The survival of Amblyomma sculptum ticks upon blood-feeding depends on the expression of an inhibitor of apoptosis protein.

BACKGROUND: The tick Amblyomma sculptum is the major vector of Rickettsia rickettsii, the causative agent of the highly lethal Brazilian spotted fever. It has been shown that R. rickettsii inhibits apoptosis in both human endothelial cells and tick cells. Apoptosis is regulated by different factors, among which inhibitors of apoptosis proteins (IAPs) play a central role. In the study reported here, we selected an IAP of A. sculptum that has not yet been characterized to assess its role in cell death and to determine the effects of its gene silencing on tick fitness and R. rickettsii infection. METHODS: An A. sculptum cell line (IBU/ASE-16) was treated with specific double-stranded RNA (dsRNA) for either IAP (dsIAP) or green fluorescent protein (dsGFP; as a control). The activity of caspase-3 and the exposure of phosphatidylserine were determined in both groups. In addition, unfed adult ticks, infected or not infected with R. rickettsii, were treated with either dsIAP or dsGFP and allowed to feed on noninfected rabbits. In parallel, noninfected ticks were allowed to feed on an R. rickettsii-infected rabbit. Ticks (infected or not with R. rickettsii) that remained unfed were used as a control. RESULTS: Caspase-3 activity and the externalization of phosphatidylserine were significantly higher in IBU/ASE-16 cells treated with dsIAP than in those treated with dsGFP. The mortality rates of ticks in the dsIAP group were much higher than those in the dsGFP group when they were allowed to feed on rabbits, independent of the presence of R. rickettsii. Conversely, lower mortality rates were recorded in unfed ticks. CONCLUSIONS: Our results show that IAP negatively regulates apoptosis in A. sculptum cells. Moreover, IAP-silenced ticks experienced higher mortality rates following the acquisition of a blood meal, suggesting that feeding may trigger the activation of apoptosis in the absence of this physiological regulator. These findings indicate that IAP is a potential antigen for an anti-tick vaccine.

Nitric Oxide Inhibition of Rickettsia rickettsii.

Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, is an enzootic, obligate, intracellular bacterial pathogen. Nitric oxide (NO) synthesized by the inducible NO synthase (iNOS) is a potent antimicrobial component of innate immunity and has been implicated in the control of virulent Rickettsia spp. in diverse cell types. In this study, we examined the antibacterial role of NO on R. rickettsii. Our results indicate that NO challenge dramatically reduces R. rickettsii adhesion through the disruption of bacterial energetics. Additionally, NO-treated R. rickettsii cells were unable to synthesize protein or replicate in permissive cells. Activated, NO-producing macrophages restricted R. rickettsii infections, but inhibition of iNOS ablated the inhibition of bacterial growth. These data indicate that NO is a potent antirickettsial effector of innate immunity that targets energy generation in these pathogenic bacteria to prevent growth and subversion of infected host cells.

Domestic dogs as amplifying hosts of Rickettsia rickettsii for Amblyomma aureolatum ticks.

Brazilian spotted fever (BSF) is an acute infectious disease caused by the bacterium Rickettsia rickettsii, which is transmitted by different tick species. Due to deleterious effects caused on ticks, the horizontal transmission of R. rickettsii through amplifying hosts is crucial for its maintenance in tick populations among BSF-endemic areas. The tick Amblyomma aureolatum is the main vector of R. rickettsii in the São Paulo metropolitan area; nevertheless, it is not known which vertebrate could act as an amplifying host for this tick species. Herein, we evaluated the potential of domestic dogs - primary hosts for A. aureolatum adults in BSF-endemic areas - to act as amplifying hosts. For this purpose, A. aureolatum non-infected adults were allowed to feed on two groups of dogs: the control group (G1), composed of one dog not exposed to R. rickettsii; and, the infected group (G2), composed of three dogs infected with R. rickettsii via tick parasitism. All G2-dogs became ill, seroconverted to R. rickettsii, and rickettsial DNA was detected in 87% of the engorged females that fed on them. Transovarial transmission rate was estimated to be 25% and infected larvae successfully transmitted R. rickettsii to guinea-pigs, confirming transovarial transmission and vector competence. No rickettsial DNA was detected in individual samples of eggs or larvae, which precluded the estimation of filial infection rate, but implies that it was low. Our results suggest that domestic dogs act as amplifying hosts of R. rickettsii for A. aureolatum ticks in BSF-endemic areas in Brazil.

Isolate-Dependent Differences in Clinical, Pathological, and Transcriptional Profiles following In Vitro and In Vivo Infections with Rickettsia rickettsii.

