Infectious Bronchitis Virus
Infectious bronchitis virus (IBV) has been isolated from trachea, oviduct, kidneys, spleen, feces, cecal tonsils, and lymphoid tissues (Alexander and Cough, 1977 and 1978; Brown el al., 1987; Chong and Apostolov, 1982; Cook, 1971; Mora, 1966). Some IBV isolates appear to be easier to recover from digestive tract than from respiratory tract (Lucio and Fabricant, 1990), however, the persistence of IBV in various organs and tissues is still not fully understood. Many questions remain to be answered, such as "What is the tissue distribution pattern of different IBV strains in chickens?" "How persistent is IBV in infected tissues?" and "How do different IBV strains compete for replication sites in organs and tissues of the same chicken?"

The indirect fluorescent antibody (IFA) and reverse transcriptase-polymerase chain reaction (RT-PCR) are currently used to detect and serotype IBV in many laboratories. The sensitivity of these two assays is dependent on the amount of virus present in the tissues collected for assays. However, misleading diagnostic and serotyping information can result if the tissues of infected birds are not collected properly. When chicken embryos were experimentally inoculated with Mass and Ark strains, both strains were detected by the RT-PCR but only Ark was detected by the IFA (Wang and Austin, unpublished). This suggests that a diagnosis based on IFA only may be misleading when there is more than one IBV strain involved. It also suggests that either the Ark strain replicates faster than the Mass strain in chicken embryos, or the RT-PCR is more sensitive than the IFA in embryos simultaneously infected with both strains.

Coronaviruses may distribute, replicate, and express differently in various tissues after the virus enter the hosts. Karaca et. al. (1990) showed that three intestinal IBV isolates were genetically related to the Conn vaccine strain, based on RNase-Tl-resistant oligonucleotide fingerprinting, but were not neutralized by Conn specific antiserum. The intestinal isolates may have been derived from the Conn vaccine strain through point mutation. Alternatively, the antigenicity and pathogenicity of the Conn vaccine strain may have been expressed differently in digestive and respiratory tract tissues. Adami et. al. (1995) showed that a variety of mutant forms of murine hepatitis virus (MHV) were from persistent CNS infections. The mutant forms included point mutants, deletion mutants, and termination mutants. Makino and Lai (1989) demonstrated RNA nucleotide changes of MHV during replication process. They found that the repeat region, close to 3'-end of leader sequence, decreased during serial passage of virus in susceptible cells. In addition, some porcine respiratory coronavirus isolates have a large in- frame deletion at the 5' end of the S gene when compared with transmissible gastroenteritis virus isolates (Rasschaert et. al., 1990; Vaughn et. al., 1995). However, the nucleotide sequences of nucleoprotein and membrane protein genes of MHV were not related to their tissue tropism (Homberger, 1994 and 1995). The roles of the S gene and the leader sequence of IBV strains in determining tissue tropism, virulence and antigenicity are still to be determined.

It was found that turkey coronavirus (TCV), an agent causing devastating enteric disease in the turkey industry, is antigenically closely related to infectious bronchitis virus (Loa et. al, 1998). The possibility of TCV being a variant of IBV requires further investigation.

Infectious Bursal Disease (IBD)
Infectious bursal disease virus (IBDV) attacks the bursa of Fabricius of young chickens resulting in bursal lymphocytolysis and immunosuppression. The immunosuppressive effects of IBD is a major component of the respiratory disease complex since immunocompromised birds are extremely susceptible to respiratory pathogens and do not respond properly to respiratory disease vaccines. Infectious bursal disease virus is a member of the Bimaviridae virus family and contains two segments (A and B) of double stranded RNA. Genome segment B encodes VP1, considered to be the viral RNA-dependent RNA polymerase (Kibenge et. al., 1988). Genome segment A encodes a 110-kilodalton polyprotein that is cleaved into three viral proteins: VP2, VP3, and VP4 (Hudson et. al., 1986). The VP2 contains antigenic regions responsible for neutralizing antibodies and serotype specificity, whereas VP3 contains the group-specific antigens (Kibenge et. al., 1988). The VP4 is considered to be a viral protease and involved in the processing of segment A into the mature viral proteins.

There are two serotypes (1 and 2) of IBDV. Serotype 1 viruses are pathogenic to chickens, whereas serotype 2 viruses are non-pathogenic to chickens (Ismail et. al., 1988). Several variant strains of serotype 1 IBDV have emerged in the field (Rosenberger and Cloud, 1986). In addition to virus isolation, electron microscopy, immunodiffusion, immunofluorescence, virus neutralization, and ELISA (Lukert et. al., 1991), molecular techniques such as DNA hybridization using radioactive and non-radioactive cDNA probes (Davis et. al., 1990; Jackwood et. al., 1990; Akin et. al., 1993), PCR (Wu et. al., 1991; Lin et. al., 1994), PCR with sequencing (Wu and Lin, 1992), and PCR with restriction enzyme analysis (Jackwood and Jackwood, 1994) have been developed for detection and/or differentiation of IBDV serotypes and strains.

