Biological properties of dissociative L- and other forms of Mycobacterium bovis

  • A. A. Tkachenko Dnіpropetrovsk State Agrarian-Economic University
  • P. O. Davydenko Dnіpropetrovsk State Agrarian-Economic University
  • V. V. Zazharskiy Dnіpropetrovsk State Agrarian-Economic University
  • V. V. Brygadyrenko Oles Honchar Dnipropetrovsk National University
Keywords: dissociative forms of Mycobacterium bovis, lipids, medium, temperature


A race of modified forms of mycobacteria with special properties that can be promising for the construction of TB vaccines was selected It was found that the persistence of the researched microorganisms (27th subculture of acid-fast bacillus and the L-form) persisted for nine months (the period of research) and longer in the bodies of guinea pigs. However, from the suspension prepared from macroscopically unchanged organs of the experimental animals we found acid-proof elementary bodies (grains) and bacilli of typical morphological forms, which formed an orange culture on the nutrient medium on the third day after suspension seeding. The inoculation of guinea pigs with isolated acid-proof mycobacteria (culture-revertant) (1 mg/cm3) was not accompanied by the development of allergic condition (allergic reaction to the tuberculin and AAM was negative at 30, 60 and 90 days); however, acid-proof bacilli, which formed an orange culture,were isolated on the the third day from experimental animals which had been subjected to euthanasia. Multiple passages through the artificial culture medium (dense), prolonged exposure (20 months) at low plus temperature changed the genetic balance, ensuring their survival as a result of the loss of some (specific to the pathogen) and the acquisition of new properties (especially atypical )which are partly inherent in other mycobacteria. At the same time, the persistence in the body of guinea pigs of typical morphological acid-proof forms (bacilli) that reverse from L-forms was not accompanied by the development of the disease. They are chromogenic and retain the ability to form colonies (culture-revertant) on dense nutrient medium from the first generation (from biological material of the guinea pigs) on the second day of cultivation. The loss of sensitization ability of Mycobacterium bovis which werepassaged many times and persistent in the body of guinea pigs can probably testify to the loss of immunogenic capacity, since the development of allergic (tuberculin) reaction, as well as its intensity, indicates an immunological restructuring of the microorganism (the development of an infection) with the parallel acquisition of the specific resistance. We observed a residual virulence of the researched modified forms of M. bovis with the possible formation of specific antiTB immunity without development of the necessary level, which was the indicator of  allergic condition and ulceration of the site where the suspension of mycobacteria was introduced. Microscopy of smears of organs of the euthanized animals (after 80 days)revealled acid-nonproof bacilli, seeding cells. In the control,the weight of the animals tended to increase while the bacterioscopic research (smears) proved negative. Mycobacteria with new, genetically fixed properties have the ability to stimulate benign infectious process, without the development of allergies to the level required for the detection by PPD- for mammals AAM. However, it should be noted that perhaps the extinction of the activity of genes responsible for the pathogenic properties, which are determined by redox processes (dehydrogenase, catalase activity, etc.) and genes which were in a dormant state, activate metabolic processes of pigment synthesis with inhibition of the action of pathogenic factors (toxins). We did not find a relationship between the rate of reproduction (duration of colony formation) and pathogenicity because the original parent culture (third generation) of the investigated modified forms of M. bovis had a high virulence and formed colonies on the second or third day without forming pigment and did not express dehydrogenase and catalase activity. In this article we discuss strains ofM. bovis which differ in biological properties from pathogenic strains. 


Anaelom, N.J., Ikechukwu, O.J., Sunday, E.W., Nnaemeka, U.C., 2010. Zoonotic tuberculosis: A review of epidemiology, clinical presentation, prevention and control. J. Public Health Epidemiol. 2(6), 118–124.

Davies, P.D.O., 2006. Tuberculosis in humans and animals: Are we a threat to each other? J. R. Soc. Med. 99(10), 539–540. >>

Davydenko, P.O., Bilan, M.V., Tkachenko, O.A., 2010. Sensybilizuval'ni, virulentni vlastyvosti ta lipidnyj sklad M. bovis, bagatorazovo pasazhovanyh cherez shhil'ne zhyvyl'ne seredovyshhe z pH 7,1 [Sensitizing, virulence properties and lipid composition of M. bovis in nutrient medium with pH 7.1]. Veterynarna Medycyna Ukrajiny 2, 20–22 (in Ukrainian).