Rickettsia rickettsii, the etiological agent of Rocky Mountain spotted fever (RMSF), a life-threatening tick-borne disease that affects humans and various animal species, has been recognized in medicine and science for more than 100 years. Isolate-dependent differences in virulence of R. rickettsii have been documented for many decades; nonetheless, the specific genetic and phenotypic factors responsible for these differences have not been characterized. Using in vivo and in vitro methods, we identified multiple phenotypic differences among six geographically distinct isolates of R. rickettsii, representing isolates from the United States, Costa Rica, and Brazil. Aggregate phenotypic data, derived from growth in Vero E6 cells and from clinical and pathological characteristics following infection of male guinea pigs (Cavia porcellus), allowed separation of these isolates into three categories: nonvirulent (Iowa), mildly virulent (Sawtooth and Gila), and highly virulent (Sheila SmithT, Costa Rica, and Taiaçu). Transcriptional profiles of 11 recognized or putative virulence factors confirmed the isolate-dependent differences between mildly and highly virulent isolates. These data corroborate previous qualitative assessments of strain virulence and suggest further that a critical and previously underappreciated balance between bacterial growth and host immune response could leverage strain pathogenicity. Also, this work provides insight into isolate-specific microbiological factors that contribute to the outcome of RMSF and confirms the hypothesis that distinct rickettsial isolates also differ phenotypically, which could influence the severity of disease in vertebrate hosts.

The intracellular bacterium Rickettsia rickettsii exerts an inhibitory effect on the apoptosis of tick cells.

BACKGROUND: Rickettsia rickettsii is a tick-borne obligate intracellular bacterium that causes Rocky Mountain spotted fever, a life-threatening illness. To obtain an insight into the vector-pathogen interactions, we assessed the effects of infection with R. rickettsii on the proteome cells of the tick embryonic cell line BME26. METHODS: The proteome of BME26 cells was determined by label-free high-performance liquid chromatography coupled with tandem mass spectrometry analysis. Also evaluated were the effects of infection on the activity of caspase-3, assessed by the hydrolysis of a synthetic fluorogenic substrate in enzymatic assays, and on the exposition of phosphatidyserine, evaluated by live-cell fluorescence microscopy after labeling with annexin-V. Finally, the effects of activation or inhibition of caspase-3 activity on the growth of R. rickettsii in BME26 cells was determined. RESULTS: Tick proteins of different functional classes were modulated in a time-dependent manner by R. rickettsii infection. Regarding proteins involved in apoptosis, certain negative regulators were downregulated at the initial phase of the infection (6 h) but upregulated in the middle of the exponential phase of the bacterial growth (48 h). Microorganisms are known to be able to inhibit apoptosis of the host cell to ensure their survival and proliferation. We therefore evaluated the effects of infection on classic features of apoptotic cells and observed DNA fragmentation exclusively in noninfected cells. Moreover, both caspase-3 activity and phosphatidylserine exposition were lower in infected than in noninfected cells. Importantly, while the activation of caspase-3 exerted a detrimental effect on rickettsial proliferation, its inhibition increased bacterial growth. CONCLUSIONS: Taken together, these results show that R. rickettsii modulates the proteome and exerts an inhibitory effect on apoptosis in tick cellsthat seems to be important to ensure cell colonization.

Capybaras (Hydrochoerus hydrochaeris) as amplifying hosts of Rickettsia rickettsii to Amblyomma sculptum ticks: Evaluation during primary and subsequent exposures to R. rickettsii infection.

