Isolation of ornithobacterium rhinotracheale from turkeys in Quebec, Canada.

In the spring of 1997, three large white hybrid turkey layer flocks of 52 wk of age experienced a severe respiratory condition. During the outbreak, the turkeys showed respiratory signs, an increased mortality rate, and an important drop in egg production. Macroscopic and histopathologic examinations were carried out on several carcasses, as well as bacteriologic analyses on different tissues. Colonies of Ornithobacterium rhinotracheale (OR) were detected after 24 hr of incubation, and the isolate appeared to be serotype A. The identification of the species was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins. Since 1993, several cases of OR infection have been diagnosed in the United States and more recently in Canada. Monitoring of this emerging infection is recommended.

Lymphoproliferative responses of specific-pathogen-free chickens to Mycoplasma gallisepticum strain PG31.

Mycoplasma gallisepticum (MG) is one of the aetiologic agents of chronic respiratory disease in chickens and infectious sinusitis in turkeys. We investigated humoral and cellular immune mechanisms following experimental infection with four different strains of MG. Peripheral blood leukocytes (PBL) obtained from chickens were examined for proliferation using antigen preparations of whole cell MG as stimuli in vitro. A consistent lymphoproliferative response was observed against the homologous whole cell antigens in the group of chickens infected with strain PG31. Significant lymphoproliferation was detected as early as 1 week post-infection. We further characterized antigen-specific proliferation by measuring the production of interferon and nitric oxide by the PBL of infected chickens. Consistent with lymphoproliferation, we also detected the presence of interferon and nitric oxide in vitro in antigen-stimulated cultures. These results indicate a possible role of cell-mediated immune responses in the development of immunity following MG infection in chickens.

Immunogenicity of E. coli-expressed sigma3 protein of avian reovirus in chickens.

An E. coli-expressed fusion protein was used to study the immunogenicity of avian reovirus sigma3 protein in chickens. The protein induced the production of specific antibodies detectable by immunofluorescence, ELISA and immunoblot. The antibody titre was low as determined by ELISA and was negative by virus neutralization test. However, when tested in passive immunization studies, the chicken antiserum specific to this protein and control antiserum from chickens vaccinated with avian reovirus SI 133 strain showed some protection against virus-induced mortality in 1-day-old chicks. The results thus confirm the importance of humoral immunity against avian reovirus infection and indicate that sigma3 protein may play a role in the induction of protective antibodies.

Evidence for the multimeric nature and cell binding ability of avian reovirus sigma 3 protein.

It has been suggested that avian reovirus sigma 3 protein is analogous to sigma 1 trimer, the mammalian reovirus attachment protein. We have investigated the multimeric nature and cell binding ability of sigma 3 protein. The data presented here demonstrate that sigma 3 protein is a multimer in its undisrupted form as determined by SDS-PAGE in non-dissociating conditions. However, virion-associated sigma 3 protein and COS-7 cell-expressed protein behaved differently in SDS-PAGE, suggesting a need for virus-associated factor(s) to control the multimerization of the protein. The data also show that Escherichia coli expressed sigma 3 fusion protein (sigma 3-MBP) in its multimeric form is capable of attaching to Vero cells. The binding was found to be specific and receptor mediated by the fact that it was inhibited by a monoclonal antibody specific for sigma 3 protein and by competition with avian reovirus particles. As determined by a reverse experiment, sigma 3-MBP was also able to reduce the virus p.f.u. in monolayer cell cultures, indicating the important role of sigma 3 protein in the initiation of virus infection.

Characterization of monoclonal antibodies against avian reovirus strain S1133.

Monoclonal antibodies (MAbs) were produced against a vaccinal S1133 strain of avian reovirus. Characterization of six MAbs in Western blotting, radioimmunoprecipitation and gold immunoelectron microscopy revealed that the MAbs were specific to the outer capsid proteins, mu2/mu2c, sigma2 and sigma3. Two of three MAbs, directed against sigma2 protein, neutralized the virus infectivity in a broadly specific manner, whereas the third had no neutralizing activity. The only MAb which reacted with sigma3 protein showed a type-specific neutralizing activity. Two MAbs recognizing the mu2/mu2c proteins failed to neutralize the virus infectivity.

Cloning, sequencing and expression of the S1 gene of avian reovirus.

