Pathogen Coxiella Burnetii Research Articles

Coxiella burnetii effector proteins that localize to the parasitophorous vacuole membrane promote intracellular replication.

The intracellular bacterial pathogen Coxiella burnetii directs biogenesis of a parasitophorous vacuole (PV) that acquires host endolysosomal components. Formation of a PV that supports C. burnetii replication requires a Dot/Icm type 4B secretion system (T4BSS) that delivers bacterial effector proteins into the host cell cytosol. Thus, a subset of T4BSS effectors are presumed to direct PV biogenesis. Recently, the PV-localized effector protein CvpA was found to promote C. burnetii intracellular growth and PV expansion. We predict additional C. burnetii effectors localize to the PV membrane and regulate eukaryotic vesicle trafficking events that promote pathogen growth. To identify these vacuolar effector proteins, a list of predicted C. burnetii T4BSS substrates was compiled using bioinformatic criteria, such as the presence of eukaryote-like coiled-coil domains. Adenylate cyclase translocation assays revealed 13 proteins were secreted in a Dot/Icm-dependent fashion by C. burnetii during infection of human THP-1 macrophages. Four of the Dot/Icm substrates, termed Coxiella vacuolar protein B (CvpB), CvpC, CvpD, and CvpE, labeled the PV membrane and LAMP1-positive vesicles when ectopically expressed as fluorescently tagged fusion proteins. C. burnetii ΔcvpB, ΔcvpC, ΔcvpD, and ΔcvpE mutants exhibited significant defects in intracellular replication and PV formation. Genetic complementation of the ΔcvpD and ΔcvpE mutants rescued intracellular growth and PV generation, whereas the growth of C. burnetii ΔcvpB and ΔcvpC was rescued upon cohabitation with wild-type bacteria in a common PV. Collectively, these data indicate C. burnetii encodes multiple effector proteins that target the PV membrane and benefit pathogen replication in human macrophages.

Identification of Coxiella burnetii genotypes in Croatia using multi-locus VNTR analysis

Although Q fever affects humans and animals in Croatia, we are unaware of genotyping studies of Croatian strains of the causative pathogen Coxiella burnetii, which would greatly assist monitoring and control efforts. Here 3261 human and animal samples were screened for C. burnetii DNA by conventional PCR, and 335 (10.3%) were positive. Of these positive samples, 82 were genotyped at 17 loci using the relatively new method of multi-locus variable number tandem repeat analysis (MLVA). We identified 13 C. burnetii genotypes not previously reported anywhere in the world. Two of these 13 genotypes are typical of the continental part of Croatia and share more similarity with genotypes outside Croatia than with genotypes within the country. The remaining 11 novel genotypes are typical of the coastal part of Croatia and show more similarity to one another than to genotypes outside the country. Our findings shed new light on the phylogeny of C. burnetii strains and may help establish MLVA as a standard technique for Coxiella genotyping.

Characterization of a lipopolysaccharide-targeted monoclonal antibody and its variable fragments as candidates for prophylaxis against the obligate intracellular bacterial pathogen Coxiella burnetii.

Our previous study demonstrated that treatment of Coxiella burnetii with the phase I lipopolysaccharide (PI-LPS)-targeted monoclonal antibody (MAb) 1E4 significantly inhibited C. burnetii infection in mice, suggesting that 1E4 is a protective MAb. To determine whether passive transfer of antibodies (Abs) can provide protection against C. burnetii natural infection, we examined if passive transfer of 1E4 would protect SCID mice against C. burnetii aerosol infection. The results indicated that 1E4 conferred significant protection against aerosolized C. burnetii, suggesting that 1E4 may be useful for preventing C. burnetii natural infection. To further understand the mechanisms of 1E4-mediated protection and to test the possibility of using humanized 1E4 to prevent C. burnetii infection, we examined whether the Fab fragment of 1E4 (Fab1E4), a recombinant murine single-chain variable fragment (muscFv1E4), and a humanized single-chain variable fragment (huscFv1E4) retained the ability of 1E4 to inhibit C. burnetii infection. The results indicated that Fab1E4, muscFv1E4, and huscFv1E4 were able to inhibit C. burnetii infection in mice but that their ability to inhibit C. burnetii infection was lower than that of 1E4. In addition, treatment of C. burnetii with Fab1E4, muscFv1E4, or huscFv1E4 can block C. burnetii infection of macrophages. Interestingly, treatment of C. burnetii with huscFv1E4 can significantly reduce C. burnetii infectivity in human macrophages. This report provides the first evidence to demonstrate that the humanized variable fragments of an LPS-specific MAb can neutralize C. burnetii infection and appears to be a promising step toward the potential use of a humanized MAb as emergency prophylaxis against C. burnetii exposure.

