The Retropepsin-Type Protease APRc as a Novel Ig-Binding Protein and Moonlighting Immune Evasion Factor of Rickettsia.

Rickettsiae are obligate intracellular Gram-negative bacteria transmitted by arthropod vectors. Despite their reduced genomes, the function(s) of the majority of rickettsial proteins remains to be uncovered. APRc is a highly conserved retropepsin-type protease, suggested to act as a modulator of other rickettsial surface proteins with a role in adhesion/invasion. However, APRc's function(s) in bacterial pathogenesis and virulence remains unknown. This study demonstrates that APRc targets host serum components, combining nonimmune immunoglobulin (Ig)-binding activity with resistance to complement-mediated killing. We confirmed nonimmune human IgG binding in extracts of different rickettsial species and intact bacteria. Our results revealed that the soluble domain of APRc is capable of binding to human (h), mouse, and rabbit IgG and different classes of human Ig (IgG, IgM, and IgA) in a concentration-dependent manner. APRc-hIgG interaction was confirmed with total hIgG and normal human serum. APRc-hIgG displayed a binding affinity in the micromolar range. We provided evidence of interaction preferentially through the Fab region and confirmed that binding is independent of catalytic activity. Mapping the APRc region responsible for binding revealed the segment between amino acids 157 and 166 as one of the interacting regions. Furthermore, we demonstrated that expression of the full-length protease in Escherichia coli is sufficient to promote resistance to complement-mediated killing and that interaction with IgG contributes to serum resistance. Our findings position APRc as a novel Ig-binding protein and a novel moonlighting immune evasion factor of Rickettsia, contributing to the arsenal of virulence factors utilized by these intracellular pathogens to aid in host colonization. IMPORTANCE Many Rickettsia organisms are pathogenic to humans, causing severe infections, like Rocky Mountain spotted fever and Mediterranean spotted fever. However, immune evasion mechanisms and pathogenicity determinants in rickettsiae are far from being resolved. We provide evidence that the highly conserved rickettsial retropepsin-type protease APRc displays nonimmune immunoglobulin (Ig)-binding activity and participates in serum resistance. APRc emerges then as a novel Ig-binding protein from Gram-negative bacteria and the first to be identified in Rickettsia. Bacterial surface proteins capable of Ig binding are known to be multifunctional and key players in immune evasion. We demonstrate that APRc is also a novel moonlighting protein, exhibiting different actions on serum components and acting as a novel evasin. This work strengthens APRc as a virulence factor in Rickettsia and its significance as a potential therapeutic target. Our findings significantly contribute to a deeper understanding of the virulence strategies used by intracellular pathogens to subvert host immune responses.

Nitric Oxide Inhibition of Rickettsia rickettsii.

Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, is an enzootic, obligate, intracellular bacterial pathogen. Nitric oxide (NO) synthesized by the inducible NO synthase (iNOS) is a potent antimicrobial component of innate immunity and has been implicated in the control of virulent Rickettsia spp. in diverse cell types. In this study, we examined the antibacterial role of NO on R. rickettsii. Our results indicate that NO challenge dramatically reduces R. rickettsii adhesion through the disruption of bacterial energetics. Additionally, NO-treated R. rickettsii cells were unable to synthesize protein or replicate in permissive cells. Activated, NO-producing macrophages restricted R. rickettsii infections, but inhibition of iNOS ablated the inhibition of bacterial growth. These data indicate that NO is a potent antirickettsial effector of innate immunity that targets energy generation in these pathogenic bacteria to prevent growth and subversion of infected host cells.

Clinical and serological evaluation of capybaras (Hydrochoerus hydrochaeris) successively exposed to an Amblyomma sculptum-derived strain of Rickettsia rickettsii.

