Mucosal immune responses in the trachea after chronic infection with Mycoplasma gallisepticum in unvaccinated and vaccinated mature chickens.

Tracheitis associated with the chronic respiratory disease in chickens caused by Mycoplasma gallisepticum is marked by infiltration of leukocytes into the mucosa. Although cytokines/chemokines are known to play a key role in the recruitment, differentiation, and proliferation of leukocytes, those that are produced and secreted into the trachea during the chronic stages of infection with M. gallisepticum have not been described previously. In this study, the levels of transcription in the trachea of genes encoding a panel of 13 cytokines/chemokines were quantified after experimental infection with the M. gallisepticum wild-type strain Ap3AS in unvaccinated chickens and chickens vaccinated 40-, 48- or 57-weeks previously with the novel attenuated strain ts-304. These transcriptional levels in unvaccinated/infected and vaccinated/infected chickens were compared with those of unvaccinated/uninfected and vaccinated/uninfected chickens. Pathological changes and subsets of leukocytes infiltrating the tracheal mucosa were concurrently assessed by histopathological examination and indirect immunofluorescent staining. After infection, unvaccinated birds had a significant increase in tracheal mucosal thickness and in transcription of genes for cytokines/chemokines, including those for IFN-γ, IL-17, RANTES (CCLi4), and CXCL-14, and significant downregulation of IL-2 gene transcription. B cells, CD3+ or CD4+ cells and macrophages (KUL01+ ) accumulated in the mucosa but CD8+ cells were not detected. In vaccinated birds, the levels of transcription of the genes for IL-6, IL-2, RANTES and CXCL-14 were significantly lower after infection than in the unvaccinated/infected and/or unvaccinated/uninfected birds, while the transcription of the IFN-γ gene was significantly upregulated, and there were aggregations of B cells in the tracheal mucosa. These observations indicated that M. gallisepticum may have suppressed Th2 responses by upregulating secretion of IFN-γ and IL-17 by CD4+ cells and induced immune dysregulation characterized by depletion of CD8+ cells and downregulation of IL-2 in the tracheas of unvaccinated birds. The ts-304 vaccine appeared to induce long-term protection against this immune dysregulation. TAKE AWAY: The ts-304 vaccine-induced long-term protection against immune dysregulation caused by M. gallisepticum Detection of B cells and plasma cells in the tracheal mucosa suggested that long-term protection is mediated by mucosal B cell memory Infection of unvaccinated birds with M. gallisepticum resulted in CD8+ cell depletion and downregulation of IL-2 in the tracheal mucosa, suggestive of immune dysregulation Infection of unvaccinated birds with M. gallisepticum resulted in upregulation of IFN-γ and infiltration of CD4+ cells and antigen presenting cells (B and KUL01+ cells) into the tracheal mucosa, suggesting enhanced antigen processing and presentation during chronic infection Th2 responses to infection with M. gallisepticum may be dampened by CD4+ cells through upregulation of IFN-γ and IL-17 during chronic infection.

Rapid typing of infectious laryngotracheitis virus directly from tracheal tissues based on next-generation sequencing.

Infectious laryngotracheitis virus (ILTV) is the causative agent of an economically important disease of chickens causing upper respiratory tract infection. Strains of ILTV are commonly identified by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and/or PCR high resolution melt (PCR-HRM) curve analysis targeting several genes. However, these techniques examine only a limited number of mutations present inside the target regions and may generate unreliable results when the sample contains more than one strain. Here, we attempted to sequence the whole genome of ILTV with known identity (class 9) directly from tracheal scrapings to circumvent in vitro culturing, which can potentially introduce variations into the genome. Despite the large number of quality reads, mapping was compromised by poor overlapping and gaps, and assembly of the complete genome sequence was not possible. In a map-to-reference alignment, the regions with low coverage were deleted, those with high coverage were concatenated and a genome sequence of 139,465 bp was obtained, which covered 91% of the ILTV genome. Sixteen single-nucleotide polymorphisms (SNPs) were found between the ILTV isolate examined and ILTV class 9 (JN804827). Despite only 91% genome coverage, using sequence analysis and comparison with previously sequenced ILTVs, we were able to classify the isolate as class 9. Therefore, this technique has the potential to replace the current PCR-HRM technique, as it provides detailed information about the ILTV isolates.

