Efficacy of spray-drying to reduce infectivity of pseudorabies and porcine reproductive and respiratory syndrome (PRRS) viruses and seroconversion in pigs fed diets containing spray-dried animal plasma1 J. Polo*2, J. D. Quigley†, L. E. Russell†, J. M. Campbell†, J. Pujols‡, and P. D. Lukert§ *APC Europe, S.A., Agda. Sant Julia 246-258. Pol. Ind. El Congost, E-08400 Granollers, Spain; †APC Inc., Ankeny, IA 50021; ‡Animal Health Institute CreSA, UAB Campus, CreSA, E-08193 Barcelona, Spain; and §Department of Medical Microbiology, College of Veterinary Medicine, University of Georgia, Athens 30602
ABSTRACT: Three experiments were conducted to evaluate viral inactivation by the spray-drying process used in the production of spray-dried animal plasma (SDAP). In Exp. 1, bovine plasma was inoculated with pseudorabies virus (PRV) grown in PK 15 cells. Three 4-L batches were spray-dried in the same manner and conditions of industrial SDAP production but with laboratory spray-drying equipment. Presence of infectivity was determined before and after spray-drying by microtiter assay in PK 15 cell cultures. Before spray-drying, all three samples contained 105.3 tissue culture infectious dose50 (TCID50)/mL of PRV. After four consecutive passages, no viable virus was detected in samples of spray-dried bovine plasma. In Exp. 2, bovine plasma was inoculated with porcine respiratory and reproductive syndrome (PRRS) virus propagated previously in MARC cell culture to provide approximately 106.3 TCID50/mL. Three 4-L batches were spray-dried in the same manner as Exp. 1. Before spray-drying, samples contained TCID50 of 104.0, 103.5, and 103.5/mL, respec-
tively. After four consecutive passages in MARC cell cultures, no viable virus was detected in spray-dried bovine plasma. In Exp. 3, 36 weaned piglets (28 d of age) were fed a common diet for 14 d and were determined to be negative for PRV, PRRS, and porcine parvovirus titer. Afterwards, pigs were allotted to six pens with six pigs per pen and fed diets containing either 0 or 8% SDAP (as-fed basis) for 63 d. The SDAP used in the feed contained antibody (titer 1:400) against porcine parvovirus. Blood samples were collected from pigs on d 0 and 63 to determine whether feeding SDAP caused seroconversion and development of antibodies against parvovirus, PRRS, or PRV. Inclusion of SDAP in the diet improved growth of pigs without seroconversion. Spray-drying conditions used in this study were effective in eliminating viable pseudorabies and PRRS viruses from bovine plasma. In this study, feeding SDAP that contained functional antibodies did not promote seroconversion in naı¨ve animals.
Key Words: Pigs, Porcine Parvovirus, Porcine Reproductive and Respiratory Syndrome Virus, Pseudorabies Virus, Spray-Dried Animal Plasma, Seroconversion 2005 American Society of Animal Science. All rights reserved.
Introduction Spray-dried animal plasma (SDAP) is a natural byproduct of the meatpacking industry. It is widely used in the diets of domestic animals to improve growth rate, feed intake, and feed efficiency (Coffey and Cromwell, 2001). Spray-dried animal plasma is produced from
1 This study was partially financed by the programs PROFIT (FIT060000-2000-188) and Eureka Euroagri (E! 2452 EUROAGRI IMMUCON). 2 Correspondence—phone: + 34 93 861 50 60; fax: +34 93 849 59 83; e-mail:
[email protected]. Received November 3, 2004. Accepted May 9, 2005.
