Veterinary Microbiology 239 (2019) 108450 Contents lists available at ScienceDirect Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic UV-C irradiation is able to inactivate pathogens found in commercially T collected porcine plasma as demonstrated by swine bioassay E. Blázqueza,b, C. Rodrígueza, J. Ródenasa, N. Navarrob, R. Rosellc, S. Pina-Pedrerob, J.M. Campbelld, M. Sibilab, J. Segalése,f, J. Pujolsb, J. Poloa,d,* a APC Europe, S.L.U., Granollers, Spain b IRTA, Centre de Recerca en Sanitat Animal (CRESA, IRTA-UAB), Campus de la Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain c Departament d’Agricultura, Ramaderia, Pesca i Alimentació (DARP) Generalitat de Catalunya, Campus de la Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain d APC Inc, Ankeny, Iowa (IA), United States e UAB, Centre de Recerca en Sanitat Animal (CRESA, IRTA-UAB), Campus de la Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain f Departament de Sanitat i Anatomia Animals, Facultat de Veterinària, UAB, 08193 Bellaterra Spain ARTICLE INFO ABSTRACT Keywords: Liquid porcine plasma is an animal origin raw material for the manufacturing process of spray-dried porcine Porcine plasma plasma that is used in pig nutrition worldwide. In previous studies we found that the application of ultraviolet Pig bioassay light C (UV-C) in liquid plasma that was inoculated with a variety of bacteria or viruses of importance in the Biosafety swine industry can be considered as redundant safety steps because in general achieve around 4 logs reduction PCV-2 for most of these pathogens. However, the final validation of the UV-C light as safety feature should be con- PRSSV ducted with commercial liquid plasma and using the pig bioassay model. As a first objective, the potential infectivity of a raw liquid plasma product collected from an abattoir was tested by means of a swine bioassay. We used Porcine circovirus 2 (PCV-2), a ubiquitous virus that has been systematically detected by PCR in porcine plasma at abattoirs as selection criteria for commercial liquid plasma lot. As a second aim of the study, the effects of different doses of UV-C irradiation on the selected raw liquid plasma were assayed in the animal bioassay. Moreover, other swine infecting agents, including Porcine reproductive and respiratory syndrome virus (PRRSV), were also determined in the original plasma and monitored in the inoculated animals. Pigs negative for PCV-2 and PRRSV genome and antibodies were allotted to one of five groups (6 to 8 pigs/ group) and injected intra- peritoneally with 10 mL of their assigned inoculum at 50 d of age. Negative control pigs (group 1) were injected with PBS. Positive control pigs (group 5) were injected with a PCV-2 inoculum. Groups 2, 3 and 4 were injected with liquid porcine plasma that had been subjected to 0 (raw plasma), 3000 or 9000 J/L UV-C irradiation, respectively. Group 2 pigs (0 J/L UV-C) got infection by PRRSV but no PCV-2 infection or seroconversion. However, one pig from group 2 seroconverted to Rotavirus A (RVA) and Hepatitis E virus (HEV) and three group 2 pigs seroconverted to Porcine parvovirus (PPV). Groups 1, 3 and 4 pigs showed no evidence of infection or seroconversion associated with the tested viruses or any other pathogens found in the liquid plasma before UV-C irradiation. Group 5 pigs developed PCV-2 infectivity as expected. UV-C irradiation of liquid plasma at 3000 and 9000 J/L was effective in preventing PRRSV and other pathogens transmission. Moreover, raw liquid plasma was non-infectious for PCV-2 in naïve pigs. 1. Introduction and survival (Coffey and Cromwell, 2001; Torrallardona, 2010). The manufacturing process of SDPP involves several biosecurity features Plasma from blood of healthy pigs is the raw material used to pro- including collection only from healthy animals at commercial abattoirs duce spray-dried porcine plasma (SDPP), which is a feed ingredient under competent authorities inspection, pooled blood which contains with functional proteins and bioactive components that is widely used antibodies with neutralizing capacity against a variety of pathogens, in pig diets due to its beneficial effects on post-weaning performance spray-drying at 80 ºC throughout its substance, and extended storage ⁎ Corresponding author at: APC Europe, S.L.U. Polígon Industrial Congost, Av. de Sant Julià, 246, 08403 Granollers, Barcelona; Spain. E-mail address: javier.polo@apc-europe.com (J. Polo). https://doi.org/10.1016/j.vetmic.2019.108450 