Construction and characterisation of glycoprotein E and glycoprotein I deficient mutants of Australian strains of infectious laryngotracheitis virus using traditional and CRISPR/Cas9-assisted homologous recombination techniques.

In alphaherpesviruses, glycoproteins E and I (gE and gI, respectively) form a heterodimer that facilitates cell-to-cell spread of virus. Using traditional homologous recombination techniques, as well as CRISPR/Cas9-assisted homologous recombination, we separately deleted gE and gI coding sequences from an Australian field strain (CSW-1) and a vaccine strain (A20) of infectious laryngotracheitis virus (ILTV) and replaced each coding sequence with sequence encoding green fluorescent protein (GFP). Virus mutants in which gE and gI gene sequences had been replaced with GFP were identified by fluorescence microscopy but were unable to be propagated separately from the wildtype virus in either primary chicken cells or the LMH continuous chicken cell line. These findings build on findings from a previous study of CSW-1 ILTV in which a double deletion mutant of gE and gI could not be propagated separately from wildtype virus and produced an in vivo phenotype of single-infected cells with no cell-to-cell spread observed. Taken together these studies suggest that both the gE and gI genes have a significant role in cell-to-cell spread in both CSW-1 and A20 strains of ILTV. The CRISPR/Cas9-assisted deletion of genes from the ILTV genome described in this study adds this virus to a growing list of viruses to which this approach has been used to study viral gene function.

Infectious bronchitis virus in Australia: a model of coronavirus evolution - a review.

Infectious bronchitis virus (IBV) was first isolated in Australia in 1962. Ongoing surveillance and characterization of Australian IBVs have shown that they have evolved separately from strains found throughout the rest of the world, resulting in the evolution of a range of unique strains and changes in the dominant wild-type strains, affecting tissue tropism, pathogenicity, antigenicity, and gene arrangement. Between 1961 and 1976 highly nephropathogenic genotype GI-5 and GI-6 strains, causing mortalities of 40% to 100%, predominated, while strains causing mainly respiratory disease, with lower mortality rates, have predominated since then. Since 1988, viruses belonging to two distinct and novel genotypes, GIII and GV, have been detected. The genome organization of the GIII strains has not been seen in any other gammacoronavirus. Mutations that emerged soon after the introduction of vaccination, incursion of strains with a novel lineage from unknown sources, recombination between IBVs from different genetic lineages, and gene translocations and deletions have contributed to an increasingly complex IBV population. These processes and the consequences of this variation for the biology of these viruses provide an insight into the evolution of endemic coronaviruses during their control by vaccination and may provide a better understanding of the potential for evolution of other coronaviruses, including SARS-CoV-2. Furthermore, the continuing capacity of attenuated IBV vaccines developed over 40 years ago to provide protection against viruses in the same genetic lineage provides some assurance that coronavirus vaccines developed to control other coronaviruses may continue to be effective for an extended period.

Preliminary comparative analysis of the genomes of selected field reisolates of the Mycoplasma synoviae vaccine strain MS-H reveals both stable and unstable mutations after passage in vivo.

