Rickettsia species identified in adult, host-seeking Dermacentor occidentalis (Acari: Ixodidae) from Baja California, Mexico, and Oregon and Washington, United States.

The Pacific Coast tick (Dermacentor occidentalis Marx, 1892) is a frequently encountered and commonly reported human-biting tick species that has been recorded from most of California and parts of southwestern Oregon, southcentral Washington, and northwestern Mexico. Although previous investigators have surveyed populations of D. occidentalis for the presence of Rickettsia species across several regions of California, populations of this tick have not been surveyed heretofore for rickettsiae from Baja California, Oregon, or Washington. We evaluated 1,367 host-seeking, D. occidentalis adults collected from 2015 to 2022 by flagging vegetation at multiple sites in Baja California, Mexico, and Oregon and Washington, United States, using genus- and species-specific assays for spotted fever group rickettsiae. DNA of Rickettsia 364D, R. bellii, and R. tillamookensis was not detected in specimens from these regions. DNA of R. rhipicephali was detected in D. occidentalis specimens obtained from Ensenada Municipality in Baja California and southwestern Oregon, but not from Washington. All ompA sequences of R. rhipichephali that were amplified from individual ticks in southwestern Oregon were represented by a single genotype. DNA of the Ixodes pacificus rickettsial endosymbiont was amplified from specimens collected in southwestern Oregon and Klickitat County, Washington; to the best of our knowledge, this Rickettsia species has never been identified in D. occidentalis. Collectively, these data are consistent with a relatively recent introduction of Pacific Coast ticks in the northernmost extension of its recognized range.

Acquisition of Rickettsia rickettsii (Rickettsiales: Rickettsiaceae) by Haemaphysalis longicornis (Acari: Ixodidae) through co-feeding with infected Dermacentor variabilis (Acari: Ixodidae) in the laboratory.

Haemaphysalis longicornis (Neumann) is an invasive ixodid tick originating from eastern Asia which recently has become established in the United States. In its native range, this tick can transmit several pathogens to animals and humans, but little is known about its ability to acquire and transmit pathogens native to the United States. Geographic overlap with ticks such as Dermacentor variabilis (Say), a known vector of Rickettsia rickettsii, makes investigation into the interactions between H. longicornis and D. variabilis of interest to the public health community. Previous studies have shown that H. longicornis can serve as a competent vector of R. rickettsii under laboratory settings, but there is little information on its ability to acquire this pathogen via other biologically relevant routes, such as co-feeding. Here, we assess the ability of H. longicornis nymphs to acquire R. rickettsii through co-feeding with infected D. variabilis adults on a vertebrate animal model under laboratory conditions. The median infection prevalence in engorged H. longicornis nymphs across 8 cohorts was 0% with an interquartile range (IQR) of 4.13%. Following transstadial transmission, the median infection prevalence in flat females was 0.7% (IQR = 2.4%). Our results show that co-feeding transmission occurs at low levels in the laboratory between these 2 species. However, based on the relatively low transmission rates, this may not be a likely mechanism of R. rickettsii introduction to H. longicornis.

Evaluating the Clinical and Immune Responses to Spotted Fever Rickettsioses in the Guinea Pig-Tick-Rickettsia System.

