Prabhu Balan, Paul J Moughan and Kyoung-Sik Han

Prabhu Balan, Paul J Moughan and Kyoung-Sik Han
Intact but not denatured ovine serum immunoglobulin positively modulate gut immunity in a rodent model. Prabhu Balan, Paul J Moughan and Kyoung-Sik Han The 80th Annual Meeting of Korean Society of Food Science and Technology (KoSFoST) 29/08/2013

Dr. Prabhu Balan, Agresearch, New Zealand
Contents Introduction Objectives Rodent model - Methods Results Possible mechanism of action Conclusion Acknowledgment 2 Dr. Prabhu Balan, Agresearch, New Zealand

Introduction Immunoglobulins (Ig) are glycoproteins produced by plasma cells (or B lymphocytes) in response to antigens. The Ig - bioactive proteins present in blood and other tissue fluids, including the milk of all mammals. Major function of Ig bind to invading pathogens activate specific functions prevent infection and get rid of disease caused by pathogens. 3 Dr. Prabhu Balan, Agresearch, New Zealand

More on Ig Research is proving that immunoglobulins (Ig) retains biological activity even when ingested. The average IgG recovery following digestion or simulated digestion in published studies is ~ 25%1. Oral IgG partly resisted digestion in the growing rat and was found throughout the digestive tract 2. No studies have reported complete or 100% loss of IgG activity. IgG fragments also retain biological activity Balan. (2012). Effects of orally administered ovine serum immunoglobulin in the normal and Salmonella enteritidis – challenged growing rat. PhD thesis, Massey University. Balan et al., (2013). Recovery of intact IgG in the gastrointestinal tract of the growing rat following ingestion of an ovine serum immunoglobulin. J Anim Physiol Anim Nutr (Berl). 4 Dr. Prabhu Balan, Agresearch, New Zealand

Why Ig ? In the last 20yrs - numerous researches – benefits of including animal plasma (AP) in the animal diets such as feed intake, growth rate and intestinal growth. Ig present in AP may bind to the potential antigens in gastrointestinal tract (GIT) and prevent their attachment to the mucosa and growth. In New Zealand ~ approximately 40 million lambs/sheep are processed annually ~ 60 million litres of ovine blood. Currently, this blood is used for low-value products or discarded as waste. It would be more beneficial if the components of ovine blood were separated and sold as high-value products. Why Sheep Ig ? 5 Dr. Prabhu Balan, Agresearch, New Zealand

Effects of oral ovine Ig
Growth Immunity Ovine Ig Microbiota Mucin 6 6 Dr. Prabhu Balan, Agresearch, New Zealand

Effects of oral ovine Ig – today’s presentation
Immunity Ovine Ig Microbiota Mucin 7 7 7 Dr. Prabhu Balan, Agresearch, New Zealand Dr. Prabhu Balan, Agresearch, New Zealand

Objectives To test the efficacy of Immunoglobulins using a rat model Question: Does orally administrated Immunoglobulins (intact) modulate gut immunity (including gut microbiota and mucin) in Salmonella enteritidis infected rats? Dr. Prabhu Balan, Agresearch, New Zealand

Animal trial design 8 weeks old Acclimatization 7 days Rat trial Salmonella enteritidis (SE) infection model SE (oral) on 15th day 14 days–BD/Test diets . BD/Test diets for 18 days Gut samples (digesta and mucosal) Groups studied (1) BD, basal diet fed rats (saline control), (2) BD-SE, basal diet fed rats (with challenge), (3) FDOI-SE, freeze dried ovine Ig fed rats (with challenge) and (4) AOI-SE, autoclaved ovine Ig fed rats (with challenge) Immunity studies - Cytokines, and Salmonella- specific and non-specific Ig’s (IgA & IgG). Microbiota studies - Denatured gradient gel electrophoresis (DGGE) and microbial DNA sequencing Mucin studies - Goblet cells, mucin protein 9 Dr. Prabhu Balan, Agresearch, New Zealand