Received 11 July 2019; Received in revised form 30 September 2019; Accepted 6 October 2019 0378-1135/ © 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/). E. Blázquez, et al. Veterinary Microbiology 239 (2019) 108450 time at controlled temperature (Blázquez et al., 2017, 2018). However Table 1 as technology develops, additional redundant safety features should be Presence of antibodies and genome (copies/mL) of PCV-2 in different porcine investigated that follows the recommendations of several international plasma batches, including the selected one (No. 9). agencies (EMEA, 1996; WHO, 2004). Sample PCV-2 genome copies/mL S/P Elisa IgG PCV-2 Ultraviolet light at 254 nm wavelength (UV-C) disrupts cellular transcription and replication, leading to death bacteria, viruses and 1 1.12E+04 0.947 2 1.65E+04 0.864 molds (Keyser et al., 2008) due to the formation of thymine-thymine 3 2.06E+04 0.907 dimers and thymine-cytosine dimers in DNA molecules and cytosine- 4 2.36E+04 0.898 uracil dimers in RNA molecules (Jagger, 1967). UV-C has been ex- 5 2.06E+04 0.880 tensively used for disinfection of water (Hijnen, 2006; Lin et al., 2012), 6 2.71E+04 0.891 7 1.76E+04 0.960 surfaces (Sizer and Balasubramaniam, 1999) and food products 8 5.08E+03 1.011 (Guerrero-Beltran and Barbosa-Cánovas, 2004). 9 2.71E+04 0.834 It is important to validate individual inactivation steps that are in- 10 2.67E+04 0.863 cluded in commercial production processes for SDPP (WHO, 2004). A Cut-off Negative 0.284 swine bioassay including intraperitoneal injection of a test material into Cut-off Positive 0.334 naïve pigs represents an extremely sensitive method to test infectivity (Christopher-Hennings et al., 2012). In a swine bioassay, it is important C radiation to all particles in an opaque liquid. Flow rate was main- to monitor the pigs after injection with the test material for viremia tained at 4000 L/h with the UV lamp switched off. After 5 min of stable and/or seroconversion, which may indicate the presence of infective flow, a positive control (1 L at 0 J/L) sample was collected into an virus in the tested material. This procedure is considered a very sensi- sterile container. Then, the UV-C lamp was switched on and irradiation tive method to test the safety of a specific product such as porcine started. One liter of treated plasma was collected into sterile containers plasma (Sampedro et al., 2015) and can determine if genome detected at 3000 and 9000 J/L, as previously described (Blázquez et al., 2017). by PCR analyses is infective. From the 1-L samples of plasma irradiated at 0 J/L, 3000 J/L and 9000 The objective of the present study was to determine if UV-C irra- J/L UV-C, 10 mL were used for intraperitoneal injection in naïve pigs. diation of liquid commercially collected porcine plasma by a specifi- cally designed UV system developed for large volumes of opaque liquids (SurePure Turbulator) could inactivate infective virus as measured in a 2.3. Animals and housing pig bioassay. The primary aim was to evaluate inactivation of Porcine circovirus 2 (PCV-2) because it is not uncommon for commercially At approximately 14 days of age, blood samples of selected pigs collected plasma to be qRT-PCR positive for this economically im- were collected at the farm of origin to verify a negative result for PRRSV portant virus of concern for the global swine industry. Furthermore, the antibodies and genome; in addition, these animals had low PCV-2 an- presence and possible transmission of other potential contaminating tibody ELISA S/P values (of maternal origin) and were negative for agents of importance for the swine industry, such as Porcine reproductive PCV-2 qRT-PCR. A total of 40 male piglets (25 ± 3 days of age; initial and respiratory syndrome virus (PRRSV) Transmissible gastroenteritis cor- average body weight 5 ± 0.5 Kg) were transported and allocated at the onavirus (TGEV), Swine Influenza A virus (SIV), Porcine parvovirus (PPV), Institut de Recerca i Tecnologia Agroalimentàries (IRTA) experimental Porcine epidemic diarrhea virus (PEDV), Swine rotavirus A (RVA), Bovine farm in Alcarràs (Lleida, Spain), in individual rooms and separated from viral diarrhea virus (BVDV), Border disease virus (BDV), Hepatitis E virus other animals for about three weeks before the start of the study. At the (HEV) and Salmonella enterica were evaluated. experimental farm, piglets were sampled at 35 and 45 days of age, and the experimental