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.

A tick cell line as a powerful tool to screen the antimicrobial susceptibility of the tick-borne pathogen Anaplasma marginale.

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 the world. After the tick bite, the bacterium invades and proliferates within the bovine erythrocytes leading to anemia, impairment of milk production and weight loss. In addition, infection can cause abortion and high mortality in areas of enzootic instability. Immunization with live and inactivated vaccines are employed to control acute bovine anaplasmosis. However, they do not prevent persistent infection. Consequently, infected animals, even if immunized, are still reservoirs of the bacterium and contribute to its dissemination. Antimicrobials are largely employed for the prophylaxis of bovine anaplasmosis. However, they are often used in sublethal doses which may select pre-existing resistant bacteria and induce genetic or phenotypic variations. Therefore, we propose a new standardized in vitro assay to evaluate the susceptibility of A. marginale strains to different antimicrobials. This tool will help health professionals to choose the more adequate treatment for each herd which will prevent the selection and spread of resistant strains. For that, we initially evaluated the antimicrobial susceptibility of two field isolates of A. marginale (Jaboticabal and Palmeira) infecting bovines. 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 enrofloxacin (ENRO) at 0.25, 1 or 4 µg/mL and oxytetracycline (OTC) at 4 or 16 µg/mL are the most efficient treatments, followed by OTC at 1 µg/mL and imidocarb dipropionate (IMD) at 1 or 4 µg/mL. In addition, this proposed tool has technical advantages compared to the previously established bovine erythrocyte culture. Thereby, it 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, prevent the spread of resistant strains and help controlling of bovine anaplasmosis.

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.

Independence of Anaplasma marginale strains with high and low transmission efficiencies in the tick vector following simultaneous acquisition by feeding on a superinfected mammalian reservoir host.

Strain superinfection occurs when a second pathogen strain infects a host already carrying a primary strain. Anaplasma marginale superinfection occurs when the second strain carries a variant repertoire different from that of the primary strain, and the epidemiologic consequences depend on the relative efficiencies of tick-borne transmission of the two strains. Following strain superinfection in the reservoir host, we tested whether the presence of two A. marginale (sensu lato) strains that differed in transmission efficiency altered the transmission phenotypes in comparison to those for single-strain infections. Dermacentor andersoni ticks were fed on animals superinfected with the Anaplasma marginale subsp. centrale vaccine strain (low transmission efficiency) and the A. marginale St. Maries strain (high transmission efficiency). Within ticks that acquired both strains, the St. Maries strain had a competitive advantage and replicated to significantly higher levels than the vaccine strain. The St. Maries strain was subsequently transmitted to naïve hosts by ticks previously fed either on superinfected animals or on animals singly infected with the St. Maries strain, consistent with the predicted transmission phenotype of this strain and the lack of interference due to the presence of a competing low-efficiency strain. The vaccine strain was not transmitted by either singly infected or coinfected ticks, consistent with the predicted transmission phenotype and the lack of enhancement due to the presence of a high-efficiency strain. These results support the idea that the strain predominance in regions of endemicity is mediated by the intrinsic transmission efficiency of specific strains regardless of occurrence of superinfection.