Results Intestinal Digesta Dr. Prabhu Balan, Agresearch, New Zealand

Intestinal Digesta Table 1 Interferon-γ, Interleukin-4, Interleukin-10, and Tumor necrosis factor-α, concentrations in ileal digesta for the unchallenged rats fed the BD diet and rats challenged with Salmonella* and fed either the BD, FDOI or AOI diets1 Unchallenged Challenged with Salmonella enteritidis Diet: BD FDOI AOI SEM P value IFN γ, pg/L 645.4b 9717.1a 743.3b 7531.7a 1331 0.05 IL-4, pg/L 42.1 44.2 40 47.4 7.4 0.96 TNFα, pg/L 323.8b 439.1a 343.8b 405.7a 10.6 0.01 IL-10, pg/L 109.3b 71.2c 144.6a 87.8c 5.88 1Values are means ± SEM, n = 8, * 3 d of challenge, Means in a row with superscripts without a common letter differ, P < 0.05. BD, Basal diet; FDOI, Freeze dried ovine immunoglobulin; AOI, Autoclaved ovine immunoglobulin Dr. Prabhu Balan, Agresearch, New Zealand

Intestinal Digesta Table 2 Interferon-γ, Interleukin-4, Interleukin-10, and Tumor necrosis factor-α, concentrations in colonic digesta for the unchallenged rats fed the BD diet and rats challenged with Salmonella* and fed either the BD, FDOI or AOI diets1 Unchallenged Challenged with Salmonella enteritidis Diet: BD FDOI AOI SEM P value IFN γ, pg/L 526.1b 7234.4a 603.3b 6531.4a 598 0.04 IL-4, pg/L 44.4 43.5 41.2 46.5 5.8 0.91 TNFα, pg/L 212.6b 380.1a 233.1b 381.7a 19.6 0.01 IL-10, pg/L 78.3b 54.3c 106.6a 60.1c 5.88 1Values are means ± SEM, n = 8, * 3 d of challenge, Means in a row with superscripts without a common letter differ, P < 0.05. BD, Basal diet; FDOI, Freeze dried ovine immunoglobulin; AOI, Autoclaved ovine immunoglobulin Dr. Prabhu Balan, Agresearch, New Zealand

Intestinal Digesta Table 3 Immunoglobulin A and G concentrations in intestinal digesta for unchallenged rats fed the BD diet and rats challenged with Salmonella* and fed either the BD, FDOI or AOI diets1 Diet : Unchallenged Challenged with Salmonella enteritidis BD FDOI AOI SEM P Value Ileal digesta IgA, mg/L 10751b 20522a 11194b 20750a 0.03 IgG, mg/L 777.3 1266.8 787 1136.4 176.42 0.16 Colonic digesta 1300.9b 3485.9a 1412.0b 2840.6ab 462.51 0.02 401.7b 819.5a 427.5b 713.7ab 92.59 0.05 1Values are means ± SEM, n = 8, * 3 d of challenge, Means in a row with superscripts without a common letter differ, P < 0.05. BD, Basal diet; BD-SE, Basal diet; FDOI, Freeze dried ovine immunoglobulin; AOI, Autoclaved ovine immunoglobulin. Dr. Prabhu Balan, Agresearch, New Zealand

Intestinal Digesta Table 4 Concentrations of specific anti-Salmonella enteritidis IgA and IgG in intestinal digesta for unchallenged rats fed the BD diet and rats challenged with Salmonella* and fed either the BD, FDOI or AOI diets Diet : Unchallenged Challenged with Salmonella enteritidis BD FDOI AOI SEM P Value Anti-Salmonella IgA2 Ileal digesta 0.09c 0.55b 0.86a 0.57b 0.03 0.003 Colonic digesta 0.45b 0.93a 0.47b 0.04 0.001 Anti- Salmonella IgG2 0.05 0.5 0.02 0.88 0.09b 0.48b 0.81a 0.08 1Values are means ± SEM, n = 8, * 3 d of challenge, Means in a row with superscripts without a common letter differ, P < 0.05. 2Data are shown in absorbance (450 nm). BD, Basal diet; FDOI, Freeze dried ovine immunoglobulin; AOI, Autoclaved ovine immunoglobulin. Dr. Prabhu Balan, Agresearch, New Zealand

