EAPA. Spray Dried Porcine Plasma. A Safe and Vital Feed Ingredient EUROPEAN ANIMAL PROTEIN ASSOCIATION

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1 IATION Spray Dried Porcine Plasma A Safe and Vital Feed Ingredient

2 Plasma is Safe represents all European blood products producers. members have manufacturing plants in EU (France, Belgium, United Kingdom, Denmark, Germany, Spain, Poland, The Netherlands, Italy, Sweden and Ireland). Blood products are not considered processed animal proteins in Europe. Blood products are in the same category of milk, egg, gelatin, etc. in the European Regulation (EU Reg. 1069/2010). Commission Regulation 55/2013 allows the use of non ruminant blood products for the feeding of non ruminant farm animals European Union has strict controls and regulations related with the manufacturing process of blood products Only blood products made in the European Union following strict manufacturing rules are used for feeding farm animals. No imported product from third countries are used for farm animals in Europe. Manufacturing process for blood products is safe for eliminating potential contamination with PEDv. In addition, the PEDv strain that are causing problems in America is not present in the European swine population.

3 Plasma Reminders Superior protein source Well researched and effective Excellent alternative to antibiotics In piglets, improve growth and survival and reduce stress and morbidity associated with the weaning step. Strategic use of plasma proteins consistently have been proved to reduce some of the negative effects of a diversity of swine diseases (PRRS, PCV 2, SI, TGE, PED?)

4 ublications available about the Bio-Safety of SDP facturing ess of y Dried ma has istently onstrated safe in a diversity ruses. PEDv epidemic present i America, however confirma proof of or feed ingredien transmis vectors e Risk Analysis of Classical Swine Fever Inefectivity related to the Use of Porcine Spray Dried Plasma and Other Blood Derivatives in Swine Feed. March 2004 EUROPEAN ANIMAL PROTEIN ASS

5 Key Topics to Review Spray-dried plasma is a safe feed ingredient Plasma products have revolutionized the industry The industry must work together to protect the biosafety of the feed supply chain

6 Spray Dried Plasma is Safe Extensive Studies Show No Link. Research by Universities, FDA and members show no link between spray dried plasma products and infective PEDv 30 Years of Safe Use. Supported by peer-reviewed research by leading Universities

7 What Have We Learned? PEDv is a low thermal stability virus, similar to other envelope viruses PEDv is inactivated by the spray drying process PEDv loses activity on spray dried plasma during storage FDA and other testing failed to detect infective virus in spray-dried plasma

8 PEDv in USA and Canada Ontario Canada January 22, 2014 First confirmed PEDv case at a farrow-finish farm. 12 of the initial farm sites fed pig starters from same feed company. CFIA reported The study demonstrated that the porcine blood plasma in question contained PED virus capable of causing disease in pigs. However, the study could not demonstrate that the feed pellets containing the blood plasma were capable of causing disease. United States April 15, 2013 First confirmed PEDv case in USA at growfinish farm. Still unknown how PEDv came to USA.

9 Canadian Case Report Based on epidemiological observations, veterinarians recommended porcine plasma and other porcine derived products be removed from feed to prevent further spread of PEDv More than 40 farms in Canada have now reported PEDv AASV L Digest Vol 56, Issue 10, Doug MacDougal, DVM, SouthWest Ontario Veterinary Services

10 Conclusion of Epidemiological Observations Based on the epidemiological observations, some industry professionals have concluded that all spray dried porcine plasma contains infective virus Epidemiological observations should be used to initially form a hypothesis, then the hypothesis should be tested with controlled trials Correlation does not equal Causation.

11 Testing the Hypothesis 1 If PCR+ spray dried porcine plasma is a vector spreading PEDv, then the virus should survive the manufacturing process

12 PEDv Inoculated in Liquid Bovine Plasma then Spray Dried Hypothesis 1 Trial conducted at CReSA using a Buchi 190 Mini Spray Dryer Inoculated liquid bovine plasma with PEDv; Ct = 13.9 Spray Drying Conditions 1 Spray Dried Plasma PEDv TCID 50 /g 70 o C throughout substance 2 Not Detected 3 80 o C throughout substance 2 Not Detected 3 1 Inlet temperature 200ºC 2 Temperature determined with thermocouple probe. 3 PEDv reduction was > TCID 50 /g

13 Conclusion Hypothesis 1 A number of viruses have been shown to be inactivated by spray drying Low: PRRSv Medium: PRv High: SVDv PEDv (low thermal stability) was also inactivated by spray drying These results are not consistent with the hypothesis that PCR+ spray dried porcine plasma contains infective PEDv.