groups were established once piglets were proven 2. Materials and methods seronegative by ELISA against PCV-2 and PRRSV at 50 days of age. Three pigs were unthrifty during this period and were excluded from 2.1. Plasma selection the study; the remaining 37 pigs were weighed, ear-tagged and ran- domly distributed in five experimental groups of 6 to 8 pigs per group The criterion for plasma selection was based on the presence of the after matching weights between groups (7 pigs in negative control PCV-2 genome. The plasma batch that presented a greater number of group, 8 pigs in each of the 3 treatment groups and 6 pigs in the positive PCV-2 genomic copies and at the same time a lower antibody titer control group). Each group of animals was allocated in separate boxes against PCV-2 was selected. Ten 10-L batches of liquid porcine plasma and also in different rooms, thus no air space was shared between collected from a commercial abattoir (each batch of plasma was col- groups. Each box had 7.5 m2 of surface area for the pigs. Environmental lected from a plasma pool from 10,000 pigs) were frozen (-20 °C) prior conditions of rooms were maintained at 20-24°C, and an area with a to pre-screen testing for PCV-2 genome and antibodies. The test batch heat lamp source at 30-35°C was included inside each box. Illumination for use in the UV-C test was selected based on the highest number of consisted of natural light. PCV-2 DNA copies measured by real-time quantitative PCR (qRT-PCR) To ensure that no cross contamination between treatment groups or using a test kit (LSI VetMAXTM Porcine Circovirus Type 2 external contamination occurred, rooms were closed, air entry was Quantification, Thermo Fisher Scientific, Massachusetts, USA) and the regulated, and strict biosafety protocols for the caretakers were im- lowest level of PCV-2 antibodies analyzed by ELISA (Ingezim Circo plemented. Caregivers were trained to wear TYVEK (DuPont, Delawe, IgG,11.PCV.K.1/5 ELISA, INGENASA, Madrid, Spain) among the pre- USA) overalls, overcoats, head coverings and gloves at the entrance of screened liquid plasma batches. each room for daily animal care. 2.2. Plasma UV-C irradiation 2.4. Experimental design and sampling Prior to UV-C irradiation, the selected plasma batch (10 L of batch Group 1 (n = 7) represented the negative control group and was #9, Table 1) was thawed and filtered to eliminate potential cryopre- injected with 10 mL of phosphate buffered saline (PBS) solution (Saline cipitate. Solution Vitulia, ERN Laboratories, Barcelona, Spain). Group 2 (n = 8) The UV-C reactor (SP1) was designed and manufactured by Sure pigs were injected with 10 mL of native non-UV-C irradiated plasma Pure Operation AG (Zug, Switzerland). The system is designed to create (raw native liquid plasma). Group 3 (n = 8) animals were injected with turbulent flow (Reynolds number > 2400) insuring the delivery of UV- 10 mL of plasma UV-C irradiated at 3000 J/L. Group 4 (n = 8) pigs 2 E. Blázquez, et al. Veterinary Microbiology 239 (2019) 108450 were injected with 10 mL of plasma UV-C treated at 9000 J/L. Group 5 Hoofddorp, The Netherlands). Antibodies against BVDV, BDV were (n = 6) was used as the positive control group and was injected with 10 evaluated by sero-neutralization tests, following the OIE protocols mL of PCV-2 at a dose of 100 TCID50/mL. All products/inoculum were (www.oie.int). administrated by intra-peritoneal route to all groups. Animal proce- After inoculation, animals were monitored for the development of dures were approved by the committee of ethics and welfare with the viremia and antibodies to pathogens identified in the plasma inoculum protocol approval number CEA-OH/9561/2. (Table 2). Serum samples taken on 50 dpi were analyzed for the pre- Piglets were monitored daily for clinical signs of infection during a sence of antibodies against PCV-2, PRRSV, TGEV, SIV, PPV, RVA, 50-day study period. Blood samples were collected on days 0, 15, 30 PEDV, HEV and S. enterica with the same commercial kits mentioned and 50 post-injection and tested for viremia and antibody seroconver- above. If antibodies were found in the serum samples 50 dpi, the pre- sion against PRRSV, PCV-2, TGEV, SIV, PPV, RVA, PEDV, BVDV, BDV, sence of virus was determined in the same serum samples (50 dpi) by HEV and S. enterica. qRT-PCR. Serum samples (50 dpi) were tested for PCV-2 and PRRSV by At the end of the study, the animals were euthanized by an overdose qRT-PCR. of sodium pentothal (Euthasol 400®; Laboratorios ECUPHAR, Serum collected at 15, 30 and 50 dpi and feces (50 dpi) were tested Barcelona, Spain). All piglets were necropsied and samples of lung, for PPV and RVA. Bile (50 dpi) was tested for the presence of HEV RNA. tonsil, lymph node, bile and feces were collected for further analysis. SIV was analyzed (qRT-PCR) in serum and lung tissue (50 dpi) samples by qRT-PCR with the technique previously cited. 