[Show abstract][Hide abstract] ABSTRACT: Arthropod-borne obligately intracellular bacteria pose a difficult challenge to the immune system. The genera Rickettsia, Orientia, Ehrlichia, and Anaplasma evolved mechanisms of immune evasion, and each interacts differently with the immune system. The roles of CD8 T cells include protective immunity and immunopathology. In Rickettsia infections, CD8 T cells are protective mediated in part by cytotoxicity toward infected cells. In contrast, TNF-α overproduction by CD8 T cells is pathogenic in lethal ehrlichiosis by induction of apoptosis/necrosis in hepatocytes. Yet, CD8 T cells, along with CD4 T cells and antibodies, also contribute to protective immunity in ehrlichial infections. In granulocytic anaplasmosis, CD8 T cells impact pathogen control modestly but could contribute to immunopathology by virtue of their dysfunction. While preliminary evidence indicates that CD8 T cells are important in protection against Orientia tsutsugamushi, mechanistic studies have been neglected. Valid animal models will enable experiments to elucidate protective and pathologic immune mechanisms. The public health need for vaccines against these agents of human disease, most clearly O. tsutsugamushi, and the veterinary diseases, canine monocytotropic ehrlichiosis (Ehrlichia canis), heartwater (Ehrlichia ruminantium), and bovine anaplasmosis (A. marginale), requires detailed immunity and immunopathology investigations, including the roles of CD8 T lymphocytes.
Seminars in Immunopathology 04/2015; 37(3). DOI:10.1007/s00281-015-0480-x · 6.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background. Ehrlichioses are emerging, tick-borne diseases distributed world-wide. Previously established animal models use needle inoculation as a mode of infection; however, there is limited representation of natural transmission in artificially inoculated models compared with transmission by the tick vector. The objective of this study was to develop a tick vector transmission animal model of ehrlichial infection using a human pathogen, Ehlichia muris-like agent (EMLA). Methods. Ixodes scapularis larvae were fed on EMLA-infected mice, and after molting infected nymphs were used to infest naïve animals. Results. Ehrlichiae were acquired by 90-100% of feeding larvae. The majority of animals fed upon by infected nymphs developed sublethal infection with 27% lethality. Bacteria disseminated to all tissues tested with greatest bacterial loads in lungs, but also spleen, lymph nodes, liver, kidneys, brain and bone marrow. Numerous foci of cellular infiltration, mitoses and hepatocellular death were observed in liver. Mice infected by tick transmission developed higher anti-ehrlichial antibody levels than needle-inoculated animals. Tick feeding site reactions were observed, but there was no observed difference between animals infested with infected or uninfected ticks. Conclusion. For the first time we were able to develop a tick transmission model with an Ehrlichia that is pathogenic for humans.
The Journal of Infectious Diseases 03/2015; DOI:10.1093/infdis/jiv134 · 5.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Twelve patients with murine typhus were identified in Galveston, Texas, USA, in 2013. An isolate from 1 patient was confirmed to be Rickettsia typhi. Reemergence of murine typhus in Galveston emphasizes the importance of vector control and awareness of this disease by physicians and public health officials.
[Show abstract][Hide abstract] ABSTRACT: The sequence of steps in the disease process caused by pathogenic microbes is known as pathogenesis. Pathogenesis encompasses the entire host–pathogen interaction and requires an understanding of the pathogen and pathogen-associated factors that contribute to the disease process as well as host responses that can be protective, circumvented, or contribute to disease pathology. During this process, microbes must evade innate and/or adaptive host defenses and often have developed molecular strategies to subvert host defenses including secretion of toxins that kill host cells or enzymes that degrade immune effectors, such as antibodies, or involve secretion of effector proteins that engage in molecular interactions with host cell targets in order to modulate cellular processes/immune responses that favor survival of the pathogen. In some cases, the damage to the host involves over-reactive response to the microbe that leads to tissue damage, such as overproduction of inflammatory cytokines as occurs in sepsis. Vaccines often do not prevent infection, but vaccine-induced antibodies can block attachment of the microbe to the host, prevent invasion, neutralize a secreted toxin, or enhance microbial killing. This chapter defines pathogenesis with respect to infectious diseases and exploresfeatures of pathogenesis and how vaccines and vaccine-induced immune mechanisms interrupt disease pathogenesis.
