Rickettsia rickettsii is a tick-borne obligate intracellular bacterium that causes Rocky Mountain Spotted Fever (RMSF). In Brazil, two species of ticks in the genus Amblyomma, A. sculptum and A. aureolatum, are incriminated as vectors of this bacterium. Importantly, these two species present remarkable differences in susceptibility to R. rickettsii infection, where A. aureolatum is more susceptible than A. sculptum. In the current study, A. aureolatum and A. sculptum ticks were fed on suitable hosts previously inoculated with R. rickettsii, mimicking a natural infection. As control, ticks were fed on non-infected animals. Both midgut and salivary glands of all positively infected ticks were colonized by R. rickettsii. We did not observe ticks with infection restricted to midgut, suggesting that important factors for controlling rickettsial colonization were produced in this organ. In order to identify such factors, the total RNA extracted from the midgut (MG) was submitted to next generation RNA sequencing (RNA-seq). The majority of the coding sequences (CDSs) of A. sculptum differentially expressed by infection were upregulated, whereas most of modulated CDSs of A. aureolatum were downregulated. The functional categories that comprise upregulated CDSs of A. sculptum, for instance, metabolism, signal transduction, protein modification, extracellular matrix, and immunity also include CDSs of A. aureolatum that were downregulated by infection. This is the first study that reports the effects of an experimental infection with the highly virulent R. rickettsii on the gene expression of two natural tick vectors. The distinct transcriptional profiles of MG of A. sculptum and A. aureolatum upon infection stimulus strongly suggest that molecular factors in this organ are responsible for delineating the susceptibility to R. rickettsii. Functional studies to determine the role played by proteins encoded by differentially expressed CDSs in the acquisition of R. rickettsii are warranted and may be considered as targets for the development of strategies to control the tick-borne pathogens as well as to control the tick vectors.

BackgroundRickettsia rickettsii, the etiological agent of Rocky Mountain spotted fever, is transmitted to humans by ticks. During tick feeding, R. rickettsii is exposed to both temperature elevation and components of the blood meal, which have previously been associated with the reactivation of its virulence. These environmental stimuli were also reported to modulate virulence genes of R. rickettsii infecting a set of organs of adult females of its natural vector, Amblyomma aureolatum.MethodsIn this study, we determined the effects of a temperature upshift, blood-feeding, and both stimuli simultaneously on the expression of 85 selected genes of R. rickettsii infecting either the midgut (MG) or salivary glands (SG) of male and female A. aureolatum by microfluidic high-throughput RT-qPCR. These two organs are key for acquisition of this bacterium by the tick and transmission to the vertebrate host, respectively.ResultsData showed that these environmental stimuli exert distinct effects on rickettsial transcription depending on the colonized organ and gender of the vector. Temperature upshift induced the majority of differentially expressed genes of R. rickettsii in tick SG, including tRNA synthetases encoding genes. On the contrary, blood-feeding downregulated most of differentially expressed genes in both organs, but induced type IV secretion system components and OmpB in tick MG. The combined effects of both stimuli resulted in a merged gene expression profile representing features of each stimulus analyzed independently, but was more similar to the profile induced by blood-feeding.ConclusionThe upregulation of the majority of differentially expressed genes in tick SG by temperature upshift suggests that this stimulus is important to prepare R. rickettsii for transmission to the vertebrate host. Blood-feeding, on the other hand, induced important virulence genes in the tick MG, which might be associated with colonization of the tick and transmission to the vertebrate host. The role of the proteins identified in this study must be addressed and might help to define future targets to block tick infection, thereby preventing RMSF. To our knowledge, this is the first transcriptional tissue-specific study of a virulent strain of R. rickettsii infecting a natural tick vector.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1581-7) contains supplementary material, which is available to authorized users.