BACKGROUND: Genomic comparison of Mycoplasma synoviae vaccine strain MS-H and the MS-H parental strain 86,079/7NS established a preliminary profile of genes related to attenuation of MS-H. In this study we aimed to identify the stability of mutations found in MS-H after passage in experimental or field chickens, and to evaluate if any reverse mutation may be associated with changes in characteristics of MS-H in vitro or in vivo. RESULTS: Whole genome sequence analysis of 5 selected MS-H field reisolates revealed that out of 32 mutations reported previously in MS-H, 28 remained stable, while four found to be reversible to the wild-type. Each isolate possessed mutations in one to three of the genes obg, oppF1 and gap and/or a non-coding region. Examination of the 4 reversible mutations by protein modeling predicted that only two of them (in obg and oppF1 genes) could potentially restore the function of the respective protein to that of the wild-type. CONCLUSIONS: These results suggest that the majority of the MS-H mutations are stable after passage in vaccinated chickens. Characterisation of stable mutations found in MS-H could be utilised to develop rapid diagnostic techniques for differentiation of vaccine from field strains or ts- MS-H reisolates.

Correction to: Preliminary comparative analysis of the genomes of selected field reisolates of the Mycoplasma synoviae vaccine strain MS-H reveals both stable and unstable mutations after passage in vivo.

An amendment to this paper has been published and can be accessed via the original article.

Analysis of antibody response to an epitope in the haemagglutinin subunit 2 of avian influenza virus H5N1 for differentiation of infected and vaccinated chickens.

The H5N1 subtype of highly pathogenic avian influenza virus has been circulating in poultry in Indonesia since 2003 and vaccination has been used as a strategy to eradicate the disease. However, monitoring of vaccinated poultry flocks for H5N1 infection by serological means has been difficult, as vaccine antibodies are not readily distinguishable from those induced by field viruses. Therefore, a test that differentiates infected and vaccinated animals (DIVA) would be essential. Currently, no simple and specific DIVA test is available for screening of a large number of vaccinated chickens. Several epitopes on E29 domain of the haemagglutinin H5N1 subunit 2 (HA2) have recently been examined for their antigenicity and potential as possible markers for DIVA in chicken. In this study, the potential of E29 as an antigen for DIVA was evaluated in detail. Three different forms of full-length E29 peptide, a truncated E29 peptide (E15), and a recombinant E29 were compared for their ability to detect anti-E29 antibodies. Preliminary ELISA experiments using mono-specific chicken and rabbit E29 sera, and a mouse monoclonal antibody revealed that the linear E29 peptide was the most antigenic. Further examination of the E29 antigenicity in ELISA, using several sera from experimentally infected or vaccinated chickens, revealed that the full-length E29 peptide had the greatest discrimination power between infected and vaccinated chicken sera while providing the least non-specific reaction. This study demonstrates the usefulness of the HPAI H5N1 HA2 E29 epitope as a DIVA antigen in HPAI H5N1-vaccinated and -infected chickens.RESEARCH HIGHLIGHTS E29 (HA2 positions 488-516) epitope is antigenic in chickens.Antibodies to E29 are elicited following live H5N1 virus infection in chickens.E29 epitope is a potential DIVA antigen for use in ELISA.

Complementation of the Mycoplasma synoviae MS-H vaccine strain with wild-type oppF1 influences its growth characteristics.

The Mycoplasma synoviae (MS) vaccine strain MS-H harbours a frameshift mutation in oppF1 (oligopeptide permease transporter) which results in expression of a truncated OppF1. The effect of this mutation on growth and attenuation of the MS-H is unknown. In this study, the impact of the mutation on the vaccine phenotype was investigated in vitro by introducing a wild-type copy of oppF1 gene in the MS-H genome. Wild-type oppF1 was cloned under the vlhA promoter into an oriC vector carrying a tetracycline resistance gene. MS-H was successfully transformed with the final construct pMS-oppF1-tetM or with a similar vector lacking oppF1 coding sequence (pMS-tetM). The MS-H transformed with pMS-oppF1-tetM exhibited smaller colony size than MS-H transformed with pMS-tetM. Monospecific rabbit sera against C-terminus of OppF1 detected bands of expected size for full-length OppF1 in the 86079/7NS parental strain of MS-H and the MS-H transformed with pMS-oppF1-tetM, but not in MS-H and MS-H transformed with pMS-tetM. Comparison of the growth curve of MS-H transformants harvested from media with/without tetracycline was conducted using vlhA Q-PCR which revealed that MS-H transformed with pMS-tetM had a higher growth rate than MS-H transformed with pMS-oppF1-tetM in the media with/without tetracycline. Lastly, the whole genome sequencing of MS-H transformed with pMS-oppF1-tetM (passage 27) showed that the chromosomal copy of the mutated oppF1 had been replaced with a wild-type version of the gene. This study reveals that the truncation of oppF1 impacts on growth characteristics of the MS-H and provides insight into the molecular pathogenesis of MS and perhaps broader mycoplasma species.RESEARCH HIGHLIGHTS The full-length OppF1 was expressed in Mycoplasma synoviae MS-H vaccine.Truncation of oppF1 impacts on growth characteristics of the MS-H.Chromosomal copy of the mutated oppF1 in MS-H was replaced with wild-type oppF1.

