Molecular detection of Rickettsia rickettsii, Ehrlichia canis and Rangelia vitalli in Rhipicephalus sanguineus senso latu collected from dogs in Brazil / [Detecção molecular de Rickettsia rickettsii, Ehrlichia canis e Rangelia vitalli em Rhipicephalus sanguineus sensu lato coletados de cães no Brasil]

ABSTRACT This study evaluated by molecular methods the presence of major canine tick-borne agents in ticks infesting domestic dogs of a hospital population in a neglected area of the southern zone of the São Paulo Metropolitan region, which is characterized by an extensive urban area surrounded and interspersed by forest remnants of the original Atlantic rainforest. During 2017, 106 tick specimens - 71 adults and 33 nymphs of Rhipicephalus sanguineus sensu lato (s.l.), and two adults of Amblyomma aureolatum - were collected from 41 dogs that were attended in a Veterinary Teaching Hospital in São Paulo City, Brazil. By molecular analyses, 4.2% (3/71) of the R. sanguineus s.l. adult ticks contained the bacterium Rickettsia rickettsii, 2.8% (2/71) contained the bacterium Ehrlichia canis, and 4.2% (3/71) contained the protozoan Rangelia vitalii. These results indicate that domestic dogs of the southern zone of the São Paulo metropolitan region might be exposed to three of the major tick-borne agents affecting dogs in Brazil, R. rickettsii, E. canis and R. vitalii. In addition, the findings reinforce the circulation of the human pathogen R. rickettsii in the study area in a likely enzootic cycle involving dogs and R. sanguineus ticks.

RESUMO Este estudo avaliou, por métodos moleculares, a presença dos principais agentes etiológicos de doenças caninas transmitidas por carrapatos que infestam cães domésticos de uma população hospitalar, em uma área negligenciada da zona sul da região metropolitana de São Paulo, caracterizada por uma extensa área urbana circundada e intercalada por remanescentes florestais de Mata Atlântica. Durante o ano de 2017, 106 espécimes de carrapatos - 71 adultos e 33 ninfas de Rhipicephalus sanguineus sensu lato (s.l.), e dois adultos de Amblyomma aureolatum - foram coletados de 41 cães atendidos em um Hospital Veterinário Universitário da cidade de São Paulo, Brasil. Por análises moleculares, 4,2% (3/71) de R. sanguineus s.l. adultos continham a bactéria Rickettsia rickettsii, 2,8% (2/71) continham a bactéria Ehrlichia canis e 4,2% (3/71) continham o protozoário Rangelia vitalii. Esses resultados indicam que cães domésticos da zona sul da região metropolitana de São Paulo podem estar expostos a três dos principais agentes carrapatos que afetam cães no Brasil, R. rickettsii, E. canis e R. vitalii. Além disso, os achados reforçam a circulação do patógeno humano R. rickettsii na área de estudo, em um provável ciclo enzoótico envolvendo cães e carrapatos R. sanguineus.

Detecção de anticorpos anti-Rickettsia rickettsii em cães residentes em área negligenciada no município de São Paulo, SP, Brasil / [Detection of anti-Rickettsia rickettsii antibodies in dogs living in a neglected area in São Paulo, SP, Brazil]

A febre maculosa brasileira (FMB), descrita inicialmente nos Estados Unidos como febre maculosa das Montanhas Rochosas, é uma antropozoonose relatada apenas no continente americano e causada pela bactéria Rickettsia rickettsii. No Brasil a transmissão ocorre sobretudo pela picada de carrapatos do gênero Amblyomma spp. A doença foi inicialmente descrita como de transmissão em áreas rurais e silvestres, no entanto áreas periurbanas e urbanas vêm apresentando casos, principalmente relacionados com a presença de humanos residindo em pequenos fragmentos de mata ciliar. O presente estudo teve por objetivo elucidar a dispersão da FMB nas proximidades dos reservatórios Guarapiranga e Billings, na cidade de São Paulo, SP. Para tanto, a presença de anticorpos anti-R. rickettsii, Rickettsia parkeri e Rickettsia bellii foi avaliada em cães atendidos nas campanhas de esterilização cirúrgica e residentes ao redor dos reservatórios. Foram coletadas amostras de 393 cães, e as amostras de soro foram analisadas pela reação de imunofluorescência indireta (RIFI), com ponto de corte de 1:64. Os títulos para R. rickettsii variaram de 256 a 4096, com positividade de 3,3% (13/393); para R. bellii, de 128 a 1024 e 4,1% (16/393) de positivos, e um único animal (0,25%) foi soropositivo para R. parkeri, com título de 128. Os achados permitem concluir que a região de estudo apresenta condições de se tornar uma possível área com casos de FMB, pois comporta fragmentação de Mata Atlântica, condições essas ideais para a manutenção do vetor do gênero Amblyomma já descrito na região, bem como para a presença da Rickettsia rickettsii circulante entre os cães, confirmada pela existência de anticorpos. Condutas referentes à conscientização da população por meio de trabalhos educacionais devem ser implantadas para a prevenção da doença na população da área.(AU)

