Childhood diarrhea is a significant problem in many developing countries and E. coli is a main causative agent of diarrhea in young children. Lysozyme is an antimicrobial protein highly expressed in human milk, but not ruminant milk, and is thought to help protect breastfeeding children against diarrheal diseases. We hypothesized that consumption of milk from transgenic goats which produce human lysozyme (hLZ-milk) in their milk would accelerate recovery from bacterial-induced diarrhea. Young pigs were used as a model for children and infected with enterotoxigenic E. coli. Once clinical signs of diarrhea developed, pigs were fed hLZ-milk or non-transgenic control goat milk three times a day for two days. Clinical observations and complete blood counts (CBC) were performed. Animals were euthanized and samples collected to assess differences in histology, cytokine expression and bacterial translocation into the mesenteric lymph node. Pigs consuming hLZ-milk recovered from clinical signs of infection faster than pigs consuming control milk, with significantly improved fecal consistency (p = 0.0190) and activity level (p = 0.0350). The CBC analysis showed circulating monocytes (p = 0.0413), neutrophils (p = 0.0219), and lymphocytes (p = 0.0222) returned faster to pre-infection proportions in hLZ-milk fed pigs, while control-fed pigs had significantly higher hematocrit (p = 0.027), indicating continuing dehydration. In the ileum, pigs fed hLZ-milk had significantly lower expression of pro-inflammatory cytokine IL-8 (p = 0.0271), longer intestinal villi (p<0.0001), deeper crypts (p = 0.0053), and a thinner lamina propria (p = 0.0004). These data demonstrate that consumption of hLZ-milk helped pigs recover from infection faster, making hLZ-milk an effective treatment of E. coli-induced diarrhea.
References
[1]
Palmiter RD, Brinster RL, Hammer RE, Trumbauer ME, Rosenfeld MG, et al. (1982) Dramatic growth of mice that develop from eggs microinjected with metallothionein–growth hormone fusion genes. Nature 300: 611–615.
[2]
Jimenez-Flores R, Richardson T (1988) Genetic engineering of the caseins to modify the behavior of milk during processing: A Review. J Dairy Sci. 71: 2640–2654.
[3]
Maga EA, Murray JD (1995) Mammary gland expression of transgenes and the potential for altering the properties of milk. Nat Biotech. 13: 1452–1457.
[4]
Maga EA, Sargent RG, Zeng H, Pati S, Zarling DA, et al. (2003) Increased efficiency of transgenic livestock production. Transgenic Res. 12: 485–496.
[5]
Yang B, Wang J, Tang B, Liu Y, Guo C, et al. (2011) Characterization of bioactive recombinant human lysozyme expressed in milk of cloned transgenic cattle. PLoS One. 16: e17593.
[6]
Maga EA, Cullor JS, Smith W, Anderson GB, Murray JD (2006) Human lysozyme expressed in the mammary gland of transgenic dairy goats can inhibit the growth of bacteria that cause mastitis and the cold-spoilage of milk. Foodborne Pathog. Dis. 3: 384–392.
[7]
Jackson KA, Berg JM, Murray JD, Maga EA (2010) Evaluating the fitness of human lysozyme transgenic dairy goats: growth and reproductive traits. Transgenic Res 19: 977–86.
[8]
Maga EA, Walker RL, Anderson GB, Murray JD (2006) Consumption of milk from transgenic goats expressing human lysozyme in the mammary gland results in the modulation of intestinal microflora. Transgenic Res. 15: 515–519.
[9]
Brundige DR, Maga EA, Klasing KC, Murray JD (2008) Lysozyme transgenic goats' milk influences gastrointestinal morphology in young pigs. J Nutr. 138: 921–926.
[10]
Brundige DR, Maga EA, Klasing KC, Murray JD (2009) Consumption of pasteurized human lysozyme transgenic goats' milk alters serum metabolite profile in young pigs. Transgenic Res. 19: 563–574.
[11]
Cooper CA, Brundige DR, Reh WA, Maga EA, Murray JD (2011) Lysozyme transgenic goats’ milk positively impacts intestinal cytokine expression and morphology. Transgenic Res. 20: 1235–1243.
[12]
Black RE, Cousens S, Johnson HL, Lawn JE, Rudan I, et al. (2010) Child Health Epidemiology Reference Group of WHO and UNICEF 2010. Global, regional, and national causes of child mortality in 2008: A systematic analysis. The Lancet 375: 1969–1987.
[13]
Turner JR (2009) Intestinal mucosal barrier function in health and disease. Nat Rev Immunol. 9: 799–809.
[14]
Direct Food Substances Affirmed As Generally Recognized As Safe Food & Drug Administration. Federal Register, Vol. 63 No.49. March 13, 1998. 21 CFR Part 184 [docket no. 89G–0393].
