Frequency and Distribution of Rickettsiae, Borreliae, and Ehrlichiae Detected in Human-Parasitizing Ticks, Texas, USA.

To describe the presence and distribution of tickborne bacteria and their vectors in Texas, USA, we screened ticks collected from humans during 2008-2014 for Rickettsia, Borrelia, and Ehrlichia spp. Thirteen tick species were identified, and 23% of ticks carried bacterial DNA from at least 1 of the 3 genera tested.

Survey of Borreliae in ticks, canines, and white-tailed deer from Arkansas, U.S.A.

BACKGROUND: In the Eastern and Upper Midwestern regions of North America, Ixodes scapularis (L.) is the most abundant tick species encountered by humans and the primary vector of B. burgdorferi, whereas in the southeastern region Amblyomma americanum (Say) is the most abundant tick species encountered by humans but cannot transmit B. burgdorferi. Surveys of Borreliae in ticks have been conducted in the southeastern United States and often these surveys identify B. lonestari as the primary Borrelia species, surveys have not included Arkansas ticks, canines, or white-tailed deer and B. lonestari is not considered pathogenic. The objective of this study was to identify Borrelia species within Arkansas by screening ticks (n=2123), canines (n=173), and white-tailed deer (n=228) to determine the identity and locations of Borreliae endemic to Arkansas using PCR amplification of the flagellin (flaB) gene. METHODS: Field collected ticks from canines and from hunter-killed white-tailed were identified to species and life stage. After which, ticks and their hosts were screened for the presence of Borrelia using PCR to amplify the flaB gene. A subset of the positive samples was confirmed with bidirectional sequencing. RESULTS: In total 53 (21.2%) white-tailed deer, ten (6%) canines, and 583 (27.5%) Ixodid ticks (252 Ixodes scapularis, 161 A. americanum, 88 Rhipicephalus sanguineus, 50 Amblyomma maculatum, 19 Dermacentor variabilis, and 13 unidentified Amblyomma species) produced a Borrelia flaB amplicon. Of the positive ticks, 324 (22.7%) were collected from canines (151 A. americanum, 78 R. sanguineus, 43 I. scapularis, 26 A. maculatum, 18 D. variabilis, and 8 Amblyomma species) and 259 (37.2%) were collected from white-tailed deer (209 I. scapularis, 24 A. maculatum, 10 A. americanum, 10 R. sanguineus, 1 D. variabilis, and 5 Amblyomma species). None of the larvae were PCR positive. A majority of the flaB amplicons were homologous with B. lonestari sequences: 281 of the 296 sequenced ticks, 3 canines, and 27 deer. Only 22 deer, 7 canines, and 15 tick flaB amplicons (12 I. scapularis, 2 A. maculatum, and 1 Amblyomma species) were homologous with B. burgdorferi sequences. CONCLUSIONS: Data from this study identified multiple Borreliae genotypes in Arkansas ticks, canines and deer including B. burgdorferi and B. lonestari; however, B. lonestari was significantly more prevalent in the tick population than B. burgdorferi. Results from this study suggest that the majority of tick-borne diseases in Arkansas are not B. burgdorferi.

Development of shuttle vectors for transformation of diverse Rickettsia species.

Plasmids have been identified in most species of Rickettsia examined, with some species maintaining multiple different plasmids. Three distinct plasmids were demonstrated in Rickettsia amblyommii AaR/SC by Southern analysis using plasmid specific probes. Copy numbers of pRAM18, pRAM23 and pRAM32 per chromosome in AaR/SC were estimated by real-time PCR to be 2.0, 1.9 and 1.3 respectively. Cloning and sequencing of R. amblyommii AaR/SC plasmids provided an opportunity to develop shuttle vectors for transformation of rickettsiae. A selection cassette encoding rifampin resistance and a fluorescent marker was inserted into pRAM18 yielding a 27.6 kbp recombinant plasmid, pRAM18/Rif/GFPuv. Electroporation of Rickettsia parkeri and Rickettsia bellii with pRAM18/Rif/GFPuv yielded GFPuv-expressing rickettsiae within 2 weeks. Smaller vectors, pRAM18dRG, pRAM18dRGA and pRAM32dRGA each bearing the same selection cassette, were made by moving the parA and dnaA-like genes from pRAM18 or pRAM32 into a vector backbone. R. bellii maintained the highest numbers of pRAM18dRGA (13.3 - 28.1 copies), and R. parkeri, Rickettsia monacensis and Rickettsia montanensis contained 9.9, 5.5 and 7.5 copies respectively. The same species transformed with pRAM32dRGA maintained 2.6, 2.5, 3.2 and 3.6 copies. pRM, the plasmid native to R. monacensis, was still present in shuttle vector transformed R. monacensis at a level similar to that found in wild type R. monacensis after 15 subcultures. Stable transformation of diverse rickettsiae was achieved with a shuttle vector system based on R. amblyommii plasmids pRAM18 and pRAM32, providing a new research tool that will greatly facilitate genetic and biological studies of rickettsiae.

