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.

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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.

Phylogenetic studies on trypanosomatid barcode using V7V8 SSU rRNA and gGAPDH gene sequences have provided support for redefining some trypanosomatid species and positioning new isolates. The genus Leishmania is a slow evolving monophyletic group and including important human pathogens. The phylogenetic relationships of this genus have been determined by the natural history of its vertebrate hosts, vector specificity, clinical manifestations, geographical distribution and molecular approaches using different markers. Thus, in an attempt to better understand the phylogenetic relationships of Leishmania species, we performed phylogenetic analysis on trypanosomatid barcode using V7V8 SSU rRNA and gGAPDH gene sequences among a large number of Leishmania species and also several Brazilian visceral Leishmania infantum chagasi isolates obtained from dogs and humans. Our phylogenetic analysis strongly suggested that Leishmania hertigi and Leishmania equatoriensis should be taxonomically revised so as to include them in the genus Endotrypanum; and supported ancient divergence of Leishmania enriettii. This, together with recent data in the literature, throws light on the discussion about the evolutionary southern supercontinent hypothesis for the origin of Leishmania ssp. and validates L. infantum chagasi from Brazil, thus clearly differentiating it from L. infantum, for the first time.

As opposed to unfed ticks, transmission of R. rickettsii occurred in <10 minutes of attachment by adult fed ticks.Feeding by A. aureolatum and Transmission of R. rickettsii

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During 2009-2012, wild animals and their ticks were sampled in two areas within the Amazon biome of Brazil, in the states of Mato Grosso and Pará. Animal tissues, blood, and ticks were molecularly tested for Rickettsia and Coxiella DNA. A total of 182 wild animals were sampled, comprising 28 mammalian, five avian, and three reptilian species. Animal tissues or blood were all negative for Rickettsia or Coxiella DNA. A total of 454 ticks (22 larvae, 226 nymphs, 127 males, 79 females) were collected from 52 (28.6%) animals, and identified into 15 species: Amblyomma cajennense, A. naponense, A. humerale, A. nodosum, A. goeldii, A. oblongoguttatum, A. longirostre, A. calcaratum, A. coelebs, A. pacae, A. geayii, A. rotundatum, A. auricularium, A. ovale, and Haemaphysalis juxtakochi. While no Coxiella DNA was identified in ticks, six Rickettsia species were detected in the ticks. "Candidatus Rickettsia amblyommii" was the most common agent, detected in four tick species, A. cajennense, A. auricularium, A. longirostre, and A. humerale. The second most common agent, R. bellii, was detected in A. humerale and A. naponense. Rickettsia rhipicephali was detected in H. juxtakochi, and R. felis in A. humerale. Two possible new Rickettsia species were detected in A. naponense ticks, namely, a novel spotted fever group agent close-related to R. africae in Pará, and a novel Canadensis group agent in Mato Grosso. Results of the present study expand our knowledge on the tick fauna, and on the yet infantile knowledge of tick-borne rickettsiae in the Amazon biome.

This study investigated rickettsial infection in Amblyomma auricularium ticks from the state of Pernambuco, northeastern Brazil. An engorged female of A. auricularium collected from a skunk (Conepatus semistriatus) was sent alive to the laboratory, where the female was found through molecular analysis to be infected by Rickettsia amblyommii. This engorged female oviposited, and its offspring was reared through three consecutive generations, always using tick-naïve rabbits to feed the ticks. PCR performed on five egg pools, 10 larvae, 10 nymphs, and 10 adults of each of the three generations always yielded rickettsial DNA, indicating maintenance of rickettsial infection in the ticks by transstadial and transovarial passages. DNA sequences of random PCR products from eggs, larvae, nymphs, and adults were identified as R. amblyommii. All infested rabbits seroconverted to R. amblyommii antigens at the 21(st) day after infestation, indicating that larvae, nymphs, and adults transmitted R. amblyommii through parasitism. However, no infested rabbit presented fever or any clinical alteration during the experimental period. Rickettsiae were successfully isolated from the two A. auricularium females, and the isolates were established in Vero cell culture. Molecular characterization of the isolates confirmed R. amblyommii by sequencing partial gltA, ompA, and ompB genes. From another sample of 15 A. auricularium adult ticks collected from two armadillos (Euphractus sexcinctus), eight (53.3%) were infected by R. amblyommii. This study reports R. amblyommii infecting the tick A. auricularium for the first time. This is also the first report of rickettsia infecting ticks in the northeastern region of Brazil.