J. Anim. Sci. 2004. 82:1942-1951
© 2004 American Society of Animal Science
ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
Vitamin E supplementation does not mitigate the acute morbidity effects of porcine reproductive and respiratory syndrome virus in nursery pigs1
T. L. Toepfer-Berg*,
J. Escobar*,2,
W. G. Van Alstine
,
D. H. Baker*,
J. Salak-Johnson* and
R. W. Johnson*,3
* Department of Animal Sciences, University of Illinois, Urbana 61801 and
and
Animal Disease and Diagnostic Laboratory, Purdue University, West Lafayette, IN 47907
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Abstract
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The objective of this study was to determine whether feeding a vitamin E–rich diet would benefit nursery pigs infected with porcine reproductive and respiratory syndrome virus (PRRSV). Sixty-four pigs were subjected to one of four treatment combinations (2 x 2 factorial) of dietary vitamin E (adequate or excess) and PRRSV (medium or inoculation with VR-2385 isolate P-129). Pigs were fed experimental diets during a 3-wk period before inoculation as well as during a 12-d period after inoculation. Growth performance was determined throughout the study, and lipid peroxidation in liver, glutathione peroxidase (GPX) activity in serum, circulating white blood cells, and serum interleukin-1ß (IL-1ß) and interferon-
(IFN-) were determined in samples collected from pigs killed 4 or 12 d after inoculation. Infection by PRRSV (P < 0.001) induced a marked decrease in both ADFI and ADG, but neither the main effect of diet nor the diet x PRRSV interaction was significant. Neither diet nor PRRSV affected feed efficiency. At 12 d after inoculation, lipid peroxidation in liver and GPX activity in serum were lower in pigs fed excess vitamin E than in those fed adequate vitamin E (P < 0.01), suggesting that the diet high in vitamin E bolstered the antioxidant status of the pigs. However, PRRSV did not affect lipid peroxidation in liver or serum GPX activity, and the diet x PRRSV interaction was not significant. White blood cell counts were decreased and IFN-, and IL-1ß were increased (P < 0.05) 4 and 12 d after inoculation in PRRSV-infected pigs, but neither diet nor the diet x PRRSV interaction was significant. Collectively, these results indicate that increasing antioxidant defenses by feeding high levels of vitamin E did not ameliorate the effects of PRRSV on decreased growth, leukopenia, and increased serum IL-1ß and IFN-. Thus, feeding nursery pigs a diet high in vitamin E may not be useful for mitigating the acute morbidity effects of PRRSV infection.
Key Words: Antioxidant • Cytokines • Growth Performance • Pig • Porcine Reproductive and Respiratory Syndrome Virus • Vitamin E
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Introduction
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The porcine reproductive and respiratory syndrome virus (PRRSV) is highly prevalent in U.S. swine herds. It preferentially infects and replicates within mononuclear phagocytic cells (i.e., macrophages) located within the mucosal surface of the respiratory tract. Infected macrophages spread the virus to regional lymphoid tissue and eventually to other macrophages in disparate tissues and to monocytes. Mononuclear phagocytic cells infected by PRRSV generate reactive oxygen species (ROS) (Chiou et al., 2000
) and produce copious amounts of inflammatory cytokines (Thanawongnuwech et al., 1997; Van Reeth et al., 1999; Van Reeth and Nauwynck, 2000). Oxidative stress and an overabundance of inflammatory cytokines, such as interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and tumor necrosis factor-
(TNF), are known to cause tissue damage in a number of diseases and likely do so in PRRSV infection as well. Furthermore, IL-1ß, IL-6, and TNF induce systemic illness, including anorexia, fever, and lethargy. If ROS and overproduction of cytokines can be regulated after infection by PRRSV, it might be possible to limit pulmonary damage and promote recovery after infection.
Recent studies in mice showed that feeding a diet high in vitamin E inhibited the increase in IL-1ß and TNF secretion but enhanced the secretion of the antiviral T helper-1 cytokine, interferon- (IFN-) (Hayek et al., 1997; Han and Meydani, 1999; Han and Meydani, 2000). Body weight loss caused by influenza infection was also inhibited by vitamin E. However, whether feeding vitamin E in excess of the NRC-recommended level is beneficial to pigs infected with PRRSV is not known. Therefore, the objective of the current study was to evaluate the effects of a vitamin E-rich diet on growth performance, circulating cytokines, and several hematological traits in nursery pigs infected with PRRSV.
