Rickettsia amblyommatis infecting Amblyomma pseudoconcolor in area of new focus of spotted fever in northeast Brazil.

In Brazil, active infections of Rickettsia spp. is confirmed in all regions, involving various species of ticks. During investigation of a new focus of Spotted Fever (SF) incidence in the state of Pernambuco, northeastern Brazil, tick species Rhipicephalus sanguineus sensu lato (Latreille), and Amblyomma pseudoconcolor were collected from one Canis familiaris and four Euphractus sexcinctus, respectively, and analized for the presence of rickettsial genes. Ten A. pseudoconcolor ticks (90.9%) were found to be infected with Rickettsia, whereas no evidence of Rickettsia spp. was found in R. sanguineus s. l. Genetic analysis based of five rickettsial genes showed that the detected strain is most closely related to Rickettsia amblyommatis (formerly Candidatus Rickettsia amblyommii). R. amblyommatis was, for the first time, detected in Amblyomma pseudoconcolor and the results pointed to this tick like a potential vector in the enzootic cycle of R. amblyommatis in a typical semiarid Brazilian savannah region. In conclusion, despite the need for further studies to confirm if R. amblyommatis was responsible for the observed case in the state of Pernambuco, the presence of this bacterium during an SF focussed investigation should be a major concern in terms of public health due the capacity of SF for rapid and extensive dispersion within Brazilian territory.

Exposure of Baixadeiro horses to Rickettsia spp. and to ticks infected by Rickettsia amblyommatis in the Baixada Maranhense micro-region, Maranhão, Brazil / Exposição dos cavalos Baixadeiro a Rickettsia spp. e carrapatos infectados pela Rickettsia amblyommatis na Baixada Maranhense, microrregião do Estado do Maranhão, Brasil

ABSTRACT: The aim of this study was to investigate exposure of Baixadeiro horses to Rickettsia spp. and to ticks infected by Rickettsia in the Baixada Maranhense (lowlands) micro-region, state of Maranhão. A total of 258 horses were tested for Rickettsia rickettsii, Rickettsia amblyommatis and Rickettsia bellii using the immunofluorescence assay (IFA). Overall, 58.91% (152/258) of the horses were seroreactive for at least one Rickettsia species, and 85.27% of the horses were infested with one or more species of tick, which were identified as Dermacentor nitens (93.63%), Amblyomma cajennense sensu stricto (4.55%) and Rhipicephalus (Boophilus) microplus (1.82%). These ticks were subjected to DNA extraction and were tested using the polymerase chain reaction (PCR), targeting two rickettsia genes: citrate synthase gene (gltA) and 190kDa outer membrane protein gene (ompA). Three specimens of A. cajennense s.s. were positive. BLAST analyses on the nucleotide sequences obtained from the PCR products showed that these were 99-100% identical to the corresponding sequences of R. amblyommatis. Thus, results indicate that R. amblyommatis and/or a strain very close to this is circulating in ticks in this micro-region.

RESUMO: O objetivo deste estudo foi verificar a exposição e a infecção de cavalos baixadeiro e carrapatos por Rickettsia spp. na micro-região da Baixada Maranhense do Estado Maranhão. Um total de 258 cavalos foi testado pelo ensaio de Imunofluorescência Indireta (IFI) para Rickettsia rickettsii, Rickettsia amblyommatis e Rickettsia bellii. Deste total, 58,91% (152/258) foram sororreativos para pelo menos uma espécie de Rickettsia sp., e 85,27% cavalos estavam infestados por uma ou mais espécies de carrapatos identificados como Dermacentor nitens (93,63%), Amblyomma cajennense sensu stricto (4,55%) e Rhipicephalus (Boophilus) microplus (1,82%). Estes carrapatos foram submetidos à extração de DNA e testados pela reação em cadeia pela polimerase (PCR) alvejando os genes citrato sintase (gltA) e o de proteína de membrana externa de 190kDa (ompA). Três espécimes de A. cajennense s.s. foram positivos para Rickettsia amblyommatis. As sequências dos nucleotídeos obtidas a partir dos produtos de PCR mostraram 99-100% de identidade com as sequências correspondentes de R. amblyommatis quando analisadas pelo BLAST. Desta forma, os resultados indicam a circulação de R. amblyommatis nos carrapatos e/ou uma cepa muito próxima circulando na microrregião.

