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The order Rickettsiales in the class Alphaproteobacteria comprises vector-borne pathogens of both medical and veterinary importance. Ticks, as a group, are second only to mosquitoes as vectors of pathogens to humans, playing a critical role in the transmission of rickettsiosis. In the present study, 880 ticks collected from Jinzhai County, Lu'an City, Anhui Province, China in 2021–2022 were identified as belonging to five species from three genera. DNA extracted from individual ticks was examined using nested polymerase chain reaction targeting the 16S rRNA gene (rrs), and the gene fragments amplified were sequenced to detect and identify Rickettsiales bacteria in the ticks. For further identification, the rrs-positive tick samples were further amplified by PCR targeting the gltA and groEL gene and sequenced. As a result, 13 Rickettsiales species belonging to the genera Rickettsia, Anaplasma, and Ehrlichia were detected, including three tentative species of Ehrlichia. Our results reveal the extensive diversity of Rickettsiales bacteria in ticks from Jinzhai County, Anhui Province. There, emerging rickettsial species may be pathogenic and cause under-recognized diseases. Detection of several pathogens in ticks that are closely related to human diseases may indicate a potential risk of infection in humans. Therefore, additional studies to assess the potential public health risks of the Rickettsiales pathogens identified in the present study are warranted.

We report a case-series study of 5 patients with Japanese spotted fever from the Three Gorges Area in China, including 1 fatal case. Seroprevalence of Rickettsia japonica was ≈21% among the local population. Our report highlights the emerging potential threat to human health of Japanese spotted fever in the area.

Between November 2021 and January 2022, four patients of community-acquired pneumonia were admitted to the hospitals in Lishui city, Zhejiang province, China. Their main clinical manifestations were fever and dry cough as well as radiographic infiltrate, but the empiric antimicrobial therapy or traditional Chinese medicine was not effective for their illness. Clinical specimens from the patients as well as environmental and poultry specimens were collected for the determination of the causative pathogen. The

Objective: Hyalomma marginatum is an important arthropod vector in the transmission of various zoonoses. The aim of this study was to identify the tick-borne pathogens (TBPs) maintained in Hy. marginatum in Tibet and to estimate the risk of human tick-borne diseases. Methods: Adult Hy. marginatum ticks (n = 14) feeding on yaks were collected. The individual DNA samples of these ticks were sequenced with metagenomic next-generation sequencing to survey the presence of TBPs. TBPs in individual ticks were identified with nested polymerase chain reaction (PCR) combined with DNA sequencing. Results: The presence of Rickettsia, Anaplasma, and Ehrlichia in individual ticks was indicated by the taxonomic profiles at the genus level, but only Rickettsia aeschlimannii (100%, 13/13) was further detected in the ticks by nested PCR. Conclusion: This study provides information on the microbial communities of Hy. marginatum in Tibet, China, and provides the first report of R. aeschlimannii found in Hy. marginatum in Tibet. The results of this study indicated that yaks in Tibet are exposed to R. aeschlimannii.

Coxiella burnetii, the causative agent of zoonotic Q fever, is characterized by replicating inside the lysosome-derived Coxiella-containing vacuole (CCV) in host cells. Some effector proteins secreted by C. burnetii have been reported to be involved in the manipulation of autophagy to facilitate the development of CCVs and bacterial replication. Here, we found that the Coxiella plasmid effector B (CpeB) localizes on vacuole membrane targeted by LC3 and LAMP1 and promotes LC3-II accumulation. Meanwhile, the C. burnetii strain lacking the QpH1 plasmid induced less LC3-II accumulation, which was accompanied by smaller CCVs and lower bacterial loads in THP-1 cells. Expression of CpeB in the strain lacking QpH1 led to restoration in LC3-II accumulation but had no effect on the smaller CCV phenotype. In the severe combined immune deficiency (SCID) mouse model, infections with the strain expressing CpeB led to significantly higher bacterial burdens in the spleen and liver than its parent strain devoid of QpH1. We also found that CpeB targets Rab11a to promote LC3-II accumulation. Intratracheally inoculated C. burnetii resulted in lower bacterial burdens and milder lung lesions in Rab11a conditional knockout (Rab11a(−/−) CKO) mice. Collectively, these results suggest that CpeB promotes C. burnetii virulence by inducing LC3-II accumulation via a pathway involving Rab11a.

Coxiella burnetiiis the etiological agent of the zoonotic disease Q fever, which is featured by its ability to replicate in acid vacuoles resembling the lysosomal network. One key virulence determinant ofC.burnetiiis the Dot/Icm system that transfers more than 150 effector proteins into host cells. These effectors function to construct the lysosome-like compartment permissive for bacterial replication, but the functions of most of these effectors remain elusive. In this study, we used an affinity tag purification mass spectrometry (AP-MS) approach to generate aC.burnetii-human protein-protein interaction (PPI) map involving 53C.burnetiieffectors and 3480 host proteins. This PPI map revealed that theC.burnetiieffector CBU0425 (designated CirB) interacts with most subunits of the 20S core proteasome. We found that ectopically expressed CirB inhibits hydrolytic activity of the proteasome. In addition, overexpression of CirB inC.burnetiicaused dramatic inhibition of proteasome activity in host cells, while knocking down CirB expression alleviated such inhibitory effects. Moreover, we showed that a region of CirB that spans residues 91–120 binds to the proteasome subunit PSMB5 (beta 5). Finally, PSMB5 knockdown promotesC.burnetiivirulence, highlighting the importance of proteasome activity modulation during the course ofC.burnetiiinfection.

BackgroundCoxiella burnetii(Cb) is the causative agent of the zoonotic disease Q fever which is distributed worldwide. Molecular typing of Cb strains is essential to find out the infectious source and prevent Q fever outbreaks, but there has been a lack of typing data for Cb strains in China. The aim of this study was to investigate the genotypes of Cb strains in wild rats in Yunnan Province, China.ResultsEighty-six wild rats (Rattus flavipectus) were collected in Yunnan Province and 8 of the 86 liver samples from the wild rats were positive in Cb-specific quantitative PCR (qPCR). The Cb strains from the 8 rats were then typed into 3 genotypes using 10-spacer multispacer sequence typing (MST), and 2 of the 3 genotypes were recognized as novel ones. Moreover, the Cb strains in the wild rats were all identified as genotype 1 using 6-loci multilocus variable number of tandem repeat analysis (MLVA).ConclusionsThis is the first report of genotypic diversity of Cb strains from wild rats in China. Further studies are needed to explore the presence of more genotypes and to associate the genotypes circulating in the wildlife-livestock interaction with those causing human disease to further expand on the epidemiological aspects of the pathogen.