Identification of Coxiella burnetii surface-exposed and cell envelope associated proteins using a combined bioinformatics plus proteomics strategy.

The Gram-negative pathogen Coxiella burnetii is an intracellular bacterium that replicates within the phagolysosomal vacuoles of eukaryotic cells. This pathogen can infect a wide range of hosts, and is the causative agent of Q fever in humans. Surface-exposed and cell envelope associated proteins are thought to be important for both pathogenesis and protective immunity. Herein, we propose a complementary strategy consisting of (i) in silico prediction and (ii) inventory of the proteomic composition using three enrichment approaches coupled with protein identification. The efficiency of classical Triton X-114 phase partitioning was compared with two novel procedures; isolation of alkaline proteins by liquid-phase IEF, and cell surface enzymatic shaving using biofunctional magnetic beads. Of the 2026 protein sequences analyzed using seven distinct bioinformatic algorithms, 157 were predicted to be outer membrane proteins (OMP) and/or lipoproteins (LP). Using the three enrichment protocols, we identified 196 nonredundant proteins, including 39 predicted OMP and/or LP, 32 unknown or poorly characterized proteins, and 17 effectors of the Type IV secretion system. We additionally identified eight proteins with moonlighting activities, and several proteins apparently peripherally associated with integral or anchored OMP and/or LP.

In Vitro and In Vivo Infectious Potential of Coxiella burnetii: A Study on Belgian Livestock Isolates

Q-fever is a zoonosis caused by the gram-negative obligate intracellular pathogen Coxiella burnetii. Since its discovery, and particularly following the recent outbreaks in the Netherlands, C. burnetii appeared as a clear public health concern. In the present study, the infectious potential displayed by goat and cattle isolates of C. burnetii was compared to a reference strain (Nine Mile) using both in vitro (human HeLa and bovine macrophage cells) and in vivo (BALB/c mice) models. The isolates had distant genomic profiles with one - the goat isolate - being identical to the predominant strain circulating in the Netherlands during the 2007–2010 outbreaks. Infective doses were established with ethidium monoazide-PCR for the first time here applied to C. burnetii. This method allowed for the preparation of reproducible and characterized inocula thanks to its capacity to discriminate between live and dead cells. Globally, the proliferative capacity of the Nine Mile strain in cell lines and mice was higher compared to the newly isolated field strains. In vitro, the bovine C. burnetii isolate multiplied faster in a bovine macrophage cell line, an observation tentatively explained by the preferential specificity of this strain for allogeneic host cells. In the BALB/c mouse model, however, the goat and bovine isolates multiplied at about the same rate indicating no peculiar hypervirulent behavior in this animal model.

Antibody kinetics in serological indication of chronic Q fever: the Greek experience

Q fever caused by the pathogen Coxiella burnetii may have both acute and chronic manifestations. Although paired sera are not required for the diagnosis of chronic Q fever, monitoring of antibody titers can be used to examine the course of treatment. Monitoring both phase II and phase I antibodies may be of limited diagnostic value, but it is a useful means of determining the response to treatment and possible disease relapses. In the current survey we determined IgG and IgM of both phase I and phase II for 35 patients suffering from chronic Q fever in an attempt to draw conclusions on the kinetics of the antibodies throughout the course of the disease. Overall, 33 cases were included in the study. Of the 33 patients, 32 had a good outcome. Our findings support the general belief that a long period of serological monitoring is required for patients with chronic Q fever.