14 Testing the Hypothesis 2 If PCR+ spray dried porcine plasma is a vector spreading PED, then the virus should remain infective in dry plasma long enough to move through the feed channel

15 PEDv Does Not Remain Infective in a Dry Environment Hypothesis 2 Spray dried plasma (SDP) is a dry powder with a moisture content of < 8% and water activity (Aw) of <0.6 SDP is typically held for: o Minimum of 1 week in dry warehouse environment pending QA analysis o Up to an additional 3 weeks to move through commercial channels

16 PEDv Inoculated in Spray Dried Bovine Plasma held at Room Temperature Virus grown in culture Hypothesis 2 Inoculate spray dried bovine plasma with infective PEDv; Ct = 25 Day 0 7 PEDv detection Positive Negative Spray-dried bovine plasma inoculated with PEDv and held at room temperature (68-72 F) for 7 d was no longer infective. Trial conducted at CReSA

17 PEDv Inoculated Spray Dried Bovine Plasma - Storage Time X Temperatur Hypothesis 2 Storage Condition Spray dried bovine plasma inoculated with PEDv and held at room temperature for 7 d was no longer infective. Trial conducted at CReSA 4C (39 F) 12 C (54 F) 21 C (70 F) Initial 1 Positive Positive Positive 7 days Positive Positive Negative 14 days Positive Negative Negative 21 days Negative Negative Negative 1 Inoculated spray dried bovine plasma; Ct = 25.9

18 Spray Dried Plasma Temperature when stored in Unheated Warehouse; January-February, Product F Inside Ambient F Outside Ambient F Hypothesis 2 F Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7

19 Spray Dried Plasma Temperature when stored in Unheated Warehouse; January-February, Outside ambient Inside ambient Product 60 Hypothesis 2 Temperature (ºC) Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7

20 Summary: Warehouse Temperature Hypothesis 2 Ambient temperature range over 7 d period Inside warehouse 1 C to 3 C Outside local temp 21 C to 8 C Spray dried plasma temperature over 7 d period 35 C at bagging to 15 C at day 7 Unheated warehouse inside temperature does not get as cold as outside temperature Temperature of spray dried plasma cools off gradually and remains higher than inside ambient warehouse temperature in winter months

21 Conclusion Hypothesis 2 Hypothesis 2 PEDv inoculated on spray dried plasma stored at 21 C was not infective by 7 days PEDv inoculated on spray dried plasma stored at 4 C was not infective by 21 days Spray dried plasma temperature from bagging to day 7 of storage stays at 59 F (15 C) for about 7 d in ambient wintertime warehouse storage conditions These results are not consistent with the hypothesis that PCR+ spray dried porcine plasma contains infective PEDv

22 CFIA Investigation Timeline Nov Dec Jan Plasma was dried Plasma picked up by customer 29 days after processing First PEDv case reported 71 days after processing Other Suppliers Plasma Plant Spray Dried Plasma Feed Mill Plasma mixed with feed Farms Fed to pigs This timeline is not consistent with the CFIA statement that the porcine blood plasma in question contained PED virus capable of causing disease in pigs. The spray dried plasma in question likely became contaminated with infective PEDv sometime after processing and just before it was fed to pigs.

