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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.

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.

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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.

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.