Brazilian Spotted Fever (BSF), initially described in the United States as Rocky Mountain Spotted Fever, is an anthropozoonosis reported only in the Americas and caused by the bacterium Rickettsia rickettsii. In Brazil, transmission occurs mainly through tick bites of the genus Amblyomma spp. The disease was initially described as transmission of rural and wild areas; however, peri-urban and urban areas have been presenting cases, mainly related to the presence of humans residing in small fragments of riparian forest. The present study aimed to elucidate the dispersal of BSF near the Guarapiranga and Billings Reservoirs, in the city of São Paulo, SP. The presence of anti-R. rickettsii, Rickettsia parkeri and Rickettsia bellii antibodies were evaluated in dogs treated in surgical sterilization campaigns and residents around the Reservoirs. Samples were collected from 393 dogs and serum samples were analyzed by indirect immunofluorescence reaction (RIFI) with a cutoff of 1:64. The titles for R. rickettsii varied from 256 to 4096 with a positivity of 3.3% (13/393); for R. bellii from 128 to 1024 and 4.1% (16/393) of positive and a single animal (0.25%) was seropositive for R. parkeri with a titre of 128. The findings allow us to conclude that the study region has conditions to become a possible area with BSF cases, as it involves Atlantic Forest, ideal conditions for the maintenance of the vector of the genus Amblyomma already described in the region and the presence of circulating Rickettsia rickettsii among dogs, confirmed by the presence of antibodies. Conducts regarding the awareness of the population through educational work should be implemented to prevent the disease in the population of the area.(AU)

New records of ticks infesting bats in Brazil, with observations on the first nymphal stage of Ornithodoros hasei.

In Brazil, at least 14 species of soft ticks (Argasidae) are associated with bats. While Ornithodoros hasei seems to be abundant among foliage-roosting bats, other groups of ticks are found exclusively inside caves. In this paper, noteworthy records of soft ticks infesting bats are documented in new localities from Bahia, Pernambuco, Piauí, and Rondônia states. Out of 201 bats examined, 25 were infested by 152 ticks belonging to seven taxa: Ornithodoros cavernicolous, O. hasei, Ornithodoros marinkellei, Ornithodoros cf. fonsecai, Ornithodoros cf. clarki, Antricola sp., and Nothoaspis amazoniensis. These findings provide new insights into the geographical distribution and host association of soft ticks occurring in the Neotropical region. Remarkably, morphological and biological observations about O. hasei are inferred based on the examination of on-host-collected first stage nymphs.

Lethal Fascioliasis in Capybaras ( Hydrochoerus hydrochaeris) in Brazil.

The liver fluke Fasciola hepatica (Trematoda: Fasciolidae) causes fascioliasis, which affects mostly domestic ruminants and humans worldwide. This parasite has an Old World origin and was introduced into the New World by European colonizers. Capybara ( Hydrochoerus hydrochaeris) is the largest living rodent species, with adults weighing over 60 kg. We report a fascioliasis outbreak caused by F. hepatica that reduced a capybara group from 21 to 2 animals within a 9-mo period. Animal infection and associated lesions were confirmed by postmortem examinations that revealed extensive liver damage associated with the presence of large number of adult and immature forms of F. hepatica. Both macroscopic and microscopic alterations in the liver were compatible with acute fascioliasis, which is characterized by a large parasite burden in the liver. Taxonomic identification of flukes collected from capybara livers were confirmed by molecular methods, which generated a mitochondrial NADH dehydrogenase I (NDI) gene partial sequence that was 100% identical to a F. hepatica NDI sequence from the United Kingdom. This is the first report of deleterious effects caused by F. hepatica in capybaras, highlighting the potential harm caused by this exotic parasite in the capybara.

A New Collection of Amblyomma parvitarsum (Acari: Ixodidae) in Peru, With Description of a Gynandromorph and Report of Rickettsia Detection.

Adult stages of Amblyomma parvitarsum Neumann parasitize wild and domestic camelids of the genera Lama and Vicugna in highlands of Andean Plateau and Patagonia. Within the Peruvian Andes, few reports have documented this tick-host association, and although reported in Chile and Argentina, Rickettsia-infected A. parvitarsum remains undocumented for this country. Here we report a new collection of A. parvitarsum from Peru, the finding of the first gynandromorph for the species and high prevalence of Rickettsia in adult stages.

