Science abhors a surveillance vacuum: Detection of ticks and tick-borne pathogens in southern New Mexico through passive surveillance.

Robust tick surveillance enhances diagnosis and prevention of tick-borne pathogens, yet surveillance efforts in the United States are highly uneven, resulting in large surveillance vacuums, one of which spans the state of New Mexico. As part of a larger effort to fill this vacuum, we conducted both active and passive tick sampling in New Mexico, focusing on the southern portion of the state. We conducted active tick sampling using dragging and CO2 trapping at 45 sites across Hidalgo, Doña Ana, Otero, and Eddy counties between June 2021 to May 2022. Sampling occurred intermittently, with at least one sampling event each month from June to October 2021, pausing in winter and resuming in March through May 2022. We also conducted opportunistic, passive tick sampling in 2021 and 2022 from animals harvested by hunters or captured or collected by researchers and animals housed in animal hospitals, shelters, and farms. All pools of ticks were screened for Rickettsia rickettsii, Rickettsia parkeri, Rickettsia amblyommatis, Ehrlichia ewingii, and Ehrlichia chaffeensis. Active sampling yielded no ticks. Passive sampling yielded 497 ticks comprising Carios kelleyi from pallid bats, Rhipicephalus sanguineus from dogs, mule deer, and Rocky Mountain elk, Otobius megnini from dogs, cats, horses, and Coues deer, Dermacentor parumapertus from dogs and black-tailed jackrabbits, Dermacentor albipictus from domesticated cats, mule deer and Rocky Mountain elk, and Dermacentor spp. from American black bear, Rocky Mountain elk, and mule deer. One pool of D. parumapterus from a black-tailed jackrabbit in Luna County tested positive for R. parkeri, an agent of spotted fever rickettsiosis. Additionally, a spotted fever group Rickettsia was detected in 6 of 7 C. kelleyi pools. Two ticks showed morphological abnormalities; however, these samples did not test positive for any of the target pathogens, and the cause of the abnormalities is unknown. Passive surveillance yielded five identified species of ticks from three domestic and six wild mammal species. Our findings update tick distributions and inform the public, medical, and veterinary communities of the potential tick-borne pathogens present in southern New Mexico.

First isolation of Rickettsia amblyommatis from Amblyomma mixtum in Colombia.

BACKGROUND: Rickettsiae are obligate intracellular Gram-negative bacteria that are the causative agent of rickettsioses and are spread to vertebrate hosts by arthropods. There are no previous reports of isolation of Rickettsia amblyommatis for Colombia. METHODS: A convenience sampling was executed in three departments in Colombia for direct collection of adult ticks on domestic animals or over vegetation. Ticks were screened for the presence of Rickettsia spp. by real-time polymerase chain reaction (qPCR) amplifying the citrate synthase gene (gltA), and the positive sample was processed for isolation and further molecular characterization by conventional PCR. The absolute and relative frequencies were calculated for several tick species variables. All products from conventional PCR were further purified and sequenced by the Sanger technique. Representative sequences of 18 Rickettsia species were downloaded from GenBank. Consensus phylogenetic trees were constructed for the gltA, ompB, ompA, and htrA genes with 1000 replicates, calculating bootstrap values through the maximum likelihood method and the generalized time reversible substitution model in the MEGA 7.0 software program. RESULTS: One female Amblyomma mixtum collected on vegetation was amplified by qPCR (gltA), indicating a frequency of 1.6% (1/61) for Rickettsia spp. INFECTION: Sequence analysis of a rickettsial isolate from this tick in BLASTn showed 100% identity with gltA (340 base pairs [bp]), 99.87% for ompB (782 bp), 98.99% for htrA (497 bp), and 100% for ompA (488 bp) to R. amblyommatis. Concatenated phylogenetic analysis confirmed these findings indicating that the isolate is grouped with other sequences of Amblyomma cajennense complex from Panama and Brazil within the R. amblyommatis clade. CONCLUSIONS: This paper describes the isolation and early molecular identification of a R. amblyommatis strain from A. mixtum in Colombia.

Spotted Fever and Typhus Group Rickettsiae in Dogs and Humans, Mexico, 2022.

