João F. Soares

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

BackgroundBrazilian 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.MethodsIn 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.ResultsA 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.ConclusionsA 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.

Experimental infection of the tick Amblyomma cajennense, Cayenne tick, with Rickettsia rickettsii, the agent of Rocky Mountain spotted fever.

In the laboratory, Amblyomma cajennense (Acari: Ixodidae) (Fabricius) larvae, nymphs and adults were exposed to Rickettsia rickettsii by feeding on needle‐inoculated animals, and thereafter reared on uninfected guinea pigs or rabbits. Regardless of the tick stage that acquired the infection, subsequent tick stages were shown to be infected (confirming transstadial and transovarial transmissions) and were able to transmit R. rickettsii to uninfected animals, as demonstrated by serological and molecular analyses. However, the larval, nymphal and adult stages of A. cajennense were shown to be partially refractory to R. rickettsii infection, as in all cases, only part of the ticks became infected by this agent, after being exposed to rickettsemic animals. In addition, less than 50% of the infected engorged females transmitted rickettsiae transovarially, and when they did so, only part of the offspring became infected, indicating that vertical transmission alone is not enough to maintain R. rickettsii in A. cajennense for multiple generations. Finally, the R. rickettsii‐infected tick groups had lower reproductive performance than the uninfected control group. Our results indicate that A. cajennense have a low efficiency to maintain R. rickettsii for successive generations, as R. rickettsii‐infection rates should decline drastically throughout the successive tick generations.

Experimental infection of Amblyomma aureolatum ticks with Rickettsia rickettsii.

We experimentally infected Amblyomma aureolatum ticks with the bacterium Rickettsia rickettsii, the etiologic agent of Rocky Mountain spotted fever (RMSF). These ticks are a vector for RMSF in Brazil. R. rickettsii was efficiently conserved by both transstadial maintenance and vertical (transovarial) transmission to 100% of the ticks through 4 laboratory generations. However, lower reproductive performance and survival of infected females was attributed to R. rickettsii infection. Therefore, because of the high susceptibility of A. aureolatum ticks to R. rickettsii infection, the deleterious effect that the bacterium causes in these ticks may contribute to the low infection rates (<1%) usually reported among field populations of A. aureolatum ticks in RMSF-endemic areas of Brazil. Because the number of infected ticks would gradually decrease after each generation, it seems unlikely that A. aureolatum ticks could sustain R. rickettsii infection over multiple successive generations solely by vertical transmission.

Cross-mating experiments with geographically different populations of Amblyomma cajennense (Acari: Ixodidae).

The present study evaluated the reproductive compatibility of the crosses between adult ticks of the following three geographically different populations of Amblyomma cajennense: State of São Paulo (SP), southeastern Brazil; State of Rondônia (RO), northern Brazil; and Colombia (CO). In addition, crosses between A. cajennense ticks from Argentina (AR) and SP ticks were also performed. The Argentinean population (AR) was compatible with SP because their crosses resulted in high % egg hatching (mean values ranging from 71.5 to 93.5%), similarly to all homologous (intrapopulational) crosses. In contrast, the tick populations SP, RO, and CO were shown to be incompatible with each other, since their heterologous (interpopulational) crosses always resulted in very low % egg hatching (range: 0–5%). The F1 larval offspring derived from some of these females that yielded 5% egg hatching were reared until the F1 adult stage. In all cases, only adult females molted from engorged nymphs. These F1 females were likely to be a product of thelytokous parthenogenesis of the SP, RO, and CO females that were used in the heterologous crosses. Reproductive incompatibility is not expected to occur between different populations of a single species. Thus, our results suggest that the taxon A. cajennense might be represented by a complex of different species, whereas SP and AR ticks might represent a single species. Further populational genetic studies, coupled with extensive morphological analyses, are needed to clarify and determine a possible complex of valid species that might have been classified under the taxon A. cajennense.

