used. The sample collected after treatment with the antibiotic continued to present protozoal DNA. The disease should be considered as a differential diagnosis and there is a great need for further studies about the therapy used.(AU)

A rangeliose canina é uma doença hemolítica extravascular causada pelo protozoário Rangelia vitalii, o qual é transmitido por carrapatos da espécie Amblyomma aureolatum. Os sinais clínicos mais comuns são apatia, hipertermia e sangramentos espontâneos. Os achados hematológicos mais comuns são anemia e trombocitopenia. Este trabalho teve como objetivo relatar um caso clínico de Rangeliose canina tratada em um hospital veterinário particular, na cidade de São Paulo no ano de 2017. Um cão foi atendido em um hospital veterinário da zona norte de São Paulo, com emagrecimento progressivo, apatia e lesão na cauda. No hemograma foram observadas anemia e trombocitopenia. Através da PCR em tempo real (qPCR) do sangue do animal constatou-se a presença de DNA de Rangelia vitalii. Além do tratamento de suporte, utilizou-se doxiciclina e aplicações subcutâneas de imidocarb. A amostra coletada após o tratamento com o antibiótico continuou apresentando DNA do protozoário. A enfermidade deve ser considerada como diagnóstico diferencial e há uma grande necessidade de maiores estudos acerca da terapia utilizada.(AU)

Rickettsia rickettsii Co-feeding Transmission among Amblyomma aureolatum Ticks.

Amblyomma aureolatum ticks are vectors of Rickettsia rickettsii, the etiologic agent of Rocky Mountain spotted fever in Brazil. Maintenance of R. rickettsii in nature depends on horizontal transmission along tick generations. Although such transmission is known to occur when uninfected and infected ticks feed simultaneously on susceptible animals (co-feeding systemic transmission), we investigated co-feeding nonsystemic transmission, which was based on R. rickettsii-infected and -uninfected A. aureolatum ticks feeding simultaneously on guinea pigs immune to R. rickettsii. Our acquisition and transmission infestations demonstrated that horizontal transmission of R. rickettsii by co-feeding ticks on immune hosts with no systemic infection did not occur when uninfected larvae fed distantly from infected nymphs but did occur in a few cases when uninfected larvae fed side-by-side with infected nymphs, suggesting that they shared the same feeding site. The co-feeding nonsystemic transmission type might have no epidemiologic importance for Rocky Mountain spotted fever.

Successful Infection of Tick Cell Cultures of Rhipicephalus sanguineus (Tropical Lineage) with Ehrlichia canis.

There are two distinct lineages of ticks, Rhipicephalus sanguineus, in South America: tropical and temperate lineages. Only the tropical lineage is recognized as competent vector for Ehrlichia canis. The epidemiological data of canine monocytic ehrlichiosis is congruent with the distribution of the two lineages of R. sanguineus. Herein, we report the infection of R. sanguineus (tropical lineage) cell cultures with E. canis, after cryopreservation. R. sanguineus (tropical lineage) cell identity was confirmed by sequencing using a 16S rDNA gene fragment. Tick cell cultures were prepared in L-15B medium supplemented with 10%, 15%, and 20% Fetal Bovine Serum (FBS), and 10% of Tryptose Phosphate Broth (TPB). Cell cultures developed better at the concentration of 20% of FBS. Cultures in the fifth harvest (approximately 7 months later) were selected for the first infections. Optimal R. sanguineus cell growth and adhesion was observed (5.0 × 106 cells/mL, and the population doubling time every 57 h). Once infected with E. canis, the cultures were maintained in L-15B medium supplemented with 2% and 5% of FBS fortified with iron and 10% TPB. Infected cells were also cryopreserved. DNA was extracted from infected and noninfected cells and analyzed using quantitative real-time PCR targeting the E. canis-dsb gene. Primary culture of the fifth passage was infected by E. canis and it maintained the pathogen for at least 40 days before partial cell destruction. Subcultures of infected cells (fresh and cryopreserved cultures) onto new tick cell cultures were successful. The E. canis infection was confirmed by real-time PCR and light and transmission electron microscopy. The R. sanguineus (tropical lineage) cells infected with E. canis successfully infected new tick cell cultures, showing that these cells could be an alternative substrate for maintenance of this pathogen.

