Intraspecies diversity of Coxiella burnetii as revealed by com1 and mucZ sequence comparison

Coxiella burnetii is classified within the γ subgroup of the Proteobacteria. All strains tested to date have an identical 16S rRNA sequence but 20 different genotypes have been determined by pulsed field gel electrophoresis (PFGE). In this study, intraspecies genetic diversity was investigated by sequence comparison of 715 bp of the Com1 encoding gene (com1) and 774 bp of the MucZ encoding gene (mucZ) in 37 strains isolated from animals and humans with acute or chronic Q fever in Europe, North America and Africa. Five and four groups were established from sequence analysis of com1 and mucZ, respectively. Neither relation of the defined groups to geographical distribution of the isolates was noted nor relation to disease form (acute/chronic). The same isolates were grouped together regardless of the gene being investigated. Comparison of the five proposed groups to previous groups, yielded after digestion by NotI PFGE, allowed for an intermediate classification of C. burnetii isolates between those obtained by using 16S rDNA (one group) and PFGE (20 groups).     

Differentiation of Coxiella burnetii isolates by sequence determination and PCR-restriction fragment length polymorphism analysis of isocitrate dehydrogenase gene

The isocitrate dehydrogenase (icd) gene of Coxiella burnetii was cloned and sequenced to differentiate between isolates with various geographic origins and phenotypic properties. Based on the gene sequences all 19 isolates studied could be divided into three groups. Group 1 contained isolates originating from acute cases of Q fever, ticks and cows. Groups 2 and 3 included isolates from chronic Q fever patients and a prototype strain from an aborted goat. Although the icd gene profiles were different among isolates of the latter two groups, there were two base differences common for both groups which could be used as markers to distinguish them from group 1 isolates. Based on one of the markers a simple method using PCR-restriction fragment length polymorphism analysis was developed for rapid differentiation of C. burnetii isolates as well as for direct detection and differentiation of the bacterium in human serum samples. Taken together, the study results suggest that the icd-based differentiation method may be useful in clinical investigation of Coxiella infections.     

Genomotyping of Coxiella burnetii using microarrays reveals a conserved genomotype for hard tick isolates

C. burnetii is a Gram-negative intracellular Y-proteobacteria that causes the zoonotic disease Q fever. Q fever can manifest as an acute or chronic illness. Different typing methods have been previously developed to classify C. burnetii isolates to explore its pathogenicity. Here, we report a comprehensive genomotyping method based on the presence or absence of genes using microarrays. The genomotyping method was then tested in 52 isolates obtained from different geographic areas, different hosts and patients with different clinical manifestations. The analysis revealed the presence of 10 genomotypes organized into 3 groups, with a topology congruent with that obtained through multi-spacer typing. We also found that only 4 genomotypes were specifically associated with acute Q fever, whereas all of the genomotypes could be associated to chronic human infection. Serendipitously, the genomotyping results revealed that all hard tick isolates, including the Nine Mile strain, belong to the same genomotype.     

Genomic analysis of phase I and II Coxiella burnetii with restriction endonucleases

Restriction endonuclease-digested DNAs from several isolates of phase I and phase II Coxiella burnetii were compared using agarose gel electrophoresis and soft-laser scanning densitometry. Our results demonstrate that the two phases are, as previously assumed, alternative phases of the same organism. Although the restriction endonuclease digestion revealed genetic differences between clonal isolates of phase I and phase II C. burnetii Nine Mile strain, these differences do not appear to be related to antigenic phase variation. However, analyses of the fragment patterns generated by restriction enzyme digestion suggest potential grouping of the different isolates.     

