Decrease of Mycoplasma gallisepticum seroprevalence and introduction of new genotypes in Dutch commercial poultry during the years 2001-2018.

Almost two decades ago, in addition to a compulsory M. gallisepticum (Mg) monitoring programme of breeding stock based on European Union regulations, the Dutch poultry industry added national regulations to further reduce the Mg prevalence in Dutch commercial poultry. Currently, all commercial chicken and turkey flocks except broilers are monitored for Mg. All breeding flocks on a farm where one or more flocks tested Mg positive are culled. Mg positive layer pullets are channelled and layer pullets placed on Mg positive multi-age farms are vaccinated. The monitoring data obtained were analysed covering a period of 17 years. Moreover, 31 Dutch Mg isolates from the same period were analysed by multilocus sequence typing (MLST) and compared to available PubMLST data. The results show that in breeding stock the seroprevalence decreased from 1.6% to 0.0%, in commercial layers from 6.3% to 1.9%, and in meat turkeys from 17.6% to 2.4%. The MLST results showed the presence of closely related and identical sequence types (STs) within the different Dutch poultry types. Similar STs were found in Northern and Southern Europe only. The results show a fast decline in the Mg prevalence since 2001, although in layers the Mg prevalence has stabilized and suggests backyard poultry might pose a risk for commercial poultry. The need for Mg control across poultry sectors and in trade was confirmed by the similarity in STs found in different types of poultry and regions. These results from the Dutch poultry industry can be extrapolated to Mg control in general.

Differential Response of the Chicken Trachea to Chronic Infection with Virulent Mycoplasma gallisepticum Strain Ap3AS and Vaxsafe MG (Strain ts-304): a Transcriptional Profile.

Mycoplasma gallisepticum is the primary etiological agent of chronic respiratory disease in chickens. Live attenuated vaccines are most commonly used in the field to control the disease, but current vaccines have some limitations. Vaxsafe MG (strain ts-304) is a new vaccine candidate that is efficacious at a lower dose than the current commercial vaccine strain ts-11, from which it is derived. In this study, the transcriptional profiles of the trachea of unvaccinated chickens and chickens vaccinated with strain ts-304 were compared 2 weeks after challenge with M. gallisepticum strain Ap3AS during the chronic stage of infection. After challenge, genes, gene ontologies, pathways, and protein classes involved in inflammation, cytokine production and signaling, and cell proliferation were upregulated, while those involved in formation and motor movement of cilia, formation of intercellular junctional complexes, and formation of the cytoskeleton were downregulated in the unvaccinated birds compared to the vaccinated birds, reflecting immune dysregulation and the pathological changes induced in the trachea by infection with M. gallisepticum Vaccination appears to protect the structural and functional integrity of the tracheal mucosa 2 weeks after infection with M. gallisepticum.

Differential house finch leukocyte profiles during experimental infection with Mycoplasma gallisepticum isolates of varying virulence.

Leukocyte differentials are a useful tool for assessing systemic immunological changes during pathogen infections, particularly for non-model species. To date, no study has explored how experimental infection with a common bacterial pathogen, Mycoplasma gallisepticum (MG), influences the course and strength of haematological changes in the natural songbird host, house finches. Here we experimentally inoculated house finches with MG isolates known to vary in virulence, and quantified the proportions of circulating leukocytes over the entirety of infection. First, we found significant temporal effects of MG infection on the proportions of most cell types, with strong increases in heterophil and monocyte proportions during infection. Marked decreases in lymphocyte proportions also occurred during infection, though these proportional changes may simply be driven by correlated increases in other leukocytes. Second, we found significant effects of isolate virulence, with the strongest changes in cell proportions occurring in birds inoculated with the higher virulence isolates, and almost no detectable changes relative to sham treatment groups in birds inoculated with the lowest virulence isolate. Finally, we found that variation in infection severity positively predicted the proportion of circulating heterophils and lymphocytes, but the strength of these correlations was dependent on isolate. Taken together, these results indicate strong haematological changes in house finches during MG infection, with markedly different responses to MG isolates of varying virulence. These results are consistent with the possibility that evolved virulence in house finch MG results in higher degrees of immune stimulation and associated immunopathology, with potential direct benefits for MG transmission. RESEARCH HIGHLIGHTS House finches show a marked pro-inflammatory response to M. gallisepticum infection. Virulent pathogen isolates produce stronger finch white blood cell responses. Among birds, stronger white blood cell responses are associated with higher infection severity.

