Immuno-histochemical, Pathological, Enzyme Linked Immunosorbent ...

Immuno-histochemical, Pathological, Enzyme Linked Immunosorbent Assay and Polymerase Chain Reaction Analysis of Experimental Ornithobacterium rhinotracheale Infection in Quails (Coturnix coturnix japonica) H. EROKSUZ1, G. OZBEY2*, A. CEVIK1, B. GENCER TARAKCI3 and D. T. BALIK4 1 2 3 4

Department of Pathology, Faculty of Veterinary Science, University of Firat, 23119 Elazig / TURKEY Department of Microbiology, Faculty of Veterinary Science, University of Firat, 23119 Elazig / TURKEY Department of Histology, Faculty of Veterinary Science, University of Firat, 23119 Elazig / TURKEY Department of Biology, Faculty of Art and Science, University of Firat, 23119 Elazig / TURKEY

* Corresponding author : Tel : +90 424 2370000/6451 Fax : +90 424 2388173 - E-mail : [email protected]

SUMMARY

RÉSUMÉ

The aims of this study were to analyse the direct pathogene role of Ornithobacterium rhinotracheale (ORT) in the respiratory tract of birds and to identify a set of techniques useful for infection diagnosis. For that, a total of 30 male 10 week old quails were infected via aerosol route by ORT strain B3263/91 (serotype A) and 30 other quails were used as negative controls. On the 1st, 3rd and the 6th weeks post infection, 10 birds of each group were slaughtered, then lung and trachea were examined by bacteriological (cultures and PCR) and immuno-histochemical methods. In addition, the occurrence of serum anti-ORT antibodies was investigated by an enzyme-linked immunosorbent assay (ELISA). The experimental ORT inoculation have induced macroscopic lesions characterised by mild pulmonary and tracheal hyperemia and pleural thickening on weeks 3 and 6. Microscopically, patchy necrosis and desquamation of tracheal epithelium associated with subepithelial hemorrhage and subserosal oedema were observed and the interalveolar septa and pleural thickening was due to inflammatory infiltration. ORT was evidenced by immuno-chemistry in temporary microscopic lung lesions at the 3rd and 6th weeks, whereas ORT was isolated and identified from trachea of four quails (10%) and from lung of only one quail (2.5%). A seroconversion was observed in all infected birds mainly during the 1st week (80%) and also during the 3rd week (20%), but the serological response was transient since antibodies were no more detected in the sera of infected quails on the 6th week. In non infected quails, no macroscopic and microscopic damage was observed, no germ could be found by culture or PCR and birds remained seronegative during all the experiment. In conclusion, among the diagnostic methods used, ELISA test was the more sensitive and for the fisrt time in Turkey, the respiratory syndrome associated with ORT was experimentally induced in quails.

Mise en évidence de l’infection par Ornithobacterium rhinotracheale induite expérimentalement chez la caille par immuno-histochimie, histologie, sérologie et PCR. Par H. EROKSUZ, G. OZBEY, A. CEVIK, B. GENCER TARAKCI et D. T. BALIK.

Keywords : Ornithobacterium rhinotracheale - culture ELISA - Immuno-histochemistry - PCR.