Molecular and serological detection of Theileria equi, Babesia caballi and Anaplasma phagocytophilum in horses and ticks in Maranhão, Brazil / Detecção molecular e serológica de Theileria equi, Babesia caballi e Anaplasma phagocytophilum em equinos e carrapatos no Maranhão

Equine piroplasmosis is a tick-borne disease caused by the intraeytrhocytic protozoans Babesia caballi and Theileria equi. It has been reported as a main equine parasitic disease. In addition, Anaplasma phagocytophilum, the causative agent of granulocytic ehrlichiosis, causes a seasonal disease in horses. Both diseases, can be detrimental to animal health. In this sense, blood samples and ticks were collected from 97 horses raised in the microregion of Baixada Maranhense, Maranhão State, Brazil. Serum samples were subjected to Indirect Fluorescence Antibody Test (IFAT) and blood samples and ticks to Polymerase Chain Reaction (PCR) to evaluate the infection by Theileria equi, Babesia caballi and Anaplasma phagocytophilum. The overall seroprevalence was 38.14%, 18.55% and 11.34% for T. equi, B. caballi and A. phagocytophilum, respectively. The results of PCR from blood samples showed 13.40% and 3.09% positive samples to T. equi and B. caballi, respectively. A total of 170 tick specimens were collected and identified as Dermacentor nitens, Amblyomma cajennense sensu lato and Rhipicephalus (Boophilus) microplus. It was detected 2.35% (4/170) and 0.59% (1/170) positive tick samples by PCR for T. equi and B. caballi, respectively. All samples were negative to A. phagocytophilum. No statically difference (p>0.05) was observed when gender, age, use of ectoparasiticide and tick presence were analyzed. A BLASTn analysis of the sequenced samples indicated 97 to 100% similarity with T. equi 18S rRNA gene sequences in GenBank and 98 to 100% with B. caballi. Genetic analysis classified the obtained sequences as T. equi and B. caballi cluster, respectively. It can be concluded that these pathogens occur and are circulating in the studied area.(AU)

A piroplasmose equina é uma doença transmitida por carrapatos causada pelos protozoários intraeritrocitários Babesia caballi e Theileria equi. É relatada como uma doença parasitária comum em equinos. Além disso, Anaplasma phagocytophilum, o agente causal da ehrlichiose granulocítica, causa uma doença sazonal em equinos. Ambas as doenças, podem ser prejudiciais para a saúde animal. Nesse sentido, amostras de sangue e carrapatos foram coletadas de 97 cavalos criados na microrregião da Baixada Maranhense, estado do Maranhão, Brasil. As amostras de soro foram submetidas ao Teste de Imunofluorescência Indireta (RIFI) e amostras de sangue e os carrapatos a Reação da Polimerase em Cadeia (PCR) para avaliar a infecção por Theileria equi, Babesia caballi e Anaplasma phagocytophilum. A prevalência foi de 38,14%, 18,55% e 11,34% para T. equi, B. caballi e A. phagocytophilum, respectivamente. Os resultados da PCR para as amostras de sangue demonstraram 13,40% e 3,09% de positividade para T. equi e B. caballi, respectivamente. Um total de 170 specimens de carrapatos foi coletado e foram identificados Dermacentor nitens, Amblyomma cajennense sensu lato and Rhipicephalus (Boophilus) microplus. Obteve-se 2,35% (4/170) e 0,59% (1/170) positivos por PCR para T. equi e B. caballi, respectivamente. Todas as amostras foram negativas para A. phagocytophilum. Não houve diferença estatística significativa (p>0.05) em relação ao sexo, idade, uso de ectoparasiticida e presença de carrapatos. A análise BLASTn das amostras sequenciadas para gene 18S rRNA indicaram 97 a 100% de similaridade com T. equi e 98-100% com B. caballi no GenBank. Análises genéticas classificaram as sequencias obtidas no mesmo clado que T. equi e B. caballi, respectivamente. Podemos concluir que estes patógenos estão circulando na área de estudo.(AU)

Rickettsia rickettsii infecting Rhipicephalus sanguineus sensu lato (Latreille 1806), in high altitude atlantic forest fragments, Ceara State, Brazil.