Specific Interferon γ Detection for the Diagnosis of Previous Q Fever

Current practice for diagnosis of Q fever, caused by the intracellular pathogen Coxiella burnetii, relies mainly on serology and, in prevaccination assessment, on skin tests (STs), which both have drawbacks. In this study, C. burnetii-specific interferon γ (IFN-γ) production was used as a new diagnostic tool for previous Q fever, circumventing most of these drawbacks. Our aim was to compare this test to serology and ST. One thousand five hundred twenty-five individuals from an endemic area with a risk for chronic Q fever were enrolled. IFN-γ production was measured after in vitro stimulation of whole blood with C. burnetii antigens. Various formats using different C. burnetii antigens were tested. Serology and ST were performed in all individuals. In all assay formats, C. burnetii-specific IFN-γ production was higher (P < .0001) in seropositive or ST-positive subjects than in seronegative and ST-negative subjects. Whole blood incubated for 24 hours with C. burnetii Nine Mile showed optimal performance. After excluding subjects with equivocal serology and/or borderline ST results, IFN-γ production was 449 ± 82 pg/mL in the positive individuals (n = 219) but only 21 ± 3 pg/mL in negative subjects (n = 908). Using Bayesian analysis, sensitivity and specificity (87.0% and 90.2%, respectively) were similar to the combination of serology and ST (83.0% and 95.6%, respectively). Agreement with the combination of serology and ST was moderate (84% concordance; κ = 0.542). Specific IFN-γ detection is a novel diagnostic assay for previous C. burnetii infection and shows similar performance and practical advantages over serology and ST. Future studies to investigate the clinical value in practice are warranted.

Effector Protein Translocation by the Coxiella burnetii Dot/Icm Type IV Secretion System Requires Endocytic Maturation of the Pathogen-Occupied Vacuole

The human pathogen Coxiella burnetii encodes a type IV secretion system called Dot/Icm that is essential for intracellular replication. The Dot/Icm system delivers bacterial effector proteins into the host cytosol during infection. The effector proteins delivered by C. burnetii are predicted to have important functions during infection, but when these proteins are needed during infection has not been clearly defined. Here, we use a reporter system consisting of fusion proteins that have a β-lactamase enzyme (BlaM) fused to C. burnetii effector proteins to study protein translocation by the Dot/Icm system. Translocation of BlaM fused to the effector proteins CBU0077, CBU1823 and CBU1524 was not detected until 8-hours after infection of HeLa cells, which are permissive for C. burnetii replication. Translocation of these effector fusion proteins by the Dot/Icm system required acidification of the Coxiella-containing vacuole. Silencing of the host genes encoding the membrane transport regulators Rab5 or Rab7 interfered with effector translocation, which indicates that effectors are not translocated until bacteria traffic to a late endocytic compartment in the host cell. Similar requirements for effector translocation were discerned in bone marrow macrophages derived from C57BL/6 mice, which are primary cells that restrict the intracellular replication of C. burnetii. In addition to requiring endocytic maturation of the vacuole for Dot/Icm-mediated translocation of effectors, bacterial transcription was required for this process. Thus, translocation of effector proteins by the C. burnetii Dot/Icm system occurs after acidification of the CCV and maturation of this specialized organelle to a late endocytic compartment. This indicates that creation of the specialized vacuole in which C. burnetii replicates represents a two-stage process mediated initially by host factors that regulate endocytic maturation and then by bacterial effectors delivered into host cells after bacteria establish residency in a lysosome-derived organelle.

