Estimated quantity of swine virus genomes based on quantitative PCR analysis in spray-dried porcine plasma samples collected from multiple manufacturing plants.

This survey was conducted to estimate the incidence and level of potential viral contamination in commercially collected porcine plasma. Samples of spray dried porcine plasma (SDPP) were collected over a 12- month period from eight spray drying facilities in Spain, England, Northern Ireland, Brazil, Canada, and the United States. In this survey, viral load for several porcine pathogens including SVA, TGEV, PRRSV (EU and US strains), PEDV, PCV-2, SIV, SDCoV and PPV were determined by qPCR. Regression of Ct on TCID50 of serial diluted stock solution of each virus allowed the estimate of potential viral level in SDPP and unprocessed liquid plasma (using typical solids content of commercially collected porcine plasma). In this survey SVA, TGEV or SDCoV were not detected in any of the SDPP samples. Brazil SDPP samples were free of PRRSV and PEDV. Samples of SDPP from North America primarily contained the PRRSV-US strain while the European samples contained the PRRSV-EU strain (except for one sample from each region containing a relatively low estimated level of the alternative PRRSV strain). Estimated viral level tended to be in the range from <1.0 log10 TCID50 to <2.5 log10 TCID50. Estimated level of SIV was the exception with a very low incidence rate but higher estimated viral load <3.9 log10 TCID50. In summary, the incidence of potential viral contamination in commercially collected porcine plasma was variable and estimated virus level in samples containing viral DNA/RNA was relatively low compared with that occurring at the peak viremia during an infection for all viruses or when considering the minimal infectious dose for each of them.

Introduction

Spray dried porcine plasma (SDPP) is a complex mixture of functional components including immunoglobulins, albumin, transferrin, fibrinogen, lipids, growth factors, bioactive peptides, enzymes, hormones, and amino acids commonly used in feed for young animals including pigs, calves, and poultry [1-4].

It has been speculated that the use of SDPP in swine feed contributed to the spread of infective viruses such as Porcine circovirus 2 (PCV-2) and Porcine epidemic diarrhea virus (PEDV) [5-7]. However, other evidence demonstrates that reduced mortality and morbidity is associated with the use of SDPP in pig diets [1, 3, 8, 9] and experimental and epidemiological evidence demonstrate that SDPP does not spread diseases [10-12].

The manufacturing process to produce SDPP includes multiple hurdles steps that have been validated to inactivate potential viral contamination. These hurdles include spray drying (SD, 80°C throughout substance), ultraviolet light (UV) treatment (3000 J/L) and post drying storage (PDS) at 20°C for 14 d [13-19]. Depending on the virus, the theoretical cumulative inactivation for SD and PDS range from 5.8 to 9.1 log10 TCID50/g liquid plasma, while SD, PDS and UV range from 11.7 to 20.9 log10 TCID50/g liquid plasma (Table 1). The World Health Organization recommends cumulative robust inactivation procedures capable of inactivating 4 log10 of virus by each of these steps in the manufacturing process for human blood and plasma products [20, 21].

Table 1

Different inactivation steps involved in the manufacturing process of spray dried porcine plasma.

Inactivation expressed as log10 reduction values (LRVs) TCID50/g for viruses.

Virus Type			Spray-Drying	UV-C*	Storage at 20°C for 14 d	Combined Theoretical Inactivation	References
RNA	Enveloped	Porcine reproductive and respiratory syndrome virus (PRRSV)	>4.0	12.9 ± 0.3	>4.0	>20.9	[13, 17, 62]
		Swine influenza virus (SIV)	2.8**	± 0.2	3.2**	13.9	[17]
		Porcine epidemic diarrhea virus (PEDV)	5.14.2	6.6 ± 0.1	3.8	14.6–15.5	[15–17]
		Classical swine fever virus (CSFV)	5.8	7.9 ± 0.2	ND	>13.7	[17, 63]
	Naked	Swine vesicular disease virus (SVDV)	6.7	3.5 ± 0.07	ND	>10.2	[14, 17]
		Senecavirus A (SVA)	ND	4.0 ± 0.08	>5.0**	>9.0	[17]
DNA	Enveloped	Pseudorabies virus (PRV)	5.3	8.1 ± 0.2	ND	>13.4	[13, 17]
		African swine fever virus (ASFV)	4.1 ± 0.2	6.8 ± 0.1	>5.7	>16.6	[17, 19, 63]
	Naked	Porcine parvovirus (PPV)	2.7**	6.0 ± 0.1	3.1**	>11.8	[17]

LRVs with symbol > results indicate the inactivated amount in the processed sample exceeded the amount inoculated in the initial sample before processing or storage.

1ND = Not determined.

*The UV log-kill estimated values were calculated commercial UV dosage (3251 J/L) by the estimated D-value from Blázquez et al., [17].

**University of Minnesota. Understanding the risk of virus transmission in spray dried porcine plasma–food safety assessment. 2020. Unpublished data.

While the inactivation capacity of the multiple hurdle manufacturing process has been validated for several economically important swine viruses, it is also important to estimate the potential virus quantity in liquid plasma used to produce SDPP. Therefore, this survey was conducted to estimate the quantity and determine the frequency of genome detection of different swine viruses in commercially produced SDPP samples collected from 8 different manufacturing plants. Results obtained from quantitative polymerase chain reaction (qPCR) analyses of the SDPP samples were used to infer the potential viral contamination in the liquid porcine plasma from which it was produced.