In Brazil, Spotted Fever (SF) is caused by Rickettsia rickettsii and Rickettsia parkeri strain Atlantic Forest. In recent years, several human cases of a milder SF have been reported from the Maciço de Baturité region of Ceará State. Previous studies in this region found R. parkeri strain Atlantic Forest to be present in Rhipicephalus sanguineus sensu lato and Amblyomma ovale ticks. The present study isolated and identified the Rickettsia spp. present in this new endemic area in Brazil. In March 2015, R. sanguineus s.l. and A. ovale were collected in rural areas of the Maciço de Baturité region, and subjected to the isolation technique. A bacterium was isolated from one R. sanguineus s.l., which phylogenetic analysis clustered to the R. rickettsii group. In conclusion, R. rickettsii bacteria is circulating in the studied area and may in future have an impact on the clinical diagnoses and consequently cause changes in the profile of the disease in the region. In addition, we suggest the increase of epidemiological and environmental surveillance in the area, in order to prevent Brazilian Spotted Fever cases.

First report of a Rickettsia asembonensis related infecting fleas in Brazil.

The present study was performed in a non-endemic area for spotted fever (SF) in Imperatriz microregion, state of Maranhão, Brazil. Blood samples and ectoparasites were collected from 300 dogs of the Imperatriz microregion. Canine serum samples were tested individually by indirect immunofluorescence assay (IFA), using five Rickettsia isolates from Brazil. Antibodies reactive to at least one of the five species of Rickettsia were detected in 1.6% of the dogs (5/300). These sera were considered reactive to Rickettsia rickettsii and Rickettsia amblyommatis or very closely related species. The ticks (Acari: Ixodidae), identified as Rhipicephalus sanguineus sensu lato (Latreille), and the fleas, identified as Ctenocephalides felis, were tested by polymerase chain reaction (PCR) for detection of rickettsial DNA. More than 78% (83/106) of the C. felis fleas were found to be infected with Rickettsia species using gltA as rickettsial PCR targets, whereas no evidence of Rickettsia spp. was found in R. sanguineus s. l. Genetic analysis based on genes gltA, htrA and ompB showed that the detected strain, is most closely related to Rickettsia asembonensis (formerly Candidatus Rickettsia asemboensis). The present study is the first report of a R. asembonensis related infecting C. felis fleas in Brazil.

Comparative growth of spotted fever group Rickettsia spp. strains in Vero cells.

In Brazil, the spotted fever group (SFG) Rickettsia rickettsii and Rickettsia parkeri related species are the etiological agents of spotted fever rickettsiosis. However, the SFG, Rickettsia rhipicephali, that infects humans, has never been reported. The study of growth dynamics can be useful for understanding the infective and invasive capacity of these pathogens. Here, the growth rates of the Brazilian isolates R. rickettsii str. Taiaçu, R. parkeri str. At#24, and R. rhipicephali HJ#5, were evaluated in Vero cells by quantitative polymerase chain reaction. R. rhipicephali showed different kinetic growth compared to R. rickettsii and R. parkeri.

Comparative growth of spotted fever group Rickettsia spp. strains in Vero cells

In Brazil, the spotted fever group (SFG) Rickettsia rickettsii and Rickettsia parkeri related species are the etiological agents of spotted fever rickettsiosis. However, the SFG, Rickettsia rhipicephali, that infects humans, has never been reported. The study of growth dynamics can be useful for understanding the infective and invasive capacity of these pathogens. Here, the growth rates of the Brazilian isolates R. rickettsii str. Taiaçu, R. parkeri str. At#24, and R. rhipicephali HJ#5, were evaluated in Vero cells by quantitative polymerase chain reaction. R. rhipicephali showed different kinetic growth compared to R. rickettsii and R. parkeri.