Brazilian spotted fever (BSF), caused by Rickettsia rickettsii, is the most lethal tick-borne disease in the western hemisphere. In Brazil, Amblyomma sculptum ticks are the main vector. Capybaras (Hydrochoerus hydrochaeris), the largest living rodents of the world (adults weighing up to 100 Kg), have been recognized as amplifying hosts of R. rickettsii for A. sculptum in BSF-endemic areas; i.e., once primarily infected, capybaras develop bacteremia for a few days, when feeding ticks acquire rickettsial infection. We conducted experimental infections of five capybaras with an A. sculptum-derived strain of R. rickettsii and performed clinical and bacteremia evaluation during primary and subsequent infections. Bacteremia was detected in all capybaras during primary infection, but not in subsequent infections. All animals seroconverted to R. rickettsii (titres range: 64-32,768), and remained seropositive throughout the study. Primary infection resulted in clinical spotted fever illness in four capybaras, of which two had a fatal outcome. Subsequent infections in seropositive capybaras resulted in no clinical signs. Capybaras developed a sustained immune response that prevented a second bacteremia. This condition may imply a high reproduction rate of capybaras in BSF-endemic areas, in order to continuously generate capybaras susceptible to bacteremia during primary infection.

Antibody Titers Reactive With Rickettsia rickettsii in Blood Donors and Implications for Surveillance of Spotted Fever Rickettsiosis in the United States.

BACKGROUND: Since 2000, the reported prevalence of tick-borne spotted fever rickettsiosis has increased considerably. We compared the level of antibody reactivity among healthy blood donors from 2 widely separated regions of the United States and evaluated the impact of antibody prevalence on public health surveillance in one of these regions. METHODS: Donor serum samples were evaluated by indirect immunofluorescence antibody assay to identify immunoglobulin G (IgG) antibodies reactive with Rickettsia rickettsii. The Georgia Department of Public Health (GDPH) analyzed characteristics of cases from 2016 surveillance data to evaluate the utility of laboratory surveillance for case assessment. RESULTS: Of the Georgia donors (n = 1493), 11.1% demonstrated antibody titers reactive with R. rickettsii at titers ≥64, whereas 6.3% of donors from Oregon and Washington (n = 1511) were seropositive. Most seropositive donors had a titer of 64; only 3.1% (n = 93) of all donors had titers ≥128. During 2016, GDPH interviewed 243 seropositive case patients; only 28% (n = 69) met inclusion criteria in the national case definition for spotted fever rickettsiosis. CONCLUSIONS: These findings suggest that a single IgG antibody titer is an unreliable measure of diagnosis and could inaccurately affect surveillance estimates that define magnitude and clinical characteristics of Rocky Mountain spotted fever and other spotted fever rickettsioses.

Rickettsia rickettsii Whole-Cell Antigens Offer Protection against Rocky Mountain Spotted Fever in the Canine Host.

Rocky Mountain spotted fever (RMSF) is a potentially fatal tick-borne disease in people and dogs. RMSF is reported in the United States and several countries in North, Central, and South America. The causative agent of this disease, Rickettsia rickettsii, is transmitted by several species of ticks, including Dermacentor andersoni, Rhipicephalus sanguineus, and Amblyomma americanum RMSF clinical signs generally include fever, headache, nausea, vomiting, muscle pain, lack of appetite, and rash. If untreated, it can quickly progress into a life-threatening illness in people and dogs, with high fatality rates ranging from 30 to 80%. While RMSF has been known for over a century, recent epidemiological data suggest that the numbers of documented cases and the fatality rates remain high in people, particularly during the last two decades in parts of North America. Currently, there are no vaccines available to prevent RMSF in either dogs or people. In this study, we investigated the efficacies of two experimental vaccines, a subunit vaccine containing two recombinant outer membrane proteins as recombinant antigens (RCA) and a whole-cell inactivated antigen vaccine (WCA), in conferring protection against virulent R. rickettsii infection challenge in a newly established canine model for RMSF. Dogs vaccinated with WCA were protected from RMSF, whereas those receiving RCA developed disease similar to that of nonvaccinated R. rickettsii-infected dogs. WCA also reduced the pathogen loads to nearly undetected levels in the blood, lungs, liver, spleen, and brain and induced bacterial antigen-specific immune responses. This study provides the first evidence of the protective ability of WCA against RMSF in dogs.

Serologic assessment for exposure to spotted fever group rickettsiae in dogs in the Arizona-Sonora border region.

