Allergic activity and specificity of tuberculin preparations with 30-50% of weakly secreted mycobacterial antigens of tuberculosis

Bovine tuberculosis remains a global problem. An intracutaneous test with tuberculin is the main method for determining the status of herds, which poses special requirements for the activity and specificity. The basis of cotemporal tuberculins are antigens of tuberculosis mycobacteria easily secreted to the liquid synthetic medium during growth, but a range of antigens with a low secretion index are in composition of tuberculins in small quantities. The purpose of the research is to obtain weakly secreted antigens from a production waste – autoclaved bacterial mass of production strain of tuberculosis mycobacteria (MTB) using ultrasound and nonionic detergent, to study the diagnostic properties of tuberculosis with 30-50% of such antigens. It has been determined that autoclaved bacterial mass of industrial MBT strain, which is a waste of tuberculin production, can be an additional source of tuberculoproteins, which are low-secreting (LS) MBT antigens, which in an equivalent dose are about 30% more active compared to standard tuberculin based on easily secreted antigens and is not inferior in terms of species specificity. Whereas, up to 50% of purified LS of tuberculoproteins from the bacterial mass can be included in tuberculin composition. The obtained preparation is not reactogenic, in an equivalent dose it does not differ in terms of activity from the international standard for PPD of tuberculin, but surpasses it in terms of species specificity. It has been shown that in herds with an undetermined tuberculosis status, 2.2 times more cows respond to tuberculins with 30-50% of purified LS tuberculoproteins compared to standard preparations based on easily secreted antigens of tuberculosis mycobacterium. Profound studies of reacting cows using methods for detecting the genome of tuberculosis mycobacterium and bacteriological markers of tuberculosis infection have confirmed the presence of latent tuberculosis infection in cow body. The inclusion of up to 50% of tuberculoproteins from the bacterial mass in tuberculin increases the diagnostic properties of the target product and significantly reduces its price cost. 

1. World Organisation for Animal Health. Terrestrial animal health code. 28th ed. Paris, OIE, 2019. 2 vol.

2. Monaghan M. L., Doherty M. L., Collins J. D., Kazda J. F., Quinn P. J. The tuberculin test. Veterinary Microbiology, 1994, vol. 40, no. 1-2, pp. 111-124. https://doi.org/10.1016/0378-1135(94)90050-7

3. Cousins D. V. Mycobacterium bovis infection and control in domestic livestock. Revue Scientifique et Technique de l’OIE, 2001, vol. 20, no. 1, pp. 71-85. https://doi.org/10.20506/rst.20.1.1263

4. World Organisation for Animal Health. Manual of diagnostic tests and vaccines for terrestrial animals 2012: OIE Terrestrial manual 2012. 7th ed. Paris, 2012. 2 vol.

5. Seibert F. B., Glenn J. T. Tuberculin purified protein derivative: preparation and analyses of a large quantity for standard. American Review of Tuberculosis, 1941, vol. 44, no. 1, pp. 9-25.

6. Pemberton J. R. Chemical, physical, and immunological characteristics of purified protein derivatives from Mycobacterium bovis: dissertations. Ames, Iowa, 1971. 115 p. https://doi.org/10.31274/rtd-180813-2247

7. Lysenko A. P., Bezgin V. M., Kozlov V. E., Pritychenko A. N. Obtaining a highly active and specific allergen for the mass diagnosis of tuberculosis in cattle using ultrafiltration. Veterinarnaya meditsina Belarusi [Veterinary Medicine of Belarus], 2001, no. 4-1 (2002), pp. 12-14 (in Russian).

8. Lysenko A. P. Antigenic composition of PPD tuberculin for mammals. Veterinariya [Veterinary], 1989, no. 5, pp. 3032 (in Russian).

9. Honda J. R., Virdi R., Chan E. D. Global environmental nontuberculous mycobacteria and their contemporaneous man-made and natural niches. Frontiers in Microbiology, 2018, vol. 9, art. 2029. https://doi.org/10.3389/fmicb.2018.02029

10. Sharov A. N. Allergic tuberculosis diagnosis of animals and increasing of its effectiveness. Abstract of Ph.D. diss. Moscow, 1989. 37 p. (in Russian).

11. Naimanov A. Kh., Kalmykov V.M., Kalmykova M.S., Mukovnin A. A. Identification of cattle indected with tuberculosis in the state of anergy to ruberculin. Veterinariya, zootekhniya i biotekhnologiya = Veterinary Medicine, Zootechnics and Biotechnology, 2019, no. 1, pp. 17-21 (in Russian). https://doi.org/10.26155/vet.zoo.bio.201901003

12. Kozlov V. E. Allergens for tuberculosis diagnosis: production improvement and standardization. Abstract of Ph.D. diss. Moscow, 2007. 43 p. (in Russian).

