A new lymphocyte proliferation assay for potency determination of bovine tuberculin PPDs.
The tuberculin skin test is the method of choice for tuberculosis surveillance in livestock ruminants. The exact definition of the biological activity of bovine tuberculin purified protein derivatives (bovine tuberculin PPDs) is essential for the reliability of a test system. PPDs consist of heterogeneous mixtures of mycobacterial antigens, making it difficult to determine their potency in vitro. The commonly used batch potency test is therefore based on the evaluation of skin reactions in mycobacteria-sensitized guinea pigs. Aim of the present study was to test an alternative in vitro method that reliably quantifies tuberculin PPD potency. This novel approach may prevent animal distress in the future. To this end a flow cytometry-based lymphocyte proliferation assay using peripheral blood mononuclear cells (PBMCs) from sensitized guinea pigs was established. Potency estimates for individual PPD preparations were calculated in comparison to an international standard. The comparison with results obtained from the guinea pig skin test revealed that the lymphocyte proliferation assay is more precise but results in systematically higher potency estimates. However, with a manufacturer specific correction factor a correlation of over 85% was achieved, highlighting the potential of this in vitro method to replace the current guinea pig skin test.
Division of Veterinary Medicine, Paul-Ehrlich-Institut, Langen, Germany.
Christina
C
Spohr
Division of Veterinary Medicine, Paul-Ehrlich-Institut, Langen, Germany.
Eva
E
Kaufmann
Division of Veterinary Medicine, Paul-Ehrlich-Institut, Langen, Germany.
Sibylle
S
Battenfeld
Division of Veterinary Medicine, Paul-Ehrlich-Institut, Langen, Germany.
Karin
K
Duchow
Division Safety of Medicinal Products and Medical Devices, Paul-Ehrlich-Institut, Langen, Germany.
Klaus
K
Cussler
Division of Veterinary Medicine, Paul-Ehrlich-Institut, Langen, Germany.
Elisabeth
E
Balks
Division of Veterinary Medicine, Paul-Ehrlich-Institut, Langen, Germany.
Max
M
Bastian
D000826:Animal Testing Alternatives; D000818:Animals; D001500:BCG Vaccine / Q000008:administration & dosage / Q000276:immunology*; D002417:Cattle; D005434:Flow Cytometry; D006168:Guinea Pigs; D066298:In Vitro Techniques; D007963:Leukocytes, Mononuclear / Q000382:microbiology; D008214:Lymphocytes / Q000502:physiology*; D009169:Mycobacterium tuberculosis / Q000276:immunology; D015203:Reproducibility of Results; D014374:Tuberculin Test; D014376:Tuberculosis / Q000276:immunology / Q000517:prevention & control*; D064166:Vaccine Potency
D001500:BCG Vaccine
Mycobacterium bovis; alternative potency assay; bovine tuberculosis; guinea pig; lymphocyte proliferation assay