VirulentCoxiella burnetiipathotypes productively infect primary human alveolar macrophages

The intracellular bacterial pathogen Coxiella burnetii is a category B select agent that causes human Q fever. In vivo, C. burnetii targets alveolar macrophages wherein the pathogen replicates in a lysosome-like parasitophorous vacuole (PV). In vitro, C. burnetii infects a variety of cultured cell lines that have collectively been used to model the pathogen's infectious cycle. However, differences in the cellular response to infection have been observed, and virulent C. burnetii isolate infection of host cells has not been well defined. Because alveolar macrophages are routinely implicated in disease, we established primary human alveolar macrophages (hAMs) as an in vitro model of C. burnetii-host cell interactions. C. burnetii pathotypes, including acute disease and endocarditis isolates, replicated in hAMs, albeit with unique PV properties. Each isolate replicated in large, typical PV and small, non-fused vacuoles, and lipid droplets were present in avirulent C. burnetii PV. Interestingly, a subset of small vacuoles harboured single organisms undergoing degradation. Prototypical PV formation and bacterial growth in hAMs required a functional type IV secretion system, indicating C. burnetii secretes effector proteins that control macrophage functions. Avirulent C. burnetii promoted sustained activation of Akt and Erk1/2 pro-survival kinases and short-termphosphorylation of stress-related p38. Avirulent organisms also triggered a robust, early pro-inflammatory response characterized by increased secretion of TNF-α and IL-6, while virulent isolates elicited substantially reduced secretion of these cytokines. A corresponding increase in pro- and mature IL-1β occurred in hAMs infected with avirulent C. burnetii, while little accumulation was observed following infection with virulent isolates. Finally, treatment of hAMs with IFN-γ controlled intracellular replication, supporting a role for this antibacterial insult in the host response to C. burnetii. Collectively, the current results demonstrate the hAM model is a human disease-relevant platform for defining novel innate immune responses to C. burnetii.

TheCoxiella burnetiitype IV secretion system substrate CaeB inhibits intrinsic apoptosis at the mitochondrial level

Manipulation of host cell apoptosis is a virulence property shared by many intracellular pathogens to ensure productive replication. For the obligate intracellular pathogen Coxiella burnetii anti-apoptotic activity, which depends on a functional type IV secretion system (T4SS), has been demonstrated. Accordingly, the C. burnetii T4SS effector protein AnkG was identified to inhibit pathogen-induced apoptosis, possibly by binding to the host cell mitochondrial protein p32 (gC1qR). However, it was unknown whether AnkG alone is sufficient for apoptosis inhibition or if additional effector proteins are required. Here, we identified two T4SS effector proteins CaeA and CaeB (C. burnetii anti-apoptotic effector) that inhibit the intrinsic apoptotic pathway. CaeB blocks apoptosis very efficiently, while the anti-apoptotic activity of CaeA is weaker. Our data suggest that CaeB inhibits apoptosis at the mitochondrial level, but does not bind to p32. Taken together, our results demonstrate that C. burnetii harbours several anti-apoptotic effector proteins and suggest that these effector proteins use different mechanism(s) to inhibit apoptosis.

Peri- and intra-operative management of the goat during acute surgical experimentation

Goats are used as animal models for surgery and trauma research. The authors discuss appropriate methods for induction of anesthetics, intubation and surgical maintenance of the goat during acute experimentation. Risks imposed by the Q fever pathogen Coxiella burnetii are described, as well as measures that have proven effective in minimizing zoonotic transmission of this pathogen to laboratory personnel. With appropriate knowledge of its applications, peri- and intra-operative management and limitations, the goat is a suitable animal model for a variety of biomedical research applications.

In silico prediction and identification of outer membrane proteins and lipoproteins from Coxiella burnetii by the mass spectrometry techniques