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Materials and Methods
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Animals, Housing, and Experimental Design
Thirty-two 3-wk-old Ausgene Line 20 dam x Line 5 sire pigs obtained from United Feeds Inc. (Sheridan, IN) were used in each of two trials. The two trials were identical but were conducted at different times. Pigs were bled by jugular venipuncture at 1 wk of age and virus isolation procedures were performed to verify they were free of PRRSV. Pigs were brought to the University of Illinois at 2 wk of age, randomly divided into two groups and placed in disease-containment chambers. Pigs received daily injections of lincomycin (11 mg/kg BW; Pharmacia & Upjohn Co., Kalamazoo, MI) as a precautionary measure for 5 d after arrival.
The disease-containment chambers were discretely ventilated with HEPA-filtrated air. The chambers provided 1.9 x 2.4 m of floor area (plastic-coated expanded metal) and were equipped with a self-feeder and nipple waterer. Pigs were maintained under an 18 h light:6 h dark lighting regimen (lights on at 0500) and had ad libitum access to water and feed. Chamber temperature was maintained at 32°C for the first 2 wk after arrival and then decreased 2°C each week until reaching a desired 24°C. Chambers were washed each day to remove fecal matter.
At 3 wk of age, pigs were divided into uniform blocks based on BW, sex, and litter of origin and were randomly assigned to one of four treatments. For each of the two trials, two pigs (one barrow and one gilt) were placed in each of 16 chambers. The four treatments were set in a 2 x 2 factorial arrangement, consisting of vitamin E (adequate or excess) and PRRSV (5 mL of Tissue Culture Infectious Dose50 of high-virulence strain VR-2385 [American Tissue Culture Collection, Manassas, VA] or sterile medium). The diets were formulated to contain the NRC-recommended level (11 mg/kg) of vitamin E in the form of a D- and DL--tocopheryl acetate mixture or a supplemental level (550 mg/kg). The vitamin E was purchased from ADM (Des Moines, IA) and consisted of 22.2% D--tocopheryl acetate, and 77.8% DL--tocopheryl acetate. Relative to DL--tocopheryl acetate, D--tocopheryl acetate has been estimated to contain 128% relative vitamin E activity in pigs (Anderson et al., 1995). The composition of each diet is shown in Table 1. Analysis for vitamin E indicated the adequate and excess diets contained (as fed) 13 mg/kg and 419 mg/kg, respectively (NP Analytical Laboratories, St. Louis, MO). Throughout the study, pigs were given ad libitum access to their respective diets unless otherwise noted. At 6 wk of age, one-half of the pigs assigned the adequate and excess vitamin E treatments were inoculated intranasally with PRRSV, whereas the other half received sterile culture medium. To minimize the chance for cross-contamination, pigs in one suite received PRRSV, whereas pigs in the other suite received sterile culture medium. The PRRSV treatment was imposed in each suite across the two trials. Biosafety Level 3 procedures (Richmond and McKinney, 1993) were employed at all times.
Pigs were fasted 12 h before measurement of BW at 0, 4, and 12 d after inoculation. After inoculation, feeders were weighed daily to determine feed intake. In each of the two trials, two chambers of pigs (four pigs total) from each treatment were randomly selected and humanely killed using sodium pentobarbital (1.1 g/kg BW, i.v.) for tissue sample collection at d 4 and 12 after inoculation. These days were chosen based on a previous study in which pigs began to show clinical signs of sickness 4 d after inoculation and began to recover 12 d afterward (Escobar et al., 2002). Whole blood was collected for leukocyte differential counts and serum was prepared for determination of GPX activity as well as levels of -tocopherol, IL-1ß, and IFN-. Serum was also subjected to virus isolation procedures to determine the presence or absence of PRRSV. Liver samples were collected, frozen in liquid nitrogen, and stored at –80°C until used to determine oxidative stress by measuring lipid peroxidation in the thiobarbituric acid–reactive substances (TBARS) assay. All procedures were approved by the campus Institutional Animal Care and Use Committee and the Environmental Health and Safety Committee.