[Show abstract][Hide abstract] ABSTRACT: Louse borne typhus (also called epidemic typhus) was one of man's major scourges, and epidemics of the disease can be reignited when social, economic, or political systems are disrupted. The fear of a bioterrorist attack using the etiologic agent of typhus, Rickettsia prowazekii, was a reality. An attenuated typhus vaccine, R. prowazekii Madrid E strain, was observed to revert to virulence as demonstrated by isolation of the virulent revertant Evir strain from animals which were inoculated with Madrid E strain. The mechanism of the mutation in R. prowazekii that affects the virulence of the vaccine was not known. We sequenced the genome of the virulent revertant Evir strain and compared its genome sequence with the genome sequences of its parental strain, Madrid E. We found that only a single nucleotide in the entire genome was different between the vaccine strain Madrid E and its virulent revertant strain Evir. The mutation is a single nucleotide insertion in the methyltransferase gene (also known as PR028) in the vaccine strain that inactivated the gene. We also confirmed that the vaccine strain E did not cause fever in guinea pigs and the virulent revertant strain Evir caused fever in guinea pigs. We concluded that a single nucleotide insertion in the methyltransferase gene of R. prowazekii attenuated the R. prowazekii vaccine strain E. This suggested that an irreversible insertion or deletion mutation in the methyl transferase gene of R. prowazekii is required for Madrid E to be considered a safe vaccine.
PLoS ONE 11/2014; 9(11):e113285. DOI:10.1371/journal.pone.0113285 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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-1β, 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.
[Show abstract][Hide abstract] ABSTRACT: Rickettsia massiliae, belonging to the spotted fever group of Rickettsia, is a human pathogen causing a similar course of disease to that caused by R. conorii, the originally recognized etiologic agent of Mediterranean spotted fever. In view of this similarity, we performed an ultrastructural study of R. massiliae in organs of Rhipicephalus sanguineus ticks, in order to advance knowledge of the complex dynamics at the tick-pathogen interface in rickettsioses. Adult R. massiliae-infected Rh. sanguineus ticks were fed on uninfected Hartley strain guinea pigs, and five females were collected daily throughout their feeding period up to day 6, and analyzed by quantitative real-time PCR and electron microscopy. An increase in rickettsial content was observed in the salivary glands, particularly in the first two days of feeding, and a plateau was observed between days 3 and 6. Rickettsial organisms were observed in all tick organs analyzed, in higher numbers in the fed state, and statistically significant differences were observed in measurements of the periplasmic layer of R. massiliae in salivary glands of fed and unfed Rh. sanguineus ticks, with increased thickness in the former case. This study provides insight into the interface between R. massiliae and Rh. sanguineus ticks, highlighting the need for analysis of R. massiliae to fully ascertain its place as an important pathogenic agent of a spotted fever rickettsiosis.
[Show abstract][Hide abstract] ABSTRACT: Abstract 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.
[Show abstract][Hide abstract] ABSTRACT: 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 1.25×106 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.
[Show abstract][Hide abstract] ABSTRACT: Background. Human ehrlichioses are emerging life-threatening diseases transmitted by ticks. Animal models have been developed to study disease development; however, there is no valid small animal model that uses a human ehrlichial pathogen. The objective of this study was to develop a mouse model for ehrlichiosis with the newly discovered human pathogen, Ehrlichia muris-like agent (EMLA). Methods.Three strains of mice were inoculated with different doses of EMLA by the intravenous, intraperitoneal, or intradermal route and evaluated for clinical and pathologic changes during the course of infection. Results.EMLA infected C57Bl/6, BALB/c, and C3H/HeN mice and induced lethal or persistent infection in a route-and dose-dependent manner. The clinical chemistry and hematologic changes were similar to those of human infection by Ehrlichia chaffeensis or EMLA. Bacterial distribution in tissues differed after intradermal infection, compared with the distribution after intravenous or intraperitoneal injection. Lethal infection did not cause remarkable pathologic changes, but it caused fluid imbalance. EMLA infection of endothelium and mononuclear cells likely plays a role in the severe outcome. Conclusions.The EMLA mouse model mimics human infection and can be used to study pathogenesis and immunity and for development of a vector transmission model of ehrlichiosis.
The Journal of Infectious Diseases 07/2014; 211(3). DOI:10.1093/infdis/jiu372 · 5.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Abstract Tick-borne diseases, such as spotted fever rickettsioses and ehrlichioses, are potentially severe and life-threatening infections. The incidences of these infections increase during warm weather months as ticks become active. Clinicians often consider outdoor activities in rural areas to be a risk factor for exposure to ticks and the pathogens they carry, but are those who live, work, and play within an urban environment excluded from this risk? In this study, we collected ticks from two urban parks in Little Rock, AR, to assess the presence of rickettsiae and ehrlichiae within an urban setting. A total of 273 ticks were collected during July, 2011. Amblyomma americanum was the predominant tick species, with 255 (93%) of those collected. The remaining 18 (7%) were Dermacentor variabilis. Ticks were separated and pooled into groups for further testing. Forty-two of the 43 (98%) A. americanum pools demonstrated molecular evidence for the presence of rickettsiae. None of the D. variabilis contained rickettsiae. Restriction enzyme fragment length polymorphism analysis and DNA sequencing revealed Rickettsia amblyommii to be the species present. One A. americanum pool from park A demonstrated the presence of Ehrlichia chaffeensis, the pathogen responsible for human monocytotropic ehrlichiosis. These results indicate that tick-borne pathogens are not limited to rural or suburban areas.