13. Abou-Zeid C., Ratliff T. L., Wiker H. G., Harboe M., Bennedsen J., Rook G. A. Characterization of fibronectin-binding antigens released by Mycobacterium tuberculosis and Mycobacterium bovis BCG. Infection and Immunity, 1988, vol. 56, no. 12, pp. 3046-3051. https://doi.org/10.1128/IAI.56.12.3046-3051.1988

14. Wiker H. G., Harboe M., Nagai S. A localization index for distinction between extracellular and intracellular antigens of Mycobacterium tuberculosis. Journal of General Microbiology, 1991, vol. 137, no. 4, pp. 875-884. https://doi. org/10.1099/00221287-137-4-875

15. Harboe M., Wiker H. G., Ulvund G., Lund-Pedersen B., Andersen Å. B., Hewinson R. G., Nagai S. MPB70 and MPB83 as indicators of protein localization in mycobacterial cells. Infection and Immunity, 1998, vol. 66, no. 1, pp. 289-296. https://doi.org/10.1128/iai.66.1.289-296.1998

16. Olds G. R., Sanson A. J., Daniel T. M. Characterization of Mycobacterium tuberculosis antigen 5 epitopes by using a panel of 19 monoclonal antibodies. Journal of Clinical Microbiology, 1987, vol. 25, no. 3, pp. 471-475. https://doi.org/10.1128/ jcm.25.3.471-475.1987

17. Wiker H. G. MPB 70 and MPB 83 - major antigens of Mycobacterium bovis. Scandinavian Journal of Immunology, 2009, vol. 69, no. 6, pp. 492-499. https://doi.org/10.1111/j.1365-3083.2009.02256.x

18. Hall M. R., Thoen C. O. Preparation of biologically active components of Mycobacterium bovis using Triton X-100 or potassium chloride. American Journal of Veterinary Research, 1983, vol. 44, no. 8, pp. 1602-1604.

19. Lysenko A. P., Vlasenko V. V., Krasnikova E. L., Van Kh. Evaluation of the efficiency of thermal disinfection of milk from tuberculin-positive cows using methods for detecting CWD forms of mycobacterium tuberculosis. Ekologiya i zhivotnyi mir [Ecology and Fauna], 2017, no. 1, pp. 41-47 (in Russian).

20. Lysenko A. P., Vlasenko V. V., Broxmeyer L., Lemish A. P., Novik T. P., Bukova N. K., Mikhalevich E. A., Bogdanovich S. V. The phenomenon of variability of mycobacterium tuberculosis and its use for the detection of tuberculosis infection. Tuberkulez - global’naya katastrofa chelovechestva: epidemiologicheskie, kliniko-diagnosticheskie, mediko-sotsial’nye i organizatsionno-pravovye aspekty protivotuberkuleznoi pomoshchi v stranakh SNG: materialy I Mezhdunarodnoi zaochnoi nauchno-prakticheskoi konferentsii, 24 marta 2014 g. [Tuberculosis as a global disaster of the mankind: epidemiological, clinic diagnostic, medico-social, organizational and legal aspects of anti-tuberculosis care in the CIS countries: proceedings of I International correspondence scientific and practical conference, March 24, 2014]. Rostov-on-Don, 2014, pp. 176-198 (in Russian).

21. Lysenko A. P., Vlasenko V. V., Krasnikova E. L., Lemish A. P., Aksenchik M. A., Pritychenko A. N. Is bovine leukemia virus a virus-like form of mycobacterium tuberculosis? Ekologiya i zhivotnyi mir [Ecology and Fauna], 2019, no. 1, pp. 15-24 (in Russian).

22. Lysenko A. P., Broxmeyer L., Vlasenko V. V, Krasochko P. A., Lemish A. P., Krasnikova E. L. Cwd tuberculosis found in spongiform disease formerly attributed to prions: its implication towards mad cow disease, scrapie and Alzheimer’S. Journal of MPE Molecular Pathological Epidemiology, 2017, vol. 3, no. 3:3, pr. 1-13. https://doi.org/10.5281/zenodo.894183

23. Vysotskii A. E., Lysenko A. P., Kuchval’skii M. V., Krasnikova E. L., Prokopenko T. M. Mycobacterium tuberculosis after lethal exposure to disinfectants can restore viability in the form of mycobacteria with a defective cell wall. Epizootologiya. Immunobiologiya. Farmakologiya. Sanitariya [Epizootology. Immunobiology. Pharmacology. Sanitation], 2019, no. 2, pp. 26-35 (in Russian).