Gut Microbial Modulation and Immunomodulation by Ovine Serum Immunoglobulins in the Growing Rat Prabhu Balan, Kyoung-Sik Han, Harjinder Singh & Paul J. Moughan Riddet Institute, Massey University, Palmerston North, New Zealand INTRODUCTION (A) (B) CF4 CF3 CF2 IL2 IL1 CB1 FDOI BD BD FDOI Table 1. Average band number, Shannon’s diversity index, peak intensity and intra-group percentage similarities of DGGE bands from ileal digesta A diverse microbiota resides in the mammalian gut and influences the physiology, biochemistry and immunology of the host.1) Immunomodulators are primarily used to prevent and treat infectious diseases. Recently, immunonutrition has been applied to improve the clinical course of severely sick or operated patients who often need a prescribed exogenous supply of nutrients through the parental or enteral routes.2) The benefits of spray dried animal plasma (SDAP) and plasma immunoglobulin (Ig) in the post-weaning diet of production animals have been demonstrated 3,4) and the effects of animal plasma appear to be more pronounced during higher pathogen challenge. A reduction in the degree of adhesion of pathogenic bacteria to the gut wall seems to be involved in the mechanism. In the lumen of the gastrointestinal tract antigens become bound by the Ig present in SDAP, thereby preventing their attachment to the mucosa. We have reported that freeze-dried ovine serum Ig selectively improves growth performance, the weight of some organs and gut morphology when compared to rats given a basal diet or inactivated ovine Ig 5), but there are no reports regarding the effects of ovine serum Ig on gut microbiota and immunomodulation in normal and infected animals. In this study, we have addressed the effects of orally administered ovine serum Ig on composition of gut microbiota and immunomodulation in growing Sprague-Dawley male rats.   Diet BD FDOI P Value DGGE Band number 7.1 (0.50) 10.2 (0.40) <0.05 Diversity index 0.27 (0.04) 0.41 (0.04) Peak intensity (%) 100.0 (12.34) 141.9 (5.80) Similarity index (%) 35.4 (7.70 ) 69.00( 6.10) 0.001 Table 2 . Identified bacterial species from DNA sequencing of the PCR-DGGE bands. ID Diet Nearest Neighbour Similarity IL1 FDOI Lactobacillus johnsonii NCC 2767 99% IL2 Lactobacillus johnsonii MH 21 CB1 BD Uncultured bacterium clone DR6-254 CF2 Lactobacillus sp BL304 CF3 Lactobacillus gasseri NCC 1741 CF4 Lactobacillus johnsonii NCC 533 MATERIALS & METHODS Figure 1. Lanes showing DGGE bands from pooled DNA samples (n = 15) of ileal (A) and colonic (B) digesta used for sequencing for the identification of bacteria Forty-five Sprague-Dawley male rats were used in a 21-d microbial and immunological study and fed a basal control diet (BD; no Ig) and 2 test diets: freeze-dried ovine Ig (FDOI) and inactivated ovine Ig (IOI). DGGE analysis was performed using the Bio-Rad DCode system with the 8% polyacrylamide gel containing the urea-formamide gradient of 30 to 55%. Electrophoresis was performed at 130V for 5 h at a constant temperature of 60oC. The phagocytic activity of peripheral blood leucocytes (PBL) was measured using flow cytometry 6) and lymphocyte proliferation of spleen cell culture was determined in the presence of a mitogen (Concanavalin A).7) The presence of interferon-gamma (IFN- gamma), interleukin (IL)-4 and tumor necrosis factor alpha (TNF-α) in the spleen cell culture supernatant was determined using a DuoSet ELISA kits (R&D systems) and Rat TNF-α Module set (Bender MedSystem). A sandwich IgA and IgG ELISA was used to quantify the IgA and IgG concentration in plasma and the intestinal digesta. The data were analysed using the GLM procedures of the SAS package (SAS Institute, Cary, NC). Values are mean ± SEM, n = 15. a,b Means across the figure and table with superscripts without a common letter differ, P < 0.05. a b Table 3. Interferon-gamma and Interleukin-4 (pg/ml) production by spleen cells of rats fed a diet containing ovine Ig with and without ConA stimulation for 21 d.   Diet BD FDOI IOI SEM P Value ConA stimulated IFN-gamma 1022.51b 1460.60a 1179.32ab 18.84 0.008 IL-4 4.43 4.48 5.03 0.22 0.977 ConA unstimulated 23.9 97.1 75.9 13.71 0.251 2.18b 4.42a 3.09ab 0.18 0.044 RESULTS Figure 2. Phagocytic activities of peripheral blood leucocytes of rats fed a diet containing ovine Ig for 21 d. Table 4. The IgA and IgG levels of intestinal contents and plasma in rats fed a diet containing ovine Ig for 21 d. The number of DGGE bands, Shannon’s diversity index and peak intensity were found to be significantly higher (P<0.05) for the rats fed the FDOI diet than for rats fed the BD diet for ileal digesta (Table 1). DNA sequencing of DGGE bands showed that the FDOI diet selectively increased beneficial bacteria such as Lactobacillus sp. in both ileum and colon (Fig. 1 & Table 2). The phagocytic activity of PBL and lymphocyte proliferation in the presence of ConA was greater (P<0.05) for the rats fed the FDOI diet than the BD- and IOI-fed groups (Fig 2 & 3). Similar results were observed in Salmonella enteridis infected model (data not shown). ConA stimulated and unstimulated spleen cell culture supernatants respectively produced higher (P<0.05) IFN-gamma and IL-4 for rats fed the FDOI than for rats fed the BD diet (Table 3). In jejunum, ileum and plasma, the rats fed the FDOI diet respectively produced larger amount of sIgA than rats fed the IOI and/or BD diet. In the jejunum, the rats fed the FDOI diet had higher amounts of IgG than rats fed the IOI or BD diet but showed a reverse tendency in plasma IgG concentration (Table 4).   Diet BD FDOI IOI SEM P Value Jejunum (μg/mL) IgA 1278.28ab 2047.33a 999.16b 29.3 0.037 IgG 177.50b 254.51a 193.02b 7.78 Ileum (μg/mL) 3156.32b 4063.20a 3576.99ab 27.64 0.05 149.51 178.34 166.12 6.49 0.416 Plasma (mg/mL) 1.14b 1.58a 1.40ab 0.04 0.041 2.17a 1.01b 2.15a 0.01 0.025 b a REFERENCES CONCLUSION Hooper et al. 2001. Sci. 291, 88 Calder et al. 2007. J. Nutr. 98 (Suppl. 1), 133. 3. Gatnau et al. 1989. J. Anim. Sci. 67 (Suppl.1), 244. 4 .Pierce et al. 2005. J. Anim. Sci. 83, 2876. 5 .Balan et al. 2009. J. Nutr. 39, 244. 6. Wan et al. 1993. J. Immunol. Methods. 62, 1. 7. Cross et al. 1999. Immunol. Cell Biol. 77, 345. Figure 3. Lymphocyte proliferative responses of spleen cells to ConA for rats fed a diet containing ovine Ig for 21 d. Orally administered ovine serum Ig selectively improved beneficial bacteria overgrowth such as Lactobacillus sp. and modulated various indices of immune function in growing rats. This is the first report of gut microbial modulation and immunomodulatory effects of dietary ovine Igs in an animal species.