Rickettsia parkeri in free-ranging wild canids from Brazilian Pampa.

Spotted fevers are tick-borne diseases associated with various Rickettsia species. Rickettsia parkeri sensu stricto (s.s.) is the agent of an emerging eschar-associated rickettsiosis in humans from the USA and South American Pampa. Considering that R. parkeri s.s. is restricted to Americas and the potential role of dogs in the epidemiology of the disease, it is thus reasonable to hypothesize that wild canids could be involved in the enzootic cycle of this rickettsiosis. The aim of this work was to investigate the potential role of the wild canids from Pampa, Cerdocyon thous (crab-eating fox) and Lycalopex gymnocercus (Pampas fox), in the ecology of R. parkeri s.s. For that, 32 live-trapped free-ranging wild canids were sampled. Ticks were observed in 30 of the 32 foxes. Of the 292 ticks collected, 22 (7.5%) were positive by PCR for the presence of R. parkeri s.s. DNA. Also, 20 (62%) wild canids showed antibodies against R. parkeri. The results suggest that wild canids are involved in the enzootic cycle of R. parkeri s.s. in the Pampa biome and could be responsible for pathogen (and its vectors) dispersal.

Diferenciação morfométrica entre larvas de Amblyomma sculptum Berlese, 1888 e Amblyomma dubitatum Neumann, 1899 / Morphometric differentiation between larvae of Amblyomma sculptum Berlese, 1888 and Amblyomma dubitatum Neumann, 1899

Os carrapatos estão envolvidos em processos biológicos de uma grande variedade de organismos patogênicos. O gênero Amblyomma é o de maior importância médica, com a espécie Amblyomma sculptum Berlese, 1888 envolvida no ciclo de transmissão da febre maculosa brasileira (FMB). Neste estudo, objetivou-se a validação molecular para uma diferenciação na característica morfométrica e no tamanho de idiossoma de larvas de duas espécies de carrapatos, Amblyomma dubitatum Neumann, 1899 e A. sculptum. Larvas não alimentadas foram coletadas em duas áreas de transmissão para FMB, por meio da técnica de armadilha atrativa de CO2. Foram identificadas em nível de espécie por morfometria comparativa, análise molecular por PCR e sequenciamento genômico, com validação pela análise de concordância pelo teste Kappa. As larvas de A. dubitatum apresentaram um comprimento significativamente maior que as larvas de A. sculptum. Embora nenhuma outra espécie do gênero Amblyomma tenha sido testada neste estudo, essa técnica poderá ser utilizada nos locais onde levantamentos acarológicos prévios, baseados nos estádios de ninfa e adultos, indicaram a presença de apenas A. sculptum e A. dubitatum, geralmente mantidos por capivaras. Digno de nota, essa condição é muito comum ao longo das áreas endêmicas para FMB na região Sudeste do Brasil.(AU)

Ticks are involved in biological processes of a wide variety of pathogenic organisms. The genus Amblyomma presents the greatest medical importance, with the species Amblyomma sculptum Berlese, 1888 involved in the transmission cycle of Brazilian Spotted Fever (BSF). In this study, we performed a molecular validation of the morphometric differentiation based on the idiosomal length of the larvae of A. dubitatum and A. sculptum. Unfed larvae were collected in two BSF-transmission areas, using the attractive CO2 trap technique. Larvae were identified at the species level by comparative morphometry, molecular analysis by PCR and genomic sequencing, with validation through agreement analysis by the Kappa test. The larvae of A. dubitatum showed a significantly longer idiosomal length than A. sculptum larvae. Although no other species of the genus Amblyomma has been tested in this study, this technique can be applied to places where previous acarological surveillances based on adult and nymphal ticks stages have indicated the presence of only A. sculptum and A. dubitatum, usually sustained by capybaras. Noteworthy, this condition is very common among many BSF-endemic areas in southeastern Brazil.(AU)

Ectoparasites of small-mammals: determinants of community structure in South American savannah.

This study aimed to assess the contribution of hosts characteristics (rodents and marsupials) in the organization of ectoparasite communities present in woodland patches in western central Brazil. We verified the effect of host species, sex, body mass and vertical strata in addition to the role of seasonality on the ectoparasite composition, richness and abundance. The total sampling effort was 22 032 trap-nights equally distributed in 54 woodland patches. Variance partition and principal coordinate analysis were used to verify the existence of significant relationships between response variables and predictors. As expected, host species was the most important variable in ectoparasite community assembly. The composition, richness and abundance of mites and lice were highly influenced by host species, although higher for mites than for lice. Host body mass had a determining role on the richness and abundance of tick species. Vertical stratification and seasonality had weak influence, while the sex of the host had no influence on the organization of these communities. The results are closely related to the evolutionary characteristics of the species involved, as well as with local environmental characteristics of the study area.

