Lysozyme as an alternative to antibiotics improves performance in nursery pigs during an indirect immune challenge1,2

Lysozyme is a 1,4-β-N-acetylmuramidase that has antimicrobial properties. The objective of this study was to determine the effect of lysozyme and antibiotics on growth performance and immune response during an indirect immune challenge. Two replicates of 600 pigs each were weaned from the sow at 26 d of age, blocked by litter and sex, and then randomly assigned to 1 of 24 pens in either a nursery room that had been fully disinfected or a nursery room left unclean since the previous group of pigs. Within a room, pigs were randomly assigned to either control diets (2 phase nursery regime), control diets + antibiotics (chlortetracycline/tiamulin hydrogen fumarate), or control diets + lysozyme (100 mg/kg diet). Pig weights and feed disappearance were measured and blood was collected on d 0, 14, and 28 of treatment. A group of 20 pigs were killed at 24 d of age for initial body composition analysis and 10 pigs of median weight were killed per diet room combination for body composition analysis after 28 d of treatment. Control + antibiotics and control + lysozyme-fed pigs grew at a faster rate for the 28-d study compared to control pigs (318 ± 14,320 ± 15 vs. 288 ± 15 g/d, respectively; P < 0.05), regardless of nursery environment (P > 0.05). The indirect immune challenge did not alter growth performance from d 0 to 14 of treatment but decreased ADG from d 14 to 28 of the study (415 ± 15 vs. 445 ± 13 g/d; P < 0.05). Feed intake was not altered by the nursery environment (P > 0.61) or dietary treatments (P > 0.10), but feed efficiency was worsened by the indirect immune challenge (P < 0.05) and improved by both control + antibiotics and control + lysozyme diets (P < 0.01). The immune challenge did not alter nutrient accretion (P > 0.25), but both control + antibiotics and control + lysozyme pigs had decreased accretion of whole-body lipid (P < 0.01) and increased accretion of protein (P < 0.09). Blood levels of tumor necrosis factor-α (TNF-α; P < 0.01), haptoglobin (P < 0.09), and C-reactive protein (CRP; P < 0.01) were higher due to the indirect immune challenge compared to pigs reared in the clean nursery (P < 0.05). In addition, pigs consuming antibiotics or lysozyme had lower TNF-α, haptoglobin, and CRP compared to control pigs, regardless of nursery environment (P < 0.04). Thus, lysozyme is a suitable alternative to antibiotics in swine nursery diets, and lysozyme ameliorates the effects of a chronic indirect immune challenge.

Key words: antibiotics, immune, lysozyme, nursery, swine

Antibiotics have been fed at subtherapeutic levels to swine as growth promoters for more than 60 yr, and the majority of swine produced in the United States received antibiotics in their feed at some point in their production cycle. These compounds benefit the producers by minimizing production losses by increasing feed efficiency and decreasing susceptibility to bacterial infection and disease (Verstegen and Williams, 2002). Lysozyme is a 1,4-β-N-acetylmuramidase that enzymatically cleaves a glycosidic linkage in the peptidoglycan component of bacterial cell walls, which results in the loss of cellular membrane integrity and cell death (Ellison and Giehl, 1991). We have identified lysozyme as a suitable alternative to antibiotics (May et al., 2012; Oliver and Wells, 2013).

It is well established that immune system activation, including proinflammatory cytokine and acute phase protein production, prevents animals from reaching their genetic growth potential (Cook, 2011). Poultry and swine reared in germ-free environments grow at a faster rate than animals reared in conventional production environments (Drew et al., 2003; Loynachan et al., 2005). In addition, utilizing a clean vs. dirty environment model elicits an immune response that decreases animal performance (Roura et al., 1992; Bassaganya-Riera et al., 2001; Renaudeau et al., 2011). In pigs, an immune response does not generally result in decreased feed conversion (Williams et al., 1997a,b; Renaudeau, 2009). However, both lysozyme (Oliver and Wells, 2013) and antibiotics (Verstegen and Williams, 2002) improve feed efficiency in nursery swine. In addition, Nyachoti et al. (2012) reported that lysozyme alleviated the piglet response to an oral challenge of Escherichia coli K88 similarly to traditional antibiotics. Thus, our objective was to determine the efficacy of lysozyme in ameliorating the effect of an immune response in pigs weaned from the sow at 26 d of age. 

