Burnetii Strains Research Articles

Genome Analysis of Coxiella burnetii Species: Insights into Pathogenesis and Evolution and Implications for Biodefense

Coxiella burnetii, the etiological agent of Q fever, is a class B biodefense agent. We are continuing the momentum of discovery generated by the first Coxiella genome sequences by extending the breadth of genomics to include four additional heterogeneous C. burnetii strains. We are also sequencing the genome of Rickettsiella grylli, an intracellular parasite of grasshoppers and the closest known phylogenetic relative to the Coxiella group. These data will enable the investigation of fundamental questions about Coxiella pathogenicity and virulence as well as broader evolutionary questions about the transition to obligate intracellular life. Specifically, sequence comparisons will permit examination of genetic differences, allowing us to address key questions: What core genes are necessary for an obligate intracellular lifestyle and developmental cycle of the genus? What specific genetic determinants can be linked to virulence properties such as host preference, disease severity, and pathology (i.e., acute vs. chronic disease)? What are the frequencies of mutation and intragenomic recombination, and levels of genome reduction? What specific factors are relevant to colonization and virulence in human hosts (based on comparisons with R. grylli)? From a public health and biodefense perspective, exposure to different strains, either natural or due to illegitimate release, may have different outcomes. With extensive genomic-level information from diverse strains, investigators can determine effective drug and vaccine targets and design methods to accurately type Coxiella based on a subset of genes, opening the way for cost-effective targeted PCR- or antibody-based tests.

Effect of vaccination with phase I and phase II Coxiella burnetii vaccines in pregnant goats

Livestock is considered to be the major “source” of human Q fever. The efficacy of two currently available vaccines (Coxevac, phase I, CEVA Santé Animale and Chlamyvax FQ, phase II, MERIAL) against Coxiella excretion was investigated in terms of risks to human health. Two months before mating, 17 goats were vaccinated subcutaneously against Coxiella burnetii with an inactivated phase I vaccine and 16 goats were vaccinated with an inactivated phase II Coxiella mixed with Chlamydophila abortus vaccine. Fourteen goats were left unvaccinated. At 84 days of gestation, the goats were subcutaneously challenged with 10 4 bacteria of C. burnetii strain CbC1. Phase I vaccine was effective and dramatically reduced both abortion and excretion of bacteria in the milk, vaginal mucus and feces. In contrast, the phase II vaccine did not affect the course of the disease or excretion.

Identification of the Coxiella sp. detected from Haemaphysalis longicornis ticks in Korea.

Two Haemaphysalis longicornis ticks were found positive in PCR assay of com-1 gene to detect Coxiella burnetii DNA from 100 ticks. The nucleotide sequences of com-1 and 16S rRNA gene were determined from 2 ticks and compared with those of other C. burnetii strains. The results suggest that H. longicornis harbor Coxiella sp. bacteria in Korea. Furthermore, icd, cbhE', and cbbE' genes are C. burnetii specific genes whereas com-1 gene is Coxiella genus specific gene. This study gives the first documentation to prove the existence of Coxiella sp. in tick collected in Korea.

Fiebre Q en Gran Canaria. Aportación de 40 nuevos casos

The aim of this study was to describe the clinical and epidemiologic features of Q fever in the southern area of the island of Gran Canaria (Spain). We conducted a retrospective analysis of the clinical and epidemiological data of the cases of Q fever diagnosed by the Microbiology Laboratory of the Hospital Universitario Insular in Gran Canaria between 1998 and 2000. Antibodies against phase II Coxiella burnetii antigens were detected using an indirect immunofluorescence test. The diagnosis of acute Q fever was established by IgG titers > or = 1:320 and IgM titers > or = 1:80, or by seroconversion. During the period of study 59 cases of acute Q fever were diagnosed, making an incidence of 5 cases/100,000 inhabitants/year. The seroprevalence (IgG > or = 1:80) in the patients for whom Q fever serology was requested during that period was 23.9%. Clinical and epidemiologic data were available for 40 patients. All were sporadic cases and 57% were hospitalized. The mean age of the patients was 40.6 6 13.3 years (range 20-74 years), 85% were males and 67.5% came from a rural background. The majority of cases (65%) clustered from April to July. The most frequent clinical presentation was an acute febrile process with elevated liver enzymes (87.5%). Pneumonia was infrequent (only three cases). In our area Q fever is mainly manifested as an acute febrile illness with subclinical hepatic involvement. This fact and the small number of cases with pneumonia and chronic forms suggest the etiological involvement of C. burnetii strains different from those in other geographic areas.

