Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are avian pathogens of worldwide significant economic impact. Definite diagnosis with pathogen identification down to sub-species level represents the first bulwark forcorrect control approach. Furthermore, the role of non-poultry birds in MG and MS epidemiology has not been extensively investigated and outbreaks of MG and MS in poultry farms continue to reappear despite implemented control measurements.The present study includes two parts. Through the first part, 104 German poultry flocks were investigated for MG and MS infection and the occurred Mycoplasma spp. were typed and compared. MG showed lower incidence and was detected in 26 flocks (25%), however MS was identified in 37 flocks (36%). MG genotyping was carried out through pvpA-restriction fragment length polymorphism (pvpARFLP) analysis, sequencing of the MG cytadhesin 2 (mgc2) gene, the MG 16S-23S intergenic spacer region (IGSR) and the phase-variable protein A (pvpA) gene that showed discrimination indexes (D-index) of 0.81, 0.52, 0.86 and 0.94, respectively. Multiple gene-targeted sequencing (GTS) analysis of mgc2/pvpA/IGSR discriminated 18 sequence types (from I to XVIII) within MG strains and showed the highest D-index (0.95). Multiple GTS revealed a characteristic genetic profile of German MG strains that could be differentiated into 4 clusters; a 6/85 like strain, a turkey breeder, in addition to two German characteristic clusters with no relationship to known MG reference strains.MS genotyping was carried out via sequencing of the MS variable lipoprotein and hemagglutinin (vlhA) gene that showed a D-index of 0.83. MS genotyping proved circulation of 15 vlhA sequence types with predomination of two sequence types. Global comparison of vlhA sequences proved genetic relationship between German and Dutch MS strains.Results demonstrated also the importance of periodical surveillance for MG and MS with species identification down to the strain level. Multiple GTS analysis (for MG) and vlhA sequencing (for MS) proved to be dependable, practical genotyping methods capable of intraspecies discrimination that offers also easier inter-laboratory data exchange enabling a better understanding of the genetic evolution of circulating pathogens. Increased MS incidence requires updating of implemented control strategies. International cooperation and coordination for MG/MS control are epidemiologically very important.Through the second part of the study, chicken and contacting free-ranging birds from two naturally MG/MS infected chicken farms (I and II) were simultaneously investigated and compared for MG and MS. From 118 captured free-ranging birds, DNA of genus Mycoplasma was detected in 41 birds. From those, MG-specific and MS-specific DNA were detected in 16 and 9 samples, respectively. The genetic profile of MG was compared through multiple genetargeted sequencing (GTS) analysis of mgc2, pvpA and MG IGSR. Free-ranging birds from both farms areas as well as chickens of farm I showed the same MG lineage that shared identical GTS profile like MG 6/85 vaccine strain. MG of chickens from farm II demonstrated a different GTS profile. The genetic profile of MS was investigated through sequencing of the MS vlhA gene. Free-ranging birds captured on both farms showed two circulating MS lineages which were also detected in chickens of farm I. However, chickens from farm II had only one MS strain in common with free-ranging birds.In conclusion, results of the present study demonstrate the importance of periodical surveillance for MG and MS. Multiple GTS analysis (for MG) and vlhA sequencing (for MS) proved to be robust, practical genotyping methods capable of intra-species discrimination. Sequencing allows also for easier data exchange between laboratories and consequently a more comprehensive understanding of the genetic relationship of internationally circulating strains. Increased MS occurrence necessitates updating of the immediate control strategies. Stronger international cooperation and coordination for MG/MS control are recommended.Moreover, results highlight also the possibility of an epidemiological role played by free-ranging birds, especially the wide spread house sparrows, in MG and MS spread between poultry farms. Hence, suitable control measures to decrease the contact between poultry and free-ranging birds should be quickly launched.
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