Development of mismatch amplification mutation assay for the rapid differentiation of Mycoplasma gallisepticum K vaccine strain from field isolates.

Mycoplasma gallisepticum causes respiratory diseases and reproduction disorders in turkeys and chickens. The infection has considerable economic impact due to reduced meat and egg production. Because elimination programmes are not feasible in a large number of poultry farms, vaccination remains the only effective measure of disease control. Differentiating vaccine strains from field isolates is necessary in the control of vaccination programmes and diagnostics. The aim of this study was to develop a polymerase chain reaction based mismatch amplification mutation assay (MAMA) for the discrimination of K vaccine strain (K 5831, Vaxxinova Japan K.K.). After determining the whole genome sequence of the K strain, primers were designed to detect seven different vaccine-specific single nucleotide polymorphisms. After evaluating preliminary results, the MAMA-K-fruA test detecting a single guanine-adenine substitution within the fruA gene (G88A) was found to be the most applicable assay to distinguish the K vaccine strain from field isolates. The detected K strain-specific single nucleotide polymorphism showed genetic stability after serial passage in vitro, but this stability test should still be evaluated in vivo as well, investigating a large number of K strain re-isolates. The MAMA-K-fruA assay was tested on a total of 280 culture and field samples. The designed assay had 102 and 103 template copy number/µl sensitivity in melt-curve analysis based and agarose-gel based assays, respectively, and showed no cross reaction with other avian Mycoplasma species. The new MAMA provides a time- and cost-effective molecular tool for the control of vaccination programmes and for diagnostics.

Evaluation in broilers of aerosolized nanoparticles vaccine encapsulating imuno-stimulant and antigens of avian influenza virus/Mycoplasma gallisepticum.

BACKGROUND: The global prevalence of economic primary infection of poultry by H9N2 virus, including the Lineage A, panzootic group ME1, and associated with secondary infection by Mycoplasma gallisepticum (MG), is alarming to the sustainability of the poultry sector. This research evaluated in broilers the immunity and protection induced by aerosolization of liposomal nanoparticles vaccine, encapsulating antigens of H9N2 virus and MG, with or without the incorporation of Echinacea extract (EE) immuno-stimulant. Six different treatments (TRTs) of broilers were included in the experimental design, with three replicate pens/TRT and stocking of 20 day-old birds/replicate. RESULTS: The tracheobronchial washings of birds subjected to aerosolization of liposomal nanoparticles, encapsulating antigens of H9N2 and MG and EE had the highest significant mean levels of each of IgA and IgG specific to H9N2 and MG, associated with lowest tracheal MG colonization, tracheal H9N2 recovery, tracheal histopathologic lesions, mortality, and best performance in body weight and feed conversion compared to all other challenged birds allocated to different treatments (P < 0.05). However, the control broilers, free from challenge with MG and H9N2, had the lowest mortality and tracheal lesions, and the highest production performance. CONCLUSION: The aerosolization of liposomal nanoparticles, encapsulating antigens of H9N2 and MG and EE resulted in enough local immunity for protection of broilers against infection, and in attaining the highest production performance in challenged birds. The potential implication of vaccinating with safe killed nanoparticle vaccines is of utmost importance to the global poultry sector.

Development of Molecular Methods for Rapid Differentiation of Mycoplasma gallisepticum Vaccine Strains from Field Isolates.