Diversidade de carrapatos (Acari: Ixodidae) em animais silvestres recebidos pelo Zoológico Municipal de Guarulhos / Tick diversity (Acari: Ixodidae) on wild animals received by the Municipal Zoo of Guarulhos

O Zoológico Municipal de Guarulhos, localizado no estado de São Paulo, recebe e recupera animais silvestres do próprio município e de alguns municípios limítrofes. Entre os anos de 1997 e 2016 foram coletados carrapatos de animais silvestres (Cariama cristata, Buteo brachyurus, Alouatta guariba, Bradypus variegatus, Tamandua tetradactyla, Puma concolor, Cerdocyon thous, Nasua nasua, Mazama gouazoubira, Didelphis aurita, Sphiggurus villosus, Myocastor coypus e Hydrochoerus hydrochaeris) atendidos pelo Zoológico. Foram identificadas as seguintes espécies de carrapatos: Amblyomma aureolatum (Pallas, 1772), Amblyomma calcaratum Neumann, 1899, Amblyomma dubitatum Neumann, 1899, Amblyomma longirostre (Koch, 1844), Amblyomma nodosum Neumann, 1899, Amblyomma parkeri Fonseca e Aragão, 1952, Amblyomma sculptum Berlese, 1888, Amblyomma varium Koch, 1844, Haemaphysalis juxtakochi Cooley, 1946, Ixodes loricatus Neumann, 1899 e Rhipicephalus sanguineus sensu lato (Latreille, 1806). Apesar de todas as associações carrapato-hospedeiro já terem sido previamente relatadas no território brasileiro, o presente estudo registra pela primeira vez um ginandromorfo de A. dubitatum.(AU)

The Municipal Zoo of Guarulhos, located in the state of São Paulo, receives and recovers wild animals from the Guarulhos municipality and from some neighboring municipalities. Between the years of 1997 and 2016 ticks were collected on wild animals (Cariama cristata, Buteo brachyurus, Alouatta guariba, Bradypus variegatus, Tamandua tetradactyla, Puma concolor, Cerdocyon thous, Nasua nasua, Mazama gouazoubira, Didelphis aurita, Sphiggurus villosus, Myocastor coypus, and Hydrochoerus hydrochaeris) attended by the Zoo. The following tick species have been identified: Amblyomma aureolatum (Pallas, 1772), Amblyomma calcaratum Neumann, 1899, Amblyomma dubitatum Neumann, 1899, Amblyomma longirostre (Koch, 1844), Amblyomma nodosum Neumann, 1899, Amblyomma parkeri Fonseca and Aragão, 1952, Amblyomma sculptum Berlese, 1888, Amblyomma varium Koch, 1844, Haemaphysalis juxtakochi Cooley, 1946, Ixodes loricatus Neumann, 1899, and Rhipicephalus sanguineus sensu lato (Latreille, 1806). Although all tick-host associations have previously been reported in Brazilian territory, the present study records for the first time a gynandromorph of A. dubitatum.(AU)

DIVERSIDADE DE CARRAPATOS (ACARI: IXODIDAE) EM ANIMAIS SILVESTRES RECEBIDOS PELO ZOOLÓGICO MUNICIPAL DE GUARULHOS

O Zoológico Municipal de Guarulhos, localizado no estado de São Paulo, recebe e recupera animais silvestres do próprio município e de alguns municípios limítrofes. Entre os anos de 1997 e 2016 foram coletados carrapatos de animais silvestres (Cariama cristata, Buteo brachyurus, Alouatta guariba, Bradypus variegatus, Tamandua tetradactyla, Puma concolor, Cerdocyon thous, Nasua nasua, Mazama gouazoubira, Didelphis aurita, Sphiggurus villosus, Myocastor coypus e Hydrochoerus hydrochaeris) atendidos pelo Zoológico. Foram identificadas as seguintes espécies de carrapatos: Amblyomma aureolatum (Pallas, 1772), Amblyomma calcaratum Neumann, 1899, Amblyomma dubitatum Neumann, 1899, Amblyomma longirostre (Koch, 1844), Amblyomma nodosum Neumann, 1899, Amblyomma parkeri Fonseca e Aragão, 1952, Amblyomma sculptum Berlese, 1888, Amblyomma varium Koch, 1844, Haemaphysalis juxtakochi Cooley, 1946, Ixodes loricatus Neumann, 1899 e Rhipicephalus sanguineus sensu lato (Latreille, 1806). Apesar de todas as associações carrapato-hospedeiro já terem sido previamente relatadas no território brasileiro, o presente estudo registra pela primeira vez um ginandromorfo de A. dubitatum.

