Hemagglutinating activity of serovar reference strains of Ornithobacterium rhinotracheale Vicente Vega, Andrea Zepeda, Sau ´l Ramı ´rez, Vladimir Morales, Pomposo Ferna ´ ndez, Roberto Montes de Oca, Fernando M. Guerra-Infante, Marı ´a de Jesu ´ s de Haro-Cruz, Patrick J. Blackall, Edgardo V. Soriano 1 Abstract. In the present study, the hemagglutinating activity of 9 reference strains (serovars A–I) of Ornithobacterium rhinotracheale was investigated by using fresh erythrocytes from 15 different species: chicken (broiler, rooster, hen), turkey, pigeon, quail, duck, Harris hawk (Parabuteo unicinctus), house finch (Carpodacus mexicanus), cow, sheep, horse, dog, rabbit, pig, human (groups A, B, AB, and O), and rainbow trout (Oncorhynchus mykiss). All 9 strains agglutinated rabbit erythrocytes. None of the strains was able to agglutinate hen, cow, horse, or rainbow trout erythrocytes. The number of positive reactions among the remaining species varied. Results indicate that the use of rabbit erythrocytes is better suited for testing the hemagglutinating activity of O. rhinotracheale. Key words: Erythrocytes; hemagglutination test; Ornithobacterium rhinotracheale; poultry. <!?show "fnote_aff1"$^!"content-markup(./author-grp[1]/aff|./author-grp[1]/dept-list)> The bacterium Ornithobacterium rhinotracheale (family Flavobacteriaceae, order Eubacteria, genus Ornithobacter- ium) has been isolated from both domestic and wild birds. 8 It is associated with respiratory disease, decreased growth, and mortality in chickens and turkeys. Lesions such as pneumonia, pleuritis, and airsacculitis are observed in diseased birds. Economic loss can be considerable when breeders are involved. 1 Identification of infected birds is based on the clinical history and bacteriological isolation of pleomorphic, Gram-negative, oxidase-positive bacteria. Confirmation is determined by routine biochemical or enzymatic tests. However, the Analytical Profile Index (API) 20NE code 0220004 is the most frequently used. 3,4,7,9 At least 18 agar gel precipitin (AGP) serovars have been recognized. 1 To date, the hemagglutinating activity of O. rhinotra- cheale has been reported in few papers. 2,4,5,7 In the first study, where hemagglutinating activity of O. rhinotracheale was reported, 15 of 25 South African isolates (serovar A) agglutinated with glutaraldehyde-fixed chicken erythro- cytes. 2 In Mexico, 10 isolates (unknown serovar) from broiler and laying chickens and 2 (unknown serovar) from turkeys agglutinated glutaraldehyde-fixed chicken erythro- cytes. 4,5 In a recent study, the hemagglutinating activity of 40 O. rhinotracheale isolates (serovar A) obtained from 28 chickens and 12 pigeons in Taiwan was tested. Only 22 agglutinated fresh chicken and pigeon erythrocytes, and none of the pigeon isolates agglutinated fresh chicken and pigeon erythrocytes. 7 The hemagglutinating activity of serovar reference strains of O. rhinotracheale is unknown. Hence, the aim of the present study was to investigate the hemagglutinating activity of 9 serovar reference strains of O. rhinotracheale by testing erythrocytes from a number of species. 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 AGP serovars A to I, respectively. All bacteria were sourced from the culture collection held at the Animal Research Institute (Moorooka, Queensland, Australia). The origin and source of strains used are shown in Table 1. Bacteria were cultivated on 10% sheep blood agar at 37uC 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 supple- mented with 1% (vol/vol) filter-sterilized, heat-inactivated horse serum. 5 All strains included in the study were tested for their ability to agglutinate fresh erythrocytes from different animal species: chicken (broiler, rooster, hen), turkey, pigeon, quail, duck, Harris hawk (Parabuteo unicinctus), house finch (Carpodacus mexicanus), cow, sheep, horse, dog, rabbit, pig, human (groups A, B, AB, and O), and rainbow trout (Oncorhynchus mykiss). Fresh erythrocyte suspensions were prepared as previously reported. 6 Also, chicken glutaraldehyde-fixed erythrocytes were used. 5 Fresh bacterial suspensions of the 9 reference strains of O. rhinotracheale were prepared from overnight cultures on 10% sheep blood agar plates at 37uC that were harvested in 1.5 ml of phosphate buffered saline (PBS; pH 7.0), washed 3 times by centrifugation, and stored at 4uC. 5 The hemagglutinating activity of all strains was investigated as previously reported. 5 In brief, hemagglutinating activity was first determined with 50-ml volumes of reagent and 1% glutaraldehyde-fixed chicken erythrocytes with a diluent of PBS that contained 0.01% thimerosal in a microdilution method. The hemagglutination titer was the reciprocal of the highest dilution of antigen showing complete aggluti- nation of the erythrocytes after incubation for 30 min at room temperature. 5 The hemagglutination titer of all From the Centro de Investigacio ´ n y Estudios Avanzados en Salud Animal, Facultad de Medicina Veterinaria y Zootecnia, Universidad Auto ´ noma del Estado de Me ´xico, Me ´xico (Vega, Zepeda, Remı ´rez, Morales, Ferna ´ ndez, Montes de Oca, Soriano), the Departamento de Microbiologı ´a Veterinaria, Escuela Nacio- nal de Ciencias Biolo ´ gicas, Instituto Polite ´cnico Nacional, Me ´xico (Guerra-Infante, de Jesu ´ s de Haro-Cruz), and the Department of Primary Industries and Fisheries Queensland, Animal Research Institute, Australia (Blackall). 1 Corresponding Author: Edgardo V. Soriano, CIESA-FMVZ- UAEM, Instituto Literario No. 100, Col. Centro, Toluca 50000, Me ´xico. soriano@uaemex.mx J Vet Diagn Invest 20:353–355 (2008) 353