Rickettsia rickettsii is the agent of Brazilian spotted fever (BSF), the most lethal tick-borne disease in the western hemisphere. Amblyomma sculptum is the main vector of R. rickettsii in southeastern Brazil. Capybaras act as primary hosts for this tick species, and as amplifying hosts for R. rickettsii, creating new infected lineages of A. sculptum in BSF-endemic areas. In a recent study, we demonstrated that capybaras successively exposed to R. rickettsii-infected A. sculptum ticks developed a mean rickettsemic period of 9.2 days during primary infection, but no rickettsemia during subsequent expositions, when capybaras were immune to R. rickettsii. During the primary and subsequent infections, capybaras were also infested with uninfected A. sculptum ticks. These infestations compose the present study, which aimed to evaluate: (i) if either rickettsemic or non-rickettsemic capybaras could serve as infection sources of R. rickettsii for A. sculptum larvae and nymphs; (ii) the vector competence of the subsequent nymphs and adults (molted from the engorged ticks collected from capybaras); and (iii) if there were R. rickettsii-acquisition by uninfected ticks co-feeding with infected ticks on immune capybaras (without rickettsemia). Through experimental infection of capybaras with R. rickettsii via tick feeding, simulating a natural condition, we demonstrated that primarily infected capybaras developed rickettsemia that resulted in successful acquisition feeding of larvae and nymphs of A. sculptum, since part of these ticks maintained R. rickettsii transstadially, and the resultant molted ticks (either nymphs or adults) successfully transmitted the bacterium by feeding on susceptible rabbits. Contrastingly, all rabbit infestations with ticks derived from acquisition feeding on R. rickettsii-immune capybaras (including when acquisition ticks fed in direct contact with donor ticks) evidenced absence of R. rickettsii transmission due to lack of clinical signs and antibody response in those rabbits. Our results indicate that capybaras could serve as R. rickettsii-amplifying hosts for A. sculptum ticks only during the capybara's primary infection, but not during subsequent infections on immune capybaras. Finally, the probable co-feeding nonsystemic transmission of R. rickettsii seems to be irrelevant in the context of BSF epidemiology, in areas where capybaras are incriminated as main amplifying hosts of R. rickettsii for A. sculptum ticks.

Comparative analysis of the midgut microbiota of two natural tick vectors of Rickettsia rickettsii.

Although the ticks Amblyomma sculptum and Amblyomma aureolatum are important vectors of Rickettsia rickettsii, causative agent of the life-threatening Rocky Mountain spotted fever, A. aureolatum is considerably more susceptible to infection than A. sculptum. As the microbiota can interfere with the colonization of arthropod midgut (MG) by pathogens, in the current study we analyzed the MG microbiota of both tick species. Our results revealed that the MG of A. aureolatum harbors a prominent microbiota, while A. sculptum does not. Remarkably, a significant reduction of the bacterial load was recorded in R. rickettsii-infected A. aureolatum. In addition, the taxonomy analysis of the MG bacterial community of A. aureolatum revealed a dominance of the genus Francisella, suggesting an endosymbiosis. This study is the first step in getting insights into the mechanisms underlying the interactions among Amblyomma species, their microbiota and R. rickettsii. Additional studies to better understand these mechanisms are required and may help the development of novel alternatives to block rickettsial transmission.

Comparative survival of the engorged stages of Amblyomma dubitatum and Amblyomma sculptum in the laboratory: Implications for Brazilian spotted fever epidemiology.

Brazilian spotted fever (BSF), caused by the bacterium Rickettsia rickettsii, is transmitted to humans mainly by the tick Amblyomma sculptum in southeastern Brazil. In most BSF-endemic areas A. sculptum populations are sustained chiefly by capybaras (Hydrochoerus hydrochaeris), which are also the host of another tick species, Amblyomma dubitatum, not implicated in R. rickettsii transmission. Herein, we evaluated the effects of relative humidity (RH), temperature, and water immersion on the successful development of the engorged stages of A. dubitatum and compared them with recently published data under the same experimental conditions for A. sculptum. We showed that free-living developmental stages (engorged larvae, nymphs and females, and eggs) of A. dubitatum had higher survival rates when these stages were submerged in water for different periods of times (24-72 h). On the other hand, higher survival rates were observed for A. sculptum when ticks were incubated at lower RH values, 65 or 78 %, regardless of summer or winter mean temperatures. These results suggest that A. dubitatum is more adapted to humid habitats and seasonally flooding soils than A. sculptum, whereas this latter species should be more adapted than A. dubitatum to drier habitats. The implications of these results for the spatial distribution of A. dubitatum and A. sculptum, in relation to BSF epidemiology, are discussed.

Minimal Duration of Tick Attachment Sufficient for Transmission of Infectious Rickettsia rickettsii (Rickettsiales: Rickettsiaceae) by Its Primary Vector Dermacentor variabilis (Acari: Ixodidae): Duration of Rickettsial Reactivation in the Vector Revisited.

It has been reported that starving ticks do not transmit spotted fever group Rickettsia immediately upon attachment because pathogenic bacteria exist in a dormant, uninfectious state and require time for 'reactivation' before transmission to a susceptible host. To clarify the length of reactivation period, we exposed guinea pigs to bites of Rickettsia rickettsii-infected Dermacentor variabilis (Say) and allowed ticks to remain attached for predetermined time periods from 0 to 48 h. Following removal of attached ticks, salivary glands were immediately tested by PCR, while guinea pigs were observed for 10-12 d post-exposure. Guinea pigs in a control group were subcutaneously inoculated with salivary glands from unfed D. variabilis from the same cohort. In a parallel experiment, skin at the location of tick bite was also excised at the time of tick removal to ascertain dissemination of pathogen from the inoculation site. Animals in every exposure group developed clinical and pathological signs of infection. The severity of rickettsial infection in animals increased with the length of tick attachment, but even attachments for less than 8 h resulted in clinically identifiable infection in some guinea pigs. Guinea pigs inoculated with salivary glands from unfed ticks also became severely ill. Results of our study indicate that R. rickettsii residing in salivary glands of unfed questing ticks does not necessarily require a period of reactivation to precede the salivary transmission and ticks can transmit infectious Rickettsia virtually as soon as they attach to the host.

