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Proteinogenic properties of the production strains of M. bovis (Valle and AN 5)
The main method of aftermortem diagnosis of tuberculosis in animals is the allergic test with the use of PPD-tuberculin for mammals. Preparations for the allergic testing of tuberculosis in animals and birds, including "Tuberculin purified (PPD) for mammals in a standard solution" (TUU 24.00497087.645-2001), PPD-tuberculin for birds (TUU 24.4.00497087-675-2002) and allergen from atypical mycobacteria (AAM) (TUU 24.400497087-697-2003) were developed by a group of authors (Kassich Y.A., Zavgorodniy A.I, Kassich V.Y., et al.) at NSC IECVM. They are manufactured by Sumy Biological Factory and used during planned diagnostic tests for tuberculosis in Ukraine.
However, it should be noted that the legislative act applied by member countries of the European Union is the Council Directive 97/12/EU from 17th March 1997. According to this document, intravital allergic studies in animals are conducted using tuberculin PPD (Protein purified derivative) or HCSM (Heat-concentrated synthetic-medium tuberculin).
According to the EU standards, which were provided by the Institute voor Dierhouderij en Diergenzondheid (ID-DLO), Lelistad, the Netherlands, PPD mammalian tuberculin must have the efficiency of 50 000 CTU/ml and be produced from M. bovis AN5 or Valle strains. At the same time "Tuberculin purified (PPD) for mammals in standard solution" TUU 24.00497087.645-2001, produced from «M. bovis IECVM-1» strain is used in the similar testing in Ukraine. Therefore the development of national dry purified tuberculin PPD from M. bovis «AN5» or «Valle» strains is an urgent task. That is why, the aim of our work was to study the proteinogenic properties of M. bovis Valle (modified KSP) and AN5 production strains with further development of national preparations for the allergic diagnosis of tuberculosis in animals that meet the EU requirements.
The test series of the purified tuberculin (PPD) for mammals in the standard solution were prepared from the culture filtrate of bovine tuberculosis causative agent M. bovis, Valle-KSP and AN5 strains, grown in a Sauton’s liquid synthetic culture medium. The PPD was obtained by autoclaving (100оС for 3 hours) and separation of bacterial mass; obtaining and sterilization of cultural filtrates (sterilizing filtration); precipitation of protein with trichloroacetic acid solution; reprecipitation with saturated solution of ammonium sulfate; removal of salts through dialysis followed by determining the concentration of protein in 1 cm3 of solution. Determination of the protein mass fraction in a standard tuberculin solution was performed by Kjeldahl method. According to current requirements, the protein mass fraction in tuberculin must be (0,8±0,2) mg/cm3. The mass fraction of protein in the tuberculin series (X1) produced from M. bovis Valle (modified KSP) strain was (0,86±0,07) mg/cm3 and in tuberculin series (X2) produced from M. bovis AN-5 strain was (0,87±0,1) mg/cm3.
Thus, the results of this study show that M. bovis Valle (modified KSP) and AN5 strains are technological, highly proteinogenic and can be used for the development, production and implementation as a national tuberculin PPD for mammals, which will meet the EU requirements.
Key words: tuberculosis, tuberculin, mycobacterium M. bovis «AN5» or «Valle».
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