The S1 genome segment of avian reovirus strain S1133 was cloned and completely sequenced. The sequence comprised 1636 bp with three distinct open reading frames (ORFs), suggesting the gene was polycistronic in nature. The three ORFs from 5' to 3' were predicted to encode polypeptides of 9.8, 3.8 and 34.9 kDa, respectively. Of the three ORFs, only the third possessed the AUG initiation codon in an optimum context for translation. The third ORF-encoded protein, 326 amino acids in length, was expressed in Escherichia coli and used as antigen in immunoblots. The protein was concluded to be sigma 3 on the basis of its recognition by a chicken anti-reovirus antiserum and due to the fact that a mouse antiserum raised against it recognized specifically the viral sigma 3 polypeptide. Sequence comparison of the avian reovirus S1 gene with its mammalian counterpart did not show any significant similarity between the two. However, amino acid sequence analysis and the predicted existence of a heptapeptide repeat pattern, as well as the relatively high frequency of alpha-helix structures in the amino terminal portion of sigma 3 suggests that this protein is structurally, and probably functionally, related to mammalian reovirus sigma 1 protein.

The 64 kDa lipoprotein of Mycoplasma gallisepticum has two distinct epitopes responsible for haemagglutination and growth inhibition.

A major Mycoplasma gallisepticum polypeptide of 64 kDa (p64) was characterized using two distinct monoclonal antibodies (MAbs), MAb KI produced in our laboratory and MAb MyG 001 produced by Avakian & Ley (1993). The p64 antigen was shown to be a lipoprotein in a radioimmunoprecipitation assay using [(3)H] palmitic acid-labelled M. gallisepticum cultures. The two MAbs inhibited the growth of M. gallisepticum in liquid medium and reacted to two distinct epitopes on the same p64 antigen in competitive enzyme-linked immunosorbent and chemiluminescence Western immunoblot assays. MAb Kl inhibited haemagglutination of chicken and turkey erythrocytes whereas MAb MyG 001 did not. The results of our study indicate that p64 has two distinct epitopes involved in haemagglutination and growth inhibition of M. gallisepticum. MAb Kl also inhibited the attachment of the mycoplasma to TLT lymphoblastoid chicken B cell line, suggesting that p64 is a cytadhesin.

Metabolic radiolabelling of Mycoplasma gallisepticum on Vero cells and radioimmunoprecipitation assay.

A monoclonal antibody (MAb) A2 was produced against a major polypeptide of Mycoplasma gallisepticum with a molecular mass of 64 kDa. MAb A2 reacted in immunoblot at a titre of 10(4.33) and had a titre of 10(4.5) in an enzyme-linked immunosorbent assay. In a radioimmunoprecipitation assay (RIPA) using metabolic [35S]methionine radiolabelling of M. gallisepticum suspension in Vero cell culture, MAb A2 was able to precipitate the 64 kDa protein and another protein of 47 kDa. The present study involving [35S]methionine labelling of M. gallisepticum in Vero cells represents a novel approach for labelling and characterizing the conformation-dependent mycoplasmal antigens in a RIPA system.

Interaction of avian reovirus with chicken lymphoblastoid cell lines.

Four chicken lymphoblastoid cell lines were inoculated with avian reovirus strain S1133 and two local isolates, 965 and 615. Of the inoculated cell lines, TLT, a B-cell line, was productively infected with the three viruses as demonstrated by immunofluorescence assay (IFA) and radioimmunoprecipitation assay. A comparative growth curve analysis of the three avian reoviruses was done at 37 degrees and 41 degrees C. Isolate 965 replicated to a higher titre at both temperatures while the replication of S1133 and 615 was found to be inhibited at 41 degrees C. IFA revealed that among the transformed T lymphoblastoid cells used in this study, only MDCC-RP1 was permissive to virus infection with isolate 965, and at 41 degrees C, but not 37 degrees C.

Flow cytometric analysis of the neutralizing immune response against infectious bursal disease virus using reticuloendotheliosis virus-transformed lymphoblastoid cell lines.