Rocky Mountain spotted fever (RMSF) is a severe tick-borne rickettsial illness. In the south-western United States and Mexico, RMSF displays unique epidemiologic and ecologic characteristics, including Rhipicephalus sanguineus sensu lato (brown dog tick) as the primary vector. Expansion and spread of the disease from hyperendemic regions of Arizona or Mexico to new areas is a key public health concern. Dogs are thought to play an important role in the emergence and circulation of R. rickettsii in these regions and are often one of earliest indicators of RMSF presence. A canine serosurvey was conducted in 2015 among owned and stray dogs at rabies clinic and animal shelters in three southern Arizona counties where RMSF had not previously been identified. Of the 217 dogs sampled, 11 (5.1%) tested positive for spotted fever group rickettsia (SFGR) IgG antibodies, with seropositivity ranging from 2.9% to 12.2% across the three counties. Large dogs were significantly more likely than small dogs to have positive titres reactive with R. rickettsii; no additional statistically significant relationships were observed between seropositivity of canine age, sex, neuter or ownership status. In addition, 17 (7.8%) dogs had ticks attached at the time of sampling, and stray dogs were significantly more likely to have ticks present than owned dogs (p < 0.001). All 57 ticks collected were identified as Rh. sanguineus s.l., and four (7%) had DNA evidence of genera-wide Rickettsia species. The results of this project demonstrated canine seroprevalence levels lower than those previously reported from dogs in highly endemic areas, indicating a low risk of SFGR transmission to humans in the southern Arizona border region at this time. Continued surveillance is critical to identify SFGR emergence in new geographic regions and to inform prevention efforts for humans and dogs in those areas.

Development of an electrochemical immunosensor for the diagnostic testing of spotted fever using synthetic peptides.

Spotted fever is a rare acute and multisystemic febrile infectious disease with a mortality rate of ≥50% without adequate antibiotic treatment, and in diagnosed and treated cases, of approximately 2.5%. Currently, the applied test to diagnose this disease is the indirect immunofluorescence reaction, however two samples of paired sera are necessary to confirm the diagnosis, since using only one sample may allow for confusion with cross reactions. OmpA is an outer membrane protein present in the R. rickettsia, the etiological agent of spotted fever, able to activate dendritic and macrophage cells. It also presents immunogenicity properties, and is considered a target for the development of diagnostic tests for spotted fever. In this context, an amperometric immunosensor was developed for the identification of sera antibodies (human IgG) from patients with spotted fever aimed at improving sensitivity and minimize sample volume. The development of the immunosensor was conducted using a synthetic peptide, derivative from the H6PGA4 R. rickettsia protein, homologous to OmpA. Amperometric responses were generated at -0.6 to 0.6V, at a scan rate of 0.025Vs-1 for 20 cycles, a limit of detection of approximately 10ngmL-1 for the synthetic peptides and 0.01µgmL-1 for the humam serum, a sensitivity of 2.59µA, adequate for the detection of spotted fever antibodies. The construction of this immunosensor, capable of identifying circulating antibodies in real time, can also be applied in the diagnosis of other infectious-parasitic diseases.

Th1 epitope peptides induce protective immunity against Rickettsia rickettsii infection in C3H/HeN mice.

Rickettsia rickettsii is the causative pathogen of Rocky Mountain spotted fever (RMSF). Adr2, YbgF and OmpB are protective antigens of R. rickettsii. In this study, 90 candidate peptides were selected from these antigens based on their high-affinity binding capacity for the MHC class II molecule H2 I-A or H2 I-E using bioinformatic methods. Six peptides were determined using ELISPOT assay to be immunodominant based on the IFN-γ recall responses of CD4+ T cells from mice immunized with R. rickettsii. Six nucleotide sequences encoding the immunodominant peptides were linked in series and inserted into a plasmid for expression in Escherichia coli cells, resulting in a new, recombinant polypeptide termed GWP. After immunization and challenge, the rickettsial load or histopathological lesions in the organs of mice immunized with GWP or pooled peptides was significantly lower than that in organs of mice immunized with PBS or the individual peptide OmpB399. An in vitro neutralization test revealed that sera from mice immunized with GWP, OmpB399, or pooled peptides reduced R. rickettsii adherence to, and invasion of, vascular endothelial cells. Furthermore, significantly higher levels of IgG, IgG1, or IgG2a were detected in sera from mice immunized with GWP or pooled peptides, and significantly higher levels of IFN-γ or TNF-α secreted by CD4+ T cells from R. rickettsii-infected mice were detected after immunization with GWP. Altogether, our results indicated that polypeptides, especially GWP, could induce a Th1-type immune response against R. rickettsii infection, which might contribute to the rational design of peptide-based vaccines for RMSF.

