Abstract: The ability of nine reference strains of Ornithobacterium rhinotracheale (serovars A-I) to raise detectable, hemagglutination-inhibition antibodies in immunized specific-pathogen-free chickens was investigated. Six of the nine O. rhinotracheale serovar (A-H) reference strains raised detectable hemagglutination-inhibition antibodies 14 and 21 days after vaccination. Detectable serum hemagglutination-inhibition antibody titers ranged from 512-16348. Furthermore, transmission electron microscopic examination of a hemagglutinating reference strain (B 3263/91, serovar A) demonstrated a close interaction between the bacterial and chicken erythrocyte surfaces. The results indicate that most of the hemagglutinating O. rhinotracheale reference strains included in this study are able to raised detectable hemagglutination-inhibition antibodies in immunized specific-pathogen-free chickens. The findings have the potential to become an alternative laboratory approach for detecting serum antibodies from chickens immunized with O. rhinotracheale.

INTRODUCTION

The bacterium Ornithobacterium rhinotracheale of the family Flavobacteriaceae (order Eubacteria, genus Ornithobacterium) has been isolated from both domestic and wild birds (Vandamme et al., 1994). It is associated with respiratory disease, decreased growth and mortality in chickens and turkeys. Lesions such as pneumonia and airsacculitis are observed in diseased birds. Economic loss can be considerable when breeders are involved (Chin et al., 2008).

Currently, at least 18 Agar Gel Precipitin (AGP) serovars in O. rhinotracheale have been recognized (Chin et al., 2008). The hemagglutinating activity of nine AGP reference strains (A-I) of O. rhinotracheale has been elsewhere reported (Vega et al., 2008). However, the bacterial structure involved in the hemagglutinating activity of this bacterium has yet to be identified. Furthermore, the ability of reference strains of O. rhinotracheale to raise serum hemagglutination-inhibition antibodies in chickens is unknown.

The aim of the present study was to investigate the ability of nine well-characterized, hemagglutinating reference strains of O. rhinotracheale to raise hemagglutination-inhibition antibodies in immunized chickens. Furthermore, the interaction of O. rhinotracheale with chicken erythrocytes was visualized by electron transmission microscopy.

MATERIALS AND METHODS

Bacterial strains: The reference strains of O. rhinotracheale used were B 3263/91, GGD 1261, ORV K91-201, ORV 94108 nr. 2, O-95029 no. 12229, ORV 94084 K858, O-95029 no. 16279, E-94063 4.2 and BAC 96-0334 #MINN 18 for serovars A-I, respectively.

Table 1:	Origin of Ornithobacterium rhinotracheale serovar reference strains included in the present study

The reference strains have been studied extensively (Van Empel et al., 1997; Soriano et al., 2003) and were all sourced from the culture collection held at the Poultry CRC, Australia. The origin and source of strains used are shown in Table 1.

Media: Bacteria were cultivated on 10% sheep blood agar at 37°C and incubated overnight in a candle jar. Brain-heart infusion broth was used for propagation and maintenance of bacterial cultures. For improved growth this medium was supplemented with 1% (v/v) filter-sterilized, heat-inactivated horse serum (Soriano et al., 2002).

Chickens: A total of 30, 7 weeks old, Specific Pathogen Free (SPF) Leghorn chickens were used in the study. All chicken were placed in isolator units at the Universidad Nacional Autonoma de Mexico (UNAM), individually identified and received antibiotic-free food and water ad libitum. To confirm no serum antibodies against O. rhinotracheale, all chickens were bled and serum samples tested by the Slide Plate Agglutination (SPA) test as elsewhere described (Back et al., 1998).

Immunization: Vaccines for each reference strain of O. rhinotracheale were produced. Briefly, bacteria were grown overnight in brain-heart infusion broth and supplemented with 1% (v/v) filter-sterilized, heat-inactivated horse serum. A viable count was performed and the culture inactivated with 0.01% (w/v) thimerosal. Once the viable counts results were available, the cells suspensions were adjusted to 5x10⁸ colony-forming units mL^-1 and aluminum hydroxide (SIGMA; St. Louis, MO, USA) added to a final concentration of 10%. Purity tests were carried out on final suspensions. Groups of three chickens were inoculated subcutaneously at 9 and 11 weeks of age with 1 mL of the relevant vaccine.

Hemagglutination-inhibition tests: Before immunization and 1 week after each immunization, all chickens were bled and sera of all chickens were examined in hemagglutination-inhibition test as previously described (Soriano et al., 2002). Hemagglutinin antigens the bacterial cells grown overnight in brain-heart infusion broth were harvested, washed 3 times in Phosphate-Buffered Saline (PBS), pH 7.0 and resuspended in PBS solution with 0.01% (w/v) thimerosal. Glutaraldehyde-fixed rabbit erythrocytes used were prepared as previously described (Soriano et al., 2002). Briefly, rabbit blood was collected into Alsever solution and the cellular packages were harvested by centrifugation and washed 3 times in 0.15 M NaCl. A 1% glutaraldehyde solution was prepared by dilution with a solution containing Na₃PO₄, pH 8.2 (1 volume) and 0.15 M NaCl (9 volumes) in distilled water (5 volumes). A 1-2% erythrocyte suspension was prepared in the 1% glutaraldehyde solution and held at 4°C for 30 min. Fixed erythrocytes were centrifuged, washed five times with 0.15 M NaCl and suspended in distilled water to 30% suspension. A 0.01% (w/v) thimerosal was added and the suspension was stored at 4°C. Hemagglutination titers of antigens were determined with 50 μL volumes of reagent and 1% glutaraldehyde-fixed erythrocytes with a diluent of PBS that contained 0.01% (w/v) thimerosal in a microdilution method. The hemagglutination titer was the reciprocal of the highest dilution of antigen causing complete agglutination of the glutaraldehyde-fixed erythrocytes.

