Experimental Rickettsia typhi Infection in Monodelphis domestica: Implications for Opossums as an Amplifying Host in the Suburban Cycle of Murine Typhus.

Murine typhus is an acute undifferentiated febrile illness caused by Rickettsia typhi. In the United States, its reemergence appears to be driven by a shift from the classic rat-rat flea cycle of transmission to one involving opossums (Didelphis virginiana) and cat fleas. Little is known of the ability of opossums to act as a reservoir and amplifying host for R. typhi. Here, we use Monodelphis domestica (the laboratory opossum) as a surrogate for D. virginiana. Opossums were inoculated via the intraperitoneal (IP) or intradermal (ID) route with 1 × 106 viable R. typhi. Blood and tissues were collected on days 6, 13, 20, and 27 or if moribund. Although one ID-infected opossum died, the remainder did not appear ill, whereas half of the IP-inoculated animals succumbed to infection. Rickettsemia was demonstrated in all animals through week 2 of infection and sporadically in weeks 3 and 4. Rickettsia typhi DNA was detected in all tissues, with most animals demonstrating the presence of bacteria into weeks 3 and 4. Histopathology and immunohistochemistry demonstrated typical findings of rickettsial infection. Akin to infection in rats, the demonstration of disseminated infection, typical inflammation, and prolonged rickettsemia with relatively few clinical effects (especially in the more natural route of ID inoculation) supports the potential of opossums to act as a competent mammalian reservoir and component of the zoonotic maintenance cycle of R. typhi. Understanding the dynamics of infection within opossums may have implications for the prevention and control of murine typhus.

Genetic characterization of a novel Ehrlichia chaffeensis genotype from an Amblyomma tenellum tick from South Texas, USA.

Ehrlichia chaffeensis is the causative agent of human monocytotropic ehrlichiosis (HME), a disease that ranges in severity from mild to fatal infection. Ehrlichia chaffeensis is maintained in a zoonotic cycle involving white-tailed deer (Odocoileus virginianus) as the main vertebrate reservoir and lone star ticks (Amblyomma americanum) as its principal vector. Through complete genomic analysis from human ehrlichial isolates and DNA sequences obtained from deer and tick specimens, nine strains of E. chaffeensis have been characterized. Few studies have examined the genetic diversity of E. chaffeensis in ticks, and some of these investigations have identified that the genetic sequences coincide with the circulating strains reported so far. Here, we report the first evidence of E. chaffeensis DNA from an unfed Amblyomma tenellum (formerly Amblyomma imitator) collected in South Texas. We characterized the genetic variation of this E. chaffeensis genotype using conserved gene markers such as rRNA, dsb, and groEL. We also used gene targets useful to distinguish genotypes, such as the variable length PCR target gene (VLPT) and 120-kDa gene, encoding the tandem-repeat proteins TRP32 and TRP120, respectively. Our results suggest a novel E. chaffeensis genotype that exhibited greater variability than other genotypes of E. chaffeensis and highlights the role for A. tenellum as a potential vector of E. chaffeensis.

Ehrlichia chaffeensis TRP120 Is a Wnt Ligand Mimetic That Interacts with Wnt Receptors and Contains a Novel Repetitive Short Linear Motif That Activates Wnt Signaling.

