Resumen de Ultraviolet light (uv-c) as a redundant biosafety step for the pathogen inactivation in the manufacturing process of spray dried plasma from animal origin
ABSTRACT Spray dried plasma (SDP) is a functional protein source obtained from blood of healthy animals, approved by the veterinary authorities to be fit for slaughter for human consumption. Blood is collected at the slaughterhouse, treated with an anticoagulant, chilled and transported to industrial facilities in which blood is centrifuged to separate the red blood cells from the plasma fraction. Plasma is then concentrated and spray dried at high temperatures (80ºC throughout its substance) to convert it in a powder. Such method preserves the biological activity of its proteins. SDP is mainly used in pig feed diets to significantly improve daily gain, feed intake, production efficiency, and to reduce post-weaning lag caused by the appearance of post-weaning diarrhea.
Although SDP is considered a safe product and its manufacturing process consists of several biosafety steps, the security of the SDP is often questioned due to its nature as raw blood by-product, especially when emergent or re-emergent pathogens appear in animal populations.
This PhD Thesis focused on the evaluation and validation of a new redundant pathogen inactivation step that may be implemented in the manufacturing process of SDP, the UV-C irradiation.
The work has consisted in evaluating the effectiveness of the UV-C irradiation treatment using a turbulent flow device, SurePure TurbulatorTM, when irradiating raw plasma artificially inoculated with different pathogens of interest for the swine industry.
In studies 1 and 2 the UV-C effect on bacterial survival was assessed on Salmonella typhimurium, Salmonella choleraesuis, Enterococcus faecium, and Escherichia coli K88 and K99 strains subjected to different UV-C doses. All tested bacteria showed non-linear inactivation kinetics with 4 log10 (4D) reduction value in all cases close to 3000 J/L.
In study 3, the effect of UV-C on different viruses of interest in the swine industry was analyzed. The selection of enveloped viruses included Pseudorabies virus, Porcine reproductive and respiratory syndrome virus (PRRSV), Porcine epidemic diarrhea virus (PEDV), Bovine viral diarrhea virus, Swine influenza A virus (SIV) and Classical swine fever virus. On the other hand, Porcine parvovirus (PPV), Swine vesicular disease virus, Porcine circovirus 2 (PCV-2) and Senecavirus A (SVA) were chosen as non-enveloped viruses. All these viruses were subjected to different UV-C doses and, by titration of the samples at each UV-C dose, the inactivation curve for each virus was constructed. In general terms, results showed that enveloped viruses have a higher sensitivity to UV-C than non-enveloped ones, being the 4D values less than 2000 J/L for enveloped viruses and around 3000 J/L or higher for non-enveloped ones.
To validate the effectiveness of the plasma UV-C irradiation measured in previous studies, a bioassay was carried out in the study 4 using different groups of piglets inoculated intraperitoneally with UV-C irradiated plasma at 0 (untreated plasma), 3000, and 9000J/L. The results showed that none of the pigs in the groups that received the plasma irradiated by UV-C were infected or seroconverted against the viruses which genome was detected in the initial plasma (PCV-2, PRRSV (European strains), SIV, PPV, Hepatitis E virus and Rotavirus A), thus confirming the efficacy of UV-C.
Furthermore, in the study 4, conventional PCR methods able to generate long amplicons were designed to amplify fragments of approximately 1.7 kb of PCV-2 and PEDV genomes. By comparison of the results with those of real time quantitative PCRs to detect the same viruses (using short amplicons), it was demonstrated that UV-C was able to damage the viral genome.
Overall results of the present PhD Thesis showed that the UV-C SurePure TurbulatorTM design was effective in inactivating a wide range of bacteria and viruses spiked and naturally present in commercially collected liquid animal plasma.