Infectious bursal disease virus (IBDV) is a lymphotropic virus with cytocidal effect on B lymphocytes of the bursa of Fabricius. We investigated the susceptibility of clonal populations of reticuloendotheliosis virus-transformed chicken B lymphocytes of both spleen and bursal origin to IBDV infection. The infected cells were metabolically-labelled and the viral polypeptides were analyzed by immunoprecipitation using monoclonal antibodies (MAbs). Virus adsorption and the effects of neutralizing convalescent antisera and MAbs on virus attachment were studied using flow cytometry. The results of the study indicate firstly that the transformed B cells support virus replication and provide an efficient system for studying IBDV-lymphocyte interactions. Secondly, results obtained also showed that the most potent neutralizing antibodies may not be those involved in preventing the receptor-mediated viral attachment but rather those involved in the inhibition of downstream events such as virus penetration or uncoating.

Radioimmunoprecipitation of avian reovirus polypeptides using virus-specific IgM and IgG murine monoclonal and chicken polyclonal antibodies.

A simple and improved procedure for radioimmunoprecipitation (RIPA) for the identification of major immunogenic proteins of avian reovirus using murine monoclonal and chicken polyclonal antibodies is described. Bacterial proteins (Staphylococcus aureus-Protein A or Streptococcus species-Protein G) commonly used in RIPA procedures lack reactivities with low avidity monoclonals belonging to immunoglobulin (Ig)G or IgM subtypes, as well as avian Ig. Hence we used an indirect approach utilizing species-specific anti-IgM, IgG or chicken Ig antibodies followed by the precipitation of the immune complexes using Protein A. This improved the sensitivity of the RIPA enabling the antigenic analysis of the major antigenic proteins of avian reovirus. The results indicated that the virus is highly immunogenic in the natural host, chicken than in mice. Furthermore, there exists a direct correlation between a strong neutralizing antibody response and an increased precipitation of the sigma (sigma) proteins of the virus. The results also demonstrate a strong association between the conformational viral epitopes of the three classes of proteins, large (lambda), medium (mu) and small (sigma).

Analysis of inter-serotypic structural relationships of infectious bursal disease virus using detergent solubilization and radioimmunoprecipitation with monoclonal antibodies.

Monoclonal antibodies (MAbs) neutralizing only the infectious bursal disease virus strains (IBDV) belonging to serotype 1 also immunoprecipitated the heterologous major antigenic proteins of serotype 2 IBDV. Detergent-solubilization followed by radioimmunoprecipitation assays (RIPA) using the MAbs revealed structural similarities between the conformation-dependent antigenic determinants of IBDV of the two existing serotypes. The presence of non-ionic detergent Triton X-100 determined the binding of altered proteins by MAbs in RIPA.

Comparison of a vaccine strain and field isolates of avian reovirus by T1-oligonucleotide mapping.

Total RNA of eight avian reovirus isolates and the S1133 strain were compared by RNase T1-oligonucleotide mapping. The viruses were propagated in Vero cell cultures, and viral genomes were extracted from purified virions for comparison. Pairwise comparisons of the oligonucleotide maps showed genetic variation among reovirus isolates ranging from 78% to 99%. The T1 fingerprints of the RNA of isolates 1103, 724, 615, and 684 differed slightly from the standard S1133 strain, suggesting that the vaccine strain might have changed and became part of the circulating reoviruses. In contrast, when compared with the vaccine strain, isolates 902, 644, and 6207 showed greater differences in the fingerprint pattern. This genomic diversity may be due to the differences in immunological status of the affected avian population and/or due to simultaneous coinfection with different reovirus strains.

Biological roles of the major capsid proteins and relationships between the two existing serotypes of infectious bursal disease virus.

Neutralizing monoclonal antibodies (n-MAbs) were produced against infectious bursal disease virus (IBDV) of serotypes 1 and 2. The n-MAbs recognizing the major antigenic proteins VP2 and VP3, were characterized using different strains of IBDV representing the existing two serotypes and a variant subtype of serotype 1. The biological properties of these viral antigens as defined by the MAbs in vitro, were studied utilizing post-adsorption virus neutralization tests and fluorescence-activated cell sorter analysis. The MAbs directed against the immunodominant epitopes on VP2 were capable of enhanced virus neutralization but did not inhibit the virus attachment to susceptible cells. These MAbs were able to neutralize the virus by interfering with an event subsequent to virus adsorption, possibly inhibiting virus penetration or uncoating. On the contrary, a MAb that immunoprecipitated the other capsid protein VP3 was able to prevent virus attachment although it possessed lower neutralization titers. Cross-immunoprecipitations of various virus strains by these MAbs and antisera revealed interrelationships between the two serotypes of IBDV.