Ehrlichia chaffeensis expresses the TRP120 multifunctional effector, which is known to play a role in phagocytic entry, on the surface of infectious dense-cored ehrlichiae, but a cognate host receptor has not been identified. We recently reported that E. chaffeensis activates canonical Wnt signaling in monocytes to promote bacterial uptake and intracellular survival and that TRP120 was involved in this activation event. To identify the specific mechanism of pathway activation, we hypothesized that TRP120 is a Wnt signaling ligand mimetic that initiates Wnt pathway activity through direct interaction with the Wnt pathway Frizzled family of receptors. In this study, we used confocal immunofluorescence microscopy to demonstrate very strong colocalization between E. chaffeensis and Fzd2, 4, 5, 7, and 9 as well as coreceptor LRP5 at 1 to 3 h postinfection. Direct binding between TRP120 and multiple Fzd receptors was further confirmed by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). Interfering RNA knockdown of Wnt receptors, coreceptors, and signaling pathway components significantly reduced E. chaffeensis infection, demonstrating that complex and redundant interactions are involved in Wnt pathway exploitation. We utilized in silico approaches to identify a repetitive short linear motif (SLiM) in TRP120 that is homologous to Wnt ligands and used mutant SLiM peptides and an α-TRP120-Wnt-SLiM antibody to demonstrate that the TRP120 Wnt SLiM activates the canonical Wnt pathway and promotes E. chaffeensis infection. This study reports the first example of bacterial mimicry of Wnt pathway ligands and highlights a pathogenic mechanism with potential for targeting by antimicrobial therapeutics.IMPORTANCE Upon infecting mammalian hosts, Ehrlichia chaffeensis establishes a replicative niche in microbe-eating immune system cells where it expertly orchestrates infection and spread. One of the ways Ehrlichia survives within these phagocytes is by activating evolutionarily conserved signaling pathways including the Wnt pathway; however, the molecular details of pathway hijacking have not been defined. This study is significant because it identifies an ehrlichial protein that directly interacts with components of the Wnt receptor complex, influencing pathway activity and promoting infection. Consequentially, Ehrlichia serves as a unique tool to investigate the intricacies of how pathogens repurpose human immune cell signaling and provides an opportunity to better understand many cellular processes in health and disease. Furthermore, understanding how this bacterium utilizes its small genome to survive within cells that evolved to destroy pathogens will facilitate the development of antibacterial therapeutics that could target Ehrlichia as well as other intracellular agents of human disease.

Susceptibility of Rickettsia rickettsii to Tigecycline in a Cell Culture Assay and Animal Model for Rocky Mountain Spotted Fever.

Rocky Mountain spotted fever (RMSF), caused by Rickettsia rickettsii, is a severe tick-borne infection endemic to the Americas. Oral doxycycline is effective, but during severe life-threatening disease, intravenous therapy is recommended. Unfortunately, intravenous formulations of doxycycline are not always available. Therefore, we aimed to determine the susceptibility of R. rickettsii to an alternative parenteral agent, tigecycline, in vitro and in vivo. To determine the minimum inhibitory concentration of tigecycline, R. rickettsii-inoculated Vero cells were incubated with medium containing tigecycline. At various time points, monolayers were collected and R. rickettsii was quantified via real-time polymerase chain reaction (PCR). The growth of R. rickettsii was inhibited in the presence of ≥ 0.5 µg/mL of tigecycline. To determine the effectiveness of tigecycline in vivo, guinea pigs were inoculated with R. rickettsii. Five days after inoculation, they were treated twice daily with subcutaneous tigecycline 3.75 mg/kg or subcutaneous doxycycline 5 mg/kg. Treated animals improved, whereas untreated controls remained ill. Tissues were collected for quantitative PCR-determined bacterial loads on day 8. Median bacterial loads in the tigecycline group were less than those in untreated animals: liver (0 versus 2.9 × 104 copies/mg), lung (0 versus 8.3 × 103 copies/mg), skin (2.6 × 102 versus 2.2 × 105 copies/mg), spleen (0 versus 1.3 × 104 copies/mg), and testes (0 versus 1.0 × 105 copies/mg, respectively). There were no significant differences in the bacterial loads between doxycycline-treated versus tigecycline-treated guinea pigs. These data indicate that tigecycline is effective against R. rickettsii in cell culture and in an animal model of RMSF.

Detection of Rickettsiae, Borreliae, and Ehrlichiae in Ticks Collected from Walker County, Texas, 2017-2018.

Cases of tick-borne diseases, including spotted fever rickettsioses, borreliosis, babesiosis, anaplasmosis and ehrlichiosis, in the United States and territories have more than doubled from 2004 to 2016 and account for 77% of all vector-borne disease reports. In an effort to inform control efforts, the presence of tick-borne pathogens and their vectors was assessed in a recreational park in Walker County, Texas. Here we report data from questing ticks collected on three dates from June 2017 to June 2018. The majority of ticks collected were Amblyomma americanum (96.69%) followed by three additional tick species: Dermacentor variabilis (2.59%), Ixodes scapularis (0.52%), and A. maculatum (0.21%). Ticks were pooled and tested for molecular evidence of bacterial and viral pathogens, respectively. All of the 68 pools of A. americanum had molecular evidence of the spotted fever group rickettsia, Rickettsia amblyommatis. Additionally, six (8.82%) of the A. americanum pools contained sequences matching Ehrlichia chaffeensis, the pathogen responsible for human monocytotropic ehrlichiosis, and 11 (16.18%) for E. ewingii. Three of the A. americanum pools demonstrated evidence of Borrelia lonestari. The presence of etiologic agents of known human disease in this study merits the continued surveillance efforts of ticks and their pathogens in areas where they could pose risks to public health.