Material and methods

Ethical statement

No animals were used for the study conducted.

Spray-dried porcine plasma sample collection

One sample per month was collected from a randomly selected commercial lot of SDPP during 12 consecutive months from eight different manufacturing plants located in Iowa, USA (IA-USA), North Carolina, USA (NC-USA), Santa Catarina, Brazil (SC-Brazil), central Spain (C-Spain), northeastern Spain (NE-Spain), central England (C-England) and Northern Ireland (N-Ireland). The N-Ireland manufacturing plant collects porcine blood from abattoirs located both, in Republic of Ireland and Northern Ireland. Samples from a manufacturing plant located in Quebec, Canada (QB-Canada), were taken biweekly during a 6 month-period.

Samples were collected from July 2018 to June 2019 (SC-Brazil), August 2018 to July 2019 (IA-USA, NE-Spain, C-Spain and N-Ireland) or September 2018 to August 2019 (NC-USA, C-England). The QB-Canada plant provided 12 samples randomly collected from March to August 2019. The collected SDPP samples represented a single point in time, not the entire month. Whole blood or plasma was chilled and stored in insulated agitated tanks at the abattoir. transported to the spray drying facility in dedicated tankers and stored and may be blended with plasma from different slaughterhouses in agitated silos before drying. In the manufacturing plants used in this study, a manufacturing lot of SDPP can range between 3,000 to 15,000 kg of plasma depending on the plant. Therefore, one lot of SDPP represented between 16,650 to 166,500 pigs. During the 12-month collection period, samples were stored in whirl packs (Whirl-Pak®, Nasco, Madison, WI) and held at each plant in the quality assurance laboratory (room temperature) during the collection period. Subsequently, all SDPP samples were sent to the IRTA-CReSA Animal Health Research Center in Barcelona, Spain, and stored (-20°C) until analyses for virus genome. One sample collected in December from the IA-USA plant was damaged during transport and was not used for analysis. Therefore, a total of 95 SDPP samples were analyzed.

Sample analysis by PCR

All SDPP samples were re-solubilized in distilled water at the ratio 1:9 of SDPP: water volume to represent the typical solid content in liquid plasma. Two hundred milliliters of diluted plasma sample were used for nucleic acid extraction using MagMAX™ Pathogen RNA/DNA Kit (Thermo Fisher Scientific, MA, USA). The recommended quantity of purified nucleic acids was amplified using real time PCR kits for PCV-2 (LSI VetMAX™ Porcine Circovirus Type 2 Quantification, Thermo Fisher Scientific, MA, USA), Porcine reproductive and respiratory syndrome virus [PRRSV] European and North American strains (LSI VetMAX™ PRRSV EU/NA Real-Time PCR Kit; Thermo Fisher Scientific, MA, USA), Swine influenza virus [SIV] (EXOone Influenza A, EXOPOL, Zaragoza, Spain), Porcine parvovirus [PPV] (VetMAX™ Porcine Parvovirus Kit, Thermo Fisher Scientific, MA, USA), PEDV, Transmissible gastroenteritis virus [TGEV] and Swine deltacoronavirus [SDCoV] (VetMAX™ PEDV/TGEV/SDCoV, Thermo Fisher Scientific, MA, USA) and Senecavirus A [SVA] (EXOone Seneca Virus Valley, EXOPOL, Zaragoza, Spain).

According to all PCR kit guidelines, virus genome results with Ct values >40 were considered negative.

Virus stock production for development of standard curves to convert PCR Ct to TCID50/g SDPP

From those viruses detected in SDPP by qPCR, a stock of each virus was produced in the laboratory. Seven serial dilutions of viral stocks (PEDV, PRRSV-1 (EU strain), PRRSV-2 (US strain), PPV-1, PCV-2 and SIV) were analyzed by quantitative PCR/RT-PCR (obtaining the corresponding Ct value) and TCID50 titration. Standard curves were established for each virus by regressing TCID50/g SDPP on Ct results [Fig 1]. Those viral stocks were used as an internal standard on each amplification run/plate and quantitative PCR/RT-PCR Ct values extrapolated to TCID50. Potential viral quantity determined on SDPP was corrected for typical solids content for each commercially collected plasma. TCID50 titers were calculated by the Reed and Muench method [22].

Fig 1

Regression curves between Ct values and tissue culture infectious dose 50 (TCID50/g) or Genome equivalent copies (GEC/g) of spray-dried porcine plasma (SDPP).

Values expressed in log10 TCID50/g SDPP or log10 GEC/g SDPP. Each box includes the spot values of the SDPP samples analyzed and the regression equation between Ct and TCID50/g or GEC/g SDPP and the r2 value. A.Regression curves for porcine epidemic diarrhea virus (PEDV); B. Regression curves for porcine circovirus type-2 (PCV-2); C. Regression curves for porcine parvovirus (PPV); D. Regression curves for swine influenza virus (SVI) H1N1; E. Regression curves for porcine reproductive and respiratory syndrome virus (PRRSV) US strain; F. Regression curves for PRRSV EU strain.

Porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus 3268 EU strain was propagated in porcine alveolar macrophages (PAM) grown in standard growth media (SGM) containing minimum essential medium eagle (MEM-E; ThermoFisher, Waltham, MA, USA) supplemented with 1% penicillin 10,000 U/mL and streptomycin 10 mg/mL (ThermoFisher), 0.5% Nystatin 10,000 IU/mL (Sigma-Aldrich, Burlington, MA, USA), 1% L-glutamine 200 mM (ThermoFisher) plus 5% fetal bovine serum (FBS). Cells were cultured in 75-cm2 flasks. When cells were confluent, the media was discarded, and the adsorption was done using the virus at 0.01 multiplicity of infection (MOI). After 1.5 hours at 37ºC, inoculum was removed, and 30 mL of medium were added. Titration was done in triplicate obtaining a final titer of 105.5±0.2 TCID50/mL.

