Rickettsiales in Ticks Removed from Outdoor Workers, Southwest Georgia and Northwest Florida, USA.

We determined the prevalence of selected Rickettsiales in 362 ticks removed from outdoor workers in southwest Georgia and northwest Florida, USA. Persons submitted an average of 1.1 ticks/month. We found Ehrlichia chaffeensis in an Amblyomma maculatum tick, and Panola Mountain Ehrlichia sp. in 2 A. maculatum ticks and 1 Dermacentor variabilis tick.

Ebola Virus Disease Diagnostics, Sierra Leone: Analysis of Real-time Reverse Transcription-Polymerase Chain Reaction Values for Clinical Blood and Oral Swab Specimens.

During the Ebola virus outbreak of 2013-2016, the Viral Special Pathogens Branch field laboratory in Sierra Leone tested approximately 26 000 specimens between August 2014 and October 2015. Analysis of the B2M endogenous control Ct values showed its utility in monitoring specimen quality, comparing results with different specimen types, and interpretation of results. For live patients, blood is the most sensitive specimen type and oral swabs have little diagnostic utility. However, swabs are highly sensitive for diagnostic testing of corpses.

Phylogeography of Rhipicephalus sanguineus sensu lato and its relationships with climatic factors.

Brown dog ticks morphologically identifiable as Rhipicephalus sanguineus sensu lato, are distributed world-wide and their systematics is controversial. Results of genetic and reproductive compatibility studies of geographically distinct populations of R. sanguineus s.l. indicate that the R. sanguineus complex is paraphyletic. To further elucidate systematic relationships within R. sanguineus s.l. and geographic boundaries of its lineages, we conducted a phylogeographical study of 136 tick specimens from 23 countries. Voucher specimens were morphologically identified. A phylogenetic tree was constructed using concatenated partial mitochondrial 12S and 16S rDNA gene sequences and analyzed by the Neighbor-Joining method. A set of 19 bioclimatic variables within the WorldClim dataset were extracted and analyzed to assess correlations between distribution of R. sanguineus s.l. lineages and climatic variables. The following four branches are clearly recognized on the phylogenetic tree: R. sanguineus s.l.-tropical and temperate clades, R. leporis, and R. turanicus. DNA sequences of Rhipicephalus ticks from Israel differ from those of other groups. Strong association between geographical locations of major clades of R. sanguineus s.l. and temperature was identified. The tropical clade of R. sanguineus s.l. occupies areas with the annual mean temperature >20 °C, whereas the temperate clade is present in areas with the annual mean temperature <20 °C. Our results indicate that ticks in two closely related phylogenetic clades are adapted to different environmental conditions and support proposals for re-classification of R. sanguineus complex. Differences in R. sanguineus s.l. ecology and human/animal pathogens transmitted by different taxa of brown dog tick need to be studied.

Ebola Virus Diagnostics: The US Centers for Disease Control and Prevention Laboratory in Sierra Leone, August 2014 to March 2015.

In August 2014, the Viral Special Pathogens Branch of the US Centers for Disease Control and Prevention established a field laboratory in Sierra Leone in response to the ongoing Ebola virus outbreak. Through March 2015, this laboratory tested >12 000 specimens from throughout Sierra Leone. We describe the organization and procedures of the laboratory located in Bo, Sierra Leone.

Multiplex qPCR assay for identification and differentiation of Amblyomma americanum, Amblyomma cajennense, and Amblyomma maculatum (Ixodida: Ixodidae) tick species in the eastern United States.