G. Flores-Rami´rez, R. Toman, Z. Sekeyova and L. SkultetyLaboratory for Diagnosis and Prevention of Rickettsial and Chlamydial Infections, Institute ofVirology, SAS, Bratislava, SlovakiaThe Gram-negative pathogen Coxiella burnetii isan obligate intracellular bacterium which growsonly within the phagolysosomes of eukaryotichost cells. The microbe is usually transmitted byaerosol and causes acute or chronic Q fever inhumans. It is well known that the outer mem-brane proteins (OMPs) play several importantroles in the host–parasite interactions of Gram-negative bacteria related to both pathogenesis andprotective immunity. In the present study, vari-ous bioinformatic tools and databases, togetherwith the mass spectrometry (MS) analyses, wereused to predict and to identify OMPs of C.burnetti,including the integral membrane proteins andlipoproteins (LPs) anchored to the outermembrane (OM) with an N-terminal lipid tail.The dataset of ORFs derived from the fullysequenced genome of C. burnetii strain Nine Mile I(RSA 493) [1] was processed for a subcellularlocalisation employing PSORTb (http://www.psort.org/psortb/index.html) with the Gram-neg-ative option. The results were compared with dataobtained from the Proteome Analyst PENCE data-base (http://www.cs.ualberta.ca/~bioinfo/PA/GOSUB) containing OMPs. The BOMP tool(http://www.bioinfo.no/tools/bomp) was usedto predict the presence of beta-barrels structures,and the TMBETA-SVM (tmbeta-svm.cbrc.jp) todiscriminate the OMPs from other folding typesof globular and membrane proteins. To predictLPs, the Lipo (http://services.cbu.uib.no/tools/lipo) tool, which detects LP sequences based on thelipobox region that is recognised by signal pepti-dase II, and the LipoP (http://www.cbs.dtu.dk/services/LipoP), which predicts LP signals pep-tides I and II in Gram-negative bacteria, have beenused. The results were further compared with thedata obtained from DOLOP database (http://www.mrc-lmb.cam.ac.uk/genomes/dolop) thatcontains LPs based on the lipobox present at theC-terminal end of the signal peptide. ORFpredicted as both OMP and LP was annotated asLP. In addition, the presence of any OMPs and LPsin the whole cell lysates of C.burnetiiRSA 493 wereconfirmed by MS techniques as describedpreviously [2].From the 2026 ORFs submitted to the bioinfor-matic tools, 160 ORFs including both low (onetool) and high confidence (at least two tools) werepredicted to be localised on the OM. Psortb,together with the PENCE database, has predicted34 OMPs, and BOMP, together with TMBETA-SVM, predicted 80 proteins with beta-barrelstructure related to OMP. Moreover, in total 46proteins were predicted as LPs, from which 33(71.73%) were predicted with a high confidence.It is noteworthy that 92% of all of them are basicproteins (pI higher than 8).Homology searches of predicted proteins sub-mitted to the Swiss-Prot database using the NCBIBLASTp tool (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) and Pfam (http://www.pfam.sanger.ac.uk/) databases showed that the highconfidence predictions are homologous to thewell-characterised OMPs. Homologs of OM effluxproteins, components of bacterial type II, IIIand IV secretion systems, membrane proteinsinvolved in drug resistance, organic solventtolerance proteins, ATPases coupled to themembrane components, peptidoglycan associatedproteins, polysaccharide biosynthesis⁄exportproteins, endoglucanases, components of synthe-sis of lipid A, surface antigens and proteinsinvolved in sporulation have been predicted.Our analyses by various MS techniques re-vealed 21 predicted OMPs (Table 1) and 9 LPsanchored to the OM till now. Among them, thehypothetical components of the LolCDE trans-portation system, LolA, a carrier protein(CBU_1190) and LolB (CBU_1829), a lipoprotein-

DETECTION OF COXIELLA BURNETII IN BOVINE MILK SAMPLES USING POLYMERASE CHAIN REACTION

Q fever is a highly contagious zoonotic disease caused by the intracellular pathogen Coxiella burnetii. It colonizes mammary glands of cattle and is shed in milk. This study was aimed to detect C.burnetii in raw bovine milk using Polymerase Chain Reaction (PCR). A total of 100 random bovine milk samples were collected from both dairy farms and shops in Assiut City, Egypt (50 samples each). A pair of primers served to amplify a targeted 448-bp fragment of genomic DNA. Our investigation showed that 22(22%) of samples were found to be positive for C. burnetii. This result proves that cattle are an important reservoir for C.burnetii organism and raw milk may be a main source of infection to humans.