A new plasmid (QpDV) common to Coxiella burnetii isolates associated with acute and chronic Q fever

Abstract Genetic studies of Coxiella burnetii strains suggested the possibility of differentiating new isolates according to their plasmid DNA content. Virulence and/or clinical manifestations ('chronic' and 'acute' Q fever) had been claimed to correlate with this plasmid typing. A new plasmid, named QpDV, was found to be common to C. burnetii isolates obtained from acute and chronic Q fever. According to the results obtained, plasmid usage for detection and differentiation of respective pathovars of C. burnetii and the correlation between gene specificity and pathovar has to be revised. Closer studies suggested a common origin of C. burnetii plasmids, but also showed some differences characteristic for each plasmid, probably reflecting divergent evolution.     

In vitro susceptibility to tetracycline and fluoroquinolones of Japanese isolates of Coxiella burnetii

Coxiella burnetii is the agent of the worldwide zoonosis, Q fever. The in vitro susceptibility to tetracycline and fluoroquinolones of Japanese isolates of C. burnetii was evaluated for the first time. The MICs against Japanese isolates were almost the same as the MICs against the foreign reference isolates. The results suggest that the common antibiotics therapy for Q fever used in other countries is also effective for Japanese Q fever patients.     

The Coxiella burnetii Dot/Icm system delivers a unique repertoire of type IV effectors into host cells and is required for intracellular replication

Coxiella burnetii, the causative agent of human Q fever, is an intracellular pathogen that replicates in an acidified vacuole derived from the host lysosomal network. This pathogen encodes a Dot/Icm type IV secretion system that delivers bacterial proteins called effectors to the host cytosol. To identify new effector proteins, the functionally analogous Legionella pneumophila Dot/Icm system was used in a genetic screen to identify fragments of C. burnetii genomic DNA that when fused to an adenylate cyclase reporter were capable of directing Dot/Icm-dependent translocation of the fusion protein into mammalian host cells. This screen identified Dot/Icm effectors that were proteins unique to C. burnetii, having no overall sequence homology with L. pneumophila Dot/Icm effectors. A comparison of C. burnetii genome sequences from different isolates revealed diversity in the size and distribution of the genes encoding many of these effectors. Studies examining the localization and function of effectors in eukaryotic cells provided evidence that several of these proteins have an affinity for specific host organelles and can disrupt cellular functions. The identification of a transposon insertion mutation that disrupts the dot/icm locus was used to validate that this apparatus was essential for translocation of effectors. Importantly, this C. burnetii Dot/Icm-deficient mutant was found to be defective for intracellular replication. Thus, these data indicate that C. burnetii encodes a unique subset of bacterial effector proteins translocated into host cells by the Dot/Icm apparatus, and that the cumulative activities exerted by these effectors enables C. burnetii to successfully establish a niche inside mammalian cells that supports intracellular replication.     

Detecting and measuring small numbers of viable Coxiella burnetii

Coxiella burnetii is an acidophilic, intracellular bacterium that causes the human disease Q fever. In some studies, it is important to distinguish between viable and nonviable C.?burnetii. We compared four methods for detecting and measuring viable C.?burnetii in biological samples as follows: growth in two different cell culture lines, infection of severe combined immunodeficient (SCID) mice (leading to death) and infection of SCID mice with detection of C.?burnetii in their spleen (after euthanasia at day 50 postinfection). Two isolates of C.?burnetii were used ('Henzerling' and 'Arandale'). Our in-house qPCR assay for C.?burnetii DNA was used as a control. SCID mouse inoculation was more sensitive than cell culture. The assay that detected C.?burnetii in SCID mouse spleens was slightly more sensitive than SCID mice deaths alone. Approximately one viable C.?burnetii cell could be detected by this method, making it suitable for determining the viability of C.?burnetii in a sample.     

Establishment of a genotyping scheme for Coxiella burnetii

Coxiella burnetii is the causative agent of Q fever. The bacterium is highly infectious and is classified as a category B biological weapon. The tools of molecular biology are of utmost importance in a rapid and unambiguous identification of C. burnetii in naturally occurring Q fever outbreaks, or in cases of a deliberate release of the infectious agent. In this work, development of a multiple locus variable number tandem repeats (VNTR) analysis (MLVA) for the characterization of C. burnetii is described. Sixteen C. burnetii isolates and five passage history/laboratory variants were characterized. The VNTR markers revealed many polymorphisms resulting in nine unique MLVA types that cluster into five different clusters. This proves that the MLVA system is highly discriminatory. The selected VNTR markers were stable. The MLVA method developed in this report is a promising tool for the characterization of C. burnetii isolates and their epidemiological study.     