Differentiation of Rickettsia felis and Rickettsia felis-Like Organisms via Restriction Fragment Length Polymorphism Analysis.

Rickettsia typhi and Rickettsia felis are flea-borne pathogens, which cause murine typhus and flea-borne spotted fever, respectively. Recently, two other flea-borne rickettsiae (phylogenetically similar to R. felis) have been discovered-Rickettsia asembonensis and Candidatus Rickettsia senegalensis. Currently, species-specific identification of detected organisms requires sequencing- or probe-based PCR assays. Our aim was to develop an efficient and inexpensive method to differentiate R. felis and R. felis-like organisms through restriction fragment length polymorphism (RFLP) analysis. Outer membrane protein B sequences of the aforementioned flea-borne rickettsiae were analyzed using DNASTAR Lasergene Core software to focus on the region amplified by the primers 120.2788 and 120.3599. Restriction enzyme digestion sites were identified, and in silico digestions of each species were compared through simulated agarose gels. The enzyme NlaIV was determined to be the most effective at creating a unique banding pattern within the area of interest. To confirm the predicted performance of NlaIV digestion, we tested the DNA of known PCR positive Ctenocephalides felis fleas collected from cats and opossums within Galveston, Texas. DNA from these fleas was amplified using the sca5 primer set 120.2788 and 120.3599. The PCR products were then digested with NlaIV, subjected to polyacrylamide gel electrophoresis, and visualized through ethidium bromide staining. The banding patterns were then compared with the computer-generated digestion patterns. All samples demonstrated a banding pattern consistent with the predicted pattern for the known species, as confirmed by previous sequencing. This RFLP assay was developed to be an efficient and cost-effective method to screen samples for R. felis, R. asembonensis, and Candidatus R. senegalensis. We believe this assay can aid in the epidemiological and ecological studies of flea-borne rickettsiae.

Rickettsiae Within the Fleas of Feral Cats in Galveston, Texas.

Murine typhus is a flea-borne typhus group rickettsiosis caused by Rickettsia typhi. Once a prevalent disease in the United States, the use of dichlorodiphenyltrichloroethane in the 1940s broke the classic rat-rat flea cycle of transmission, and the remaining endemic foci are now believed to be associated with opossums and the cat flea (Ctenocephalides felis). In Galveston, Texas murine typhus has re-emerged as a cause of febrile illness, and 7% of fleas collected from opossums are infected with R. typhi. In this study, we sought to explore the prevalence of rickettsiae associated with fleas on cats, as these animals have been speculated to play a role in the epidemiology of murine typhus. Fleas were collected from feral cats entering a local veterinary clinic as part of a trap, spay, neuter, and release program. Fleas were identified and subjected to analysis by PCR and sequencing. An estimation of the minimum infection rate (MIR) of pooled samples was performed. Three hundred fourteen fleas (all C. felis) were collected from 24 cats. Sequences for the outer membrane protein B gene revealed R. typhi in one pool (MIR 0.3%), Rickettsia felis in four pools (MIR 1.3%), Rickettsia asembonensis in one pool (MIR 0.3%), and "Candidatus R. senegalensis" in six pools (MIR 2.0%). Results were confirmed by sequencing portions of the rickettsial citrate synthase and 17-kD protein gene. In this study, the presence of R. typhi in fleas from cats suggests that in Galveston, there exists a small but measurable risk to humans who come into contact with flea-infested cats. Despite this, we believe that the low prevalence from cat-collected fleas, compared with that previously detected from opossums, makes cats less likely to play a role in the maintenance of R. typhi in this region. The significance of other identified flea-borne rickettsiae is yet to be elucidated.