23 Testing the Hypothesis 3 If PCR+ spray dried porcine plasma is a vector spreading PEDv, then retained samples from the lot of spray dried porcine plasma collected from the field and investigated by CFIA should also contain infective PEDv

24 Results of FDA Bioassay FDA Comment Hypothesis 3 Please share, In summary: NVSL received ten (10) porcine plasma samples collected by the FDA. These samples were tested by PCR for the presence of PEDV RNA and nine of the samples were positive, while one is still pending. Three of these samples, corresponding to the CFIA samples, were prepared and inoculated into day old piglets (5 pigs/lot). All three samples were negative by bioassay. Bioassay Sample PCR PCR Serology Sample 1 Positive Negative Negative Sample 2 Positive Negative Negative Sample 3 Positive Negative Negative All three samples were negative by bioassay

25 Member Bioassay of Retained Spray Dried Porcine Plasma Initial PEDv Clinical Score 72 hr post exposure Jejunum PEDv PCR CT IHC Hypothesis 3 Sample PCR CT Pig 1 Pig 2 Pig 3 Pig 1 Pig 2 Pig 3 Sample neg neg neg neg Sample neg neg neg neg Sample neg neg neg neg Sample neg neg neg neg Pos Cont Pos Neg Cont neg neg neg neg Score 0 = well formed feces Score 1 = soft poorly formed feces Score 2 = watery feces Score 3 = watery diarrhea and dehydration Trial conducted at University of Minnesota 1 Retained samples from product investigated by CFIA 2 Commercial product produced in December and January

26 Bioassays of Commercial Spray Dried Plasma Hypothesis 3 Retained samples from the lot of PCR+ spray dried porcine plasma investigated by CFIA did not contain infective PEDv per the FDA and Member bioassays. Samples from two different commercial lots of PCR+ spray dried porcine plasma did not contain infective PEDv. These results are not consistent with the hypothesis that PCR+ spray dried porcine plasma contains infective PED virus.

27 Testing the Hypothesis 4 The design of bioassays includes limited number of pigs fed a small amount of test material for a short time. Therefore, a negative does not mean negative

28 Nursery Trials Traditionally, plasma is added to the first diet(s) in the nursery. Hypothesis 4 Diet Plasma, % Feed schedule, lb/pig Nursery 1 2.5%to 6.0% 2 lb to 5 lb Nursery 2 0% to 2.5% 5 lb to 10 lb

29 Field Trial with Spray Dried Porcine Plasma PCR+ for PEDv Genome Hypothesis 4 Treatments D 0-14 days post weaning D days post weaning Control feed Control diet Control diet SDPP PCR+ for PEDv 5.0% SDPP Control diet (Plasma = 26.2 Ct) Diets were fed in meal form 24 pigs per treatment

30 Field trial with Spray Dried Porcine Plasma PCR+ for PEDv Genome Hypothesis 4 Analysis Pigs in Control Group Feeding pigs a diet with commercial spray dried porcine plasma PCR+ for PEDv did not result in PEDv infectivity. Pigs in Plasma Group PCR in fecal samples at day 0 Negative Negative PCR in fecal samples at day 3 Negative Negative PCR in fecal samples at day 7 Negative Negative PCR in fecal samples at day 14 Negative Negative PCR in fecal samples at day 21 Negative Negative Serum antibodies against PEDv at day 21 Negative Negative Immunohistochemistry Negative Negative

31 Iowa State University Study with Feed Containing SDPP PCR+ for PEDv Hypothesis 4 Treatments D 0-28 post weaning PEDv Inoculation Negative Control Control diet No SDPP (Ct=30.0) 5.0% SDPP No Positive Control Control diet Yes SDPP+PEDv 5% SDPP Yes Diets fed in meal form 6 pigs/trt Necropsy on 3 pigs/trt at d 7 and d 28

32 Iowa State University Study with Feed Containing SDPP PCR+ for PEDv Hypothesis 4 Feed Control 5% SDPP Control 5% SDPP PEDv Inoculation No No Yes Yes PCR in fecal samples at day 0 Neg. Neg. Neg. Neg. PCR in fecal samples at day 3 Neg. Neg. Pos. Pos. PCR in fecal samples at day 7 Neg. Neg. Pos. Pos. PCR in fecal samples at day 14 Neg. Neg. Pos. Pos. PCR in fecal samples at day 21 Neg. Neg. Pos. Pos. PCR in fecal samples at day 28 Neg. Neg. Pos. Pos. Serum antibodies against PEDv at d 28 Neg. Neg. Pos. Pos. Preliminary Results from ISU study Feeding pigs a diet with PEDv PCR+ commercial SDPP did not cause PEDv infectivity. PEDv inoculated pigs fed SDPP had less intestinal lesions than positive control pigs Dietary SDPP may reduce some negative effects associated with PEDv