Les buts de cette étude sont d’une part d’analyser le rôle pathogène direct de Ornithobacterium rhinotracheale (ORT) dans le tractus respiratoire des oiseaux et d’autre part, de proposer des techniques utiles au diagnostic. Les bactéries ORT (souche B3263/91, serotype A) sous forme d’aérosols ont été inoculées à trente cailles mâles âgées de 10 semaines tandis que 30 autres oiseaux ont servi de contrôles négatifs. Une, 3 et 6 semaines après inoculation, 10 oiseaux de chaque groupe ont été sacrifiés : l’examen des trachées et des poumons a été réalisé par des techniques bactériologiques (cultures et PCR) et immuno-histochimiques. De plus, la présence des anticorps sériques anti -ORT a été recherchée par un test immuno-enzymatique de type ELISA. L’inoculation de ORT a induit des lésions macroscopiques caractérisées par une hyperhémie modérée pulmonaire et trachéale et un épaississement pleural visibles lors de la 3ème et la 6ème semaines. Microscopiquement, quelques foyers de nécrose et une desquamation de l’épithélium trachéal associée à des hémorragies sub-épithéliales et des oedèmes sub-séreux ont été observés. Les épaississements des septa interalvéolaires et de la plèvre ont été dus à des infiltrats inflammatoires. Les bactéries ORT ont été mises en évidence par immuno-histochimie dans les lésions pulmonaires lors de la 3ème et la 6ème semaines, tandis qu’elles n’ont été isolées et identifiées qu’à partir des trachées de 4 cailles (10%) et des poumons d’un seul animal (2.5%). Tous les oiseaux infectés ont présenté une séroconversion, principalement durant la 1ère semaine (80%) mais aussi durant la 3ème semaine (20%), mais cette réponse sérologique n’a été que transitoire puisque les anticorps n’ont plus été détectés au bout de la 6ème semaine. Aucune lésion macroscopique et microscopique n’a été décelée, aucun germe n’a été mis en évidence par culture ou PCR chez les animaux indemnes, et ils sont restés séronégatifs pendant la durée de l’étude. En conclusion, parmi les méthodes de diagnostic utilisées, le test ELISA a été le plus sensible et pour la 1ère fois en Turquie, ce syndrome respiratoire associé à la bactérie ORT a été induit expérimentalement chez la caille.

Mots-clés : Ornithobacterium rhinotracheale - culture ELISA - Immuno-histochimie - PCR.

Introduction The recently identified germ, Ornithobacterium rhinotracheale (ORT) [27] has been isolated from patridge, pheasant, pigeon, rook, quail, duck, chukar patridge, ostrich, goose, guinea fowl, chicken and turkey [1, 4, 5, 8, 22, 27]. ORT is a Revue Méd. Vét., 2006, 157, 4, 197-202

Gram negative pleomorphic rod shaped bacterium, which has been isolated from domestic birds with respiratory problems [5, 12, 22, 27]. Clinically, relatively mild respiratory symptoms were accompanied by an increased mortality and growth retardation. At post-mortem examination, the most striking feature was a foamy, yellow-white, “yogourt-like”

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exudate in the air sacs, predominantly in the abdominal air sacs and sometimes accompanied with a purulent pneumonia, often unilateral, and/or exudate in the trachea [25]. As many researchers have been unable to experimentally reproduce lesions or death in turkeys with ORT, the microorganism has been suggested to be a secondary pathogen [19]. For reproducing this disease syndrome, agents such as Newcastle disease virus, infectious bursal disease virus, infectious bronchitis virus, chicken anemia virus, turkey rhinotracheitis virus, Bordetella avium, and Escherichia coli were inoculated in combination with ORT with varying degrees of success [7, 9, 10, 21]. The clinical signs and lesions caused by ORT are very similar to those caused by other respiratory infectious agents; therefore, an accurate diagnostic test is necessary to identify the infection [16]. For rapid diagnosis and surveillance studies of this newly recognised disease, a serologic test would be out of a great benefit [2]. ELISA could detect the presence of antibodies against ORT in 1-day-old birds and in egg-yolks, as well as in birds with clinical signs, confirming that the ELISA can be useful for diagnostic purposes. Microbiological tests give inconsistent results, because not all ORT strains can grow in the liquid media, which are normally used for identification purposes [24]. Consequently, a reliable identification method that can be used in laboratory investigations may be useful. For this purpose, PCR assays appear to be out of interest [13, 26]. The aims of this study were 1) to reproduce the disease syndrome associated with ORT in quails (Coturnix coturnix japonica) in Turkey for the first time and 2) to identify a set of technics (bacteriological, immuno-histochemical, serological and PCR assays), which can be used for routine ORT infection diagnosis.

Material and methods PREPARATION OF BACTERIAL INOCULUM The bacterial inoculum was prepared from ORT strain B3263/91 (serotype A). Bacteria were grown on blood agar plates with 7% sheep blood and they were incubated in a 510% CO2 atmosphere at 37°C for at least 48 h. Bacterial suspension adjusted to 3.8x108 viable colony forming units (cfu/ml) were prepared and used to infect quails.

