Life cycle of Ixodes schulzei (Acari: Ixodidae) in the laboratory, and demonstration of reproduction by parthenogenesis.

Ixodes schulzei is an ixodid tick that parasitizes Cricetidae rodents, chiefly the South American water rat, Nectomys squamipes, in Brazil and Argentina. In the present study, we evaluated the life cycle of I. schulzei by exposing larvae and nymphs to feed on two rodent species, N. squamipes and Calomys callosus (large vesper mouse),while adult ticks were exposed to feed on N. squamipes. Off-host developmental periods were observed in an incubator at 27 °C, 95% relative humidity, and 0:24 (light:dark) regimen. Larvae and nymphs successfully fed on either C. callosus or N. squamipes. Mean larval and nymphal feeding periods were 8.8 and 8.7 days on N. squamipes and 8.5 and 9.7 days on C. callosus. The majority of engorged larvae (79.0-80.8%) and nymphs (67.0-86.0%) successfully molted to nymphs and adults, respectively. Mean premolt periods were 11.5-11.7 days for engorged larvae and 22.5-23.7 days for engorged nymphs. Only adult females emerged from engorged nymphs, regardless of host species, i.e., none of 120 engorged nymphs molted to male. Around 18% of the unfed females presented teratologies compatible with the metagynander type of gynandromorphism. Ixodes schulzei adult females successfully fed (mean feeding period, 9.4 days), oviposited, and presented high reproductive performance (high engorged weight, egg mass weight, and % egg mass hatching), in the absence of male ticks. Our results showed that I. schulzei successfully reproduces by parthenogenesis, and corroborate field data that indicate N. squamipes as the most important host for this tick species. The male of I. schulzei remains unknown.

Survey of ticks and tick-borne agents in maned wolves (Chrysocyon brachyurus) from a natural landscape in Brazil.

This study evaluated ticks and tick-borne agents in 104 captures of the maned wolf Chrysocyon brachyurus (50 different individuals and 54 recaptures) in the Serra da Canastra National Park (SCNP), a Cerrado preserved area in southeastern Brazil, from 2005 to 2012. From the 104 capture events, a total of 1,206 ticks were collected on 94 occasions (90.4 %), and identified into five species: Amblyomma tigrinum (77.3 % of all collected ticks), Amblyomma sculptum (16.6 %), Amblyomma ovale (0.1 %), Amblyomma brasiliense (0.1 %), Rhipicephalus microplus (0.1 %), and Amblyomma spp. larvae (5.8 %). Molecular analyses of A. tigrinum adult ticks revealed the presence of 'Candidatus Rickettsia andeanae', Rickettsia parkeri sensu stricto, two different haplotypes of 'Ca. Midichloria sp.', and a Hepatozoon canis haplotype. Molecular analyses of maned wolf blood samples revealed two distinct haplotypes of Hepatozoon spp., one identical to the H. canis genotype that was detected in the A. tigrinum ticks, and a Hepatozoon americanum-like haplotype. None tick or blood samples yielded amplicons through PCR assays targeting the genera Ehrlichia, Anaplasma, Babesia, Rangelia, Cytauxzoon, and Theileria. Maned wolf serum samples were tested by immunofluorescence assay against antigens of five Rickettsia species (R. parkeri, R. rickettsii, R. amblyommatis, R. rhipicephali, and R. bellii) and Ehrlichia canis. Among 78 serum samples (45 captures plus 33 recaptures), 74 (95 %) were reactive to at least one Rickettsia species, with R. parkeri eliciting the highest endpoint titers. Some maned wolves that were recaptured during the study were shown to seroconvert to R. parkeri. Serum-reactiveness to E. canis was detected in 36 % (16/45) maned wolves. During the study, general clinical signs of tick-borne diseases were not found in any of the captured animals, indicating that they were under a good health status in the SCNP, despite of been exposed to ticks (mostly A. tigrinum) and some tick-borne agents (Rickettsia, Hepatozoon, Ehrlichia). The results of the present study might represent baseline data for the conservation of the maned wolf in its natural habitat, which should be used to interpret further studies about ticks and tick-borne diseases in maned wolves within human-modified landscapes.