Porcine reproductive and respiratory syndrome virus RV2332 US strain was propagated in MARC145 cells (ATCC No. CRL-12231) (kindly provided by Dr. Enric Mateu, Universitat Autònoma de Barcelona, Barcelona, Spain) using SGM supplemented with 10% FBS as explained above until a viral stock solution with a final titer of 104.9±0.4 TCID50/mL was obtained.

Porcine epidemic diarrhea virus

Porcine epidemic diarrhea virus CV777 strain [23], kindly provided by Dr. Hans Nauwynck (University of Ghent, Belgium), was propagated in VERO cells (ATCC CCL-81) grown in SGM with 10% FBS. Cells were cultured in 175-cm2 flask and when they were confluent, the media was removed, and cells were rinsed twice with phosphate buffered saline (PBS). Finally, inoculum was added at 0.001 MOI and adsorption was done for 1 hour at 37ºC. Subsequently, the inoculum was discarded, flasks were rinsed twice with PBS and SGM supplemented with 10 mg/mL trypsin, and 0.3% tryptose (Sigma-Aldrich, Burlington, MA, USA). The viral stock was produced in the same cells and was titrated in triplicate obtaining a suspension with a viral titer of 105.4±0.1 TCID50 /mL.

Swine influenza virus

Swine influenza virus strain H1N1 A/Swine/Spain/SF11131/2017 [24] was propagated in MDCK cell line (ATCC CCL-34) grown in DMEM (ThermoFisher, Waltham, MA, USA) supplemented with 1% penicillin (10,000 U/mL), 1% streptomycin (10 mg/mL; ThermoFisher), 0.5% Nystatin (10,000 U/mL) (Sigma-Aldrich, Burlington, MA, USA), 1% L-glutamine 200mM (ThermoFisher) and 5% FBS. Cells were cultured in 175-cm2 flask. When cells were confluent, the media was discarded, and the adsorption was done at 0.1 MOI. After 1 hour at 37ºC, inoculum was removed, and 30 mL of medium were added. The viral suspension was titrated in triplicate and the final virus titer was 107.6±0.2 TCID50 /mL.

Porcine circovirus 2

Porcine circovirus 2 genotype b isolate Sp-10-7-54-13 [25] was cultured in the PK-15 cell line (provided by the Institute of Virology UE and OIE Reference Laboratory for CSFV, Hannover), grown in SGM with 10% FBS. A mix of 6 mL of virus stock and 7 x 106 PK-15 cells resuspended in 50 mL of MEM-E (MOI 0.1) were added in 175 and 25 cm2 flasks. At 24 hours cells were treated with glucosamine (Sigma-Aldrich, Burlington, MA, USA) to facilitate the virus infection. Forty-eight hours later, viral infection was checked by immunoperoxidase monolayer assay (IPMA) [26] in the 25 cm2 flask. If more than 25 positive cells were counted in a microscope field, the 175 cm2 flask was trypsinized and the cells were transferred to 3 new 175 cm2 flasks. The virus stock was titrated in triplicate with a final titer of 105.5±0.04 TCID50 /mL.

Porcine parvovirus

Porcine parvovirus strain NADL-2 was kindly provided by Dr Albert Bosch (Department of Genetics, Microbiology and Statistics School of Biology, University of Barcelona, Spain). It was propagated in SK-RST cells (ATCC CRL-2842), grown in SGM supplemented with 5% FBS. One mL of virus stock and 9 mL of MEM-E supplemented with 1% pyruvate (Merck KGaA, Darmstadt, Germany) were added to a conical tube with 16 x 106 SK-6 cells and shaken for 30 minutes at 104 rpm and 37ºC. After that time, the contents of the tube were transferred to a 175 cm2 flask, in which 40 mL of MEM-E supplemented with 1% pyruvate were added. Inoculated flasks were incubated for four days at 37ºC until CPE was observed. A viral suspension was obtained and titrated in triplicate, obtaining a final viral solution of 106.6±0.2 TCID50 /mL.

Estimation of TCID50 and genomic equivalent copies (GEC) from Ct values obtained from q-PCR results

To establish equivalence of positive qPCR results (measured as Ct values) with TCID50/mL and viral genome equivalent copies (GEC) content, seven serial dilutions of abovementioned titrated virus stocks were performed, and virus genome amplified with a second set of PCR kits (GPS, Genetic PCR Solutions Alicante, Spain). Each kit contained a genome quantified standard for the different viruses tested: PRRSV (PRRSV-I dtec-RT-qPCR, PRRSV-II dtec-RT-qPCR), PEDV (PEDV dtec-RT-qPCR), PPV (PPV-1 dtec-RT-qPCR) and SIV (SIV dtec-RT-qPCR).

Statistical analysis

Dilutions of titrated viral stocks were included as an internal standard on each amplification PCR run containing SDPP samples. The Excel software was used to obtain the equation correlating TCID50 and Ct values as well as GEC and Ct values. Then, results of the different PCR techniques originally expressed as Ct values for each SDPP sample tested were extrapolated to virus infectious particles and GEC based on the obtained regression formulae.