Rhipicephalus microplus, a vector that can transmit many pathogens to humans and domestic animals, is widely distributed in Yunnan province, China. However, few reports on the prevalence of tick-borne pathogens (TBPs) in Rh. microplus in Yunnan are available. The aim of this study was to detect TBPs in Rh. microplus in Yunnan and to analyze the phylogenetic characterization of TBPs detected in these ticks. The adult Rh. microplus (n = 516) feeding on cattle were collected. The pooled DNA samples of these ticks were evaluated using metagenomic next-generation sequencing (mNGS) and then TBPs in individual ticks were identified using genus- or group-specific nested polymerase chain reaction (PCR) combined with DNA sequencing assay. As a result, Candidatus Rickettsia jingxinensis (24.61%, 127/516), Anaplasma marginale (13.18%, 68/516), Coxiella burnetii (3.10%, 16/516), and Coxiella-like endosymbiont (CLE) (8.33%, 43/516) were detected. The dual coinfection with Ca. R. jingxinensis and A. marginale and the triple coinfection with Ca. R. jingxinensis, A. marginale, and CLE were most frequent and detected in 3.68% (19/516) and 3.10% (16/516) of these ticks, respectively. The results provide insight into the diversity of TBPs and their coinfections in Rh. microplus in Yunnan province of China, reporting for the first time that C. burnetii had been found in Rh. microplus in China. Multilocus variable number tandem repeat analysis with 6 loci (MLVA-6) discriminated the C. burnetii detected in Rh. microplus in Yunnan into MLVA genotype 1, which is closely related to previously described genotypes found primarily in tick and human samples from different regions of the globe, indicating a potential public health threat posed by C. burnetii in Rh. microplus in Yunnan.

Background Hard ticks act as arthropod vectors in the transmission of human and animal pathogens and are widely distributed in northern China. The aim of this study is to screen the important tick-borne pathogens (TBPs) carried by hard ticks in Inner Mongolia using metagenomic next-generation sequencing (mNGS) and to estimate the risk of human infection imposed by tick bites. Methods The adult Dermacentor nuttalli (n = 203) and Ixodes persulcatus (n = 36) ticks feeding on cattle were collected. The pooled DNA samples prepared from these ticks were sequenced as the templates for mNGS to survey the presence of TBPs at the genus level. Individual tick DNA samples were detected by genus--specific or group-specific nested polymerase chain reaction (PCR) of these TBPs and combined with DNA sequencing assay to confirm the results of mNGS. Results R. raoultii (45.32%, 92/203), Candidatus R. tarasevichiae (5.42%, 11/203), Anaplasma sp. Mongolia (26.60%, 54/203), Coxiella-like endosymbiont (CLE) (53.69%, 109/203), and Babesia venatorum (7.88%, 16/203) were detected in D. nuttalli, while R. raoultii (30.56%, 11/36), Anaplasma sp. Mongolia (27.80%, 10/36), and CLE (27.80%, 10/36) were detected in I. persulcatus. The double- and triple-pathogen/endosymbiont co-infections were detected in 40.39% of D. nuttalli and 13.89% of I. persulcatus, respectively. The dual co-infection with R. raoultii and CLE (14.29%, 29/203) and triple co-infection with R. raoultii, Anaplasma sp. Mongolia, and CLE (13.79%, 28/203) were most frequent in D. nuttalli. Conclusions This study provides insight into the microbial diversity of D. nuttalli and I. persulcatus in Inner Mongolia, China, reporting for the first time that Candidatus R. tarasevichiae had been found in D. nuttalli in China, and for the first time in the world that Anaplasma sp. Mongolia has been detected in I. persulcatus. This study proves that various vertically transmitted pathogens co-inhabit D. nuttalli and I. persulcatus, and indicates that cattle in Inner Mongolia are exposed to several TBPs. Graphical Abstract

Q fever is a worldwide zoonosis caused by Coxiella burnetii (Cb). From January 2018 to November 2019, plasma samples from 2,382 patients with acute fever of unknown cause at a hospital in Zhuhai city of China were tested using metagenomic next-generation sequencing (mNGS). Of those tested, 138 patients (5.8%) were diagnosed with Q fever based on the presence of Cb genomic DNA detected by mNGS. Among these, 78 cases (56.5%) presented from Nov 2018 to Mar 2019, suggesting an outbreak of Q fever. 55 cases with detailed clinical information that occurred during the outbreak period were used for further analysis. The vast majority of plasma samples from those Cb-mNGS-positive patients were positive in a Cb-specific quantitative polymerase chain reaction (n = 38) and/or indirect immunofluorescence assay (n = 26). Mobile phone tracing data was used to define the area of infection during the outbreak. This suggested the probable infection source was Cb-infected goats and cattle at the only official authorized slaughterhouse in Zhuhai city. Phylogenic analysis based on genomic sequences indicated Cb strains identified in the patients, goat and cattle were formed a single branch, most closely related to the genomic group of Cb dominated by strains isolated from goats. Our study demonstrates Q fever was epidemic in 2018–2019 in Zhuhai city, and this is the first confirmed epidemic of Q fever in a contemporary city in China., Author summary Generally, the clinical diagnosis of acute Q fever, which is caused by Coxiella burnetii, is based on serologic methods that detect the presence antibodies produced by the body to fight the infection. However, the lag time between becoming infected and production of antibodies limits early diagnosis using this method. Here, we confirmed an epidemic of human Q fever in Zhuhai, a contemporary city in China, using clinical metagenomic next-generation sequencing (mNGS) and cell phone location data. Our results indicate that Cb-infected goats and cattle at the only official authorized slaughterhouse in Zhuhai were the likely infection source for the Q fever epidemic. More importantly, we demonstrate that mNGS is a useful tool for rapid and effective public health responses to acute bacterial infections.

BackgroundCoxiella burnetiiis an obligate intracellular Gram-negative bacterium that causes a zoonotic disease commonly called Q fever globally. In this study, an up-converting phosphor technology-based lateral flow (UPT-LF) assay was established for the rapid and specific detection of phase I strains ofC. burnetii.ResultsSpecific monoclonal antibodies (10B5 and 10G7) againstC. burnetiiphase I strains were prepared and selected for use in the UPT-LF assay by the double-antibody-sandwich method. The detection sensitivity of theCoxiella-UPT-LF was 5 × 104GE/ml for a purifiedC. burnetiiphase I strain and 10 ng/ml for LPS ofC. burnetiiNine Mile phase I (NMI). Good linearity was observed forC. burnetiiphase I and NMI LPS quantification (R2 ≥ 0.989). The UPT-LF assay also exhibited a high specificity toC. burnetii, without false-positive results even at 108GE/ml of non-specific bacteria, and good inclusivity for detecting different phase I strains ofC. burnetii. Moreover, the performance of theCoxiella-UPT-LF assay was further confirmed using experimentally and naturally infected samples.ConclusionsOur results indicate thatCoxiella-UPT-LF is a sensitive and reliable method for rapid screening ofC. burnetii,suitable for on-site detection in the field.