Leukocyte Differential Counts
Complete white blood cell (WBC) and differential counts were determined from blood samples collected in Vacutainer tubes containing EDTA (BD Vacutainer, Franklin Lakes, NJ). Complete WBC counts were performed using a Z1 Coulter Counter (Coulter Corp., Miami, FL), and differential counts were performed manually on Hema 3 (Fisher Scientific Co., Pittsburgh, PA) stained blood smears. Two counts of 100 cells each were taken on each slide. The average number of cells present was determined by averaging the two counts, and the percentage of neutrophils, lymphocytes, monocytes, and eosinophils was determined.
Thiobarbituric Acid–Reactive Substances
Thiobarbituric acid–reactive substances (TBARS) were assayed as previously described (Ohkawa et al., 1979; Kikugawa et al., 1992) with the following modifications. In brief, liver samples were suspended in PBS containing 0.01% butyl hydroxytolune (BHT) and homogenized using a Dounce homogenizer to achieve a 30% wt/vol solution. Resulting liver homogenates were incubated with 8.1% SDS (wt/vol), 20% acetic acid (vol/vol, pH 3.5), and 0.8% thiobarbituric acid (wt/vol) for 60 m at 95°C. Cooled samples were extracted with 15:1 butanol:pyridine and the upper organic phase was recovered. Absorbance was determined using a plate reader (540 nm) with reference to a standard curve prepared with 1,1,3,3-tetramethoxypropane, and results were expressed as nanomoles of malondialdehyde (MDA) formed per milligram of protein. Total proteins were determined from liver tissue solubilized in lysis buffer (50 mM Tris•HCl pH 7.4, containing 10% glycerol, 1.0% Triton-X-100, 100 mM NaCl, 50 mM NaF, 1 mM EDTA, 1 mM EGTA, and 2 mM phenylmethanesulfonyl fluoride, and leupeptin, aprotinin, and pepstatin at 1 µg/mL each) by the Bio-Rad Dc protein assay according to the manufacturer’s instructions (Bio-Rad Laboratories, Hercules, CA).
Glutathione Peroxidase (GPX) Activity
Glutathione peroxidase activity was quantified in serum samples using a kinetic colorimetric assay from Cayman Chemical (Ann Arbor, MI). The kit measured all of the glutathione-dependent peroxidases. In brief, frozen serum samples (–80°C) were thawed and diluted in sample buffer before analysis. Each sample well contained 100 µL of assay buffer ([50 mM Tris•HCl, pH 7.6, containing 5 mM EDTA], 50 µL of co-substrate mixture [reconstituted lyophilized powder of NADPH, glutathione, and glutathione reductase]), and 20 µL of sample. The serum samples were diluted to 1:20 in sample buffer (50 mM Tris•HCl, pH 7.6, containing 5 mM EDTA and 1 mg/mL BSA) before assaying. To initiate the reaction, 20 µL of cumene hydroperoxide was added to the wells. Samples were monitored once per minute over 7 min for a decrease in absorbance at 340 nm using a Molecular Devices OPTImax tunable microplate reader (Sunnyvale, CA). Background well absorbance was subtracted from sample well absorbance, and positive control (bovine erythrocyte GPX) wells were used to confirm that the assay was working properly.
HPLC Analysis of -Tocopherol
Serum and liver -tocopherol levels were measured using a Shimadzu HPLC system (SCL-10A VPSystem controller, LC-10AT VP pump, FCV-10AL VP mixer, and a DGU-14A degasser, Shimadzu, Kyoto, Japan) equipped with a manual injector and fluorescence detector. The column used was a Supelco Discovery C18, and a Discovery C18 guard column was attached online before the main column (Supelco, Bellefonte, PA). All solvents were HPLC grade and mobile phase solvents were filtered before analysis. The mobile phase used was 99:1 methanol to water (vol/vol). The flow rate used was 1.2 mL/min.
Serum -Tocopherol Extraction.
-Tocopherol (Sigma Chemical, St. Louis, MO) and tocol (Matreya Inc., Pleasant Gap, PA) were used as external and internal standards, respectively. Briefly, 200 µL of serum and 200 µL of ethanol plus 0.1% BHT were added to a 1.5-mL microcentrifuge tube. Next, 10 µL of 10 µg/mL tocol and 500 µL of hexane were added and vortexed for 30 s. The tubes were centrifuged for 1 min at 2,400 x g at 4°C, the top hexane layer was removed, pipetted into a microcentrifuge tube, and placed under argon to dry. The hexane extraction was repeated. Once dried, 500 µL of hexane was used to rinse the sides of the tube, vortexed for 30 s, and dried. For injection, 15 µL of methanol was added to reconstitute the sample, vortexed for 30 s, and 10 µL of the sample was subjected to HPLC analysis.
