New real-time PCR tests for species-specific detection of Chlamydophila psittaci and Chlamydophila abortus from tissue samples.

Chlamydophila psittaci and Chlamydophila abortus are the causative agents of avian chlamydiosis (psittacosis) and ovine enzootic abortion, respectively. Both pathogens are known to possess zoonotic potential. Due to their close genetic relatedness, direct and rapid species identification is difficult. In the present study, new real-time PCR assays are reported for both species. The tests are based on highly specific probes targeting the ompA gene region and were conducted as duplex PCRs including an internal amplification control. The Cp. psittaci assay successfully passed a proficiency test at national level. Examination of field samples revealed Cp. psittaci as the dominating species in birds, but also Cp. abortus in a few psittacines. Real-time PCR assays for species-specific detection of Cp. psittaci and Cp. abortus are suited for routine diagnosis, which renders them important tools for the recognition of outbreaks of psittacosis and ovine enzootic abortion.

Highly sensitive real-time PCR for specific detection and quantification of Coxiella burnetii.

BACKGROUND: Coxiella burnetii, the bacterium causing Q fever, is an obligate intracellular biosafety level 3 agent. Detection and quantification of these bacteria with conventional methods is time consuming and dangerous. During the last years, several PCR based diagnostic assays were developed to detect C. burnetii DNA in cell cultures and clinical samples. We developed and evaluated TaqMan-based real-time PCR assays that targeted the singular icd (isocitrate dehydrogenase) gene and the transposase of the IS1111a element present in multiple copies in the C. burnetii genome. RESULTS: To evaluate the precision of the icd and IS1111 real-time PCR assays, we performed different PCR runs with independent DNA dilutions of the C. burnetii Nine Mile RSA493 strain. The results showed very low variability, indicating efficient reproducibility of both assays. Using probit analysis, we determined that the minimal number of genome equivalents per reaction that could be detected with a 95% probability was 10 for the icd marker and 6.5 for the IS marker. Plasmid standards with cloned icd and IS1111 fragments were used to establish standard curves which were linear over a range from 10 to 10(7) starting plasmid copy numbers. We were able to quantify cell numbers of a diluted, heat-inactivated Coxiella isolate with a detection limit of 17 C. burnetii particles per reaction. Real-time PCR targeting both markers was performed with DNA of 75 different C. burnetii isolates originating from all over the world. Using this approach, the number of IS1111 elements in the genome of the Nine Mile strain was determined to be 23, close to 20, the number revealed by genome sequencing. In other isolates, the number of IS1111 elements varied widely (between seven and 110) and seemed to be very high in some isolates. CONCLUSION: We validated TaqMan-based real-time PCR assays targeting the icd and IS1111 markers of C. burnetii. The assays were shown to be specific, highly sensitive and efficiently reproducible. Cell numbers in dilutions of a C. burnetii isolate were reliably quantified. PCR quantification suggested a high variability of the number of IS1111 elements in different C. burnetii isolates, which may be useful for further phylogenetic studies.

Evaluation of a real-time PCR assay to detect Coxiella burnetii.

We evaluated real-time PCR assays for the detection of C. burnetii which targets sequences that are present either in one (icd) or in several copies (transposase of IS1111a) on the chromosome. The assays are highly sensitive, with reproducible detection limits of approximately 10 copies per reaction, at least 100 times more sensitive than capture ELISA, when performed on infected placenta material and specific for C. burnetii. The numbers of IS1111 elements in the genomes of 75 C. burnetii isolates were quantified by real-time PCR and proved to be highly variable.

Immunization experiments with recombinant Coxiella burnetii proteins in a murine infection model.

Previous attempts to develop Q fever vaccines were less successful in that the vaccines caused unacceptable side effects or failed to be protective. In this study, we tested the efficacy of a mixture of eight recombinant Coxiella burnetii (C. b.) proteins in sublethal challenge infections with mice. Eight potential C. b. virulence genes (Omp, Pmm, HspB, Fbp, Orf410, Crc, CbMip, and MucZ) were overexpressed in E. coli as his-tagged fusion proteins and partially purified. All recombinant proteins but rPmm proved to be antigenic in BALB/c mice when administered as protein mixtures. For efficacy testing, mice were immunized with an adjuvanted mixture of the eight recombinant proteins and subsequently challenged intraperitoneally with the C. b. isolate Nine Mile RSA493 (1.8 x 10(8) C. b.). Only animals vaccinated with the licensed Q fever vaccine Q-Vax (vaccination control) exhibited milder symptoms and minor gain of spleen and liver weights. In summary, clinical examinations and dissection of mice immunized with the eight recombinant C. b. proteins did not indicate a protective immune response after test infection.

Differences in cytokine mRNA profiles between naïve and in vivo-primed ovine PBMC after exposure to heat-inactivated Coxiella burnetii.

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-gamma 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-gamma in 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 T(H)2-like pattern and this action thereby represents the first clue for the involvement of ovine immune cells in the response to C. burnetii infections.

The genome of Coxiella burnetii Z3055, a clone linked to the Netherlands Q fever outbreaks, provides evidence for the role of drift in the emergence of epidemic clones.

