Inactivation of African swine fever virus inoculated in liquid plasma by spray drying and storage for 14 days at 4°C or 20°C.

African swine fever virus (ASFV) is a dsDNA virus that can cause high mortality in pigs of all ages. Spray-dried porcine plasma (SDPP) is a highly digestible ingredient used in feed because it benefits performance, gut function and immunity. The objectives were to test if the spray-drying (SD) conditions along with post-drying storage of product for 14 days can inactivate ASFV inoculated in liquid plasma. Fresh liquid porcine plasma was inoculated with ASFV (BA71V) to a final concentration of 105.18 ±0.08 TCID50/mL of liquid plasma. Triplicate 2-L samples of spiked plasma were SD in a lab drier set at an outlet temperature of 80°C or 71°C. The final dried samples were stored at 4°C or 20°C for 14 d. Liquid and SD samples were analyzed for ASFV infectivity in two mirror 24-well plaques containing VERO cells monolayers. Wells were inoculated with different dilutions of SDPP dissolved 1:9 in PBS. One plaque was immediately frozen at -80°C and the other was incubated at 37°C for 3 d. Each dilution was replicated 9 times. After incubation both plaques were analyzed for ASFV by qRT-PCR. Results indicated that the SD process inactivated between 3.2 to 4.2 Logs ASFV TCID50/mL and 2.53 to 2.75 Logs TCID50/mL when the outlet temperature were 80°C and 71°C respectively. All SD samples stored at 4°C or 20°C for 14 d were absent of infectious ASFV. The combination of SD and post drying storage at both temperatures for 14 d was able to inactive >5.18 ±0.08 Log10 of ASFV inoculated in liquid porcine plasma, demonstrating that the manufacturing process for SDPP can be considered safe regarding ASFV.

Effect of spray-drying and ultraviolet C radiation as biosafety steps for CSFV and ASFV inactivation in porcine plasma.

Spray-dried animal plasma (SDAP) is widely used in diets of domestic animals to improve health status and increase growth and feed efficiency. Individual steps in the SDAP manufacturing process, including spray-drying, have been validated to inactivate potential pathogens. Manufacturing standards have established a minimum exit temperature of 80°C and a minimum post-drying storage period of 14 days at 20°C for production of SDAP. Also, UV-C irradiation has been evaluated as another inactivation step that could be included in the manufacturing process. The aim of this study was to assess the inactivation effectiveness of spray-drying on Classical swine fever virus (CSFV) and African swine fever virus (ASFV) and the effect of UV-C inactivation on ASFV as redundant biosafety steps of the manufacturing process for producing spray-dried porcine plasma (SDPP). This study demonstrated that UV-C treatment of liquid porcine plasma can inactivate more than 4 Log10 TCID50/mL of ASFV at 3000 J/L. Spray-drying effectively inactivated at least 4 Log10 TCID50/mL of both CSFV and ASFV. Incorporating UV-C technology within the SDAP manufacturing process can add another biosafety step to further enhance product safety.

Commercial feed containing porcine plasma spiked with African swine fever virus is not infective in pigs when administered for 14 consecutive days.

The objective of this study was to determine if commercially collected liquid porcine plasma contaminated with African swine fever virus (ASFV) and fed for 14 consecutive days would infect pigs. Commercially collected liquid porcine plasma was mixed with the serum from an ASFV experimentally infected pig. To simulate the potential of pigs slaughtered being ASFV viremic but asymptomatic and passing antemortem inspection, the ratio of liquid plasma from healthy animals to serum from an ASFV infected pig used in this study represented 0.4% or 2.0% of the pigs slaughtered being viremic (Studies 1 or 2, respectively). The contaminated liquid plasma was mixed on commercial feed and pigs were fed for 14 consecutive days providing to each pig 104.3 or 105.0 TCID50 ASFV daily (Studies 1 or 2, respectively). Pigs were observed for an additional 5 or 9 days (Studies 1 or 2, respectively). In both experiments, the pigs did not become infected with ASFV during the 14d feeding period or during the subsequent observation period. In these experiments, unprocessed liquid plasma contaminated with ASFV mixed on commercial feed and fed for 14 consecutive days did not infect pigs. From our results we can conclude that the infectious dose of ASFV on feed is much higher than that previously reported, at least with ASFV-spiked raw plasma.