Mycoplasma gallisepticum is among the most economically significant mycoplasmas causing production losses in poultry. Seven melt-curve and agarose gel-based mismatch amplification mutation assays (MAMAs) and one PCR are provided in the present study to distinguish the M. gallisepticum vaccine strains and field isolates based on mutations in the crmA, gapA, lpd, plpA, potC, glpK, and hlp2 genes. A total of 239 samples (M. gallisepticum vaccine and type strains, pure cultures, and clinical samples) originating from 16 countries and from at least eight avian species were submitted to the presented assays for validation or in blind tests. A comparison of the data from 126 samples (including sequences available at GenBank) examined by the developed assays and a recently developed multilocus sequence typing assay showed congruent typing results. The sensitivity of the melt-MAMA assays varied between 101 and 104M. gallisepticum template copies/reaction, while that of the agarose-MAMAs ranged from 103 to 105 template copies/reaction, and no cross-reactions occurred with other Mycoplasma species colonizing birds. The presented assays are also suitable for discriminating multiple strains in a single sample. The developed assays enable the differentiation of live vaccine strains by targeting two or three markers/vaccine strain; however, considering the high variability of the species, the combined use of all assays is recommended. The suggested combination provides a reliable tool for routine diagnostics due to the sensitivity and specificity of the assays, and they can be performed directly on clinical samples and in laboratories with basic PCR equipment.

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.

TLR2/MyD88/NF-κB signaling pathway regulates IL-1ß production in DF-1 cells exposed to Mycoplasma gallisepticum LAMPs.

Mycoplasma gallisepticum (M. gallisepticum) is one of the most important pathogens that cause chronic respiratory disease in chickens. M. gallisepticum-derived lipid-associated membrane proteins (LAMPs) are thought to be one of the major factors in mycoplasma pathogenesis and are potent inducers of the host innate immune response. However, the interaction of pathogenic M. gallisepticum-derived LAMPs with Toll-like receptors (TLRs) and the signaling pathways responsible for activating inflammation and NF-κB have not been fully elucidated. In this study, we found that IL-1ß expression was induced in DF-1 cells stimulated with M. gallisepticum LAMPs. Subcellular localization experiments using immunofluorescence assays (IFAs) showed p65 translocation from the cytoplasm to the nucleus in DF-1 cells following stimulation with M. gallisepticum LAMPs. Phosphorylation of p65 was detected in LAMP-stimulated DF-1 cells. Treatment with an NF-κB-specific inhibitor showed that NF-κB is required for M. gallisepticum LAMP-induced IL-1ß expression. In addition, the results indicated that TLR2 and myeloid differentiation primary-response protein 88 (MyD88)-dependent signaling pathways were involved in the activation of NF-κB by M. gallisepticum LAMPs. Together, these results provide evidence that M. gallisepticum LAMPs activate IL-1ß production through the NF-κB pathway via TLR2 and MyD88.

Assessment of Mycoplasma gallisepticum vaccine efficacy in a co-infection challenge model with QX-like infectious bronchitis virus.

Mycoplasma gallisepticum (MG) is the primary cause of chronic respiratory disease in poultry. We investigated the protective efficacy of the live-attenuated ts-11 and 6/85 MG vaccines against a local MG strain and, in order to enhance signs and mimic a typical field situation, we co-infected birds with a virulent strain of QX-like infectious bronchitis virus (IBV). Both vaccines showed similar ability to protect infected chickens from clinical signs, although ts-11 performed slightly better. Despite the lower protection against clinical disease, 6/85-vaccinated birds had significantly (P ≤ 0.05) lower tracheal lesion scores and mucosal thickness at day 28 post-vaccination (7 days post-challenge [dpc] with MG, 2 dpc IBV) and day 31 post-vaccination (10 dpc MG challenge, 5 dpc IBV) compared to ts-11 vaccinated birds, but these difference was not significant at day 33 (12 dpc MG, 7 dpc IBV). Pathogen infection and replication was assessed by qPCR, and the 6/85 vaccine produced a more significant (P ≤ 0.05) reduction in MG replication in the lungs, kidneys and livers but enhanced late replication in bursae and caecal tonsils. In contrast, the ts-11 vaccine had a more pronounced reductive effect on replication in tracheas, air sacs, bursae and heart at days 28 and 31, yet increased replication in lungs. Interestingly, both vaccines provided non-specific protection against IBV challenge. The co-challenge model provided useful data on vaccine efficacy, especially on days 31 and 33, and tracheas, lungs, air sacs, kidneys, liver and caecal tonsils were the best organs to assess.