Results Intestinal mucosal samples 15
Dr. Prabhu Balan, Agresearch, New Zealand

Intestinal Mucosa Figure 1Concentrations of INFγ, IL-4, IL-10, & TNFα in ileal mucosa from unchallenged rats fed the BD diet and rats challenged with Salmonella for 3 d and fed the BD, FDOI, or AOI diet BD BD-S FOI-S AOI-S c b a IFNγ IL4 IL10 TNFα 1Values are means ± SEM, n = 15, Means in a row with superscripts without a common letter differ, P < 0.05. BD, Basal diet; BD-SE, Basal diet; FOI, Freeze dried ovine immunoglobulin; AOI, Autoclaved ovine immunoglobulin. 16 Dr. Prabhu Balan, Agresearch, New Zealand

Intestinal Mucosa Figure 2 Concentrations of INFγ, IL-4, IL-10, & TNFα in colonic mucosa from unchallenged rats fed the BD diet and rats challenged with Salmonella for 3 d and fed the BD, FDOI, or AOI diet BD BD-S FOI-S AOI-S b a c IFNγ IL4 IL10 TNFα 1Values are means ± SEM, n = 15, Means in a row with superscripts without a common letter differ, P < 0.05. BD, Basal diet; BD-SE, Basal diet; FOI, Freeze dried ovine immunoglobulin; AOI, Autoclaved ovine immunoglobulin. 17 Dr. Prabhu Balan, Agresearch, New Zealand

Intestinal Mucosa Figure 3 Concentrations of IgA & IgG in ileal mucosa from unchallenged rats fed the BD diet and rats challenged with Salmonella for 3 d and fed the BD, FDOI, or AOI diet BD BD-S FOI-S AOI-S a b IgA IgG 1Values are means ± SEM, n = 15, Means in a row with superscripts without a common letter differ, P < 0.05. BD, Basal diet; BD-SE, Basal diet; FOI, Freeze dried ovine immunoglobulin; AOI, Autoclaved ovine immunoglobulin. 18 Dr. Prabhu Balan, Agresearch, New Zealand

Intestinal Mucosa Figure 4 Concentrations of IgA & IgG in colonic mucosa from unchallenged rats fed the BD diet and rats challenged with Salmonella for 3 d and fed the BD, FDOI, or AOI diet BD BD-S FOI-S AOI-S a b IgA IgG 1Values are means ± SEM, n = 15, Means in a row with superscripts without a common letter differ, P < 0.05. BD, Basal diet; BD-SE, Basal diet; FOI, Freeze dried ovine immunoglobulin; AOI, Autoclaved ovine immunoglobulin. 19 Dr. Prabhu Balan, Agresearch, New Zealand

Intestinal Mucosa Table 1 Intestinal mucosal ASE IgA and ASE IgG concentrations (absorbance) in rats challenged with Salmonella for 3 d and fed the BD, FDOI, or AOI diet1 Challenged with Salmonella enteritidis BD FDOI AOI SEM P-value Ileal mucosa ASE IgA, OD (450nm) 0.44b 0.91a 0.49b 0.08 0.003 ASE IgG, OD (450nm) 0.22 0.27 0.24 0.06 0.325 Colonic mucosa 0.50b 0.92a 0.46b 0.04 0.001 0.48b 0.85a 0.006 1Values are means ± SEM, n = 15. ASE, Anti-Salmonella enteritidis. Means in a row with superscripts without a common letter differ, P <0.05. Dr. Prabhu Balan, Agresearch, New Zealand

Microbiota results 21 Dr. Prabhu Balan, Agresearch, New Zealand

Figure 1 Lanes showing DGGE bands from pooled DNA samples of ileal (A) and colonic (B) digesta used for sequencing for the identification of bacteria in ungavaged rats fed the BD diet and rats gavaged with Salmonella and fed either the BD, FDOI or AOI diets. Bands were selected for sequencing only when they were showing an obvious difference between the diets. BD, Basal diet; FDOI, Freeze dried ovine immunoglobulin; AOI, Autoclaved ovine immunoglobulin. A B ILFS5 ILFS3 ILFS2 ILFS1 ILFS4 CFS1 CFS2 CFS3 CFS5 CFS4 Challenged Unchallenged BD FDOI AOI BD AOI FDOI Dr. Prabhu Balan, Agresearch, New Zealand