Biosafety steps in the manufacturing process of spray-dried plasma: a review with emphasis on the use of ultraviolet irradiation as a redundant biosafety procedure.

Spray dried plasma (SDP) is a functional protein source obtained from blood of healthy animals, approved by the veterinary authorities from animals declared to be fit for slaughter for human consumption. Blood of these animals 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 (80 °C throughout its substance) to convert it in a powder. Such method preserves the biological activity of its proteins, mainly albumins and globulins. 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. This review provides an evaluation and validation of the different safety steps present in the manufacturing process of SDP, with special focus on a new redundant pathogen inactivation step, the UV-C irradiation, that may be implemented in the manufacturing process of the SDP. Overall results showed that the manufacturing process of SDP is safe and the UV-C radiation was effective in inactivating a wide range of bacteria and viruses spiked and naturally present in commercially collected liquid animal plasma and it can be implemented as a redundant biosafety step in the manufacturing process of the SDP.

Evaluation of the effectiveness of the SurePure Turbulator ultraviolet-C irradiation equipment on inactivation of different enveloped and non-enveloped viruses inoculated in commercially collected liquid animal plasma.

The objective of this study was to evaluate the effectiveness of the SurePure Turbulator ultraviolet-C (UV-C, 254 nm wavelength) irradiation equipment on inactivation of different enveloped and non-enveloped viruses in commercially collected liquid animal plasma. Specifically, Pseudorabies virus (PRV), Porcine reproductive and respiratory syndrome virus (PRRSV), Porcine epidemic diarrhea virus (PEDV), Bovine viral diarrhea virus (BVDV), Classical swine fever virus (CSFV), Swine influenza virus (SIV) as enveloped viruses and Porcine parvovirus (PPV), Swine vesicular disease virus (SVDV), Porcine circovirus type 2 (PCV-2) and Senecavirus A (SVA) as non-enveloped viruses, were inoculated in bovine or porcine plasma and subjected to different UV-C irradiation doses (0, 750, 1500, 3000, 6000 and 9000 J/L) using an UV-C device developed for opaque liquid working under turbulent flow. The enveloped viruses tested were inactivated at < 3000 J/L of UV-C, being the dose needed to inactivate 4 log TCID50 (4D) of 1612 J/L for PRV,1004 J/L for PRRSV, 1953 J/L for PEDV, 1639 J/L for SIV, 1641 J/L for CSFV and 1943 J/L for BVDV. The non-enveloped viruses tended to have higher 4D values: 2161 J/L for PPV, 3223 J/L for SVA and 3708 J/L for SVDV. Because the initial viral concentration was <4.0 Log for PCV-2, it was not possible to calculate the 4D value for this virus. In conclusion, these results demonstrated that the SurePure Turbulator UV-C treatment system is capable of inactivating significant levels of swine viruses inoculated in commercially collected porcine or bovine plasma. It was concluded that irradiation with UV-C can provide an additional redundant biosafety feature in the manufacturing process of spray-dried animal plasma.

Ultraviolet (UV-C) inactivation of Enterococcus faecium, Salmonella choleraesuis and Salmonella typhimurium in porcine plasma.