Infectious Laryngotracheitis Virus Viral Chemokine-Binding Protein Glycoprotein G Alters Transcription of Key Inflammatory Mediators In Vitro and In Vivo.

Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that infects chickens, causing upper respiratory tract disease and significant losses to poultry industries worldwide. Glycoprotein G (gG) is a broad-range viral chemokine-binding protein conserved among most alphaherpesviruses, including ILTV. A number of studies comparing the immunological parameters between infection with gG-expressing and gG-deficient ILTV strains have demonstrated that expression of gG is associated with increased virulence, modification of the amount and the composition of the inflammatory response, and modulation of the immune responses toward antibody production and away from cell-mediated immune responses. The aims of the current study were to examine the establishment of infection and inflammation by ILTV and determine how gG influences that response to infection. In vitro infection studies using tracheal organ tissue specimen cultures and blood-derived monocytes and in vivo infection studies in specific-pathogen-free chickens showed that leukocyte recruitment to the site of infection is an important component of the induced pathology and that this is influenced by the expression of ILTV gG and changes in the transcription of the chicken orthologues of mammalian CXC chemokine ligand 8 (CXCL8), chicken CXCLi1 and chicken CXCLi2, among other cytokines and chemokines. The results from this study demonstrate that ILTV gG interferes with chemokine and cytokine transcription at different steps of the inflammatory cascade, thus altering inflammation, virulence, and the balance of the immune response to infection.IMPORTANCE Infectious laryngotracheitis virus is an alphaherpesvirus that expresses gG, a conserved broad-range viral chemokine-binding protein known to interfere with host immune responses. However, little is known about how gG modifies virulence and influences the inflammatory signaling cascade associated with infection. Here, data from in vitro and in vivo infection studies are presented. These data show that gG has a direct impact on the transcription of cytokines and chemokine ligands in vitro (such as chicken CXCL8 orthologues, among others), which explains the altered balance of the inflammatory response that is associated with gG during ILTV infection of the upper respiratory tract of chickens. This is the first report to associate gG with the dysregulation of cytokine transcription at different stages of the inflammatory cascade triggered by ILTV infection of the natural host.

Development and application of high-resolution melting analysis for the classification of infectious laryngotracheitis virus strains and detection of recombinant progeny.

Live attenuated vaccines against infectious laryngotracheitis virus (ILTV) are widely used in the poultry industry to control disease and help prevent economic losses. Molecular epidemiological studies of currently circulating strains of ILTV within poultry flocks in Australia have demonstrated the presence of highly virulent viruses generated by genomic recombination events between vaccine strains. In this study, high-resolution melting (HRM) analysis was used to develop a tool to classify ILTV isolates and to investigate ILTV recombination. The assay was applied to plaque-purified progeny viruses generated after co-infection of chicken embryo kidney (CEK) monolayers with the A20 and Serva ILT vaccine strains and also to viruses isolated from field samples. The results showed that the HRM analysis is a suitable tool for the classification of ILTV isolates and can be used to detect recombination between ILTV vaccine strains in vitro. This method can be used to classify a broad range of ILTV strains to facilitate the classification and genotyping of ILTV and help to further understand recombination in these viruses.