Diagnosis of quinolone-resistantCoxiella burnetii strains by PCR-RFLP

A total of 12 strains of Coxiella burnetii (8 Greek isolates from acute Q-fever patients, two reference strains-Nine Mile and Q212-and two pefloxacin-resistant laboratory strains) were examined for the presence of point mutations in the quinolone resistance determining region (QRDR) of gyrA gene by direct DNA sequencing of the polymerase chain reaction (PCR)-amplified fragments. The gene sequences of all eight Greek isolates and the two reference strains Nine Mile and Q212 [minimal inhibitory concentration (MIC)</= 4 microg/ml] were identical. Direct DNA sequencing of the in vitro-selected resistant strains (MICs to pefloxacin, 8-32 microg/ml) revealed a transition (G-->A) at the corresponding codon 87 of E. coli. This mutation lead to the substitution of Glu (codon GAG) by Lys (codon AAG ). Restriction maps of amplified gyrA gene sequences were determined by GCG Wisconsin PACKAGE, and the MnlI restriction enzyme was found to cut only the sensitive strains sequences and not the resistant ones. The present PCR-RFLP analysis has proved to be a simple, rapid, and useful method for the detection of Coxiella burnetii and, at the same time, for the diagnosis of quinolone-resistant Coxiella burnetii strains.

Diagnosis of quinolone-resistant Coxiella burnetii strains by PCR-RFLP

Diagnosis of quinolone-resistant Coxiella burnetii strains by PCR-RFLP

Investigation on the Susceptibility of Coxiella Burnetii Strains to Antibiotics Depending on Their Virulence

ABSTRACTThe objective of the present study has been to establish the species belonging, the phase of the antigen complexes and the virulent properties of C. burnetii strains isolated from active Q-rickettsious foci in cows and goats. Three strains of C. burnetii, isolated from placenta of an aborted cow (strain HP), of a cow with normal parturition (strain MP) and from the milk of a goat having a Q-rickettsious mastitis (strain PM) have been studied. It has been established that after their successive cultivation on chick embryos strain ITP (7th-8th passage) is in I-phase, strain MP (11th-13th passage)—in a transitional phase and strain PM (16th-17thpassage)—in II-phase. For the first time have been studied the immunologic belonging, the serologic activity and the virulent properties of the C. burnetii strain, isolated from the milk of a goat with Q-rickettsious mastitis, It has been found that they are similar to those of a museum human-isolated Henzerling strain.

A comparative study of lipopolysaccharides from two Coxiella burnetii strains considered to be associated with acute and chronic Q fever

Coxiella burnetii strains Nine Mile and Priscilla, considered to be associated with acute and chronic forms of Q fever, were investigated for variation in composition of their lipopolysaccharides. Though SDS-PAGE profiles of the lipopolysaccharides were distinct, chemical analyses showed only small differences in their overall composition. Further studies on lipid A-deprived O-polysaccharide fractions of both lipopolysaccharides, obtained by steric-exclusion chromatography, revealed noticeable differences in distribution and chemical composition of the O-polysaccharide chains. It is likely that C. burnetii strains are capable of synthesizing chemically distinct subclasses of O-specific polysaccharide molecules differing in their antigenic reactivities. The results provide suggestive evidence that virulence of C. burnetii may be modulated through lipopolysaccharide composition and structure.

Antigenic characteristics of polypeptides of Coxiella burnetii isolates.

Eighteen Coxiella burnetii strains from a variety of clinical and geographical sources were screened for antigenic variation of polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) coupled with Coomassie brilliant blue (CBB) staining or immunoblotting. These polypeptide profiles showed the greatest variability in the region from 33 to 8.1 kDa. Such differences in the antigenicity of the polypeptides were also recognized by immunoblotting with 15 various mouse anti-C. burnetii antisera. In addition, we detected a polypeptide at about 28 kDa which was immunodominant in strains from human cases of acute Q fever, milk and ticks but not immunogenic in strains from human cases of chronic Q fever. These findings suggest that this polypeptide is a marker to distinguish between acute and chronic strains.

Determination of Coxiella burnetii rpoB sequence and its use for phylogenetic analysis

The nucleotide sequence of the rpoB, encoding the β-subunit of RNA polymerase of the obligate intracellular bacterium Coxiella burnetii, was determined using a polymerase chain reaction amplification and direct sequencing methodology. Comparison between C. burnetii and other eubacterial rpoB sequences indicated sequence similarity ranging from 53.6% to 67.6%. Coxiella burnetii rpoB consists of 4128 base pairs with a 45.3% GC content encoding 1375 amino acids with a calculated molecular mass of 153.67 kDa. Comparison of 512 bases of the rpoB variable region I, from eight C. burnetii strains isolated from various sources, revealed fewer than four base differences, although the distribution of these did not correlate with previously determined genotypic groupings with the species. Phylogenetic analysis of C. burnetii based on comparison of its rpoB sequence with sequences available for other bacteria is consistent with those previously derived from 16S rRNA gene sequence, and indicate that C. burnetii belongs to the γ-group of Proteobacteria. Furthermore, phylogeny inferred from comparison of RpoB, or homologous sequences including Archae, Bacteria and Eukarya, concurred with these results.

