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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.
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Ciervos , Ehrlichia chaffeensis , Rhipicephalus sanguineus , Rickettsia , Rickettsiosis Exantemáticas , Animales , Gatos , Perros , Caballos , Vacio , New Mexico/epidemiología , EquidaeRESUMEN
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.
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Amblyomma , Rickettsia , Animales , Colombia/epidemiología , Filogenia , Rickettsia/genética , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
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.
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Rickettsia , Rickettsiosis Exantemáticas , Tifus Epidémico Transmitido por Piojos , Humanos , Animales , Perros , Rickettsia/genética , México/epidemiología , Anticuerpos Antibacterianos , Rickettsiosis Exantemáticas/diagnóstico , Rickettsiosis Exantemáticas/epidemiología , Rickettsiosis Exantemáticas/veterinariaRESUMEN
Whether increases in typhus group rickettsiosis in Galveston County, Texas, USA, are caused by increased recognition or true reemergence is unclear. We conducted a serosurvey that demonstrated Rickettsia typhi antibodies increased from 1.2% in 2013 to 7.8% in 2021 (p<0.001). These findings support pathogen reemergence rather than enhanced recognition alone.
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Tifus Endémico Transmitido por Pulgas , Tifus Epidémico Transmitido por Piojos , Humanos , Tifus Endémico Transmitido por Pulgas/diagnóstico , Tifus Endémico Transmitido por Pulgas/epidemiología , Rickettsia typhi , Tifus Epidémico Transmitido por Piojos/epidemiología , Tifus Epidémico Transmitido por Piojos/microbiología , Texas/epidemiología , Estudios SeroepidemiológicosRESUMEN
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.
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Didelphis , Monodelphis , Infecciones por Rickettsia , Rickettsia , Siphonaptera , Tifus Endémico Transmitido por Pulgas , Animales , Didelphis/microbiología , Ratones , Ratas , Rickettsia/genética , Infecciones por Rickettsia/microbiología , Rickettsia typhi , Siphonaptera/microbiología , Tifus Endémico Transmitido por Pulgas/microbiologíaRESUMEN
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.
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Ciervos , Ehrlichia chaffeensis , Ehrlichiosis , Garrapatas , Amblyomma , Animales , Ehrlichia chaffeensis/genética , Ehrlichiosis/epidemiología , Ehrlichiosis/veterinaria , Genotipo , Humanos , TexasRESUMEN
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.
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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.
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Orientia tsutsugamushi infection can cause acute lung injury and high mortality in humans; however, the underlying mechanisms are unclear. Here, we tested a hypothesis that dysregulated pulmonary inflammation and Tie2-mediated endothelial malfunction contribute to lung damage. Using a murine model of lethal O. tsutsugamushi infection, we demonstrated pathological characteristics of vascular activation and tissue damage: 1) a significant increase of ICAM-1 and angiopoietin-2 (Ang2) proteins in inflamed tissues and lung-derived endothelial cells (EC), 2) a progressive loss of endothelial quiescent and junction proteins (Ang1, VE-cadherin/CD144, occuludin), and 3) a profound impairment of Tie2 receptor at the transcriptional and functional levels. In vitro infection of primary human EC cultures and serum Ang2 proteins in scrub typhus patients support our animal studies, implying endothelial dysfunction in severe scrub typhus. Flow cytometric analyses of lung-recovered cells further revealed that pulmonary macrophages (MΦ) were polarized toward an M1-like phenotype (CD80+CD64+CD11b+Ly6G-) during the onset of disease and prior to host death, which correlated with the significant loss of CD31+CD45- ECs and M2-like (CD206+CD64+CD11b+Ly6G-) cells. In vitro studies indicated extensive bacterial replication in M2-type, but not M1-type, MΦs, implying the protective and pathogenic roles of M1-skewed responses. This is the first detailed investigation of lung cellular immune responses during acute O. tsutsugamushi infection. It uncovers specific biomarkers for vascular dysfunction and M1-skewed inflammatory responses, highlighting future therapeutic research for the control of this neglected tropical disease.