Comparison of the short-term and long-term efficacies of the Mycoplasma gallisepticum vaccines ts-11 and 6/85.

In order to compare the short-term efficacies of the live attenuated Mycoplasma gallisepticum (MG) vaccine strains ts-11 and 6/85, four groups of SPF chickens were vaccinated with each of the vaccines using eye drop and aerosol inoculations, and were subsequently challenged with a wild-type MG strain. When administered by the recommended routes (eye drop for ts-11 and fine aerosol for 6/85), both vaccines induced substantial and comparable levels of protection against airsacculitis and tracheitis caused by wild-type MG. The long-term efficacies of the two vaccines administered by the recommended route were also assessed. Serum antibody responses and colonization of the vaccines in the upper respiratory system were monitored at different time points after vaccination, and protective efficacies of the vaccines were evaluated at 36 weeks post vaccination as above. Systemic antibody response following ts-11 eye drop vaccination was initially strong but reduced gradually over time while, in contrast, that to 6/85 spray vaccination was initially weak but increased over time. Kinetics of the antibody response to the vaccines appeared to be correlated with the number of birds harbouring each vaccine in their upper respiratory system throughout the sampling timepoints. Regardless of the levels of serum antibodies or number of birds harbouring the vaccine, both vaccines induced substantial and comparable levels of protection against airsacculitis and tracheitis caused by wild-type MG. Therefore, kinetics of systemic antibody response and persistence in the upper respiratory system varies between vaccine strains; however, the levels of protection may not, at least up to 36 weeks post vaccination. RESEARCH HIGHLIGHTS The kinetics of systemic antibody response and persistence of the vaccine in the upper respiratory system varies between vaccine strains ts-11 and 6/85. The levels of protection induced by the two vaccines against virulent MG strain challenge are comparable when they are administered by the route recommended by their manufacturers.

Vaccination with FAdV-8a induces protection against inclusion body hepatitis caused by homologous and heterologous strains.

Fowl aviadenoviruses (FAdV) are important avian pathogens, responsible for several poultry diseases prevalent worldwide, including inclusion body hepatitis (IBH). FAdV intraspecies cross-protection has been clearly demonstrated, but there is little evidence that any interspecies cross-protection exists. The present study aimed to assess the inter- and intraspecies protection between three FAdV field isolates (FAdV-8a, FAdV-8b, FAdV-11) identified in association with severe IBH outbreaks. Inocula prepared using inactivated plaque-purified virus with adjuvant Montanide™ ISA 71VG, were injected intramuscularly into 3-week-old SPF chickens. At 6-weeks of age, the birds were challenged with 106 TCID50 of homologous or heterologous virus intraperitoneally, and full post mortem examination performed at 4 days post-challenge. Various tissues were examined for gross and histological lesions and assessed for the presence of virus by PCR-HRM. All homologous-type vaccine/challenge groups exhibited protection against IBH lesions with no virus detected in the tissues. Unvaccinated groups challenged with virus showed evidence of FAdV-induced lesions; however, FAdV-8a demonstrated lower pathogenicity compared with FAdV-8b and FAdV-11. In the heterologous-type vaccine/challenge groups, FAdV-8a vaccine was shown to protect against challenge with both FAdV-8b and FAdV-11. FAdV-8a and 8b belong to species E and were therefore anticipated to cross-protect. However, FAdV-11 belongs to species D and therefore cross-protection by FAdV-8a was an uncharacteristic and unique finding of this study. Further research is required to disseminate the molecular basis for the interspecies cross-protection between FAdV-8a and FAdV-11. Nonetheless, the FAdV-8a isolate was shown to have substantial potential as a vaccine candidate in countries where FAdV-8a, 8b or 11 are prevalent.

Comparative genomic analyses of Mycoplasma synoviae vaccine strain MS-H and its wild-type parent strain 86079/7NS: implications for the identification of virulence factors and applications in diagnosis of M. synoviae.