Transformation of Coxiella burnetii to ampicillin resistance.

A 5.8-kb chromosomal fragment isolated from Coxiella burnetii initiates plasmid replication in Escherichia coli and was characterized as an autonomous replication sequence, ars (M. Suhan, S.-Y. Chen, H.A. Thompson, T.A. Hoover, A. Hill, and J.C. Williams, J. Bacteriol. 176:5233-5243, 1994). In the present study, an ars replicon was used to transform C. burnetii to ampicillin resistance. Plasmid pSKO(+)1000 contained the C. burnetii ars sequence cloned into a ColE1-type replicon encoding beta-lactamase. pSKO(+)1000 was introduced into C. burnetii by electroporation. Ampicillin-resistant cells were selected, and survivors were examined for the transformed genotype by Southern hybridization. Transformants stably maintained the pSKO(+)1000 bla DNA sequence in the chromosome as a result of homologous recombination. The recombination event resulted in the duplication of the 5.8-kb ars sequence in the C. burnetii chromosome. The bla gene was also located in an episome. However, an ampicillin resistance plasmid lacking the C. burnetii ars sequence did not stably transform C. burnetii. A biological assay analyzing beta-lactamase activity of C. burnetii transformants during acid activation in vitro provided evidence for expression of the bla (beta-lactamase) gene.     

水稻条叶枯病毒NS2基因遗传多样性分析

应用单链构象多态性 (SSCP)和序列分析方法研究了来自我国 9个省份的水稻条叶枯病毒(RSV) 80个田间分离物的NS2基因遗传结构特征 .SSCP分析结果表明 ,我国RSVNS2基因遗传结构符合准种 (quasispecies)结构特征 .部分分离物的序列分析结果表明 ,RSV上述分离物和已报道的日本 2个分离物可以归入 2个组 :云南的部分分离物划分为 1个组 ;其它分离物及日本T、O的 2个分离物为另 1组 .组与组之间 ,NS2蛋白基因核苷酸同源性为 94 %～ 95 % ,氨基酸同源性为 95 %～ 97% .遗传多样性分析结果表明 ,RSV种群存在地理隔离但在种群间可能发生了基因漂移 (geneflow) .NS2蛋白可能的运动蛋白功能所造成的负选择压力和介体传播引起的奠基者效应可能是RSV种群内和种群间遗传多样性差别的主要因素     

Differentiation of Coxiella burnetii isolates by analysis of restriction-endonuclease-digested DNA separated by SDS-PAGE.

Thirty-two isolates of Coxiella burnetii collected from various hosts ranging from arthropods to man were compared by restriction endonuclease (RE) digestion patterns of chromosomal DNA using SDS-PAGE. SDS-PAGE provided better DNA fragment separation than agarose gel electrophoresis and enabled the differentiation of these isolates into six distinct groups on the basis of DNA restriction fingerprints. Two groups of chronic disease isolates could be distinguished, each having unique RE digestion patterns of chromosomal DNA. Three similar but distinct RE digestion patterns were seen among the group of acute disease isolates. Three additional isolates included in this study exhibited a unique RE digestion pattern and also had a unique plasmid type, designated QpDG. DNA-DNA hybridization on selected isolates quantified the relatedness between several groups and supported the classification of these groups as distinct strains.     