BIRDS A total of sixty 10-week-old male quails obtained from Firat University (Quails Breeding Farm of Bingol Vocational Training School, Elazig-Turkey) were used in this study. The animals did not receive any vaccination or treatment. Two groups of each 30 quails were housed in two seperate rooms. The first group was infected by aerosol route with 1 ml of a pure culture of ORT strain B3263/91 (serotype A) containing 3.8x108 CFU per ml. The second group was non-infected and served as a control group. Ten birds of each group were slaughtered at the 1st, 3rd and 6th weeks after inoculation and samples from lung and trachea were collected and examined

EROKSUZ (H.) AND COLLABORATORS

by bacteriological and immuno-histochemical methods. In addition, blood samples were taken from birds, serum was removed from the clotted blood samples by centrifugation (10 min, 2.000 g, 4°C) and serologically analysed by ELISA.

BACTERIOLOGICAL ANALYSIS For cultural isolation, swabs from the lung and trachea samples were aseptically inoculated on Blood Agar supplemented with 7% sheep blood and 10 µg/ml gentamicin (to inhibit growth of other bacteria). The plates were incubated in a 5-10% CO2 atmosphere at 37°C for at least 48 h. Suspected colonies were subcultured and pure cultures were firstly stored in 10 ml of nutrient broth with 15% glycerol and freezing at -20°C. Identification or confirmation of bacterial species was assessed by observation of the colonial morphology and Gram staining results by biochemical methods [6].

ELISA TEST ORT strain B3263/91(serotype A) was used as antigen for coating the ELISA plates. The antisera against serotype A (obtained from Paul van Empel, Intervet-International, Boxmeer, The Netherlands) were used as positive controls and sera from specific pathogen free (SPF) quails were used as negative controls. The extraction of the antigen with sodium dodecyl sulphate (SDS-antigen), the coating of ELISA plates as well as the procedure for ELISA test were carried out according to the method described previously by HAFEZ and STING [14]. The ELISA cut off value for positive reaction was calculated as the average optical density (OD) of five negative serum samples plus three times the standard deviation (SD).

DNA EXTRACTION AND PCR A few colonies from cultures were transferred into an eppendorf tube containing 300 (l distilled water and the tubes vortexed. Lysis was achieved by the addition of TNES buffer (300 µl) (20 mM Tris pH 8.0, 150 mM NaCl, 10 mM EDTA, 0.2% SDS) and 200 µg/ml Proteinase K, following by incubation at 37°C for 2 hours and boiling for 30 min. Bacterial DNA was extracted by phenol/chloroform-isoamylalchool. DNA was precipitated with ethanol and 0.3 M sodium acetate at -20°C for one hour or overnight. The DNA pellet was washed with 300 µl of 90% and 70% ethanol, successively, each step followed by centrifugation (5 min, 7 000g, 4°C). The pellet was dried, and dissolved in 50 µl of distilled water, then used as template DNA for PCR. PCR was performed in a Touchdown Thermocycler (Eppendorf, Mastercycler personal, Germany) in a total reaction volume of 50 µl containing 5 µl of 10xPCR buffer (10 mM Tris-HCl, pH 9.0, 50 mM KCl, 0.1% Triton® X100), 5 µl of 25 mM MgCl2, 250 µM of each deoxynucleotide triphosphate, 2U of Taq DNA Polymerase (Promega) and 40 pmol of each of primers [OR16S-F1 (5’-GAG AAT TAA TTT ACG GAT TAA G-3’) and OR16S-R1 (5’-TTC GCT TGG TCT CCG AAG AT-3’)] derived from 16S rRNA gene (24)] and 5 µl of template sample DNA. Amplification Revue Méd. Vét., 2006, 157, 4, 197-202

DIAGNOSIS OF ORNITHOBACTERIUM RHINOTRACHEALE INFECTION IN QUAILS

was obtained with an initial denaturation step at 94°C for 5 minutes, followed by 45 cycles at 94°C for 30 s, and 52°C for 1 min and 72°C for 1 min 30 s. The final cycle was at 72°C for 7 min [26]. Ten microlitres of PCR products were analysed on a 1.5% agarose gel in 1XTAE buffer. The DNA fragments were visualized by UV illumination and photographed with Polaroid film. A 100 bp DNA ladder (Hyperladder I, Bioline) was used as a molecular size standard.

