Salmonella typhimurium and Escherichia coli are two of the most frequently isolated pathogens from pig farms, inducing poor performance in the post-weaning period (Bhandari et al., 2008) and food-borne illnesses in humans (Rugbjerg et al., 2004). Generally, antibiotics are used at sub-therapeutic levels to treat Salmonella and E. coli infection as well as to protect host animals from weaning stress such as poor growth, reduced immunity, and an imbalanced microbial ecosystem. However, due to the appearance of resistant bacteria and residues in animal products, the use of antibiotics has been prohibited by the European Union (Regulation (EC) No. 1831/2003). This ban is expected to result in reduced livestock performance as well as higher incidence of intestinal disorders due to increased proliferation of gut pathogens (Hughes et al., 2005). Therefore, a natural, safe, and inexpensive alternative with activities similar to antibiotics should be developed in order to improve the health and performance of animals.

Phytogenics, a relatively new class of feed additives, comprise a wide variety of herbs, spices, and products derived thereof, such as essential oils, polyphenols (resveratrol), etc. The possible mechanisms of action of herbal extracts in animals are related to alteration of the intestinal microbiota, increased digestibility and absorbance of nutrients (Hernandez et al., 2004), as well as antioxidative and immunomodulatory activities (Chang et al., 1995). The rapid metabolization and short half-life of bioactive compounds suggest a minimum risk of accumulation and cross-resistance in tissues (Kohlert et al., 2000). However, experimental research on the addition of synthetic antimicrobials and vegetable extracts to swine diets has shown conflicting results. In this study, two commercial products, ResPig^® (containing resveratrol from mulberry and Japanese knotweed) and Biomin^® PEP (containing oregano, anise, orange peel, and chicory essential oils), were used as phytogenic feed additives. Resveratrol (trans-3,5,4′-trihydroxystilbene) is a stilbenoid, a type of natural polyphenol and aromatic phytoalexin found predominantly in grapes, berries (mulberries), and Japanese knotweed. Trans-resveratrol is found to be stable under 75% humidity and 40°C in the presence of air (Baur et al., 2006). It is thought to possess strong antimicrobial activity (Paulo et al., 2010) and has gained wide attention due to its ability to inhibit various animal diseases (Baur et al., 2006). On the other hand, essential oils are volatile, oily liquids that have been used to replace antibiotic supplements in weanling pigs without affecting performance (Cho et al., 2006). Some essential oils display strong antimicrobial activity, selectivity against Gram-negative over Gram-positive bacteria (Lin et al., 2000). De Souza et al. (2008) found no significant interference on the essential oil antimicrobial activity and chemical composition under different heating treatments (room temp. to 120°C). Further, most essential oils are classified as Generally Recognized as Safe (GRAS; FDA, 2004). Both of the phytogenics assumed to improve livestock performance as well as may be added to the set of non-antibiotic growth promoters. Therefore, this study evaluated the effects of resveratrol and an essential oil blend made from medicinal plants on growth performance, immunity, nutrient digestibility, and fecal microbial shedding in E. coli and Salmonella enterica serover Typhimurium challenged piglets.

In the present study, we evaluated whether or not resveratrol and an essential oils blend can influence growth performance, humoral immune function, nutrient digestibility, as well as fecal shedding of microorganisms as alternatives to antibiotics in challenged weaned piglets. The results of our study showed that PC diet significantly improved ADG and ADFI during the post-weaning period in piglets, consistent with the findings of Cromwell (1991) and Oetting et al. (2006). An improved FCR was found in T1 group, whereas the growth performance of T2 group was poor. Antibiotic growth promoters exert positive effects on animal growth performance by reducing the pathogenic microbial load, thereby increasing nutrient utilization in the gastrointestinal tract (Cromwell, 2001). The observed decrease in feed intake in T1 group during 0 to 2 wks period may be due to the aversive palatability of resveratrol leading to an immediate reduction in feed intake (Alexandre et al., 2010). The improved FCR in piglets fed T1 diet could be attributed to the effects of resveratrol, which mediated reduction of pathogenic microbes (E. coli and Salmonella) and improved utilization of nutrients in the digestive tract. Similar improvements in FCR have been observed upon treatment with resveratrol (Paulo et al., 2010) to pigs or concentrated grape pomace containing resveratrol (Viveros et al., 2011) to broilers. The poor growth performance in T2 group can be explained as low feed intake by the effect of strong flavor of essential oil which reduced the palatability of the diet. Jugl-Chizzola et al. (2006) previously found dose-related depressions of palatability in pigs fed essential oils from thyme and oregano. Stress induced by pathogenic challenge could be another reason and more positive results can be obtained under normal conditions. Kroismayr et al. (2008) previously found no significant effects of essential oils on growth performance parameters of weaned piglets compared to antibiotic and negative control. The oral challenge of pigs with pathogenic E. coli 2571 and Salmonella has been used widely as a model of postweaning lag phase (Wellock et al., 2008). The feed intake and growth rate responses have proven to be useful measures of the clinical manifestations in pigs with enteric disease (Balaji et al., 2000). The poor growth performance in this experiment compared to the target performance of pigs of the same age (ADG: 234 g vs 460 g; ADFI: 478 g vs 620 g; FCR: 2.05 vs 1.35) can be explained as the effect of challenging with pathogenic E. coli and Salmonella (Close, 2002).

