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Short Communications Diagnosis and incidence of Ornithobacterium rhinotracheale infections in commercial broiler chickens at slaughter L. van Veen, J. Nieuwenhuizen, D. Mekkes, M. Vrijenhoek, P. van Empel

Veterinary Record (2005) 156, 315-317 L. van Veen, DVM, J. Nieuwenhuizen, D. Mekkes, Animal Health Service, PO Box 9, 7400 AA Deventer, The Netherlands M. Vrijenhoek, P van Empel, PhD, Intervet International, PO Box 31, 5830 AA Boxmeer, The Netherlands Correspondence to Dr van Empel

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 The Veterinary Record, March 5, 2005

TABLE 1: Lesions found at postmortem examination, and bacteriology, ELISA and peroxidase-anti-peroxidase (PAP) test results for Ornithobacterium rhinotracheale in commercial broilers at six weeks of age (study 1) Flock 1 2 3 4 5 6

Lesion

Bacteriology

Airsacculitis, pneumonia, pericarditis Airsacculitis Airsacculitis, pneumonia Airsacculitis, pneumonia None Pneumonia

7 Airsacculitis 8 Airsacculitis 9 None 10 Pneumonia Total positive

ELISA

PAP

Escherichia coli, Staphylococcus aureus

–

+*

O rhinotracheale‡ –

+ +

+* +*

E coli

+

+*

E coli E coli, Mannheimia haemolytica E coli, O rhinotracheale* E coli – E coli, O rhinotracheale* 3

+ _

– _

– – – – 4

+* +† – +* 7

* Serotype A † Serotype C ‡ Serotype F – Negative, + Positive

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 (bioMérieux) 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

TABLE 3: European survey of the occurrence of respiratory Ornithobacterium rhinotracheale infections in broiler flocks at slaughter age using the peroxidase-anti-perixodase (PAP) test on infected organs (study 3)

TABLE 2: Lesions found at postmortem examination, and bacteriology, ELISA immunofluorescence assay (IFA) and peroxidase-anti-peroxidase (PAP) test results for Ornithobacterium rhinotracheale in condemned broilers at slaughter (study 2) Flock Lesion 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Airsacculitis, pneumonia Airsacculitis, pneumonia, pericarditis Airsacculitis, pneumonia Airsacculitis, pneumonia Airsacculitis Airsacculitis Airsacculitis, pericarditis Airsacculitis, pericarditis, pneumonia Airsacculitis, pneumonia Airsacculitis, pneumonia

Airsacculitis Airsacculitis Airsacculitis Airsacculitis, pneumonia, pericarditis 15 Airsacculitis Total positive

Bacteriology –

ELISA

–

IFA

Country

–

–

+

+*

– –

+ +

+* +*

– ND

+ +

+* +*

E coli

+

+

+*

E coli

–

–

–

ND

–

–

+

+

+*

+ + –

+ + –

+* +* –

M haemolytica

ND

+

+*

E coli 8

ND

+ 11

+* 11

Escherichia coli O rhinotracheale* O rhinotracheale* Mannheimia haemolytica O rhinotracheale* O rhinotracheale* Mannheimia haemolytica O rhinotracheale*

– E coli O rhinotracheale* O rhinotracheale* O rhinotracheale* –

4

Flocks

Number positive (%)

PAP

* Serotype A ND Not done, – Negative, + Positive

lesions at slaughter age in 2003, and these were tested using the PAP test at Intervet International, Boxmeer. In study 1, O rhinotracheale was isolated from three of the eight flocks with respiratory lesions (Table 1). Two strains were typed as serotype A and one strain, from a pure culture, was typed as serotype F. E coli was isolated from six of the eight flocks with respiratory lesions. O rhinotracheale was clearly detected within the affected tissues by the PAP test in seven of the eight flocks with lesions. Most of the positive staining was associated with accumulations of macrophages present within the infected organs. In the two flocks without respiratory lesions, no positive staining could be found. Serology was positive in four flocks, one of which had no lesions. Three of these flocks were positive by the PAP test as well, of which one flock was also positive by culture. The PAP test detected more positive flocks than the conventional methods. In study 2, both the IFA and the PAP test determined the presence of O rhinotracheale in the respiratory lesions of birds of the same 11 of the 15 flocks (Table 2). Isolation of O rhinotracheale was successful in eight of the 11 flocks, while E coli or M haemolytica were isolated from the remaining three flocks. In four flocks, a pure culture of O rhinotracheale was obtained; serology was also positive in four flocks. These were also positive by the IFA and PAP test and three of these flocks were positive bacteriologically. In more than 70 per cent of these cases O rhinotracheale was detected in infected tissues in abundance and mainly in macrophages. Four of the 15 flocks were negative for all tests. In the European survey (study 3), of 89 flocks for which samples were submitted, 23 (26 per cent) were found to be positive for O rhinotracheale (Table 3). The results from studies 1 and 2 on the comparative sensitivity of diagnostic tests show that with the use of the con-