HISTOLOGICAL AND IMMUNOHISTOCHEMICAL ANALYSES Sections of lung, trachea, air sac, heart, kidneys and pectoral muscle were collected at necropsy and fixed in 10% neutral formalin. The tissues were embedded in paraffin, sectioned at 5 µm thickness, and stained with haematoxylin and eosin [17]. Immunohistochemical staining was carried out by using a peroxidase anti peroxidase (PAP) method. The primary antiserum against ORT used in the PAP method, was a monovalent rabbit antiserum against strain B3263/91 (serotype A) (obtained from Paul van Empel, Intervet-International, Boxmeer, The Netherlands). Blocking of endogenous peroxidase was carried out with 0.008% hydrogen peroxid (H2O2) in methanol for 5 min. In order to the block unspecific binding, an incubation step with (1:10) normal goat serum in 0.1 M phosphate buffered saline (PBS), pH 7.2 was performed. Sections were incubated for 16-20 h at 4°C in rabbit anti-ORT. Antibodies were diluted to 1:500 in PBS containing 0.25% sodium azide and 2.5% bovine serum albumin respectively. Sections were then incubated in goat anti-rabbit IgG (Dako, ZO421, Denmark), followed by rabbit peroxidase anti-peroxidase complex (Zymed Lab., 61.2003, San Francisco), both at dilution of 1:50 in PBS, for 1 hour at room temperature. Sections were washed in PBS for 30 min and counterstaining with eosin, sections were dehydrated and coverslips mounted with DPX [20]. Sections were examined with light microscope and photographs were taken.

Results CULTURE RESULTS ORT was re-isolated from trachea samples of four birds (10%) during the 1st week after ORT exposure and from only one lung sample (2.5%) at the 3rd week, whereas no ORT culture was obtained from trachea or lungs removed at the

6th week. The lung and trachea samples from control quails remained always negative for ORT (Table I).

PCR RESULTS In this study, PCR amplification of genomic bacterial DNA using OR16S-F1 and OR16S-R1 primer pairs gave bands of 784 bp, corresponding to the expected size of amplified genomic ORT DNA (Figure 1). All ORT isolates that were positive for ORT in culture were also positive by PCR. No amplified product was obtained from the negative controls.

HISTOPATHOLOGY Quails exposed to ORT in this study did not show any clinical sign. Macroscopic lesions were characterised by mild pulmonary and tracheal hyperemia and pleural thickening and were observed at the 3rd and the 6th weeks after inoculation. Microscopically, patchy necrosis and desquamation in tracheal epithelium associated with subepithelial hemorrhage, subserosal oedema and alveolar hyperemia were evidenced on the 3rd and the 6th weeks post-inoculation. Thickening of the interalveolar septa and of pleural was also noticed and was due to mononuclear inflammatory cell infiltration (figure 2).

IMMUNOHISTOCHEMICAL RESULTS Some positive staining for ORT cells were evidenced in the lung parenchyma with no specific localisation, on weeks 3 and 6 after inoculation (figure 3), while in trachea no positive staining for ORT cells was detected.

SEROLOGICAL RESULTS At the 1st week post ORT exposure, antibodies against ORT were detected in the sera of 8 quails (80%). A delayed seroconversion was obtained for 2 infected birds on the 3rd week after inoculation. But, serum anti-ORT antibodies became undetectable in all infected quails on the 6th week after ORT exposure. On the other hand, control quails did not produce anti-ORT antibody whatever the time of blood collection (Table II).