33 Conclusion Hypothesis 4 Pigs fed nursery diets containing PCR+ spray dried plasma for PEDv, typical of commercial feeding programs, did not become infected with PEDv These results are not consistent with the hypothesis that PCR+ spray dried porcine plasma contains infective PEDv

34 Testing the Hypothesis 5 If PCR+ spray dried plasma is a vector spreading PEDv, then feeding PCR+ spray dried plasma should result in PEDv outbreaks in other regions of the world

35 Spray Dried Porcine Plasma Made in Brazil Hypothesis 5.1 Manufacturing Date December 29, 2013 December 31, 2013 January 3, 2014 January 7, 2014 January 8, 2014 PEDv PCR Results Negative Negative Negative Negative Negative Spray dried porcine plasma produced from slaughter pigs in Brazil were negative for PEDv even though PCR+ spray dried porcine plasma from the USA has been fed in Brazil since the summer of 2013

36 Conclusion Hypothesis 5.1 Between April 2013 and December 2013 enough PCR+ spray dried porcine plasma was shipped from the US to Brazil to feed between 2.5M to 3.5M pigs. The US sourced spray dried porcine plasma was PCR+ for PEDv. PEDv cases have not been detected in Brazil. These results are not consistent with the hypothesis that PCR+ spray dried porcine plasma contains infective PED virus.

37 Spray Dried Porcine Plasma Manufactured in Calgary, AB Manufacturing Date PEDv PCR Results Hypothesis 5.2 August, 2013 Negative September, 2013 Negative October, 2013 Negative November, 2013 Negative December, 2013 Negative January, 2014 Negative February, 2014 Negative March, 2014 Negative Spray dried porcine plasma produced from slaughter pigs in Western Canada were negative for PEDv even though PCR+ spray dried porcine plasma from the USA has been fed in Canada since early summer of 2013

38 Conclusion Hypothesis 5.2 Between April 2013 and December 2013 enough PCR+ spray dried porcine plasma was shipped from the US to Western Canada to feed between 4.5M to 5.0M pigs. The US sourced spray dried porcine plasma was PCR+ for PEDv. PEDv cases have not been detected in Western Canada during this time period. PEDv PCR results remained negative for porcine plasma produced in Western Canada through March 2014 These results are not consistent with the hypothesis that PCR+ spray dried porcine plasma contains infective PED virus.

39 Summary: Spray Dried Plasma is Safe Data from controlled trials does not support the hypothesis that PCR+ spray dried porcine plasma is a vector spreading PEDv PEDv does not remain active after spray drying PEDv does not remain active in dry plasma PEDv infectivity was not detected in retained samples of the spray dried plasma investigated by the CFIA Feeding PCR+ spray dried porcine plasma simulating typical nursery feed programs did not result in infected pigs US sourced PCR+ spray dried porcine plasma was fed to 2.5M to 3.5M pigs in Brazil and 4.5M to 5.0M in Western Canada, and both of these regions remain PEDv free These results are not consistent with the hypothesis that PCR+ spray dried porcine plasma contains infective PEDv

40 Spray Dried Plasma Products are Revolutionary Plasma products help supply proper nutrition to sick animals and help healthy animals thrive Plasma products can t cure PEDv, but the nutrition they provide can help with the duration and severity of PEDv symptoms

41 Stands with the Pork Industry PEDv is a real threat to millions of pigs and our pork industry We continue to collaborate with industry to provide additional research and support

42 hanks for Your ime Questions?

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44 Appendix Supplementary Information

45 Appendix PCR Testing

46 PCR Testing of Spray-Dried Plasma PCR detects presence of PED genome in spray dried plasma, but it does not determine if it causes infectivity Other methods must be used to confirm if tested material is infective o Virus isolation in cell culture o Bioassay RT PCR showing virus replication in feces Confirmation by IHC o Feeding trials

47 Correlation of TCID 50 to Ct 6,0 5,0 y = 0,0096x 2 0,6959x + 12,474 R² = 0,9878 4,0 TCID 50 /ml 3,0 2,0 1,0 0,0 1,0 0,0 5,0 10,0 15,0 20,0 25,0 30,0 35,0 40,0 C t Data provided by Joan Pujols, CReSA