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Angiopoyetina 2/metabolismo , Células Endoteliales/patología , Orientia tsutsugamushi/crecimiento & desarrollo , Neumonía/patología , Receptor TIE-2/metabolismo , Tifus por Ácaros/patología , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Humanos , Macrófagos/inmunología , Macrófagos/microbiología , Ratones Endogámicos C57BL , Neumonía/inmunología , Tifus por Ácaros/inmunologíaRESUMEN
Introduction: During pandemic situations like the one caused by the emergent coronavirus SARS-CoV-2, healthcare systems face the challenge of limited personal protective equipment and impaired supply chains. This problem poses a threat to healthcare workers, first responders, and the public, which demands solutions that can span the gap between institutional shortages and resupplies. Objectives: To examine the efficacy of autoclave-based decontamination for the reuse of single-use surgical masks and N95 filtering facepiece respirators (FFRs). This method is the most readily available form of decontamination in the hospital and laboratory settings. Methods: Three models of N95 FFRs and two procedural masks were evaluated in this study. A moist heat autoclave using four different autoclave cycles: 115°C for one hour, 121.1°C for 30 minutes, 130°C for two minutes, and 130°C for four minutes was used. After the autoclave process, the FFRs were NIOSH fit tested and particle counting was performed for both coarse particles of 5 micrometers (µM) and fine particles from 0.1µM to 1.0µM. Results: We observed negligible alterations in the functionality and integrity of 3M 1805 and 3M 1870/1870+ N95 FFRs after three autoclave cycles. Surgical masks also showed minimal changes in functionality and integrity. The 3M 1860 FFR failed fit test after a single autoclave decontamination cycle. Discussion and Conclusion: The study finds that specific surgical masks and N95 FFR models can withstand autoclave decontamination for up to three cycles. Additionally, the autoclave cycles tested were those that could be readily achieved by both clinical and research institutions.
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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 (ROS) as a means of decontamination. Objectives: The purpose of this study is to evaluate the diffusion effect of iHP and its decontamination capabilities using biological and enzyme indicators. Methods: A gas-tight fumigation room with a volume of 880 ft3 was used for the decontamination trials. During the decontamination process, empty animal cages were placed inside to create fumigant distribution restrictions. Spore and enzyme indicators were placed in eleven locations throughout the decontamination room. Generation of iHP was done with the use of TOMI's SteraMist Environmental System and the SteraMist Solution, with 7.8% H2O2 at a dose of 0.5 ml per ft3. Results: For the decontamination of 1hr, 2hrs, 6hrs, and 12hrs, the biological indicators of B. atrophaeus in Stainless Steel (SS) Disk in Tyvek envelope have an inactivation rate of 94%, 97%, 100%, and 100%, respectively. For G. stearothermophilus in SS disk and Tyvek envelope, it has 82%, 68%, 100%, and 100%, respectively and, for G. stearothermophilus in SS strips it has an effective rate of 88%, 67%, 91%, and 100%, respectively. Conclusion: iHP inactivates spores, and the residual tAK activity indicates a gas-like fumigant diffusion due to the uniformity of the inactivation without the use of oscillating fans as the contact time is extended.
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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.
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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.
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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.
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Contención de Riesgos Biológicos , Modelos Animales de Enfermedad , Rickettsia/crecimiento & desarrollo , Rickettsia/patogenicidad , Rickettsiosis Exantemáticas/patología , Animales , Masculino , Ratones Endogámicos C3H , Análisis de SupervivenciaRESUMEN
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.
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Polimorfismo de Longitud del Fragmento de Restricción , Infecciones por Rickettsia/veterinaria , Rickettsia felis/aislamiento & purificación , Rickettsia/aislamiento & purificación , Animales , Gatos , Ctenocephalides/microbiología , ADN Bacteriano , Didelphis , Electroforesis en Gel de Poliacrilamida , Infestaciones por Pulgas/veterinaria , Insectos Vectores/microbiología , Rickettsia/genética , Infecciones por Rickettsia/microbiología , Rickettsia felis/genética , TexasRESUMEN
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.