2.5. Laboratory procedures 3. Results The selected plasma batch was tested for the presence of PCV-2 3.1. The selected raw plasma as inoculum was positive for several pathogens genome and antibodies, as indicated previously, and was also analyzed for the presence of other pathogens by commercial qPCR or qRT-PCR Among the 10 porcine plasma batches tested (Table 1), the one with techniques, including PRRSV (LSI VetMAXTM PRRSV EU/NA Real- highest PCV-2 load and lowest PCV-2 antibody values was selected (No. Time PCR Kit. Thermo Fisher Scientific, Massachusetts, USA), TGEV 9). This batch contained 2.71 x 104 PCV-2 DNA copies/mL (Ct = 30.96) (EXOone TGEV, EXOPOL, Zaragoza, Spain), SIV (EXOone Influenza A, and a Ct value of 35.35 for PRRSV European strain (no number of RNA EXOPOL, Zaragoza, Spain), PPV (EXOone Parvovirus, EXOPOL, copies were provided by the technique). Also, it had ELISA S/P ratios of Zaragoza, Spain), PEDV (EXOone PEDV, EXOPOL, Zaragoza, Spain), 0.83 (low to moderate) for PCV-2 and 2.99 (high) for PRRSV anti- RVA (EXOone Rotavirus A, EXOPOL, Zaragoza, Spain), HEV (Path-HEV bodies. Furthermore, the selected plasma used as inoculum had anti- advance from Genesig-Primerdesign, Cambridge, UK), and S. enterica bodies against SIV, RVA, PPV, HEV and S. enterica and presence of low (EXOone Salmonella enterica, EXOPOL, Zaragoza, Spain). BVDV, and amounts of nucleic acid of SIV (Ct = 36.41), RVA (Ct = 37.6), PPV (Ct BDV were analyzed by previously published PCR techniques (Blázquez = 39.44) and HEV (Ct = 37.8) were detected (Table 2). et al., 2019). Besides for PCV-2, the presence of antibodies for the following agents were also evaluated in the plasma batch: PRRSV (IDEXX PRRS 3.2. UV-irradiated plasma did not transmit any virus X3 Ab Test ELISA, IDEXX, Hoofddorp, The Netherlands), TGEV (Ingezim TGEV, INGENASA, Madrid, Spain), SIV (CIVTEST SUIS INF- During the experimental period and at necropsy no clinical signs of LUENZA, Hipra, Amer, Spain), PPV (Ingezim PPV, INGENASA, infection were observed in any of the piglets. Madrid,Spain), RVA (Ingezim Swine Rotavirus, INGENASA, Madrid, The negative control group (group 1) remained free from PRRSV Spain), PEDV (Ingezim PEDV, INGENASA, Madrid, Spain), HEV (ID and PCV-2 and showed absence of seroconversion against those viruses Screen Hepatitis E Indirect Multi-species, IDvet Genetics, Grabels, during the entire study period. In contrast, all piglets from group 5 France) and S. enterica (IDEXX Swine Salmonella Ab, IDEXX, (positive control group, inoculated with PCV-2) became infected with Table 2 Presence of antibodies (Ab) and genome (PCR) of different infectious agents tested in the porcine plasma inoculum and in the different animal groups. Group 1 Group 2 Group 3 Group 4 Group 5 Plasma Inoculum Negative control Non-UV plasma UV 3000J/L plasma UV 9000J/L plasma Positive control PCV-2 Pathogen Ab PCR Ab PCR Ab PCR Ab PCR Ab PCR Ab PCR PCV-2 + + - - - - - - - - + (6/6) + (6/6) PRRSV EU + + - - + (8/8) + (8/8) - - - - - - PRRSV US - - - - - - - - - - - - TGEV - - - na - na - na - na - na SIV + + - na - na - na - na - na PPV + - - - + (3/8) Feces - - - - - - - RVA + + - - + (1/8) Feces - - - - - - - PEDV - na - na - na - na - na - na PRV - na - na - na - na - na - na HEV + + - - + (1/8) Bile - - - - - - - SVDV - na - na - na - na - na - na CSFV - na - na - na - na - na - na BDV - na - na - na - na - na - na S. enterica + - - na - na - na - na - na Plasma inoculum (untreated abattoir collected porcine plasma used for bioassay); Group 1 (negative control group of pigs injected ip with saline); Group 2 (pigs injected ip with untreated abattoir plasma; 0 J/L); Group 3 (pigs injected ip with 3000 J/L UV-C treated plasma); Group 4 (pigs injected ip with 9000 J/L UV-C treated plasma); Group 5 (positive control group of pigs inoculated with PCV-2). Ab (serum antibody); PCR (serum genome); + (positive result); - (negative result); na (not analyzed); (n/n) number of pigs with positive antibodies or genome respect to the total number of pigs in the treatment; Feces (negative fecal result); Bile (negative bile result). 