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blood collected at veterinary-inspected abattoirs from animals designated as fit for slaughter for human consumption. Blood is collected into containers with anticoagulant, followed by chilling and centrifugation to separate plasma from the cellular fraction. Individual fractions are then spray-dried to produce ingredients used in food, feed, and industrial applications. Blood from healthy animals is normally sterile, except in the case of animals with subclinical bacteremia or virema (Bourgeois and Le Roux, 1982; Ockerman and Hansen, 1994; Carretero and Pare´s, 2000). Microbial contamination also may occur due to contamination on the animals’ skin at time of slaughter (Swingler, 1982; Pare´s and Carretero, 1997). In humans, viral inactivation methods for plasma used in transfusion have been reviewed (Cuthbertson
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et al. 1991; Pamphilon, 2000); however, spray-drying methods used in the manufacturing of SDAP have not been evaluated for inactivation of economically important pathogens. Rapid changes in temperature and pressure during spray-drying cause immediate evaporation of water, leading to decreased numbers of viable microorganisms (Lievense, 1991; Linders et al., 1996; To and Etzel, 1997a). Nonetheless, specific spray-drying conditions are critical to ensure decreases in microbial load (Lian et al., 2002). Therefore, the objective of these experiments was to evaluate the efficacy of pseudorabies virus (PRV) and porcine respiratory and reproductive syndrome (PRRS) virus inactivation under typical industrial spray-drying conditions. An additional objective was to determine whether naı¨ve pigs fed diets containing SDAP with antibody titer against porcine parvovirus (PPV) developed titers to this pathogen.
Materials and Methods Experiment 1 A virus stock of PRV (supplied by A. W. Roberts, Athens Diagnostic Lab, Univ. of Georgia, Athens) was propagated in porcine kidney cell (PK) grown in modified Eagle medium, supplemented with 10% tryptose phosphate broth, fetal bovine serum, and antibiotic cocktail containing penicillin, streptomycin, and amphotericin B. Porcine PRV was propagated for 48 h in the PK15 cell line on PK15 monolayers. The supernatant fraction was centrifuged and divided into 4-mL aliquots containing tissue culture infectious dose50 (TCID50) of 107.3/mL. Twenty milliliters of stock virus (five vials) were added to 4 L of liquid bovine plasma to approximate 105.0 TCID50/mL. Bovine plasma was derived from whole blood collected from a USDA-inspected slaughter facility. Animals were inspected and approved for slaughter for human consumption. Blood was collected into stainless steel pans containing anticoagulant and was transported to the laboratory, where plasma was separated by centrifugation. Bovine plasma was used to ensure porcine pseudorabies antibodies were not present. A 10-mL sample of the inoculated 4L batch was frozen (−20°C) for later determination of viable viral particles. Three 4-L batches were dried using a pilot-plant spray-drier (compact spray dryer, Anhydro A/S, Copenhagen, Denmark). Inlet temperature was 240 ± 1°C and outlet temperature was 90 ± 1°C. The suspension flow to the nozzle was 10 L/h. Airflow through the feeding nozzle was set at 15 m3/h at 20°C. The estimated dwell time was 0.41 s. Samples of inoculated liquid plasma and spray-dried plasma were frozen with dry ice before analysis of infectivity in PK 15 cell cultures using the microtiter assay procedure (Burleson et al., 1992). Dried samples were reconstituted by adding 25 mL of distilled water to each sample. To determine viral survival, 5 mL of each of the reconstituted spray-dried samples were inoculated in a 75-
cm2 flask of PK 15 cells. After 3 d, cell cultures were harvested and passed to new PK 15 cell cultures. A total of four consecutive passages were made as a standard procedure to ensure that any viable virus had ample opportunity to adapt, grow, and multiply in the PK 15 cell cultures. A tissue culture virus-neutralizing (VN) test was performed on the three spray-dried samples to verify whether the bovine plasma contained neutralizing antibodies to PRV.