Detecção de proteínas imunorreativas de Rickettsia sp. cepa Mata Atlântica / Detection of immunoreactive proteins of Rickettsia sp. Atlantic Forest strain

A Febre Maculosa Brasileira (FMB) é uma doença infecciosa, transmitida por carrapatos ao homem. Uma nova riquetsiose humana foi descrita como causadora de Febre Maculosa no Estado de São Paulo, sendo denominada de Rickettsia sp. cepa Mata Atlântica. O presente trabalho teve como objetivo detectar e identificar proteínas com potencial de estimular o sistema imune de hospedeiro mamífero, desta nova cepa descrita. Para tanto, foi realizado a extração proteica total de Rickettsia sp. cepa Mata Atlântica. As proteínas extraídas foram fracionadas por eletroforese. As bandas proteicas foram transferidas para membranas de nitrocelulose por migração elétrica e submetidas à técnica de Western-blot, para detecção proteica. Ao todo sete proteínas imunorreativas foram detectadas. Duas proteínas apresentaram maior abundancia, com peso molecular, de 200 e 130 kDa respectivamente. Através da comparação de mapas proteômicos existentes e pelo peso molecular que estas proteínas apresentaram, sugere-se que as duas proteínas detectadas representem rOmpA (200 kDa) e rOmpB (130 kDa). As demais proteínas detectadas apresentaram menor ocorrência e peso molecular inferior a 78 kDa, podendo representar membros da família de antígenos de superfície celular (Sca - Surface cell antigen). As proteínas detectadas poderão servir como base de estudo na elaboração de métodos diagnósticos sensíveis e específicos, no desenvolvimento de vacinas, além de possibilitarem novos estudos para terapias mais eficazes.(AU)

Brazilian Spotted Fever (BSF) is an infectious disease transmitted by ticks to humans. A new human rickettsial infection was reported to cause spotted fever in the State of São Paulo and was named Rickettsia sp. Strain Atlantic Forest. This study aimed to detect and identify proteins with potential to stimulate the immune system of mammalian host of this new strain described. Therefore, we performed total protein extraction Rickettsia sp. Strain Atlantic Forest. The extracted proteins were fractionated by electrophoresis. The protein bands were transferred to nitrocellulose membrane by electrical migration and subjected to Western blot for protein detection. In all, seven immunoreactive proteins were detected. Two proteins showed higher abundance, with molecular weight of 200 and 130 kDa respectively. By comparing existing proteomic maps and the molecular weight of these proteins showed that, it is suggested that the two proteins detected representing rOmpA (200 kDa) and rOmpB (130 kDa). The other detected proteins had lower occurrence and molecular weight less than 78 kDa, which may represent members of the cell surface antigens Family (Sca - Surface cell antigen). The detected proteins may serve as a study based on the development of sensitive and specific diagnostic methods in the development of vaccines and they enable further studies to more effective therapies.(AU)

Failure of a heterologous recombinant Sca5/OmpB protein-based vaccine to elicit effective protective immunity against Rickettsia rickettsii infections in C3H/HeN mice.

Spotted fever group (SFG) rickettsial species are obligate intracellular tick-borne pathogens that are responsible for important human diseases. Previous reports have demonstrated the feasibility of using recombinant surface cell antigen Sca5/OmpB to elicit protective immunity against homologous challenges using murine models of Mediterranean spotted fever and Rocky Mountain spotted fever. In addition, the feasibility of generating cross-protective immunity against related rickettsial species has also been established, but the molecular basis for these phenomena was not explored. Here, we demonstrate that vaccination of C3H/HeN mice with a recombinant OmpB domain derived from Rickettsia conorii induced high titer humoral immune responses that are capable of recognizing the native OmpB protein at the R. rickettsii outer membrane, but this immunization was not sufficient to induce effective protective immunity. In contrast, animals vaccinated with a corresponding OmpB domain derived from R. rickettsii protected animals from fatal outcomes. These results demonstrate that vaccination with nearly identical antigens may not be an effective strategy to induce wide-ranging protective immunity against related SFG Rickettsia species.