The scallop Chlamys farreri is an important bivalve species cultured in the north of China. With the large expansion of culturing, mass mortalities have occurred persistently and caused great economic loss since 1996. An intracellular Rickettsiales-like prokaryote (RLP) was proposed as the causative agent. To identify the RLP, a partial 16S rDNA sequence of 1319 base pairs was amplified, cloned and sequenced. Phylogenetic analysis indicates that the prokaryote is a member of the α subclass of the proteobacteria. In the phylogenetic tree, it forms an independent and novel clade, which shares an evolutionary line of descent with a group of uniquely obligate intracellular organism comprised of Caedibacter caryophilus, an endosymbiont of Acanthamoeba sp., and the necrotizing hepatopancreatitis (NHP) bacterium of shrimp. Based on the obtained 16S rDNA sequence, three specific RNA targeted oligonucleotide DNA probes were designed, synthesized and labeled with digoxigenin for in situ hybridization detection. Results showed that the probes specifically bonded to the RLP inclusion in infected C. farreri tissues, indicating the amplified, cloned and sequenced 16S rDNA originated from the intracellular RLP parasitizing the C. farreri. We propose the name Sinorickettsia chlamys sp.nov. to C. farreri RLP.

Background: Q fever is a worldwide zoonosis caused by Coxiella burnetii and mainly transmitted by aerosols. This study aims at establishing a systematic and efficient mouse model of acute Q fever via intratracheal (IT) inoculation of aerosolized C. burnetii. Methods: BALB/c mice were infected with C. burnetii via IT route using a non-invasive aerosol pulmonary delivery device to directly place the living C. burnetii organisms into their tracheas. The bacterial loads, pathological lesions, and serological responses were analyzed in mice, and compared with those of mice infected via intraperitoneal (IP) route. Results: As early as at day three post-infection (pi) with a low dose of C. burnetii (1×10⁴ per mouse), a large amount of C. burnetii organisms were determined in blood, lungs, hearts, livers, and spleens of the mice. The inflammatory infiltration was observed in hearts and lungs of mice. Compared with mice infected via IP route, the mice infected via IT route exhibited a higher level of bacterial loads and more severe pathological lesions in hearts and lungs at day 3 and day 7 pi. Conclusions: These data indicated that IT route is more efficient than IP route to cause acute C. burnetii infection in mice. Overall, we successfully established a mouse model of C. burnetii infection via IT route, which is useful for investigations of pathogenesis and immunity of acute C. burnetii infection as well as evaluation of therapeutic drugs and preventive vaccines of Q fever.

Human Q fever is recognized as a worldwide public health problem. It often occurs by inhalation of airborne aerosols contaminated with Coxiella burnetii, a gram-negative intracellular bacterium, mainly from domestic livestock. In this study, we analyzed the possibility to establish mucosal and systemic immunity against C. burnetii infection using a pulmonary delivery of chloroform-methanol residue of C. burnetii (CMR) vaccine. Mice were immunized by the intratracheal inoculation of CMR (IT-CMR) or the subcutaneous injection of CMR (SC-CMR), and the immunized mice were challenged with C. burnetii by the intratracheal route. The levels of IFN-γ, IL-12p70, IL-5, and IL-4 in the IT-CMR group in splenic T cells stimulated ex vivo were significantly higher than in the SC-CMR group. Significantly elevated sIgA to C. burnetii was detected in the bronchoalveolar lavage fluid of mice immunized by IT-CMR but not by SC-CMR, which might have contributed to the significant reduction in C. burnetii load and pathological lesions in the lungs of the mice after the challenge of C. burnetii. These results suggest that compared with SC-CMR in mice, IT-CMR was more efficient to elicit cellular and lung mucosal immune responses against aerosol infection of C. burnetii.

Q fever is a worldwide zoonosis caused by Coxiella burnetii. Human Q fever is typically acquired through inhalation of contaminated aerosols, resulting in an initial pulmonary infection. In this study, BALB/c mice were infected with C. burnetii via an intratracheal (IT) route using a non-invasive aerosol pulmonary delivery device to directly place the living C. burnetii organisms into the lungs of the mice. The bacterial loads, pathological lesions, and antibody and cellular responses were analyzed and compared with those of mice infected via an intraperitoneal (IP) route. Compared with mice infected via an IP route, mice infected via an IT route exhibited a higher bacterial load and more severe pathological lesions in the heart and lungs at days 3 and 7 post-infection (pi). The levels of interferon-γ and IL-12p70 in the serum of mice infected via the IT route were significantly higher than those of mice infected via the IP route at day 3 pi. In conclusion, this murine model of acute C. burnetii infection via IT inoculation closely resembles the natural route of C. burnetii infection than that of IP injection. Thus, this newly developed model will be useful for investigating the pathogenesis and immunity of C. burnetii aerosol infection, as well as for the evaluation of therapeutic drugs and preventive vaccines of Q fever.

The obligate intracellular Gram-negative bacterium Coxiella burnetii causes Q fever, a worldwide zoonosis. Here we labelled Cox . burnetii with biotin and used biotin-streptavidin affinity chromatography to isolate surface-exposed proteins (SEPs). Using two-dimensional electrophoresis combined with mass spectrometry, we identified 37 proteins through bioinformatics analysis. Thirty SEPs expressed in Escherichia coli (recombinant SEPs, rSEPs) were used to generate microarrays, which were probed with sera from mice experimentally infected with Cox. burnetii or sera from Q fever patients. Thirteen rSEPs were recognized as seroreactive, and the majority reacted with at least 50 % of the sera from mice infected with Cox. burnetii but not with sera from mice infected with Rickettsia rickettsii, R. heilongjiangensis, or R. typhi. Further, 13 proteins that reacted with sera from patients with Q fever did not react with sera from patients with brucellosis or mycoplasma pneumonia. Our results suggest that these seroreactive SEPs have potential as serodiagnostic antigens or as subunit vaccine antigens against Q fever.

© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. Coxiella burnetii is a gram-negative bacterium that causes acute and chronic Q fever. Because of the severe adverse effect of whole-cell vaccination, identification of immunodominant antigens of C. burnetii has become a major focus of Q fever vaccine development. We hypothesized that secreted C. burnetii type IV secretion system (T4SS) effectors may represent a major class of CD8 + T-cell antigens, owing to their cytosolic localization. Twenty-nine peptides were identified that elicited robust CD8 + T-cell interferon 3 (IFN-3) recall responses from mice infected with C. burnetii. Interestingly, 22 of 29 epitopes were derived from 17 T4SS-related proteins, none of which were identified as immunodominant antigens by using previous antibody-guided approaches. These epitopes were expressed in an attenuated Listeria monocytogenes vaccine strain. Immunization with recombinant L. monocytogenes vaccines induced a robust CD8 + T-cell response and conferred measurable protection against C. burnetii infection in mice. These data suggested that T4SS effectors represent an important class of C. burnetii antigens that can induce CD8 + T-cell responses. We also showed that attenuated L. monocytogenes vaccine vectors are an efficient antigen-delivery platform that can be used to induce robust protective CD8 + T-cell immune responses against C. burnetii infection.