Diversidade de carrapatos (Acari: Ixodidae) em animais silvestres recebidos pelo Zoológico Municipal de Guarulhos / Tick diversity (Acari: Ixodidae) on wild animals received by the Municipal Zoo of Guarulhos

O Zoológico Municipal de Guarulhos, localizado no estado de São Paulo, recebe e recupera animais silvestres do próprio município e de alguns municípios limítrofes. Entre os anos de 1997 e 2016 foram coletados carrapatos de animais silvestres (Cariama cristata, Buteo brachyurus, Alouatta guariba, Bradypus variegatus, Tamandua tetradactyla, Puma concolor, Cerdocyon thous, Nasua nasua, Mazama gouazoubira, Didelphis aurita, Sphiggurus villosus, Myocastor coypus e Hydrochoerus hydrochaeris) atendidos pelo Zoológico. Foram identificadas as seguintes espécies de carrapatos: Amblyomma aureolatum (Pallas, 1772), Amblyomma calcaratum Neumann, 1899, Amblyomma dubitatum Neumann, 1899, Amblyomma longirostre (Koch, 1844), Amblyomma nodosum Neumann, 1899, Amblyomma parkeri Fonseca e Aragão, 1952, Amblyomma sculptum Berlese, 1888, Amblyomma varium Koch, 1844, Haemaphysalis juxtakochi Cooley, 1946, Ixodes loricatus Neumann, 1899 e Rhipicephalus sanguineus sensu lato (Latreille, 1806). Apesar de todas as associações carrapato-hospedeiro já terem sido previamente relatadas no território brasileiro, o presente estudo registra pela primeira vez um ginandromorfo de A. dubitatum.

The Municipal Zoo of Guarulhos, located in the state of São Paulo, receives and recovers wild animals from the Guarulhos municipality and from some neighboring municipalities. Between the years of 1997 and 2016 ticks were collected on wild animals (Cariama cristata, Buteo brachyurus, Alouatta guariba, Bradypus variegatus, Tamandua tetradactyla, Puma concolor, Cerdocyon thous, Nasua nasua, Mazama gouazoubira, Didelphis aurita, Sphiggurus villosus, Myocastor coypus, and Hydrochoerus hydrochaeris) attended by the Zoo. The following tick species have been identified: Amblyomma aureolatum (Pallas, 1772), Amblyomma calcaratum Neumann, 1899, Amblyomma dubitatum Neumann, 1899, Amblyomma longirostre (Koch, 1844), Amblyomma nodosum Neumann, 1899, Amblyomma parkeri Fonseca and Aragão, 1952, Amblyomma sculptum Berlese, 1888, Amblyomma varium Koch, 1844, Haemaphysalis juxtakochi Cooley, 1946, Ixodes loricatus Neumann, 1899, and Rhipicephalus sanguineus sensu lato (Latreille, 1806). Although all tick-host associations have previously been reported in Brazilian territory, the present study records for the first time a gynandromorph of A. dubitatum.

Vectorial competence of Amblyomma tonelliae to transmit Rickettsia rickettsii.

The aim of this work was to test the vectorial competence of Amblyomma tonelliae (Ixodida: Ixodidae) to transmit Rickettsia rickettsii (Rickettsiales: Rickettsiaceae), the agent of Rocky Mountain spotted fever (RMSF). All parasitic stages of A. tonelliae were exposed to R. rickettsii by allowing each stage to feed on hosts inoculated with this pathogen. Thereafter, ticks were fed on uninfected hosts. All stages of A. tonelliae were able to acquire the R. rickettsii infection and maintain it by transstadial and transovarial transmission. When infected ticks fed on uninfected hosts, the hosts developed rickettsiosis disease. This study demonstrates the vectorial competence of A. tonelliae to transmit R. rickettsii. These results have epidemiological relevance because A. tonelliae is one of the tick species most likely to infest humans in Argentina, including in areas in which RMSF has been reported.

Dynamics of Exposure to Rickettsia parkeri in Cattle in the Paraná River Delta, Argentina.