Rickettsia rickettsii outer membrane protein YbgF induces protective immunity in C3H/HeN mice.

Rickettsia rickettsii is the etiological agent of Rocky Mountain spotted fever (RMSF). YbgF and TolC are outer membrane-associated proteins of R. rickettsii that play important roles in its interaction with host cells. We investigated the immunogenicity of YbgF and TolC for protection against RMSF. We immunized C3H/HeN mice with recombinant R. rickettsii YbgF (rYbgF) or TolC (rTolC). Rickettsial burden and impairment in the lungs, spleens, and livers of rYbgF-immunized mice were significantly lower than in rTolC-immunized mice. The ratio of IgG2a to IgG1 in rYbgF-immunized mice continued to increase over the course of our experiments, while that in rTolC-immunized mice was reduced. The proliferation and cytokine secretion of CD4(+) and CD8(+) T cells isolated from R. rickettsii-infected mice were analyzed following antigen stimulation. The results indicated that proliferation and interferon (IFN)-γ secretion of CD4(+) or CD8(+) T cells in R. rickettsii-infected mice were significantly greater than in uninfected mice after stimulation with rYbgF. YbgF is a novel protective antigen of R. rickettsii. Protection conferred by YbgF is dependent upon IFN-γ-producing CD4(+) and CD8(+) T cells and IgG2a, which act in synergy to control R. rickettsii infection.

Enhanced protection against Rickettsia rickettsii infection in C3H/HeN mice by immunization with a combination of a recombinant adhesin rAdr2 and a protein fragment rOmpB-4 derived from outer membrane protein B.

BACKGROUND: Two surface proteins of Rickettsia rickettsii, outer membrane protein B (OmpB) and adhesion 2 (Adr2), have been recognized as protective antigens. Herein, the immunization with both OmpB and Adr2 was performed in mice so as to explore whether their combination could induce an enhanced immunoprotection against R. rickettsii infection. METHODS: C3H/HeN mice were immunized with recombinant protein rAdr2 or/and rOmp-4, a fragment derived from OmpB, and then mice were challenged with R. rickettsii. After which rickettsial loads in mice were measured by quantitative PCR. The specific antibodies in mouse sera were determined by ELISA and antigen-specific cytokines secretion by mouse T cells were analyzed in vitro. RESULTS: After challenge with R. rickettsii, the mice immunized with rAdr2 or/and rOmpB-4 had significant lower rickettsial load in livers, spleens, or lungs compared to PBS mock-immunized mice. Particularly, the load in lungs of mice immunized with both rAdr2 and rOmpB-4 was significantly lower than that with either of them. High levels of specific antibodies were detected in sera from mice immunized with rAdr2 or/and rOmpB-4, but the ratios of specific IgG2a to IgG1 induced by their combination were significantly higher than that by either rAdr2 or rOmpB-4. Following stimulation with rAdr2 or/and rOmpB-4, the INF-γ secreted by CD4(+) T cells from infected mice was significantly higher than that by cognate cells from uninfected mice. And the TNF-α secreted by CD4(+) or CD8(+) T cells from infected mice was markedly greater than that by cognate cells from uninfected mice after stimulation by their combination but not either of them. CONCLUSION: The combination of rAdr2 and rOmpB-4 conferred an enhanced protection against R. rickettsii infection in mice, which was mainly dependent on a stronger Th1-oriented immunoresponse with greater INF-γ and TNF-α secretion by antigen-specific T cells and specific IgG2a elicited by the combination.

A cluster of Rickettsia rickettsii infection at an animal shelter in an urban area of Brazil.