Average, number of observations, standard deviation, minimum value, maximum value, and ranges were calculated within each virus and for each SDPP producing plant using LSMEANS (SAS 9.4, 2016).

Results and discussion

In this survey, viral loads for several porcine pathogens including SVA, TGEV, PRRSV (EU and US strains), PEDV, PCV-2, SIV, SDCoV and PPV were determined by qPCR in reconstituted commercial SDPP. First, the Ct values from serial dilutions of a stock solution for each virus allowed the development of a regression equation between Ct and TCID50 that allowed an estimate of the viral titers in the SDPP samples. Finally, using typical solids content of unprocessed liquid plasma, the viral level in liquid plasma was adjusted per gram (TCID50/g liquid plasma). The relationships between Ct and TCID50 of serial diluted stock solutions were linear with a correlation coefficient from 0.95 to 0.995 (Fig 1). Similar correlation coefficients were found when regressing Ct on log10 GEC/g on the tested samples (Fig 1). The slope of the lines for either TCID50 or GEC/g were similar, while the intercepts were different (Fig 1), consistent with the fact that not all viral genome copies are infective [27]. There was variability between infectious particles and genome copy numbers observed among tested viruses, with less than 1 log difference for SIV to around 4 log differences for PCV-2.

Previous research has shown PCR/RT-PCR Ct values in SDPP to be relatively stable during normal storage conditions [19, 28, 29]. Similar levels of viral genome were detected in plasma inoculated with PCV-2 or SIV before and after spray drying (E. Blázquez, personal communication). The stability of PCR Ct values, the linear relationship between Ct and TCID50 and the linear relationship between Ct and GEC provides additional assurance that estimated viral contamination of commercially collected SDPP and estimates of liquid plasma are accurate.

Frequency of detection and estimated quantity of virus in SDPP samples mimicking unprocessed liquid plasma samples collected at different plants is presented in Tables 2 and 3.

Table 2

Ct values and estimated viral genome presence expressed in log10 genome equivalent copies (GEC) and log10 TCID50/g spray dried porcine plasma in manufacturing plants located in different swine production areas around the world during the years 2018–2019.

Values expressed as Average ± SD for positive samples.

Plant	US-IA(n = 11)	US-NC(n = 12)	Canada(n = 12)	Spain-NE (n = 12)	Spain-C(n = 12)	England(n = 12)	NI(n = 12)	Brazil(n = 12)
PEDV
Ct	33 ± 3	34 ± 2	34	35 ± 1	35 ± 1	Neg	Neg	Neg
log10 GEC/g	2.9 ± 0.9	2.7 ± 0.6	2.7	2.4 ± 0.3	2.4 ± 0.4
log10 TCID50/g	0.3 ± 0.9	0.1 ± 0.6	0.3	0.01 ± 0.33	-0.05 ± 0.38
% Positive samples	82	50	8	83	67	0	0	0
PCV-2
Ct	32 ± 1	31 ± 2	30 ± 1	30 ± 1	30 ± 1	31 ± 1	31 ± 1	31.0 ± 0.4
log10 GEC/g	5.3 ± 0.2	5.5 ± 0.5	5.7 ± 0.3	5.5 ± 0.2	5.6 ± 0.3	5.4 ± 0.4	5.4 ± 0.2	5.3 ± 0.1
log10 TCID50/g	1.4 ± 0.2	1.6 ± 0.5	1.8 ± 0.3	1.6 ± 0.2	1.7 ± 0.3	1.5 ± 0.4	1.5 ± 0.2	1.4 ± 0.1
% Positive samples	100	100	100	100	100	100	100	100
PPV
Ct	30 ± 1	32 ± 2	31 ± 1	31 ± 3	31 ± 1	30 ± 1	28.4 ± 0.5	31 ± 1
log10 GEC/g	4.0 ± 0.3	3.5 ± 0.6	3.9 ± 0.3	3.9 ± 0.8	3.9 ± 0.3	4.0 ± 0.3	4.4 ± 0.1	3.8 ± 0.2
log10 TCID50/g	2.8 ± 0.3	2.4 ± 0.6	2.8 ± 0.4	2.7 ± 0.8	2.8 ± 0.3	2.9 ± 0.3	3.3 ± 0.1	2.6 ± 0.3
% Positive samples	100	100	100	100	100	100	100	100
SIV
Ct	38	Neg	35	23 ± 4	19.6 ± 0.3	24 ± 11	21	28 ± 10
log10 GEC/g
log10 TCID50/g	-1.3		0.4	3.9 ± 1.1	5.0 ± 0.1	3.8 ± 3.0	4.6	2.7 ± 2.7
% Positive samples	9	0	8	17	17	25	8	25
PRRS-US
Ct	33 ± 2	34 ± 1	34 ± 2	Neg	36	Neg	Neg	Neg
log10 GEC/g	2.4 ± 0.5	2.1 ± 0.4	2.2 ± 0.7		1.6
log10 TCID50/g	-1.3 ± 0.5	-1.5 ± 0.4	-1.5 ± 0.7		-2.1
% Positive samples	100	17	50	0	8	0	0	0
PRRS-EU
Ct	36	Neg	Neg	35 ± 1	34 ± 2	34 ± 1	34 ± 1	Neg
log10 GEC/g	2.1			2.4 ± 0.3	2.6 ± 0.5	2.7 ± 0.4	2.6 ± 0.3
log10 TCID50/g	-0.3			0.03 ± 0.24	0.2 ± 0.4	0.3 ± 0.4	0.2 ± 0.3
% Positive samples	9	0	0	33	58	50	83	0

Table 3

Estimated quantification of different viruses’ genomes expressed in log10 TCID50/g ± SD (percentage of positive samples) in unprocessed raw liquid plasma from PCR or RT-PCR analyses of spray dried porcine plasma samples collected at different plants.