Serum IFN- and IL-1ß
Serum of one pig selected from each chamber (n = 8; four barrows and four gilts) was assayed for IFN- and IL-1ß. Serum IFN- and IL-1ß were measured using porcine-specific ELISA kits purchased from Pierce Endogen (Rockford, IL) and R & D systems (Minneapolis, MN), respectively. The IFN- assay had a sensitivity of <2 pg/mL, and the minimum detectable concentration of IL-1ß was 10 pg/mL. For each cytokine, all samples were assayed in a single run. The intraassay variation was <10%.
Statistical Analyses
All data were initially subjected to three-way (trial x vitamin E x PRRSV) ANOVA procedures using the GLM procedure of the SAS (SAS Inst. Inc., Cary, NC). The effect of trial was not significant and was excluded from subsequent analyses. The chamber was considered the experimental unit for growth performance, whereas the pig was the experimental unit for other measurements. One pig developed a bacterial dermatitis with Staphylococcus hyicus and was removed from the study, and hematological data from one pig were excluded because several values differed from the treatment mean by more than three standard deviation units. All data are presented as means ± SEM.
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Results
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PRRSV Infection
Pigs were tested before the study and determined to be PRRSV-free. They were retested at the end of the study to verify the efficacy of the disease model. As expected, PRRSV was isolated from sera of all pigs inoculated with PRRSV regardless of dietary treatment, whereas PRRSV was not detected in sera of pigs receiving sterile culture medium. There were no gross pulmonary lesions evident in either control or PRRSV pigs.
Growth Performance
Growth performance of pigs from wk 3 to 6 (before inoculation) was not affected by diet. During the 3-wk preinoculation period, ADFI and ADG of pigs fed adequate vitamin E was 0.58 kg ± 0.02 and 0.40 kg ± 0.02, respectively; ADFI and ADG of pigs fed excess vitamin E was 0.58 kg ± 0.02 and 0.37 kg ± 0.02, respectively. Thus, at the time of inoculation body weight of pigs fed adequate and excess vitamin E was similar (14.4 kg ± 0.30 and 13.7 kg ± 0.30, respectively).
The effect of diet and PRRSV on ADFI, ADG, and gain:feed (G:F) are summarized in Figures 1, 2, and 3, respectively. There was a main effect of PRRSV for ADFI and ADG (P < 0.001), but neither diet nor the interaction between diet and PRRSV were significant. For pigs on diets with either adequate or excess vitamin E, PRRSV decreased ADFI beginning 3 d after inoculation and throughout the remainder of the 12-d study period (Figure 1). The reduction in feed intake caused by PRRSV was paralleled by a reduction in ADG, irrespective of diet. Compared with uninfected control pigs, ADG of PRRSV pigs was depressed 17.9 and 31.8% at d 0 to 4 and d 5 to 12 after inoculation, respectively (Figure 2). The decrease in ADG seemed to be due to the reduction in ADFI because G:F ratio was not affected by PRRSV (Figure 3). Collectively, these data indicate that feeding a high level of vitamin E did not alter the growth performance of pigs infected with PRRSV.
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Figure 1. Average daily feed intake (as fed) of pigs fed diet with adequate or excess vitamin E (Low E and High E, respectively) and either given medium or inoculated with porcine reproductive and respiratory syndrome virus (Control or PRRSV, respectively). Asterisks indicate PRRSV pigs decreased (P < 0.001) feed intake compared with control pigs from d 3 to 12. Values are least squares means ± SEM (0 to 4 d, n = 8; 5 to 12 d, n = 4).
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Figure 2. Average daily gain by pigs fed diet with adequate or excess vitamin E (Low E and High E, respectively) and either given medium or inoculated with porcine reproductive and respiratory syndrome virus (Control and PRRSV, respectively). Asterisks indicate PRRSV pigs had a decreased (P < 0.002) ADG compared with control pigs. Values are least squares means ± SEM (0 to 4 d, n = 8; 5 to 12 d, n = 4).