Studies have shown antigenic subtypes (classical and variant) exist within serotype 1 IBDV. Vaccination of chicks with one of these subtypes does not ensure birds protected from infection and disease from another serotype 1 subtype. Ismail and Saif(1991) demonstrated that vaccination with one serotype 1 subtype did not always protect chickens from challenge with another serotype 1 subtype particularly if a vaccine containing low virus titers, was used. The failure of some vaccination programs may be due to differences between antigenic subtypes of serotype 1 IBDV.

Other studies have revealed that there is a large number of nucleotide sequence variation and amino acid divergence in VP2 among different IBDV strains or isolates in natural populations, particularly in the central hyper-variable region (Bayliss et. al., 1990). Two prominent hydrophilic regions were found in the hypervariable region of VP2 and deletion of either of these hydrophilic regions completely abolished binding with neutralizing monoclonal antibody (Azad et. al., 1987). In addition, a serine-rich heptapeptide motif located adjacent to the second hydrophilic region was found to be conserved in highly pathogenic strains and have serine substitutions in less virulent strains (Heine et. al., 1991; Lana et. al., 1992).

Passage of IBDV in chick-embryo-fibroblast (CEF) cells changed both the properties and structural proteins of the virus. When virus was passaged at a high multiplicity of infection (MOI), a small plaque variant was observed and at a low MOI, large plaque variant was observed. The small and large plaque variant populations were stable after the ninth passage in CEF cells. The small plaque morphology remained even when the virus was further passaged at a low MOI. The emergence of small plaque variants was also reported. Recently, small and large plaque variants were purified and stable populations were observed through 10 passages in CEF cells. Differences were noted in structural proteins between a population of viruses grown in bursa tissue compared to small and large plaque viruses grown in CEF cells. It was speculated that cellular factors such as proteases were important in the maturation of IBDV particles and differences in the micro-environment among different host cells could account for the development of defective virus particles. The antigenicity of the small and large plaque viruses was reported to be similar. These studies were conducted using a VN assay. The pathogenicity and immunogenicity of two antigenic variant IBDV strains was studied following 30-40 passages in BGM-70 cells. Although both viruses lost pathogenicity, their immunogenicity was altered. Since no attempt was made to separate large and small plaque viruses both sizes were present in the population of virus used for these vaccination/challenge studies. It was reported that both plaque sizes retained immunogenicity in chickens but the large plaque virus had a higher potency compared to the small plaque virus. The large plaque viruses were less pathogenic for chicks compared to the wild type virus grown in bursa tissue and the small plaque virus was more attenuated than the large plaque virus.

When IBDV was passaged at a high MOI, a small plaque was observed. At a low MOI, a large plaque was noted (Muller et. al., 1986). The small and large plaque populations were stable after the ninth passage in CEF cells. When the virus was further passaged at low MOI, the small plaque remained stable. The immunogenicity and pathogenicity of the two IBDV variant strains serially passaged in BGM-70 cells for 30 to 40 passages were studied (Tsai and Saif, 1992). Although both viruses lost pathogenicity, their immunogenicity was unchanged.

Commercially available IBDV ELISA kits detect antibodies to both serotypes 1 and 2. Identification of antibodies to different antigenic subtypes of IBDV is currently possible using the VN assay. ELISA systems employ whole virus particles as antigen. One approach to the development of a more specific assay includes expression the IBDV structural protein VP2 for use as an ELISA antigen. It was reported that VP2 contains at least three independent epitopes. Using monoclonal antibodies, differences were shown in neutralizing epitopes on VP2 exist between variant and classic viruses. These results suggest that it may be possible to express IBDV epitope which will only bind antibodies to specific subtypes of IBDV.

Several expression systems have been utilized to produce IBDV proteins. The VP2 was expressed in Escherichia coli. A recombinant fowl pox virus expressing VP2 protected birds challenged with IBDV against mortality, but not against damage to the bursa. Vaccination of specific-pathogen-free (SPF) hens with a VP2 fusion protein produced in yeast provided passive (maternal antibodies) protection to IBDV challenged progeny. IBDV proteins have also been expressed using the baculovirus system. Proteins expressed in baculovirus have been produced in amounts of 1 to 500 mg/ml.

Other strategies in combating IBDV infection including DNA vaccines and antiviral approach are being developed and preliminary data are very encouraging. (Chang et. al 1998, Wu et. al 1998)

Infectious laryngotracheitis Virus
Infectious laryngotracheitis viruses (ILTVs) of variable pathogenicity have been used as vaccines. However, some of the vaccine viruses have been associated with outbreaks of infectious laryngotracheitis. In order to differentiate vaccine viruses from the field strains as well as to reduce the virulence of the vaccine virus, a "marker" recombinant ILTV has been created. This normally pathogenic strain of the virus was attenuated by insertional inactivation of its thymidine kinase gene with an expressible bacterial B-galactosidase gene (Schnitzlein et. al, 1995).