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.

Rickettsia heilongjiangensis, the causative agent of far eastern spotted fever, is an obligate intracellular gram-negative bacterium that belongs to the spotted fever group rickettsiae. To understand the evolution and pathogenesis of R. heilongjiangensis, we analyzed its genome and compared it with other rickettsial genomes available in GenBank. The R. heilongjiangensis chromosome contains 1333 genes, including 1297 protein coding genes and 36 RNA coding genes. The genome also contains 121 pseudogenes, 54 insertion sequences, and 39 tandem repeats. Sixteen genes encoding the major components of the type IV secretion systems were identified in the R. heilongjiangensis genome. In total, 37 β-barrel outer membrane proteins were predicted in the genome, eight of which have been previously confirmed to be outer membrane proteins. In addition, 266 potential virulence factor genes, seven partially deleted antibiotic resistance genes, and a genomic island were identified in the genome. The codon usage in the genome is compatible with its low GC content, and the amino acid usage shows apparent bias. A comparative genomic analysis showed that R. heilongjiangensis and R. japonica share one unique fragment that may be a target sequence for a diagnostic assay. The orthologs of 37 genes of R. heilongjiangensis were found in pathogenic R. rickettsii str. Sheila Smith but not in non-pathogenic R. rickettsii str. Iowa, which may explain why R. heilongjiangensis is pathogenic. Pan-genome analysis showed that R. heilongjiangensis and 42 other rickettsiae strains share 693 core genes with a pan-genome size of 4837 genes. The pan-genome-based phylogeny showed that R. heilongjiangensis was closely related to R. japonica.

Background Surface proteins YbgF and PrsA are major seroreactive antigens of Rickettsia heilongjiangensis, the etiological agent of Far-Eastern spotted fever. This study investigated their potential immunogenicity for protective immunity. Methods Recombinant YbgF and PrsA were used to immunize C3H/HeN mice and rickettsial loads in immunized mouse organs were assessed after R. heilongjiangensis challenge. Anti-sera from immunized mice were applied to neutralize rickettsiae. CD4+ and CD8+ T cells isolated from R. heilongjiangensis-infected mice were stimulated with YbgF or PrsA, and proliferation and cytokine production assessed. Results The IgG2a/IgG1 ratio of sera was markedly increased in YbgF-immunized mice but was unaltered in PrsA-immunized mice after immunization. The rickettsial load in YbgF-immunized mice was significantly lower than in PrsA-immunized mice after R. heilongjiangensis challenge. Incubation with anti-serum to YbgF, but not PrsA, significantly reduced the number of rickettsiae adhering to and invading endothelial cells. The proliferation level and tumor necrosis factor-α secretion level of CD4+ T cells from R. heilongjiangensis-infected mice were significantly higher than in uninfected mice after stimulation with YbgF but not PrsA. Conclusion YbgF is a novel protective antigen that induces a Th1-type of protective immune response against R. heilongjiangensis infection.

Coxiella burnetii is the etiological agent of human Q fever. In this study, adaptive transfer of mouse bone marrow-derived dendritic cells (BMDCs) stimulated with C. burnetii antigen, phase I whole-cell antigen (PIAg), lipopolysaccharide (LPS)-removed PIAg (PIIAg), protein antigen Com1, or SecB significantly reduced coxiella burden in recipient mice compared with control mice. Mice that received PIIAg-pulsed BMDCs displayed substantially lower coxiella burden than recipient mice of PIAg-pulsed BMDCs after C burnetii challenge. The protection offered by the antigen-activated BMDCs was correlated with the increased proliferation of helper T (T(H)) T(H)1 CD4(+) cells, preferential development of T(H)17 cells, and impaired expansion of regulatory T lymphocytes. Our results suggest that PIIAg is far superior to PIAg in activating BMDCs to confer protection against C. burnetii in vivo, whereas Com1 and SecB are protective antigens because Com1- or SecB-pulsed BMDCs confer partial protection.

Rickettsia heilongjiangensis is the pathogen of Far eastern spotted fever, and T-cell immunoglobulin and mucin domain protein 3 (Tim-3) is expressed in human vascular endothelial cells, the major target cells of rickettsiae. In the present study, we investigated the effects of altered Tim-3 expression in vivo in mice and in vitro in human endothelial cells, on day 3 after R. heilongjiangensis infection. Compared with corresponding controls, rickettsial burdens both in vivo and in vitro were significantly higher with blocked Tim-3 signaling or silenced Tim-3 and significantly lower with overexpressed Tim-3. Additionally, the expression of inducible nitric oxide synthase and interferon γ in endothelial cells with blocked Tim-3 signaling or silenced Tim-3 was significantly lower, while the expression of inducible nitric oxide synthase, interferon γ, and tumor necrosis factor α in transgenic mice with Tim-3 overexpression was significantly higher. These results reveal that enhanced Tim-3 expression facilitates intracellular rickettsial killing in a nitric oxide-dependent manner in endothelial cells during the early phase of rickettsial infection.

Respiratory syncytial virus (RSV) is a major pathogen in infants and the elderly, causing pneumonia and bronchiolitis. Despite decades of research, to date there is still no approved RSV vaccine available. In this study, we developed RSV virus-like particle (VLP) vaccines containing an RSV fusion (F) and/or attachment (G) protein with Newcastle disease virus (NDV) as the platform. The VLPs were expressed in a baculovirus system and purified by sucrose gradient centrifugation. BALB/c mice immunized intranasally (i.n.) with rNDV/RSV/F plus rNDV/RSV/G developed robust humoral, mucosal RSV-specific antibodies and cellular immune responses. Furthermore, rNDV/RSV/F plus rNDV/RSV/G provided better protection than did rNDV/RSV/F or rNDV/RSV/G alone, as shown by an obvious decrease in viral replication together with alleviation of histopathological changes in the lungs of the challenged mice. Our data demonstrate that the intranasal vaccination of combined RSV virus-like particle vaccine candidates has great potential for protection against RSV infection.

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.