The objective of this study was to assess the effectiveness of an ultraviolet (UV-C, 254 nm) irradiation system on reducing the load of Salmonella typhimurium (S. typhimurium), Salmonella choleraesuis (S. choleraesuis) resistant to streptomycin and Enterococcus faecium (E. faecium) inoculated in sterile porcine plasma and then subjected to different UV-C irradiation doses (750, 1500, 3000, 6000 and 9000 J/L) using a pilot plant UV-C device working under turbulent flow. Results indicated that UV-C treatment induced a viability reduction of 0.38, 1.18, 3.59, 4.72 and 5.06 log10 S. typhimurium when irradiated at 750, 1500, 3000, 6000 and 9000 J/L, respectively. The observed log10 reduction of S. choleraesuis was 1.44, 2.68, 5.55, 7.07 and 7.97 at 750, 1500, 3000, 6000 and 9000 J/L, respectively. The best-fit inactivation for S. choleraesuis was the Weibull distribution curve, while the best-fit curve for S. typhimurium was the Weibull plus tail model, indicating that around 102 cfu/mL resistant S. typhimurium was detected when the liquid plasma was UV-C irradiated at doses up to 9000 J/L. Viability reduction for E. faecium was 0.44, 1.01, 3.70, 5.61 and 6.22 log10 when irradiated at 750, 1500, 3000, 6000 and 9000 J/L, respectively, with no bacterial resistance observed with UV-C doses of 6000 J/L or higher. The biphasic model was the best fit model for the inactivation curve for E. faecium. For the three microorganisms tested, about a 4 log-unit reduction was achieved when the liquid plasma was irradiated at 3000J/L. Overall results demonstrate the usefulness of the UV-C system to inactivate bacteria in liquid plasma before spray-drying. We conclude that the UV-C system can provide an additional biosafety feature that can be incorporated into the manufacturing process for spray-dried animal plasma.

Ultraviolet Light (UV) Inactivation of Porcine Parvovirus in Liquid Plasma and Effect of UV Irradiated Spray Dried Porcine Plasma on Performance of Weaned Pigs.

A novel ultraviolet light irradiation (UV-C, 254 nm) process was designed as an additional safety feature for manufacturing of spray dried porcine plasma (SDPP). In Exp. 1, three 10-L batches of bovine plasma were inoculated with 10(5.2 ± 0.12) tissue culture infectious dose 50 (TCID50) of porcine parvovirus (PPV) per mL of plasma and subjected to UV-C ranging from 0 to 9180 J/L. No viable PPV was detected in bovine plasma by micro-titer assay in SK6 cell culture after UV-C at 2295 J/L. In Exp. 2, porcine plasma was subjected to UV-C (3672 J/L), then spray dried and mixed in complete mash diets. Diets were a control without SDPP (Control), UV-C SDPP either at 3% (UVSDPP3) or 6% (UVSDPP6) and non-UV-C SDPP at 3% (SDPP3) or 6% (SDPP6). Diets were fed ad libitum to 320 weaned pigs (26 d of age; 16 pens/diet; 4 pigs/pen) for 14 d after weaning and a common diet was fed d 15 to 28. During d 0 to 14, pigs fed UVSDPP3, UVSDPP6, or SDPP6 had higher (P < 0.05) weight gain and feed intake than control. During d 0 to 28, pigs fed UVSDPP3 and UVSDPP6 had higher (P < 0.05) weight gain and feed intake than control and SDPP3, and SDPP6 had higher (P < 0.05) feed intake than control. Also, pigs fed UVSDPP had higher (P < 0.05) weight gain than pigs fed SDPP. In conclusion, UV-C inactivated PPV in liquid plasma and UVSDPP used in pig feed had no detrimental effects on pig performance.

Determination of the presence of bovine immunoglobulin G in liquid or spray-dried porcine plasma and whole blood by agar gel immunodiffusion.

There is currently urgent interest in identifying the species of origin of the components of different animal by-products. In Europe, this interest is expected to increase with authorization of the re-introduction of these proteins into animal feed formulations. The number of validated methods to differentiate the species of origin for most of these products is limited. An easy, inexpensive, and accurate test was developed to determine the cross-contamination of bovine blood or plasma in porcine whole blood and plasma, both before and after spray drying. Agar gel immunodiffusion (AGID), the studied technique, detected the presence of bovine immunoglobulin G (IgG) in porcine plasma and in whole blood at inclusion levels >0.5% (v/v) in all cases. However, detectability was lower in liquid plasma (0.3%, v/v) and in liquid whole blood (0.5%, v/v). No differences were found when cross-contamination was simulated before or after whole blood centrifugation. The method described is reliable and inexpensive, and the samples are easy to prepare. Both minimal laboratory equipment and expertise are required for detection of bovine IgG in porcine blood products at inclusion levels of >0.5% (v/v).