Many ticks of the genus Amblyomma are vectors of human pathogens, and the correct species identification is medically and epidemiologically important. Morphological identification is time-consuming and requires a high level of expertise. Identification of engorged, immature, or damaged ticks and the differentiation of closely related species remain problematic. Here, we report the development of a real-time TaqMan assay for the genomic identification and differentiation of Amblyomma americanum (L.), Amblyomma cajennense (F.), and Amblyomma maculatum (Koch), which are human-biting species found in the eastern United States. New species-specific sets of oligonucleotides for the multiplex reaction that detect and differentiate the ITS2 genomic regions of three target species were designed using Visual OMP; the previously published A. americanum oligonucleotide set was also incorporated into our assay. Specificity and sensitivity tests for two multiplex master mixes using different A. americanum sets were performed using individual and pooled samples of adult, nymphal, and larval ticks, and optimization procedures were applied. The multiplex assay successfully differentiates between genomes of three target species and does not cross-react with DNAs of ticks from other genera. Rare cases of nonspecific amplification occurred with DNAs of A. imitator and Amblyomma triste Koch misidentified as A. americanum and A. maculatum, respectively. However, this cross-reaction does not diminish the usefulness of the developed assay east of the 95th meridian, where neither A. imitator nor A. triste are found. Two master mixes incorporating the previously published or newly developed A. americanum sets are being recommended for identification of individual ticks or pooled samples, respectively.

Detection of a novel spotted fever group Rickettsia in the gophertortoise tick.

The gophertortoise tick, Amblyomma tuberculatum (Marx), is distributed throughout the southeastern United States, and its immature life stages have been reported to occasionally bite humans. Here we report detection of a novel spotted fever group (SFG) Rickettsia in A. tuberculatum ticks collected in the southern United States. Among questing ticks collected in Georgia, 10 pools of larvae were identified as gophertortoise ticks, A. tuberculatum. Each of these samples was positive for SFG Rickettsiae. The restriction fragment-length polymorphism profiles were identical to each other, but distinct from those of other rickettsiae previously found in Amblyomma spp. ticks. Partial genetic characterization of the novel agent was achieved by sequencing the 17 kDa, gltA, ompB, ompA, rpoB, and sca4 genes. Analysis of a concatenated tree of four genes (gltA, ompB, ompA, and sca4) demonstrates close relatedness of the detected Rickettsia to several SFG Rickettsia spp. The identical rickettsial DNA was detected in 50 and 70% of adult A. tuberculatum ticks from Mississippi and Florida, respectively. The results indicate wide distribution of a novel Rickettsia, capability for transovarial transmission, and high prevalence in tested tick populations.

Frequent Prescribed Fires Can Reduce Risk of Tick-borne Diseases.

Recently, a two-year study found that long-term prescribed fire significantly reduced tick abundance at sites with varying burn regimes (burned surrounded by burned areas [BB], burned surrounded by unburned areas [BUB], and unburned surrounded by burned areas [UBB]). In the current study, these ticks were tested for pathogens to more directly investigate the impacts of long-term prescribed burning on human disease risk. A total of 5,103 ticks (4,607 Amblyomma americanum, 76 Amblyomma maculatum, 383 Ixodes scapularis, two Ixodes brunneus, and 35 Dermacentor variabilis) were tested for Borrelia spp., Rickettsia spp., Ehrlichia spp., and Anaplasma phagocytophilum. Long-term prescribed fire did not significantly impact pathogen prevalence except that A. americanum from burned habitats had significantly lower prevalence of Rickettsia (8.7% and 4.6% for BUB and UBB sites, respectively) compared to ticks from control sites (unburned, surrounded by unburned [UBUB])(14.6%). However, during the warm season (spring/summer), encounter rates with ticks infected with pathogenic bacteria was significantly lower (98%) at burned sites than at UBUB sites. Thus, despite there being no differences in pathogen prevalence between burned and UBUB sites, risk of pathogen transmission is lower at sites subjected to long-term burning due to lower encounter rates with infected ticks.

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.

Comparative value of blood and skin samples for diagnosis of spotted fever group rickettsial infection in model animals.