Several cases of human rickettsiosis caused by Rickettsia parkeri were recently documented in the Paraná River delta of Argentina, where the tick vector is Amblyomma triste Koch. As cattle suffer recurrent A. triste infestations, they are at risk of becoming infected with R. parkeri Herein we investigated the dynamics of R. parkeri and its A. triste vector in a herd of beef cattle. Cattle were followed for 18 mo and samples were analyzed for the presence of antibodies against four Rickettsia species (R. parkeri, Rickettsia bellii, Rickettsia amblyommii, and Rickettsia felis) and also for the presence of rickettsial DNA. Additionally, cattle were examined for attached ticks and questing adult ticks were collected. All ticks were analyzed for the presence of rickettsial DNA. No evidence of rickettsemia was found in any cow, but the high R. parkeri infection rate documented in A. triste both questing in the study area (13.9%) and feeding on cattle (19.8%) and the identification of antibodies against R. parkeri antigen in 90% of cattle are evidence that infection is taking place. Altogether, our data suggest that A. triste ticks are capable of naturally exposing cattle to R. parkeri However, the progress of R. parkeri infection and its impact on bovine health and production remain to be established.

A survey of tick-borne pathogens in dogs and their ticks in the Pantanal biome, Brazil.

Tick and blood samples collected from domestic dogs in the Brazilian Pantanal were tested by molecular methods for the presence of tick-borne protozoa and bacteria. Among 320 sampled dogs, 3.13% were infected by Babesia vogeli (Piroplasmida: Babesiidae), 8.75% by Hepatozoon canis (Eucoccidiorida: Hepatozoidae), 7.19% by Anaplasma platys (Rickettsiales: Anaplasmataceae), and 0.94% by an unclassified Anaplasma sp. In three tick species collected from dogs, the following tick-borne agents were detected: (a) B. vogeli, An. platys and Ehrlichia canis (Rickettsiales: Anaplasmataceae), infecting Rhipicephalus sanguineus sensu lato (Ixodida: Ixodidae) ticks; (b) H. canis, an unclassified Anaplasma sp. and Rickettsia amblyommii (Rickettsiales: Rickettsiaceae), infecting Amblyomma cajennense sensu lato (Ixodida: Ixodidae) ticks, and (c) Rickettsia sp. strain Atlantic rainforest, an emerging human pathogen, infecting Amblyomma ovale ticks. Molecular analysis, based on a mitochondrial gene, revealed that the Am. cajennense s.l. ticks of the present study corresponded to Amblyomma sculptum, a member of the Am. cajennense species complex, and that Rh. sanguineus s.l. belonged to the tropical lineage. Whereas dogs are exposed to a number of tick-borne bacterial and protozoan agents in the Pantanal biome, humans are potentially exposed to infection by spotted fever group rickettsiae (e.g. R. amblyommii and Rickettsia sp. strain Atlantic rainforest) because both Am. sculptum and Am. ovale are among the most important human-biting ticks in Brazil.

Rickettsial Infection in Animals, Humans and Ticks in Paulicéia, Brazil.

A previous study in Paulicéia Municipality, south-eastern Brazil, reported 9.7% of the Amblyomma triste ticks to be infected by Rickettsia parkeri, a bacterial pathogen that causes spotted fever in humans. These A. triste ticks were shown to be associated with marsh areas, where the marsh deer Blastocerus dichotomus is a primary host for this tick species. During 2008-2009, blood serum samples were collected from 140 horses, 41 dogs, 5 opossums (Didelphis albiventris) and 26 humans in farms from Pauliceia Municipality. Ticks were collected from these animals, from vegetation and from additional wildlife in these farms. Overall, 25% (35/140) of the horses, 7.3% (3/41) of the dogs, 3.8% (1/26) of the humans and 100% (5/5) of the opossums were seroreactive (titre ≥64) to spotted fever group (SFG) Rickettsia spp. Multivariate statistical analysis indicated that horses that were allowed to forage in the marsh were 4.8 times more likely to be seroreactive to spotted fever group (SFG) Rickettsia spp than horses that did not forage in the marsh. In addition, horses that had been living in the farm for more than 8.5 years were 2.8 times more likely to be seroreactive to SFG Rickettsia spp than horses that were living for ≤8.5 years. Ticks collected from domestic animals or from vegetation included Amblyomma cajennense, Amblyomma coelebs, Amblyomma dubitatum, Dermacentor nitens and Rhipicephalus microplus. By PCR analyses, only one pool of A. coelebs ticks from the vegetation was shown to be infected by rickettsiae, for which DNA sequencing revealed to be Rickettsia amblyommii. Ticks (not tested by PCR) collected from wildlife encompassed A. cajennense and Amblyomma rotundatum on lizards (Tupinambis sp), and A. cajennense and A. triste on the bird Laterallus viridis. Our results indicate that the marsh area of Paulicéia offers risks of infection by SFG rickettsiae.

