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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 10 1 and 10 4 M. gallisepticum template copies/reaction, while that of the agarose-MAMAs ranged from 10 3 to 10 5 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.

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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.

Case Report An outbreak of systemic isosporosis caused mortalities in greenfinches (Carduelis chloris) and goldfinches (Carduelis carduelis) kept in an aviary in the western suburbs of Melbourne. The following year, a further outbreak in the same aviary occurred in a different flock of goldfinches. At the time of the second outbreak, dead and sick common sparrows (Passer domesticus) discovered near the aviary were also found to have systemic isosporosis. Method The systemic isosporosis was investigated and described using histopathology, electron microscopy and sequence analysis of the 18s gene. Results Isospora spp. infecting the greenfinch and the goldfinch caused significant thickening of the duodenal lamina propria. Measurements in the goldfinches showed an inverse correlation coefficient between the thickening of the duodenum and the weightof the birds. Electron microscopy confirmed the presence of Isospora spp. within lymphocytes migrating into the lamina propria of the duodenum. Analysis of the 18s sequence discovered two different gene sequences across the three species of birds that didn't completely match any sequences previously deposited in GenBank. Conclusion Although the sparrows were found to have died from causes other than systemic Isospora, molecular studies of samples from their liver revealed the presence of an Isospora with 18s gene sequence identical to that found in the captive greenfinches.

Preventative measures, such as vaccination, are commonly used for the control of mycoplasmal infections in poultry. A live attenuated vaccine strain (Vaxsafe MS; MS-H; Bioproperties Pty.