Serological and pathogenic characterization of avian reoviruses isolated in Quebec.

Reoviruses were isolated from intestinal contents of broiler chickens from nine flocks in Quebec with malabsorption syndrome. Serum neutralization test demonstrated the existence of antigenic differences between the isolates and the reference vaccine strain. The isolated reoviruses were inoculated orally and into the foot pad in one-day-old chicks, resulting in a transient, but significant depression in body weight gains. Chickens infected with isolate 615, showed in addition to growth problems, clinical signs and tissue lesions similar to those observed in field cases. When isolate 615 was inoculated into SPF chicks at one day of age, intestinal absorption of D-xylose in infected chicks at 7 days post-infection was significantly lower (P <0.05) than for corresponding controls. This study suggests the implication of some reovirus isolates, such as 615 which was serologically distinct from the vaccine strain S1133, as infectious agents associated with pathological conditions other than viral arthritis.

Comparison of neutralizing antigens of recent isolates of infections bursal disease virus.

Monoclonal antibodies (MAbs) to a local turkey isolate (QT-1) of infectious bursal disease virus (IBDV) were produced to identify the virus-specific neutralizing proteins. Radioimmunoprecipitation assays showed that all the MAbs were specific for major viral protein, VP2. Two of the MAbs neutralized the local turkey and chicken isolates along with a reference strain belonging to serotype 1 but not the reference strain of serotype 2. The reactivities of the neutralizing MAbs against two reference strains and some recent field strains of IBDV isolated in the province of Québec were studied by indirect enzyme-linked immunosorbent assay and virus neutralization tests. The variations in the reactivities of the MAbs observed suggest differences in the neutralizing epitopes of the different isolates. Competitive binding assay using the MAbs revealed the presence of a third epitope involved in the neutralization of IBDV belonging to serotype 1.

Isolation of infectious bursal disease virus from turkeys with arthritic and respiratory symptoms in commercial farms in Quebec.

Infectious bursal disease viruses (IBDVs) were isolated from turkeys showing symptoms of arthritis and respiratory disease in commercial poultry farms in the province of Quebec, Canada. Synovial fluids collected from hock joints of arthritic birds and peripheral blood leukocytes obtained from the birds with respiratory problems were used for virus isolation in embryonated chicken eggs, and Vero and BGM-70 cell cultures. The infected cells were evaluated for the presence of IBDV by indirect immunofluorescence assay using monoclonal antibodies. The viruses were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of viral genome and by electron microscopy. Although one of these turkey isolates tested was neutralized by serotype 1-specific commercial chicken antisera, preliminary results indicated that there are antigenic differences between the Quebec isolate, IBDV QT-1, and the existing strains of IBDV belonging to serotype 1.

Characteristics and analysis of electropherotypes of avian reovirus field isolates.

Genomic segments of 10 selected isolates of avian reoviruses recovered from the intestine of birds affected with malabsorption syndrome or runting/stunting syndrome were separated by polyacrylamide gel electrophoresis. Different electropherotypes were observed and analysed, depending on the period of recovery and particular geographic locations. The analysis showed great variability in the dsRNA profiles of the isolates and higher mobility of the segments L1, S1, S2, S3 and S4. There was no correlation between electropherotype and geographic origin of the isolate. The analysis also showed the emergence of electropherotypically distinct strains since the introduction of modified live reovirus vaccines.

Comparison of egg-yolk and serum antibody titers to four avian viruses by enzyme-linked immunosorbent assay using paired field samples.

Eggs and blood were collected from 11 hens in each of nine broiler-breeder flocks in Quebec. Serum and egg-yolk extracts were assayed for antibody titers to infectious bursal disease virus (IBDV), infectious bronchitis virus (IBV), Newcastle disease virus (NDV), and reovirus (RV) by a commercial enzyme-linked immunosorbent assay (ELISA) kit. Comparison was made between egg-yolk and serum antibody titers by a regression analysis. A high correlation was observed between serum and yolk antibody titers to all the viruses tested (r = 0.9 for IBDV, 0.84 for IBV, 0.84 for NDV, and 0.91 for RV). Antibody monitoring of commercial breeder flocks using egg yolk instead of serum with commercial ELISA plates is thus feasible and is recommended.