Group I Introns and Inteins: Disparate Origins but Convergent Parasitic Strategies

Genomes of most organisms harbor DNA of foreign origin that has no known function. Since these elements may not contribute to a host’s fitness but utilize host resources for their perpetuation, it is appropriate to consider them genetic parasites (4). With the advent of sequencing technologies, a wide variety of parasitic elements have been discovered in bacteria from all environments, including obligate intracellular pathogens (100), which were thought to be shielded from horizontal gene transfer (HGT). Detection of a parasitic genetic element in a genome represents only a snapshot of the continuing and dynamic interplay between the host’s attempts to purge the element and the element’s ability to persist. These adaptable genetic parasites have evolved mechanisms to overcome defenses (75) of the cellular machinery to ultimately invade, colonize, and replicate within the host. Their success is very evident in the human genome, which consists mostly of such apparently superfluous DNA (65). Even compact bacterial genomes packed with functional genes contain mobile genetic elements (100), underscoring their universality in nature. A number of parasitic genetic elements are found in bacterial genomes, including transposons, insertion sequences, prophages, introns, inteins, and intervening sequences. While bacteria, especially pathogenic bacteria, are well studied, their parasitic genetic elements have not received as much attention. In the past few years, while studying the obligate intracellular pathogen Coxiella burnetii, we came to appreciate the intimate relationship between bacterial hosts and parasitic elements (92, 93). In addition, interesting new studies have shed light on the evolutionary histories of group I introns, inteins, and homing endonucleases (HEs) (9, 109) and infused excitement into the field. This minireview, which focuses on the biology and evolution of group I introns and inteins found in bacteria, is an attempt to catalyze interest among bacteriologists in these fascinating genetic parasites. GROUP I INTRONS

Antibody-mediated immunity to the obligate intracellular bacterial pathogen Coxiella burnetii is Fc receptor- and complement-independent

BackgroundThe obligate intracellular bacterial pathogen Coxiella burnetii causes the zoonosis Q fever. The intracellular niche of C. burnetii has led to the assumption that cell-mediated immunity is the most important immune component for protection against this pathogen. However, passive immunization with immune serum can protect naïve animals from challenge with virulent C. burnetii, indicating a role for antibody (Ab) in protection. The mechanism of this Ab-mediated protection is unknown. Therefore, we conducted a study to determine whether Fc receptors (FcR) or complement contribute to Ab-mediated immunity (AMI) to C. burnetii.ResultsVirulent C. burnetii infects and replicates within human dendritic cells (DC) without inducing their maturation or activation. We investigated the effects of Ab opsonized C. burnetii on human monocyte-derived and murine bone marrow-derived DC. Infection of DC with Ab-opsonized C. burnetii resulted in increased expression of maturation markers and inflammatory cytokine production. Bacteria that had been incubated with naïve serum had minimal effect on DC, similar to virulent C. burnetii alone. The effect of Ab opsonized C. burnetii on DC was FcR dependent as evidenced by a reduced response of DC from FcR knockout (FcR k/o) compared to C57Bl/6 (B6) mice. To address the potential role of FcR in Ab-mediated protection in vivo, we compared the response of passively immunized FcR k/o mice to the B6 controls. Interestingly, we found that FcR are not essential for AMI to C. burnetii in vivo. We subsequently examined the role of complement in AMI by passively immunizing and challenging several different strains of complement-deficient mice and found that AMI to C. burnetii is also complement-independent.ConclusionDespite our data showing FcR-dependent stimulation of DC in vitro, Ab-mediated immunity to C. burnetii in vivo is FcR-independent. We also found that passive immunity to this pathogen is independent of complement.