Discussion Several ORT strains, isolated from turkey, chicken and patridge, have been used for aerosol infectious challenges in both turkeys and broilers of various ages [21]. In these stu-

TABLE I. — Culture results of lungs and trachea removed from ORT infected quails on the 1st, 3rd and the 6th weeks after inoculation. Group 1 : ORT infected quails ; group 2 : non infected quails. Revue Méd. Vét., 2006, 157, 4, 197-202

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TABLE II. — Serological response of ORT infected quails on the 1st, 3rd and the 6th weeks after inoculation. Group 1 : ORT infected quails; group 2 : non infected quails.

dies, no airsacculitis or pneumonia was evidenced in the absence of virus (Newcastle disease or turkey rhinotracheitis (TRT) virus) [10, 21]. BACK et al. [3] showed that intravenous, intra-tracheal and intranasal ORT administrations in turkeys failed to induce any clinical sign of disease or any marked pathological changes in tissues [3]. Probably, the low virulence of the strain used may explain the failure of experimental ORT infection. By contrast, in some other studies, airsacculitis, pneumonia and increased mortality were observed after aerosol, intra-tracheal, intravenous and/or intra-thoracic infections without a viral priming [18, 19]. Nevertheless, in these studies, commercial birds, whose their microbiological and/or immunological status was not controlled, were used. It was probable that their low resistance to infections or the presence of other non-identified germs have increased their sensitivity to ORT infection. Up to now, no pneumonia or airsacculitis has been induced by aerosol, intra-tracheal or intra-thoracic ORT administrations in specific-pathogen-free (SPF) birds [25]. Only an intravenous ORT inoculation was able to induce up to 20% mortality and clinical reactions, such as meningitis and osteitis in SPF chickens, but no airsacculitis [11]. Consequently, these different and contradictory results did not allow knowing if ORT should be regarded as a primary pathogen [24]. In our study, quails exposed to ORT did not show severe pneumonic lesions and mortality. In addition, no macroscopic and microscopic lesion in the air sacs was observed. This might be due to aerosol ORT challenge without viral priming and to the modalities of bacterial inoculum preparation (number of passages, growth medium, and dose of the bacterial inoculum used). Growing the organism on solid medium and using low passages may have prevented loss of virulence factors that can occur with organisms that have been passed many times or have been grown in broth medium [19]. Immunohistochemical investigations have confirmed that aerosol ORT exposure of chickens without viral priming did not induce significant pathological changes in the respiratory tract [21, 25]. ORT bacterium is most commonly isolated from trachea and lungs of naturally or experimentally infected birds, but little information about ORT localisation in tissues other than the respiratory tract is available {3]. VAN EMPEL et al [25] reported that the bacteria could be isolated from trachea and air sacs of chickens exposed to ORT without viral priming only during the first two days post exposure, whereas these germs were present in air sacs and lungs of chickens exposed to ORT with viral priming during the first 10 days post infection. BACK et al. [3] demonstrated that ORT was essentially recovered from tissues on days 3 and 7 after inoculation than on day 14 and this may be due to the ability of the bird’s immune response to clear the infection from most

of the tissues on day 14 [3]. In agreement with these previous studies, ORT was mainly isolated from trachea of infected quails during the 1st week in our study, whereas on week 3, germs could be isolated from the lung of only one infected quail. The OR16S-F1 and OR16S-R1 primer combination was very specific in amplifying a 784 bp fragment of the 16S rRNA gene of ORT but not of other closely related bacteria with which ORT could be confused [15, 23]. In this study, we detected ORT DNA from all the isolates by PCR. Consequently, the microbiological and PCR methods only allowed a precocious and specific diagnostic of ORT infection with a moderate sensitivity because the germ was probably quickly removed from the respiratory tract. Anti ORT antibodies were detectable on the 5th day and their titres peaked the 7th day post aerosol ORT exposure and no difference was found between chickens exposed to ORT after Newcastle Disease priming and chickens exposed to ORT without priming. Moreover, LOPES et al. [16] showed that ELISA detected up to 100% of infected turkeys during the 8 weeks post inoculation. In our study, a seroconversion has occurred in infected quails mainly during the 1st week after ORT exposure and, furthermore all infected quails were seropositive for ORT during the first 3 weeks after ORT inoculation. Consequently, the ELISA test offers high sensitivity and specificity with no false positive or false negative results. This study reported the experimental reproduction of the disease syndrome associated with ORT with no viral priming in quails in Turkey for the first time and emphasised the necessity to associate several diagnostic tests for direct and indirect detection of ORT. However, further studies are needed to clarify the potential pathogen role of ORT.