[Show abstract][Hide abstract] ABSTRACT: We report here the complete genome sequence of Ehrlichia muris strain AS145(T), which was isolated from a wild mouse in 1983 in Japan. E. muris establishes persistent infections in laboratory mice and is widely used as a surrogate pathogen in a murine model of ehrlichiosis.
[Show abstract][Hide abstract] ABSTRACT: Severe fever with thrombocytopenia syndrome virus (SFTSV) is a newly discovered Phlebovirus causing an emerging hemorrhagic fever in East Asia with reported case fatality rates up to 30%. Despite the high case fatality rate and large number of persons at risk of infection, the pathobiology of the disease is unknown, and no effective animal model has been available for investigating the pathogenesis of the disease. We have studied mice and hamsters as potential small animal models of SFTSV infection following subcutaneous, intraperitoneal or intracerebral inoculation. Animal tissues were processed for viral load, histopathology, immunohistochemistry, confocal and electron microscopic studies. We found that immunocompetent adult mice and hamsters did not become ill after SFTSV infection. However, alpha/beta interferon receptor (IFNAR(-/-)) knockout mice were highly susceptible to SFTSV infection, and all mice died within 3-4 days after subcutaneous inoculation of 10(6) ffu of SFTSV. Histologic examination of tissues of IFNAR(-/-)mice infected with SFTSV showed no detectable lesions. In contrast, by immunohistochemistry virus antigen was found in liver, intestine, kidney, spleen, lymphoid tissue, and brain, but not in the lungs. Mesenteric lymph nodes and spleen were the most heavily infected tissues. Quantitative RT-PCR confirmed the presence of virus in these same tissues. Confocal microscopy showed that SFTSV colocalized with reticular cells, but did not colocalize with dendritic cells, monocytes/macrophages, neutrophils or endothelium. Our results indicate that SFTSV multiplied in all organs except for lungs and that mesenteric lymph nodes and spleen were the most heavily infected tissues. The major target cells of SFTSV appear to be reticular cells in lymphoid tissues of intestine and spleen.
Journal of Virology 11/2013; 88(3). DOI:10.1128/JVI.02277-13 · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Rickettsiae are responsible for some of the most devastating human infections. A high infectivity and severe illness after inhalation make some rickettsiae bioterrorism threats. We report that deletion of the exchange protein directly activated by cAMP (Epac) gene, Epac1, in mice protects them from an ordinarily lethal dose of rickettsiae. Inhibition of Epac1 suppresses bacterial adhesion and invasion. Most importantly, pharmacological inhibition of Epac1 in vivo using an Epac-specific small-molecule inhibitor, ESI-09, completely recapitulates the Epac1 knockout phenotype. ESI-09 treatment dramatically decreases the morbidity and mortality associated with fatal spotted fever rickettsiosis. Our results demonstrate that Epac1-mediated signaling represents a mechanism for host-pathogen interactions and that Epac1 is a potential target for the prevention and treatment of fatal rickettsioses.
Proceedings of the National Academy of Sciences 11/2013; DOI:10.1073/pnas.1314400110 · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ehrlichioses are emerging tick-borne bacterial diseases of humans and animals for which no vaccines are available. The diseases are caused by obligately intracellular bacteria belonging to the genus Ehrlichia. Several immunoreactive proteins of ehrlichiae have been identified based on their reactivity with immune sera from human patients and animals. These include the major outer membrane proteins, ankyrin repeat proteins and tandem repeat proteins (TRP). Polyclonal antibodies directed against the tandem repeats (TRs) of Ehrlichia chaffeensis TRP32, TRP47 and TRP120 have been shown to provide protection in mice. In the present study, we evaluated E. muris P29, which is the ortholog of E. chaffeensis TRP47 and E. canis TRP36, as a subunit vaccine in a mouse model of ehrlichiosis. Our study indicated that unlike E. chaffeensis TRP47 and E. canis TRP36, orthologs of E. muris (P29) and E. muris-like agent (EMLA) do not contain tandem repeats. Immunization of mice with recombinant E. muris P29 induced significant protection against a challenge infection. The protection induced by E. muris P29 was associated with induction of strong antibody responses. In contrast to development of P29-specific IgG antibodies following immunization, development of P29-specific IgG antibodies, but not IgM antibodies, was impaired during persistent E. muris infection. Furthermore, our study indicated that CD4+ T cells target P29 during E. muris infection and differentiate into IFN-γ-producing Th1 effector/memory cells. In conclusion, our study indicated that orthologs of E. muris P29 showed considerable variation in the central tandem repeat region among different species, induction of P29-specific IgG antibody response was impaired during persistent E. muris infection, and rP29 induced protective immune responses.