The guinea pig was the original animal model developed for investigating spotted fever rickettsiosis (SFR). This model system has persisted on account of the guinea pig's conduciveness to tick transmission of SFR agents and ability to recapitulate SFR in humans through clinical signs that include fever, unthriftiness, and in some cases the development of an eschar. The guinea pig is the smallest animal model for SFR that allows the collection of multiple blood and skin samples antemortem for longitudinal studies. This unit provides the basic protocols necessary to establish, maintain, and utilize a guinea pig-tick-Rickettsia model for monitoring the course of infection and immune response to an infection by spotted fever group Rickettsia (SFGR) that can be studied at biosafety level 2 (BSL-2) and arthropod containment level 2 (ACL-2); adaptations must be made for BSL-3 agents. The protocols cover methods for tick feeding and colony development, laboratory infection of ticks, tick transmission of Rickettsia to guinea pigs, and monitoring of the course of infection through clinical signs, rickettsial burden, and immune response. It should be feasible to adapt these methods to study other tick-borne pathogens. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Tick transmission of SFGR to guinea pigs Support Protocol 1: Laboratory infection of ticks by injection Alternate Protocol 1: Needle inoculation of SFGR to guinea pigs Basic Protocol 2: Monitoring the course of guinea pig rickettsial infection: clinical signs Basic Protocol 3: Monitoring the course of guinea pig rickettsial infection: collection of biological specimens Support Protocol 2: Guinea pig anesthesia Basic Protocol 4: Monitoring rickettsial burden in guinea pigs by multiplex qPCR Basic Protocol 5: Monitoring guinea pig immune response to infection: blood leukocytes by flow cytometry Basic Protocol 6: Monitoring immune response to guinea pig rickettsial infection: leukocyte infiltration of skin at the tick bite site by flow cytometry Basic Protocol 7: Monitoring the immune response to guinea pig rickettsial infection: antibody titer by ELISA Support Protocol 4: Coating ELISA Plates Alternate Protocol 2: Monitoring immune response to guinea pig rickettsial infection: antibody titer by immunofluorescence assay.

Predicting the northward expansion of tropical lineage Rhipicephalus sanguineus sensu lato ticks in the United States and its implications for medical and veterinary health.

The tropical lineage within the Rhipicephalus sanguineus species complex is cause for growing concern in the U.S. based on its prominent role in creating and perpetuating multiple recently identified outbreaks of Rocky Mountain spotted fever in the southwestern United States and northern Mexico. This lineage is undergoing a northward range expansion in the United States, necessitating the need for enhanced surveillance for Rh. sanguineus. To inform more focused surveillance efforts we use species distribution models (SDMs) to predict current (2015-2019) and future (2021-2040) habitat for the tropical lineage. Models using the MaxEnt algorithm were informed using geolocations of ticks genetically confirmed to be of the tropical lineage, for which data on 23 climatic and ecological variables were extracted. Models predicted that suitability was optimal where temperatures are relatively warm and stable, and there is minimal precipitation. This translated into habitat being predicted along much of the coast of southern states including California, Texas, Louisiana, and Florida. Although the endophilic nature of tropical Rh. sanguineus somewhat violates the assumptions of SDMs, our models correctly predicted known locations of this tick and provide a starting point for increased surveillance efforts. Furthermore, we highlight the importance of using molecular methods to distinguish between ticks in the Rh. sanguineus species complex.

Duration of tick attachment necessary for transmission of Anaplasma phagocytophilum by Ixodes scapularis (Acari: Ixodidae) nymphs.

This study assessed the duration of tick attachment necessary for a successful transmission of Anaplasma phagocytophilum by an infected I. scapularis nymph. Individual nymphs were placed upon BALB/c mice and allowed to feed for predetermined time intervals of 4 to 72 h. Ticks removed from mice at predetermined intervals were tested by PCR for verification of infection and evaluation of the bacterial load. The success of pathogen transmission to mice was assessed by blood-PCR at 7, 14 and 21 days postinfestation, and IFA at 21 days postinfestation. Anaplasma phagocytophilum infection was documented in 10-30 % of mice, from which ticks were removed within the first 20 h of feeding. However, transmission success was ≥70% if ticks remained attached for 36 h or longer. Notably, none of the PCR-positive mice that were exposed to infected ticks for 4 to 8 h and only half of PCR-positive mice exposed for 24 h developed antibodies within 3 weeks postinfestation. On the other hand, all mice with detectable bacteremia after being infested for 36 h seroconverted. This suggests that although some of the ticks removed prior to 24 h of attachment succeed in injecting a small amount of A. phagocytophilum, this amount is insufficient for stimulating humoral immunity and perhaps for establishing disseminated infection in BALB/c mice. Although A. phagocytophilum may be present in salivary glands of unfed I. scapularis nymphs, the amount of A. phagocytophilum initially contained in saliva appears insufficient to cause sustainable infection in a host. Replication and, maybe, reactivation of the agent for 12-24 h in a feeding tick is required before a mouse can be consistently infected.