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Enfermedades de los Gatos/parasitología , Infestaciones por Pulgas/veterinaria , Rickettsia/aislamiento & purificación , Siphonaptera/microbiología , Animales , Enfermedades de los Gatos/epidemiología , Gatos , Femenino , Infestaciones por Pulgas/epidemiología , Infestaciones por Pulgas/parasitología , Masculino , Texas/epidemiologíaRESUMEN
Working with virological samples requires validated inactivation protocols for safe handling and disposal. Although many techniques exist to inactivate samples containing viruses, not all procedures have been properly validated or are compatible with subsequent assays. To aid in the development of inactivation protocols for Alphaviruses, and specifically Venezuelan equine encephalitis virus (VEEV), a variety of methods were evaluated for their ability to completely inactivate a high titer sample of the vaccine strain VEEV TC-83. The methods evaluated include reagents used in RNA extraction, fixation, treatment with a detergent, and heat inactivation. Most methods were successful at inactivating the sample; however, treatment with only Buffer AVL, SDS, and heat inactivation at 58⯰C for one hour were not capable of complete inactivation of the virus in the sample. These results provide a substantial framework for identifying techniques that are safe for complete inactivation of Alphaviruses and to advise protocol implementation.
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Desinfectantes/farmacología , Desinfección , Virus de la Encefalitis Equina Venezolana/efectos de los fármacos , Virus de la Encefalitis Equina Venezolana/efectos de la radiación , Calor , Animales , Línea Celular , Chlorocebus aethiops , Efecto Citopatogénico Viral/efectos de los fármacos , Efecto Citopatogénico Viral/efectos de la radiación , Desinfección/métodos , Células VeroRESUMEN
[This corrects the article DOI: 10.1371/journal.pntd.0005763.].
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BACKGROUND: Scrub typhus is a life-threatening disease, due to infection with O. tsutsugamushi, a Gram-negative bacterium that preferentially replicates in endothelial cells and professional phagocytes. Meningoencephalitis has been reported in scrub typhus patients and experimentally-infected animals; however, the neurological manifestation and its underlying mechanisms remain poorly understood. To address this issue, we focused on Orientia tsutsugamushi Karp strain (OtK), and examined host responses in the brain during lethal versus self-healing scrub typhus disease in our newly established murine models. PRINCIPLE FINDINGS: Following inoculation with a lethal dose of OtK, mice had a significant increase in brain transcripts related to pathogen-pattern recognition receptors (TLR2, TLR4, TLR9), type-1 responses (IFN-γ, TNF-α, CXCL9, CXCR3), and endothelial stress/damage such as angiopoietins, but a rapid down-regulation of Tie2. Sublethal infection displayed similar trends, implying the development of type 1-skewed proinflammatory responses in infected brains, independent of time and disease outcomes. Focal hemorrhagic lesions and meningitis were evident in both infection groups, but pathological changes were more diffuse and frequent in lethal infection. At 6-10 days of lethal infection, the cortex and cerebellum sections had increased ICAM-1-positive staining in vascular cells, as well as increased detection of CD45+ leukocytes, CD3+ T cells, IBA1+ phagocytes, and GFAP+ astrocytes, but a marked loss of occludin-positive tight junction staining, implying progressive endothelial activation/damage and cellular recruitment in inflamed brains. Orientia were sparse in the brains, but readily detectable within lectin+ vascular and IBA-1+ phagocytic cells. These CNS alterations were consistent with type 1-skewed, IL-13-suppressed responses in lethally-infected mouse lungs. SIGNIFICANCE: This is the first report of type 1-skewed neuroinflammation and cellular activation, accompanied with vascular activation/damage, during OtK infection in C57BL/6 mice. This study not only enhances our understanding of the pathophysiological mechanisms of scrub typhus, but also correlates the impact of immune and vascular dysfunction on disease pathogenesis.
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Encéfalo/patología , Meningoencefalitis/fisiopatología , Tifus por Ácaros/complicaciones , Tifus por Ácaros/patología , Animales , Encéfalo/inmunología , Citocinas/análisis , Modelos Animales de Enfermedad , Regulación hacia Abajo , Femenino , Humanos , Inflamación , Pulmón/patología , Meningoencefalitis/microbiología , Ratones , Ratones Endogámicos C57BL , Orientia tsutsugamushiRESUMEN
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.