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.

Exploring Leishmania infantum cathepsin as a new molecular marker for phylogenetic relationships and visceral leishmaniasis diagnosis.

BACKGROUND: Leishmania infantum, the etiological agent of visceral leishmaniasis, is a neglected zoonosis that requires validation and standardization of satisfactory diagnostic methodologies. Thus, the aim of the present study was to evaluate the effectiveness of cathepsin L-like protease as a target for making molecular diagnoses and as a phylogenetic marker enabling to understand the intraspecies variations and evolutionary history of L. infantum in Brazil. METHODS: We used 44 isolates of L. infantum. The cathepsin L-like gene fragments were amplified, sequenced, manually aligned and analyzed using inference methods. The sequences generated were used to search and design oligonucleotide primers to be used in reactions specific to the target parasite. RESULTS: The cathepsin L-like gene did not show any intraspecies variability among the isolates analyzed. The pair of primers proposed amplified the target deoxyribonucleic acid (DNA) of L. infantum isolates and were effective for DNA amplification at concentrations of as low as 10- 11 ng/µl. The proposed marker did not present cross-reactions with other hemoparasites. When used for making the diagnosis in a panel of clinical samples from dogs, a positivity rate of 49.03% (102/208) was obtained, versus 14.42% (30/208) for a ribosomal internal transcribed spacer (ITS) marker. In samples from sandflies, the rate was 6.25% and from humans, 14.28%. CONCLUSIONS: The results described in this work allow us to infer that CatLeish-PCR is a sensitive and specific marker for use in diagnostic trials of L. infantum and in clinical and epidemiological surveys.

Occurrence of autogeny in a population of Ornithodoros fonsecai (Acari: Argasidae).

Ornithodoros fonsecai is an argasid tick that is endemic to Brazil and has been described in the municipality of Bonito, state of Mato Grosso do Sul. Some specimens of this species were found in a cave in the municipality of Nobres, state of Mato Grosso. The specific identification of this population was confirmed by means of morphology and molecular biology. The mitochondrial 16S rDNA partial sequence of this species from Nobres has been deposited in GenBank (MK158949). The objective of this study was to elucidate the biology of O. fonsecai from Nobres, and to report autogeny in this tick population. Along three laboratory generations was observed molting of first nymphal instar to the second instar without feeding, a typical behavior of species included in the subgenus Alectorobius. The first generation (F1) presented five nymphal instars (N1 to N5), and most of adults emerged through molting of N5. The last nymphal instar of second generation (F2) was N4, but most of adults emerged from N3. In the third generation (F3) the last nymphal instar was N5, with most of the adults emerging from N4. In F2, some females (n = 20) originated from N3 began laying eggs without a blood meal. It was observed that those N3 fed twice before they molted to autogenic females. However, autogenic behavior occurred in relation to third generation females (F3) with specimens originating from N4 (n = 12) that were fed only once as nymphs. This behavior has already been reported as obligatory for the genera Otobius and Antricola, while it is facultative for one species of genus Argas and for four species of genus Ornithodoros. However, the present report provides the first record of facultative autogeny for a species of Ornithodoros in Brazil.