Borrelia, Ehrlichia, and Rickettsia spp. in ticks removed from persons, Texas, USA.

Data regarding the type, frequency, and distribution of tick-borne pathogens and bacterial agents are not widely available for many tick species that parasitize persons in the southern United States. We therefore analyzed the frequency and identity of pathogens and bacterial agents in ticks removed from humans and subsequently submitted to the Texas Department of State Health Services, Zoonosis Control Program, from October 1, 2004, through September 30, 2008. The data showed associations of bacterial agents and potential vectors. Tick-related illnesses may pose unidentified health risks in areas such as Texas, where incidence of human disease related to tick bites is low but well above zero and where ticks are not routinely suspected as the cause of disease. Cause, treatment, and prevention strategies can be better addressed through collecting sufficient data to establish baseline assessments of risk.

Wide dispersal and possible multiple origins of low-copy-number plasmids in rickettsia species associated with blood-feeding arthropods.

Plasmids are mobile genetic elements of bacteria that can impart important adaptive traits, such as increased virulence or antibiotic resistance. We report the existence of plasmids in Rickettsia (Rickettsiales; Rickettsiaceae) species, including Rickettsia akari, "Candidatus Rickettsia amblyommii," R. bellii, R. rhipicephali, and REIS, the rickettsial endosymbiont of Ixodes scapularis. All of the rickettsiae were isolated from humans or North and South American ticks. R. parkeri isolates from both continents did not possess plasmids. We have now demonstrated plasmids in nearly all Rickettsia species that we have surveyed from three continents, which represent three of the four major proposed phylogenetic groups associated with blood-feeding arthropods. Gel-based evidence consistent with the existence of multiple plasmids in some species was confirmed by cloning plasmids with very different sequences from each of two "Ca. Rickettsia amblyommii" isolates. Phylogenetic analysis of rickettsial ParA plasmid partitioning proteins indicated multiple parA gene origins and plasmid incompatibility groups, consistent with possible multiple plasmid origins. Phylogenetic analysis of potentially host-adaptive rickettsial small heat shock proteins showed that hsp2 genes were plasmid specific and that hsp1 genes, found only on plasmids of "Ca. Rickettsia amblyommii," R. felis, R. monacensis, and R. peacockii, were probably acquired independently of the hsp2 genes. Plasmid copy numbers in seven Rickettsia species ranged from 2.4 to 9.2 per chromosomal equivalent, as determined by real-time quantitative PCR. Plasmids may be of significance in rickettsial evolution and epidemiology by conferring genetic plasticity and host-adaptive traits via horizontal gene transfer that counteracts the reductive genome evolution typical of obligate intracellular bacteria.

Rickettsia amblyommii infecting Amblyomma americanum larvae.

Polymerase chain reaction analysis of Amblyomma americanum adults, nymphs, and larvae from Aberdeen Proving Ground, MD (APG), revealed a very high prevalence of a spotted fever group (SFG) rickettsia. Restriction fragment length polymorphism (RFLP) and sequence analysis identified "Rickettsia amblyommii." This organism is not yet described or well studied, and its pathogenicity is unknown; however, investigations of the organism are warranted because of its high prevalence in A. americanum. This tick is extremely abundant at military training facilities in the south, central, and Mid-Atlantic United States, and many soldiers experience multiple concurrent tick bites. Bites by R. amblyommii-infected A. americanum may account for rates of SFG rickettsia seropositivity that are higher than reported rates of Rocky Mountain spotted fever (RMSF) cases from the same location. Seroconversion to SFG rickettsia following bites of A. americanum may suggest that R. amblyommii is infectious in humans. Subclinical infection in the numerous A. americanum tick bite victims could contaminate donated blood and compromise immunodeficient recipients. Detection of R. amblyommii in questing A. americanum larvae suggests transovarial transmission. The absence of R. rickettsii, the agent of RMSF, in A. americanum may be due to transovarial interference by R. amblyommii. The likelihood of pathogen transmission by larvae is magnified by their habit of mass attack. The very small size of the larvae is also a risk factor for pathogen transmission. High R. amblyommii prevalence in populations of A. americanum presage co-infection with other A. americanum-borne pathogens. A. americanum nymphs and adults from APG were found to be co-infected with R. amblyommii and Borrelia lonestari, Ehrlichia chaffeensis and Ehrlichia ewingii, respectively, and larval pools were infected with both R. amblyommii and B. lonestari. Co-infections can compound effects and complicate diagnosis of tick-borne disease.