Tick extracellular vesicles enable arthropod feeding and promote distinct outcomes of bacterial infection.

Extracellular vesicles are thought to facilitate pathogen transmission from arthropods to humans and other animals. Here, we reveal that pathogen spreading from arthropods to the mammalian host is multifaceted. Extracellular vesicles from Ixodes scapularis enable tick feeding and promote infection of the mildly virulent rickettsial agent Anaplasma phagocytophilum through the SNARE proteins Vamp33 and Synaptobrevin 2 and dendritic epidermal T cells. However, extracellular vesicles from the tick Dermacentor andersoni mitigate microbial spreading caused by the lethal pathogen Francisella tularensis. Collectively, we establish that tick extracellular vesicles foster distinct outcomes of bacterial infection and assist in vector feeding by acting on skin immunity. Thus, the biology of arthropods should be taken into consideration when developing strategies to control vector-borne diseases.

Incompetence of the Asian Longhorned Tick (Acari: Ixodidae) in Transmitting the Agent of Human Granulocytic Anaplasmosis in the United States.

The Asian longhorned tick, Haemaphysalis longicornis Neumann (Acari: Ixodidae), was recently introduced into the United States and is now established in at least 15 states. Considering its ability for parthenogenetic propagation and propensity for creating high-density populations, there is concern that this tick may become involved in transmission cycles of endemic tick-borne human pathogens. Human granulocytic anaplasmosis (HGA) caused by Anaplasma phagocytophilum is one of the more common tick-borne diseases in the United States, especially in the northeastern and midwestern states. There is considerable geographical overlap between HGA cases and the currently known distribution of H. longicornis, which creates a potential for this tick to encounter A. phagocytophilum while feeding on naturally infected vertebrate hosts. Therefore, we evaluated the ability of H. longicornis to acquire and transmit the agent of HGA under laboratory conditions and compared it to the vector competence of I. scapularis. Haemaphysalis longicornis nymphs acquired the pathogen with the bloodmeal while feeding on infected domestic goats, but transstadial transmission was inefficient and PCR-positive adult ticks were unable to transmit the pathogen to naïve goats. Results of this study indicate that the Asian longhorned tick is not likely to play a significant role in the epidemiology of HGA in the United States.

Assessment of the Pathogenicity of Rickettsia amblyommatis, Rickettsia bellii, and Rickettsia montanensis in a Guinea Pig Model.

Members of the genus Rickettsia range from nonpathogenic endosymbionts to virulent pathogens such as Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever. Many rickettsiae are considered nonpathogenic because they have been isolated from ticks but not vertebrate hosts. We assessed the ability of three presumed endosymbionts: Rickettsia amblyommatis, Rickettsia bellii, and Rickettsia montanensis, to infect a guinea pig animal model. These species were chosen because of their high prevalence in respective tick vectors or published reports suggestive of human or animal pathogenicity. Following intraperitoneal (IP) inoculation of cell culture suspensions of R. rickettsii, R. amblyommatis, R. bellii, or R. montanensis into guinea pigs, animals were monitored for signs of clinical illness for 13 days. Ear biopsies and blood samples were taken at 2- to 3-day intervals for detection of rickettsial DNA by PCR. Animals were necropsied and internal organ samples were also tested using PCR assays. Among the six guinea pigs inoculated with R. amblyommatis, fever, orchitis, and dermatitis were observed in one, one, and three animals respectively. In R. bellii-exposed animals, we noted fever in one of six animals, orchitis in one, and dermatitis in two. No PCR-positive tissues were present in either the R. amblyommatis- or R. bellii-exposed groups. In the R. montanensis-exposed group, two of six animals became febrile, two had orchitis, and three developed dermatitis in ears or footpads. R. montanensis DNA was detected in ear skin biopsies collected on multiple days from three animals. Also, a liver specimen from one animal and spleen specimens of two animals were PCR positive. The course and severity of disease in the three experimental groups were significantly milder than that of R. rickettsii. This study suggests that the three rickettsiae considered nonpathogenic can cause either subclinical or mild infections in guinea pigs when introduced via IP inoculation.