3 E. Blázquez, et al. Veterinary Microbiology 239 (2019) 108450 PCV-2 within 15 dpi as measured by qRT-PCR. These pigs had already confirms that a positive PCR test does not imply that the pathogen is seroconverted against PCV-2 by the following sampling at 30 dpi. infective. Conversely, group 2 pigs, who received untreated liquid All piglets in group 2 injected with raw native plasma that was not plasma, seroconverted against PRRSV, being present in the initial subjected to UV-C (0 J/L) were found PRRSV positive by qRT-PCR and inoculum at a Ct value of 35.35. seroconverted by 15 dpi. No evidence of PCV-2 infection by qRT-PCR or SIV RNA was detected in the raw plasma by qRT-PCR, but with a ELISA was detected in group 2 (0 J/L UV-C) piglets. Three piglets of this high Ct number. Since no piglets seroconverted to the virus and no group seroconverted against PPV at 50 dpi, without evidence of viral presence of SIV was found in the analyzed samples (sera and lung qRT- excretion in feces at 50 dpi. One piglet also seroconverted against RVA PCR), it is very likely that the very low load of SIV present in the in- at 50 dpi, having a negative qRT-PCR in feces at 50 dpi and in sera at 15 oculum was unable to cause seroconversion. The low load of SIV pre- and 30 dpi. By 50 dpi, one group 2 piglet (0 J/L) seroconverted against sent in the inoculum was likely a result of contamination at the abattoir. HEV without presence of virus in bile. No seroconversion against SIV Due to the administration route selected, infection of the animals was was observed in group 2 (0 J/L) piglets. not expected because SIV is an exclusively respiratory pathogen. Piglets in groups 3 and 4 injected intraperitoneally with UV-C ir- However, if the amount of virus in the inoculum would have been radiated plasma at 3000 J/L and 9000 J/L, respectively did not become greater, a seroconversion to the level of immunization could have viremic (PCR or RT-PCR) for PCV-2, PRRSV, SIV, PPV, RVA and HEV happened. and they did not seroconvert at any time during the 50 dpi period. The current data are consistent with previously published research in which different enveloped and non-enveloped viruses of interest for 4. Discussion the swine industry (PRV, PRRSV, SIV, PPV, PEDV, Swine vesicular dis- ease virus (SVDV), Classical swine fever virus (CSFV), Bovine viral diarrhea The swine bioassay was used to demonstrate the effectiveness of the virus (BVDV), Senecavirus A (SVA) and PCV-2) inoculated in bovine or UV-C to reduce the viral load that may be present in liquid commercial porcine plasma were inactivated after UV-C treatment with the same plasma in an in vivo model. Intraperitoneal administration was selected Sure-Pure Turbulator used in the present study (Blázquez et al., 2019). as an administration route to avoid the possible inactivation or loss of These in vitro assays for virus quantification allowed determining the viral particles due to the hydrochloric acid and digestive enzymes in the inactivation capacity of the UV system for the different viruses tested gastrointestinal tract (Kasorndorkbua et al., 2002). By the in- and confirmed that this step was able to inactivate more than 4 logs for traperitoneal route, the main route of absorption of an inoculated ma- most of abovementioned viruses (Blázquez et al., 2019). terial is through the mesenteric blood vessels, which drain into the The World Health Organization (WHO) (WHO, 2004) guidelines on portal vein and pass through the liver (Lukas et al., 1971). In con- viral inactivation and removal procedures intended to assure the viral sequence, a certain amount of the injected liquid may pass directly safety of human blood plasma products establishes that a robust in- across the diaphragm through small lacunae and into the thoracic activation process should be capable of removing or inactivating at lymph (Abu-Hijleh et al., 1995) and then to systemic circulation. This least 4 logs of a wide range of viruses (WHO, 2004). In previous studies, technique has been shown to be a very sensitive model to detect in- the UV-C irradiation of liquid plasma was able to inactive 4 logs of fective pathogens (Gerber et al., 2014; Dee et al., 2015; Sampedro et al., different bacteria and viruses (Polo et al., 2015; Blázquez et al., 2017, 2015). 