Experiment 2 Stock PRRS virus (supplied by A. W. Roberts as in Exp. 1) was propagated in MARC cells and harvested 5 d after infection. The stock was assayed by infecting MARC cell cultures with 10-fold dilutions of the virus and determining the TCID50 by the indirect fluorescent antibody procedure (Nelson et al., 1993). A monoclonal antibody against PRRS virus (provided by A. W. Roberts) was the primary antibody and fluorescein isothiocyanate (FITC)-labeled anti-mouse globulin was the secondary antibody. Stock virus (107.9 TCID50/mL) was mixed with liquid bovine plasma at a ratio of 100 mL in 4 L of plasma to approximate 106.3 TCID50/mL. After the stock virus and bovine plasma were mixed, an aliquot of 10 mL was removed before spray-drying. Three 4-L batches of plasma were spray-dried as in Exp. 1. Samples of inoculated liquid plasma and spraydried plasma were frozen on dry ice until analyzed for infectivity. Dried samples were reconstituted as 9 g of powder to 100 mL of phosphate-buffered saline (pH 7.2) and stirred until dissolved. Inoculated liquid plasma and spray-dried plasma samples were analyzed for infectivity in MARC cell cultures using the microtiter assay procedure (Burleson et al., 1992). Two milliliters of each reconstituted spray-dried sample were inoculated in a 250-cm2 flask of MARC cell. After 5 d, cell cultures were harvested and passed to new MARC cell cultures. A total of four consecutive passages were made. The media from each passage were assayed for infectivity using the microtiter assay procedure.
Experiment 3 Thirty-six Landrace × Duroc weanling pigs (28 d of age) were obtained from the breeding center at the Experimental Farm of Institut de Recerca i Tecnologia Agroalimenta`ries, in Prat de Llobregat, Spain, and were maintained in conditions free of PPV, PRRS, PRV, and several other pathogens. Before the start of the study, a blood sample was obtained by venipuncture of the anterior vena cava to assure the absence of antibodies to specific pathogens including PPV (Ingezim PPV, Ingenasa, Spain), PRRS (PRRSV test kit, IDEXX, Netherlands), and PRV (Ingelvac Aujeszky diagnostic kit, Svanova, Uppsala, Sweden). Animals were housed in accordance with the rules provided in the laws protecting laboratory animals. Ani-
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Table 2. Calculated nutrient content of the diets, as-fed basis
Table 1. Composition of the diets, as-fed basis Dietsa Ingredients, g/kg Corn Wheat Lard Soybean meal, 36% CP Spray-dried animal plasmab Extruded soybeans Sweet milk whey DL-Methionine L-Lysine-HCl L-Threonine Limestone Dicalcium phosphate, 18.5% P Salt Vitamin and mineral premixc
Diets
Common starter
Control
SDAP
152.90 360.45 14.00 203.30 — 130.00 107.10 1.37 3.31 1.10 2.93 18.59 0.95 4.00
640.00 — 9.40 314.30 — — — 0.88 1.68 0.23 6.56 19.56 3.39 4.00
591.81 121.90 — 162.10 80.00 — — 7.24 — — 10.78 15.95 6.22 4.00
b
a
All pigs fed the common starter diet from d 28 to 42 of age. SDAP = spray-dried animal plasma (Appetein, APC-Europe, Barcelona, Spain). c Providing per kilogram of diet: vitamin A, 10,000 IU; vitamin D3, 2,000 IU; vitamin E, 15 mg; vitamin B1, 1.3 mg; vitamin B2, 3.5 mg; vitamin B12, 0.025 mg; vitamin B6,1.5 mg; calcium panthothenate, 10 mg; nicotinic acid, 15 mg; biotin, 0.1 mg; folic acid, 0.6 mg; vitamin K3, 2 mg; Fe, 80 mg; Cu, 140 mg; Co, 0.75 mg; Zn, 185 mg; Mn, 60 mg; I, 0.75 mg; Se, 0.10 mg; ethoxyquin, 0.15 mg. b
mal Experimentation Ethics Committee of IRTA approved the animal experimental procedures. Weaned pigs were housed in the experimental facility and fed a common diet (Table 1) for 14 d before initiation of the experiment. All pigs tested negative for antibody titers to PPV, PRRS, and PRV at the end of the 14-d acclimation period. Pigs were allocated into six pens according to a randomized complete block design with three BW blocks of two pens each. Special care was taken to ensure an equal distribution of pigs by weight, sex, and maternal origin among the pens of each block. Each pen had six pigs and was assigned randomly within block to one of two experimental treatments (18 pigs and three pens per treatment). Experimental treatments were diets containing either 0 or 8% SDAP (asfed basis; Table 1). Appetein (APC-Europe, Barcelona, Spain) was the source of SDAP, and it contained a mixture of bovine and porcine plasma. The common starter diet and the experimental diets were formulated to meet or exceed the nutrient requirements of swine (NRC, 1998), and contained 12.6 and 12 g of total lysine and 3.3 and 3.2 Mcal of ME/kg, respectively (as-fed basis; Table 2). Feed and water were available ad libitum throughout the study. The pens (3 m2) had slatted floors with single-pen feeders and an automated water supply. Room temperature was maintained between 18 to 24°C during the experimental period by mechanical ventilation and a thermostat control system. Artificial light was provided 12 h/d. Pigs were weighed individually on d 0, 21, 42, and 63. Average daily feed intake and feed efficiency were calculated for d 0 to 21, 21 to 42, and 42 to 63.