Reproductive incompatibility between Amblyomma maculatum (Acari: Ixodidae) group ticks from two disjunct geographical regions within the USA.

The Amblyomma maculatum Koch group of ixodid ticks consists of three species: A. maculatum, A. triste, and A. tigrinum. However, since Koch described this group in 1844, the systematics of its members has been the subject of ongoing debate. This is especially true of A. maculatum and A. triste; recent molecular analyses reveal insufficient genetic divergence to separate these as distinct species. Further confounding this issue is the discovery in 2014 of A. maculatum group ticks in southern Arizona (AZ), USA, that share morphological characteristics with both A. triste and A. maculatum. To biologically evaluate the identity of A. maculatum group ticks from southern Arizona, we analyzed the reproductive compatibility between specimens of A. maculatum group ticks collected from Georgia (GA), USA, and southern Arizona. Female ticks from both Arizona and Georgia were mated with males from both the Georgia and Arizona Amblyomma populations, creating two homologous and two heterologous F1 cohorts of ticks: GA ♀/GA ♂, AZ ♀/AZ ♂, GA ♀/AZ ♂, and AZ ♀/GA ♂. Each cohort was maintained separately into the F2 generation with F1 females mating only with F1 males from their same cohort. Survival and fecundity parameters were measured for all developmental stages. The observed survival parameters for heterologous cohorts were comparable to those of the homologous cohorts through the F1 generation. However, the F1 heterologous females produced F2 egg clutches that did not hatch, thus indicating that the Arizona and Georgia populations of A. maculatum group ticks tested here represent different biological species.

Vector competence of Rhipicephalus sanguineus sensu stricto for Anaplasma platys.

Anaplasma platys is a Gram-negative, obligate intracellular bacteria that causes canine infectious cyclic thrombocytopenia in dogs. The brown dog tick Rhipicephalus sanguineus sensu lato is presumed to be the vector of A. platys based on the overlap in distribution of R. sanguineus and A. platys infections, detection of A. platys DNA in both flat and engorged field-collected R. sanguineus, and the fact that dogs are primary hosts of both brown dog ticks and A. platys. However, the only study evaluating the vector competence of R. sanguineus for A. platys under controlled laboratory conditions reported an apparent inability of ticks to acquire or maintain the pathogen. In 2016, engorged female Rhipicephalus sanguineus sensu stricto ticks were collected off dogs to start a laboratory tick colony. After one generation of colony maintenance on tick-naïve and pathogen-free New Zealand White rabbits, a rabbit used to feed F1 adults seroconverted to Anaplasma phagocytophilum antigen. PCR and subsequent DNA sequencing identified the presence of A. platys in both the adult ticks fed on this rabbit and their resulting F2 progenies. Retrospective testing of all previous (P and F1) life stages of this colony demonstrated that the infection originated from one field-collected A. platys-infected female whose progeny was propagated in the laboratory and produced the PCR-positive F1 adults. Over the following (F2-F4) generations, the prevalence of A. platys in this colony reached 90-100 % indicating highly efficient transovarial and horizontal transmission, as well as transstadial maintenance, of this pathogen by R. sanguineus s.s.

The Ability of the Invasive Asian Longhorned Tick Haemaphysalis longicornis (Acari: Ixodidae) to Acquire and Transmit Rickettsia rickettsii (Rickettsiales: Rickettsiaceae), the Agent of Rocky Mountain Spotted Fever, Under Laboratory Conditions.

The invasive Asian longhorned tick, Haemaphysalis longicornis Neumann, was first detected in the United States in 2017. It has since been found in 12 states, and there is concern that the tick's parthenogenetic ability and wide variety of host species may allow for broader dissemination. Of the tick-borne diseases endemic to the United States, Rocky Mountain spotted fever (RMSF), a rapidly progressive and potentially fatal disease caused by Rickettsia rickettsii, is the most severe. There is considerable geographical overlap between spotted fever rickettsioses cases, which include RMSF, and the currently known distribution of H. longicornis, providing the potential for this tick to encounter this pathogen. We have evaluated the ability of H. longicornis to acquire and transmit R. rickettsii under laboratory conditions. Haemaphysalis longicornis as larvae and nymphs acquired the pathogen while feeding on infected guinea pigs. The infection persisted through every life stage, all of which were able to transmit R. rickettsii to naïve hosts. The pathogen was also transmitted at a low frequency between generations of H. longicornis through the ova. While H. longicornis was demonstrated to be a competent vector for R. rickettsii under laboratory conditions, the probability of its involvement in the maintenance and transmission of this pathogen in nature, as well as its potential impact on human health, requires further study.