Identification of rickettsiae isolated in Japan as Coxiella burnetii by 16S rRNA sequencing.

The 16S rRNA genes of Japanese Coxiella isolates obtained from various sources and geographical areas were directly sequenced by dideoxynucleotide chain termination methods in which Taq DNA polymerase was used. The levels of sequence similarity among Japanese, European, and American isolates were more than 99%, and the Japanese isolates were identified as Coxiella burnetii, C. burnetii strains isolated worldwide, including Japan, were found to be very similar.

Analysis of immunoglobulin classes and subclasses in response to infection of Balb/cJ and C57BL/6J mice with Coxiella burnetii.

In order to establish defined immunological parameters for Q fever infection models, a microtitre enzyme-linked immunosorbent fluorescence assay (ELISA) was used for the first time to analyse the humoral immune response of Balb/cJ and C57BL/6J mice after experimental infection with Coxiella burnetii strain 'Nine Mile' in phase I. The experimental infection evoked a seroconversion in all mice within 10 days. Typically, the immune response measured against the whole-cell antigen showed an early increase of immunoglobulin (Ig) M followed by a later increase of the IgG subclasses. The IgA was low during the entire investigation period. Within the IgG subclasses only IgG2a and IgG2b gained higher values, whereby C57BL/6J mice produced high IgG2b titres and significantly lower IgG2a titres. In contrast, Balb/cJ mice developed IgG2a and IgG2b at equal levels. The use of partial antigens of C. burnetii demonstrated that the dominating IgG2b reaction of the C57BL/6J mice was directed against the lipopolysaccharide (LPS) of C. burnetii. This reaction was almost absent in Balb/cJ mice. In contrast, the SP27 protein antigen did not evoke different IgG2b reactions within the two breeds. No significant influence was observed within the two breeds in regard to sex or between hormone synchronized and non-hormone synchronized animals.

Study of the 16S-23S ribosomal DNA internal spacer of Coxiella burnetii.

The complete 16S-23S ribosomal DNA (rDNA) internal transcribed spacer (ITS) of 22 isolates of the obligate intracellular bacterium Coxiella burnetii, the agent of Q fever, were amplified by the polymerase chain reaction (PCR) and sequenced using an automated laser fluorescent DNA sequencer. The ITS measured 497 base pairs (bp) and encoded isoleucine-tRNA and alanine-tRNA. The comparison of the sequence alignments of the 22 C. burnetii strains revealed very high levels of sequence similitary (> 99%) although they had different geographic origins and phenotypic characteristics. Sequencing of the 16S-23S rDNA ITS of C. burnetii could be utilized for identification of the bacterium but is not applicable to studies of epidemiology, virulence and taxonomy.

Possible sexual transmission of Q fever among humans.

Direct transmission of Q fever between persons who have been exposed to Coxiella burnetii and their family members has been hypothesized on the basis of the results of serological surveillance. We studied nine shepherds who were employed in Spain during the sheep shearing season. After they returned to Poland, Q fever was detected in these shepherds and their wives. The titers of serum antibodies to phase I C. burnetii antigens ranged from 0 to 64 in patients with Q fever and in their spouses, and the titer of serum antibodies to phase II antigens ranged from 0 to 1.024 in patients and their spouses. Other family members were seronegative for antibodies to C. burnetii. C. burnetii strains were isolated from urine and semen samples obtained from patients with Q fever. Attached bacteria have been detected in spermatozoal cells observed with use of scanning electron microscopy.

Prevalence of Coxiella burnetti Infection in Military Training Areas in the Czech Republic and Slovakia

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Isolation ofCoxiella burnetii and of an unknown rickettsial organism fromIxodes ricinus ticks collected in Austria

Two strains of Coxiella burnetii and two strains of an unidentified rickettsial organism were isolated for the first time from Ixodes ricinus ticks collected in the Alpine region of Tirol, Austria. The C. burnetii strains belong to the group of agents causing acute forms of Q fever. The other two strains of isolated rickettsial agent share some antigenic epitopes with C. burnetii and R. prowazekii but they differ from them by their high sensitivity to freezing and refreezing and by poor multiplication in yolk sacs of chick embryos. There is at present no evidence that these organisms cause human illness and no ecological information is available. We suggest they may be some new species of rickettsiae or rickettsia-like organisms.

