Molecular genetic and bacteriological methods of bovine mycoplasmosis diagnosis

Mycoplasmas are bacteria that are extremely unstable in vitro as they lack a rigid cell wall. They are most often detected in association with other pathogens, including those that can become L-forms if treated with antibiotics. Mycoplasma colonies, as well as colonies of L-form bacteria, have a typical «fried egg» appearance, therefore it is necessary to differentiate them for the accurate diagnosis and choice of treatment. The paper presents data on mycoplasma infection diagnosis in cattle and results of differentiation of isolated mycoplasma and L-form bacteria colonies using multiple passaging and real-time polymerase chain reaction. For that, 177 samples were collected from animals with mycoplasmosis clinical signs, 45 of them were tested using molecular genetic method, 132 samples were subjected to bacteriological testing. Mycoplasma DNA was detected in 71.1% of samples, and specific colonies were detected in 3.8% of samples. Such biochemical tests of mycoplasma species identification as arginine hydrolysis, blood serum liquefaction, film and grain formation, inoculation into Tween-80-containing medium, hemadsorption and hemolysis of erythrocytes do not allow an objective assessment of the species belonging to mycoplasmas, but, according to the results obtained, the isolated species most likely belongs to Mycoplasma dispar, which is pathogenic for cattle. Real-time polymerase chain reaction is undoubtedly the most accurate and rapid diagnostic method for mycoplasmosis, but a preliminary diagnosis can also be established bacteriologically within 2–7 days. In addition, during microbiological testing, it is possible to assess the antibiotic resistance of mycoplasma isolates, thereby developing an optimal and high-quality scheme of the disease treatment and prevention.

1. Koromyslov G. F., Mesarosh Ya., Shtipkovich L., et al. Mycoplasma species in animal pathology. Мoscow: Аgropromizdat; 1987. 256 p. (in Russ.)

2. Holt J. G., Krieg N. R., Sneath P. H. A., Staley J. T., Williams S. T. Bergey’s Manual of Determinative Bacteriology. 9th ed. Baltimore: Williams & Wilkins; 1994. 787 p.

3. Skorodumov D. I., Subbotin V. V., Sidorov M. A., Kostenko T. S. Microbiological diagnosis of bacterial animal diseases: Handbook. Moscow: Izograf; 2005. 656 p. (in Russ.)

4. Krasikov A. P., Rudakov N. V. Mycoplasmoses in humans and animals and their epidemiological significance: Monograph. Omsk: Omsk Scientific Bulletin; 2015. 717 p. (in Russ.)

5. Eissa S. I., Hassan A. M., Hashem Y. M., Shaker M. M. Comparative molecular study of Mycoplasma bovis isolates from Egyptian buffaloes and cows suffered from mastitis. European J. Biol. Sci. 2012; 4 (4): 114–120. DOI: 10.5829/idosi.ejbs.2012.4.4.6668.

6. Kovalenko Ya. R. Novye dannye v izuchenii mikoplazm i mikoplazmozov zhivotnykh = New study data on animal mycoplasmas and mycoplasmoses. Trudi VIEV. 1977; 46: 3–7. eLIBRARY ID: 22403215. (in Russ.)

7. Vasiliev R. M., Vasilieva S. V. Immuno-biological properties of vaginal discharge in healthy and mycoplasmosis-infected cows. Medical Immunology (Russia). 2021; 23 (4): 987–990. DOI: 10.15789/1563-0625-IBP-2278.

8. Pudovkin D. N., Shchepetkina S. V., Karpenko L. Y., Rishko O. A. Diseases of young cattle: practical recommendations. 2nd ed. supplemented. Saint Petersburg: FSBEI HPE SPbGAVM; 2019. 204 p. (in Russ.)

9. Grechaniy V. S., Klyuchnikov A. G., Kartashov S. N. Patomorfologichesky features of the pneumonia at the calfs caused by association virus parainfluenza-3, Mycoplasma bovis and Нaemophilus somnus. Veterinarnaya patologiya. 2010; 3 (34): 85–89. eLIBRARY ID: 16701725. (in Russ.)

10. Smirnova L. I., Temnicova L. V. Laboratory diagnostic of association urogenetalis infections by Micoplasma of large horned livestock. International Bulletin of Veterinary Medicine. 2008; 4: 6–9. eLIBRARY ID: 23066526. (in Russ.)

11. Krasikov A. P., Vologodskaya O. V., Alekseeva I. G., Vodop’yanova N. I. Profilaktika nositel’stva assotsiatsii patogennykh mikroorganizmov u beremennykh korov i netelei = Prevention of carriage of pathogenic microorganisms associations in pregnant cows and heifers. Veterinarnaya patologiya. 2006; 3 (18): 144–147. eLIBRARY ID: 16823125. (in Russ.)

12. Krasikov A. P., Trofimov I. G., Alekseeva I. G., Zabolotnysh M. V. Complex diagnosis of mixed infective diseases in cattle. Veterinarnaya patologiya. 2014; 1 (47): 13–21. eLIBRARY ID: 21757161. (in Russ.)