The Balb/c mice were infected with Coxiella burnetii and samples of blood and major organs from the infected mice were collected on days 1 to 14 after infection. The DNAs extracted from the samples were detected by a developed real-time quantitative PCR specific for C. burnetii and high loads of C. burnetii were found in spleens, livers, and lungs, particularly in spleens. The Balb/c mice were immunized with whole cell antigen (WCA) of C. burnetii and coxiella loads in spleens of mice were assessed by the real-time quantitative PCR on day 7 after challenge with C. burnetii. The analysis suggested that phase I whole cells were excellent immunogen that elicited complete protection against coxiella infection by two-booster but not one-booster immunization. The results suggest that the combination of Balb/c model and the real-time quantitative PCR assay is a reliable and sensitive way to evaluate the efficiency of vaccines against Q fever.

The gene fragments encoding outer membrane protein 1 (P1) and heat-shock protein B (HspB) amplified from genomic DNA of Coxiella burnetii Xinqiao by PCR were inserted into prokaryotic expression vector pQE30 to construct recombinant expression plasmids pQE30/p1 and pQE30/hspB, respectively. The p1 fragment from pQE30/p1 was ligated with hspB of pQE30/hspB to construct pQE30/p1-hspB. Recombinant proteins, P1, HspB, and P1-HspB, were expressed in Escherichia coli cells transformed with pQE30/p1, pQE30/hspB, and pQE30/p1-hspB, respectively. The purified recombinant proteins and whole-cell antigen (WCA) of C. burnetii were used to immunize BALB/c mice. The antibody detection, T-cell proliferation assay, and cytokine detection demonstrated that the animals immunized with P1-HspB or WCA exhibited stronger humoral and cellular immune responses compared with animals immunized with P1 or HspB individually. Seven days after challenge of 10-fold 50% infection dose of C. burnetii, mice were euthanized and their spleens were collected. The splenic weights of mice immunized with P1-HspB or WCA were significantly lighter than that of mice immunized with P1 or HspB. By real-time PCR assay, the coxiella loads of spleens of mice immunized with P1-HspB or WCA were also significantly lower than that of mice immunized with P1 or HspB. The data from this study indicate that fusion antigen P1-HspB is a good immunogen for eliciting immunoresponses against C. burnetii, and it may be a more suitable candidate for preparing subunit vaccine against Q fever.

A truncated gene ompA was amplified from Rickettsia heilongjiangensis isolated in China and a 56-kDa truncated OmpA protein was expressed in E. coli cells transformed with the ompA-recombined expression plasmid. High levels of serum antibodies to R. heilongjiangensis and proliferation of the splenic cells were found in mice immunized with the truncated OmpA. After challenge with R. heilongjiangensis or R. rickettsii, fever and pathological damages of the guinea pigs immunized with the truncated OmpA were significantly slighter as compared with those of nonimmunized guinea pigs. These results suggest that the truncated OmpA of R. heilongjiangii is immunogenic for effectively inducing humoral and cell-mediated immune responses against homologous and heterologous species in the spotted fever group.

A partial gene sequence encoding the 56-kD scrub typhus antigen (Sta56) was amplified from genomic DNA of the Orientia tsutsugamushi Karp strain by a polymerase chain reaction (PCR). The PCR product was ligated with the 47-kD scrub typhus antigen (Sta47) gene in the pQE30/47 expression vector, and the resulting recombinant expression vector was designated pQE30/56-47. A fusion antigen (Sta56-47) was expressed in Escherichia coli cells transformed with pQE30/56-47 after induction with isopropyl-beta-d-thiogalactopyranoside. The Sta56-47 antigen was recognized by both Sta47 and Sta56 immune sera and by immune serum to Sta56-47 in an immunoblot assay. This antigen was purified and used to immunize BALB/c mice. The animals immunized with Sta56-47 exhibited profound humoral and cellular immune responses, as well as increased resistance to O. tsutsugamushi Karp compared with mice immunized with Sta56 or Sta47. These results strongly suggest that Sta56-47 contains antigenic epitopes of the Sta56 and Sta47 antigens of O. tsutsugamushi Karp, and is a more suitable candidate for replacing whole-cell antigen of O. tsutsugamushi Karp to induce protective immunity against scrub typhus.

An epidemic of tremor disease has been a serious problem in Chinese mitten crabs,Eriocheir sinensis, in China in recent years. The disease-causing agent was previously considered to be a rickettsia-like organism. Here, analysis of the 16S rRNA gene sequence, light and electron microscopy and cultivationin vitrowere used to identify the agent. Sequence analysis of the 16S rRNA gene found it to have 98 % identity with that ofSpiroplasma mirum. The agent was able to be passed through membrane filters with pores 220 nm in diameter and could be cultivated by inoculating the yolk sac of embryonated chicken eggs and M1D medium. Rotary motion and flexional movement were seen by light microscopy, and electron microscopy showed that the organism had a helical morphology and lacked a cell wall. The organism produced small colonies with a diameter of 40–50 μm after 17–25 days of incubation on solid M1D medium. The agent was found in blood cells, muscles, nerves and connective tissues of crabs inoculated with a filtrate of yolk sacs or with cultures grown in M1D medium, and it was similar in structure to those grown in eggs and cultivation broth. Disease was reproduced by experimental infection with the cultivated organisms. This study has demonstrated that the causative agent of tremor disease in the Chinese mitten crab is a member of the genusSpiroplasma. This is believed to be the first time a spiroplasma has been found in a crustacean. These findings are not only significant for studies on pathogenic spiroplasmas, but also have implications for studies of freshwater ecology.

The gene encoding the 56-kDa protein of Orientia tsutsugamushi Shanxi was amplified by a nested PCR and cloned into the expression vector pQE30. The 56-kDa protein of O. tsutsugamushi Shanxi (Sxh56) was expressed as a fusion protein with the His 6 -binding protein of Escherichia coli by deleting the signal peptide-encoding sequence from the 5′ end of the open reading frame. The recombinant protein formed inclusion bodies when expressed in E. coli M15. The recombinant protein was examined for reactivity with mouse sera against three antigenic prototypes of O. tsutsugamushi by an immunoblot assay. The recombinant Sxh56 reacted only to polyclonal antiserum to O. tsutsugamushi Gilliam in an enzyme-linked immunosorbent assay (ELISA) and in an immunoblot assay. Recombinant Sxh56 was purified by Ni-nitrilotriacetic acid affinity chromatography and injected into mice to evaluate its ability to stimulate immune responses. High levels of immunoglobulin G and T-cell proliferation appeared in mice immunized with the recombinant protein. The recombinant Sxh56 was used in an ELISA to evaluate the ability of the method to detect antibodies to O. tsutsugamushi in human and animal sera. Thirty sera from mice infected with O. tsutsugamushi Gilliam or Shanxi and 55 sera from normal mice were detected in the ELISA with recombinant Sxh56, and the sensitivity and specificity were 96.67 and 100%, respectively. One hundred fifty-one positive sera and 412 negative sera to O. tsutsugamushi Gilliam were detected in an indirect immunofluorescence assay with the recombinant protein, and the sensitivity and specificity were 96.36 and 88.08%, respectively. These results strongly suggest that the recombinant Sxh56 is a suitable type-specific immunodiagnostic antigen and vaccine candidate.