Preliminary Evaluation of Human Personal Protective Measures Against the Nymphal Stage of the Asian Longhorned Tick (Acari: Ixodidae).

The invasive, human-biting Asian longhorned tick, Haemaphysalis longicornis Neumann, is establishing in the United States. This tick is a threat to public health in its native range in Asia, serving as a vector of severe fever with thrombocytopenia syndrome virus and Rickettsia japonica, the agent of Japanese spotted fever. However, there is a lack of published information specifically for H. longicornis concerning the efficacy of generally recommended personal tick bite prevention measures. We, therefore, evaluated permethrin-treated clothing and formulated human skin repellent products, representing the six repellent active ingredients generally recommended for tick bite prevention by the Centers for Disease Control and Prevention (CDC), against H. longicornis nymphs from a colony established with adult ticks collected in New York state. Reluctance of H. longicornis nymphs to stay in contact with nontreated human skin precluded the use of a human skin bioassay to optimally evaluate repellency. In a Petri dish choice bioassay, all tested product formulations were highly effective with estimated repellencies ranging from 93 to 97%. In addition, we observed strong contact irritancy of a summer-weight permethrin-treated garment against H. longicornis nymphs, with 96% of introduced ticks dislodging from the vertically oriented textile within 3 min. These preliminary studies indicate that personal tick bite prevention measures currently recommended by the CDC are effective against the invasive H. longicornis. However, additional studies are needed to explore the efficacy of the evaluated products against different life stages of H. longicornis, as well as ticks collected in the field rather than reared in the laboratory.

Minimal Duration of Tick Attachment Sufficient for Transmission of Infectious Rickettsia rickettsii (Rickettsiales: Rickettsiaceae) by Its Primary Vector Dermacentor variabilis (Acari: Ixodidae): Duration of Rickettsial Reactivation in the Vector Revisited.

It has been reported that starving ticks do not transmit spotted fever group Rickettsia immediately upon attachment because pathogenic bacteria exist in a dormant, uninfectious state and require time for 'reactivation' before transmission to a susceptible host. To clarify the length of reactivation period, we exposed guinea pigs to bites of Rickettsia rickettsii-infected Dermacentor variabilis (Say) and allowed ticks to remain attached for predetermined time periods from 0 to 48 h. Following removal of attached ticks, salivary glands were immediately tested by PCR, while guinea pigs were observed for 10-12 d post-exposure. Guinea pigs in a control group were subcutaneously inoculated with salivary glands from unfed D. variabilis from the same cohort. In a parallel experiment, skin at the location of tick bite was also excised at the time of tick removal to ascertain dissemination of pathogen from the inoculation site. Animals in every exposure group developed clinical and pathological signs of infection. The severity of rickettsial infection in animals increased with the length of tick attachment, but even attachments for less than 8 h resulted in clinically identifiable infection in some guinea pigs. Guinea pigs inoculated with salivary glands from unfed ticks also became severely ill. Results of our study indicate that R. rickettsii residing in salivary glands of unfed questing ticks does not necessarily require a period of reactivation to precede the salivary transmission and ticks can transmit infectious Rickettsia virtually as soon as they attach to the host.

Failure of the Asian longhorned tick, Haemaphysalis longicornis, to serve as an experimental vector of the Lyme disease spirochete, Borrelia burgdorferi sensu stricto.