Experimental infection with Rangelia vitalii in dogs: Acute phase, parasitemia, biological cycle, clinical-pathological aspects and treatment

Recently we conducted the molecular characterization of Rangelia vitalii, a protozoan with high pathogenicity for young dogs in southern Brazil. To date, the descriptions of the disease have been restricted to natural infection cases. Therefore, this study aimed to evaluate the parasitemia, biological cycles and clinical-pathological findings in dogs experimentally infected with R. vitalii in the acute phase of disease, and also aimed to test a therapeutic protocol based on the diminazene aceturate. For this study, we used 12 young dogs (females), separated into two groups. Group A was composed of healthy dogs, not-infected (n=5), and Group B consisted of animals infected with R. vitalii (n=7). After infection, the animals were monitored by blood smear examinations, which showed intra-erythrocytic forms of the parasite 5 days post-infection (PI). Parasitemia increased progressively in these animals and had the highest peak of circulating parasites between 9 and 11 days PI. Subsequently, the parasitemia reduced and the protozoan was seen inside the leukocytes in days 17, 19 and 21 PI. The most prominent clinical signs observed at the 20 day PI of experiment were lethargy, fever and anorexia. We observed a decrease of hematocrit of infected animals compared with not-infected dogs, featuring a moderate anemia. Pathological evaluation of one dog in Group B at day 21 PI revealed splenomegaly, hepatomegaly, lymphadenopathy, and hemorrhages at necropsy. Histological examination showed only follicular hyperplasia in the spleen and lymph nodes, and the etiologic agent in the vascular endothelium. At 21 days PI, it was performed the treatment of dogs in Group B (n=6) with a single dose of diminazene aceturate, which showed a curative efficacy of 100% in cleaning R. vitalii from blood of infected dogs.

Protective efficacy of bacterial membranes containing surface-exposed BM95 antigenic peptides for the control of cattle tick infestations

The Rhipicephalus (Boophilus) microplus BM86 and BM95 glycoproteins are homologous proteins that protect cattle against tick infestations. In this study, we demonstrated that the recombinant chimeric protein comprising tick BM95 immunogenic peptides fused to the A. marginale MSP1a N-terminal region for presentation on the Escherichia coli membrane was protective against R. microplus infestations in rabbits. This system provides a novel and simple approach for the production of tick protective antigens by surface display of antigenic protein chimera on live E. coli and suggests the possibility of using recombinant bacterial membrane fractions for vaccination against cattle tick infestations.

Experimental infection with Rickettsia rickettsii in an Amblyomma dubitatum tick colony, naturally infected by Rickettsia bellii

Amblyomma dubitatum engorged females, naturally infected by Rickettsia bellii, were used to establish a laboratory colony. Larvae, nymphs, and adults were exposed to two strains of Rickettsia rickettsii by feeding on needle-inoculated guinea pigs, and thereafter reared on uninfected guinea pigs. After acquisition feeding, engorged larvae and nymphs molted to nymphs and adults, respectively, which were shown to be infected (confirming transstadial perpetuation), and were able to transmit both strains of R. rickettsii to uninfected animals, as demonstrated by clinical, serological, and molecular analyses. However, the larval, nymphal, and adult stages of A. dubitatum showed to be only partially susceptible to R. rickettsii infection, since in all cases, only part of the ticks became infected by this agent, after being exposed to rickettsemic animals. While transovarial transmission of R. rickettsii was inefficient in the A. dubitatum engorged females of the present study, 100% of these females passed R. bellii transovarially. Because it has been reported that a primary infection by a Rickettsia species would preclude transovarial transmission of a second Rickettsia species, it is likely that the ineffectiveness of A. dubitatum to perpetuate R. rickettsii by transovarial transmission was related to its primary infection by R. bellii; however, it could also be related to unknown factors inherent to A. dubitatum. The relevance of A. dubitatum as a natural vector of R. rickettsii to humans or animals is discussed.