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)

Occurrence of autogeny in a population of Ornithodoros fonsecai (Acari: Argasidae)

Ornithodoros fonsecai is an argasid tick that is endemic to Brazil and has been described in the municipality of Bonito, state of Mato Grosso do Sul. Some specimens of this species were found in a cave in the municipality of Nobres, state of Mato Grosso. The specific identification of this population was confirmed by means of morphology and molecular biology. The mitochondrial 16S rDNA partial sequence of this species from Nobres has been deposited in GenBank (MK158949). The objective of this study was to elucidate the biology of O. fonsecai from Nobres, and to report autogeny in this tick population. Along three laboratory generations was observed molting of first nymphal instar to the second instar without feeding, a typical behavior of species included in the subgenus Alectorobius. The first generation (F1) presented five nymphal instars (N1 to N5), and most of adults emerged through molting of N5. The last nymphal instar of second generation (F2) was N4, but most of adults emerged from N3. In the third generation (F3) the last nymphal instar was N5, with most of the adults emerging from N4. In F2, some females (n?=?20) originated from N3 began laying eggs without a blood meal. It was observed that those N3 fed twice before they molted to autogenic females. However, autogenic behavior occurred in relation to third generation females (F3) with specimens originating from N4 (n?=?12) that were fed only once as nymphs. This behavior has already been reported as obligatory for the genera Otobius and Antricola, while it is facultative for one species of genus Argas and for four species of genus Ornithodoros. However, the present report provides the first record of facultative autogeny for a species of Ornithodoros in Brazil.

Occurrence of autogeny in a population of Ornithodoros fonsecai (Acari: Argasidae)

Ornithodoros fonsecai is an argasid tick that is endemic to Brazil and has been described in the municipality of Bonito, state of Mato Grosso do Sul. Some specimens of this species were found in a cave in the municipality of Nobres, state of Mato Grosso. The specific identification of this population was confirmed by means of morphology and molecular biology. The mitochondrial 16S rDNA partial sequence of this species from Nobres has been deposited in GenBank (MK158949). The objective of this study was to elucidate the biology of O. fonsecai from Nobres, and to report autogeny in this tick population. Along three laboratory generations was observed molting of first nymphal instar to the second instar without feeding, a typical behavior of species included in the subgenus Alectorobius. The first generation (F1) presented five nymphal instars (N1 to N5), and most of adults emerged through molting of N5. The last nymphal instar of second generation (F2) was N4, but most of adults emerged from N3. In the third generation (F3) the last nymphal instar was N5, with most of the adults emerging from N4. In F2, some females (n?=?20) originated from N3 began laying eggs without a blood meal. It was observed that those N3 fed twice before they molted to autogenic females. However, autogenic behavior occurred in relation to third generation females (F3) with specimens originating from N4 (n?=?12) that were fed only once as nymphs. This behavior has already been reported as obligatory for the genera Otobius and Antricola, while it is facultative for one species of genus Argas and for four species of genus Ornithodoros. However, the present report provides the first record of facultative autogeny for a species of Ornithodoros in Brazil.

First report of African tick-bite fever in a South American traveler.

We report a clinical case of African tick-bite fever in a Brazilian traveler right after his return from South Africa. Definitive diagnosis was supported by seroconversion between acute-phase and convalescent-phase serum samples, detection of rickettsial DNA in skin lesions, and in vitro culture of Rickettsia africae from the patient's skin. Most of the previous reported cases of African tick-bite fever were confirmed solely by serological or/and molecular methods. Through this first confirmed case of African tick-bite fever in Brazil, it is quite possible that other cases are occurring unnoticed by the health authorities, requiring a greater vigilance in traveler's medicine in South America.

Phylogenetic Evidence for the Existence of Multiple Strains of Rickettsia parkeri in the New World.