The invasive, human-biting Asian longhorned tick, Haemaphysalis longicornis, was detected in New Jersey in the eastern United States in August of 2017 and by November of 2018 this tick had been recorded from 45 counties across 9 states, primarily along the Eastern Seaboard. The establishment of H. longicornis in the United States has raised the questions of how commonly it will bite humans and which native pathogens may naturally infect this tick. There also is a need for experimental vector competence studies with native pathogens to determine if H. longicornis can acquire a given pathogen while feeding, pass it transstadially, and then transmit the pathogen in the next life stage. In this experimental study, we evaluated the vector competence of a population of H. longicornis originating from the United States (New York) for a native isolate (B31) of the Lyme disease spirochete, Borrelia burgdorferi sensu stricto (s.s.). In agreement with a previous experimental study on the vector competence of H. longicornis for Borrelia garinii, we found that uninfected H. longicornis larvae could acquire B. burgdorferi s.s. while feeding on infected Mus musculus mice (infection prevalence >50% in freshly fed larvae) but that the infection was lost during the molt to the nymphal stage. None of 520 tested molted nymphs were found to be infected, indicating that transstadial passage of B. burgdorferi s.s. is absent or rare in H. longicornis; and based on the potential error associated with the number of nymphs testing negative in this study, we estimate that the upper 95% limit for infection prevalence was 0.73%. An Ixodes scapularis process control showed both effective acquisition of B. burgdorferi s.s. from infected mice by uninfected larvae and transstadial passage to the nymphal stage (infection prevalence of 80-82% for both freshly fed larvae and molted nymphs). We also observed that although H. longicornis larvae could be compelled to feed on mice by placing the ticks within feeding capsules, attachment and feeding success was minimal (<0.5%) when larvae were placed freely on the fur of the mice. We conclude that H. longicornis is unlikely to contribute more than minimally, if at all, to transmission of Lyme disease spirochetes in the United States.

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.

Extensive Ethnic Variation and Linkage Disequilibrium at the FCGR2/3 Locus: Different Genetic Associations Revealed in Kawasaki Disease.

The human Fc-gamma receptors (FcγRs) link adaptive and innate immunity by binding immunoglobulin G (IgG). All human low-affinity FcγRs are encoded by the FCGR2/3 locus containing functional single nucleotide polymorphisms (SNPs) and gene copy number variants. This locus is notoriously difficult to genotype and high-throughput methods commonly used focus on only a few SNPs. We performed multiplex ligation-dependent probe amplification for all relevant genetic variations at the FCGR2/3 locus in >4,000 individuals to define linkage disequilibrium (LD) and allele frequencies in different populations. Strong LD and extensive ethnic variation in allele frequencies was found across the locus. LD was strongest for the FCGR2C-ORF haplotype (rs759550223+rs76277413), which leads to expression of FcγRIIc. In Europeans, the FCGR2C-ORF haplotype showed strong LD with, among others, rs201218628 (FCGR2A-Q27W, r2 = 0.63). LD between these two variants was weaker (r2 = 0.17) in Africans, whereas the FCGR2C-ORF haplotype was nearly absent in Asians (minor allele frequency <0.005%). The FCGR2C-ORF haplotype and rs1801274 (FCGR2A-H131R) were in weak LD (r2 = 0.08) in Europeans. We evaluated the importance of ethnic variation and LD in Kawasaki Disease (KD), an acute vasculitis in children with increased incidence in Asians. An association of rs1801274 with KD was previously shown in ethnically diverse genome-wide association studies. Now, we show in 1,028 European KD patients that the FCGR2C-ORF haplotype, although nearly absent in Asians, was more strongly associated with susceptibility to KD than rs1801274 in Europeans. Our data illustrate the importance of interpreting findings of association studies concerning the FCGR2/3 locus with knowledge of LD and ethnic variation.

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.

Effects of Rickettsia amblyommatis Infection on the Vector Competence of Amblyomma americanum Ticks for Rickettsia rickettsii.