The various ticks collected from different areas of China were examined for the existence of ehrlichial agents by polymerase chain reaction (PCR) with genus- or species-specific primers designed on the basis of ehrlichial 16S rRNA genes and sequence analyses. In southern China, E. chaffeensis was detected in Amblyomma testudinarium ticks from infested cattle, Haemaphysalis yeni ticks from hare, and Ixodes ovatus ticks from Muntiacus reevesi. E. canis was identified in Rhipicephalus sanguineus ticks from dogs and Boophilus microplus ticks from goats. A new species of the genus Ehrlichia, closely related to E. chaffeensis, and Anaplasma marginale were found in B. microplus ticks from cattle in Tibet. In northern China, E. chaffeensis was detected in Dermacentor silvarum and I. persulcatus ticks; the granulocytic ehrlichial agents were detected in I. persulcatus ticks from an area where Lyme disease is endemic. Canine ehrlichiosis was found in southern China and E. canis and E. platys were identified in dogs; human ehrlichioses were demonstrated by amplifying the 16S rRNA genes of E. chaffeensis and granulocytic ehrlichial agents from patients' blood specimens. In comparison of 16S rRNA gene sequences, the sequences of E. chaffeensis, E. canis, and E. platys in China were found to be different from that in other countries at certain nucleotide positions. These results reveal that a variety of tick-borne ehrlichial agents and diseases exist in China, and the ehrlichial agents and their tick-vectors are same as or different from that in other countries at species or strain levels.

In this study, the fragment of 47 kDa gene (301 bp-1428 bp) was cloned into a prokaryotic expression vector pBV220 to construct a recombinant plasmid pBV-47. The E. coli cells were transformed with pBV-47 and the transformants were induced to express the recombinant protein at 42 degrees C. The expression product (40 kDa) was detected by SDS-PAGE analysis and the 40kDa protein was recognized by mouse polyclonal antibodies against O. tsutsugamushi Karp strain in western blot analysis. The entire 47 kDa protein gene was inserted into an eukaryotic expression vector pcDNA3.1(+) to construct a recombinant plasmid pcDNA3.1/47 and Balb/c mice were immunized with recombinant pcDNA3.1/47, control vector pcDNA3.1, PBS buffer, 40 kDa protein, and recombinant pcDNA3.1/47 plus 40 kDa protein (pcDNA3.1/47/40), respectively. The results showed that spleen cells from pcDNA3.1/47/40-immunized mice gave higher proliferation than other groups. A significant IgG rise was detected in mice immunized with 40 kDa protein, but it was less strong than that in mice immunized with pcDNA3.1/47/40. The results suggested that immunization with pcDNA3.1/47 and 40 kDa protein simultaneously could induce a strong immune response.

To identify ehrlichial agents in Boophilus microplus ticks, DNA samples of B. microplus collected from the Tibet Autonomous Region and Sichuan Province of China were screened by a nested PCR. Sixteen of 43 (37%) DNA samples of B. microplus from Tibet were positive in nested PCR analysis. All 27 samples from Sichuan were negative. The screen identified two ehrlichial agents based on different 16S rRNA genes that were found after amplifying and sequencing the 5′-end fragments of the 16S rRNA genes. One sequence was identical to that of the gene of Anaplasma marginale , an etiological agent of animal anaplasmosis. The other sequence was most similar to that of the gene of Ehrlichia chaffeensis , an etiological agent of human monocytic ehrlichiosis. The sequence of 1,501 bases from the novel ehrlichial agent was obtained and showed the greatest levels of sequence similarity (97 to 98%) to 16S rRNA gene sequences of the members of the E. canis group of the genus Ehrlichia . Sequence comparison of the 16S rRNA gene with the members of the genus Ehrlichia reveals that the novel ehrlichial agent detected in B. microplus ticks is a new species of the genus Ehrlichia and is most closely related to E. chaffeensis .

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.

Background Far-eastern spotted fever (FESF) is an important emerging infectious disease in Northeast Asia. The laboratory diagnosis of FESF in hospitals is mainly based on serological methods. However, these methods need to cultivate rickettsial cells as diagnostic antigens, which is both burdensome and dangerous. Methods Eleven surface-exposed proteins (SEPs) were identified in our previous study and their recombinant proteins (rSEPs) fabricated on a microarray were serologically analyzed with seventeen paired sera from patients suffered from FESF in this study. Results All the rSEPs showed sensitivities of between 53% and 82% to acute-phase sera and of between 65% and 82% to convalescent-phase sera, and all the rSEPs except rRplA showed specificities of between 80% and 95%. The combination assay of two, three, or four of the four rSEPs (rOmpA-2, rOmpB-3, rRpsB, and rSdhB) showed better sensitivities of between 76% and 94% to the acute-phase sera or between 82% and 100% to the convalescent-phase sera and acceptable specificities of between 75% and 90%. Conclusions Our results suggest that the four rSEPs are more likely candidate antigens for serological diagnosis of FESF.

Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, is the most pathogenic member among Rickettsia spp. Surface-exposed proteins (SEPs) of R. rickettsii may play important roles in its pathogenesis or immunity. In this study, R. rickettsii organisms were surface-labeled with sulfo-NHS-SS-biotin and the labeled proteins were affinity-purified with streptavidin. The isolated proteins were separated by two-dimensional electrophoresis, and 10 proteins were identified among 23 protein spots by electrospray ionization tandem mass spectrometry. Five (OmpA, OmpB, GroEL, GroES, and a DNA-binding protein) of the 10 proteins were previously characterized as surface proteins of R. rickettsii. Another 5 proteins (Adr1, Adr2, OmpW, Porin_4, and TolC) were first recognized as SEPs of R. rickettsii herein. The genes encoding the 5 novel SEPs were expressed in Escherichia coli cells, resulting in 5 recombinant SEPs (rSEPs), which were used to immunize mice. After challenge with viable R. rickettsii cells, the rickettsial load in the spleen, liver, or lung of mice immunized with rAdr2 and in the lungs of mice immunized with other rSEPs excluding rTolC was significantly lower than in mice that were mock-immunized with PBS. The in vitro neutralization test revealed that sera from mice immunized with rAdr1, rAdr2, or rOmpW reduced R. rickettsii adherence to and invasion of vascular endothelial cells. The immuno-electron microscopic assay clearly showed that the novel SEPs were located in the outer and/or inner membrane of R. rickettsii. Altogether, the 5 novel SEPs identified herein might be involved in the interaction of R. rickettsii with vascular endothelial cells, and all of them except TolC were protective antigens.

