Lindsay F. Killmaster

Domestic Dogs (Canis familiaris) as Reservoir Hosts for Rickettsia conorii

Rickettsia conorii is the causative agent of Mediterranean spotted fever (MSF) and Israeli spotted fever (ISF) transmitted by the brown dog tick Rhipicephalus sanguineus. In areas where MSF or ISF are prevalent, dogs have high prevalence of R. conorii -neutralizing antibodies. However, the true role of dogs in the persistence of the R. conorii transmission cycle is unknown, and their reservoir competence for this pathogen has remained untested. We assessed the ability of dogs infected with R. conorii to transmit the pathogen to previously uninfected Rh. sanguineus ticks. Dogs were infected either via needle-inoculation of cultured rickettsiae or naturally via infected tick bite. Dogs were monitored for clinical signs of infection, for rickettsemia by PCR, and for seroconversion and were subjected to infestation with uninfected ticks at different time points. Rh. sanguineus larvae and nymphs successfully acquired the agent from both needle-inoculated and tick-infected dogs and transmitted it transtadially. Tick-infected dogs remained infectious to ticks for at least a month postinfection. The molted ticks were, in turn, infectious to naïve dogs. These results demonstrate that dogs are capable of acquiring R. conorii from infected Rh. sanguineus ticks and transmitting infection to cohorts of uninfected ticks, thus confirming for the first time that dogs are indeed competent reservoirs for R. conorii. In addition, dogs with different genetic backgrounds appear to differ in their susceptibility to R. conorii infection.

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.

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

ABSTRACT 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 hosts 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.

Experimental infection by capillary tube feeding of Rhipicephalus sanguineus with Bartonella vinsonii subspecies berkhoffii.

It has been speculated that ticks may serve as vectors of Bartonella species. Circumstantial, clinical, epidemiological and serological evidence suggest that B. vinsonii subspecies berkhoffii (B. v. berkhoffii) might be transmitted by Rhipicephalus sanguineus. The purpose of the present study was to determine whether adult R. sanguineus ticks can be infected with a B. v. berkhoffii genotype II isolate via capillary tube feeding and whether the infection can then be transmitted from adult females to their eggs via trans-ovarial transmission. Furthermore, tick fecal material was also collected and screened as a possible source of infectious inoculum for canine infections. B. v. berkhoffii DNA was detected in 50% (7 of 14) of females that did not oviposit and in 14.3% (2 of 14) of female ticks that laid eggs, but not detected in egg clutches (100 eggs/female). DNA was also detected in tick feces collected on days 2 through 6 post-capillary tube feeding, however, dogs (n=3) did not become bacteremic or seroconvert when inoculated with tick fecal material. Therefore, trans-ovarial transmission of B. v. berkhoffii by R. sanguineus is unlikely, but further studies are needed to determine if tick fecal material can serve as a source of infection to canines.

Isolation of a Rickettsia slovaca-Like Agent from Dermacentor variabilis Ticks in Vero Cell Culture

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.

First Report of Rickettsia Identical to R. slovaca in Colony-Originated D. variabilis in the United States: Detection, Laboratory Animal Model, and Vector Competence of Ticks

Ticks of the genus Dermacentor are known vectors of rickettsial pathogens in both the Old World and New World. 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 spotted fever group (SFG) Rickettsia by PCR. Analysis of 17 kDa gltA, rpoB, ompA, ompB, and sca4 genes revealed 100% identity to R. slovaca sequences available in the GenBank. New Zealand white 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 SFG Rickettsia. 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.

Apparent Disappearance of Vesicular Stomatitis New Jersey Virus from Ossabaw Island, Georgia

Ossabaw Island, Georgia, is the only reported endemic focus of Vesicular Stomatitis New Jersey Virus (VSNJV) in the United States. Based on recent negative serologic results of white-tailed deer and feral swine and the failure to isolate VSNJV from Lutzomyia shannoni, it appears that VSNJV is no longer present at this site. This apparent disappearance does not appear to be related to a change in L. shannoni habitat, specifically to the density of tree holes in the maritime and mixed hardwood forests. We believe that the disappearance of VSNJV from Ossabaw Island is directly related to a reduction in the feral swine population and a subsequent increase in the utilization of white-tailed deer by the known vector, L. shannoni.

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.

Isolation and Short-Term Persistence of Ehrlichia ewingii in Cell Culture.

Ehrlichia ewingii is the causative agent of human and canine granulocytic ehrlichiosis. Since its discovery in 1970, little work has been done to characterize the pathogen or study the transmission dynamics due to the inability to grow the agent in vitro. The aim of this study was to assess the suitability of multiple cell lines and media formulations for propagation of E. ewingii in cell culture. In this study, we present an overview of attempts to isolate E. ewingii from the buffy coat of goats naturally infected by Amblyomma americanum ticks, as well as a methodology for maintaining the pathogen for up to 16 weeks in culture. The most promising results were seen with HL-60 cells differentiated by the addition of 1.5% DMSO to the media and supplemented with 8 mM l-glutamine. Cultures were passaged multiple times, and fluorescence and morulae were observed by indirect fluorescent antibody test and Diff-Quik staining. It is our hope that this information will provide a foundation for future attempts to propagate and maintain E. ewingii in cell culture.