In this study, we report the development and validation of a duplex real-time polymerase chain reaction (PCR) assay with an internal control using TaqMan-labelled probes for the detection of Mycoplasma gallisepticum and Mycoplasma synoviae (duplex MGMS PCR). The MGMS PCR was highly specific with a sensitivity of 7 and 1 colony-forming units/ml for M. gallisepticum and M. synoviae, respectively, using dilution of pure culture that corresponds to 34 and 29 DNA copies per reaction. Validation of the assay was completed with 260 and 27 pooled samples (tracheal swabs) from commercial chickens and turkeys, respectively, with potential M. gallisepticum and M. synoviae involvement and 42 samples (palatine cleft swabs) from backyard geese and ducks. Using isolation as the gold standard, the MGMS PCR was more sensitive than isolation and the analytical sensitivity was 0.944 and 0.958 for M. gallisepticum and M. synoviae, respectively. In comparison with a gapA-based assay (gapA PCR) and a 16S rRNA-based assay (16S PCR) for M. gallisepticum and M. synoviae, respectively, the results agreed for 94.5% and 96.6%, respectively. The use of the internal control allowed monitoring of proper extraction and inhibition of amplification that was detected in 12 samples. The duplex MGMS PCR was shown to be superior to the presently reported real-time PCR assays in terms of combination of sensitivity, specificity and capacity of detection of more than one target in a single tube. In conclusion, the duplex MGMS PCR was highly specific, sensitive, and reproducible and could be used on clinical samples from commercial chickens, turkeys and backyard poultry including ducks and geese.

An earlier study on commercial chickens and turkeys with a history of respiratory disease established Mycoplasma gallisepticum infection rates on 164 poultry farms of the Russian Federation. Forty-seven (29%) of these poultry farms were M. gallisepticum-positive by polymerase chain reaction but isolation of the mycoplasma was successful only on 10 farms. Five field isolates from different farms were selected for pathogenicity studies in specific pathogen-free chicks. Clinical signs, seroconversion, culture rates, air sac and tracheal lesions and mean tracheal mucosal thickness were all assessed in comparison with the reference strain, S6. Of the five isolates, MG140905 and MG070607 appeared to be slightly more pathogenic than the other three, as indicated by clinical signs, culture-positive rates and lesions, but only isolate MG140905 differed statistically (P < 0.05) from them, thus proving to be the most pathogenic. However, none of the Russian field isolates was as pathogenic as the S6 strain by the parameters measured. Stress or other factors such as concurrent bacterial or viral infections may have served as exacerbating factors for the disease seen in the naturally affected flocks. Sequence analysis of the gapA and mgc2 genes showed that MG140905 clustered with M. gallisepticum R(low) and was more distant from the majority of the Russian isolates.

The genetic diversity of the pvpA gene of Mycoplasma gallisepticum (MG) samples originating from commercial chickens was investigated. In the present study, we evaluated the genetic variability of 26 field samples of MG detected in commercial chickens and turkeys from 18 regions of Russia and compared them to the reference strains of MG available in GenBank. Genetic variability was evaluated by partial nucleotide sequencing of the pvpA gene, which encodes a putative cytadhesin protein. Comparisons with MG strains and isolates from the United States, Australia, China, and Iran using sequence analysis of PCR products showed that Russian MG field samples clustered more closely to each other than to the international reference MG strains. The MG pvpA sequences were found to be highly variable with a discrimination index of 0.975 for Russian field samples. No apparent cluster was found using the criteria of year or location of detection. DNA sequence polymorphism and size variation in the pvpA gene were shown among the Russian MG field samples and could be used for MG typing. These findings might help better understand the relationship among MG isolates from Russia and other countries.

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The paper demonstrates examination results of antigenic properties ofAvibacterium paragallinarumreference strain and ten isolates thereof recovered in the Russian Federation and Republic of Belarus. Nine isolates and the reference strain demonstrated type L hemagglutinin (thermoliable, trypsin-sensitive, active against fresh and glutaraldehyde-treated RBC). One isolate was marked by type Hl hemagglutinin (thermoliable, trypsin-resistant, active only against glutaraldehydetreated RBC). The reference strain antigen proved to be inagglutinable by homologous antiserum, and unable to agglutinate RBCs due to hyaluronic acid in the capsular substance. Serological and hemagglutination non-reactivity was removed through the cell treatment with hyaluronidase. The agglutination test demonstrated that seven out of ten tested isolates belonged to the same serological group; herewith, the proportion of the bilateral antigenic relatedness amounted to ≥78.4%. HI results demonstrated ≥92.6% antigenic relatedness of the tested isolates being indicative of the fact that they belonged not only to the same serological group but also to the same serotype. No serological relatedness was identifed between the tested isolates and reference strain of serogroup A both using agglutination test (≤23.6%) and HI (≤12.2%). Polymerase chain reaction demonstrated that all the isolates recovered in the Russian Federation and Republic of Belarus in 2014-2016 belonged to serogroup B.