The definitive diagnosis of spotted fever group (SFG) rickettsioses in humans is challenging due to the retrospective nature and cross reactivity of the serological methods and the absence of reliable and consistent samples for molecular diagnostics. Existing data indicate the transient character of bacteremia in experimentally infected animals. The ability of arthropod vectors to acquire rickettsial infection from the laboratory animals in the absence of systemic infection and known tropism of rickettsial agents to endothelial cells of peripheral blood vessels underline the importance of local infection and consequently the diagnostic potential of skin samples. In order to evaluate the diagnostic sensitivity of rickettsial DNA detection in blood and skin samples, we compared results of PCR testing in parallel samples collected from model laboratory animals infected with Rickettsia rickettsii, Rickettsia parkeri and Rickettsia slovaca-like agent at different time points after infection. Skin samples were collected from ears - away from the site of tick placement and without eschars. Overall, testing of skin samples resulted in a higher proportion of positive results than testing of blood samples. Presented data from model animals demonstrates that testing of skin samples from sites of rickettsial proliferation can provide definitive molecular diagnosis of up to 60-70% of tick-borne SFG rickettsial infections during the acute stage of illness. Detection of pathogen DNA in cutaneous samples is a valuable alternative to blood-PCR at least in model animals.

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.

Relative sensitivity of conventional and real-time PCR assays for detection of SFG Rickettsia in blood and tissue samples from laboratory animals.

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.

Clinical presentation, convalescence, and relapse of rocky mountain spotted fever in dogs experimentally infected via tick bite.

Rocky Mountain spotted fever (RMSF) is a tick-borne disease caused by R. rickettsii in North and South America. Domestic dogs are susceptible to infection and canine RMSF can be fatal without appropriate treatment. Although clinical signs of R. rickettsii infection in dogs have been described, published reports usually include descriptions of either advanced clinical cases or experimental infections caused by needle-inoculation of cultured pathogen rather than by tick bite. The natural progression of a tick-borne R. rickettsii infection has not been studied in sufficient detail. Here, we provide a detailed description of clinical, hematological, molecular, and serological dynamics of RMSF in domestic dogs from the day of experimental exposure to infected ticks through recovery. Presented data indicate that neither the height/duration of fever nor detection of rickettsial DNA in dogs' blood by PCR are good indicators for clinical prognosis. Only the apex and subsequent subsidence of neutrophilia seem to mark the beginning of recovery and allow predicting a favorable outcome in Rickettsia-infected dogs, even despite the continuing persistence of mucosal petechiae and skin rash. On the other hand the appropriate (doxycycline) antibiotic therapy of sufficient duration is crucial in prevention of RMSF relapses in dogs.

The phenology of ticks and the effects of long-term prescribed burning on tick population dynamics in southwestern Georgia and northwestern Florida.

Some tick populations have increased dramatically in the past several decades leading to an increase in the incidence and emergence of tick-borne diseases. Management strategies that can effectively reduce tick populations while better understanding regional tick phenology is needed. One promising management strategy is prescribed burning. However, the efficacy of prescribed burning as a mechanism for tick control is unclear because past studies have provided conflicting data, likely due to a failure of some studies to simulate operational management scenarios and/or account for other predictors of tick abundance. Therefore, our study was conducted to increase knowledge of tick population dynamics relative to long-term prescribed fire management. Furthermore, we targeted a region, southwestern Georgia and northwestern Florida (USA), in which little is known regarding tick dynamics so that basic phenology could be determined. Twenty-one plots with varying burn regimes (burned surrounded by burned [BB], burned surrounded by unburned [BUB], unburned surrounded by burned [UBB], and unburned surrounded by unburned [UBUB]) were sampled monthly for two years while simultaneously collecting data on variables that can affect tick abundance (e.g., host abundance, vegetation structure, and micro- and macro-climatic conditions). In total, 47,185 ticks were collected, of which, 99% were Amblyomma americanum, 0.7% were Ixodes scapularis, and fewer numbers of Amblyomma maculatum, Ixodes brunneus, and Dermacentor variabilis. Monthly seasonality trends were similar between 2010 and 2011. Long-term prescribed burning consistently and significantly reduced tick counts (overall and specifically for A. americanum and I. scapularis) regardless of the burn regimes and variables evaluated. Tick species composition varied according to burn regime with A. americanum dominating at UBUB, A. maculatum at BB, I. scapularis at UBB, and a more even composition at BUB. These data indicate that regular prescribed burning is an effective tool for reducing tick populations and ultimately may reduce risk of tick-borne disease.

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.