The experimental protocol was approved by the Animal Care and Use Committee of the U.S. Meat Animal Research Center.

Two replicates of 600 pigs (Landrace Duroc, Yorkshire composite) were weaned from sows at 26 d of age. Pigs were blocked by litter and sex and then randomly assigned to 1 of 24 pens (n = 16 pens per treatment for the entire experiment) in either a nursery room that had been fully cleaned and disinfected or a nursery room left unclean since the previous group of pigs. Within a room, pigs were randomly assigned to either control diets (2 phase nursery regime), control diets + antibiotics (chlortetracycline [CTC]/tiamulin hydrogen fumarate), or control diets + lysozyme (100 mg/kg diet; Entegard, Neova Technologies, Abbotsford, BC, Canada) and allowed to consume diets ad libitum for 4 wk. All diets met or exceeded NRC recommendations for required nutrients (Table 1; NRC, 1998). Pig weights and feed disappearance were measured on d 0, 14, and 28 of treatment. After 4 wk in the nursery, pigs were moved to finishing facilities where weights were recorded every 2 wk until 120-kg body weight.

On d 0, 14, and 28 of treatment, 5 mL of blood was collected from the same 14 pigs per diet room combination (n = 28 per treatment for the entire experiment) into syringes via jugular venipuncture. After collection and blood coagulation, blood samples were centrifuged at 800 × g for 20 min at 4°C, with serum collected and frozen at -20°C until further analyses. Serum was analyzed for blood urea N (BUN), NEFA, cytokines (tumor necrosis factor-α [TNF- α] and IL-6), and acute phase proteins (C-reactive protein [CRP], haptoglobin, and pig major acute phase protein [Pig-MAP]). Blood urea N was measured (Marsh et al., 1965) using a Technicon Autoanalyzer System (Technicon Autoanalyzer Systems, Tarrytown, NY). The sample mean for BUN pools was 8.2 ± 0.3 mM and the intraassay CV was 4.1%. Serum NEFA concentrations were determined by an enzymatic colorimetric method (Zen-Bio, Inc., Research Triangle Park, NC). The sample mean for NEFA pools was 24.1 ± 1.2 µM and the intraassay CV was 4.8%. Acute phase proteins (CRP and haptoglobin, Life Diagnostics, Inc., West Chester, PA; Pig-MAP, Pigchamp Pro Europa, S.L., Segovia, Spain) and cytokines (TNF- α and IL-6, R&D Systems, Inc., Minneapolis, MN) were measured by commercially available ELISA. The sample mean and intraassay CV for pools were: CRP, 112.4 ± 3.9 ng/mL and 5.9%; haptoglobin, 46.2 ± 1.4 ng/mL and 3.8%; Pig-MAP, 0.76 ± 0.16 µg/mL and 9.8%; TNF-α, 186.9 ± 4.6 pg/mL and 5.2%; and IL-6, 45.6 ± 0.9 pg/mL and 4.8%.

An initial group of pigs (24 d of age; n = 10 per replicate) were killed (Beuthanasia-D, Shering-Plough Animal Health Corp., Union, NJ) for initial body composition analysis (proximate analysis; AOAC, 1997). After 4 wk of treatment, 10 pigs of median weight per replicate (n = 20 per treatment for the entire experiment) were killed per diet room combination for body composition analysis. The contents of the gastrointestinal tract, urinary bladder, and gall bladder were removed before the carcasses were stored at -20°C until further analysis.

This experiment was designed as a split-plot with pig within block being the experimental unit for dietary treatment (subplot) and pen within room being the experimental unit for the nursery environment (whole plot). Data was analyzed as a 2 × 3 factorial arrangement of treatments within the split-plot design using the GLM procedure (Minitab Inc., State College, PA). The model included replicate, sex, day, treatment, and all appropriate interactions. Where no statistically significant sex differences were observed, sex data were combined. The significance level for all tests was set at P < 0.05 tendencies set at P < 0.10. 