13. Lukjanova I., Ermakova T., Pleshakova V., Hatko N. Associative respiratory viral-bacterial infections of cattle in Omsk region. Vestnik of Buryat State Academy of Agriculture named after V. Philippov. 2012; 2 (27): 7–10. eLIBRARY ID: 17784122. (in Russ.)

14. Trukhonenko А. А., Stroganova I. Ya. Mikoplazmy krupnogo rogatogo skota v khozyaistve, neblagopoluchnom po virusnym boleznyam = Bovine mycoplasmas in a holding infected with viral diseases. Innovatsionnye tendentsii razvitiya rossiiskoi nauki: materialy VII Mezhdunarodnoi nauchno-prakticheskoi konferentsii molodykh uchenykh (24–26 marta, 2014 g.) = Innovative trends in the development of Russian science: proceedings of the VII International Scientific and Practical Conference of Early Career Researchers (March 24–26, 2014). Krasnoyarsk: KrasGAY; 2014. 117–118. (in Russ.)

15. Sviridova A. N. Treatment regimens and laboratory testing methods of their efficiency applied for calves affected with mycoplasmosis-associated infection. Siberian Herald of Agricultural Science. 2007; 6: 69–74. eLIBRARY ID: 9497384. (in Russ.)

16. Stroganova I. Ya., Trukhonenko A. A., Gumennaya E. Yu. Polymerase chain reaction in diagnosis of the cattle mycoplasmosis in the Eastern Siberia animal farms. The Bulletin of KrasGAU. 2014; 12: 147–150. eLIBRARY ID: 22895357. (in Russ.)

17. Bezborodova N. A., Kozhukhovskaya V. V. The importance of molecular- biological methods for diagnosis of cattle infectious diseases. Actual Questions of Veterinary Biology. 2018; 4 (40): 22–25. eLIBRARY ID: 36702271. (in Russ.)

18. Shneyder Е. D., Makavchik S. A. Identification and diagnostics of pathogens of bovine mycoplasma mastitis by bacteriological and molecular genetic methods. Bacteriology. 2018. 3 (1): 22–25. DOI: 10.20953/2500-1027-2018-1-22-25. (in Russ.)

19. Kozlov D. A., Volkov M. S., Chernyayeva T. Yu., Sorokina M. I., Irza V. N. Influence of bovine blood serum on growth properties of nutrient media for Mycoplasma gallisepticum and Mycoplasma synoviae cultivation. Veterinary Science Today. 2022; 11 (2): 156–162. DOI: 10.29326/2304-196X-2022-11-2-156-162.

20. Kovalenko Ya. R., Shegidevich E. F., Yablonskaya I. A., et al. Guidelines for isolation, cultivation and identification of mycoplasmas, achholeplasmas and ureaplasmas. Moscow: VASKhNIL; 1982. 47 p. (in Russ.)

21. Sukhanova S. M., Berdnikova Z. E., Tikhonova A. S. Ways to improve quality control of culture medium used for Mycoplasma detection. BIOpreparations. Prevention, Diagnosis, Treatment. 2019; 19 (3): 161–168. DOI: 10.30895/2221-996X-2019-19-3-161-168. (in Russ.)

22. Klieneberger E. The natural occurrence of pleuropneumonia-like organism in apparent symbiosis with Streptobacillus moniliformis and other bacteria. J. Pathol. 1935; 40 (1): 93–105. DOI: 10.1002/PATH.1700400108.

23. Timakov V. D., Kagan G. Ya. Biology of L-form bacteria. Moscow: Medgiz; 1961. 235 p. (in Russ.)

24. Plackett P., Buttery S. H., Cottew G. S. Carbohydrates of some Mycoplasma strains. In: Recent Progress in Microbiology VIII: Symposia Held at the VIII International Congress for Microbiology Montreal. Ed. N. E. Gibbons. University of Toronto Press; 1962; 535–548. Available at: http://www.jstor.org/stable/10.3138/j.ctt1vgw69r.74.

25. Timakov V. D., Kagan G. Ya. Family Mycoplasmataceae and L-form bacteria. Мoscow: Meditsina; 1967. 336 с. (in Russ.)

26. Kozlova A. D., Gorbacheva N. S., Hayerova R. F., Krasnikova M. S., Lazareva E. A., Yatsentyuk S. P. Differentiation of Mycoplasma bovis, Mycoplasma bovigenitalium, Mycoplasma californicum and identification of Ureaplasma diversum by real-time PCR. Agricultural Biology. 2019; 54 (2): 378–385. DOI: 10.15389/agrobiology.2019.2.378eng.

27. Doronin M. I., Lozovoy D. A., Shcherbakov A. V., Makarov V. V. Application of the real-time PCR in veterinary practice. Russian veterinary journal. 2020; 2 (6): 5–12. DOI: 0.32416/2500-4379-2020-2-5-12. (in Russ.)