Development and evaluation of novel recombinant adenovirus-based vaccine candidates for infectious bronchitis virus and Mycoplasma gallisepticum in chickens.

Avian infectious bronchitis caused by the infectious bronchitis virus (IBV), and mycoplasmosis caused by Mycoplasma gallisepticum (MG) are two major respiratory diseases in chickens that have resulted in severe economic losses in the poultry industry. We constructed a recombinant adenovirus that simultaneously expresses the S1 spike glycoprotein of IBV and the TM-1 protein of MG (pBH-S1-TM-1-EGFP). For comparison, we constructed two recombinant adenoviruses (pBH-S1-EGFP and pBH-TM-1-EGFP) that express either the S1 spike glycoprotein or the TM-1 protein alone. The protective efficacy of these three vaccine constructs against challenge with IBV and/or MG was evaluated in specific pathogen free chickens. Groups of seven-day-old specific pathogen free chicks were immunized twice, two weeks apart, via the oculonasal route with the pBH-S1-TM-1-EGFP, pBH-S1-EGFP, or pBH-TM-1-EGFP vaccine candidates or the commercial attenuated infectious bronchitis vaccine strain H52 and MG vaccine strain F-36 (positive controls), and challenged with virulent IBV or MG two weeks later. Interestingly, by days 7 and 14 after the booster immunization, pBH-S1-TM-1-EGFP-induced antibody titre was significantly higher (P < 0.01) compared to attenuated commercial IBV vaccine; however, there was no significant difference between the pBH-S1-TM-1-EGFP and attenuated commercial MG vaccine groups (P > 0.05). The clinical signs, the gross, and histopathological lesions scores of the adenovirus vaccine constructs were not significantly different from that of the attenuated commercial IBV or MG vaccines (positive controls) (P > 0.05). These results demonstrate the potential of the bivalent pBH-S1-TM-1-EGFP adenovirus construct as a combination vaccine against IB and mycoplasmosis.

Transcriptional Profiling of the Chicken Tracheal Response to Virulent Mycoplasma gallisepticum Strain Rlow.

Mycoplasma gallisepticum, the primary etiologic agent of chronic respiratory disease (CRD) in poultry, leads to prolonged recruitment and activation of inflammatory cells in the respiratory mucosa. This is consistent with the current model of immune dysregulation that ostensibly allows the organism to evade clearance mechanisms and establish chronic infection. To date, studies using quantitative reverse transcription-PCR (qRT-PCR) and microarrays have shown a significant transient upregulation of cytokines and chemokines from tracheal epithelial cells (TECs) in vitro and tracheal tissue ex vivo in response to virulent strain Rlow that contributes to the infiltration of inflammatory cells into the tracheal mucosa. To expand upon these experiments, RNA was isolated from tracheas of 20 chickens infected with M. gallisepticum Rlow and 20 mock-infected animals at days 1, 3, 5, and 7 postinoculation, and samples were analyzed for differential gene expression using Illumina RNA sequencing. A rapid host response was observed 24 h postinfection, with over 2,500 significantly differentially expressed genes on day 3, the peak of infection. Many of these genes have immune-related functions involved in signaling pathways, including Toll-like receptor (TLR), mitogen-activated protein kinase, Jak-STAT, and the nucleotide oligomerization domain-like receptor pathways. Of interest was the increased expression of numerous cell surface receptors, including TLR4 and TLR15, which may contribute to the production of cytokines. Metabolic pathways were also activated on days 1 and 3 postinfection, ostensibly due to epithelial cell distress that occurs upon infection. Early perturbations in tissue-wide gene expression, as observed here, may underpin a profound immune dysregulation, setting the stage for disease manifestations characteristic of M. gallisepticum infection.