We found serologic evidence of spotted fever group Rickettsia in humans and dogs and typhus group Rickettsia in dogs in Reynosa, Mexico. Our investigation revealed serologic samples reactive to spotted fever group Rickettsia in 5 community members, which highlights a potential rickettsial transmission scenario in this region.

A scrub typhus vaccine presents a challenging unmet need.

Scrub typhus caused by the obligately intracellular bacterium, Orientia tsutsugamushi, is a major cause of life-threatening acute undifferentiated febrile illness in eastern Asia and the islands of the Western Pacific and Indian oceans. Since the estimation of an incidence of 1 million cases annually two decades ago, the number of cases has increased substantially in endemic regions, reappeared where the disease was forgotten, and spread northward. Trombiculid mites are both reservoir and vector. Despite 80 years of efforts to develop a vaccine, there is none. Protective immunity is mediated by antibodies and CD8 and CD4 T cells. Previous efforts have failed because of gaps in understanding immunity to O. tsutsugamushi, particularly the requirements for vaccine-induced immunity, lack of knowledge regarding immune memory in scrub typhus, and lack of attention to addressing the issue of cross-protection between strains. There are numerous strains of O. tsutsugamushi, and modestly durable immunity is strain-specific. Antibodies to the strain that caused infection are protective against challenges with the homologous strain but, despite reactivity with other immunodominant antigens, the immune serum does not protect against heterologous strains. Among the antigens detected by western immunoblot in immune sera (22-, 47-, 56-, 58-, and 110 kDa proteins), only the 56 kDa protein stimulates strong protection. This protein contains four hypervariable regions which are likely, on the basis of limited data, to be the targets of neutralizing antibodies. However, a method that definitively detects neutralizing antibody has yet to be developed. Only one study has used genomic data to pursue the discovery of protective antigens. Three conserved autotransporters were identified, and only immunization with ScaA provided protection against the homologous strain, but only 40% of animals were protected against challenge with a heterologous strain. A multiplex vaccine containing conformational antigens of the hypervariable regions of the 56 kDa protein of the strains of the greatest clinical and epidemiological importance, as well as conserved regions of the 56 kDa protein, ScaA, and other protective antigens identified by future genomic and bioinformatics methods should be developed and tested.

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.

A Murine Model of Waning Scrub Typhus Cross-Protection between Heterologous Strains of Orientia tsutsugamushi.

Orientia tsutsugamushi, the etiologic agent of the life-threatening febrile disease scrub typhus, is an obligately intracellular small coccobacillary bacterium belonging to the family Rickettsiaceae and is transmitted by the parasitic larval stage of trombiculid mites. Progress towards a vaccine for protection against scrub typhus has been impeded by characteristics of the pathogen and the infection. There are numerous strains of O. tsutsugamushi in the Asia-Pacific region with geographical overlap. In human cases immunity has been described as poor against heterologous strains of the pathogen, as well as short-lived against the homologous strain, with a mean antibody reversion rate of less than one year. Animal models of cross-protection as well as of deterioration of this cross-protection are needed to enhance understanding of transient immunity to scrub typhus. To build upon current understanding of this ineffective protection we sought to utilize our recently developed models, sublethal intradermal infection followed by challenge via ordinarily lethal hematogenous dissemination. Mice that were initially infected sublethally with O. tsutsugamushi Gilliam strain and were challenged with an ordinarily lethal dose of heterologous Karp strain were protected from death by a robust immune response at one month after the primary infection as evidenced by an abundance of mononuclear cellular infiltrates in target organs such as lung, liver, and kidney; maintenance of body weight; and low bacterial loads in the organs. Waning protection from lethal Karp strain challenge indicated by weight loss mirroring that observed in naïve mice was observed as early as 9 months after primary Gilliam strain infection, and higher bacterial loads, severe disease, and eventual death in some mice was observed after challenge with Karp strain at 14 months post-initial heterologous infection.

Mechanisms of Sporicidal Activity Induced by Ionized Hydrogen Peroxide in the Spores of Bacillus atrophaeus.

