Cornelie Jäger

Molecular characterization of Coxiella burnetii isolates.

Restriction fragment length polymorphism (RFLP) was used for the differentiation of 80 Coxiella burnetii isolates derived from animals and humans in Europe, USA, Africa and Asia. After NotI restriction of total C. burnetii DNA and pulsed field gel electrophoresis (PFGE) 20 different restriction patterns were distinguished. The index of discrimination for this typing system was 0.86. Comparison and phylogenetic analysis of the different RFLP patterns revealed evolutionary relationships among groups that corresponded to the geographical origin of the isolates. This finding was confirmed by genetic mapping. No correlation between restriction group and virulence of isolates was detected.

Detection of Chlamydia suis from clinical specimens: comparison of PCR, antigen ELISA, and culture.

Cell culture is still widely regarded as the gold standard in chlamydial diagnosis despite its well-known limitations in terms of sensitivity. On the other hand, the polymerase chain reaction (PCR) has emerged as a promising alternative because of rapidity and high sensitivity. However, validation of methodologies is required before the issue of standardization can be addressed. In the present study, 109 clinical samples (organ tissue, nasal, and faecal swabs) from pigs experimentally infected with Chlamydia suis were examined by cell culture, nested PCR in the ompA gene region, and two different antigen enzyme-linked immunosorbent assays (ELISAs) in order to compare the diagnostic performance of these methods. Culture and PCR produced the highest proportion of concordant results (kappa coefficient 0.712). Among 99 samples, 34 were positive in both assays, 51 were negative in both assays, 12 culture-negatives were positive in PCR, and only 2 culture-positives were negative in PCR. Thus, the sensitivity and specificity of PCR vs. culture as standard were 94.4% and 81.0%, respectively, whereas the corresponding values for culture vs. PCR as standard were 73.9% and 96.2%, respectively. Both ELISA tests performed considerably weaker. The data underline the potential of PCR as a powerful detection method for chlamydiae.

Coxiella burnetii plasmid types QpDG and QpH1 are closely related and likely identical.

Strains and isolates of Coxiella burnetii, an obligate intracellular bacterium, carry a single plasmid or a plasmid-homologous sequence integrated into the chromosome. The plasmids QpH1, QpRS, QpDV and the chromosome-integrated plasmid-homologous region have been completely sequenced, whereas no sequence data are available for the QpDG plasmid. In this study, we used total genomic DNA from reference strain C. burnetii Dugway 5J108-111 to demonstrate and characterize the QpDG plasmid by pulsed-field gel electrophoresis (PFGE) and Southern hybridization. Primers derived from regions shared among C. burnetii plasmids were used to construct a physical map of the QpDG plasmid by extra long (XL) PCR. Both approaches, Southern hybridization and XL PCR indicated that QpDG and QpH1 represent a closely related and likely identical plasmid.

Multiplex-PCR-based differentiation and characterization of Candida-isolates derived from tortoises (Testudinidae).

Candida isolates (n=23) derived from Testudinidae were investigated by multiplex-polymerase chain reaction (PCR). The isolates comprised 13 Candida (C.) tropicalis, nine C. albicans and one C. parapsilosis. In addition, five reference strains C. albicans (ATCC 10231), C. tropicalis (DSM 4238), C. parapsilosis (DSM 4237), C. glabrata (ATCC 70614) and C. krusei (ATCC 70075) were investigated. PCR revealed easily distinguishable species-specific amplicons of the chs1-gene and resulted in a clear identification in all cases. No discrepancies between conventional identification techniques and the PCR-based system were seen. The described PCR offers a rapid alternative to conventional techniques for the identification of C. albicans, C. glabrata, C. tropicalis, C. parapsilosis and C. krusei.

Differences in Cytokine mRNA Profiles between Naïve and in Vivo-Primed Ovine PBMC after Exposure to Heat-Inactivated Coxiella burnetii

Abstract: During human Coxiella burnetii (C. burnetii) infections, high IL‐10 levels favor replication of C. burnetii in monocytes and development of chronic Q fever, whereas IFN‐γ promotes intracellular killing. Sheep are a common source for human C. burnetii infections, but in contrast to man become transiently infected only. In a first approach to unravel the role of cytokines during ovine C. burnetii infections, we investigated by semiquantitative RT‐PCR whether heat‐inactivated C. burnetii affects the transcription of genes coding for IL‐2, IL‐4, IL‐10, and INF‐γin vitro in PBMC from sheep seropositive or seronegative for C. burnetii. By computer‐assisted evaluation of band intensities the transcription rate of the cytokine genes was quantified in relation to transcription in Concanavalin A‐stimulated and nonstimulated controls. Transcription rates in PBMC from seropositive animals after incubation with C. burnetii for 4 hours strongly resembled those found in PBMC from seronegative sheep. However, upon prolonged incubation (24 h) C. burnetii induced an increased IL‐10 transcription in PBMC from 2 of 5 seronegative, but in PBMC from 5 of 5 seropositive animals. The data suggest that natural C. burnetii infections prime the ovine immune system towards a TH2‐like pattern and this action thereby represents the first clue for the involvement of ovine immune cells in the response to C. burnetii infections.