Pigs were weaned at 26.3 ± 0.1 d of age and weighed 8.6 ± 0.1 kg, which did not differ with nursery or dietary

treatment (P > 0.22). From d 0 to 14, pigs consuming antibiotics or lysozyme grew at a faster rate compared to pigs consuming control diets (Table 2; P < 0.05) regardless of nursery environment (P > 0.80). Similarly, pigs fed antibiotic or lysozyme grew at a faster rate from d 14 to 28 of the study (P < 0.001). In addition, pigs reared in the dirty nursery environment grew at a slower rate than pigs reared in the clean nursery from d 14 to 28 (P < 0.05). This resulted in an ADG for pigs consuming antibiotics or lysozyme that was 10% greater than pigs consuming control diets (P < 0.001). However, overall ADG was not affected by nursery environment (P > 0.11). Due to the changes in ADG, pigs consuming antibiotics or lysozyme were heavier at the completion of the 28-d study compared to control pigs (P < 0.001). The improved growth response observed in the nursery carried into the remainder of production in that days to market (120 kg) were reduced by 5 d in antibiotic and lysozyme pigs compared to control pigs (P < 0.04). Pigs reared in the dirty nursery tended to have an increase in days to market (P < 0.09). In addition, male pigs reached market weight 6 d earlier than female pigs (data not shown; P < 0.01), regardless of dietary treatment or nursery environment (P > 0.43). No differences in ADFI were observed during the course of the study, regardless of dietary treatment or nursery environment (P > 0.11). Pigs reared in the dirty nursery had decreased feed efficiency (G:F) from d 14 to 28 (P < 0.04) but not from d 0 to 14 (P > 0.15), which led to an overall 5% worsening of G:F (P < 0.05). Pigs consuming antibiotics or lysozyme had improved feed efficiency compared to control fed pigs from d 0 to 14 of treatment (P < 0.003) but not from d 14 to 28 (P > 0.16). For the entire 28-d experiment, antibiotics and lysozyme improved feed efficiency by approximately 6.5% (P < 0.006). 

Table 1. Composition and calculated analysis of the dietary treatments¹

Regardless of dietary treatment or nursery environment, pigs had lower lipid and ash and higher water, as a proportion of whole body, after 28 d of treatment compared to the initial reference group (Table 3; P < 0.05). No interactions were observed between dietary treatment and nursery environment (P > 0.05). Protein percentage of all pigs was similar to the reference group (P > 0.10). Nursery environment did not alter body composition (P > 0.68), but both antibiotic- and lysozymefed pigs had less whole-body lipid (P < 0.01), as a proportion of whole body, compared to the control group. No differences were observed in water or ash accretion rates during the 28-d study, regardless of dietary treatment or nursery environment (Table 3; P > 0.25). No differences due to nursery environment on lipid or protein accretion rates were observed during the study (P > 0.65). However, lipid accretion was decreased in pigs consuming antibiotics or lysozyme compared to control pigs (P < 0.02). In addition, pigs consuming antibiotics or lysozyme tended to accrue more protein (P < 0.09). 

Table 2. Effect of an indirect immune challenge on pigs weaned at 26 d of age and fed control, control + antibiotics (C + A), or control + lysozyme (C + Lyso) diets for 28 d¹

Table 3. Effect of an indirect immune challenge on the composition of the empty body and tissue accretion rates of pigs weaned at 26 d of age and fed control, control + antibiotics (C + A), or control + lysozyme (C + Lyso) diets for 28 d¹

Dietary treatment (P > 0.90) and nursery environment (P > 0.31) had no effect on circulating NEFA concentrations (Fig. 1A). In addition, there were no sex differences on NEFA (P > 0.74). Circulating NEFA decreased from d 0 to 14 (P < 0.001), and NEFA concentrations at d 14 did not differ from concentrations at d 28 (P > 0.85). Sex had no effect on circulating BUN (Fig. 1B; P > 0.12). Overall, BUN was greater in control pigs than both antibiotic- and lysozyme-fed pigs (P < 0.01). In addition, antibiotic-fed pigs tended to have a greater BUN compared to pigs consuming lysozyme (P < 0.10). Blood urea nitrogen increased during the course of the study (P < 0.001) but a dietary treatment × day interaction was observed (P > 0.001). Control-fed pigs had a greater BUN on d 14 of treatment compared to antibiotic- or lysozyme-fed pigs, but there were no differences between dietary treatment on d 28 (P > 0.14). In addition, pigs in the dirty nursery had greater BUN compared to pigs in the clean nursery, regardless of dietary treatment (P < 0.05). 