Nutrients, g/kga DM CP Crude fiber Fat Ash Ca P Cl Na Methionine Methionine + cysteine Lysine Tryptophan Threonine ME, Mcal/kg
Common starterb
Control
SDAPc
894.0 215.0 33.5 46.8 60.0 7.5 4.8 3.0 1.5 4.2 7.4 12.6 2.4 7.8 3.3
892.2 210.0 32.4 38.8 56.0 8.0 7.0 2.8 1.5 4.0 7.2 12.0 2.3 7.8 3.2
893.5 210.0 28.3 28.6 54.0 8.5 7.0 2.0 2.8 3.6 8.3 12.1 2.6 8.9 3.2
a The experimental diets were formulated to meet or exceed the nutrient requirements of swine (NRC, 1998). b All pigs fed the common starter diet from d 28 to 42 of age. c SDAP = spray-dried animal plasma (Appetein, APC-Europe, Barcelona, Spain).
A blood sample was obtained from all the animals on d 0 and 63. Serum was collected for determination of presence of antibodies against PPV, PRRS, and PRV. Additional samples were collected on d 21 and 42 for determination of antibodies against PPV. Antibody assays also performed on SDAP used in the study. The product was diluted to a concentration of 8% (wt/vol) in sterile distilled water. Body weight gain, feed intake, and feed efficiency were analyzed as a randomized complete block design with two treatments and three BW blocks (six pens total), with the pen as the experimental unit. Analysis of variance was performed using the GLM of procedure of SAS (SAS Inst., Inc., Cary, NC).
Results Experiments 1 and 2 PRV Assay. Neutralizing antibodies to PRV were not detected in the bovine plasma used in Exp. 1. After inoculation with PRV, infectivity in each of the three lots of liquid bovine plasma samples was 105.3 TCID50/ mL (Table 3). Spray-dried samples contained no detectable viable PRV in any of the four consecutive passages in PK 15 cell cultures (Table 3). PRRS Assay. Neutralizing antibodies to PRRS were not detected in the bovine plasma used in Exp. 2. After inoculation of liquid plasma with PRRS, infectivity of the three batches was 104.0, 103.5, and 103.5 TCID50/mL, respectively (Table 3). This was 200 to 500 times less than the expected infectivity (106.3 TCID50/mL). The PRRS virus can be unstable in some environmental conditions (Van Alstine et al., 1993), which may explain
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Table 3. Pseudorabies virus (PRV) and porcine reproductive and respiratory syndrome (PRRS) virus infectivity of inoculated bovine plasma samples before and after spray-dryinga Lot No. sample identification PRV assay Lot No. 1 Lot No. 2 Lot No. 3 PRRS assay Lot No. 1 Lot No. 2 Lot No. 3
Table 4. Performance by pigs fed diets containing 0 or 8% (as-fed basis) SDAP (means ± SEM)a,b SDAP Item
Inoculated liquid plasma samples, TCID50/mLb
Spray-dried samples, TCID50/mL
105.3 105.3 105.3
Not detectedc Not detected Not detected
104.0 103.5 103.5
Not detected Not detected Not detected
a
Spray-dried samples were passed four consecutive times in PK 15 cells or MARC cells for PRV and PRRS virus, respectively, to amplify any viable virus that might have been undetected in the first pass. b TCID50 = tissue culture infection dose as determined by microtiter assay procedure (Burleson et al., 1997). c The theoretical limit of detection of the method used was estimated to be able to detect as little as 0.2 viral particles/mL.