Mycoplasma synoviae is an economically important avian pathogen worldwide, causing respiratory disease, infectious synovitis, airsacculitis and eggshell apex abnormalities in commercial chickens. Despite the widespread use of MS-H as a live attenuated vaccine over the past two decades, the precise molecular basis for loss of virulence in this vaccine is not yet fully understood. To address this, the whole genome sequence of the vaccine parent strain, 86079/7NS, was obtained and compared to that of the MS-H vaccine. Except for the vlhA expressed region, both genomes were nearly identical. Thirty-two single nucleotide polymorphisms (SNPs) were identified in MS-H, including 11 non-synonymous mutations that were predicted, by bioinformatics analysis, to have changed the secondary structure of the deduced proteins. One of these mutations caused truncation of the oppF-1 gene, which encodes the ATP-binding protein of an oligopeptide permease transporter. Overall, the attenuation of MS-H strain may be caused by the cumulative and complex effects of several mutations. The SNPs identified in MS-H were further analyzed by comparing the MS-H and 86079/7NS sequences with the strains WVU-1853 and MS53. In the genomic regions conserved between all strains, 30 SNPs were found to be unique to MS-H lineage. These results have provided a foundation for developing novel biomarkers for the detection of virulence in M. synoviae and also for designing new genotyping assays for discrimination of MS-H from field strains.

Genome analysis of Mycoplasma synoviae strain MS-H, the most common M. synoviae strain with a worldwide distribution.

BACKGROUND: The bacterial pathogen Mycoplasma synoviae can cause subclinical respiratory disease, synovitis, airsacculitis and reproductive tract disease in poultry and is a major cause of economic loss worldwide. The M. synoviae strain MS-H was developed by chemical mutagenesis of an Australian isolate and has been used as a live attenuated vaccine in many countries over the past two decades. As a result it may now be the most prevalent strain of M. synoviae globally. Differentiation of the MS-H vaccine from local field strains is important for epidemiological investigations and is often required for registration of the vaccine. RESULTS: The complete genomic sequence of the MS-H strain was determined using a combination of Illumina and Nanopore methods and compared to WVU-1853, the M. synoviae type strain isolated in the USA 30 years before the parent strain of MS-H, and MS53, a more recent isolate from Brazil. The vaccine strain genome had a slightly larger number of pseudogenes than the two other strains and contained a unique 55 kb chromosomal inversion partially affecting a putative genomic island. Variations in gene content were also noted, including a deoxyribose-phosphate aldolase (deoC) fragment and an ATP-dependent DNA helicase gene found only in MS-H. Some of these sequences may have been acquired horizontally from other avian mycoplasma species. CONCLUSIONS: MS-H was somewhat more similar to WVU-1853 than to MS53. The genome sequence of MS-H will enable identification of vaccine-specific genetic markers for use as diagnostic and epidemiological tools to better control M. synoviae.

Characterisation of the antigenic epitopes in the subunit 2 haemagglutinin of avian influenza virus H5N1.

Monitoring avian influenza (AI) infection and detecting silent infection in vaccinated chickens has been challenging due to the lack of effective serological diagnostic assays to differentiate between vaccinated and infected animals. Very few studies have identified suitable proteins in AI virus that can be used in successfully differentiating infected from vaccinated animals (DIVA). An HA2 peptide: HA2 position 197-201 (HA position 488-516) described by Khurana et al. (J Virol 85(23):12455-12463, 2011), was shown to have DIVA ability by differentiating H5N1-infected human sera in ELISA. In order to explore the capacity of the HA2 protein, as a DIVA reagent in chickens, four overlapping recombinant HA2 proteins, were expressed in E. coli and tested for reaction with H5N1 sera obtained from infected and vaccinated chickens. Recombinant protein HA2_B2 (380-461) was able to generate a detectable reaction with both H5N1 infected and vaccinated chicken sera but recombinant protein HA2_B4 (483-565) reacted strongly only with sera obtained from chickens infected with live virus, confirming its suitability as a DIVA antigen. Further analysis of the HA2 using several overlapping peptides suggested that positions 380-461 and 483-565 were antigenic in mouse and chicken. This study, for the first time, identified novel antigenic epitopes on the H5N1 HA2 subunit. Two epitopes, found in the HA2 ectodomain, have never been described for AIV infection in any animal species. Also one HA2 epitope was found to have high potential as a DIVA antigen.