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.

Interaction of Mycoplasma gallisepticum with Chicken Tracheal Epithelial Cells Contributes to Macrophage Chemotaxis and Activation.

Mycoplasma gallisepticum colonizes the chicken respiratory mucosa and mediates a severe inflammatory response hallmarked by subepithelial leukocyte infiltration. We recently reported that the interaction of M. gallisepticum with chicken tracheal epithelial cells (TECs) mediated the upregulation of chemokine and inflammatory cytokine genes in these cells (S. Majumder, F. Zappulla, and L. K. Silbart, PLoS One 9:e112796, http://dx.doi.org/10.1371/journal.pone.0112796). The current study extends these observations and sheds light on how this initial interaction may give rise to subsequent inflammatory events. Conditioned medium from TECs exposed to the virulent Rlow strain induced macrophage chemotaxis to a much higher degree than the nonvirulent Rhigh strain. Coculture of chicken macrophages (HD-11) with TECs exposed to live mycoplasma revealed the upregulation of several proinflammatory genes associated with macrophage activation, including interleukin-1ß (IL-1ß), IL-6, IL-8, CCL20, macrophage inflammatory protein 1ß (MIP-1ß), CXCL-13, and RANTES. The upregulation of these genes was similar to that observed upon direct contact of HD-11 cells with live M. gallisepticum. Coculture of macrophages with Rlow-exposed TECs also resulted in prolonged expression of chemokine genes, such as those encoding CXCL-13, MIP-1ß, RANTES, and IL-8. Taken together, these studies support the notion that the initial interaction of M. gallisepticum with host respiratory epithelial cells contributes to macrophage chemotaxis and activation by virtue of robust upregulation of inflammatory cytokine and chemokine genes, thereby setting the stage for chronic tissue inflammation.

Global Changes in Mycoplasma gallisepticum Phase-Variable Lipoprotein Gene vlhA Expression during In Vivo Infection of the Natural Chicken Host.

Mycoplasma gallisepticum is the primary etiologic agent of chronic respiratory disease in poultry, a disease largely affecting the respiratory tract and causing significant economic losses worldwide. Immunodominant proteins encoded by members of the variable lipoprotein and hemagglutinin (vlhA) gene family are thought to be important for mechanisms of M. gallisepticum-host interaction, pathogenesis, and immune evasion, but their exact role and the overall nature of their phase variation are unknown. To better understand these mechanisms, we assessed global transcriptomic vlhA gene expression directly from M. gallisepticum populations present on tracheal mucosae during a 7-day experimental infection in the natural chicken host. Here we report differences in both dominant and minor vlhA gene expression levels throughout the first week of infection and starting as early as day 1 postinfection, consistent with a functional role not dependent on adaptive immunity for driving phase variation. Notably, data indicated that, at given time points, specific vlhA genes were similarly dominant in multiple independent hosts, suggesting a nonstochastic temporal progression of dominant vlhA gene expression in the colonizing bacterial population. The dominant expression of a given vlhA gene was not dependent on the presence of 12-copy GAA trinucleotide repeats in the promoter region and did not revert to the predominate vlhA gene when no longer faced with host pressures. Overall, these data indicate that vlhA phase variation is dynamic throughout the earliest stages of infection and that the pattern of dominant vlhA expression may be nonrandom and regulated by previously unrecognized mechanisms.

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.

Evaluation of the egg transmission and pathogenicity of Mycoplasma gallisepticum isolates genotyped as ts-11.