the decreased infectivity in samples before drying. Spray-dried samples had no detectable viable PRRS virus in any of the four consecutive passages in MARC cell cultures (Table 3).
Experiment 3 Blood samples collected from pigs on d 0, 21, 42 and 63 were negative for all antibodies tested, indicating a lack of seroconversion during the trial. The SDAP used in the experimental diet contained an antibody titer for PPV (1:400) but not for PRRS or PRV. Clinical symptoms of disease were not observed in pigs fed the SDAP diet; however, during the fifth week of the experiment, a watery diarrhea was observed in pigs fed the control diet. Average daily gain from d 22 to 42 tended to be greater (P < 0.07) for pigs fed SDAP than for those fed the control diet (Table 4).
Discussion Plasma is separated from whole blood collected primarily from pigs and cattle slaughtered for human consumption (Russell, 2001). Spray-dried animal plasma is considered to be an essential ingredient in the diet for weanling piglets (Nelssen et al., 1999), and it is commonly used in the diet for calves, dogs, cats, and aquatic species. Therefore, it is important to determine whether SDAP contains viable viruses. The Office International des Epizooties (OIE) List A diseases include transmissible diseases that have the potential for very serious and rapid spread, irrespective of national borders, which are of serious socioeconomic or public health consequence, and which are of major importance in the international trade of animals and animal products. For bovine and porcine diseases, the
BW, kg d0 d 21 d 42 d 63 ADG, g/d d 0 to 21 d 22 to 42 d 43 to 63 d 0 to 63 ADFI (as-fed basis), g/d d 0 to 21 d 22 to 42 d 43 to 63 d 0 to 63 Feed:gain d 0 to21 d 22 to 42 d 43 to 63 d 0 to 63
0%
8%
P-value
13.6 26.4 42.9 61.4
± ± ± ±
2.46 3.33 4.16 5.28
13.6 27.5 45.5 64.0
± ± ± ±
2.38 3.09 3.76 3.72
NSc NS NS NS
611 783 877 761
± ± ± ±
40.9 40.9 47.8 47.1
663 856 884 801
± ± ± ±
34.4 32.2 13.6 21.5
NS 0.07 NS NS
1,056 1,603 2,120 1,593
± ± ± ±
76.0 131 153 116
1,126 1,738 2,279 1,714
± ± ± ±
92.8 121 105 106
NS NS NS NS
1.73 2.04 2.42 2.09
± ± ± ±
0.01 0.07 0.05 0.02
1.69 2.03 2.58 2.14
± ± ± ±
0.05 0.06 0.13 0.08
NS NS NS NS
a Pigs (42 d of age; 36 total) were distributed in six pens containing six pigs per pen (three pens and 18 pigs per treatment). b SDAP = spray-dried animal plasma (Appetein, APC-Europe, Barcelona, Spain). c NS = P > 0.10.