Introduction: Ionized hydrogen peroxide (iHP) is a new technology used for the decontamination of surfaces or laboratory areas. It utilizes a low concentration of hydrogen peroxide (H2O2) mixed with air and ionized through a cold plasma arc. This technology generates reactive oxygen species as a means of decontamination. Objectives: The purpose of this study is to review the effects of iHP on the structure of the spores of Bacillus atrophaeus by observing its effects using transmission electron microscopy (TEM) and also by evaluating the existence of DNA damage by fluorescence-based quantitative polymerase chain reaction (qPCR). Methods: Spore samples of B. atrophaeus decontaminated using iHP at different exposure times (Control, 1, 2, 6, and 12 h) were fixed for TEM. In addition, DNA was extracted for evaluation of DNA damages using fluorescence-based qPCR assays. Results: Damages to the spore structures of B. atrophaeus caused by the decontamination process with iHP at different exposure times (Control, 1, 2, 6, and 12 h) can be observed in micrographs. The effects of the decontamination to short DNA segment (132 base pairs [bp]) of the yaaH gene using qPCR present a linear degradation, and for the long DNA segment (680 bp), it presents a biphasic mode. Conclusion: The results of the qPCR analysis show two initial stages of damage to DNA with very noticeable damage at 12 h contact time, which confirms the observations of the TEM micrographs for the B. atrophaeus spores. The study demonstrates damage to the spore core DNA.

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.

Whole-Genome Sequence of Rickettsia parkeri Strain Atlantic Rainforest, Isolated from a Colombian Tick.

Rickettsia parkeri is classified as a member of the alphaproteobacterial microorganisms, genus Rickettsia Here, we report the complete genome sequence of Rickettsia parkeri strain Atlantic Rainforest, which was isolated from an Amblyomma ovale tick collected in the municipality of Necoclí, Colombia.

A biosafety level-2 dose-dependent lethal mouse model of spotted fever rickettsiosis: Rickettsia parkeri Atlantic Rainforest strain.

BACKGROUND: The species of the Rickettsia genus are separated into four groups: the ancestral group, typhus group, transitional group and spotted fever group. Rickettsia parkeri, a spotted fever group Rickettsia, has been reported across the American continents as infecting several tick species and is associated with a relatively mild human disease characterized by eschar formation at the tick feeding site, regional lymphadenopathy, fever, myalgia and rash. Currently, there are several mouse models that provide good approaches to study the acute lethal disease caused by Rickettsia, but these models can only be performed in an animal biosafety level 3 laboratory. We present an alternative mouse model for acute lethal rickettsial disease, using R. parkeri Atlantic Rainforest strain and C3H/HeN mice, with the advantage that this model can be studied in an animal biosafety level 2 laboratory. PRINCIPAL FINDINGS: In the C3H/HeN mouse model, we determined that infection with 1x106 and 1x107 viable R. parkeri Atlantic Rainforest strain organisms produced dose-dependent severity, whereas infection with 1x108 viable bacteria resulted in a lethal illness. The animals became moribund on day five or six post-infection. The lethal disease was characterized by ruffled fur, erythema, labored breathing, decreased activity, and hunched posture, which began on day three post-infection (p.i.) and coincided with the peak bacterial loads. Significant splenomegaly (on days three and five p.i.), neutrophilia (on days three and five p.i.), and thrombocytopenia (on days one, three and five p.i.) were observed. SIGNIFICANCE: Since R. parkeri is used at biosafety level 2, the greatest advantage of this inbred mouse model is the ability to investigate immunity and pathogenesis of rickettsiosis with all the tools available at biosafety level 2.

Correction: CD8+ T cells provide immune protection against murine disseminated endotheliotropic Orientia tsutsugamushi infection.

[This corrects the article DOI: 10.1371/journal.pntd.0005763.].

CD8+ T cells provide immune protection against murine disseminated endotheliotropic Orientia tsutsugamushi infection.