Live Mycoplasma gallisepticum vaccines are used for the control of respiratory disease, egg production losses and egg transmission associated with M. gallisepticum infection in long-lived poultry. The first field case of apparent increased virulence and vertical transmission of ts-11, a live M. gallisepticum vaccine, has been reported. In that study a M. gallisepticum isolate from the broiler progeny of ts-11-vaccinated breeders was genotyped as ts-11 by sequence analysis of four different genetic targets and Random Amplified Polymorphic DNA and found to be significantly more virulent than ts-11 vaccine. The objective of the current study was to evaluate the rate of egg transmission and pathogenicity of ts-11 vaccine and isolates recovered from ts-11-vaccinated breeders (K6222B) and their broiler progeny (K6216D) which had been genotyped as ts-11. Groups of 28-week-old specific pathogen-free chickens at 87% average weekly egg production were inoculated with sterile broth media (negative controls), ts-11 vaccine, K6222B, K6216D or R strain (positive controls) by eye-drop and aerosol. K6216D transmitted via the egg at an average rate of 4.0% in the third and fourth weeks post-infection, while egg transmission of K6222B and ts-11 vaccine was not detected. M. gallisepticum was isolated from the air sacs, ovaries and oviducts of hens infected with K6216D and K6222B, but not from those infected with ts-11 vaccine. K6216D and K6222B both induced respiratory signs and significantly more tracheal colonization and more severe tracheal and air sac lesions than ts-11 vaccine (P ≤ 0.05). There were no substantial differences in the egg production of ts-11, K6216D and K6222B infected groups. These results provide the first conclusive evidence of transovarian transmission of an isolate genotyped as ts-11 and indicate that isolates genotyed as ts-11 vary in their virulence and ability to transmit via the egg.

The efficacy of Mycoplasma gallisepticum K-strain live vaccine in broiler and layer chickens.

The efficacy of a live Mycoplasma gallisepticum (MG) vaccine candidate (K-strain) was compared to commercially available vaccines in broiler-type chickens (Trial 1) and layer-type chickens (Trial 2). In Trial 1, three-week-old broiler-type chickens were vaccinated via aerosol with K-strain or an F-strain vaccine. The vaccinated chickens and 10 non-vaccinated controls were subsequently challenged with virulent R-strain via aerosol at six weeks post vaccination; both K-strain and F-strain vaccination resulted in significant protection from air sac and tracheal lesions, as well as R-strain colonization (P ≤ 0.05). In Trial 2, commercial layer-type chickens were vaccinated with ts-11 (via eye drop) or K-strain (via aerosol) at 12 weeks of age. At 25 weeks of age these birds were challenged with R-strain via aerosol. The ts-11 and K-strain vaccinated groups both had significantly lower air sac lesion scores and a lower prevalence of ovarian regression after challenge as compared to non-vaccinated chickens (P ≤ 0.05). K-strain vaccination also prevented significant tracheal lesions and R-strain colonization (P ≤ 0.05). K-strain shows great potential as a highly efficacious live MG vaccine in broiler and layer-type chickens for protection of the respiratory and reproductive systems as well as prevention of infection with field strains.

Effects of different vaccine combinations against Mycoplasma gallisepticum on the digestive and reproductive organ characteristics of commercial egg-laying hens.

Mycoplasma gallisepticum (MG) is a major and economically significant pathogen of avian species. When administered before lay, F-strain MG (FMG) can reduce egg production during lay, but the ts-11 strain of MG (ts11MG) does not exert this effect. Two trials were conducted to determine the effects of pre-lay vaccinations of ts11MG, MG-Bacterin (MGBac), or their combination, in conjunction with an FMG vaccination overlay after peak production on the digestive and reproductive organ characteristics of Hy-Line W-36 layers housed in biological isolation units (4 units per treatment, 10 birds per unit). The following vaccination treatments were administered at 10 wk of age (woa): 1) Control (no vaccinations); 2) MGBac; 3) ts11MG; and 4) ts11MG and MGBac combination (ts11MG+MGBac). At 45 woa, half of the birds were vaccinated with a laboratory stock of high passage FMG. In both trials, parameters determined in 4 birds per unit at 55 woa included: BW; fatty liver hemorrhagic syndrome incidence; mean number of mature ovarian follicles; ovarian, oviduct, and small intestine weights; and the weights and lengths of the various portions of the oviduct and small intestine. Treatment effects were observed for the weights of the entire small intestine and the duodenum, jejunum, and ileum, as percentages of BW; and for vagina weight as a percentage of total oviduct weight. In general, the weights of the small intestine and its 3 components were increased in response to the FMG vaccine that was administered at 45 woa. An FMG vaccination at 45 woa may increase relative intestine weight in layers; however, use of a prelay MGBac vaccine alone or in combination with ts11MG, with or without an FMG overlay, does not affect the gross characteristics of their digestive and reproductive organs, and may be used without having an adverse effect on their performance, as was observed in a previous companion study.

