Diagnosis and incidence of Ornithobacterium rhinotracheale infections in commercial broiler chickens at slaughter

Ornithobacterium rhinotracheale infections cause economic losses in the poultry industry due to increased mortality rates, increased medication costs, growth suppression and high condemnation rates at processing (van Beek and others 1994, van Empel and others 1996a, Odor and others 1997, Travers and others 1996, van Veen and others 2000a). Postmortem examination of condemned birds from the initial slaughter round reveals an airsacculitis in diseased flocks. However, this kind of airsacculitis differs from that traditionally associated with secondary infection with Escherichia coli (Odor and others 1997, van Veen, personal communication), and further examination is required. O rhinotracheale is difficult to culture, since it grows slowly under specialised conditions, and it is often overlooked or is overgrown by other bacteria (Vandamme and others 1994, Travers and others 1996). In addition, experimental O rhinotracheale infections show only low re-isolation rates (van Empel and others 1999). Antibody levels after infection are low (van Empel and others 1996b) and antibodies are only detectable five days or more after infection, which implies that infections in the last week of the fattening period are not detected by serology. This suggests that both the impact of O rhinotracheale infections on respiratory disease and the number of infected broiler flocks might be underestimated. This short communication describes three studies on O rhinotracheale infection. The aim of the first study (study 1) was to determine if bacteriology and serology are sensitive enough for the diagnosis of O rhinotracheale infections in commercial broilers, because in the case of a suspected O rhinotracheale infection, it is customary to postpone the slaughter date. Therefore, it is necessary to detect diseased flocks just before slaughter. Conventional methods were compared with the peroxidase-anti-peroxidase (PAP) test, an immunohistochemical test on paraffin sections. In the second study (study 2), an immunofluorescence assay (IFA) on cryostat sections for routine investigation was added to the comparative tests. It had been developed because the PAP test on paraffin sections is laborious and less suitable for rapid routine diagnosis. The study would indicate whether the IFA had the same sensitivity as the PAP test. Comparison was performed on birds that were condemned because of respiratory lesions at slaughter. In this way it could also be determined whether O rhinotracheale influenced condemnation of carcases at slaughter. The third study (study 3) was a survey carried out in 10 different European countries to obtain an indication of the prevalence of O rhinotracheale in broiler chickens with respiratory lesions at slaughter age. Study 1 involved 10 farms with commercial broilers of different breeds. The birds were raised under field conditions, with each farm having its own management programme. Blood samples from the flocks were collected at the end of the fattening period and were submitted simultaneously with four live birds to the Animal Health Service, Deventer. Postmortem examination showed respiratory lesions in birds from eight of the 10 flocks. For bacteriological examination, swabs were taken from the air sacs and lungs and inoculated on to sheep blood agar containing thymidinephosphorylase. The agar plates were incubated for 48 hours at 37°C under 5 to 10 per cent carbon dioxide. Suspected colonies were subcultured under the same conditions and identified as described by van Empel and others (1996b), using the API identification system (bioMerieux) and an agar gel precipitation test. PAP staining was performed on paraffin sections of the affected air sacs and/or lungs. The primary antiserum used was mono-specific rabbit antiserum against O rhinotracheale serotypes A and C (96 per cent of the strains found in chickens are of serotype A). The sera were prepared and the PAP test was performed as described by van Empel and others (1999). Paraffin sections were examined microscopically and were considered to be positive when O rhinotracheale was clearly present in the affected tissues. A commercial indirect ELISA kit (BioCheck) was used for the detection of antibodies to O rhinotracheale. ELISA log2 titres of at least 10 were regarded as being positive, which is suggested to be useful for screening field sera (Sakai and others 2000). In study 2, birds condemned because of respiratory disease from 15 flocks were sent to the Animal Health Service from the processing plant for postmortem examination. Gross lesions were recorded and samples for investigation by bacteriology, PAP and IFA were taken from the air sacs, lungs and trachea. The IFA was performed on cryostat sections. The primary antiserum was a polyvalent rabbit antiserum against O rhinotracheale serotypes A, D, F and H. The sections were rinsed in phosphate-buffered saline and incubated with the rabbit antiserum for 45 minutes. After washing, the sections were incubated with fluorescein-labelled goat-anti-rabbit immunoglobulin G and washed again. Subsequently, the sections were stained with Evans blue and examined with a fluorescent microscope. The IFA was tested in vitro on pure cultures and tissues of birds that were experimentally infected with Pasteurella multocida, Mannheimia haemolytica, Riemerella anatipestifer, E coli or Bordetella avium. Minor cross-reactions were seen with R anatipestifer but these reactions were not strong enough to influence the diagnosis. No cross-reactions were seen with the other bacteria. For study 3, 10 European countries were asked to send in samples of air sacs taken from broiler flocks with respiratory Short Communications