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Trivalent live attenuated influenza vaccines whose type A components are based on cold-adapted A/Leningrad/134/17/57 (H2N2) (caLen17) master donor virus (MDV) have been successfully used in Russia for decades to control influenza. The vaccine virus comprises hemagglutinin and neuraminidase genes from the circulating viruses and the remaining six genes from the MDV. The latter confer temperature-sensitive (ts) and attenuated (att) phenotypes. The ts phenotype of the vaccine virus is a critical biological determinant of attenuation of virulence. We developed a plasmid-based reverse genetics system for MDV caLen17 to study the genetic basis of its ts phenotype. Mutations in the polymerase proteins PB1 and PB2 played a crucial role in the ts phenotype of MDV caLen17. In addition, we show that caLen17-specific ts mutations could impart the ts phenotype to the divergent PR8 virus, suggesting the feasibility of transferring the ts phenotype to new viruses of interest for vaccine development.

Ornithobacterium rhinotracheale was found to cause growth retardation in both turkeys and chickens after experimental intra air sac administration and to cause growth retardation together with airsacculitis and pneumonia after aerosol administration. Both turkey and chicken isolates of O. rhinotracheale were able to induce the same kind of respiratory inflammations and weight-gain losses in chickens as well as turkeys. Turkey rhinotracheitis virus was found to have a triggering effect on the O. rhinotracheale infection in turkeys while Newcastle disease virus and to a lesser extent Infectious Bronchitis virus showed triggering effects on the O. rhinotracheale infection in chickens. O. rhinotracheale could be reisolated from affected organs of experimentally infected birds.

In the present study 443 strains of Ornithobacterium rhinotracheale, a causative agent of respiratory disease in fowl, were investigated biochemically and serologically. In both ways O. rhinotracheale could be differentiated from other gram-negative rods and, more particularly, from the Pasteurella-like bacteria potentially pathogenic for fowl. For the biochemical characterization of O. rhinotracheale the API 2ONE identification strip proved to be useful, although O. rhinotracheale is not included in the API system. Serologically, by using monovalent antisera in agar gel precipitation (AGP) tests and enzyme-linked immunosorbent assays (ELISAs), seven serotypes (serotypes A to G) of O. rhinotracheale could be discriminated. The AGP test was chosen as the preferred method to be used for serotyping. Isolates of serotype A were found to be the most prevalent, especially in chickens. Isolates from turkeys were more heterogeneously divided over the serotypes. Some strains showed cross-reactivity between serotypes A, B, and E. Five O. rhinotracheale strains could not be serotyped with the available antisera. Relationships between the geographic origin and the serotypes were found. By the ELISA the presence of antibodies against O. rhinotracheale could be detected in 1-day-old birds as well as in birds with clinical signs, and therefore, it might be useful for diagnostic purposes. Respiratory problems, together with purulent pneumonia, airsacculitis, severe growth retardation, and rapidly increasing mortality, were reported in meat turkeys and broilers in South Africa, Germany, the United States, France, and The Netherlands (1, 2, 4, 6). A gram-negative, pleomorphic rod could repeatedly be isolated from affected organs. This Pasteurellalike organism has recently been referred to as Ornithobacterium rhinotracheale gen. nov. sp. nov. (7). In experimental infections, it was possible to evoke severe growth retardation and airsacculitis in turkeys and chickens with this bacterium (8). O. rhinotracheale strains from different countries reporting fowl infected with the organism were investigated for their bacteriological, biochemical, and serological relationships and their differences from other gram-negative rods.

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