De la Rua-Domenech, R., 2006. Human Mycobacterium bovis infection in the United Kingdom: Incidence, risks, control measures and review of the zoonotic aspects of bovine tuberculosis. Tuberculosis 86, 77–109. >>

Downing, K.J., Mischenko, V.V., Shleeva, M.O., Young, D.I., Young, M., Kaprelyants, A.S., Apt, A.S., Mizrahi, V., 2005. Mutants of Mycobacterium tuberculosis lacking three of the five rpf-like genes are defective for growth in vivo and for resuscitation in vitro. Infect. Immun. 73, 3038–3043. >>

Glickman, M.S., Jacobs, Jr, W.R., 2001. Microbial pathogenesis review of Mycobacterium tuberculosis: Dawn of a discipline. Cell 104, 477–485. >>

Haddad, N., Masselot, M., Durand, B., 2004. Molecular differentiation of Mycobacterium bovis isolates. Review of main techniques and applications. Res. Vet. Sci. 76, 1–18. >>

Krauss, H., Weber, A., Appel, M., Enders, B., Isenberg, D.H., Schiefer, G.H., Slenczka, W., von Graevenitz, A., Zahner, H., 2003. Zoonoses: Infectious diseases transmissible from animals to humans. 3rd ed. Amer. Society for Microbiology.

Model, L.M., 1952. Byologyja y byohymyja tuberkuleznyh mykobakteryj [Biology and biochemistry of Mycobacterium]. AMN SSSR, Moscow (in Russian).

O’Reilly, L.M., Daborn, C.J., 1995. The epidemiology of Mycobacterium bovis infections in animals and man: A review. Tuber. Lung. Dis. 76, 1–46. >>

Pavlik, I., Jahn, P., Dvorska, L., Bartos, M., Navotny, L., Halouzka, R., 2004. Mycobacterial infections in horses: A re-view of the literature. Vet. Med. Czech. 49(11), 427–440.

Perez-Lago, L., Navarro, Y., Garcia-de-Viedma, D., 2014 Current knowledge and pending challenges in zoonosis caused by Mycobacterium bovis: A review. Res. Vet. Sci. 97, S94S100. >>

Sherman, D.R., Voskuil, M., Schnappinger, D., Liao, R., Harrell, M.I., Schoolnik, G.K., 2001. Regulation of the Mycobacterium tuberculosis hypoxic response gene encoding alphacrystallin. Proc. Natl. Acad. Sci. USA 98, 7534–7539. >>

Thoen, C.O., Steele, J.H., Kaneene, J.B. (eds.), 2014. Zoonotic tuberculosis Mycobacterium bovis and other pathogenic mycobacteria. 3rd ed. John Wiley and Sons, Chichester, UK.

Tkachenko, O.A., Bilan, M.V., Miskiv, V.V., Davydenko, P.O., Zazhars'kyj, V.V., 2010. Biologichni vlastyvosti dysociatyvnyh form M. bovis: Kul'tural'ni osoblyvosti za temperatur 3 i 37 °C [Biological properties of dissociative forms of M. bovis: Cultural features at 3 and 37 °C]. Veterynarna Medycyna Ukrajiny 3, 33–35 (in Ukrainian).

Tkachenko, O.A., Bilan, M.V., Zazhars'kyj, V.V., Davydenko, P.O., Miskiv, V.V., Koval'ov, A.V., 2010. Biologichni vlastyvosti dysociatyvnyh form M. bovis: Morfologichni oznaky ta tynktorial'ni vlastyvosti za temperatur 3 ta 37 °C [Biological properties of dissociative forms of M. bovis: Morphological characteristics and properties at 3 and 37 °C]. Veterynarna Medycyna Ukrajiny 12, 27–30 (in Ukrainian).

Tkachenko, O.A., Galatjuk, O.J., Bilan, M.V., 2008. Morfologichni aspekty reversiji nekyslotostijkyh nytkopodibnyh M. bovis v bakterial'nu kyslotostijku formu [Morphological aspects of M. bovis reversion in bacterial acid form]. Suchasni problemy tuberkul'ozu v Ukrajini: Prychyny ta shljahy jih podolannja [Modern problems of tuberculosis in Ukraine: Causes and ways to overcome them]. Kyiv. P. 149–153 (in Ukrainian).

Tufariello, J.M., Jacobs, W.R., Chan, J., 2004. Individual Mycobacterium tuberculosis resuscitation-promoting factor homologues are dispensable for growth in vitro and in vivo. Infect. Immun. 72, 515–526. >>

Voskuil, M.I., Schnappinger, D., Visconti, K.C., Harrell, M.I., Dolganov, G.M., Sherman, D.R., Schoolnik, G.K., 2003. Inhibition of respiration by nitric oxide induces a Mycobacterium tuberculosis dormancy program. J. Exp. Med. 198, 705–713. >>

Yuan, Y., Crane, D.D., Barry, C.E. 3rd, 1996. Stationary-phaseassociated protein expression in Mycobacterium tuberculosis: Function of the mycobacterial alphacrystallin homolog. J. Bacteriol. 178, 4484–4492.