Successful infection of tick cell cultures of Rhipicephalus sanguineus (Tropical Lineage) with Ehrlichia canis

There are two distinct lineages of ticks, Rhipicephalus sanguineus, in South America: tropical and temperate lineages. Only the tropical lineage is recognized as competent vector for Ehrlichia canis. The epidemiological data of canine monocytic ehrlichiosis is congruent with the distribution of the two lineages of R. sanguineus. Herein, we report the infection of R. sanguineus (tropical lineage) cell cultures with E. canis, after cryopreservation. R. sanguineus (tropical lineage) cell identity was confirmed by sequencing using a 16S rDNA gene fragment. Tick cell cultures were prepared in L-15B medium supplemented with 10%, 15%, and 20% Fetal Bovine Serum (FBS), and 10% of Tryptose Phosphate Broth (TPB). Cell cultures developed better at the concentration of 20% of FBS. Cultures in the fifth harvest (approximately 7 months later) were selected for the first infections. Optimal R. sanguineus cell growth and adhesion was observed (5.0?×?106 cells/mL, and the population doubling time every 57?h). Once infected with E. canis, the cultures were maintained in L-15B medium supplemented with 2% and 5% of FBS fortified with iron and 10% TPB. Infected cells were also cryopreserved. DNA was extracted from infected and noninfected cells and analyzed using quantitative real-time PCR targeting the E. canis-dsb gene. Primary culture of the fifth passage was infected by E. canis and it maintained the pathogen for at least 40 days before partial cell destruction. Subcultures of infected cells (fresh and cryopreserved cultures) onto new tick cell cultures were successful. The E. canis infection was confirmed by real-time PCR and light and transmission electron microscopy. The R. sanguineus (tropical lineage) cells infected with E. canis successfully infected new tick cell cultures, showing that these cells could be an alternative substrate for maintenance of this pathogen.

Successful Infection of Tick Cell Cultures of Rhipicephalus sanguineus (Tropical Lineage) with Ehrlichia canis

There are two distinct lineages of ticks, Rhipicephalus sanguineus, in South America: tropical and temperate lineages. Only the tropical lineage is recognized as competent vector for Ehrlichia canis. The epidemiological data of canine monocytic ehrlichiosis is congruent with the distribution of the two lineages of R. sanguineus. Herein, we report the infection of R. sanguineus (tropical lineage) cell cultures with E. canis, after cryopreservation. R. sanguineus (tropical lineage) cell identity was confirmed by sequencing using a 16S rDNA gene fragment. Tick cell cultures were prepared in L-15B medium supplemented with 10%, 15%, and 20% Fetal Bovine Serum (FBS), and 10% of Tryptose Phosphate Broth (TPB). Cell cultures developed better at the concentration of 20% of FBS. Cultures in the fifth harvest (approximately 7 months later) were selected for the first infections. Optimal R. sanguineus cell growth and adhesion was observed (5.0?×?106 cells/mL, and the population doubling time every 57?h). Once infected with E. canis, the cultures were maintained in L-15B medium supplemented with 2% and 5% of FBS fortified with iron and 10% TPB. Infected cells were also cryopreserved. DNA was extracted from infected and noninfected cells and analyzed using quantitative real-time PCR targeting the E. canis-dsb gene. Primary culture of the fifth passage was infected by E. canis and it maintained the pathogen for at least 40 days before partial cell destruction. Subcultures of infected cells (fresh and cryopreserved cultures) onto new tick cell cultures were successful. The E. canis infection was confirmed by real-time PCR and light and transmission electron microscopy. The R. sanguineus (tropical lineage) cells infected with E. canis successfully infected new tick cell cultures, showing that these cells could be an alternative substrate for maintenance of this pathogen.