Rickettsia rickettsii Co-feeding Transmission among Amblyomma aureolatum Ticks.

Amblyomma aureolatum ticks are vectors of Rickettsia rickettsii, the etiologic agent of Rocky Mountain spotted fever in Brazil. Maintenance of R. rickettsii in nature depends on horizontal transmission along tick generations. Although such transmission is known to occur when uninfected and infected ticks feed simultaneously on susceptible animals (co-feeding systemic transmission), we investigated co-feeding nonsystemic transmission, which was based on R. rickettsii-infected and -uninfected A. aureolatum ticks feeding simultaneously on guinea pigs immune to R. rickettsii. Our acquisition and transmission infestations demonstrated that horizontal transmission of R. rickettsii by co-feeding ticks on immune hosts with no systemic infection did not occur when uninfected larvae fed distantly from infected nymphs but did occur in a few cases when uninfected larvae fed side-by-side with infected nymphs, suggesting that they shared the same feeding site. The co-feeding nonsystemic transmission type might have no epidemiologic importance for Rocky Mountain spotted fever.

Descriptions of two new cases of Rocky Mountain spotted fever in Panama, and coincident infection with Rickettsia rickettsii in Rhipicephalus sanguineus s.l. in an urban locality of Panama City, Panama.

The clinical and pathologic characterisation of two fatal cases of tick-borne rickettsiosis in rural (El Valle) and urban (City of Panama) Panama are described. Clinical and autopsy findings were non-specific, but the molecular analysis was used to identify Rickettsia rickettsii in both cases. No ticks were collected in El Valle, while in the urban case, R. rickettsii was detected in Rhipicephalus sanguineus s.l., representing the first molecular finding in this tick in Panama and Central America.

The Distinct Transcriptional Response of the Midgut of Amblyomma sculptum and Amblyomma aureolatum Ticks to Rickettsia rickettsii Correlates to Their Differences in Susceptibility to Infection.

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.

Vector competence of Amblyomma americanum (Acari: Ixodidae) for Rickettsia rickettsii.

Rickettsia rickettsii - the etiologic agent of Rocky Mountain spotted fever (RMSF) - is widely spread across the Americas. In the US, Dermacentor spp. ticks are identified as primary vectors of R. rickettsii and Rhipicephalus sanguineus s.l. has been implicated in transmission of this pathogen in several locations in the Southwest. Conversely, ticks of the genus Amblyomma are recognized vectors of RMSF in Central and South America, but not in the US. A. americanum is one of the most aggressive human-biting ticks in the US, whose geographical range overlaps with that of reported RMSF cases. Despite sporadic findings of R. rickettsii DNA in field-collected A. americanum and circumstantial association of this species with human RMSF cases, its vector competence for R. rickettsii has not been appropriately studied. Therefore, we assessed the ability of A. americanum to acquire and transmit two geographically distant isolates of R. rickettsii. The Di-6 isolate of R. rickettsii used in this study originated in Virginia and the AZ-3 isolate originated in Arizona. Under laboratory conditions, A. americanum demonstrated vector competence for both isolates, although the efficiency of acquisition and transovarial transmission was higher for Di-6 than for AZ-3 isolate. Uninfected larvae acquired the pathogen from systemically infected guinea pigs, as well as while feeding side by side with Rickettsia-infected ticks on non-rickettsiemic hosts. Once acquired, R. rickettsii was successfully maintained through the tick molting process and transmitted to susceptible animals during subsequent feedings. Guinea pigs and dogs infested with infected A. americanum developed fever, scrotal edema and dermatitis or macular rash. R. rickettsii DNA was identified in animal blood, skin, and internal organs. The prevalence of infection within tick cohorts gradually increased due to side-by-side feeding of infected and uninfected individuals from 33 to 49% in freshly molted nymphs to 71-98% in engorged females. Moreover, R. rickettsii was transmitted transovarially by approximately 28% and 14% of females infected with Di-6 and AZ-3 isolates, respectively. Hence, A. americanum is capable of acquiring, maintaining and transmitting R. rickettsii isolates originating from two different geographical regions of the US, at least under laboratory conditions. Its role in ecology and epidemiology of RMSF in the US deserves further investigation.