Intestinal Digesta Table 1 Identified bacterial species from DNA sequencing of the PCR-DGGE bands for the challenged* FDOI-fed rats (refer Figure 1). Sample ID Nearest neighbour Similarity with nearest neighbour Accession no. of nearest neighbour ILFS1 Leuconostoc citreum 95% AB ILFS2 Weissella cibaria 99% AB ILFS3 Lactobacillus johnsonii strain NCC2767 FJ ILFS4 ILFS5 Uncultured bacterium clone EU CFS1 CFS2 100% HM CFS3 Akkermansia muciniphila ATCC BAA-835, NC_ CFS4 EU CFS5 96% EU * 3 d of challenge, IL, Ileal digesta; C, Colonic digesta. Dr. Prabhu Balan, Agresearch, New Zealand

Mucin results 24 Dr. Prabhu Balan, Agresearch, New Zealand

Table 1 Mean goblet cell counts in the intestine of growing rats after challenging with Salmonella enteritidis. Unchallenged Challenged with Salmonella enteritidis Diet: BD FDOI AOI SEM P value Villus goblet cells2 Duodenum 104.57b 25.71d 137.86a 51.86c 7.96 <.0001 Jejunum 99.14a 18.71c 107.14a 37.14b 6.16 Ileum 83.43b 29.57c 100.00a 38.43c 4.73 Crypt goblet cells2 64.33b 33.33c 76.00a 29.67c 3.1 0.001 46.57b 22.00c 54.29a 21.71c 2.45 34.00a 18.86b 39.00a* 18.57b 1.87 Colon 212.43a 138.00b 224.00a 138.14b 8.27 11Values are means ± SEM, n = 15, Means in a row with superscripts without a common letter differ, P < BD, Basal diet; FDOI, Freeze dried ovine immunoglobulin; AOI, Autoclaved ovine immunoglobulin. 2 Number of goblet cells per five complete villi and their associated crypts. Dr. Prabhu Balan, Agresearch, New Zealand

Intestinal Digesta Table 2 Mean concentration of mucin protein in the ileal and colonic luminal digesta for the growing rat following challenging with Salmonella enteritidis. Unchallenged Challenged with Salmonella enteritidis* Diet: BD FDOI AOI SEM P value Ileum (μg/mL) 10.69a 3.03b 9.95a 3.58b 1.85 0.007 Colon (ng/mL) 46.39a 5.95b 50.22a 9.51b 11.56 0.015 1Values are means ± SEM, n = 15, * 3 d of challenge, Means in a row with superscripts without a common letter differ, P < 0.05. Dr. Prabhu Balan, Agresearch, New Zealand

Intestinal Mucosa Table 3 Intestinal mucosal mucin protein concentrations for the unchallenged rats fed the BD diet and rats challenged with Salmonella for 3 d and fed the BD, FDOI, or AOI diet1. Unchallenged Challenged with Salmonella enteritidis Diet: BD FDOI AOI SEM P Value Ileum, mg/L 3.72a 0.75b 4.01a 0.98b 1.01 0.04 Colon, μg/L 12.17a 5.41b 13.65a 5.65b 2.01 0.02 1Values are means ± SEM, n = 15. Means in a row with superscripts without a common letter differ, P < 0.05. Dr. Prabhu Balan, Agresearch, New Zealand

Possible mechanism of action An understanding of the detailed mechanistic basis of the effects of ovine serum Ig awaits future research. However, the results reported in this presentation suggest few mechanisms. Dr. Prabhu Balan, Agresearch, New Zealand

Possible mechanism of action An understanding of the detailed mechanistic basis of the effects of ovine serum Ig awaits future research. However, the results reported in this presentation suggest few mechanisms. Sheep are exposed to a much harsher environment than the average human - more serum antibodies against pathogens. Dr. Prabhu Balan, Agresearch, New Zealand

Possible mechanism of action An understanding of the detailed mechanistic basis of the effects of ovine serum Ig awaits future research. However, the results reported in this presentation suggest few mechanisms. Sheep are exposed to a much harsher environment than the average human - more serum antibodies against pathogens. Passive inhibition of pathogenic bacteria (entering host gut lumen) - by antibodies present in FDOI. Dr. Prabhu Balan, Agresearch, New Zealand