Further, we observed improved DM and N digestibility in the PC and T2 groups compared to the NC and T1 groups. Similar improvement in digestibility has been shown in pigs fed an essential oils blend (Huang et al., 2010). The antibiotic effects of essential oil supplementation may influence the formation of intestinal bacterial colonies as well as promote production of digestive enzymes, which could influence DM and N digestibility (Stoni et al., 2006). However, Cho et al. (2006) found negative effect of essential oils on N digestibility.

Plasma IgG is the major serum immunoglobulin that protects extra vascular compartments against pathogenic microorganisms, resulting in long-lasting immunity. In this study, resveratrol and essential oils blend showed the ability to boost IgG levels in weaned piglets under challenged conditions (p<0.05). Similar increases in IgG concentration upon treatment with resveratrol or different plant extracts such as cinnamon, thyme, and oregano have been confirmed in mice and pigs (Liu et al., 2003; Namkung et al., 2004). In contrast, the IgG level in pigs remained the same as control following treatment with oregano oil mixed with anis and citrus oils (Kommera et al., 2006). The mechanism by which phytogenic feed additives increase IgG levels is largely unknown and should be elucidated. It is possible that active molecules from phytogenics (polyphenols, essential oils, etc.) act as additional ligands of Fc receptors, which bind to IgG and stimulate the immune response (Nimmerjahn and Ravetch, 2010). Cytokines such as IL-1β and TNF-α, which are known to mediate the inflammatory response (Dinarello, 1988), have been linked to alterations in nutrient uptake and utilization (Spurlock, 1997). In our results, the TNF-α level was higher in the NC group compared to the PC group, whereas it was intermediate in the T1 and T2 groups, consistent with the findings of Trevisi et al. (2007).

Pathogenic microorganisms reduce the growth rate and health status of pigs by producing toxins, utilizing the nutrients within host animals (Gaggia et al., 2010), and suppressing microbes that synthesize vitamins or other growth factors (Falaki et al., 2010). To evaluate the effects of various dietary treatments on fecal shedding of microorganisms (S. typhimurium, E. coli, Lactobacillus spp., and Bacillus spp.), bacterial concentrations were determined at 1st, 2nd, 3rd, and 4th week post challenge. The order of inhibitory effects of the dietary treatments on fecal shedding of microorganisms was: S. typhimurium; PC>T1>T2>NC, E. coli; PC>T2>T1>NC, Lactobacillus spp.; T1>NC>PC>T2, Bacillus spp.; T1>NC>PC>T2.

The PC diet showed the highest inhibitory effect on S. typhimurium and E. coli which is likely due to the bactericidal action of Apramycin. The T1 diet showed the highest inhibitory effect on Lactobacillus spp. and Bacillus spp., in contrast T2 diet increased their number. Manzanilla et al. (2004) and Castillo et al. (2006) also found an increase in the number of Lactobacillus spp. upon supplementation with mixed essential oils. The main bioactive compound of the essential oil blend used in this study was the phenolic carvacrol. Si et al. (2006) reported that carvacrol is less active against Lactobacilli and Bifidobacteria in comparison to pathogenic bacteria such as E. coli and S. typhimurium, which is partially consistent with our observations. Resveratrol is a phytoalexin of low molecular weight that has been shown to possess bacteriostatic activity specifically against Gram-positive bacteria such as Bacillus spp. (Paulo et al., 2010) rather than Gram-negative bacteria such as S. typhimurium and E. coli.

Although it is generally accepted that antibiotics administered at growth-promoting doses reduce the number of bacteria in the gut, the response in the literature is not always consistent, depending on the type of antibiotic and dosage. Indeed, resistance against aminoglycoside type antibiotics may be responsible for the increased fecal shedding of Lactobacillus spp. observed in our study (Zhou et al., 2005; Castillo et al., 2006).

In total, the above findings suggest that supplementing resveratrol to weaned piglets could have beneficial effects on growth performance, immune function and the microbial ecosystem following challenge. The growth performance parameters investigated in this study were not positively affected by the essential oil blend. Further feeding trials using resveratrol and essential oils at different inclusion rates are required in order to better understand the effects of such additives as antibiotic alternatives as well as to elucidate their mechanisms of action.