Adaptive immunity to the obligate intracellular pathogen Coxiella burnetii

Coxiella burnetii is an obligate intracellular bacterial pathogen that causes the zoonosis Q fever. While an effective whole-cell vaccine (WCV) against Q fever exists, the vaccine has limitations in being highly reactogenic in sensitized individuals. Thus, a safe and effective vaccine based on recombinant protein antigen (Ag) is desirable. To achieve this goal, a better understanding of the host response to primary infection and the precise mechanisms involved in protective immunity to C. burnetii are needed. This review summarizes our current understanding of adaptive immunity to C. burnetii with a focus on recent developments in the field.

P1736 A proteomics approach to understanding the pathogenesis mechanisms of the obligate intracellular pathogen Coxiella burnetii

P1736 A proteomics approach to understanding the pathogenesis mechanisms of the obligate intracellular pathogen Coxiella burnetii

Q FEVER: A CONTINUING DIAGNOSTIC PROBLEM IN WEST TEXAS AND EASTERN NEW MEXICO.

Background Q fever is a widespread zoonotic infection caused by the pathogen Coxiella burnetii that has both acute and chronic manifestations. Q fever may present as a fever of unknown origin (FUO). We examined three cases of Q fever in west Texas and eastern New Mexico: two chronic and one acute that presented as FUO. Q fever usually occurs following exposure to infected animals. Q fever continues to be a diagnostic possibility in patients with FUO and animal exposure in the southwestern United States. Methods Cases review from medical records at our institution 2000-2006. Results: Discussion Q fever may present as an acute or chronic febrile illness, either of which can have nonspecific symptoms and signs. Serologic studies are necessary for the diagnosis. Phase I and phase II serology will differentiate between acute and chronic disease. Although somewhat slow and costly, these studies are key to appropriate diagnosis and therapy. Conclusion Q fever continues to be a diagnostic problem in west Texas and eastern New Mexico.

Rickettsiella-like bacteria in Ixodes woodi (Acari: Ixodidae).

We examined a parthenogenetic strain of the hard tick Ixodes woodi Bishopp for the presence of endosymbiotic bacteria. Electron microscopic examination revealed the ovarian tissues and Malpighian tubules were infected with pleomorphic bacteria. Two basic types were observed: a larger granular cell and a smaller condensed cell. Cloning and sequence analysis of polymerase chain reaction (PCR) amplified 16S rRNA gene yielded a single sequence from bacteria present in I. woodi tissues. Phylogenetic analysis of the nearly complete 16S rDNA indicated that the ticks were infected with an endosymbiont belonging to the gamma subdivision of the Proteobacteria. It clustered with the insect pathogenic species Rickettsiellagrylli (Vago and Martoja 1963) and the animal pathogen Coxiella burnetii (Derrick 1939) Philip 1948. Our results suggest that the I. woodi females harbored a single endosymbiotic bacterium related to selected Rickettsiella species and to C burnetii.

Comparative efficacy and immunogenicity of Q fever chloroform:methanol residue (CMR) and phase I cellular (Q-Vax) vaccines in cynomolgus monkeys challenged by aerosol

Preliminary evidence gathered in rodents and livestock suggested that a phase I chloroform:methanol residue (CMR) extracted vaccine was safe and efficacious in protecting these animals from challenge with the obligate phagolysosomal pathogen ( Coxiella burnetii). Prior to the initiation of phase II studies in human volunteers, we compared, in non-human primates ( Macaca fascicularis), the efficacy of CMR vaccine with Q-Vax, a licensed cellular Australian Q fever vaccine that has been demonstrated to provide complete protection in human volunteers. Vaccine efficacy was assessed by evaluating thoracic radiographs and the presence of fever and bacteremia in monkeys challenged by aerosol with Coxiella burnetii. Changes in blood chemistries, hematology, behavior and pulmonary function were also examined. CMR, whether administered in single 30 or 100 μg doses or two 30 μg subcutaneous doses, gave equivalent protection in vaccine recipients as a single 30 μg dose of Q-Vax. In addition, vaccination resulted in significant, although temporary, increases in specific antibody titers against C. burnetii phases I and II antigens. The C. burnetii CMR vaccine may be an efficacious alternative to cellular Q fever vaccines in humans.