The bacterium Rickettsia parkeri has been reported to infect ticks of the "Amblyomma maculatum species complex" in the New World, where it causes spotted fever illness in humans. In South America, three additional rickettsial strains, namely, Atlantic rainforest, NOD, and Parvitarsum, have been isolated from the ticks Amblyomma ovale, Amblyomma nodosum, and Amblyomma parvitarsum, respectively. These three strains are phylogenetically closely related to R. parkeri, Rickettsia africae, and Rickettsia sibirica Herein, we performed a robust phylogenetic analysis encompassing 5 genes (gltA, ompA, virB4, dnaA, and dnaK) and 3 intergenic spacers (mppE-pur, rrl-rrf-ITS, and rpmE-tRNAfMet) from 41 rickettsial isolates, including different isolates of R. parkeri, R. africae, R. sibirica, Rickettsia conorii, and strains Atlantic rainforest, NOD, and Parvitarsum. In our phylogenetic analyses, all New World isolates grouped in a major clade distinct from the Old World Rickettsia species (R. conorii, R. sibirica, and R. africae). This New World clade was subdivided into the following 4 clades: the R. parkerisensu stricto clade, comprising the type strain Maculatum 20 and all other isolates of R. parkeri from North and South America, associated with ticks of the A. maculatum species complex; the strain NOD clade, comprising two South American isolates from A. nodosum ticks; the Parvitarsum clade, comprising two South American isolates from A. parvitarsum ticks; and the strain Atlantic rainforest clade, comprising six South American isolates from the A. ovale species complex (A. ovale or Amblyomma aureolatum). Under such evidences, we propose that strains Atlantic rainforest, NOD, and Parvitarsum are South American strains of R. parkeriIMPORTANCE Since the description of Rickettsia parkeri infecting ticks of the "Amblyomma maculatum species complex" and humans in the New World, three novel phylogenetic close-related rickettsial isolates were reported in South America. Herein, we provide genetic evidence that these novel isolates, namely, strains Atlantic rainforest, NOD, and Parvitarsum, are South American strains of R. parkeri. Interestingly, each of these R. parkeri strains seems to be primarily associated with a tick species group, namely, R. parkerisensu stricto with the "Amblyomma maculatum species group," R. parkeri strain NOD with Amblyomma nodosum, R. parkeri strain Parvitarsum with Amblyomma parvitarsum, and R. parkeri strain Atlantic rainforest with the "Amblyomma ovale species group." Such rickettsial strain-tick species specificity suggests a coevolution of each tick-strain association. Finally, because R. parkerisensu stricto and R. parkeri strain Atlantic rainforest are human pathogens, the potential of R. parkeri strains NOD and Parvitarsum to be human pathogens cannot be discarded.

Ehrlichia canis and Rickettsia spp. in dogs from urban areas in Paraiba state, northeastern Brazil

Abstract The aims of our study was to identify Ehrlichia canis and antibodies against Rickettsia spp. belonging to the spotted fever group (SFG) in dogs sampled from Paraiba state, northeastern Brazil. Blood and serum samples collected by convenience from dogs in urban areas of five municipalities were analyzed by real-time PCR for the detection of E. canis DNA and by immunofluorescence assay test (IFAT) for the identification of antibodies against Rickettsia rickettsii, R. felis, R. parkeri, R. amblyommii and R. rhipicephali antigens. E. canis DNA was detected in 8.9% (64/719) of the blood samples, whereas 5.63% (43/763) of the serum samples were positive for at least one of the Rickettsia antigens tested by IFAT. This study showed for the first time the occurrence of E. canis and suggested the circulation of SFG Rickettsia in dogs in the study region of Paraiba state, northeastern Brazil.

Resumo Os objetivos do nosso estudo foram identificar Ehrlichia canis e anticorpos contra Rickettsia spp. pertencentes ao Grupo da Febre Maculosa (GFM) em cães amostrados no estado da Paraíba, nordeste do Brasil. As amostras de sangue e soro, coletados por conveniência, de cães em áreas urbanas de cinco municípios foram analisadas por PCR em tempo real para a detecção de DNA de E. canis e pela Reação de Imunofluorescência Indireta (RIFI) para identificação de anticorpos contra Rickettsia rickettsii, R. felis, R. parkeri, R. amblyommii e R. rhipicephali. O DNA de E. canis foi detectado em 8,9% (64/719) das amostras de sangue, enquanto que 5,63% (43/763) das amostras de soro foram positivas para pelo menos um dos antígenos de Rickettsia testados por RIFI. Este estudo mostrou pela primeira vez a ocorrência de E. canis e sugere a circulação de Rickettsia do GFM em cães na região em estudo do estado da Paraíba, Nordeste do Brasil.