Effects of different vaccine combinations against Mycoplasma gallisepticum on the internal egg and eggshell characteristics of commercial layer chickens 1,2,3.

Live F-strain Mycoplasma gallisepticum (FMG) vaccines are presently being used to help control field-strain MG outbreaks. However, they may exert some adverse effects on egg production. Live strains of MG of lesser virulence as well as killed vaccines have little or no effect on egg production, but afford lower levels of protection. This has led to research investigating their use in combination with a subsequent overlay vaccination of FMG given later in the production cycle. In the present study, 2 trials were conducted to investigate the effects of prelay vaccinations of live and killed MG vaccines or their combination, in conjunction with an FMG vaccine overlay after peak production, on the egg characteristics of commercial layers. The following vaccination treatments were administered at 10 wk of age (woa): 1) unvaccinated (Control), 2) MG-Bacterin (MGBac) vaccine, 3) ts-11 strain MG (ts11MG) vaccine, and 4) MGBac and ts11MG combination (MGBac + ts11MG). At 45 woa, half of the birds were overlaid with an FMG vaccine. In each trial, internal egg and eggshell parameters including egg weight (EW), Haugh unit score (HU), eggshell breaking strength (EBS), percentage yolk weight (PYW), percentage albumen weight (PAW), percentage eggshell weight (PSW), eggshell weight per unit surface area (SWUSA), percentage yolk moisture (PYM), and percent total lipids (PYL) were determined at various time periods between 21 and 52 woa. At 28 woa, SWUSA was lower in the ts11MG and MGBac + ts11MG groups compared to the Control group. Conversely, at 43 woa, SWUSA was higher in the ts11MG than in the MGBac group. Between 23 and 43 woa, PYL was higher in the MGBac and ts11MG groups in comparison to the Control group. In conclusion, vaccination with MGBac alone or in combination with ts11MG at 10 woa with or without an FMG vaccine overlay at 45 woa does not adversely affect the internal egg or eggshell quality of commercial layers throughout lay.

Cloning, expression, and antigenic characterization of recombinant protein of Mycoplasma gallisepticum expressed in Escherichia coli.

Mycoplasma gallisepticum (MG) is a member of the most important avian mycoplasmas, causing chronic respiratory disease in chickens and leading to important economic losses in the poultry industry. Recombinant technology represents a strategic approach used to achieve highly reliable and specific diagnostic tests in veterinary diseases control: in particular this aspect is crucial for confirming mycoplasma infection and for maintaining mycoplasma-free breeder flocks. In this study, we identified a component of the pyruvate dehydrogenase dihydrolipoamide acetyltransferase (i.e., E2) protein by 2-dimensional electrophoresis (2-DE), characterized it in immunoblotting assays, and analyzed its recombinant (r-E2) in a rec-ELISA test. For full-length protein expression in Escherichia coli (EC) a point mutation was introduced. A rabbit antiserum produced against r-E2 was tested in a Western Blot using different samples of Mycoplasma species. The results showed the applicability of site-directed mutagenesis, with a good yield of the r-E2 after purification. Also, anti-E2 serum reacted with all the tested MG strains showing no cross reaction with other mycoplasmas. The developed E2 ELISA test was capable of detecting MG antibodies in the sera examined. Those results demonstrate the antigenic stability of the E2 protein which could represent a recombinant antigen with potential diagnostic applications.