FIGURE 1. — An agarose gel stained with ethidium bromide, with PCR products of ORT isolates (M : 100 bp DNA ladder, P : positive control, N : negative control, lanes 1,3,4,6,7 : ORT isolates). Revue Méd. Vét., 2006, 157, 4, 197-202

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FIGURE 2. — Irregularly scattered ORT positive staining cells in the lung paranchym of quail at the 3rd week post inoculation (x100).

FIGURE 3. — Pleural thickening and intraalveolar hyperemia at the 3rd week post ORT inoculation in quail. H&E (x100).

Acknowledgment

6. — CHIN R.P., VAN EMPEL P.C.M., HAFEZ H. M. : Ornithobacterium rhinotracheale infection. In : Saif, Y. M. et. al (Ed), Diseases of Poultry, 11th Edition, Iowa State University Press, Ames, Iowa, USA, 2003, 683-690. 7. — DE ROSA M., DROUAL R., CHIN R.P., SHIVAPRASAD H.L. : Interaction of.Ornithobacterium rhinotracheale and Bordetella avium in turkey poults. In Proceedings of the 46th Western Poultry Disease Conference, Sacramento, 1997, 52-53. 8. — DEVRIESE L.A, HOMMEZ J., VANDAMME P., KERSTERS K., HAESEBROUCK F. : In vitro antibiotic sensitivity of Ornithobacterium rhinotracheale strains from poultry and wild birds. Vet. Rec., 1995, 137, 435-6. 9. — DROUAL R., CHIN R.P. : Interaction of.Ornithobacterium rhinotracheale and Escherichia coli O78 H9 when inoculated into the air sac in turkey poults. In Proceedings of the 46th Western Poultry Disease Conference, Sacramento, 1997, 11. 10. — FRANZ G., HEIN R., BRICKER J., WALLS P., ODOR E., SALEM M., SAMPLE B. : Experimental studies in broilers with a Delmarva Ornithobacterium rhinotracheale isolate. In Proceedings of the 46th Western Poultry Disease Conference, Sacramento, 1997, 46-48. 11. — GOOVAERTS D., VRIJENHOEK M., VAN EMPEL P. : Immunohistochemical and bacteriological investigation of the pathogenesis of Ornithobacterium rhinotracheale infection in South Africa in chickens with osteitis and encephalitis syndrome. In Proceedings of the 16th Meeting of the European Society of Veterinary Pathology, Lillehammer, 1998, 81. 12. — HAFEZ H.M. : Current status on the role of (ORT) in respiratory disease complexes in poultry. Archiv. Geflügelkunde, 1996, 61, 208211. 13. — HAFEZ H.M., BEYER W. : Preliminary investigation on Ornithobacterium rhinotracheale (ORT) isolates using PCR-fingerprints. In Proceedings of the XIth International Congress of the World Veterinary Poultry Association, Budapest, 1997, 51. 14. — HAFEZ H.M., STING R. : Investigations on different Ornithobacterium rhinotracheale “ORT” isolates. Avian Dis., 1999, 43, 1-7. 15. — HUNG A.L., ALVARADO A. : Phenotypic and molecular characterization of isolates of Ornithobacterium rhinotracheale from Peru. Avian Dis., 2001, 45, 999-1005.

We thank Dr. Paul van Empel, Intervet International, Boxmeer, The Netherlands for supplying the ORT strain B3263/91, specific antisera and a monovalent rabbit antiserum against strain B 3263/91 (serotype A), Dr. Hafez Mohamed Hafez, Institute of Poultry Diseases, Free University Berlin, Germany for supplying the conjugate and Dr. Armando, Hung, Laboratory of Avian Pathology, Faculty of Veterinary, Universidad Peruana Cayetano Heredia, Peru for technical assistance.