Isolation of the Pathogen Rickettsia sp. Strain Atlantic Rainforest From Its Presumed Tick Vector, Amblyomma ovale (Acari: Ixodidae), From Two Areas of Brazil.

In this study, Amblyomma ovale Koch ticks were collected from domestic dogs in two localities of the Atlantic rainforest biome of Brazil: 1) the Paty Valley of the Chapada Diamantina National Park, Bahia state (northeastern Brazil), and 2) Adrianópolis, Paraná state (southern Brazil). Ticks were screened for the presence of Rickettsia-like structures by the hemolymph test with Giménez staining, and then processed for isolation of rickettsiae in Vero cell culture by the shell-vial technique. Rickettsiae were isolated from one A. ovale tick of each of the two localities. The two isolates were successfully established in the laboratory with several passages, each one reaching >90% infection of the cells. The two isolates were identified as the spotted fever group (SFG) agent Rickettsia sp. strain Atlantic rainforest, as their gltA (350 bp), ompB (781 bp), and ompA (567 bp) gene fragments were 100% equal to GenBank corresponding sequences of the original strain Atlantic rainforest, reported to be infecting a human in southeastern Brazil, and also 100% equal to the available ompA sequence of strain Bahia, reported to be infecting a human in Paty Valley, the same area of the present study in Bahia state. Ten dogs from Paty Valley were serologically tested against rickettsial antigens by the indirect immunofluorescence antibody test. At least 60% of them were seroreactive to SFG rickettsiae. The role of A. ovale as vector of Rickettsia sp. strain Atlantic rainforest in the Paty Valley area, as well as in other parts of Latin America, is discussed.

A novel spotted fever group Rickettsia infecting Amblyomma parvitarsum (Acari: Ixodidae) in highlands of Argentina and Chile.

The tick Amblyomma parvitarsum (Acari: Ixodidae) has established populations in Andean and Patagonic environments of South America. For the present study, adults of A. parvitarsum were collected in highland areas (elevation >3500 m) of Argentina and Chile during 2009-2013, and tested by PCR for rickettsial infection in the laboratory, and isolation of rickettsiae in Vero cell culture by the shell vial technique. Overall, 51 (62.2%) out of 82 A. parvitarsum adult ticks were infected by spotted fever group (SFG) rickettsiae, which generated DNA sequences 100% identical to each other, and when submitted to BLAST analysis, they were 99.3% identical to corresponding sequence of the ompA gene of Rickettsia sp. strain Atlantic rainforest. Rickettsiae were successfully isolated in Vero cell culture from two ticks, one from Argentina and one from Chile. DNA extracted from the third passage of the isolates of Argentina and Chile were processed by PCR, resulting in partial sequences for three rickettsial genes (gltA, ompB, ompA). These sequences were concatenated and aligned with rickettsial corresponding sequences available in GenBank. Phylogenetic analysis revealed that the A. pavitarsum rickettsial agent grouped under high bootstrap support in a clade composed by the SFG pathogens R. sibirica, R. africae, R. parkeri, Rickettsia sp. strain Atlantic rainforest, and two unnamed SFG agents of unknown pathogenicty, Rickettsia sp. strain NOD, and Rickettsia sp. strain ApPR. The pathogenic role of this A. parvitarsum rickettsia cannot be discarded, since several species of tick-borne rickettsiae that were considered nonpathogenic for decades are now associated with human infections.

Toxoplasma gondii antibodies in wild rodents and marsupials from the Atlantic Forest, state of São Paulo, Brazil.

