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Ornithobacterium rhinotracheale (O. rhinotracheale, ORT) is a gram-negative staining rod. In chickens and turkeys O. rhinotracheale causes a respiratory disease. Isolation of the bacterium from infected flocks is necessary for serotyping, to produce autogenous vaccines, and to determine the antimicrobial susceptibility for an effective therapy.
Therefore, in the first part of the thesis, a series of experiments was carried out to determine optimal conditions for storage of swabs soaked in O. rhinotracheale suspension to simulate the transport of swabs for isolation of O. rhinotracheale to the laboratory. Swabs were immersed in O. rhinotracheale suspensions with different bacterial counts and then stored under different conditions. At several time points the viable O. rhinotracheale count in the swabs was determined. Dry cotton swabs as well as three transport media, namely Amies gel medium, Amies gel medium with charcoal (AC), and Stuart gel medium were compared. O. rhinotracheale was reisolated from dry swabs stored at room temperature for up to five days and from swabs stored in the media at room temperature for more than seven days. Differences among the transport media were minor. The minimal number of cfu in the O. rhinotracheale suspension in which the swabs were soaked was 105 cfu/ml for successful reisolation of O. rhinotracheale one day post immersion from swabs stored at room temperature in Amies gel medium with charcoal, and 106 cfu/ml was successful for reisolation from dry swabs. Higher inoculation doses and storage at 4°C prolonged the period in which O. rhinotracheale could be reisolated. Storage of dry swabs at -20°C allowed reisolation of O. rhinotracheale at a constant level for at least five days. Inoculation of swabs with O. rhinotracheale and E. coli reduced the period during which O. rhinotracheale was reisolated.
In the second part of the thesis information about diagnosis and serotyping of O. rhinotracheale isolates at the Institute of Poultry Diseases of the Free University Berlin was compiled and analyzed. Between 2009 and 2011 714 dry swabs taken from diseased turkeys, broilers, broiler breeders, layers, or from unknown origin were investigated by PCR for the presence of O. rhinotracheale. One hundred ninety seven out of 481 swabs from turkeys (41.0 %), 10 out of 144 swabs from broilers or broiler breeders (6.9 %), 17 out of 28 swabs from layers (60.7 %), and 26 out of 61 swabs from unknown origin (42.6%) were tested positive. The results of three swabs from turkeys were suspect.
Furthermore 310 isolates from turkeys and 62 isolates from unknown origin were typed using agar gel precipitation test with antisera prepared for this study. 56.1 % of isolates from turkeys belonged to serotype A and 20.6 % to serotype E. The prevalence of other serotypes was below 10 %. Serotypes D, F, and K were not detected. Eleven isolates were not typable with reference sera against serotypes A – L. The three serotypes most often found in the isolates from unknown origin were A (35.5 %), B (19.4 %), and C (12.9 %). The prevalence of other isolates was below 10 %. Serotypes F and K were not detected. Seven isolates were not typable with reference sera A – L. Cross reactions, especially of serotype A isolates with serotypes I, H and J, were common.
Further the partial 16S ribosomal RNA (rRNA) and the complete Or01 genes of reference strains A – H and of nine field isolates were cloned and sequenced. Identity scores of 16S rRNA fragments were between 98 % and 100 %. Identities of the Or01 sequences were between 94 % and 100 %. Phylogenetic trees of both genes showed similarities. However, there was no apparent correlation between reference strains and isolates belonging to one serotype, so sequencing of 16S rRNA or of the Or01 gene does not seem to be a suitable method to replace the AGP for serotyping.