Pathological and microbiological investigations into cases of bacterial chondronecrosis and osteomyelitis in broiler poultry.

Bacterial chondronecrosis and osteomyelitis (BCO) is increasingly recognized as a major cause of lameness in commercial broilers chickens worldwide, but the pathogenesis of the condition is incompletely understood. This was a longitudinal study of 20 commercial broiler farms in Victoria, Australia, to investigate the aetiology and pathology of BCO. Thorough postmortem examination was performed on culled and dead birds (n = 325) from 20 different flocks at either 1 week, 4 weeks or 5 weeks of age and samples were analysed by conventional bacteriology, molecular identification of infectious organisms detected, serology and histopathology. BCO occurs throughout the life of broiler flocks at a very high rate, with lesions detected in 28% (95% CI 23-34%) of the mortalities and culls. The condition occurs with similar prevalence in both the femur and tibiotarsus. BCO is an infectious process that appears to result from bacteraemia and haematological spread of bacterial pathogens, especially Escherichia coli, to the bones, with 65.3% bacterial isolates from histologically confirmed BCO identified as E. coli, 11.5% as Staphylococcus and the remainder composed of mixed infections or a range of other minor isolates. We observed that almost all E. coli isolated from cases of BCO are avian pathogenic E. coli, suggesting that preventative measures should be directed at this organism.

Immune responses to vaccination and infection with Mycoplasma gallisepticum in turkeys.

Infection with Mycoplasma gallisepticum induces severe lymphoproliferative lesions in multiple sites along the respiratory tract in chickens and turkeys. These immunopathological responses have been well-characterized in chickens, but have not been studied closely in turkeys. The aim of the study described here was to examine the immune responses of turkeys after live vaccination and infection with M. gallisepticum. In a strain comparison study, the mean log10 antibody titre of birds exposed to an aerosol culture of M. gallisepticum strain Ap3AS was found to be significantly higher at day 14 than that of birds exposed to strain 100809/31. In a dose-response study, there was a significant difference in the mean log10 antibody titre between birds exposed to mycoplasma broth and birds exposed to the highest dose of strain Ap3AS at day 7 after exposure. Immunohistochemical analysis of the tracheal mucosa and the air sacs revealed similar patterns of distribution of CD4+ and CD8+ lymphocytes to those seen in the tracheal mucosa of chickens, implicating these cell types in the pathogenesis of respiratory mycoplasmosis in turkeys. Turkeys that had been vaccinated with M. gallisepticum GapA+ ts-11 had significantly higher antibody titres than unvaccinated birds at both 7 and 14 days after challenge with strain Ap3AS. Vaccination with GapA+ ts-11 protected against the lymphoproliferative response to infection with virulent M. gallisepticum in both the tracheal mucosa and the air sacs, suggesting that this strain may be a useful vaccine candidate for use in turkeys.

Evidence of apoptosis induced by viral protein 2 of chicken anaemia virus.