48 PCR cycle time appears related to ambient temperatures 45 Spray Dried Plasma PEDv PCR CT Result CT count South Eastern US Midwestern US Each data point represents plasma from at least 30,000 pigs Poly. (South Eastern US) Poly. (Midwestern US) Mar 13 6 May Jun Aug 13 3 Oct Nov Jan 14 2 Mar Apr Jun 14

49 Appendix Past Research with other Viruses

50 ding PCV2 PCR+ Porcine Plasma Does Not Infect Pig PCV2 wo studies emonstrated that naïve igs did not become nfected with PCV2 when ed a diet containing igh levels of SDPP with igh concentration of CV2 DNA. Shen et al., 2011; Pujols et al., 2008

51 A PEDv PCR+ Test Result Does Not Reflect Infectivity The results from this bioassay indicate that feed can contain PEDv genetic material that is detectable by PCR but is not infectious (Rovira, 2013)

52 Past Research with PCR+ Plasma A PCR+ result in spray dried plasma does not reflect infectivity PCR+ spray dried plasma has consistently been demonstrated to be a safe ingredient for the feed industry

53 Appendix Inactivation by Spray Drying

54 Inoculate liquid plasma then spray dry The virus is grown. Liquid plasma (free of target virus) is inoculated with live virus. Spray dry the inoculated plasma in lab drier. Determine the reduction in infective units.

55 Inactivation of PRRSv Inoculated in Liquid Bovine Plasma by Spray Drying Sample ID Liquid plasma Spray dried PRRS TCID 50 /ml TCID 50 /ml Lot # Not detected Lot # Not detected Lot # Not detected Polo et al., 2005, J. Anim. Sci. 83: Inlet temp. 240 C; Exit temp. 90 C.

56 ctivation of PRRSv Inoculated in Liquid Bovine Plasma by Spra Drying Sample ID Liquid plasma Spray dried PRRS TCID 50 /ml TCID 50 /ml Spray Drying inactivated a low heat resistant virus Lot # Not detected Lot # Not detected Lot # Not detected Polo et al., 2005, J. Anim. Sci. 83: Inlet temp. 240 C; Exit temp. 90 C.

57 tivation of Pseudorabies Virus Inoculated in Liquid Bovine Plas pray Drying Sample ID Liquid plasma Spray dried PRV TCID 50 /ml TCID 50 /ml Lot # Not detected Lot # Not detected Lot # Not detected Polo et al., 2005, J. Anim. Sci. 83: Inlet temp. 240 C; Exit temp. 90 C.

58 tivation of Pseudorabies Virus Inoculated in Liquid Bovine Plas pray Drying Sample ID Liquid plasma Spray dried PRV TCID 50 /ml TCID 50 /ml Lot # Not detected Spray Drying inactivated a medium Lot #2 heat resistant virusnot detected Lot # Not detected Polo et al., 2005, J. Anim. Sci. 83: Inlet temp. 240 C; Exit temp. 90 C.

59 Inactivation of SVD Inoculated in Porcine Plasma by Spray-Drying Sample ID Exit Temp, 90 o C Inoculated liquid plasma TCID 50 /ml Spray dried samples, TCID 50 /ml 0 90 o C Not detected o C Not detected o C Not detected Exit Temp, 80 o C 0 80 o C Not detected o C Not detected o C Not detected Pujols et al., 2007, Proc. AASV, p

60 Inactivation of SVDv Inoculated in Porcine Plasma by Spray-Drying Sample ID Exit Temp, 90 o C Inoculated liquid plasma TCID 50 /ml Spray dried samples, TCID 50 /ml 0 90 o C Not detected o C Not detected o C Not detected Spray Drying inactivated a high heat resistant virus Exit Temp, 80 o C 0 80 o C Not detected o C Not detected o C Not detected Pujols et al., 2007, Proc. AASV, p

61 Conclusion A number of viruses have been shown to be inactivated by spray drying Low: PRRSv Medium: PRv High: SVDv PEDv (low thermal stability) was also inactivated by spray drying These results are not consistent with the hypothesis that PCR+ spray dried porcine plasma contains infective PEDv.