Plant	PEDV	PCV-2	PPV	SIV	PRRS- US	PRRS-EU
US-IA	-0.8 ± 0.9	0.3 ± 0.2	1.7 ± 0.3	-2.5	-2.4 ± 0.5	-1.4
US-NC	-0.9 ± 0.6	0.6 ± 0.5	1.3 ± 0.6	Neg	2.6 ± 0.4	Neg
Canada	-0.8	0.6 ± 0.3	1.7 ± 0.4	-0.7	-2.5 ± 0.7	Neg
Spain-NE	-1.0 ± 0.3	0.6 ± 0.2	1.7 ± 0.8	2.9 ± 1.1	Neg	-1.0 ± 0.3
Spain-C	-1.2 ± 0.4	0.5 ± 0.3	1.7 ± 0.3	3.8 ± 0.1	-3.2	-0.9 ± 0.4
England	Neg	0.5 ± 0.4	1.9 ± 0.3	2.8 ± 3	Neg	-0.8 ± 0.4
Northern Ireland	Neg	0.4 ± 0.2	2.2 ± 0.1	3.5	Neg	-0.9 ± 0.3
Brazil	Neg	0.3 ± 0.1	1.5 ± 0.3	1.6 ± 2.7	Neg	Neg
Range	-1.8–0.5	-0.3–1.4	-0.2 –-2.6	-2.5–4.6	-3.2 –-1.5	-1.5 –-0.2

The S1 Table -SDPP includes monthly (during the years 2018–2019) Ct values and estimated virus levels reported as log10 GEC/g and log10 TCID50/g in reconstituted SDPP from the different manufacturing plants located in different swine production areas around the world. The S2 Table (Raw Plasma) includes estimated viral levels in unprocessed plasma reported as log10 TCID50/g. It is important to recognize that a positive PCR/RT-PCR does not imply infectivity [16], a fact that was observed for all the viruses studied in the present work.

In this survey neither SVA, TGEV nor SDCoV were detected in any of the SDPP samples. SVA infection has been detected in the Americas and Asia, but not in Europe [30]. Viremia and clinical signs in SVA infected pigs appear within 2 to 3 days post-inoculation and last for few days [31, 32]; therefore, there was minimal chance of an infected pig being undetected at the farm or during antemortem inspection. Despite SVA infected animals have been sporadically detected on-farm and at abattoirs during ante-mortem inspection [33], effective identification of farm outbreaks and surveillance system in place probably contributed to the absence of SVA genome in the tested SDPP samples. Further supporting this hypothesis, a US survey reported only 1.2% of oral samples from 25 states being RT-PCR positive for SVA [34]. On the other hand, the inability to detect TGEV in these samples is also consistent with a very low incidence in the US and European swine population [35-37]. In case of SDCoV, the current data agree with prevalence results from Puente et al. [38] that indicated absence of SDCoV and TGEV in 106 Spanish pig farms analyzed between 2017–2019. Furthermore, Ajayi et al. [39] indicated that the presence of SDCoV in Ontario farms decreased from 1.14% in 2014 to 0.08% in 2016, matching with our results of very low presence of SDCoV in the North American pig population analyzed in 2018–19. Noteworthy, samples from Brazil were negative for both PRRSV and PEDV, which is consistent with other reports indicating that these viruses are not present in this country [40-45].

All SDPP samples were tested for both the EU and US strains of PRRSV independently of the geographical origin of the SDPP. Samples from the US contained PRRSV genotype 2, except for one sample from US-IA that had a PRRSV genotype 1 RT-PCR positive result (Ct of 36, equivalent to -0.3 log10 TCID50/g SDPP). Similarly, the samples from EU contained the PRRSV genotype 1, except for one sample from Spain-C that had PRRSV genotype 2 positivity (Ct of 36, equivalent to -2.1 log10 TCID50/g SDPP). The detection frequency of positive samples differed between plants, with 100% in those from US-IA, 17% in US-NC and 50% in Canada production plants. In Europe, the RT-PCR positivity against PRRSV was 33% for Spain-NE, 58% for Spain-C, 50% for England and 83% for N-Ireland. However, in both the US and in the EU, the estimated PRRSV TCID50 in SDPP was < 2 virus particle/g SDPP, with an average Ct of 34 ± 2 and 34 ± 1 for genotype 2 and 1, respectively. Other works have reported low incidence of PRRSV viremia in slaughtered aged pigs [46] and differences in infection prevalence among US geographical areas [47], which is aligned with the results obtained in the present survey.

Estimated PEDV levels in SDPP was <2.0 log10 PEDV/g SDPP. The detection frequency of positive samples was 82% in US-IA, 50% in US-NC and 8% in Canada. These results indicated that PEDV genome distribution was low in Eastern Canada compared with the USA and agrees with surveillance of PEDV cases reported in North America [48, 49]. In Europe, the incidence of positive PEDV samples was 83% in Spain-NE, and 67% in Spain-C while in England and N-Ireland the samples were negative. Although the present study was not designed to elucidate seasonal differences in the estimated quantity for PEDV genome in the different parts of the world, the results suggest a higher frequency of detection and viral loads during the winter, while it was lower in summertime (S1 and S2 Tables). These results are consistent with the observation that PEDV is more stable in cold environments [50] and has a lower incidence of clinical diarrhea cases at farms during the summer season [51].