Belgium Denmark Great Britain Germany Greece Ireland Hungary Poland France Total

11 1 10 5 10 10 20 15 7 89

3 (27) 0 2 (20) 1 (20) 3 (30) 4 (40) 1 (5) 4 (27) 5 (71) 23 (26)

ventional diagnostic methods for O rhinotracheale infections (serology and/or bacteriology), infected broiler flocks are regularly missed. In these tests the results of the IFA proved to be equal to those of the PAP test and therefore the IFA test, because it is easier and simpler, is the test of choice for O rhinotracheale infection in chickens in field situations. E coli and other easy growing potential pathogens will often be present at the infection site and therefore may be erroneously designated to be the (only) causal agent. However, because O rhinotracheale was mainly found within the macrophages in the infected tissue and because it has been proven that O rhinotracheale can be a primary pathogen (van Veen and others 2000b), these observations suggest that O rhinotracheale may be the causal agent of the diseases. In addition, when comparing the O rhinotracheale detection levels by serology and/or bacteriology with those of the IFA and/or PAP test, it can be concluded that the impact of O rhinotracheale infections has been underestimated. This underestimation can even be greater taking into account the fact that the vaccination of breeders with O rhinotracheale significantly reduces the ‘normal’ mortality of their offspring even under a low infection burden (De Herdt and others 2001). The results of the survey (study 3) show that in Europe more than one-third of the respiratory lesions in broiler chickens at slaughter age are associated with O rhinotracheale, indicating that the bacterium appears to be widespread throughout the European broiler industry. References DE HERDT, P., CAUWERTS, K., VERBLOESEM, J. & DUCATELLE, R. (2001) The relevance and efficacy of Ornithobacterium rhinotracheale control in chickens. World Poultry 17, 32-33 ODOR, E. M., SALEM, M., POPE, C. R., SAMPLE, B., PRIMM, M., VANCE, K. & MURPHY, M. (1997) Isolation of Ornithobacterium rhinotracheale from commercial broiler flocks on the Delmarva Peninsula. Avian Diseases 41, 257260 SAKAI, E., TOKUYAMA, Y., NONAKA, F., OHISHI, S., ISHIKAWA, Y., TANAKA, M. & TANENO, A. (2000) Ornithobacterium rhinotracheale infection in Japan: preliminary investigations. Veterinary Record 146, 502-503 TRAVERS, A., COETZEE, L. & GUMMOW, G. (1996) Pathogenicity differences between South African isolates of Ornithobacterium rhinotracheale. Onderstepoort Journal of Veterinary Research 63, 197-207 VAN BEEK, P., VAN EMPEL, P., VAN DEN BOSCH, G., STORM, P., BONGERS, J. & DUPREEZ, J. (1994) Ademhalingsproblemen, groeivertraging en gewrichtsontsteking bij kalkoenen en vleeskuikens door een Pasteurellaachtige bacterie: Ornithobacterium rhinotracheale of ‘Taxon 28’. Tijdschrift voor Diergeneeskunde 119, 99-101 VANDAMME, P., SEGERS, P., VANCANEYT, M., VAN HOVER, K., MUTTERS, R., HOMMEZ, J., DEWIRST, F., PASTER, B., KERSTERS, K., FALSEN, E., DEVRIEZE, L., BISGAARD, M., HINZ, K-H. & MANNHEIM, W. (1994) Description of Ornithobacterium rhinotracheale gen nov sp nov isolated from the avian respiratory tract. International Journal of Systematic Bacteriology 44, 24-37 VAN EMPEL, P., VAN DEN BOSCH, H., GOOVAERTS, D. & STORM, P. (1996a) Experimental infection in turkeys and chickens with Ornithobacterium rhinotracheale. Avian Diseases 40, 858-864

The Veterinary Record, March 5, 2005


Short Communications

VAN EMPEL, VAN DEN BOSCH, H., LOEFFEN, P. & STORM, P. (1996b) Identification and serotyping of Ornithobacterium rhinotracheale. Journal of Clinical Microbiology 35, 418-421 VAN EMPEL, P., VRIJENHOEK, M., GOOVAERTS, D. & VAN DEN BOSCH, H. (1999) Immunohistochemical and serological investigation of experimental Ornithobacterium rhinotracheale infection in chickens. Avian

The Veterinary Record, March 5, 2005

Pathology 28, 187-193 VAN VEEN, L., GRUYS, E., FRIK, K. & VAN EMPEL, P. (2000a) Increased condemnation of broilers associated with Ornithobacterium rhinotracheale. Veterinary Record 147, 422-423 VAN VEEN, L., VAN EMPEL, P. & FABRI, T. (2000b) Ornithobacterium rhinotracheale, a primer pathogen in broilers. Avian Diseases 44, 896-900


Diagnosis and incidence of Ornithobacterium rhinotracheale infections in commercial broiler chickens at slaughter L. van Veen, J. Nieuwenhuizen,, D. Mekkes,, et al. Veterinary Record 2005 156: 315-317

doi: 10.1136/vr.156.10.315

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