Rickettsia rickettsii infection is being increasingly recognized as an important cause of fatal acute illness in Brazil, where this tick-borne disease is designated Brazilian spotted fever (BSF). In this study we report five fatal cases of BSF in employees of an animal shelter in an urban area in the municipality of Rio de Janeiro in southeast Brazil after a natural disaster on 11 January 2011. Four of the cases occurred from 27 January to 11 April 2011, while the fifth fatal case was identified in April 2012. Three cases were confirmed by molecular analysis and two by epidemiological linkage. An investigation of BSF was performed in the animal shelter, and blood samples were collected from 115 employees and 117 randomly selected dogs. The presence of high levels (1024-4096) of antibodies against spotted fever group rickettsiae was found in three (2·6%) employees and 114 (97·5%) dogs. These findings emphasize the need to consider BSF as a possible cause of undifferentiated febrile illness, especially dengue and leptospirosis, in patients occupationally exposed to dogs heavily infested by ticks, mainly working at kennels and animal shelters that have inadequate space for the animals housed and frequently providing an environment conducive to exposure to pathogens such as R. rickettsii.

Rickettsial infection among military personnel deployed in Northern Sri Lanka.

BACKGROUND: Military personnel deployed in field actvities report on frequent tick bites. Therefore they may run the risk of exposure to rickettsial organisms. METHODS: In order to assess the risk of exposure to rickettsial organisms, two groups of military personnel who were deployed in field activities of Nothern Sri Lanka were investigated. The first group was studied in order to assess the sero-prevalence of rickettsioses and consisted of soldiers who were admitted following injuries during field activities. The second group was studied to identify the incidence of acute rickettsioses during their acute febrile presentations. They were tested with IFA-IgG against spotted fever group rickettsioses (SFG), scrub typhus (ST) and murine typhus. RESULTS: In the first group, 48/57 (84%) military personnel had serological evidence of exposure to rickettsioses (in all, IFA-IgG titer ≥ 1:128): 33/50 (66%) to SFG rickettsioses, 1/50 (2%) to ST and 14/50 (28%) had mixed titers for both (in all, titers were higher for SFG). While all of them were in military uniform most of the time and frequently slept on scrub land, 35/57 (61.4%) had never used insect repellents and none were on doxycycline prophylaxis. 48/57 (84%) had experienced tick bites during field activity. In the second group, there were 49 who presented with acute febrile illness with a mean duration of 8.5 days (SD 3.2). 33/49 (67.3%) were serologically positive for acute rickettsioses (IgG ≥1:256); 26 (79%) due to ST and 7 (21%) due to SFG rickettsioses, CONCLUSIONS: Exposure to rickettsial disease was common among soldiers who were deployed in Northern Sri Lanka. Scrub typhus was the predominent species accounting for acute febrile illness. Further studies are needed to understand the reasons for very high sero-prevalence for SFG rickettsioses with no anticedent febrile illness. Use of preventive measures was not satisfactory. The high sero-prevelence of SFG rickettsioses is likely to interfere with serological diagnosis of acute SFG rickettsioses in this population.

"Rickettsia amblyommii" induces cross protection against lethal Rocky Mountain spotted fever in a guinea pig model.

Rocky Mountain spotted fever (RMSF) is a severe illness caused by Rickettsia rickettsii for which there is no available vaccine. We hypothesize that exposure to the highly prevalent, relatively nonpathogenic "Rickettsia amblyommii" protects against R. rickettsii challenge. To test this hypothesis, guinea pigs were inoculated with "R. amblyommii." After inoculation, the animals showed no signs of illness. When later challenged with lethal doses of R. rickettsii, those previously exposed to "R. amblyommii" remained well, whereas unimmunized controls developed severe illness and died. We conclude that "R. amblyommii" induces an immune response that protects from illness and death in the guinea pig model of RMSF. These results provide a basis for exploring the use of low-virulence rickettsiae as a platform to develop live attenuated vaccine candidates to prevent severe rickettsioses.

Investigation of an outbreak of rickettsial febrile illness in Guatemala, 2007.

