Detection of bacterial agents in Amblyomma americanum (Acari: Ixodidae) from Georgia, USA, and the use of a multiplex assay to differentiate Ehrlichia chaffeensis and Ehrlichia ewingii.

Amblyomma americanum, the lone star tick, is the most common and most aggressive human biting tick in the Southeastern United States. It is known to transmit the agents of human ehrlichioses, Ehrlichia chaffeensis and Ehrlichia ewingii. In addition, it carries agents of unspecified pathogenicity to humans, including Rickettsia amblyommii, Borrelia lonestari, and the newly emerging Panola Mountain Ehrlichia (PME). Surveillance of these ticks for recognized or emerging pathogens is necessary for assessing the risk of human infection. From 2005 to 2009, we surveyed A. americanum ticks from four locations in the state of Georgia. Ticks (1,183 adults, 2,954 nymphs, and 99 larval batches) were tested using a multiplex real-time polymerase chain reaction (PCR) assay designed to detect and discriminate DNA from Rickettsia spp., E. chaffeensis, and E. ewingii. This assay was capable of detecting as few as 10 gene copies of the aforementioned agents. Ticks were also tested for PME and B. lonestari by nested PCR. The prevalence of infection ranged from 0 to 2.5% for E. chaffeensis, 0 to 3.9% for E. ewingii, 0 to 2.2% for PME, 17 to 83.1% for R. amblyommii, and 0 to 3.1% for B. lonestari. There were 46 (4.1%) individual adults positive for two agents, and two females that were each positive for three agents. Two larval batches were positive for both B. lonestari and R. amblyommii, indicating the potential for transovarial transmission of both agents from a single female. Although infrequent in occurrence, the dynamics of coinfections in individual ticks should be explored further, given the potential implications for differential diagnosis and severity of human illness.

Effects of homologous and heterologous immunization on the reservoir competence of domestic dogs for Rickettsia conorii (israelensis).

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.

Crossbreeding between different geographical populations of the brown dog tick, Rhipicephalus sanguineus (Acari: Ixodidae).

Brown dog ticks are distributed world-wide, and their systematics and phylogeny are the subject of an ongoing debate. The present study evaluates the reproductive compatibility between Rhipicephalus sanguineus ticks from North America, Israel, and Africa. Female ticks of the parent generation were mated with males from the same and alternate colonies. Every pure and hybrid cohort was maintained separately into the F2 generation with F1 females being allowed to mate only with males from the same cohort. The following survival parameters were measured and recorded for every developmental stage: feeding duration and success; engorgement weight, fertility, and fecundity of females; molting and hatching success. Ticks from North American and Mediterranean populations hybridized successfully. The survival parameters of all their hybrid lines were similar to those in pure lines throughout the F1 generation, and F1 adults were fully fertile. Parent adult ticks from the African population hybridized with either North American or Mediterranean ticks and produced viable progenies whose survival parameters were also similar to those in pure lines throughout the F1 generation. However, F1 adults in the four hybrid lines that included African ancestry were infertile. No parthenogenesis was observed in any pure or hybrid lines as proportion of males in F1 generation ranged from 40 to 60 %. Phylogenetic analysis of the 12S rDNA gene sequences placed African ticks into a separate clade from those of the North American or Mediterranean origins. Our results demonstrate that Rh. sanguineus ticks from North America and Israel represent the same species, whereas the African population used in this study is significantly distant and probably represents a different taxon.

Co-feeding as a route for transmission of Rickettsia conorii israelensis between Rhipicephalus sanguineus ticks.

Rickettsia conorii is widely distributed in Europe, Asia, and Africa. The brown dog tick, Rhipicephalus sanguineus, is the recognized vector of R. conorii. In this study, we assessed the efficiency of R. conorii israelensis transmission between co-feeding Rh. sanguineus ticks. Infected Rh. sanguineus adults and uninfected nymphs were fed simultaneously upon either naïve dogs or a dog previously exposed to this agent. When ticks were placed upon naïve dogs, 92-100% of nymphs acquired the infection and 80-88% of infected engorged nymphs transmitted it transstadially. When ticks were placed upon a seropositive dog, only 8-28.5% of recipient nymphs became infected. Our results establish the first evidence for efficient natural transmission of R. conorii israelensis between co-feeding ticks upon both naïve and seropositive dogs. This route of transmission can ensure continuous circulation of R. conorii israelensis in tick vectors even in the absence of naïve reservoir hosts.

Incongruent effects of two isolates of Rickettsia conorii on the survival of Rhipicephalus sanguineus ticks.

Rickettsia conorii, the etiologic agent of Mediterranean spotted fever is widely distributed in Southern Europe, the Middle East, Africa, India and the Caspian region. In the Mediterranean region, the brown dog tick, Rhipicephalus sanguineus, is the recognized vector of R. conorii. To study tick-pathogen relationships and pathogenesis of infection caused in model animals by the bite of an infected tick, we attempted to establish a laboratory colony of Rh. sanguineus persistently infected with R. conorii. Rhipicephalus sanguineus ticks of North American and Mediterranean origin were exposed to R. conorii isolates of African (R. conorii conorii strain Malish) and Mediterranean (R. conorii israelensis strain ISTT) origin. Feeding of ticks upon infected mice and dogs, intra-hemocoel inoculation, and submersion in suspensions of purified rickettsiae were used to introduce the pathogen into uninfected ticks. Feeding success, molting success and the longevity of molted ticks were measured to assess the effects of R. conorii on the survival of Rh. sanguineus. In concordance with previously published results, Rh. sanguineus larvae and nymphs from both North American and Mediterranean colonies exposed to R. conorii conorii Malish experienced high mortality during feeding and molting or immediately after. The prevalence of infection in surviving ticks did not exceed 5%. On the other hand, exposure to ISTT strain had lesser effect on tick survival and resulted in 35-66% prevalence of infection. Rh. sanguineus of Mediterranean origin were more susceptible to infection with either strain of R. conorii than those from North America. Previous experimental studies had demonstrated transovarial and transstadial transmission of R. conorii in Rh. sanguineus; however, our data suggest that different strains of R. conorii may employ different means of maintenance in nature. The vertebrate host may be a more important reservoir than previously thought, or co-feeding transmission between different generations of ticks may obviate or lessen the requirement for transovarial maintenance of R. conorii.