For rapid and sensitive detection of fowlpox and infectious laryngotracheitis, cloned genomic fragments have been used to diagnose these viruses. In addition to specific PCR for each virus infection, single step PCR for diagnosis of these two diseases was developed.

Ornithobacterium Rhinotracheale (ORT)
Ornithobacterium rhinotracheale has recently been reported as a pathogen causing respiratory tract infections in turkeys and other birds. Clinical signs associated with O. rhinotracheale include tracheitis, airsacculitis, pericarditis, sinusitis, and exudative pneumonia. In late 1995, widespread outbreaks of acute respiratory disease associated with O.rhinotracheale were reported in turkeys for the first time in the major turkey growing states in the US, and resulted in substantial economic loss to the turkey industry. The disease typically appeared in birds 11 to 26 weeks of age, with mortality rates ranging from 3 to 7%. Diagnosis of O. rhinotracheale infections based on clinical signs and postmortem examination is difficult because of the generalized nature of the signs and lesions. There were many unanswered questions regarding O. rhinotracheale infections in poultry flocks. It was not known whether the disease outbreaks resulted from the dissemination of a single clone or from multiple clones. The provenance of this organism and the overall genetic relationship of O. rhinotracheale isolates recovered from avian sources in the U.S.A. to those from other countries were also unknown. Further, there was a critical need to develop diagnostic tests for the agent, experimentally reproduce the disease, and develop vaccines and other immunoprophylactic agents that would protect birds against infection and disease associated with this agent.

Substantial progress has been made in the study of O. rhinotracheale infection in turkeys. Studies have been carried out with the goals of understanding the epidemiology of O. rhinotracheale infections and developing a comprehensive strategy for the prevention and control of this disease. Progress in this area includes: (a) O. rhinotracheale infection and disease has been experimentally reproduced in turkeys without the need for any primary viral infection. This experimental infection model suggests that the disease is systemic and the organism can be recovered from several organs and tissues, including the ovaries and oviducts, (b) A serological test has been developed for the detection of 0. rhinotracheale infected flocks. This polyvalent antigen test can detect infection by all of the known serotypes of O. rhinotracheale. (c) We have used MLEE and rep-PCR to determine that there is relatively restricted diversity amongst O. rhinotracheale isolates recovered from worldwide sources implying that this agent has recently been introduced into commercial poultry, probably from wild bird populations, (d) A killed bacterin has been prepared and shown to protect birds from 0. rhinotracheale clinical signs and infection on subsequent challenge.

Pasteurella multocida
Pasteurella multocida causes fowl cholera in domestic and wild birds. Any of sixteen different serotypes can be associated with the disease, but in poultry the disease is usually produced by serotype 1, 3, or 4. Protection conferred by bacterins is largely related to specific serotype. However, vaccination with live-attenuated strains can produce cross-protection. The failure of bacterins to confer cross-protection is due to minimal or complete lack of expression of unique antigens. Subunits derived from P. multocida grown in turkeys can induce cross-protection in poultry. The cross-protection antigen(s) are mainly associated with insoluble components of the bacteria. Passive and active immune protection studies in turkeys with vaccines of purified antigens and antigenic analyses with SDS-PAGE indicated that cross-protection was due to a 39 kDa protein antigen. Although a strong correlation was shown between active and passive immune protection using partially purified antigens, removal of precipitates which formed at pH 4.6 from solubilized membrane antigens resulted in vaccines that produced good active immunity but poor passive immunity. The latter finding suggested that cell-mediated immunity may be involved in cross-protection or that certain bacterial components may act as adjuvants. However, in experiments in which spleen cells were adoptively transferred from vaccinated to unvaccinated turkeys, a role for cell-mediated immune cross-protection was not demonstrated. A peptone-based laboratory medium was formulated to provide bacteria for isolation of cross-protection antigens. Field trials with aqueous bacterins of P. multocida grown in this medium indicated that the bacteria would induce cross-protection. However, cross-protective immunity was of short duration and lasted less than 8 weeks. A test to determine when vaccinated-turkey flocks are cross protected against P. multocida is needed. A monoclonal antibody was produced against the 39 kDa cross-protective antigen that may be useful in ELISA tests to determine vaccinated-flock cross-protective immunity.

Riemerella anatipestifer
Riemerella anatipestifer infection is primarily a disease of domestic ducks, but it may affect other avian species such as geese and turkeys. It is unknown how R. anatipestifer infection is introduced into a flock, but adverse environmental conditions often predispose birds to outbreaks. Differentiation of R. anatipestifer from similar bacteria is based upon its biochemical characteristics. Diversity amongst R. anatipestifer isolates has been demonstrated by serologic typing and 19 serotypes have been described using agglutination tests. A restriction endonuclease analysis (REA) method was developed for resolving relationships and differences among R. anatipestifer strains within and without the same serotype. Use of Hinf I in REA with 89 strains from various hosts and geographic origins showed that considerable genetic diversity occurs. REA was successful in detecting differences in R. anatipestifer that can not be observed by biochemical and serological tests.