OBJECTIVES: We describe an outbreak of spotted fever group (SFG) rickettsiosis that occurred in 2007 in a farming community in southeastern Guatemala. We identified 17 cases of an acute febrile illness, among which 10, including two fatalities, were confirmed or probable cases of rickettsial disease (case-fatality proportion 12%). METHODS: PCR, a microimmunofluorescence assay (IFA), and Western blotting were performed on patient samples, and PCR was performed on ticks. RESULTS: Using an indirect IFA, seven of 16 (44%) ill persons tested had both IgM and IgG antibodies reacting with one or more Rickettsia spp antigens; the other nine (56%) had only IgM titers or were seronegative. Antibodies to SFG protein and lipopolysaccharide were detected by Western blotting with antigens of Rickettsia typhi, Rickettsia rickettsii, and Rickettsia akari. Only one sample, from an ill person who died, tested positive by PCR for a SFG Rickettsia. PCR analysis of Amblyomma cajennense ticks from domestic animals in the area detected the presence of SFG Rickettsia DNA in one of 12 ticks collected. CONCLUSIONS: Further studies in Guatemala are warranted to establish the prevalence of rickettsioses and to fully characterize the identity of the etiologic agents and vectors.

A fatal urban case of rocky mountain spotted fever presenting an eschar in San Jose, Costa Rica.

This study reports the first urban human case of Rocky Mountain spotted fever caused by Rickettsia rickettsii, in Costa Rica. An 8-year-old female who died at the National Children's Hospital 4 days after her admission, and an important and significant observation was the presence of an "eschar" (tache noire), which is typical in some rickettsial infections but not frequent in Rocky Mountain spotted fever cases.

Life-threatening ANCA-positive vasculitis associated with rickettsial infection.

We present a 47-year-old Caucasian fire fighter who developed multisystem organ failure in the setting of a positive antineutrophil cytoplasmic autoantibody (myeloperoxidase) as well as confirmed Rocky Mountain spotted fever by skin biopsy PCR. This case provided a diagnostic challenge, a rare association of a Rickettsia infection and autoimmune vasculitis as well as a unique management approach.

Identification of potential hosts and vectors of scrub typhus and tick-borne spotted fever group rickettsiae in eastern Taiwan.

Scrub typhus and tick-borne spotted fever group (SFG) rickettsioses are transmitted by chiggers (larval trombiculid mites) and hard ticks, respectively. We assessed exposure to these disease vectors by extensively sampling both chiggers and ticks and their small mammal hosts in eastern Taiwan during 2007 and 2008. The striped field mouse Apodemus agrarius Pallas (Rodentia: Muridae) was the most common of the small mammals (36.1% of 1393 captures) and presented the highest rate of infestation with both chiggers (47.8% of 110 760) and ticks (78.1% of 1431). Leptotrombidium imphalum Vercammen-Grandjean & Langston (Trombidiformes: Trombiculidae) and immature Rhipicephalus haemaphysaloides Supino (Ixodida: Ixodidae) were the most abundant chiggers (84.5%) and ticks (>99%) identified, respectively. Immunofluorescent antibody assay revealed high seropositive rates of rodents against Orientia tsutsugamushi Hyashi (Rickettsiales: Rickettsiaceae), the aetiological agent of scrub typhus (70.0% of 437 rodents), and tick-borne SFG rickettsiae (91.9% of 418 rodents). The current study represents a first step towards elucidating the potential hosts and vectors in the enzootic transmission of O. tsutsugamushi and tick-borne SFG rickettsiae in Taiwan. Further studies should focus on characterizing pathogens in L. imphalum and R. haemaphysaloides, as well as the proclivity of both vectors to humans. Uncovering the main hosts of adult ticks is also critical for the prevention of SFG rickettsial infections.

A survey of antibodies against Rickettsia rickettsii and Ehrlichia chafeensis in domestic animals from a rural area of Colombia.

In a rural area of Colombia endemic for Rocky Mountain spotted fever, we performed indirect immunofluorescent antibody assays for Rickettsia spp. and Ehrlichia spp. using sera from randomly sampled dogs and horses to test the use of domestic animals as possible sentinels. Antibodies against Ehrlichia spp. were detected in 8 dogs (31.8%). Antibody titers against Rickettsia rickettsii antigen were positive in 4 dogs (18.2%) and 26 horses (16.3%). These values, albeit not directly comparable, are lower than those previously reported for humans in this region. A systemic approach to understanding dynamics of transmission is needed before implementing the use of domestic animals for disease surveillance activities.