Possible mechanism of action An understanding of the detailed mechanistic basis of the effects of ovine serum Ig awaits future research. However, the results reported in this presentation suggest few mechanisms. Sheep are exposed to a much harsher environment than the average human - more serum antibodies against pathogens. Passive inhibition of pathogenic bacteria (entering host gut lumen) - by antibodies present in FDOI. Enhanced intestinal function may be through supporting elevated numbers of lactic acid bacteria (Lactobacillus sp, L. johnsonii, Leuconostoc citreum, Weissella cibaria). Dr. Prabhu Balan, Agresearch, New Zealand

Possible mechanism of action An understanding of the detailed mechanistic basis of the effects of ovine serum Ig awaits future research. However, the results reported in this presentation suggest few mechanisms. Sheep are exposed to a much harsher environment than the average human - more serum antibodies against pathogens. Passive inhibition of pathogenic bacteria (entering host gut lumen) - by antibodies present in FDOI. Enhanced intestinal function may be through supporting elevated numbers of lactic acid bacteria (Lactobacillus sp, L. johnsonii, Leuconostoc citreum, Weissella cibaria). The increased colonization of lactic acid bacteria into the gut may prevent S. enteritidis pathogenesis in four ways: firstly, by reducing the intestinal pH by producing various organic acids, such as lactic acid, citric acid, etc., which may inhibit the growth of S. enteritidis by bacteriocidal action, Dr. Prabhu Balan, Agresearch, New Zealand

Possible mechanism of action An understanding of the detailed mechanistic basis of the effects of ovine serum Ig awaits future research. However, the results reported in this presentation suggest few mechanisms. Sheep are exposed to a much harsher environment than the average human - more serum antibodies against pathogens. Passive inhibition of pathogenic bacteria (entering host gut lumen) - by antibodies present in FDOI. Enhanced intestinal function may be through supporting elevated numbers of lactic acid bacteria (Lactobacillus sp, L. johnsonii, Leuconostoc citreum, Weissella cibaria). The increased colonization of lactic acid bacteria into the gut may prevent S. enteritidis pathogenesis in four ways: firstly, by reducing the intestinal pH by producing various organic acids, such as lactic acid, citric acid, etc., which may inhibit the growth of S. enteritidis by bacteriocidal action, secondly, by producing antimicrobial substances such as bateriocins, Dr. Prabhu Balan, Agresearch, New Zealand

Possible mechanism of action An understanding of the detailed mechanistic basis of the effects of ovine serum Ig awaits future research. However, the results reported in this presentation suggest few mechanisms. Sheep are exposed to a much harsher environment than the average human - more serum antibodies against pathogens. Passive inhibition of pathogenic bacteria (entering host gut lumen) - by antibodies present in FDOI. Enhanced intestinal function may be through supporting elevated numbers of lactic acid bacteria (Lactobacillus sp, L. johnsonii, Leuconostoc citreum, Weissella cibaria). The increased colonization of lactic acid bacteria into the gut may prevent S. enteritidis pathogenesis in four ways: firstly, by reducing the intestinal pH by producing various organic acids, such as lactic acid, citric acid, etc., which may inhibit the growth of S. enteritidis by bacteriocidal action, secondly, by producing antimicrobial substances such as bateriocins, thirdly, by competitive exclusion of S. enteritidis from GIT epithelial cell contact sites by lactic acid bacteria and other beneficial bacteria, Dr. Prabhu Balan, Agresearch, New Zealand