Scrub typhus, caused by a Gram-negative obligately intracellular coccobacillus, Orientia tsutsugamushi, is a long neglected but important tropical disease. Orientia tsutsugamushi causes illness in one million people each year, and 1 billion people are at risk. Without appropriate diagnosis and treatment, the disease can cause severe multiorgan failure with a case fatality rate of 7-15%. The current gaps in knowledge of immunity include the unknown mechanisms of host immunity to O. tsutsugamushi. Using an intravenous (i.v.) disseminated infection mouse model, we observed that more CD8+ T cells than CD4+ T cells were present in the spleen of infected mice at 12 dpi. We also determined that Treg cells and the proportion of T cells producing IL-10 were significantly increased from 6 dpi, which correlated with the onset of illness, body weight loss, and increased bacterial loads. We further studied CD8-/-, MHC I-/- and wild type control (WT) C57BL/6J mice to determine the importance of CD8+ T cells and MHC I molecules. After infection with an ordinarily sub-lethal dose of O. tsutsugamushi, all CD8-/- and MHC I-/- mice were moribund between 12 and 15 dpi, whereas all WT mice survived. Bacterial loads in the lung, kidney, liver and spleen of CD8-/- and MHC I-/- mice were significantly greater than those in WT mice. Interferon-γ (IFN-γ) and granzyme B mRNA levels in the liver of CD8-/- and MHC I-/- mice were significantly greater than in WT mice. In addition, more severe histopathologic lesions were observed in CD8-/- mice. Finally, adoptive transfer confirmed a major role of immune CD8+ T cells as well as a less effective contribution by immune CD8 T cell-depleted splenocytes in protection against O. tsutsugamushi infection. These studies demonstrated the critical importance of CD8+ T cells in the host immune response during O. tsutsugamushi infection.

Murine models of scrub typhus associated with host control of Orientia tsutsugamushi infection.

BACKGROUND: Scrub typhus, a febrile illness of substantial incidence and mortality, is caused by infection with the obligately intracellular bacterium Orientia tsutsugamushi. It is estimated that there are more than one million cases annually transmitted by the parasitic larval stage of trombiculid mites in the Asia-Pacific region. The antigenic and genetic diversity of the multiple strains of O. tsutsugamushi hinders the advancement of laboratory diagnosis, development of long-lasting vaccine-induced protection, and interpretation of clinical infection. Despite the life-threatening severity of the illness in hundreds of thousands of cases annually, 85-93% of patients survive, often without anti-rickettsial treatment. To more completely understand the disease caused by Orientia infection, animal models which closely correlate with the clinical manifestations, target cells, organ involvement, and histopathologic lesions of human cases of scrub typhus should be employed. Previously, our laboratory has extensively characterized two relevant C57BL/6 mouse models using O. tsutsugamushi Karp strain: a route-specific intradermal model of infection and persistence and a hematogenously disseminated dose-dependent lethal model. PRINCIPAL FINDINGS: To complement the lethal model, here we illustrate a sublethal model in the same mouse strain using the O. tsutsugamushi Gilliam strain, which resulted in dose-dependent severity of illness, weight loss, and systemic dissemination to endothelial cells of the microcirculation and mononuclear phagocytic cells. Histopathologic lesions included expansion of the pulmonary interstitium by inflammatory cell infiltrates and multifocal hepatic lesions with mononuclear cellular infiltrates, renal interstitial lymphohistiocytic inflammation, mild meningoencephalitis, and characteristic typhus nodules. SIGNIFICANCE: These models parallel characteristics of human cases of scrub typhus, and will be used in concert to understand differences in severity which lead to lethality or host control of the infection and to address the explanation for short duration of heterologous immunity in Orientia infection.

An Intradermal Inoculation Mouse Model for Immunological Investigations of Acute Scrub Typhus and Persistent Infection.

Scrub typhus is a neglected tropical disease, caused by Orientia tsutsugamushi, a Gram-negative bacterium that is transmitted to mammalian hosts during feeding by Leptotrombidium mites and replicates predominantly within endothelial cells. Most studies of scrub typhus in animal models have utilized either intraperitoneal or intravenous inoculation; however, there is limited information on infection by the natural route in murine model skin or its related early host responses. Here, we developed an intradermal (i.d.) inoculation model of scrub typhus and focused on the kinetics of the host responses in the blood and major infected organs. Following ear inoculation with 6 x 104 O. tsutsugamushi, mice developed fever at 11-12 days post-infection (dpi), followed by marked hypothermia and body weight loss at 14-19 dpi. Bacteria in blood and tissues and histopathological changes were detected around 9 dpi and peaked around 14 dpi. Serum cytokine analyses revealed a mixed Th1/Th2 response, with marked elevations of MCP-1/CCL2, MIP-1α/CCL3 and IL-10 at 9 dpi, followed by increased concentrations of pro-inflammatory markers (IL-6, IL-12, IFN-γ, G-CSF, RANTES/CCL5, KC/CCL11, IL-1α/ß, IL-2, TNF-α, GM-CSF), as well as modulatory cytokines (IL-9, IL-13). Cytokine levels in lungs had similar elevation patterns, except for a marked reduction of IL-9. The Orientia 47-kDa gene and infectious bacteria were detected in several organs for up to 84 dpi, indicating persistent infection. This is the first comprehensive report of acute scrub typhus and persistent infection in i.d.-inoculated C57BL/6 mice. This is a significant improvement over current murine models for Orientia infection and will permit detailed studies of host immune responses and infection control interventions.