Coxiella burnetii is a pathogen causing Q fever. The aim of our work was to study Z3055, a strain that is genotypically related to the strain causing the Netherlands outbreak. We compared Z3055 to 5 other completed genomes available in GenBank. We calculated the blast score ratio (BSR) to analyze genetic differences among the strains. The ratio core genome/pangenome was 98% likely other bacteria with closed pangenomes. Differences between Z3055 and the reference NMI consisted only of point mutations and insertion/deletion (INDELs). Non-synonymous mutations significantly increased in genes coding for membrane proteins (16/156 vs 103/1757, bilateral Chi(2) test, p<0.05), ankyrin repeat domains containing proteins (2/9 vs 117/1904, bilateral Chi(2) test, p<0.05), transcription factors (7/53 vs 112/1860, bilateral Chi(2) test, p<0.05) and translation proteins (15/144 vs 109/1655, bilateral Chi(2) test, p<0.05). The evolution of this strain may have been driven by mutations in critical genes.

Use of competition ELISA for monitoring of West Nile virus infections in horses in Germany.

West Nile virus (WNV) is a mosquito-borne viral pathogen of global importance and is considered to be the most widespread flavivirus in the World. Horses, as dead-end hosts, can be infected by bridge mosquito vectors and undergo either subclinical infections or develop severe neurological diseases. The aim of this study was to detect WNV specific antibodies in horses in Germany as an indicator for an endemic circulation of WNV. Sera from more than 5,000 horses (primarily fallen stock animals) were collected in eight different federal states of Germany from 2010 to 2012. Sera were screened by a competitive ELISA and positive reactions were verified by an indirect IgM ELISA and/or by virus neutralization tests (VNT) for WNV and Tick-borne encephalitis virus (TBEV) in order to exclude cross-reacting antibody reactions. In essence WNV specific antibodies could not be detected in any of the horse sera. Not surprisingly, a small number of sera contained antibodies against TBEV. It is noteworthy that equine sera were often collected from horse carcasses and therefore were of poor quality. Nonetheless, these sera were still suitable for WNV ELISA testing, i.e., they did not produce a high background reaction which is a frequently observed phenomenon. According to these data there is no evidence for indigenous WNV infections in horses in Germany at present.

[Epidemiological enquiries in two Q fever outbreaks in a community of Baden-Württemberg during 2008 and 2009]. / Epidemiologische Untersuchungen zu zwei Q-Fieber-Ausbrüchen in einer Gemeinde Baden-Württembergs in den Jahren 2008 und 2009.

In 2008 and 2009, two consecutive outbreaks of Q fever in humans were recorded in the district of Freudenstadt, northern Black Forrest, Baden-Württemberg, Germany. In 2008, a total of 41 persons from a single local community fell ill and were found infected with Coxiella burnetii. Although comprehensive diagnostic and epidemiological outbreak investigations were conducted and control measures taken which included vaccination of ruminants at risk in three parts of the affected community, re-occurrence of the disease in 2009 with further 29 confirmed human Q fever cases could not be prevented. While the origin of infection of the first outbreak was probably a flock of 550 sheep moved in the surrounding of the affected villages, the source of infection for the consecutive outbreak in 2009 could not be identified. It seems possible that meadows contaminated with infectious placenta or birth fluids represented the sources of infection.

Detection of all Chlamydophila and Chlamydia spp. of veterinary interest using species-specific real-time PCR assays.

The aim of the present study was to analyse the occurrence of chlamydiae in several mammalian host species. Clinical samples that previously tested positive in a Chlamydiaceae-specific real-time PCR were retested using six species-specific real-time PCR assays to identify the chlamydial species involved. Chlamydophila (Cp.) abortus was the agent most frequently found in cattle, sheep, horses, goats, and pigs. Detection in cattle of Cp. psittaci (11% of samples) and Chlamydia (C.) suis (9%), as well as Cp. psittaci in a goat sample was somewhat unexpected. DNA of two different chlamydiae was identified in 56 (12.7%) of 440 samples tested. Cp. felis was the predominant species found in cats, while in guinea pigs and rabbits only Cp. caviae was detected. Interestingly, the latter two pathogens were also identified in samples from dogs. The data show that mixed chlamydial infections are not rare and suggest an extended host range of individual species.

Coxiella burnetii genotyping.

Coxiella burnetii is a strict intracellular bacterium with potential as a bioterrorism agent. To characterize different isolates of C. burnetii at the molecular level, we performed multispacer sequence typing (MST). MST is based on intergenic region sequencing. These regions are potentially variable since they are subject to lower selection pressure than the adjacent genes. We screened 68 spacers in 14 isolates and selected the 10 that exhibited the most variation. These spacers were then tested in 159 additional isolates obtained from different geographic areas or different hosts or were implicated in different manifestations of human disease caused by C. burnetii. The sequence analysis yielded 30 different allelic combinations. Phylogenic analysis showed 3 major clusters. MST allows easy comparison and exchange of results obtained in different laboratories and could be a useful tool for identifying bacterial strains.