Although viral protein 3 (VP3) of chicken anaemia virus (CAV) has been well recognised as an inducer of apoptosis, viral protein 2 (VP2) of the virus has only been speculated to have apoptotic activity. This has not been verified because the open reading frame (ORF) encoding VP2 completely encompasses that encoding VP3, and thus the possibility of expression of VP3 cannot be excluded. The aim of this study was to elucidate the potential role of VP2 as an inducer of apoptosis. Site-directed mutagenesis was used to generate a point mutation that knocked out VP3 by early termination of its translation with a stop codon without imposing any change in the amino acid sequence of VP2. The mutated sequence was inserted into the pCAT plasmid preceded by a favorable Kozak's consensus sequence to create pCAT-VP2(+)VP3(-). The absence of VP3 expression in MSB1 cells transfected with this plasmid was confirmed using Western blotting, and DNA strand breaks and nuclear morphological changes were assessed to detect apoptosis. There was an increased level of apoptotic death in cells transfected with pCAT-VP2(+)VP3(-) compared to those transfected with the vector alone. This provides evidence that CAV VP2 can induce apoptosis.

Chronological analysis of gross and histological lesions induced by field strains of fowl adenovirus serotypes 1, 8b and 11 in one-day-old chickens.

Fowl adenoviruses (FAdVs) cause diseases in domestic chickens, including inclusion body hepatitis (IBH), with immunosuppression believed to play a role in their pathogenesis. To gain a better understanding of the pathogenesis and chronology of disease caused by FAdVs, the gross pathology, histopathology and dissemination of virus were examined at several different time points, after inoculation of one-day-old specific pathogen-free chickens with FAdV-1, FAdV-8b or FAdV-11 via the ocular route. FAdV-8b had a slightly greater virulence than FAdV-11, but both were primary pathogens. The presence and severity of hepatic lesions were used to define the three stages of the disease: incubation (1-3 days post-inoculation, PI), degeneration (4-7 days PI) and convalescence (14 days PI). Both viruses were detected in the liver, kidney, bursa, thymus and gizzard of most birds during the degenerative stage, and persisted in the gizzard into convalescence. The FAdV-1 isolate was found to be apathogenic, but virus was detected in the bursa and/or gizzard of several birds between 2 and 7 days PI. This is the first study examining the chronology of gross and microscopic lesions of pathogenic and apathogenic FAdVs in association with viral presence in multiple tissues. It was concluded that both FAdV-8b and FAdV-11 are primary pathogens, and that these strains may play a role in immunosuppression.

Development of a Mycoplasma gallisepticum infection model in turkeys.

Mycoplasma gallisepticum causes chronic respiratory disease in chickens and is also highly pathogenic in turkeys. Several live attenuated M. gallisepticum vaccines are available for prevention of disease in chickens but they are considered to be either not safe or not efficacious in turkeys. The studies presented here aimed to develop a suitable infection model in turkeys, a prerequisite for development of a vaccine against M. gallisepticum for turkeys. Two wild-type Australian M. gallisepticum strains, Ap3AS and 100809/31, were used and their capacity to induce lesions was evaluated in 5-week-old to 6-week-old turkeys exposed to aerosols of these strains. Gross air sac lesion scores in the group exposed to Ap3AS were significantly greater than those in the group exposed to 100809/31 (P < 0.05). Histological tracheal lesion scores and tracheal mucosal thicknesses were significantly greater in birds exposed to either strain than in the unexposed birds (P < 0.05), but no significant differences were observed between the two infected groups. In a subsequent experiment, 6-week-old to 7-week-old turkeys were exposed to different doses of M. gallisepticum Ap3AS. Serology and M. gallisepticum re-isolation performed 14 days after infection showed that all birds exposed to Ap3AS were positive by rapid serum agglutination and by culture. Gross air sac lesion scores in the groups exposed to the highest dose, 8.17 × 10(8) colour-changing units Ap3AS/ml, as well as a 10-fold lower dose were significantly more severe than in the uninfected control group. Lesion scores and tracheal mucosal thicknesses were significantly greater in birds exposed to Ap3AS than in the unexposed birds (P < 0.05). However, no significant differences were seen in tracheal mucosal thicknesses or lesion scores between the groups exposed to the different doses of Ap3AS. This study has established a reliable challenge model for M. gallisepticum infection in turkeys, which will be useful for evaluation of potential M. gallisepticum vaccine candidates for this species.