Figure 1. Effect of an indirect immune challenge and antibiotics or lysozyme in nursery pig diets on A) NEFA and B) blood urea N (BUN) in pigs weaned from the sow at 26 d of age. Values shown are means ± SEM; n = 31 or 32. Dietary treatment (P > 0.90) and nursery environment (P > 0.31) had no effect on circulating NEFA concentrations. The NEFA decreased from d 0 to 14 (P < 0.001) but was similar between d 14 and d 28 (P > 0.85). Overall, BUN was greater in control pigs than both antibiotic- and lysozyme-fed pigs (P < 0.01). Antibiotic-fed pigs had larger BUN than lysozyme-fed pigs (P < 0.10). The BUN increased with day (P < 0.001), but a dietary treatment × day interaction was observed (P > 0.001). Control-fed pigs had a higher BUN on d 14, but there were no differences between dietary treatment on d 28 (P > 0.14). Pigs in the dirty nursery had higher BUN compared to pigs in the clean nursery (P < 0.05) 

 

No IL-6 or TNF-α differences due to sex were observed (P > 0.75). In addition, circulating IL-6 decreased over the course of the experiment (Fig. 2A; P < 0.001) to undetectable levels at d 28, regardless of dietary treatment (P > 0.13) or nursery environment (P > 0.50). Similarly, TNF-α decreased from d 0 to 14 of treatment (Fig. 2B; P < 0.001), but levels were similar between d 14 and 28 (P > 0.68). TNF-α was lower in pigs consuming antibiotics or lysozyme compared to control pigs (P < 0.04), and this was more evident in pigs reared in the dirty nursery environment (dietary treatment × nursery environment; P = 0.003).

Sex had no effect on acute phase proteins (P > 0.82). Circulating Pig-MAP increased from d 0 to 14 (Fig. 3A; P < 0.001) but was unchanged from d 14 to 28 (P > 0.24). Pig-MAP was not affected by dietary treatment (P > 0.20) or nursery environment (P > 0.97). Haptoglobin (Fig. 3B; P < 0.001) and CRP (Fig. 3C; P < 0.001) both increased from d 0 to 14 of dietary treatment and were unchanged (P > 0.62) from d 14 to 28. Haptoglobin concentration was lower in antibiotic- and lysozymefed pigs compared to pigs consuming the control diet (P < 0.02). Pigs reared in the dirty nursery environment tended to have higher haptoglobin concentrations (P < 0.09). Similarly, CRP concentrations were lower for pigs consuming antibiotic or lysozyme compared with control diets (P < 0.01) and higher for pigs reared in the dirty nursery environment (P < 0.001). 

Antibiotics are used in feed as growth promotants for several species, including swine (Schwarz et al., 2001; Cromwell, 2002; Thymann et al., 2007). In addition, antibiotics improve feed efficiency and reduce the susceptibility to bacterial infections in swine (Verstegen and Williams, 2002). As a result, the use of antibiotics improves the profitability of production for swine producers. However, the use of antibiotics is banned in some countries and there is pressure in the United States to eliminate or reduce their use as growth promotants in animal production. Finding safe and effective alternatives to traditional antibiotics will allow swine producers to keep the competitive advantage of antibiotics without the stigma associated with their use.