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Figure 3. Gain:feed ratio of pigs fed diet with adequate or excess vitamin E (Low E and High E, respectively) and either given medium or inoculated with porcine reproductive and respiratory syndrome virus (Control and PRRSV, respectively). The main effects of PRRSV and diet, and the diet x virus interaction were not significant. Values are least squares means ± SEM (0 to 4 d, n = 8; 5 to 12 d, n = 4).
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Vitamin E Status and Oxidative Stress
Pigs were fed diets with adequate or excess vitamin E before (for a 3-wk period)and after (12 d) inoculation with either PRRSV or sterile culture medium. In our study, serum vitamin E concentration was determined 4 and 12 d after inoculation. Pigs fed the diet containing excess vitamin E had significantly greater amounts of vitamin E in serum compared with the pigs fed the diet containing adequate vitamin E (Table 2). The main effect of diet (P < 0.001) 4 and 12 d after inoculation, but not PRRSV, was significant.
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Table 2. Serum -tocopherol (µg/mL) in pigs fed diet with adequate or excess vitamin E (vit. E) and inoculated with porcine reproductive and respiratory syndrome virus (PRRSV)a
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Lipid peroxidation in liver and GPX activity in serum were used as indicators of systemic oxidative stress. Lipid peroxidation in liver samples from pigs killed 4 d after inoculation was not affected by either diet or PRRSV (Figure 4). However, hepatic lipid peroxidation 12 d after inoculation was reduced (P < 0.006) by feeding the high level of vitamin E (Figure 4). Consistent with the effect of diet on lipid peroxidation, serum GPX activity 12 d after inoculation was reduced (P < 0.05) in pigs fed the high level of vitamin E, but neither the PRRSV nor diet x PRRSV interaction were significant (Figure 5). Thus, these data suggest that feeding a high level of vitamin E improved the pig’s defenses against oxidative stress. However, hepatic lipid peroxidation and GPX activity in serum suggest that PRRSV infection did not induce systemic oxidative stress.
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Figure 4. Effects of medium or inoculation with porcine reproductive and respiratory syndrome virus (Control and PRRSV, respectively) and dietary vitamin E (Low E and High E, respectively) on lipid peroxidation (thiobarbituric acid–reactive substances; TBAR) in liver. The TBAR values are expressed as nanomoles of malondialdehyde (MDA) formed per milligram of protein and were determined from samples collected 4 and 12 d after inoculation. Asterisks indicate pigs fed the Low E diet differed (P < 0.01) from pigs fed the High E diet. Values are least squares means ± SEM (4 d, n = 15 or 16; 12 d, n = 7 or 8).
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Figure 5. Effects of medium or inoculation with porcine reproductive and respiratory syndrome virus (Control and PRRSV, respectively) and dietary vitamin E (Low E and High E, respectively) on glutathione peroxidase (GPX) activity in serum. The GPX activity was determined from samples collected 4 and 12 d after inoculation. Asterisks indicate pigs fed the Low E diet differed (P < 0.01) from pigs fed the High E diet. Values are least squares means ± SEM (4 d, n = 15 or 16; 12 d, n = 7 or 8).
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Serum IFN- and IL-1ß
Interferon- and IL-1ß were measured in serum collected 4 and 12 d after inoculation (Figure 6 and 7). The PRRSV-infected pigs had higher (P < 0.01) levels of both IFN- and IL-1ß than control pigs at both 4 and 12 d postinoculation (P < 0.01). It appeared that vitamin E supplementation may have decreased IL-1ß in PRRSV-infected pigs (but not in control pigs), but the variability in this measurement did not allow a significant interaction (diet x PRRSV) to be manifest (Figure 7).
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Figure 7. Serum IL-1ß concentrations in pigs fed a diet with adequate or excess vitamin E (Low and High E, respectively) and given medium or inoculated with porcine reproductive and respiratory syndrome virus (Control and PRRSV, respectively). Asterisks indicate pigs inoculated with PRRSV had higher (P < 0.01) IL-1ß in serum than Controls. Values are least squares means ± SEM (n = 8; ND = not detectable).