Toxoplasma gondii is a protozoan parasite that infects a large spectrum of warm-blooded animals, including humans. Small rodents and marsupials play an important role in the epidemiology of T. gondii because they are sources of infection for domestic and feral cats. Serum samples from 151 rodents and 48 marsupials, captured in the Atlantic Forest, São Paulo State, southeastern Brazil, were analyzed for the presence of T. gondii antibodies. Antibodies detected by the modified agglutination test (MAT ≥ 25) were found in 8.6% (13/151) of the rodents and 10.4% (5/48) of the marsupials, with titers ranging from 25 to 6400 and from 25 to 3200, respectively for the rodents and marsupials. Three of the eight species of rodents (Akodon spp., Oligoryzomys nigripesand Rattus norvegicus), and one from the four marsupial species (Didelphis aurita) presented positive animals. T. gondii was described for the first time in the rodent Oligoryzomys nigripes.

Babesia canis vogeli infection in dogs and ticks in the semiarid region of Pernambuco, Brazil / Infecção por Babesia canis vogeli em cães e carrapatos de uma região semiárida de Pernambuco

This study aimed to report the prevalence of Babesia canis vogeli in dogs and ticks in the urban and rural areas of Petrolina, Pernambuco. Serum and peripheral blood samples of 404 dogs were tested by indirect immunofluorescence assay (IFA) and by blood smears, respectively. The presence of tick infestation was evaluated, and some specimens were submitted to DNA amplification by polymerase chain reaction (PCR). The presence of antibodies anti-B. canis vogeli was determinate in 57.9% (234/404) of dogs. The direct detection of Babesia spp was obtained in 0.5% (2/404) dogs by visualization of intraerythrocytic forms. Infestation by Rhipicephalus sanguineus sensu lato was observed in 54.5% (220/404) of dogs in both urban and rural areas. DNA of Babesia canis vogeli were obtained by PCR in 6% individual (3/50) and 8.7% of pool of ticks (7/80). The risk factors for the presence of anti-B. canis vogeli antibodies, as determined through the application of logistic regression models (P<0.05), were the following: medium breed size variables (P<0.001); contact with areas of forest (P=0.021); and access on the street (P=0.046). This study describes, for the first time, the confirmation of infection of B. canis vogeli in dogs and ticks in the semiarid region of Pernambuco, Brazil.

Este trabalho objetivou avaliar a prevalência de Babesia canis vogeli em cães e carrapatos de áreas urbanas e rurais do município de Petrolina, Pernambuco, Nordeste do Brasil. Amostras de soro e sangue periférico de 404 cães foram testadas pela Reação de Imunoflorescência Indireta (RIFI), e por esfregaço sanguíneo. A presença de infestação por carrapatos foi avaliada, e alguns espécimes foram submetidos à amplificação do DNA pela Reação em Cadeia pela Polimerase (PCR). A presença de anticorpos anti-B. canis vogeli foi determinada em 57,9% (234/404) dos cães. A soroprevalência em áreas urbanas e rurais foi 48,5% e 67,3%, respectivamente. A detecção direta de Babesia spp foi obtida em 0,5% dos cães pela visualização de formas intraeritrocitárias. A infestação pelo carrapato Rhipicephalus sanguineus foi observada em 54,5% (220/404) dos cães. DNA de Babesia canis vogeli obtido pela PCR foi 6% (3/50) em carrapatos processados individualmente e 8,7% (7/80) em pools. Os fatores de risco para presença de anticorpos anti- B. canis vogeli utilizando modelo de regressão logística (P < 0,05) foram porte médio (P <0,001), contato com áreas de floresta (P = 0,021), e acesso dos cães à rua (P = 0,046). Este estudo descreve pela primeira vez a confirmação da infecção de Babesia canis infectando cães e carrapatos em uma região semiárida de Pernambuco, Brasil.

Rickettsia parkeri infecting free-living Amblyomma triste ticks in the Brazilian Pantanal.