In the current study, we showed that pigs consuming diets with either chlortetracycline/Denegard or lysozyme had improved growth rates and feed efficiency. This was similar to our previous work in young, milk-fed pigs (May et al., 2012) and nursery pigs (Oliver and Wells, 2013). In addition, an indirect disease challenge model was used in the current study to assess the effect of antibiotics or lysozyme on performance during a subclinical immune response. The chronic immune stimulation did not decrease growth rate or daily feed intake in pigs over the 28-d study. This contradicts previous work that showed modest decreases in ADG and ADFI in pigs utilizing a similar model (Williams et al., 1997b; Bassaganya-Riera et al., 2001; Renaudeau, 2009). However, ADG was decreased due to chronic immune stimulation during the second 2-wk period of the current study. Due to this difference, feed efficiency was worsened in chronically stimulated pigs, similarly to previous work (Williams et al., 1997a). These changes, as well as changes in cytokines and acute phase proteins, confirmed that the clean vs. dirty challenge model produced pigs with a subclinical immune response. In the current experiment, both antibiotics and lysozyme improved growth rate and feed efficiency in pigs with a chronic immune stimulation. It is well established that antibiotics improve growth rate during bacterial infections in swine (Verstegen and Williams, 2002), but this is the first evidence of lysozyme improving growth rate during a chronic immune stimulation. These data differ from Nyachoti et al. (2012), who did not observe a change in growth rate in pigs consuming antibiotics or lysozyme. However, those pigs were given a more acute challenge of Escherichia coli K88 and only on study for 7 d, whereas the current study used a chronic challenge for 4 wk. 

Figure 2. Effect of an indirect immune challenge and antibiotics or lysozyme in nursery pig diets on A) Interleukin-6 (IL-6) and B) Tumor necrosis factor-α (TNF-α) in pigs weaned from the sow at 26 d of age. Values shown are means ± SEM; n = 30 to 32. Dietary treatment (P > 0. 13) and nursery environment (P > 0.50) had no effect on circulating IL-6 concentrations and IL-6 decreased over the course of the experiment (P < 0.001) to undetectable levels at d 28. The TNF-α decreased from d 0 to 14 of treatment (P < 0.001), but levels were similar between d 14 and 28 (P > 0.68). The TNF-α was lower in pigs consuming antibiotics or lysozyme compared to control pigs (P < 0.04), which was more evident in pigs reared in the dirty nursery environment (dietary treatment × nursery environment; P = 0.003).

Previous work has shown that antibiotic supplementation in feed may influence body composition in pigs, depending on the experiment. Van Lunen (2003) observed greater P2 lean but no difference in P2 fat depths in pigs consuming tylosin phosphate. However, Song et al. (2011) did not observe differences in LM area or backfat depth in pigs fed bacitracin methylene disalicylate.

In the current study, accretion rates of both fat and protein as measured by the slaughter balance technique were altered in pigs consuming either antibiotics or lysozyme compared to control. Lipid accretion was decreased and protein accretion tended to increase in these pigs. The data for antibiotic pigs may differ from previous work due to the different antibiotics use (CTC/Denegard vs. tylosin or bacitracin). To our knowledge, this is the first report of lysozyme altering the partitioning of nutrients in pigs. In the current study, a chronic immune stimulation did not alter accretion rates. This differs from BassaganyaRiera et al. (2001), who observed lower fat, as a proportion, in pigs reared in a dirty nursery environment. However, Williams et al. (1997a) determined that, in spite of differences in N retention, chronic immune stimulation did not alter the efficiency of lysine utilization. Nitrogen retention differences would indicate differences in the partitioning of nutrients for protein and fat but Williams et al. (1997a) determined this was due to the low immune stimulation allowing pigs greater biological capacity for accretion. Although protein and lipid accretion was lower due to an elevated immune status (Williams et al., 1997a), this is likely due to the greater difference in growth rates observed in that study. Lack of accretion rate differences between nursery groups in the current study is likely due to lower, more chronic immune system activation. Nonetheless, due to performance, cytokine, and acute phase protein differences in the dirty nursery, we conclude that lysozyme decreases fat and increases protein deposition in both clean nursery environments and pigs under a chronic immune stimulation.