Phylogenetic analysis of coat protein gene of BYDV-MAV strain from wheat

Barley yellow dwarf disease is globally the most important viral disease of wheat. The full-length nucleotide sequence of coat protein (CP) gene of 12 isolates revealed the presence of three distinct clusters. Pakistani isolate of MAV (MAV-PK) has maximum similarity of 99.23% with MAV isolate of Morocco and PAV-Australia following 99.22 and 99.22% with PAV-France. Similar degree of similarity was found in comparison of amino acid sequence. The finding of this study is that MAV-PK has similarity with both MAV-France and PAV-Australia, which is due to the reason that both MAV and PAV belong to the same group and both share maximum nucleotide homology. Low genetic diversity was found not only between MAV isolates but also between MAV and PAV isolates because phylogenetic analysis was done on the CP gene which is highly conserved region in genome of Barley yellow dwarf viruses (BYDVs). Divergence in MAV-PK was due to this recombination which is now most prevalent in Pakistan. MAV-PK has maximum similarity with MAV-Morocco followed by MAV-Sweden and MAV-Cz, which seems to indicate that Pakistani isolate of MAV evolved as the result of recombination between MAV isolates of the USA and PAV isolates of Australia and France. At the same time, recombination of MAV-CZ and MAV-Sweden also occur. This work can be successfully utilised in epidemiological studies of MAV isolate in Pakistan. Further analysis of variation level in these isolates will help scientists to formulate appropriate management strategies like incorporation of BdV 2 gene in wheat against BYDVs.     

Genetic variation analyses of nsp2 gene of PRRSV in Ningxia Hui Autonomous Region of China

To gain a better understanding of the genetic diversity and evolution of PRRSV in the Ningxia Hui Nationality Autonomous Region (Ningxia) of China, the nsp2 genes from a series of PRRSV strains collected from the region in 2007 were partially sequenced. These sequences were then analyzed along with the classical strain (ch-la) and two other epidemic strains SD (3) and SD2006. Comparison of the nucleotide sequence with ch-la indicated that nsp2 genes of seventeen Ningxia isolates (NX strain) have deletions of 87 nucleotides. Sequence analysis indicated that homology between the Ningxia strain and ch-la was 60.3%-79.9% in the nucleotide sequence, and homology between the NX strains and SD strains was 80.3%-98.8% in the nucleotide sequence. The nsp2 genes of the seventeen isolates had 74.9%-100% nucleotide sequence identities with each other. This study was undertaken to assess the regional variation of prevalent PRRSV and to establish a sequence database for PRRSV molecular epidemiological studies.     

The unusual 23S rRNA gene of Coxiella burnetii: two self-splicing group I introns flank a 34-base-pair exon, and one element lacks the canonical omegaG

We describe the presence and characteristics of two self-splicing group I introns in the sole 23S rRNA gene of Coxiella burnetii. The two group I introns, Cbu.L1917 and Cbu.L1951, are inserted at sites 1917 and 1951 (Escherichia coli numbering), respectively, in the 23S rRNA gene of C. burnetii. Both introns were found to be self-splicing in vivo and in vitro even though the terminal nucleotide of Cbu.L1917 is adenine and not the canonical conserved guanine, termed OmegaG, found in Cbu.L1951 and all other group I introns described to date. Predicted secondary structures for both introns were constructed and revealed that Cbu.L1917 and Cbu.L1951 were group IB2 and group IA3 introns, respectively. We analyzed strains belonging to eight genomic groups of C. burnetii to determine sequence variation and the presence or absence of the elements and found both introns to be highly conserved (>/=99%) among them. Although phylogenetic analysis did not identify the specific identities of donors, it indicates that the introns were likely acquired independently; Cbu.L1917 was acquired from other bacteria like Thermotoga subterranea and Cbu.L1951 from lower eukaryotes like Acanthamoeba castellanii. We also confirmed the fragmented nature of mature 23S rRNA in C. burnetii due to the presence of an intervening sequence. The presence of three selfish elements in C. burnetii's 23S rRNA gene is very unusual for an obligate intracellular bacterium and suggests a recent shift to its current lifestyle from a previous niche with greater opportunities for lateral gene transfer.     