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Leukocyte Differential Counts
Leukocyte differential counts on d 4 and 12 after inoculation indicated that PRRSV-infected pigs had a lower (P < 0.01) number of circulating neutrophils, lymphocytes, and monocytes 4 d after PRRSV inoculation (Table 3). Also, PRRSV caused the neutrophil-to-lymphocyte (N:L) ratio to be elevated (P < 0.001) 12 d after inoculation. Neither diet nor a diet x virus interaction was detected for the hematological traits measured at 4 or 12 d after inoculation, although there was a trend for total leukocyte count to be elevated at 12 d after inoculation in pigs fed excess vitamin E.
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Table 3. White blood cell distribution for nursery pigs fed diet with adequate or excess vitamin E (vit. E) and inoculated with porcine reproductive and respiratory syndrome virus (PRRSV)a
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Discussion
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In endemically infected herds, predominant signs of PRRSV infection occur in nursery-grower pigs in which acute infection of immunologically naïve pigs induces signs of illness, including fever and lethargy. The clinical signs of PRRSV infection are accompanied by anorexia and depressed growth. For example, a recent study reported that nursery pigs experimentally infected with PRRSV had reduced feed intake and weight gain for 1 wk, with a lesser reduction for another 2 wk (Greiner et al., 2000). The serum concentration of virus was negatively correlated with feed intake and weight gain. Another study suggested that the effects of PRRSV infection on growth performance might persist because a higher PRRSV antibody titer was consistently associated with a lower growth rate in pigs 16 and 24 wk of age (Regula et al., 2000). Pigs that were seropositive for PRRSV gained 40 g/d less than pigs that were seronegative for the virus (Regula et al., 2000). Thus, preventing PRRSV infection or mitigating the detrimental effects of infection on growth performance is of paramount importance.
We investigated the potential of feeding high levels of vitamin E for the purpose of reducing the detrimental impact of PRRSV infection on growth performance of nursery pigs. We chose to evaluate vitamin E because of its capacity to protect cells from oxidative damage and its purported immunomodulatory effects. Vitamin E is a major lipid-soluble antioxidant that prevents lipid peroxidation of cell membranes. It is present in high concentrations in leukocytes because they are high in polyunsaturated fatty acids and at high risk for oxidative damage (Coquette et al., 1986). Damage to membranes by ROS or peroxides can inhibit immune cell function. Thus, vitamin E has been shown to decrease superoxide anion production and to enhance random migration, chemotaxis, and phagocytic activity of mouse peritoneal macrophages (Del Rio et al., 1998). Moreover, Han et al. (Han et al., 2000) showed that, after influenza infection, viral titers in lung as well as IL-1ß and TNF secretion by splenocytes were reduced in mice that had received a diet high in vitamin E (500 mg/kg -tocopherol acetate) compared with a control diet (30 mg/kg -tocopherol acetate). Also, splenocyte production of IFN-, a T helper-1 cytokine that inhibits viral replication, was enhanced in mice fed the diet high in vitamin E. Finally, mice fed the vitamin E–supplemented diet maintained their body weight whereas those on the control diet lost weight when infected with influenza. In pigs, vitamin E supplementation in excess of minimal requirements has been shown to increase antibody production and lymphocyte proliferation (Ellis and Vorhies, 1976; Peplowski et al., 1980; Larsen and Tollersrud, 1981). Injecting pigs with vitamin E before lipopolysaccharide challenge markedly decreased the production of IL-6 (Webel et al., 1998). Taken together, these studies made it reasonable to postulate that vitamin E would reduce the severity of PRRSV infection and improve growth performance of infected pigs.
The diets fed in this study were formulated to contain either the NRC-recommended level (NRC, 1998) of vitamin E (11 mg/kg) or 50 times the recommended level (550 mg/kg). The high level of vitamin E was chosen because this level has been demonstrated to be nontoxic in pigs and to markedly increase serum and liver vitamin E levels (Bonnette et al., 1990; Moreira and Mahan, 2002). As expected, there was no effect of vitamin E supplementation on growth performance (Figure 1
), even though pigs fed the diet with excess vitamin E exhibited significantly higher circulating levels of -tocopherol than pigs fed the control diet. In other studies, circulating levels of -tocopherol were correlated with levels found in liver (Martin et al. 1999; Moreira and Mahan, 2002) and lung (Meydani et al., 1987; Redlich et al., 1996). Therefore, although not measured here, it is reasonable to assume that pigs fed excess vitamin E also had increased liver and lung -tocopherol levels. Pigs fed the high level of vitamin E had reduced lipid peroxidation in liver and GPX activity in serum at the end of the study, indicating a reduction in oxidative stress. Thus, these data suggest that the two dietary treatments yielded pigs with different antioxidant capacities, which was the goal of these dietary treatments.