Isolation and characterization of microsatellite loci from the tick Amblyomma aureolatum (Acari: Ixodidae).

Amblyomma aureolatum (Pallas) is the main vector of the bacterium Rickettsia rickettsii, the etiological agent of Brazilian spotted fever. This disease is the most lethal human spotted fever rickettsiosis in the world. Microsatellite loci were isolated from a dinucleotide-enriched library produced from A. aureolatum sampled in Southeastern Brazil. Eight polymorphic microsatellites were further characterized among 38 individuals sampled from São Paulo metropolitan region. The number of observed alleles ranged from 2 to 9, observed heterozygosity was 0.184-0.647, and expected heterozygosity was 0.251-0.747. Cross-species amplifications suggested that these loci will be useful for other Amblyomma species.

The spreading process of Ehrlichia canis in macrophages is dependent on actin cytoskeleton, calcium and iron influx and lysosomal evasion.

Ehrlichia canis is an obligate intracellular microorganism and the etiologic agent of canine monocytic ehrlichiosis. The invasion process has already been described for some bacteria in this genus, such as E. muris and E. chaffeensis, and consists of four stages: adhesion, internalisation, intracellular proliferation and intercellular spreading. However, little is known about the spreading process of E. canis. The aim of this study was to analyse the role of the actin cytoskeleton, calcium, iron and lysosomes from the host cell in the spreading of E. canis in dog macrophages in vitro. Different inhibitory drugs were used: cytochalasin D (actin polymerisation inhibitor), verapamil (calcium channel blocker) and deferoxamine (iron chelator). Our results showed a decrease in the number of bacteria in infected cells treated with all drugs when compared to controls. Lysosomes in infected cells were cytochemically labelled with acid phosphatase to allow the visualisation of phagosome-lysosome fusion and were further analysed by transmission electron microscopy. Phagosome-lysosome fusion was rarely observed in vacuoles containing viable E. canis. These data suggest that the spreading process of E. canis in vitro is dependent on cellular components analysed and lysosomal evasion.

Occurrence of Toxoplasma gondii antibodies in birds from the Atlantic Forest, state of São Paulo, Brazil.

Avian are considered important intermediate hosts for Toxoplasma gondii because they serve as source of infection for Felidae, which shed environmentally resistant oocysts after ingesting infected tissues. Little is known of epidemiology of toxoplasmosis in wild birds. In the present study, antibodies to T. gondii were determined in 202 wild birds of 37 species captured in seven small areas of the Atlantic Forest, in the state of São Paulo, Brazil, and provided information on possible associated risk factors. Antibodies to T. gondii were assayed by the modified agglutination test (MAT) and found in 73 (36.1%) of 202 birds with titers of 1:5 in 16 samples, 1:10 in 26 samples, 1:20 in 17 samples, 1:40 in 10 samples, 1:80 in three samples, and 1:160 in one sample. No association was observed between T. gondii seropositivity and the local where the birds were collected. Seropositivity was higher in birds that lived on the forest floor (p<0.001; U=1230.0), and in omnivorous birds (p=0.007; U=3939.0). T. gondii antibodies were reported for the first time in 23 species of birds enlarging the host range of this parasite. Notably, T. gondii antibodies were found in 83.3% (15/18) of the Rufous-bellied Thrush (Turdus rufiventris).

Antibodies against rickettsiae from spotted fever groups in horses from two mesoregions in the state of Santa Catarina, Brazil / Anticorpos contra rickettsias do grupo da febre maculosa em equinos de duas mesorregiões de Santa Catarina, Brasil

Bacteria of the Rickettsia genus are agents of Brazilian Spotted Fever (BSF), a zoonotic disease which is difficult to diagnose, evolves quickly and can result in death. Antibodies against Rickettsia spp. in horses were studied, by means of Indirect Immunofluorescence Assay (IFAT ≥64), in 150 blood samples taken from animals in two Santa Catarina mesoregions (Planalto Serrano and Vale do Itajaí). The overall occurrence of Rickettsia spp. antibodies in horses was 18.66%, with cross-reactivity occurring in all positive samples for at least two of the species tested. Separately, according to the species, 25 (16.66%) samples were positive for R. rickettsii, 15 (10%) for R. parkeri, 22 (14.66%) for R. amblyommii, 23 (15.33%) for R. rhipicephali, 16 (10.66%) for R. bellii and 19 (12.66%) for R. felis. Only two animals resulted in a conclusive serodiagnosis, one for R. bellii and the other for R. rickettsii, at maximum dilutions of 1:4096 and 1:512, respectively. The occurrence of antibodies against Rickettsia spp. in horses from two mesoregions in the state of Santa Catarina indicates the movement of BSF agents in these sentinel animals and confirms the importance of studying spotted fever in the state of Santa Catarina.

