Bulletin "Veterinary biotechnology"

Veterynarna biotehnologija – Veterinary biotechnology, 2016, 28, 217-225 [in Ukrainian].

RYZHENKO G.F., GORBATYUK O.I., ANDRIYASCHUK V.A., ZHOVNIR O.M., KAMENCHUK P.P., TIUTIUM S.M., e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it.

Institute of Veterinary Medicineof NAAS

DYBKOVA S.М., RIEZNICHENKO L.S., GRUZINA Т.G., e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it.

F.D.Ovcharenko Institute of Biocolloidal Chemistry


Introduction. Vaccines’ testing using the in vitro genotoxicity and cytotoxicity estimation and mutagenicity in vivo is very informative and high prognostic because of the possibility to predict malignant degeneration of the eukaryotic cells as well as risk for posterity health in the case of changes in the DNA of animals’ reproductive cells.

The goal of the work was the estimation of biosafety and biocompatibility of the multicomponent vaccine “Multisuisan”.

Materials and methods of research. A series of dilutions of different concentrations of veterinary vaccine “Multisuisan” (10 concentrations in total) were prepared in a nutrient medium for the incubation of cells at a 1.5-fold step.

 Genotoxicity estimation in vitro for “Multisuisan” vaccine has been analyzed by the Comet assay method (alkaline gel-electrophoresis of isolated eukaryotic cells) using eukaryotic cell culture PTP (testicles of pigs) from the Bank of Cell Lines from Human and Animal Tissues of R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology of NAS of Ukraine.

The estimation cytotoxicity in vitro for “Multisuisan” vaccine has been tested by the method of trypan blue using eukaryotic cell culture PTP.

For the in vivo estimation of mutagenicity of “Multisuisan” vaccin was performed using laboratory mice. All experiments with laboratory animals have been carried out in compliance with “Guide for the Care and Use of Laboratory Animals”. Mouses were challenged with intravenous and intramuscular injection with 0.5 ml of “Multisuisan” vaccine. Vaccination was performed thrice, according to the specification of the vaccine. Experimental group consisted of 5 animals. The animals were kept in the vivarium in accordance with the appropriate sanitary regulations on a standard diet with 12-hour light regime and free access to food and water. Micronucleus test in vivo has been performed according to standard protocols.

Results of research and discussion. Vaccine “Multisuisan” showed a tendency to 100% cell survival at a 13-fold dilution. IC50 was observed at a 6-fold dilution for this vaccine.  The experimental investigations of veterinary vaccine“Multisuisan”showed the low level of cytotoxic effect.

When using the Comet assay method for testing of veterinary vaccine  “Multisuisan” genotoxicity the electrophoretic tracks of "DNA-comets" type have been obtained in the positive control samples, where PTP cell culture have been treated by 1 mM N-nitrosomethylurea and ethidium bromide. In the negative control samples the electrophoretic tracks of “DNA-comets” type have been absent. The genotoxicity indexes «ІDNA» for all dilutions of different concentrations of investigated veterinary vaccine were close to the level of these data for negative control.

By the micronucleus test in vivo, absence of the mutagenic effect of vaccine “Multisuisan”, has been shown.

Conclusions and prospects for further research. Investigated “Multisuisan” vaccine are biosafe and biocompatibility according to the genotoxicity, cytotoxicity and mutagenicity parameters. Fulfilled investigations open perspectives for improvement of the system for estimation of vaccines safety according to the parameters of their influence on animals’ genetic apparatus.

Keywords: “Multisuisan”, biosafety, biocompatibility, cytotoxicity, genotoxicity, mutagenicity.


  1. Kamenchuk, P.P, Tiutiun, S.M. Ukhovska, T.M et al. (2015). Rozroblenna multicomponentnogo profilaktychnogo preparaty proty naiposhyrenishich bacterioziv svyney v Ukrayni [The development of multicomponent preventive preparation against common bacterial diseases of pigs in Ukraine – the vaccine “Multisuisan”]. Bjuleten’ “Veterynarna biotehnologija” – Bulletin “Veterinary Biotechnology”, 27, 121-131 [in Ukrainian].
  2. Trachtenberg, I.M., Ulberg, Z.R., Chekman, I.S. et al. (2013). Ocinka bezpeki likarskich nanopreparativ: Metodychni rekomendacii’ [Safety assessment of drugs nanopreparation: Мethodological guidelines]. Kyi’v, 108 [in Ukrainian].
  3. Dybkova, S.M., Gruzina, T.G., Ulberg, Z.R. et al. (2010). Ocinka bezpeki nanomaterialiv organichnoi ta neorganichnoi prirody metodom vyznachenna genotoksychnosti lujnim gel-electroforezom izoluovanich eukariotychnych clityn: Metodychni rekomendacii’, [Biosafety assessment of nanomaterials of organic and inorganic nature by definition of genotoxicity by alkaline gel electrophoresis of isolated eukaryotic cells: Guidelines]. Kyi’v, 24 [in Ukrainian].
  4. Pinaev, G.P. (1988). Metody cyltivirovania cletok [Methods of cells culture: Guidelines] L:Nauka [in Russian].
  5. Benites, C.I. at al. (2006). Micronucleus test on gas station attendants. Genet. Mol. Res.,5, 45-54.
  6. Dybkova, S.M., Rieznichenko, L.S., Gruzina, T.G. et al. (2011). Ocinka mutagennosti nanomaterialiv: Metodychni rekomendacii’ [Assessment of nanomarerials’ mutagenicity: Guidelines]. Kyi’v, 23 [in Ukrainian].
  7. Test Method Protocol for the NHK Neutral Red Uptake Cytotoxicity Assay Phase III - Validation Study: November 4, 2003.
  8. Dybkova, S.M., Hodovskyi, O. V., Romanko, M.Y et al. Sposib ocinki genotoxychnych vlastyvostey nanomaterialiv. [Method for evaluation of genotoxic properties of nanomaterials] Patene UA on useful modelМPК (2009.01) G01N33/00 G01N33/48 2010.
  9. Didenko, V.V. (Eds) (2002). Methods in Molecular Biology. In Situ Detection of DNA Damage. Methods and protocols. Totowa: Humana press.

Download full text in PDF