Both PPV and PCV-2 are stable non-enveloped DNA viruses [52, 53]. Frequency of detection of both PPV and PCV-2 was 100%, since all samples tested positive for genetic material. In all regions, the estimated level of PCV-2 was <2.0 log10 TCID50/g SDPP, while PPV presence was <3.0 log10 TCID50/g SDPP. Other studies have reported low levels of PCV-2 viremia in finishing swine [54, 55], in part due to the widespread use of PCV-2 vaccine [56, 57]. In addition, PCV-2 infections typically occur during the nursery and growing periods, so, most of animals reach slaughterhouse immunized and with low levels or no circulating virus [58]. On the other hand, PPV vaccines are commonly used in sows globally; considering the duration of PPV maternally derived immunity [53], it was expected to have evidence of natural infection in late finisher pigs. This was confirmed with the present study.

Detection frequency of SIV RNA was very sporadic and the range of potential viral contamination was variable. In IA, NC and Canada, 9%, 0% and 8% of samples yielded positive results, respectively, and estimated amount of viable virus was <1.0 log10 TCID50/g SDPP. Similarly, the frequency of detection of SIV in Spain-C, Spain-NE, England, N-Ireland and Brazil was 17%, 17%, 25%, 8% and 25%, respectively. However, when SIV was present, a very wide range of viral loads were obtained, from 0.3 to 5.6 log10 TCID50/g SDPP (corresponding to -0.7 to 4.6 log10 TCID50/g liquid raw plasma). It is speculated that slower line speed of abattoirs in Europe and Brazil compared to that in US and Canada, resulting in longer time for blood collection that may contribute to increased levels of SIV contamination.

Estimated levels of infectious viruses in commercially collected porcine plasma was significantly lower than viral levels at peak viremia of pigs [31, 46, 56, 59]. Commercially collected porcine plasma is harvested from animals that have been inspected and passed as fit for slaughter for human consumption, precluding collection of blood from clinically sick animals. Typically, market hogs have been vaccinated for many of the economically important diseases and have developed effective immunity [60, 61]. Combined inactivation by multiple hurdles for the viruses analyzed in this study would be >6 log10 TCID50/g SDPP for spray drying and post drying storage and >10 log10 TCID50/g SDPP if UV-C if also included (Table 1).

In summary, the data from this survey allowed the estimation of potential viral contamination in commercially collected porcine plasma. Estimated level of viral contamination in commercially collected porcine plasma ranged from <2.0 log10 TCID50 for most viruses with infrequent SIV levels as high as 4.5 log10 TCID50/g liquid plasma. The multiple hurdles in the manufacturing process (UV-C, spray drying and post drying storage) are theoretically capable of inactivating much higher levels of virus (11 to 20 log10 TCID50). These data suggest that the multiple hurdles in the manufacturing process of SDPP should be sufficient to inactivate much higher loads of viruses than the potential viral contamination that can be detected in commercially collected porcine plasma.

SDPP.

Ct values and estimated virus genome presence in SDPP per months during the years 2018–2019.

(XLSX)

Click here for additional data file.

Raw plasma.

Estimated virus genome presence in raw plasma per months during the years 2018–2019.

(XLSX)

Click here for additional data file.

2 Feb 2022

Submitted filename: PONE-D-21-33668_reviewer.pdf

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23 Feb 2022

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We apologized for the misunderstanding. In fact, we provide the data of all the information included in the manuscript. We clarified this subject in the revised version.

4. We note you have included a table to which you do not refer in the text of your manuscript. Please ensure that you refer to Table 3 in your text; if accepted, production will need this reference to link the reader to the Table.

The Table 3 was already included in the text of the initial submission. Lines 234-235 as Tables 2 and 3. In the new revised version appears in line 236-237 as Table 2 and Table3.

5. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

Captions have been included for the Supporting information files. We have identified the supplementary tables as:

S1 Table -SDPP. Ct values and estimated virus genome presence in SDPP per months during the years 2018-2019.

S2 Table – Raw Plasma. Estimated virus genome presence in raw plasma per months during the years 2018-2019.

Reviewer’s Comments to the Author

Reviewer #1: This manuscript provides valuable information on the presence of genomic information for porcine viruses of concern in spray dried porcine plasma. The estimation of TCID50 based on standard curves is also of interest, as is the % positive sample information for SDPP from different geographical regions.

The authors appreciated the comments from Reviewer #1 because confirms our believe that the information provided in the manuscript can be of relevance for the swine sector, not only for understanding the presence of swine viral genome in raw or SDPP but also as a picture of the presence of different viruses in the different geographical areas studied. This information can be of importance for surveillance studies because provide quantitative information about the incidence of the different virus of concern for the swine industry.

I have annotated the manuscript with suggestions for improving English language usage and reader understanding. Please use these suggestions if you agree. I have also asked some questions that can be addressed in your revision.

The authors appreciated all the grammar suggestions provided by the Reviewer #1. We incorporated all of them in the revised manuscript.

Regarding the questions from reviewers #1. Find below the authors response to these questions.

Line # 55. “…multiple hurdles that…”. The reviewer is suggesting to change the word “hurdles” by “steps” throughout the manuscript.