Bactérias do gênero Rickettsia são agentes da Febre Maculosa Brasileira (FMB), uma doença zoonótica, de difícil diagnóstico, rápida evolução e que pode levar o indivíduo à morte. Anticorpos contra Rickettsia spp. em equinos foram pesquisados, por meio da Reação de Imunofluorescência Indireta (RIFI≥64), em 150 amostras de sangue colhidas de animais em duas mesorregiões de Santa Catarina (Planalto Serrano e Vale do Itajaí). A ocorrência de anticorpos contra Rickettsia spp. observada em equinos de duas mesorregiões de Santa Catarina foi de 18,66%, ocorrendo reações cruzadas em todas as amostras positivas para, no mínimo, duas das espécies testadas. Isoladamente, de acordo com as espécies, 25 (16,66%) amostras foram positivas para R. rickettsii, 15 (10%) para R. parkeri, 22 (14,66%) para R. amblyommii, 23 (15,33%) para R. rhipicephali, 16 (10,66%) para R. bellii e 19 (12,66%) para R. felis. Somente dois animais resultaram em um sorodiagnóstico conclusivo, um para Rickettsia bellii e outro para R. rickettsii, nas diluições máximas de 1:4096 e 1:512, respectivamente. A ocorrência de anticorpos contra Rickettsia spp. em equinos de duas mesorregiões de Santa Catarina indica a circulação de agentes da FMB nestes animais sentinela e ratificam a importância do estudo da febre maculosa no estado de Santa Catarina.

Antibodies against rickettsiae from spotted fever groups in horses from two mesoregions in the state of Santa Catarina, Brazil / Anticorpos contra rickettsias do grupo da febre maculosa em equinos de duas mesorregiões de Santa Catarina, Brasil

Bacteria of the Rickettsia genus are agents of Brazilian Spotted Fever (BSF), a zoonotic disease which is difficult to diagnose, evolves quickly and can result in death. Antibodies against Rickettsia spp. in horses were studied, by means of Indirect Immunofluorescence Assay (IFAT ≥64), in 150 blood samples taken from animals in two Santa Catarina mesoregions (Planalto Serrano and Vale do Itajaí). The overall occurrence of Rickettsia spp. antibodies in horses was 18.66%, with cross-reactivity occurring in all positive samples for at least two of the species tested. Separately, according to the species, 25 (16.66%) samples were positive for R. rickettsii, 15 (10%) for R. parkeri, 22 (14.66%) for R. amblyommii, 23 (15.33%) for R. rhipicephali, 16 (10.66%) for R. bellii and 19 (12.66%) for R. felis. Only two animals resulted in a conclusive serodiagnosis, one for R. bellii and the other for R. rickettsii, at maximum dilutions of 1:4096 and 1:512, respectively. The occurrence of antibodies against Rickettsia spp. in horses from two mesoregions in the state of Santa Catarina indicates the movement of BSF agents in these sentinel animals and confirms the importance of studying spotted fever in the state of Santa Catarina.(AU)

Bactérias do gênero Rickettsia são agentes da Febre Maculosa Brasileira (FMB), uma doença zoonótica, de difícil diagnóstico, rápida evolução e que pode levar o indivíduo à morte. Anticorpos contra Rickettsia spp. em equinos foram pesquisados, por meio da Reação de Imunofluorescência Indireta (RIFI≥64), em 150 amostras de sangue colhidas de animais em duas mesorregiões de Santa Catarina (Planalto Serrano e Vale do Itajaí). A ocorrência de anticorpos contra Rickettsia spp. observada em equinos de duas mesorregiões de Santa Catarina foi de 18,66%, ocorrendo reações cruzadas em todas as amostras positivas para, no mínimo, duas das espécies testadas. Isoladamente, de acordo com as espécies, 25 (16,66%) amostras foram positivas para R. rickettsii, 15 (10%) para R. parkeri, 22 (14,66%) para R. amblyommii, 23 (15,33%) para R. rhipicephali, 16 (10,66%) para R. bellii e 19 (12,66%) para R. felis. Somente dois animais resultaram em um sorodiagnóstico conclusivo, um para Rickettsia bellii e outro para R. rickettsii, nas diluições máximas de 1:4096 e 1:512, respectivamente. A ocorrência de anticorpos contra Rickettsia spp. em equinos de duas mesorregiões de Santa Catarina indica a circulação de agentes da FMB nestes animais sentinela e ratificam a importância do estudo da febre maculosa no estado de Santa Catarina.(AU)