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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.

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The paper presents results of avian influenza epidemiological monitoring in the Republic of Crimea in 2019–2020. The attention was focused on the study of water basins of the Azov and Black Seas, the Sivash Lagoon and freshwater lakes in the Feodosia Urban Okrug, Leninsky, Sovetsky, Nizhnegorsky, Chernomorsky and Saksky Raions to detect the avian influenza virus circulation. Examination of the above mentioned areas showed that some freshwater reservoirs became shallow and dry, and aquatic vegetation degraded. The natural biotope analysis conducted in 2019 and 2020 showed a decreased number of semiaquatic wild birds. The pathological material was sampled from semiaquatic and migratory wild birds, as well as from poultry kept in poultry farms and backyards. The collected samples were tested using real-time RT-PCR. In 2019, the AIV type A (H9) genome was detected in one fecal sample taken from wild birds near Kuchuk-Adzhigol Lake in Feodosia Urban Okrug. The AIV type A (H5) genome was detected in 2020 during laboratory testing of pathological material taken from the remains of a mute swan within the shoreline of a freshwater lake near the Ermakovo settlement of the Dzhankoysky Raion. The genetic analysis was performed in the FGBI “ARRIAH” (Vladimir), and the N8 subtype neuraminidase of the influenza virus isolate was determined. The comparative genetic analysis of 258 bp nucleic acid sequences of the AIV H gene fragment showed that the identified isolate belongs to the Asian genetic lineage of highly pathogenic AIV subtype H5 (clade 2.3.4.4) associated with the epidemic spread in Asia, Europe, the Middle East and Africa in 2016–2020.