To the authors, the term “multiple hurdles” is a common term to describe food manufacturing systems as can be found in the publication of Leistner (2000). Int. J. Food Microb.

55:181-186. https://edisciplinas.usp.br/pluginfile.php/128773/mod_resource/content/1/Leistner.2000.pdf

Therefore, since this is a usual wording for the field being here studied, the authors prefer to keep the term “multiple hurdles” throughout the article.

Line #56. “…SD, 80oC throughout substance…”. How much time?

Throughout substance means that the particle achieves 80ºC during the drying process but the time that the particle is at that temperature is unknown and is variable depending on the driers design and capacity. The safety heat treatment is to achieve this 80ºC. It is the same safety concept that in case of meat for human consumption, the heat treatment at a minimum temperature of 80°C, which must be reached throughout the meat but without specifying the time at that temperature (European Council Directive 2002/99/EC. Annex III).

Line #56. “…ultraviolet light (UV) treatment (3000 J/L)…”. J/m2

No, the values are correctly expressed as J/L and not J/m2 as suggested by the reviewer #1. We refer to the paper of Blázquez et al., 2019. Reference [17] in the manuscript. In the case of treating liquid plasma, the UV system is a continuous flow reactor designed for exposing a liquid to UV light. Therefore, J/L is the appropriate unit of measure.

Line #139. “…After 1.5 hours at 37ºC, inoculum was removed, and 30 mL of medium were added. Titration was done in triplicate obtaining a final titer of 105.48 TCID50/mL.”. Two significant figures is enough. The titration assay is not accurate to three significant figures, I think.

The authors did not fully understand the comment from the reviewer #1. If he/she is referring that expressing the virus titer with maximum two decimals and not three as appear in some cases, we corrected this mistake in the revised version of the manuscript.

We assume that some were expressed with three significant figures due to the effect of the mathematical mean. We apologize for the mistake and thank you for correcting it.

Line #186. “…A viral suspension was obtained and titrated in triplicate, obtaining a final viral solution of 106.64 TCID50 /mL. “ Provide incubation duration, is CPE the endpoint?

Sorry, we forgot to provide this information. Thank you for your observation. Once the flasks were inoculated, they were incubated at 37ºC for four days, at which time a clear CPE is observed and the infection was stopped.

We add this information on line 188 of the revised manuscript:

“After that time, the contents of the tube were transferred to a 175 cm2 flask, in which 40 mL of MEM-E supplemented with 1% pyruvate were added. Inoculated flasks were incubated for four days at 37ºC until CPE was observed. A viral suspension was obtained and titrated in triplicate, obtaining a final viral solution of 106.64 TCID50 /mL. “

Line #197. “… Each kit contained a genome quantified standard for the different vi 195 ruses tested: PRRSV (PRRSV-I dtec-RT-qPCR, PRRSV-II dtec-RT-qPCR), PEDV (PEDV dtec-RT-qPCR), PPV (PPV-1 dtec-RT-qPCR) and SIV (SIV dtec-RT-qPCR).” Add information for PCV?

We do not provide information about PCV-2 in this section since in the case of PCV-2 it was not necessary to perform a quantitative PCR again due that the first PCR performed (PCV-2 (LSI VetMAXTM Porcine Circovirus Type 2 Quantification, Thermo Fisher Scientific, MA, USA)) was already quantitative.

Line #247. “…effective identification of non-symptomatic animals probably contributed to the absence of SVA genome in the tested SDPP samples.”. How are non-symptomatic animals identified?

SVA has been associated with lameness and cutaneous vesicles, as well as increased mortality in the first weeks of age. Therefore, the absence of these clinical signs, at least the ones affecting finishers would be one of the arguments by which SVA might not be detected. On the other hand, animals at abattoir are inspected ante-mortem, and presumably only animals non-displaying clinical signs are sacrificed and used for human consumption.

The reviewer is right that non-symptomatic animals is difficult to identified. We changed this paragraph in the revised version of the manuscript as following

“Despite SVA infected animals have been sporadically detected on-farm and at abattoirs during ante-mortem inspection [33], effective identification of farm outbreaks and surveillance system in place probably contributed to the absence of SVA genome in the tested SDPP samples”

Line #291. “…In all regions, the estimated level of PCV2- was low (<2 log10 TCID50/g SDPP that corresponds to less than 5 virus particles per g of raw plasma), while PPV presence was slightly higher (<2.0 log10 TCID50/g liquid plasma).” This value is the same as that for PCV-2. Am I missing something?

The value for PCV-2 was indicated per g of SDPP while the value for PPV was refer to liquid plasma. In the revised version we put both values as g of SDPP. This sentence has been changed in the revised version of the manuscript as following:

“In all regions, the estimated level of PCV-2 was <2 log10 TCID50/g SDPP, while PPV presence was <3.0 log10 TCID50/g SDPP.”

Line #308. “…It is speculated that differences in stunning method, design of collection trough or slower line speed of abattoirs in Europe and Brazil compared to that in US and Canada may contribute to different levels of SIV contamination.” How?

Slower line speed of abattoirs in Europe and Brazil may affect to have higher residence time during the collection period and therefore more time for potential contamination of blood with SIV at the slaughter line.

We changed this paragraph in the revised version of the manuscript as following:

“…It is speculated that slower line speed of abattoirs in Europe and Brazil compared to that in US and Canada resulting in longer time for blood collection that may contribute to increased levels of SIV contamination.”