Although Dermacentor spp. ticks are considered the primary vectors of Rickettsia rickettsii in the United States, other North American tick species are also capable of transmitting the agent, including the lone star tick-Amblyomma americanum. The lone star tick is an aggressive human-biting tick abundant in the South, Central, and Mid-Atlantic United States, which has been shown to be a competent vector of R. rickettsii in laboratory studies. However in nature, A. americanum frequently carry Rickettsia amblyommatis-another member of the spotted fever group-with the prevalence of infection reaching 84% in some populations. It has been postulated that the presence of an endosymbiotic Rickettsia in a significant proportion of a vector population would diminish or even block transmission of pathogenic Rickettsia in ticks from generation to generation due to transovarial interference. We measured the ability of R. amblyommatis-infected A. americanum to acquire R. rickettsii from an infected host with a bloodmeal, and transmit it transstadially, horizontally (to a susceptible host), and vertically to the next generation. Larvae from both the R. amblyommatis-infected and R. amblyommatis-free cohorts acquired R. rickettsii from infected guinea pigs, but the presence of the symbiont diminished the ability of coinfected engorged larvae to transmit R. rickettsii transstadially. Conversely, acquisition of R. rickettsii by cofeeding was unaffected in R. amblyommatis-infected nymphs and adults; prevalence of R. rickettsii in engorged adults reached 97% in both R. amblyommatis-infected and R. amblyommatis-free cohorts. In guinea pigs exposed to dually infected nymphs, R. rickettsii infection was milder than in those fed upon nymphs infected with R. rickettsii only. The frequency of transovarial transmission of R. rickettsii in the R. amblyommatis-infected cohort (31%) appeared lower than that in the R. amblyommatis-free cohort (48%), but the difference was not statistically significant. Larval progenies of dually infected A. americanum females transmitted R. rickettsii to naïve guinea pigs confirming viability of the pathogen. Thus, the vector competence of A. americanum for R. rickettsii was not significantly affected by R. amblyommatis.

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.

Unique Strain of Rickettsia parkeri Associated with the Hard Tick Dermacentor parumapertus Neumann in the Western United States.

In 1953, investigators at the Rocky Mountain Laboratories in Hamilton, MT, described the isolation of a spotted fever group Rickettsia (SFGR) species from Dermacentor parumapertus ticks collected from black-tailed jackrabbits (Lepus californicus) in northern Nevada. Several decades later, investigators characterized this SFGR (designated the parumapertus agent) by using mouse serotyping methods and determined that it represented a distinct rickettsial serotype closely related to Rickettsia parkeri; nonetheless, the parumapertus agent was not further characterized or studied. To our knowledge, no isolates of the parumapertus agent remain in any rickettsial culture collection, which precludes contemporary phylogenetic placement of this enigmatic SFGR. To rediscover the parumapertus agent, adult-stage D. parumapertus ticks were collected from black-tailed jackrabbits shot or encountered as roadkills in Arizona, Utah, or Texas from 2011 to 2016. A total of 339 ticks were collected and evaluated for infection with Rickettsia species. Of 112 D. parumapertus ticks collected in south Texas, 16 (14.3%) contained partial ompA sequences with the closest identity (99.6%) to Rickettsia sp. strain Atlantic rainforest Aa46, an SFGR that is closely related or identical to an SFGR species that causes a mild rickettsiosis in several states of Brazil. A pure isolate, designated strain Black Gap, was cultivated in Vero E6 cells, and sequence analysis of the rrs, gltA, sca0, sca5, and sca4 genes also revealed the closest genetic identity to Rickettsia sp. Atlantic rainforest Aa46. Phylogenetic analysis of the five concatenated rickettsial genes place Rickettsia sp. strain Black Gap and Rickettsia sp. Atlantic rainforest Aa46 with R. parkeri in a distinct and well-supported clade.IMPORTANCE We suggest that Rickettsia sp. Black Gap and Rickettsia sp. Atlantic rainforest Aa46 represent nearly identical strains of R. parkeri and that Rickettsia sp. Black Gap or a very similar strain of R. parkeri represents the parumapertus agent. The close genetic relatedness among these taxa, as well as the response of guinea pigs infected with the Black Gap strain, suggests that R. parkeri Black Gap could cause disease in humans. The identification of this organism could also account, at least in part, for the remarkable differences in severity ascribed to Rocky Mountain spotted fever (RMSF) among various regions of the American West during the early 20th century. We suggest that the wide variation in case fatality rates attributed to RMSF could have occurred by the inadvertent inclusion of cases of milder disease caused by R. parkeri Black Gap.