The present study evaluated the infection of rickettsiae in 151 Rhipicephalus sanguineus, 59 Amblyomma ovale, 166 Amblyomma triste, one Amblyomma dissimile and four Amblyomma dubitatum ticks collected in the municipality of Poconé, State of Mato Grosso, within the Pantanal biome of Brazil. Ticks were individually processed by the hemolymph test with Gimenez staining, isolation of rickettsia in Vero cell culture by the shell vial technique, and polymerase chain reaction (PCR) targeting the citrate synthase rickettsial gene. Through the shell vial technique, rickettsiae were successfully isolated and established in Vero cell culture from one free-living A. triste female tick, which previously showed to contain Rickettsia-like organisms by the hemolymph test. Molecular characterization of the rickettsial isolate was achieved through DNA partial sequences of three rickettsial genes (gltA, ompA, ompB), which showed to be all 100% identical to Rickettsia parkeri. After testing all ticks by PCR, the frequency of R. parkeri infection was 7.23% (12/166) in A. triste adult ticks. The remaining ticks were negative by PCR. This is the first report of in vitro isolation of R. parkeri in the Pantanal biome, confirming the occurrence of this emerging rickettsial pathogen in this natural area of South America.

Toxoplasma gondii antibodies in wild rodents and marsupials from the Atlantic Forest, state of São Paulo, Brazil

Toxoplasma gondii is a protozoan parasite that infects a large spectrum of warm-blooded animals, including humans. Small rodents and marsupials play an important role in the epidemiology of T. gondii because they are sources of infection for domestic and feral cats. Serum samples from 151 rodents and 48 marsupials, captured in the Atlantic Forest, São Paulo State, southeastern Brazil, were analyzed for the presence of T. gondii antibodies. Antibodies detected by the modified agglutination test (MAT 25) were found in 8.6% (13/151) of the rodents and 10.4% (5/48) of the marsupials, with titers ranging from 25 to 6400 and from 25 to 3200, respectively for the rodents and marsupials. Three of the eight species of rodents (Akodon spp., Oligoryzomys nigripesand Rattus norvegicus), and one from the four marsupial species (Didelphis aurita) presented positive animals. T. gondii was described for the first time in the rodent Oligoryzomys nigripes.

Toxoplasma gondii é um protozoário parasita que infecta animais de sangue quente, incluindo seres humanos. Pequenos roedores e marsupiais têm papel importante na epidemiologia do T. gondii, pois são fontes de infecção para os felídeos domésticos e selvagens. Amostras de soro de 151 roedores e 48 marsupiais, capturados na Mata Atlântica, Estado de São Paulo, Sudeste do Brasil, foram analisadas para a pesquisa de anticorpos anti-T. gondii. Os anticorpos foram detectados pelo Teste de Aglutinação Modificada (MAT 25), com 8,6% (13/151) dos roedores e 10,4% (5/48) dos marsupiais soropositivos, com títulos variando de 25 a 6.400 e de 25 a 3.200, respectivamente, para os roedores e os marsupiais. Três das oito espécies de roedores (Akodon spp., Oligoryzomys nigripes e Rattus norvegicus) e uma das quatro espécies de marsupiais (Didelphis aurita) apresentaram animais positivos. A presença de anticorpos anti-T. gondii foi descrita pela primeira vez no roedor Oligoryzomys nigripes.

Epidemiology of Rickettsia sp. strain Atlantic rainforest in a spotted fever-endemic area of southern Brazil.