Coxiella burnetii is a Gram-negative bacterium that causes Q fever in humans. In the present study, 131 candidate peptides were selected from the major immunodominant proteins (MIPs) of C. burnetii due to their high-affinity binding capacity for the MHC class II molecule H2 I-A(b) based on bioinformatic analyses. Twenty-two of the candidate peptides with distinct MIP epitopes were well recognized by the IFN-γ recall responses of CD4(+) T cells from mice immunized with parental proteins in an ELISPOT assay. In addition, 7 of the 22 peptides could efficiently induce CD4(+) T cells from mice immunized with C. burnetii to rapidly proliferate and significantly increase IFN-γ production. Significantly higher levels of IL-2, IL-12p70, IFN-γ, and TNF-α were also detected in serum from mice immunized with a pool of the 7 peptides. Immunization with the pool of 7 peptides, but not the individual peptides, conferred a significant protection against C. burnetii infection in mice, suggesting that these Th1 peptides could work together to efficiently activate CD4(+) T cells to produce the Th1-type immune response against C. burnetii infection. These observations could contribute to the rational design of molecular vaccines for Q fever.

Background Rickettsia rickettsii is the pathogen of Rocky Mountain spotted fever (RMSF), a life-threatening tick-transmitted infection. Adr2 was a surface-exposed adhesion protein of R. rickettsii and its immunoprotection against RMSF was investigated in mice. Methods Recombinant Adr2 (rAdr2) was used to immunize C3H/HeN mice, and the rickettsial loads in organs of the mice were detected after challenge with R. rickettsii . The levels of specific antibodies of sera from the immunized mice were determined and the sera from immunized mice were applied to neutralize R. rickettsii . Proliferation and cytokine secretion of CD4 + and CD8 + T cells isolated from R. rickettsii -infected mice were also assayed after rAdr2 stimulation. Results After R. rickettsii challenge, the rickettsial loads in spleens, livers, and lungs were significantly lower and the impairment degrees of these organs in rAdr2-immunized mice were markedly slighter, compared with those in negative control mice. The ratio of specific IgG2a/IgG1 of rAdr2-immunized mice kept increasing during the immunization. After treatment with rAdr2-immunized sera, the total number of R. rickettsii organisms adhering and invading host cells was significantly lower than that treated with PBS-immunized sera. Interferon-γ secretion by CD4 + or CD8 + T cells and tumor necrosis factor-α secretion by CD4 + T cells from R. rickettsii -infected mice were respectively significantly greater than those from uninfected mice after rAdr2 stimulation. Conclusion Adr2 is a protective antigen of R. rickettsii . Protection offered by Adr2 is mainly dependent on antigen-specific cell-mediated immune responses, including efficient activity of CD4 + and CD8 + T cells to produce great amount of TNF-α and/or IFN-γ as well as rapid increase of specific IgG2a, which synergistically activate and opsonize host cells to killing intracellular rickettsiae.

Summary A disease entity known as the "Shasta River crud" (SRC) has been recognized for at least 25 years in areas of Siskiyou and Shasta counties, in far northern California. It is characterized by anorexia, lethargy, diarrhea, variable fever, and in some cases laminitis, and is tetracycline responsive. The incidence is seasonal, with cases occurring in the spring and summer months near the Shasta and Klamath rivers. Limited serologic testing has provided some suggestion that the illness may be linked to Ehrlichia risticii , the agent of Potomac horse fever. Here we report the identification of E. risticii genomic DNA in blood buffy-coat cells from horses with SRC, and so provide the first definitive evidence that this enzootic diarrhea is in fact Potomac horse fever.

A partial 16S rRNA gene was amplified in Ehrlichia canis-infected cells by nested PCR. The assay was specific and did not amplify the closely related Ehrlichia chaffeensis, Ehrlichia muris, Neorickettsia helminthoeca, and SF agent 16S rRNA genes. The assay was as sensitive as Southern hybridization, detecting as little as 0.2 pg of E. canis DNA. By this method, all blood samples from four dogs experimentally infected with E. canis were positive as early as day 4 postinoculation, which was before or at the time of seroconversion. One hundred five blood samples from dogs from Arizona and Texas (areas of E. canis endemicity) and 30 blood samples from dogs from Ohio (area of E. canis nonendemicity) were examined by nested PCR and immunofluorescent-antibody (IFA) test. Approximately 84% of dogs from Arizona and Texas had been treated with doxycycline before submission of blood specimens. Among Arizona and Texas specimens, 46 samples were PCR positive (44%) and 80 were IFA positive (76%). Forty-three of 80 IFA-positive samples (54%) were PCR positive, and 22 of 25 IFA-negative samples (88%) were negative in the nested PCR. None of the Ohio specimens were IFA positive, but 5 specimens were PCR positive (17%). Our results indicate that the nested PCR is highly sensitive and specific for detection of E. canis and may be more useful in assessing the clearance of the organisms after antibiotic therapy than IFA, especially in areas in which E. canis is endemic.

Infectious agents were isolated from the spleens of three wild mice (Apodemus argenteus) by intraperitoneal inoculation of the spleen homogenate into laboratory mice. The laboratory mice developed clinical signs and splenomegaly, and three isolates were maintained by passage in mice. Tetracyclines were effective in preventing infection of mice with these agents, but streptomycin and penicillin were ineffective. The agents did not grow in bacterial growth media or chicken embryos. In smears of blood from infected mice stained by the Giemsa or the indirect immunofluorescence method, numerous organisms were found on the surfaces of erythrocytes. Electron microscopy revealed cell wall-less pleomorphic cocci of 350 to 700 nm in diameter. On the basis of these results, the isolates were identified as Haemobartonella muris. There was no antigenic cross-reactivity with Rickettsia or Ehrlichia spp. or other related organisms. Western immunoblot analysis of three strains of H. muris with mouse antisera to H. muris revealed identical major antigens of 118, 65, 53, 45, and 40 kDa. By heteroduplex analysis of the three PCR-amplified segments of the 16S rRNA genes, the three strains of H. muris were found to be identical. The 16S rRNA genes of one of the H. muris strains, four strains of H. felis, and two strains of Eperythrozoon suis were sequenced and compared. The sequences of two strains of H. felis from cats in California were identical, as were the sequences of a strain from a cat in Ohio and a strain from a cat in Florida, but the similarity of sequences between the California and the Ohio-Florida strains was only 85%. The sequence of an H. muris strain was unique and was more closely related to that of the Ohio-Florida strain of H. felis (89%) than to that of the California strain of H. felis (84%). The sequence of E. suis from a pig in Illinois was identical to that from another pig from Taiwan. The similarity of the 16S rRNA gene sequence of E. suis with those of three Haemobartonella strains was 84 to 92%, with that of E. suis being most similar to that of the H. felis strain from California. In the phylogenetic analysis based on 16S rRNA gene sequences, the Haemobartonella spp. and E. suis formed a distinct clade more closely related to Mycoplasma spp. (79 to 83% similarity) than to Anaplasma marginale (72 to 75% similarity). Our results suggest that the Haemobartonella spp. and E. suis may be reclassified in the same genus in the family Mycoplasmataceae.