Possible mechanism of action An understanding of the detailed mechanistic basis of the effects of ovine serum Ig awaits future research. However, the results reported in this presentation suggest few mechanisms. Sheep are exposed to a much harsher environment than the average human - more serum antibodies against pathogens. Passive inhibition of pathogenic bacteria (entering host gut lumen) - by antibodies present in FDOI. Enhanced intestinal function may be through supporting elevated numbers of lactic acid bacteria (Lactobacillus sp, L. johnsonii, Leuconostoc citreum, Weissella cibaria). The increased colonization of lactic acid bacteria into the gut may prevent S. enteritidis pathogenesis in four ways: firstly, by reducing the intestinal pH by producing various organic acids, such as lactic acid, citric acid, etc., which may inhibit the growth of S. enteritidis by bacteriocidal action, secondly, by producing antimicrobial substances such as bateriocins, thirdly, by competitive exclusion of S. enteritidis from GIT epithelial cell contact sites by lactic acid bacteria and other beneficial bacteria, and fourthly, by augmentation of the immune system by lactic acid bacteria - might have conferred enhanced immune-mediated protection against S. enteritidis. Dr. Prabhu Balan, Agresearch, New Zealand

Possible mechanism of action An understanding of the detailed mechanistic basis of the effects of ovine serum Ig awaits future research. However, the results reported in this presentation suggest few mechanisms. Sheep are exposed to a much harsher environment than the average human - more serum antibodies against pathogens. Passive inhibition of pathogenic bacteria (entering host gut lumen) - by antibodies present in FDOI. Enhanced intestinal function may be through supporting elevated numbers of lactic acid bacteria (Lactobacillus sp, L. johnsonii, Leuconostoc citreum, Weissella cibaria). The increased colonization of lactic acid bacteria into the gut may prevent S. enteritidis pathogenesis in four ways: firstly, by reducing the intestinal pH by producing various organic acids, such as lactic acid, citric acid, etc., which may inhibit the growth of S. enteritidis by bacteriocidal action, secondly, by producing antimicrobial substances such as bateriocins, thirdly, by competitive exclusion of S. enteritidis from GIT epithelial cell contact sites by lactic acid bacteria and other beneficial bacteria, and fourthly, by augmentation of the gut immunity by lactic acid bacteria - might have conferred enhanced immune-mediated protection against S. enteritidis. Oral Ig supported the gut mucusal barrier by increasing mucin level. Dr. Prabhu Balan, Agresearch, New Zealand

Overall conclusion Feeding ovine serum Ig (intact and not denatured) positively modulate gut health in the growing rat, by boosting the immunity by altering the composition of gut microbiota by increasing mucin Dr. Prabhu Balan, Agresearch, New Zealand

Publications Han et al., (2009) The in vitro pathogenic activity of immunoglobulin concentrates extracted from ovine blood. Appl Biochem Biotech. 157, Balan et al., (2009) Orally administered ovine serum immunoglobulins influence growth performance, organ weights, and gut morphology in growing rats. J Nutr.139, Balan et al., (2010) Immunomodulatory effects of ovine serum immunoglobulin in the growing rat. Animal. 4, Balan et al., (2011). Dietary supplementation with ovine serum immunoglobulin attenuates acute effects on growth, organ weights, gut morphology and intestinal mucin production in the growing rat challenged with Salmonella enteritidis. Animal. 5, Balan et al., (2011). Immunomodulatory effects of ovine serum immunoglobulin in growing rats challenged with Salmonella enteritidis J Nutr. 141, Balan et al., (2011). Dietary supplementation with ovine serum immunoglobulin is associated with increased gut mucin secretion in the growing rat. Animal. Balan. (2012). Effects of orally administered ovine serum immunoglobulin in the normal and Salmonella enteritidis – challenged growing rat. PhD thesis, Massey University. Balan et al., (2012). Oral healthcare product for companion animals. New Zealand patent (pending). Balan et al., (2013). Orally administered ovine serum immunoglobulins modulate the intestinal levels of Lactobacillus and enterobacteria in the growing rat. J Anim Sci. 91, Balan et al., (2013). Intact but not denatured ovine serum immunoglobulin positively modulate mucosal immune mediators in the growing rat challenged with Salmonella enteritidis Br J Nutr. 22, 1-9. Balan et al., (2013). Recovery of intact IgG in the gastrointestinal tract of the growing rat following ingestion of an ovine serum immunoglobulin. J Anim Physiol Anim Nutr (Berl). Dr. Prabhu Balan, Agresearch, New Zealand

Acknowledgment Special thanks to Riddet Institute (funding research – PhD & postdoc) Agresearch Dr. Prabhu Balan, Agresearch, New Zealand

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