Effects of Coxiella burnetii on MAPKinases phosphorylation

Q fever is a disease caused by Coxiella burnetii, an obligate intracellular bacterium. Acute Q fever is characterized by efficient immune response, whereas chronic Q fever is characterized by dysregulated immune response as demonstrated by the lack of granulomas, the failure of C. burnetii to induce lymphoproliferation, and interferon-γ production. The mitogen-activated protein kinase (MAPK) signaling pathway plays crucial roles in innate immune responses and control of bacterial infections. However, its role in Q fever has not been addressed. First, we investigated the activation of MAPKs p38, c-jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) 1/2 in murine macrophages stimulated with C. burnetii. Coxiella burnetii NM phase I (virulent) and NM phase II (avirulent) induced the activation of JNK and ERK1/2. Avirulent C. burnetii activate p38, whereas C. burnetii did not induce the phosphorylation of p38. Second, the level of p38 activation was studied in Q fever patients. We found that p38 was activated in monocyte-derived macrophages from healthy donors and patients with acute Q fever in response to a potent agonist such as lipopolysaccharide. Interestingly, p38 was not activated in patients with active chronic Q fever and was activated in patients with cured chronic Q fever. These results suggest that the determination of p38 activation may serve as a tool for measuring Q fever activity.     

Differentiation of Coxiella burnetii by Sequence Analysis of the Gene (com1) Encoding a 27-kDa Outer Membrane Protein

The gene (com1) encoding a 27-kDa outer membrane protein in 21 strains of Coxiella burnetii from a variety of clinical and geographical sources was sequenced for strain differentiation. The com1 gene was highly conserved among all the strains tested but there were several differences in nucleotide and deduced amino acid sequences. Based on the com1 gene-specific nucleotides and deduced amino acids, the 21 strains were divided into four groups. Group 1 contained 14 strains originating from ticks, cattle and human cases of acute Q fever. Groups 2 and 3 included 2 and 3 strains, respectively, originating from human cases of chronic Q fever. Group 4 contained 2 strains originating from a human case of acute Q fever and a goat with abortion. The results indicated that the strains originating from ticks, cattle and human cases of acute Q fever differed at the molecular level from those of human chronic Q fever. This study suggests that a sequence analysis of the com1 gene can be used for strain differentiation of C. burnetii.     

Genetic diversity of the Q fever agent, Coxiella burnetii, assessed by microarray-based whole-genome comparisons

Coxiella burnetii, a gram-negative obligate intracellular bacterium, causes human Q fever and is considered a potential agent of bioterrorism. Distinct genomic groups of C. burnetii are revealed by restriction fragment-length polymorphisms (RFLP). Here we comprehensively define the genetic diversity of C. burnetii by hybridizing the genomes of 20 RFLP-grouped and four ungrouped isolates from disparate sources to a high-density custom Affymetrix GeneChip containing all open reading frames (ORFs) of the Nine Mile phase I (NMI) reference isolate. We confirmed the relatedness of RFLP-grouped isolates and showed that two ungrouped isolates represent distinct genomic groups. Isolates contained up to 20 genomic polymorphisms consisting of 1 to 18 ORFs each. These were mostly complete ORF deletions, although partial deletions, point mutations, and insertions were also identified. A total of 139 chromosomal and plasmid ORFs were polymorphic among all C. burnetii isolates, representing ca. 7% of the NMI coding capacity. Approximately 67% of all deleted ORFs were hypothetical, while 9% were annotated in NMI as nonfunctional (e.g., frameshifted). The remaining deleted ORFs were associated with diverse cellular functions. The only deletions associated with isogenic NMI variants of attenuated virulence were previously described large deletions containing genes involved in lipopolysaccharide (LPS) biosynthesis, suggesting that these polymorphisms alone are responsible for the lower virulence of these variants. Interestingly, a variant of the Australia QD isolate producing truncated LPS had no detectable deletions, indicating LPS truncation can occur via small genetic changes. Our results provide new insight into the genetic diversity and virulence potential of Coxiella species.