list includes foot and mouth disease, African swine fever, classical swine fever, bluetongue, contagious bovine pleuropneumonia, lumpy skin disease, swine vesicular disease, vesicular stomatitis, Rift Valley disease, and pestes des petits ruminants. These diseases are a primary concern. A comprehensive risk assessment was conducted to evaluate the potential for transmission of List A diseases by oral consumption of SDAP (Hueston and Rhodes, 1999). Risk factors evaluated included animal sourcing, harvesting, processing techniques, and product uses. The conclusion was that “there is a remote to minuscule risk that spray-dried plasma for animal consumption that is sourced from E.U., U.S., Canada, and Argentina will contain OIE List A disease infectivity” (Hueston and Rhodes, 1999). The spray-drying process involves atomization of the liquid into a heated chamber. To and Etzel (1997b) showed that outlet temperature was the most critical factor in the inactivation of Brevibacterium linens. Minimal reduction in cell viability was reported when bacterial suspensions were atomized into either a cool or a heated chamber; however, a significant decrease in bacterial numbers was reported as the liquid was atomized into a heated chamber and allowed to dry. A linear decrease in bacterial survival was reported as the outlet temperature was increased from 60 to 90°C, demonstrating the importance of the outlet temperature in the bacteria inactivation. Thermal inactivation or inactivation by dehydration have been suggested as mechanisms that may contrib-
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ute to microbial mortality during the spray-drying process (Lievense et al., 1990, 1992; To and Etzel, 1997a). Both mechanisms occur simultaneously and affect the microorganism differently depending on the microorganism’s resistance to heat or dehydration. Some microorganisms adapt to high temperature; however, the short drying time with the almost immediate increase in temperature does not allow the microorganism time to adapt to this high temperature (Linders et al., 1996). Lievense (1991) suggested that the cell damage or mortality caused by thermal inactivation is due to an effect on DNA, RNA (including ribosomal RNA), proteins (enzymes), and the cell membrane. Inactivation by dehydration leads to the loss of cell components including cations, nucleotides, enzymes, proteins, and amino acids (Wagman, 1960; Brennan et al., 1986; Beker and Rapoport, 1987). Zimmermann (1987) suggested the rapid removal of water was important in microbial inactivation. Damage to the cytoplasmic membrane also is considered to be a primary mechanism leading to microbial inactivation (Lievense, 1991; Lievense and Van’t Riet, 1994). In these experiments, PRV and PRRS virus were added to bovine plasma and subsequently spray-dried. Before spray-drying, PRV titer was 105.3 TCID50/mL, but no viable virus was detected after spray-drying and after four consecutive passages on PK 15 cell cultures. Before spray-drying, PRRS titer was between 104.0 and 103.5 TCID50/mL, and PRRS could not be detected after spray-drying and after four consecutive passages on MARC cell cultures. These data indicate that the spraydrying process inactivated both viruses. Polo et al. (2002) reported that the spray-drying process decreased Escherichia coli over five log units. Over the past 20 yr, random samples of commercially produced SDAP have been submitted to Iowa State University Diagnostics Laboratory for viral screening (bovine reovirus, IBR virus, PI3, PPV, PRRS, TGE, SIV, rabies virus, blue tongue; CFR 9, 1996). Viral contamination has never been reported (M. Vanden Berg, APC Inc., Ankeny, IA, personal communication). Typically, SDAP is added to the diet for weaning pigs at a rate of 3 to 5% and fed for the first 1 to 2 wk after weaning (Coffey and Cromwell, 2001). In the present experiment, specific pathogen-free pigs housed in isolated facilities were fed a diet containing 8% spray-dried plasma for 63 d. A typical SDAP effect was observed as indicated by improved ADG over the period of d 22 to 42 (P < 0.07; Table 4). According to Torrallardona et al. (2002), the positive effects of SDAP on growth performance appears to be higher in pigs weaned at younger age. Our results of growth performance starting with 42-d-old pigs fed SDAP for 63 d confirm this suggestion. To determine whether the pigs were or became infected with PPV, PRV, or PRRS, blood samples were taken before, at the beginning, and throughout the experiment to determine the presence of antibodies. Antibodies were not detected for any of these viruses, indicating that the pigs did not become infected.
In summary, spray-drying techniques used in this study were effective in eliminating infectivity of PRV and PRRS viruses when added to bovine plasma. Pigs fed a diet with 8% SDAP (as-fed basis), which contained antibody titers for PPV during 63 d, did not develop clinical or serological symptoms of viral exposure. In conclusion, these data suggest that with respect to viral contamination, SDAP is a safe feed ingredient for use in swine diets.
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