Coxiella burnetii is the etiological agent of Q fever. To identify its major seroreactive proteins, a subgenomic protein array was developed. A total of 101 assumed virulence-associated recombinant proteins of C. burnetii were probed with sera from mice experimentally infected with C. burnetii and sera from Q fever patients. Sixteen proteins were recognized as major seroreactive antigens by the mouse sera. Seven of these 16 proteins reacted positively with at least 45% of Q fever patient sera. Notably, HspB had the highest fluorescence intensity value and positive frequency of all the proteins on the array when probed with both Q fever patient sera and mouse sera. These results suggest that these seven major seroreactive proteins, particularly HspB, are potential serodiagnostic and subunit vaccine antigens of Q fever.

The recombinant membrane-associated proteins of Coxiella burnetii, Com1, Mip and GroEL, were used in vitro to stimulate BALB/c mouse bone marrow-derived dendritic cells (BMDCs). The antigen-activated BMDCs were transferred into naive BALB/c mice. Seven days after challenge of C. burnetii, the bacterial loads of mice receiving BMDCs activated with Com1 or Mip, but not GroEL, were significantly lower than that of mice receiving BMDCs pulsed with TrxA (Esherichia coli thioredoxin) in a quantitative polymerase chain reaction assay. After in vitro interaction with cognate antigen-pulsed BMDCs, the percentages of CD69-positive cells and TNF-α-positive cells in CD4(+) and CD8(+) T cells isolated from the spleens of mice receiving Com1-, Mip-, or GroEL-pulsed BMDCs were significantly higher than that of mice receiving mock-pulsed BMDCs in flow cytometric analysis. The percentages of IFN-γ-positive cells in CD4(+) and CD8(+) T cells from mice receiving Com1- or Mip-pulsed BMDCs were significantly greater than that of mice receiving GroEL-pulsed BMDCs. However, the percentage of IL-4-positive cells in CD4(+) T cells of mice receiving GroEL-pulsed BMDCs was obviously higher than that of mice receiving Com1- or Mip-pulsed BMDCs. Our results demonstrate that Com1 and Mip are protective antigens and strongly indicate that they favor to induce IFN-γ-producing Th1 and Tc1 cells, whereas the non-protective antigen GroEL is biased to induce a Th2 response. Therefore, Com1 and Mip are key antigens to induce a protective immune response against C. burnetii infection.

Rickettsia heilongjiangensis is an emerging tick-transmitted human pathogen causing far-Eastern spotted fever. Here we report the complete sequence and the main features of the genome of R. heilongjiangensis (strain 054).

Far-eastern spotted fever is an emerging disease caused by Rickettsia heilongjiangensis, a tick-borne obligate intracellular bacterium. In this study, R. heilongjiangensis was used to infect BALB/c mice by inoculation of retro-orbital venous plexus to imitate a blood infection caused by tick biting. We found that R. heilongjiangensis rapidly entered the circulation for systemic dissemination and the pathogen existed in liver, spleen, lungs, and brain of the mice at least 9 days post-infection (p.i.). Severe pathological lesions were observed in liver, lungs, and brain at Day 6 p.i. In addition, the elevated levels of inflammatory cytokines, including interferon-γ, tumor necrosis factor, and CC chemokine, were detected in the infected organs at Day 3 p.i. Our results reveal that R. heilongjiangensis may cause an infection in BALB/c mice and the pathological lesions in the infected mice are associated with host inflammatory response induced by R. heilongjiangensis.

The obligate intracellular bacteria, Rickettsia rickettsii and Coxiella burnetii, are the potential agents of bio-warfare/bio-terrorism. Here C3H/HeN mice were immunized with a recombinant protein fragment rOmp-4 derived from outer membrane protein B, a major protective antigen of R. rickettsii, combined with chloroform-methanol residue (CMR) extracted from phase I C. burnetii organisms, a safer Q fever vaccine. These immunized mice had significantly higher levels of IgG1 and IgG2a to rOmpB-4 and interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), two crucial cytokines in resisting intracellular bacterial infection, as well as significantly lower rickettsial loads and slighter pathological lesions in organs after challenge with R. rickettsii, compared with mice immunized with rOmpB-4 or CMR alone. Additionally, after challenge with C. burnetii, the coxiella loads in the organs of these mice were significantly lower than those of mice immunized with rOmpB-4 alone. Our results prove that CMR could markedly potentiate enhance the rOmpB-4-specific immunoprotection by promoting specific and non-specific immunoresponses and the immunization with the protective antigen of R. rickettsii combined with CMR of C. burnetii could confer effective protection against infection of R. rickettsii or C. burnetii.

A rickettsia-like organism (RLO) has been suggested to be the etiological agent responsible for heavy losses of the oyster Crassostrea ariakensis Gould in China. Because of the lack of molluscan cell lines for in vitro culture of intracellular prokaryotes, antigenic analysis of RLOs has been limited by the inherent difficulties of their purification. In this report, we describe the use of differential speed centrifugation and renografin density gradient centrifugation to purify the RLO directly from infected oyster tissues. The purity and integrity of purified prokaryotes were validated by transmission electron microscopy. Thirteen major constituent proteins, with molecular weights ranging between 17 and 99 kDa, were electrophoretically identified by silver staining, and 8 major proteins were identified with Coomassie blue R staining. Specific mouse polyclonal antiserum was prepared for serological characterization of the RLO and was used in an immunoblot assay, and 3 major antigen groups were identified. The present results advance our knowledge of RLO protein antigens, and several proteins have been identified that could potentially be useful for diagnostic assays or for production of experimental immunostimulants.

A human granulocytic ehrlichiosis (HGE) agent with 16S rDNA sequence identical to the published sequence of HGE agents was isolated from a patient from New York State by inoculation of the blood leukocyte fraction directly into a human promyelocytic leukemia cell line HL-60. The HGE agent was also isolated from the leukocyte fraction of the blood and bone marrow of a mouse inoculated with the leukocyte fraction of the patient's blood. The isolate has been passaged in tissue culture 30 times over 8 months. Electron microscopy revealed pleomorphic coccobacilli with a thin and highly rippled outer membrane in the clear inclusion matrix. Comparison of IFA reactivity of antisera obtained from a variety of sources with the cell-cultured HGE agent revealed that 3 HGE agent strains (New York isolate, Wisconsin [BDS] isolate, and a tick-derived isolate) are highly cross-reactive and there are diverse antigenic cross-reactivities between HGE agent and Ehrlichia chaffeensis.