Studies on the natural transmission cycles of zoonotic pathogens and the reservoir competence of vertebrate hosts require methods for reliable diagnosis of infection in wild and laboratory animals. Several PCR-based applications have been developed for detection of infections caused by Spotted Fever group Rickettsia spp. in a variety of animal tissues. These assays are being widely used by researchers, but they differ in their sensitivity and reliability. We compared the sensitivity of five previously published conventional PCR assays and one SYBR green-based real-time PCR assay for the detection of rickettsial DNA in blood and tissue samples from Rickettsia- infected laboratory animals (n = 87). The real-time PCR, which detected rickettsial DNA in 37.9% of samples, was the most sensitive. The next best were the semi-nested ompA assay and rpoB conventional PCR, which detected as positive 18.4% and 14.9% samples respectively. Conventional assays targeting ompB, gltA and hrtA genes have been the least sensitive. Therefore, we recommend the SYBR green-based real-time PCR as a tool for the detection of rickettsial DNA in animal samples due to its higher sensitivity when compared to more traditional assays.

A number of spotted fever group (SFG) rickettsiae cause serious infections in humans. Several antigenically related rickettsial agents may coexist within the same geographical area, and humans or vertebrate hosts may be sequentially exposed to multiple SFG agents. We assessed whether exposure of a vertebrate reservoir to one SFG Rickettsia will affect the host’s immune response to a related pathogen and the efficiency of transmission to uninfected ticks. Two pairs of dogs were each infected with either Rickettsia massiliae or Rickettsia conorii israelensis, and their immune response was monitored twice weekly by IFA. The four immunized dogs and a pair of naïve dogs were each challenged with R. conorii israelensis-infected Rhipicephalus sanguineus nymphs. Uninfected Rh. sanguineus larvae were acquisition-fed on the dogs on days 1, 7, and 14 post-challenge. These ticks were tested for the presence of rickettsial DNA after molting to the nymphal stage. The naive dogs became infected with R. conorii israelensis and were infectious to ticks for at least 3 weeks, whereas reservoir competence of dogs previously infected with either R. massiliae or R. conorii was significantly diminished. This opens an opportunity for decreasing the efficiency of transmission and propagation of pathogenic Rickettsia in natural foci by immunizing the primary hosts with closely related nonpathogenic SFG bacteria. However, neither homologous immunization nor cross-immunization significantly affected the efficiency of R. conorii transmission between cofeeding infected nymphs and uninfected larvae. At high densities of ticks, the efficiency of cofeeding transmission may be sufficient for yearly amplification and persistent circulation of a rickettsial pathogen in the vector population.

Rickettsia slovaca is transmitted by Dermacentor marginatus ticks, and is the causative agent of tick-borne lymphadenopathy and Dermacentor-borne necrosis erythema lymphadenopathy throughout Europe. It has not been found in New World ticks, nor have tick-borne lymphadenopathy or Dermacentor-borne necrosis erythema lymphadenopathy been reported in humans in the Americas. Here we describe the isolation of a R. slovaca-like agent from D. variabilis nymphs from a colony of ticks derived from field collected adults.

Ticks of the genus Dermacentor are known vectors of rickettsial pathogens in both the Old and New Worlds. In North America, Dermacentor variabilis and D. andersoni are vectors of Rickettsia rickettsii while in Europe, D. marginatus and D. reticulatus transmit R. slovaca and R. raoultii, respectively. Neither the presence of R. slovaca in the Americas, nor the ability of American tick species to maintain this pathogen have been reported. Here we describe detection of Rickettsia genetically identical to R. slovaca in D. variabilis, its molecular characterization, assessment of pathogenicity to guinea pigs, and vector competence of D. variabilis ticks. Ticks from a laboratory colony of D. variabilis, established from wild ticks and maintained on naïve NZW rabbits, tested positive for SFGR Rickettsia by PCR. Analysis of 17kDa, gltA, rpoB, ompA, ompB, and sca4 genes revealed 100% identity to R. slovaca sequences available in the Gene Bank. NZW rabbits fed upon by infected ticks seroconverted to SFG Rickettsia. Guinea pigs inoculated with the Rickettsia culture or infested by the infected ticks developed antibodies to SFGR. The intensity of clinical signs and immune response were dependent on dose and route of infection. The identified Rickettsia was detected in all life stages of D. variabilis ticks, confirming transstadial and transovarial transmission. Thirty six percent of uninfected larvae co-fed with infected nymphs on guinea pigs were PCR-positive and able to pass rickettsia to at least 11.7% of molted nymphs. To our knowledge this is a first report of identification of a European pathogen R. slovaca or a highly similar agent in the American dog tick - D. variabilis. Considering pathogenicity of R. slovaca in humans, further laboratory and field studies are warranted to assess the relevance of the above findings to the public health and epidemiology of SFG rickettsioses in the United States.