Human Infections by Multiple Spotted Fever Group Rickettsiae in Tennessee.

Rocky Mountain spotted fever is the most common tick-borne disease in Tennessee. However, Rickettsia rickettsii has rarely been isolated from endemic ticks, suggesting rickettsioses may be caused by other species. A total of 56 human serum samples that were serologically positive for exposure to Rickettsia were obtained from commercial laboratories in 2010 and 2011. In addition, 20 paired sera from patients with encephalitis and positive Rickettsia serology were obtained from the Tennessee Unexplained Encephalitis Surveillance (TUES) study. Using an immunofluorescence assay, reactivity of the sera to R. rickettsii, Rickettsia montanensis, Rickettsia parkeri, and Rickettsia amblyommii was tested, and a comparison of endpoint titers was used to determine the probable antigen that stimulated the antibody response. Cross-absorption was conducted for 94.8% (N = 91) of the samples due to serologic cross-reactivity. Of the commercial laboratory samples, 55.4% (N = 31) had specific reactivity to R. amblyommii and 44.6% (N = 25) were indeterminate. Of the paired TUES samples, 20% (N = 4) had specific reactivity to R. amblyommii, 5% (N = 1) to R. montanensis, and 5% (N = 1) to R. parkeri Patients with specific reactivity to R. amblyommii experienced fever (75%), headache (68%) and myalgia (58%). Rash (36%) and thrombocytopenia (40%) were less common. To our knowledge, this is the first time R. amblyommii has been reported as a possible causative agent of rickettsioses in Tennessee.

IL-33-Dependent Endothelial Activation Contributes to Apoptosis and Renal Injury in Orientia tsutsugamushi-Infected Mice.

Endothelial cells (EC) are the main target for Orientia tsutsugamushi infection and EC dysfunction is a hallmark of severe scrub typhus in patients. However, the molecular basis of EC dysfunction and its impact on infection outcome are poorly understood. We found that C57BL/6 mice that received a lethal dose of O. tsutsugamushi Karp strain had a significant increase in the expression of IL-33 and its receptor ST2L in the kidneys and liver, but a rapid reduction of IL-33 in the lungs. We also found exacerbated EC stress and activation in the kidneys of infected mice, as evidenced by elevated angiopoietin (Ang) 2/Ang1 ratio, increased endothelin 1 (ET-1) and endothelial nitric oxide synthase (eNOS) expression. Such responses were significantly attenuated in the IL-33-/- mice. Importantly, IL-33-/- mice also had markedly attenuated disease due to reduced EC stress and cellular apoptosis. To confirm the biological role of IL-33, we challenged wild-type (WT) mice with a sub-lethal dose of O. tsutsugamushi and gave mice recombinant IL-33 (rIL-33) every 2 days for 10 days. Exogenous IL-33 significantly increased disease severity and lethality, which correlated with increased EC stress and activation, increased CXCL1 and CXCL2 chemokines, but decreased anti-apoptotic gene BCL-2 in the kidneys. To further examine the role of EC stress, we infected human umbilical vein endothelial cells (HUVEC) in vitro. We found an infection dose-dependent increase in the expression of IL-33, ST2L soluble ST2 (sST2), and the Ang2/Ang1 ratio at 24 and 48 hours post-infection. This study indicates a pathogenic role of alarmin IL-33 in a murine model of scrub typhus and highlights infection-triggered EC damage and IL-33-mediated pathological changes during the course of Orientia infection.

Strong type 1, but impaired type 2, immune responses contribute to Orientia tsutsugamushi-induced pathology in mice.