Circulating urea N is a reliable indirect measurement to show the oxidation of dietary AA in young pigs (Oliver and Miles, 2010). Blood urea N was increased in control pigs compared to pigs consuming lysozyme or antibiotics. This contradicts earlier work from our lab (Oliver and Wells, 2013). However, the greater BUN was expected due to increased protein accretion observed in lysozymeor antibiotic-fed pigs and feed intake was similar between all 3 treatment groups. Presumably, those pigs consuming lysozyme or antibiotics utilized more of their dietary amino acids for protein deposition than control pigs. It is likely that our earlier work had too few animals to detect a response in BUN. In addition, the growth response observed due to antibiotics and lysozyme was slightly larger in the current study. Similar to other work (BassaganyaRiera et al., 2001), chronically immune-stimulated pigs had lower BUN than pigs in the clean nursery. Circulating NEFA is an indirect measure of lipolysis, fatty acids available for uptake into tissues, or both, and lipolysis rates should be relatively low in nursery pigs. In spite of differences in lipid accretion, no differences in NEFA due to diet or immune activation were observed. Unlike previous work in 10- to 20-d-old pigs accruing more protein and less lipid (Oliver and Miles, 2010), the rates of lipolysis in 26- to 54-d-old pigs were likely fast enough to preclude the detection of differences due to lipid use in the current study.

In addition to regulating the immune response, cytokines have a profound effect on nutrient metabolism. Proinflammatory cytokines such as IL-6 and TNF-α redirect nutrients toward the immune response and away from growth processes (Johnson, 1997; Spurlock, 1997). This is due to, among other factors, increased muscle protein degradation and acute phase protein production (Elsasser et al., 2008). Cytokines and acute phase proteins were measured to both confirm the chronic immune stimulation and to determine the effect of antibiotics and lysozyme on the immune response. Interleukin-6 and PigMAP were unaffected by immune status. However, the variability of PigMAP concentrations likely prohibited the ability to detect any differences between nursery environments or dietary treatments. In contrast, circulating levels of the cytokine TNF-α and the acute phase proteins haptoglobin and CRP were higher in chronically immune-stimulated pigs compared to pigs reared in the clean nursery. These changes in cytokines and acute phase proteins, as well as the performance changes observed, indicate that an acceptable level of immune response was generated in pigs reared in the dirty nursery to make inferences into the effect of antibiotics and lysozyme on chronically immunestimulated pigs. In the current study, pigs consuming antibiotics or lysozyme had lower TNF-α, haptoglobin, and CRP compared to control pigs. This was true of pigs under chronic immune stimulation or reared in the clean nursery. These data agreed with others in that both antibiotics and lysozyme can ameliorate an immune response. Lee et al. (2012) observed lower haptoglobin levels in antibiotic-fed pigs compared to nonmedicated controls. In addition, Nyachoti et al. (2012) showed lower circulating TNF-α postchallenge in pigs consuming lysozyme. Both of these studies used acute Escherichia coli challenges but demonstrated that antibiotic- and lysozyme-fed to pigs reduced immune response when exposed to pathogens. 

Figure 3. Effect of an indirect immune challenge and antibiotics or lysozyme in nursery pig diets on A) pig major acute phase protein (PigMAP), B) haptoglobin, and C) C-reactive protein (CRP) in pigs weaned from the sow at 26 d of age. Values shown are means ± SEM; n = 30 to 32. The PigMAP increased from d 0 to14 (P < 0.001) but was unchanged from d 14 to 28 (P > 0.24). The PigMAP was not affected by dietary treatment (P > 0.20) or nursery environment (P > 0.97). Haptoglobin (P < 0.001) and CRP (P < 0.001) both increased from d 0 to 14 of dietary treatment and were unchanged (P > 0.62) from d 14 to 28. Haptoglobin and CRP were lower in antibiotic-and lysozyme-fed pigs compared to control pigs (P < 0.02). Pigs reared in the dirty nursery environment had higher haptoglobin (P < 0.09) and CRP (P < 0.001) concentrations.

The use of subtherapeutic levels of antibiotics in diets increased the growth performance of swine. However, the industry is under increased pressure to reduce or remove antibiotics from swine diets due the perceived danger of their use. This study confirms that lysozyme improves growth rates and feed efficiency in nursery diets. In addition, lysozyme, as well as antibiotics, increase protein deposition at the expense of lipid accretion in the current study. An immune response in pigs redirects nutrients away from growth and toward the immune system, which decreases the efficiency of nutrient use. In the current study, both lysozyme and antibiotics decreased the severity of the immune response in pigs under a chronic immune challenge. Thus, we conclude that lysozyme is a suitable alternative to antibiotics in swine nursery diets and that lysozyme ameliorated the effects of a chronic immune challenge. 

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