References 1. — ANONYMOUS : Ornithobacterium rhinotracheale in ostrich. Khangela Quarterly Report on Animal Disease Surveillance, South Africa, January-March, 1995, 11. 2. — BACK A., HALVORSON D., RAJASHEKARA G., NAGARAJA K.V. : Development of a serum plate agglutination test to detect antibodies to Ornithobacterium rhinotracheale. J. Vet. Diagn. Invest., 1998a, 10, 84-86. 3. — BACK A., RAJASHEKARA G., JEREMIAH R.B., HALVARSON D.A., NAGARAJA K.V. : Tissue distribution of Ornithobacterium rhinotracheale in experimentally infected turkeys. Vet. Rec., 1998b, 143, 52-53. 4. — BUYS S. : Ornithobacterium rhinotracheale an emerging disease in South Africa. Aerosols, Newslett. World Vet. Poultry Assoc. 1996, 810. 5. — CHARLTON B.R., CHANNING-SANTIAGO S.E., BICKFORD A.A., CARDONA C.J., CHIN R.P., COOPER G.L., DROUAL R., JEFFREY J.S., METEYER C.U., SHIVAPRASAD H.L., WALKER R.L. : Preliminary characterisation of a pleomorphic gram-negative rod associated with avian respiratory disease. J. Vet. Diagn. Invest., 1993, 5, 47-51. Revue Méd. Vét., 2006, 157, 4, 197-202

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16. — LOPES V., RAJASHEKARA G., BACK A., SHAW D.P., HALVORSON D.A., NAGARAJA K.V. : Outer membrane proteins for serologic detection of Ornithobacterium rhinotracheale infection in turkeys. Avian Dis., 2000, 44, 957-962. 17. — LUNA L.G. : Manuel of Histologic Staining Methods of Armed Forces Institute of Pathology. McGraw-Hill Book Co, New York, 1968. 18. — RYLL M., HINZ K-H., BEHR K-P., KRUSE W., LÖHREN U., SÜDBECK M. : Zu pathogenität und verbreitung der Ornithobacterium rhinotracheale-infection bei der pute. In Proceedings of the 52nd Meeting of the Fachgruppe ‘Geflügelkrankheiten’ der Deutsche Veterinärmedizinische Gesellschaft, Hannover, 1997, 39-60. 19. — SPRENGER S.J., BACK A., SHAW D.P., NAGARAJA K.V., ROEPKE D.C., HALVORSON D.A. : Ornithobacterium rhinotracheale infection in turkeys : experimental reproduction of the disease. Avian Dis., 1998, 42, 154-161. 20. — STENBERGER L.A. : Immunohistochemystry. 3rd. ed. John Wiley, New York, 1986. 21. — VAN EMPEL P., VAN DEN BOSCH H., GOOVAERTS D., STORM P. : Experimental infection in turkeys and chickens with

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Ornithobacterium rhinotracheale. Avian Dis., 1996, 40, 858-864. 22. — VAN EMPEL P., VAN DEN BOSCH H., LOEFFEN P., STORM P. : Identification and serotyping of Ornithobacterium rhinotracheale. J. Clin. Microbiol., 1997, 35, 418-421. 23. — VAN EMPEL P. : Ornithobacterium rhinotracheale. PhD Thesis, University of Utrecht, Utrecht, Netherlands, 1998. 24. — VAN EMPEL P., HAFEZ H.M. : Ornithobacterium rhinotracheale : a review. Avian Pathol., 1999a, 28, 217-227. 25. — VAN EMPEL P., VRIJENHOEK M., GOOVAERTS D., VAN DEN BOSCH H. : Immuno-histochemical and serological investigation of experimental Ornithobacterium rhinotracheale infection in chickens. Avian Pathol., 1999b, 28, 187-193. 26. — VAN EMPEL P., SAVELKOUL P., SEGERS R., STOOF J., LOEFFEN P., VAN DEN BOSCH H. : Molecular characterization of Ornithobacterium rhinotracheale. Avian Dis., 1999c, 42, 567-578. 27. — VANDAMME P., SEGERS P., VANCANEYT M., VAN HOVER K., MUTTERS R., HOMMEZ J., DEWIRST F., PASTER B., KERSTERS K., FALSEN E., DEVRIESE L., BISGAARD M., HINZ K.H., MANNHEIM W. : Description of Ornithobacterium rhinotracheale gen. nov., sp. nov., isolated from the avian respiratory tract. Int. J. Syst. Bacteriol., 1994, 44, 24-37.

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