The growth performance data of pigs infected with PRRSV was consistent with previous studies from our laboratory (Escobar et al., 2002). A decrease in feed intake was evident by d 3 after inoculation and ADG was reduced d 0 to 4 after inoculation. These effects of PRRSV persisted to the end of the study 12 d later. Feeding a diet high in vitamin E did not improve growth performance of PRRSV-infected pigs. Although there may be several reasons as to why vitamin E was not beneficial, one possible explanation is that PRRSV did not induce systemic oxidative stress. For example, lipid peroxidation in liver and GPX activity in serum were not affected by PRRSV. Pigs fed the diet high in vitamin E had decreased lipid peroxidation in liver and GPX activity in serum compared with pigs fed the control diet. Because vitamin E quenches oxygen free radicals, lipid peroxidation was reduced. Furthermore, it is possible that GPX activity was not increased in pigs fed the high vitamin E diet because vitamin E prevented the accumulation of oxygen free radicals, which are precursors of hydrogen peroxide.
Pigs infected by PRRSV have weakened immunological defenses (Molitor et al., 1997) and are more susceptible to other endemic diseases (Nakamine et al., 1998; Thacker et al., 1999; Brockmeier et al., 2000). Consistent with this notion, PRRSV-infected pigs exhibited a reduction in total white blood cells, which may leave them more vulnerable to other infectious diseases. Thus, although vitamin E has immunomodulatory properties, it was not advantageous in preventing PRRSV-induced leukopenia.
The circulating levels of IL-1ß and IFN- measured provide further evidence that feeding a high level of vitamin E did not alter pig’s immune responses to PRRSV. Interleukin-1ß is an inflammatory cytokine produced primarily by activated macrophages and monocytes, whereas IFN- is a T helper-1 cytokine that inhibits viral replication. Both cytokines were elevated 4 and 12 d after PRRSV inoculation. The levels measured at d 4 were more variable, probably due to the progression of the infection, which can vary from one animal to the next. In any case, had vitamin E inhibited or enhanced the immune responses to PRRSV, one would have anticipated a concomitant change in circulating cytokines. The cytokine IL-1ß is known to regulate feed intake and metabolism in sick animals (Johnson, 1998; Johnson, 2002). Thus, because feeding a diet high in vitamin E did not alter IL-1ß, it was not surprising that growth performance was not improved in PRRSV-infected pigs by the high level of vitamin E.
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Implications
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Feeding vitamin E at nearly 50 times the NRC (1998) requirement did not afford pigs protection from the effects of porcine reproductive and respiratory syndrome virus infection on growth performance and certain hematological traits. Thus, our study indicates that the decreased feed intake and growth rate caused by porcine reproductive and respiratory syndrome virus infection cannot be overcome by increasing the dietary concentration of vitamin E. However, it should be noted that our study focused on the acute response to porcine reproductive and respiratory syndrome virus. Had the study been extended beyond 12 d, it is possible that pigs fed the high level of vitamin E might have recovered faster than pigs fed the control diet. Furthermore, pigs in our study were housed in a highly sanitized environment, so exposure to secondary pathogens was minimized. In commercial settings, pigs are subjected to multiple pathogens and may experience more oxidative insults. Therefore, in commercial settings, it may be beneficial to bolster antioxidant defenses of pigs by feeding vitamin E in excess of the NRC-recommended level.
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Footnotes
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1 This research was supported by Animal Health and Disease Research Funds (ILLU-35-0913) and a grant from the Illinois Council for Food and Agricultural Research (ILLU-35-0203). Appreciation is expressed to United Feeds Inc., Sheridan, IN, for furnishing the pigs for this study. 
2 Current address: Dept. of Pediatrics–Nutrition, Baylor College of Medicine, Houston, TX 77030.
3 Correspondence: 390 ASL, 1207 W. Gregory Dr. (phone: 217-333-2118; fax: 217-333-8286; e-mail: rwjohn{at}uiuc.edu).
Received for publication October 7, 2003.
Accepted for publication April 1, 2004.
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Abstract
Introduction