The hemagglutination-inhibition tests were performed with four hemagglutinin units, the serum hemagglutination-inhibition antibody titers were expressed as the reciprocal of the highest dilution of serum sample that showed complete inhibition of the hemagglutinating activity (Soriano et al., 2002).

Transmission Electron Microscopy (TEM): Transmission electron microscopy study was performed as previously described (Zepeda et al., 2009). A mixed suspension of strain O-95029 no. 16279 and fresh chicken erythrocytes was harvested by centrifugation and fixed in a 3% glutaraldehyde solution. Thin sections (approximately 70-100 nm) for transmission electron microscopy analysis were visualized with a JEOL-1200EXII microscope with a field emission gun at an accelerating voltage of 65 kV at the Instituto de Fisiologia Celular, Universidad Nacional Autonoma de Mexico.

RESULTS AND DISCUSSION

Hemagglutination-inhibition antibody titers values of serum samples of experimental immunized chickens with different O. rhinotracheale vaccines are shown Table 2:Hemagglutination-inhibition antibody titers against Ornithobacterium rhinotracheale detected in immunized chickens included in the study in Table 2.

Table 2:	Hemagglutination-inhibition antibody titers against Ornithobacterium rhinotracheale detected in immunized chickens included in the study

The serum samples collected from all the experimental groups before the immunization with any type of vaccine showed no positivity in both SPA and hemagglutination-inhibition tests. Similarly, serum samples collected from the unimmunized control group showed no positive reactions in both SPA and hemagglutination-inhibition tests. All immunized chickens raised detectable, agglutinating antibodies 12 and 21 days after vaccination.

Transmission electron micrographs showed O. rhinotracheale strain B 3263/91 in close association with the surface of a fresh chicken erythrocyte (Fig. 1).

The present study appears to be the first published investigation on the hemagglutination-inhibition antibodies raised by O. rhinotracheale in immunized chickens. A previous study showed the ability of an isolate to raise hemagglutination-inhibition antibodies in immunized rabbits (Soriano et al., 2002).

The results confirmed that O. rhinotracheale reference strains possess hemagglutinating antigens as elsewhere reported (Vega et al., 2008). Furthermore, most of the O. rhinotracheale reference strains included in the study raised detectable, serum hemagglutination-inhibition antibodies. It is unknown why the remaining three O. rhinotracheale strains with hemagglutinating activity did not raised detectable, serum hemagglutination-inhibition antibodies. A similar result was observed when glutaraldehyde-fixed chicken erythrocytes were used in the test.

Fig. 1:	Transmission electron micrograph of a cross-section of Ornithobacterium rhinotracheale B 3263/91 reference strain (A) adhered to the surface of a fresh chicken erythrocyte (B). Bar = 50 nm

However, all reference strains raised detectable antibodies in immunized chickens when tested by the SPA test. Future studies focused on immunogenicity of hemagglutinating O. rhinotracheale strains need to be performed.

The TEM examination showed a close interaction between bacterial and fresh chicken erythrocytes surfaces, yet structural adhesions could not be visualized. Hemagglutination mainly depends of the expression of hemagglutinins (adhesins) and the interaction with erythrocyte receptors (Goldhar, 1995). Specific bacterial hemagglutinins or erythrocyte surface receptors involved in O. rhinotracheale hemagglutination have yet to be identified. Also, the effect of sera raised in this study, on the close interaction between the bacterium and erythrocytes, need to be studied.

In this study, a reduced number of chickens per O. rhinotracheale reference strain were immunized. However, we were able to detect serum hemagglutination-inhibition antibody titers raised by most of the O. rhinotracheale reference strains. The findings have the potential to become an alternative laboratory approach for detecting serum antibodies from chickens immunized with O. rhinotracheale.

CONCLUSION

In this study, the results confirmed the hemag glutinating ability of a reference strain of O. rhinotracheale and suggest that most of the O. rhinotracheale reference strains included in this study have the ability to raise hemagglutination-inhibition antibodies in immunized SPF chickens.

ACKNOWLEDGEMENTS

Dr. Pat Blackall (Poultry CRC, Australia) is greatly acknowledged for providing Ornithobacterium rhinotracheale reference strains. This research was supported by Consejo Nacional de Ciencia y Tecnología (CONACYT), project 52244-Z (UAEM 1774/2007C).