Line #338. “…The authors have read the journal's policy and the authors of this manuscript have the following competing interests: EB, CR, and JPolo are employed by APC Europe, S.L.U.” JPujols?

Dr. Joan Pujols is a researcher of IRTA-CReSA and his never has been employed of APC Europe, S.L.U. or APC companies.

Line #584. Table 3. The reviewer #1 is asking why the range of the values of PEDV, PPV and PPRS-US are negative.

We appreciate the comment from reviewer #1. As indicated in the title of the Table 3, these values are expressed in log10 and therefore a negative log10 values do not mean that the virus particle is negative, only that is less than 1 particle/g of liquid plasma. For example, log10 -1.76 TCID50/ g liquid plasma in fact, means 0.017 TCID50/g liquid plasma

Reviewer #2: This is a well-written manuscript that contributes greatly to the literature and warrants publication. However, there is heavy discussion and conclusions that the estimated level of contamination was very low or low, whereas the data suggest otherwise. There was no prior establishment of what constitutes 'high' 'moderate' or 'low' levels of contamination, so these conclusions are entirely that of the authors, which have a conflict of interest in the data interpretation to be low. In reality, up to 100% contamination and relatively low Ct (below that of the established infectious dose) suggest there is risk in this ingredient, but the abstract, discussion, and conclusions try to convince the reader otherwise. It would be more appropriate to recognize there are a high number of positive samples with Ct below the known infectious dose, but that it is likely these are nonviable gene fragments that were inactivated during the manufacturing process.

We appreciated the comment received from the reviewer #2. It is true that the term high, moderate, or low levels of contamination is not defined and is subjective for each expert in the field. However, from the authors, having average values of log10 0.1 for PEDV, 1.56 for PCV-2, -1.4 for PRRSV US strain or 0.17 for PRRSV EU strain are relatively low, especially when compare with values of these viruses as the peak of viremia. Importantly, those values are considered low from the point of view of their potential infectiousness in an animal model. For example, 5.6 × 101 TCID50/g (1.75 log10 TCID50/g) was the minimum PEDV dose in feed to be infective for pigs (Schumacher et al., 2016, Am J Vet Res 77(10):1108-13). For PRRSV USA, a 2-mL inoculum containing 101 fluorescent foci units (equivalent to TCID50) of virus per milliliter was found sufficient to achieve infection by either route, but it was the very minimal dose to get it (Yoon et al., 1999 Vet Res 30(6):629-38). Therefore, all values obtained in plasma at slaughter should be considered within the low range of viral loads, most of them below the infectiousness levels as demonstrated when using one of the most susceptible routes of inoculation, the intraperitoneal one (Blázquez et al., 2019 Vet Microbiol 239:108450). Moreover, the likelihood of infection through the oral route is usually much lower compared to other routes such as drinking water or intranasal/oropharyngeal exposure as demonstrated with ASFV for example (Niederwerder et al., 2021 AnimaIs 11, 792). Therefore, all together would point out to low levels of contamination, also assuming this can be considered a subjective appraisal.

In any case, throughout the manuscript we have tried to eliminate this ambiguity of low or high levels of contamination. You can find these changes in the revised version of the manuscript.

Finally, although it is true that some co-authors have conflict of interest, all the data from the study is readily available in tables and supplementary material for the readers and it cannot be underestimated that other co-authors signing this manuscript are worldwide very well-recognized virology researchers in the field. Therefore, the authors consider that there is no bias with the information provided in this manuscript.

Submitted filename: Response to reviewers-R1bis.docx

Click here for additional data file.

14 Mar 2022

23 Mar 2022

Additional Editor Comments (if provided):

Thank you for addressing the reviewers comments. I ask that you fix the significant figures and add errors for your stock titers.

For all of the titers given in the methods, the significant figures are too high and errors are needed. Example, it says for PRRS (3268 EU strain) that the final titer was 10^5.48 TCID50/ml and done in triplicate. An error needs to be associated with the final titer and then the significant digits are so that there is only one significant digit in the error. So, if the error is 10^0.5, then the titer is 10^5.5 ± 10^0.5.

The authors appreciated the suggestion from the Academic Editor. We have included in the new version of the manuscript the error for the titers in the inoculum. As example the new reported information for the PRRS EU strain (3268EU) inoculum is as follow: ”… final titer of 105.5±0.2 TCID50/mL.”

The error should also be fixed in Table 2 and Table 3. The error should have one significant digit and this will tell you how many digits you can use in the number reported. See https://www.ruf.rice.edu/~bioslabs/tools/data_analysis/errors_sigfigs.html. Error and significant figures need also be fixed in the supplemental data.

The authors really appreciated the help from the Academic Editor to clarify the request and provide the significant digit for all data included in Table2 and Table 3 and Supplementary Table 1 and Suplementary Table 2. We are deeply in debt for her willing to review all the data in advance to the new submission.

The new revised version of the manuscript includes the correct format for all the data in the mentioned tables.

Submitted filename: Response to reviewers-2.docx

Click here for additional data file.

6 Apr 2022

Estimated quantity of swine virus genomes based on quantitative PCR analysis in spray-dried porcine plasma samples collected from multiple manufacturing plants.

PONE-D-21-33668R2

Dear Dr. Polo Pozo,

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Additional Editor Comments (optional):

Reviewers' comments:

11 Apr 2022

PONE-D-21-33668R2

Estimated quantity of swine virus genomes based on quantitative PCR analysis in spray-dried porcine plasma samples collected from multiple manufacturing plants.

Dear Dr. Polo Pozo:

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