Scrub typhus is a neglected, but important, tropical disease, which puts one-third of the world's population at risk. The disease is caused by Orientia tsutsugamushi, an obligately intracellular Gram-negative bacterium. Dysregulation in immune responses is known to contribute to disease pathogenesis; however, the nature and molecular basis of immune alterations are poorly defined. This study made use of a newly developed murine model of severe scrub typhus and focused on innate regulators and vascular growth factors in O. tsutsugamushi-infected liver, lungs and spleen. We found no activation or even reduction in base-line expression for multiple molecules (IL-7, IL-4, IL-13, GATA3, ROR-γt, and CXCL12) at 2, 6 and 10 days post-infection. This selective impairment in type 2-related immune responses correlated with a significant activation of the genes for IL-1ß, IL-6, IL-10, TNF-α, IFN-γ, as well as CXCR3- and CXCR1-related chemokines in inflamed tissues. The elevated angiopoietin (Ang)-2 expression and Ang-2/Ang-1 ratios suggested excessive inflammation and the loss of endothelial integrity. These alterations, together with extensive recruitment of myeloperoxidase (MPO)-expressing neutrophils and the influx of CD3+ T cells, contributed to acute tissue damage and animal death. This is the first report of selective alterations in a panel of immune regulators during early O. tsutsugamushi infection in intravenously inoculated C57BL/6 mice. Our findings shed new light on the pathogenic mechanisms associated with severe scrub typhus and suggest potential targets for therapeutic investigation.

Seroepidemiologic study of human infections with spotted fever group Rickettsiae in North Carolina.

Increasing entomologic and epidemiologic evidence suggests that spotted fever group rickettsiae (SFGR) other than Rickettsia rickettsii are responsible for spotted fever rickettsioses in the United States. A retrospective seroepidemiologic study was conducted on stored acute- and convalescent-phase sera that had been submitted for Rocky Mountain spotted fever testing to the North Carolina State Laboratory of Public Health. We evaluated the serologic reactivity of the paired sera to R. rickettsii, Rickettsia parkeri, and Rickettsia amblyommii antigens. Of the 106 eligible pairs tested, 21 patients seroconverted to one or more antigens. Cross-reactivity to multiple antigens was observed in 10 patients, and seroconversions to single antigens occurred in 11 patients, including 1 against R. rickettsii, 4 against R. parkeri, and 6 against R. amblyommii. Cross-absorption of cross-reactive sera and/or Western blots identified two presumptive cases of infection with R. parkeri, two presumptive cases of infection with R. rickettsii, and one presumptive case of infection with R. amblyommii. These findings suggest that species of SFGR other than R. rickettsii are associated with illness among North Carolina residents and that serologic testing using R. rickettsii antigen may miss cases of spotted fever rickettsioses caused by other species of SFGR.

Hematogenously disseminated Orientia tsutsugamushi-infected murine model of scrub typhus [corrected].

Orientia tsutsugamushi, the etiologic agent of scrub typhus, is a mite-borne rickettsia transmitted by the parasitic larval stage of trombiculid mites. Approximately one-third of the world's population is at risk of infection with Orientia tsutsugamushi, emphasizing its importance in global health. In order to study scrub typhus, Orientia tsutsugamushi Karp strain has been used extensively in mouse studies with various inoculation strategies and little success in inducing disease progression similar to that of human scrub typhus. The objective of this project was to develop a disease model with pathology and target cells similar to those of severe human scrub typhus. This study reports an intravenous infection model of scrub typhus in C57BL/6 mice. This mouse strain was susceptible to intravenous challenge, and lethal infection occurred after intravenous inoculation of 1.25 × 10(6) focus (FFU) forming units. Signs of illness in lethally infected mice appeared on day 6 with death occurring ∼ 6 days later. Immunohistochemical staining for Orientia antigens demonstrated extensive endothelial infection, most notably in the lungs and brain. Histopathological analysis revealed cerebral perivascular, lymphohistiocytic infiltrates, focal hemorrhages, meningoencephalitis, and interstitial pneumonia. Disseminated infection of endothelial cells with Orientia in C57BL/6 mice resulted in pathology resembling that of human scrub typhus. The use of this model will allow detailed characterization of the mechanisms of immunity to and pathogenesis of O. tsutsugamushi infection.