The present study was performed in Vila Itoupava, an area of the state of Santa Catarina, southern Brazil, in which a tick-borne spotted fever illness has been endemic since 2003. Notably, both the etiological agent and the vector of these spotted fever cases remain unknown. During January 2011, humans, domestic dogs, and their ticks were sampled in households that are typically surrounded by highly preserved Atlantic rainforest fragments. Ticks collected from dogs were Amblyomma ovale (34% prevalence), Amblyomma aureolatum (18.9%), and Rhipicephalus sanguineus (3.8%). A total of 7.8% (6/77) A. ovale and 9.3% (4/43) A. aureolatum were infected by Rickettsia sp. strain Atlantic rainforest, a Rickettsia parkeri-like agent recently shown to cause spotted fever illness in southeastern Brazil. Overall, 67.3% (35/52) of the dogs were seroreactive to spotted fever group rickettsiae, mostly with highest endpoint titers to R. parkeri. Among humans, 46.7% (7/15) reacted serologically to rickettsiae at low to moderate endpoint titers. Because canine seroreactivity to R. parkeri was strongly associated with frequent contact with forests (the preferred habitat for A. ovale and A. aureolatum), it is concluded that sampled dogs have been infected by strain Atlantic rainforest through the parasitism of these tick species. The present study provides epidemiological evidence that the spotted fever in the study area has been caused by Rickettsia sp. strain Atlantic rainforest, transmitted to humans by either A. ovale or A. aureolatum. Further studies encompassing direct diagnostic methods on clinical specimens from patients are needed to confirm the above epidemiological evidence.

Candidatus Rickettsia andeanae, a spotted fever group agent infecting Amblyomma parvum ticks in two Brazilian biomes.

Adult ticks of the species Amblyomma parvum were collected from the vegetation in the Pantanal biome (state of Mato Grosso do Sul) and from horses in the Cerrado biome (state of Piauí) in Brazil. The ticks were individually tested for rickettsial infection via polymerase chain reaction (PCR) targeting three rickettsial genes, gltA, ompA and ompB. Overall, 63.5% (40/63) and 66.7% (2/3) of A. parvum ticks from Pantanal and Cerrado, respectively, contained rickettsial DNA, which were all confirmed by DNA sequencing to be 100% identical to the corresponding fragments of the gltA, ompA and ompB genes of Candidatus Rickettsia andeanae. This report is the first to describe Ca. R. andeanae in Brazil.

Rickettsial infection in Amblyomma cajennense ticks and capybaras (Hydrochoerus hydrochaeris) in a Brazilian spotted fever-endemic area.

BACKGROUND: Brazilian spotted fever (BSF), caused by the bacterium Rickettsia rickettsii, is the deadliest spotted fever of the world. In most of the BSF-endemic areas, capybaras (Hydrochoerus hydrochaeris) are the principal host for the tick Amblyomma cajennense, which is the main vector of BSF. METHODS: In 2012, a BSF case was confirmed in a child that was bitten by ticks in a residential park area inhabited by A. cajennense-infested capybaras in Itú municipality, southeastern Brazil. Host questing A. cajennense adult ticks were collected in the residential park and brought alive to the laboratory, where they were macerated and intraperitoneally inoculated into guinea pigs. A tick-inoculated guinea pig that presented high fever was euthanized and its internal organs were macerated and inoculated into additional guinea pigs (guinea pig passage). Tissue samples from guinea pig passages were also used to inoculate Vero cells through the shell vial technique. Infected cells were used for molecular characterization of the rickettsial isolate through PCR and DNA sequencing of fragments of three rickettsial genes (gltA, ompA, and ompB). Blood serum samples were collected from 172 capybaras that inhabited the residential park. Sera were tested through the immunofluorescence assay using R. rickettsii antigen. RESULTS: A tick-inoculated guinea pig presented high fever accompanied by scrotal reactions (edema and marked redness). These signs were reproduced by consecutive guinea pig passages. Rickettsia was successfully isolated in Vero cells that were inoculated with brain homogenate derived from a 3rd passage-febrile guinea pig. Molecular characterization of this rickettsial isolate (designated as strain ITU) yielded DNA sequences that were all 100% identical to corresponding sequences of R. rickettsii in Genbank. A total of 83 (48.3%) out of 172 capybaras were seroreactive to R. rickettsii, with endpoint titers ranging from 64 to 8192. CONCLUSIONS: A viable isolate of R. rickettsii was obtained from the tick A. cajennense, comprising the first viable R. rickettsi isolate from this tick species during the last 60 years. Nearly half of the capybara population of the residential park was seroreactive to R. rickettsii, corroborating the findings that the local A. cajennense population was infected by R. rickettsii.