2019) and the present study also demonstrated the lack of transmission Although the commercially collected liquid porcine plasma was of any virus after being UV irradiated at a minimum of 3000 J/L. selected for the presence of PCV-2 genome, it also contained antibodies Therefore, UV-C irradiation complies with the WHO definition of a and presence of small amounts of nucleic acid of different viruses robust inactivation process. Furthermore, the UV-C mechanism of in- (PRRSV, SIV, RVA, PPV and HEV), which was consistent with endemic activation (DNA or RNA damage) is different than the thermal in- pathogens present in commercial swine population in the Catalonia, activation provided by the spray-drying step. Spain region (de Deus et al., 2007; López-Soria et al., 2010). Overall results indicate that liquid commercial plasma without any All the piglets in group 2, injected intraperitoneally with 10 mL of safety treatment and administered intraperitoneally to naïve suscep- untreated liquid porcine plasma (0 J/L), seroconverted against PRRSV tible pigs was infective for some viruses for which the initial plasma had and, to a lesser percentage, against PPV, RVA and HEV. In the case of virus genome. However, UV irradiation with the SurePure Turbulator PPV, 3 piglets seroconverted against the virus but no excretion of PPV system at 3000 J/L or more was enough to avoid transmission of any of was found in feces at the end of the study or its detection in sera during the tested viruses to the animals. The irradiation at 3000 J/L was the experimental period. Similarly, one piglet seroconverted to RVA chosen as the level of irradiation for liquid plasma that is used com- while sera and feces remained PCR negative. Furthermore, one piglet mercially by a specialized blood plasma producer. The opaqueness of a seroconverted against HEV, while the bile remained PCR negative. This liquid can limit the penetration of UV-C irradiation and therefore affect low seroconversion rate could be a result of the extremely low quan- the efficiency of pathogen inactivation (Matak et al., 2005; Groenewald tities of virus in commercially collected porcine plasma. However, pigs et al., 2013). However, if sufficient turbulence is introduced into the in groups 3 and 4 injected with liquid porcine plasma treated with UV-C liquid, all material is exposed to the surface of the UV source therefore (3000 J/L or 9000 J/L, respectively) did not seroconvert or become overcoming the limited penetration of the UV irradiation (Alberini PCR positive demonstrating that the viral particles present in com- et al., 2015). The Sure Pure Turbulator (EP-1255444B1) is designed to mercially collected porcine plasma were inactivated. create turbulent flow and has been successfully used to pasteurize Interestingly, the commercially collected porcine plasma con- opaque liquids like fruit juices, wine (Keyser et al., 2008; Fredericks tained 104 DNA copies/mL PCV-2. However, piglets injected with et al., 2011) and milk (Donaghy et al., 2009). In addition, in case of untreated plasma (0 J/L) did not become viremic or seroconvert with animal plasma, a UV dose of 3000 J/L has been demonstrated to have PCV-2 indicating that the PCV-2 virus particles were not infective. no effect on the functionality of proteins present in SDPP (Polo et al., The absence of PCV-2 infection could be either associated to the 2015; Cottingim et al., 2017). presence of neutralizing antibodies in commercial pooled plasma, an In summary, the results of this study demonstrated that the swine inadequate amount of PCV-2 in the intraperitoneal injection to infect bioassay is a sensitive technique able to detect infectivity of raw liquid the animals, or that the virus present in raw plasma was already plasma, confirming the infectiousness of PRRSV but not PCV-2. inactivated despite a positive PCR result. Neutralizing antibodies Moreover, the UV-C treatment seems to be a useful step to inactivate naturally present in pooled plasma were reported to inactivate up to potential pathogen contamination of commercially collected animal 4 log TCID50 of PCV-2 (Polo et al., 2013). This observation also plasma subsequently used to produce spray-dried animal plasma. 4 E. Blázquez, et al. Veterinary Microbiology 239 (2019) 108450 Acknowledgment drying process is sufficient to inactivate infectious porcine epidemic diarrhea virus in plasma. Vet. Microbiol. 174, 86–92. https://doi.org/10.1016/j.vetmic.2014.09.008. Groenewald, W.H., Gouws, P.A., Cilliers, F.P., Witthuhn, R.C., 2013. 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