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.

Virulence genes of Rickettsia rickettsii are differentially modulated by either temperature upshift or blood-feeding in tick midgut and salivary glands.

BACKGROUND: Rickettsia 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. METHODS: In 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. RESULTS: Data 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. CONCLUSION: The 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.

Characterization of Genetic Variability and Population Structure of the Tick Amblyomma aureolatum (Acari: Ixodidae).

The hard tick Amblyomma aureolatum (Pallas) is a vector of the bacterium Rickettsia rickettsii, the etiologic agent of Brazilian spotted fever (BSF) in parts of Brazil. Despite its wide distribution in southeastern South America and its public health importance, there is no information about genetic variation of this species that might help to understand the epidemiology of BSF. Using data from eight microsatellite markers and ticks from six localities, we used a population genetics approach to test the hypothesis that tick populations from areas with the presence of R. rickettsii are genetically different from ticks from areas without R. rickettsii Contrary to expectations, we found low genetic structure between studied regions. Thus, the presence of R. rickettsii in the specific area is more likely correlated with ecological and the environmental conditions or due to unknown gene coding regions of A. aureolatum genome that would be related to R. rickettsii infection resistance.

Effect of Rickettsia rickettsii (Rickettsiales: Rickettsiaceae) Infection on the Biological Parameters and Survival of Its Tick Vector-Dermacentor variabilis (Acari: Ixodidae).

Rocky Mountain spotted fever, caused by Rickettsia rickettsii, is a potentially fatal tick-borne disease spread from North America to Argentina. The major vectors of R. rickettsii in the United States are Dermacentor andersoni Stiles and Dermacentor variabilis (Say). It is generally believed that vector ticks serve as major reservoirs of R. rickettsii in nature; however, the ability of ticks to support the indefinite perpetuation of R. rickettsii has been challenged by reports of deleterious effects of rickettsial infection on D. andersoni. To better elucidate the relationship of the pathogen with D. variabilis, we assessed the effects of R. rickettsii on the survival, fertility, and fecundity of D. variabilis. We used an isolate of R. rickettsii (Di-6), originally acquired from an opossum caught in Virginia, and ticks from a laboratory colony established from adult D. variabilis also collected in Virginia. Overall, infection with R. rickettsii protracted the feeding periods of all life stages of ticks. Infected nymphal and adult ticks experienced a slight decrease in feeding success compared with the uninfected colony, but neither larval nor nymphal molting success was affected. Infected females reached smaller engorgement weights, were less efficient in conversion of bloodmeal into eggs, and produced smaller egg clutches with a lower proportion of eggs hatching. However, no sudden die-off was observed among infected ticks, and longevity was not decreased due to R. rickettsii infection in any stage. Although infection with the studied isolate of R. rickettsii caused slight decrease in fecundity in sympatric vector ticks, no obvious deleterious effects were observed.

Altitudinal Assessment of Amblyomma aureolatum and Amblyomma ovale (Acari: Ixodidae), Vectors of Spotted Fever Group Rickettsiosis in the State of São Paulo, Brazil.

Amblyomma aureolatum (Pallas) and Amblyomma ovale Koch are common ectoparasites of domestic dogs in São Paulo state, southeastern Brazil, where they are vectors of distinct spotted fever group rickettsioses, one caused by Rickettsia rickettsii (transmitted by A. aureolatum), and the other caused by Rickettsia sp. strain Atlantic rainforest (transmitted by A. ovale). For the present study, we performed an altitudinal assessment of all 1992-2012 records of A. aureolatum and A. ovale retrieved from a tick collection. The municipalities with A. ovale records presented significantly (P < 0.05) lower altitude than the ones with A. aureolatum records; the higher the altitude, the lower the chances for the occurrence of A. ovale and the greater the likelihood for the occurrence of A. aureolatum. Regarding A. aureolatum, the chances of finding it in municipalities between 101 and 700 m are nine times higher than in municipalities at ≤ 100 m, or 31.5 times higher in municipalities above 700 m, when compared with municipalities at ≤ 100 m. The reverse was observed for A. ovale, which had its odds ratio diminishing at higher altitudes. These findings have a major role to public health, as A. aureolatum is associated with the transmission of a highly lethal spotted fever (caused by R. rickettsii), whereas A. ovale is associated with the transmission of a milder spotted fever (caused by Rickettsia sp. strain Atlantic rainforest, a R. parkeri-like agent), both in the state of São Paulo.

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.