Viral safety of biologicals includes certification of freedom from extraneous
agents. In particular poultry vaccines – where embryonated hen eggs and primary cell cultures are used for vaccine production – impose the risk of contamination with unwanted microorganisms. The current official requirements also involve testing in animals. To replace the use of animals for the quality control of batches of avian viral vaccines (the Three Rs approach) an alternative testing scheme using polymerase chain reaction (PCR) has been developed. Replacing the current protocols for identification of Mareks disease virus (MDV) serotypes as extraneous agents by PCR could be advantageous.
The object of this project was to establish and validate an in-vitro method to detect possible viral contaminants in the quality control of biologicals. The European Pharmacopoeia (Ph. Eur.) allows the use of alternative methods if they are validated and in agreement with the national authorities. Two novel independent PCR assays have been designed to amplify parts of the UL19 and gA regions of MDV serotype 1, serotype 2 and HVT. All MDV vaccine strains CVI988/Rispens, SB-1 and PB-HTV1 as well as field isolates from Germany, The Netherlands and Hungary could be specifically detected. The identity of the amplified fragments could be confirmed by sequence analysis.
The sensitivities of the PCR assays were demonstrated by serial dilutions series with vaccine virus strains and in spiking and recovery experiments using various poultry vaccines as matrices.
Thus, the single-tube PCR has been shown to be a rapid, sensitive and specific alternative testing method for extraneous agent testing in the quality control of poultry vaccines.