Natural Blood Feeding and Temperature Shift Modulate the Global Transcriptional Profile of Rickettsia rickettsii Infecting Its Tick Vector

Rickettsia rickettsii is an obligate intracellular tick-borne bacterium that causes Rocky Mountain Spotted Fever (RMSF), the most lethal spotted fever rickettsiosis. When an infected starving tick begins blood feeding from a vertebrate host, R. rickettsii is exposed to a temperature elevation and to components in the blood meal. These two environmental stimuli have been previously associated with the reactivation of rickettsial virulence in ticks, but the factors responsible for this phenotype conversion have not been completely elucidated. Using customized oligonucleotide microarrays and high-throughput microfluidic qRT-PCR, we analyzed the effects of a 10°C temperature elevation and of a blood meal on the transcriptional profile of R. rickettsii infecting the tick Amblyomma aureolatum. This is the first study of the transcriptome of a bacterium in the genus Rickettsia infecting a natural tick vector. Although both stimuli significantly increased bacterial load, blood feeding had a greater effect, modulating five-fold more genes than the temperature upshift. Certain components of the Type IV Secretion System (T4SS) were up-regulated by blood feeding. This suggests that this important bacterial transport system may be utilized to secrete effectors during the tick vector’s blood meal. Blood feeding also up-regulated the expression of antioxidant enzymes, which might correspond to an attempt by R. rickettsii to protect itself against the deleterious effects of free radicals produced by fed ticks. The modulated genes identified in this study, including those encoding hypothetical proteins, require further functional analysis and may have potential as future targets for vaccine development.

Hematologic and bone marrow changes in dogs experimentally infected with Rangelia vitalii.

BACKGROUND Rangelia vitalii is a tick-transmitted piroplasm that causes both hemolytic and hemorrhagic disease in dogs in Brazil. OBJECTIVE The aim of this study was to evaluate the response of the bone marrow in dogs experimentally infected with R vitalii during the acute stage of the disease. METHODS For this study, 2 groups of a total of 12 young dogs were used. Group A was composed of healthy dogs (n = 5), and group B consisted of animals infected with R vitalii (n = 7). Blood samples were collected on days 0, 10, 20, and 30 post-inoculation and stored in EDTA tubes for a full hematology profile, including a reticulocyte count. On days 10 and 20, bone marrow samples were collected, stained, and examined. RESULTS In infected dogs anemia was identified on days 10 and 20 post-inoculation (P < .01), and on day 20 reticulocytosis was present. Infected dogs had leukopenia due to neutropenia and eosinopenia, along with lymphocytosis and monocytosis, when compared with control animals. In bone marrow, the myeloid:erythroid ratio was significantly decreased (P < .05) in infected dogs due to increased erythroid precursors. CONCLUSIONS Dogs experimentally infected with R vitalii develop regenerative extravascular hemolytic anemia accompanied by erythroid hyperplasia in the bone marrow. During the acute phase of the disease, leukopenia due to neutropenia and eosinopenia suggests intense tissue recruitment of these cells in response to the endothelial damage caused by this parasite.

Thrombocytopenia and platelet activity in dogs experimentally infected with Rangelia vitalii

The aim of this study was to evaluate the platelet count, coagulation time and platelet activity in dogs experimentally infected with Rangelia vitalii during the acute phase of the disease. For this study, 12 young dogs (females) were used, separated in two groups. Group A (uninfected control) was composed by healthy dogs (n=5), and group B consisted of R. vitalii-infected animals (n=7). After being inoculated with R. vitalii-infected blood, animals were monitored by blood smear examinations, which showed intra-erythrocytic forms of the parasite five days post-inoculation (PI). Blood samples were collected on days 0, 10, 20 and 30 PI. The material collected was placed in tubes containing EDTA for quantification of platelets, citrate anticoagulant platelet aggregation, and measuring the clotting time. Right after blood collection on days 10 and 20 PI, dogs were anesthetized for collecting bone marrow samples. A significant reduction (P<0.01) of the number of platelets was observed in R. vitalii-infected blood, when compared with uninfected dogs on days 10 and 20 PI. Additionally, macro-platelets were observed only in infected dogs. Prothrombin time and activated partial thromboplastin time did not differ between infected and uninfected dogs. The megakaryocyte count increased (P<0.01) significantly in infected dogs when compared with uninfected ones on days 10 and 20 PI. Platelet aggregation decreased (P<0.01) significantly in infected dogs in comparison to the control on days 10 and 20 PI. Therefore, rangeliosis in dogs causes a severe thrombocytopenia during the acute phase of infection. This platelets reduction probably occurred due to splenic sequestration and/or immune-mediated thrombocytopenia.