Analysis of the cbhE' plasmid gene from acute disease-causing isolates of Coxiella burnetii

A gene termed cbhE' was cloned from the QpH1 plasmid of Coxiella burnetii. Expression of recombinants containing cbhE' in vitro and in Escherichia coli maxicells, produced an insert-encoded polypeptide of approx. 42 kDa. The CbhE protein was not cleaved when intact maxicells were treated with trypsin. Hybridizations of total DNA isolated from the six strains of C. burnetii indicate that this gene is unique to C. burnetii strains associated with acute disease, i.e., Hamilton[I], Vacca[II], and Rasche[III]. The cbhE' gene was not detected in strains associated with chronic disease (Biotzere[IV] and Corazon[V]) or the Dod[VI] strain. The cbhE' open reading frame (ORF) is 1022 bp in length and is preceded by a predicted promoter/Shine-Dalgarno (SD) region of TCAACT(-35)-N16-TAAAAT(-10)-N14-AGAAGGA (SD) located 10 nucleotides (nt) before the presumed AUG start codon. The ORF ends with a single UAA stop codon and has no apparent Rho-factor-independent terminator following it. The cbhE' gene codes for the CbhE protein of 341 amino acid (aa) residues with a deduced Mr of 39,442. CbhE is predominantly hydrophilic with a predicted pI of 4.43. The function of CbhE is unknown. No nt or aa sequences with homology to cbhE' or CbhE, respectively, were found in searches of a number of data bases.

Epidemic of Q fever in Leszno district in Poland

In 1988 an epidemic of Q fever was detected in Leszno district. During 1973-1985 all 28,066 cattle tested for C. burnetii antibodies were found to be negative. The first seroconversions were found in cows which produced stillborn young. In the following years the number of seropositive cattle increased from 8.4% in 1987 to 21.6% in 1989. In 1988 all animal workers in the district were tested for C. burnetii antibodies. Of 4,264 persons tested, 1,451 (34%) were seropositive. A detailed study of workers and animals on one farm were performed. A herd of animals was found to be seropositive (32.1%), 68% of workers in direct contact with infected animals were seropositive and 29% of persons drinking raw milk. C. burnetii strains have been isolated from ticks, wild animals and birds hunted in close proximity of the farm. The dynamics of C. burnetii infection among animals and humans in this district and the fact that there is no importation of animal herds suggests that the possible route of introduction of Q fever in this area may be with imported semen or breeding bulls. The presence of C. burnetii in ticks and wild animals indicates the transfer of Q fever to the natural environment and its maintenance in this territory.

Experimental Q fever in sheep.

The aim of the present investigation is the complex study of experimental infection in pregnant ewes by means of clinical, serological, biological, histological and Electron microscopy methods. Four ewes, pregnant from the 2nd to 5th month, were infected by intravenous (in one case by intraperitoneal) routes with a C. burnetii strain at 10(6) ID 50/ml. The clinical illness in all of the animals was characterized by fever and two-phase temperature reaction on the 5th and 12th days. The clinical symptoms were as follows: torpidity, reduced appetite, thirst, conjuctivitis, rhinitis, rapid breathing. As a result of the developed latent infection, after the acute stage, the animals gave birth to three unviable lambs who died within 24 h. Another lamb was still-born. The lambs showed cachexia, arthritis, ataxia, wrinkled skin. The highest CF-titers (1:256-1:512) were reached on the 40th day, but serum antibodies (1:8-1:32) first appeared on the 8th day. The titers began to decrease on the 60th day. The pathomorphological changes testify to a latent infection characterized by placentitis, lymphocellular proliferation of the lamb's parenchymal organs and lymph nodes, multiple thromboses, interstitial pneumonia and plural proliferative changes. The EM exam showed rickettsiae in placentas mainly in the form of inclusions in cytoplasm of leukocytes and epithelial cells.

Approaches to the molecular epidemiology of rickettsioses

This review deals with the developments of molecular approaches to the investigation of rickettsial disease epidemiology. The data presented include changes in the incidence and geographic distribution of endemic rickettsioses. Use of the DNA restriction enzyme technique, in combination with DNA probe analysis, for the molecular genetic differentiation of tick spotted fever--and typhus fever--group rickettsiae and correlation between with the analysis of polypeptide composition of the above group of rickettsiae are discussed. The data are presented on progress in the identification of various Coxiella burnetii strains as a result of restriction analysis of plasmid DNA as well as chromosomal DNA in combination with DNA probe. New and detailed characteristics of classified and newly isolated strains of rickettsiae and Coxiella burnetii revealed by molecular genetic differentiation techniques are discussed. New identification techniques using DNA probes in combination with restriction analysis of chromosomal from rickettsiae and both plasmid and chromosomal DNA from Coxiella burnetii are considered to have good prospects for future use in epidemiological assessment. The establishment of reference file banks containing restriction endonuclease data on the available typical and atypical strains of rickettsiae and Coxiella burnetii is suggested.