The salivary glands (SG) of ixodid ticks play a pivotal role in blood feeding, producing both the cement and the saliva. The cement is an adhesive substance that helps the attachment of the tick to the host skin, while the saliva contains a rich mixture of antihemostatic, anti-inflammatory, and immunomodulatory substances that allow ticks to properly acquire the blood meal. The tick saliva is also a vehicle used by several pathogens to be transmitted to the vertebrate host, including various bacterial species from the genus Rickettsia . Rickettsia rickettsii is a tick-borne obligate intracellular bacterium that causes the severe Rocky Mountain spotted fever. In Brazil, the dog yellow tick Amblyomma aureolatum is a vector of R. rickettsii . In the current study, the effects of an experimental infection with R. rickettsii on the global gene expression profile of A. aureolatum SG was determined by next-generation RNA sequencing. A total of 260 coding sequences (CDSs) were modulated by infection, among which 161 were upregulated and 99 were downregulated. Regarding CDSs in the immunity category, we highlight one sequence encoding one microplusin-like antimicrobial peptide (AMP) (Ambaur-69859). AMPs are important effectors of the arthropod immune system, which lack the adaptive response of the immune system of vertebrates. The expression of microplusin was confirmed to be significantly upregulated in the SG as well as in the midgut (MG) of infected A. aureolatum by a quantitative polymerase chain reaction preceded by reverse transcription. The knockdown of the microplusin expression by RNA interference caused a significant increase in the prevalence of infected ticks in relation to the control. In addition, a higher rickettsial load of one order of magnitude was recorded in both the MG and SG of ticks that received microplusin-specific dsRNA. No effect of microplusin knockdown was observed on the R. rickettsii transmission to rabbits. Moreover, no significant differences in tick engorgement and oviposition were recorded in ticks that received dsMicroplusin, demonstrating that microplusin knockdown has no effect on tick fitness. Further studies must be performed to determine the mechanism of action of this AMP against R. rickettsii .

Título da Tese: Efeitos da temperatura e da alimentação sanguínea sobre o perfil de expressão gênica de Rickettsia rickettsii durante a infecção do carrapato-vetor Amblyomma aureolatum.

Background Anaplasma marginale is the causative agent of the severe bovine anaplasmosis. The tick Rhipicephalus microplus is one of the main vectors of A. marginale in tropical and subtropical regions of world. After the tick bite, the bacterium invades and proliferates within the bovine erythrocytes, causing anemia and impairing milk production and weight gain. In addition, infection can cause abortion and high mortality in areas of enzootic instability. The immunization with live and inactivated vaccines are employed to the control acute bovine anaplasmosis. However, they do not prevent persistent infection. Therefore, infected animals, even if immunized, are reservoirs of the bacterium and contribute to the dissemination of the disease. Antimicrobials are also largely employed for the prophylaxis of bovine anaplasmosis. However, they are often used in subtlethal doses, what can select pre-existing resistant bacteria and induce genetic or phenotypic variations. Therefore, the standardization of an in vitro assay to evaluate the susceptibility of A. marginale strains to different antimicrobials is important to allow the prescription of the more effective treatment, preventing both the selection and spread of resistant strains. Results Initially the antimicrobial susceptibility of two eld isolates of A. marginale (Jaboticabal and Palmeira) infecting bovines was evaluated. The least susceptible strain (Jaboticabal) was used for the standardization of an antimicrobial assay using a culture of Ixodes scapularis-derived tick cell line, ISE6.Results showed that enro oxacin (ENRO) at 0.25, 1 or 4 μg/mL and oxytetracycline (OTC) at 4 or 16 μg/mL are the most e cient treatments, followed by OTC at 1 μg/mL and imidocarb dipropionate (IMD) at 1 or 4 μg/mL. Conclusion In the current study, we present a new in vitro assay using a tick cell line to evaluate the susceptibility of A. marginale to antimicrobials. The maintenance of such culture is much easier than the maintenance of bovine erythrocyte culture, which depends on continuous cell replacement. This assay may be used to guide cattle farmers to the correct use of antimicrobials. The choice of the most suitable antimicrobial is essential to eliminate persistent infections, preventing the spread of resistant strains and helping in the control of bovine anaplasmosis.