[15]
Chemical Safety Information from Intergovernmental Organizations website. Available http://www.inchem.org/documents/jecfa/je？cmono/v30je04.htm. Accessed 2013 January 13.
[16]
Hettinga K, van Valenberg H, de Vries S, Boeren S, van Hooijdonk T, et al. (2011) The host defense proteome of human and bovine milk. PLoS One 6: 19433.
[17]
Proctor VA, Cunningham FE, Fung DYC (1988) The chemistry of lysozyme and its use as a food preservative and a pharmaceutical. Crit Rev Food Sci Nutr. 26: 359–395.
[18]
Gordon LI, Douglas SD, Kay NE, Yamada O, Osserman EF, et al. (1979) Modulation of neutrophil function by lysozyme. Potential negative feedback system of inflammation. J Clin Invest. 64: 226–232.
[19]
Ogundele MO (1998) A novel anti-inflammatory activity of lysozyme: modulation of serum complement activation. Mediators Inflamm. 7: 363–365.
[20]
Newburg DS, Walker WA (2007) Protection of the neonate by the innate immune system of developing gut and of human milk. Pediatr Res. 61: 2–8.
[21]
Actor JK, Hwang SA, Kruzel ML (2009) Lactoferrin as a Natural Immune Modulator. Cur. Pharma. Design 15: 1956–1973.
[22]
Rockova S, Rada V, Marsik P, Vlkova E, Bunesova V, et al. (2011) Growth of bifidobacteria and clostridia on human and cow milk saccharides Anaerobe. 17: 223–225.
[23]
Nyachoti CM, Kiarie E, Bhandari SK, Zhang G, Krause DO (2012) Weaned pig responses to Escherichia coli K88 oral challenge when receiving a lysozyme supplement. J Anim Sci. 90: 252–260.
Awad WA, Ghareeb K, Abdel-Raheem S, B？hm J (2009) Effects of dietary inclusion of probiotic and synbiotic on growth performance, organ weights, and intestinal histomorphology of broiler chickens. Poult Sci. 88: 49–56.
[26]
Liu P, Piao XS, Thacker PA, Zeng ZK, Li PF, et al. (2010) Chito-oligosaccharide reduces diarrhea incidence and attenuates the immune response of weaned pigs challenged with Escherichia coli K88. J Anim Sci. 88: 3871–3879.
[27]
Maga EA, Shoemaker CF, Rowe JD, Bondurant RH, Anderson GB, et al. (2006) Production and processing of milk from transgenic goats expressing human lysozyme in the mammary gland. J Dairy Sci. 89: 518–524.
[28]
Schulze F, Müller G (1980) Lysozyme in sow's milk and its importance to bacterial population of the gastrointestinal tract in suckling piglets. Arch Exp Veterinarmed. 34: 317–324.
[29]
Maga EA, Desai PT, Weimer BC, Dao N, Kültz D, et al. (2012) Consumption of lysozyme-rich milk can alter microbial fecal populations. Appl. Environ. Microbiol. 78: 6153–6160.
[30]
Jensen GM, Frydendahl K, Svendsen O, J？rgensen CB, Cirera S, et al. (2006) Experimental infection with Escherichia coli O149:F4ac in weaned piglets. Vet Microbiol. 115: 243–249.
[31]
Pfaffl MW, Horgan GW, Dempfle L (2002) Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res. 30: 36.
[32]
Dwinell MB, Johanesen PA, Smith JM (2003) Immunobiology of epithelial chemokines in the intestinal mucosa. Surgery 133: 601–607.
[33]
Lessard M, Dupuis M, Gagnon N, Nadeau E, Matte JJ, et al. (2008) Administration of Pediococcus acidilactici or Saccharomyces cerevisiae boulardii modulates development of porcine mucosal immunity and reduces intestinal bacterial translocation after Escherichia coli challenge. J Anim Sci. 87: 922–934.
[34]
Matkowskyj KA, Danilkovich A, Marrero J, Savkovic SD, Hecht G, et al. (2000) Galanin-1 receptor up-regulation mediates the excess colonic fluid production caused by infection with enteric pathogens. Nat Med. 6: 1048–1051.
Savkovic SD, Koutsouris A, Hecht G (1996) Attachment of a noninvasive enteric pathogen, enteropathogenic Escherichia coli, to cultured human intestinal epithelial monolayers induces transmigration of neutrophils. Infect Immun. 64: 4480–4487.
[37]
Liu D, Guo Y, Wang Z, Yuan J (2010) Exogenous lysozyme influences Clostridium perfringens colonization and intestinal barrier function in broiler chickens. Avian Pathol. 39: 17–24.
[38]
Liao Y, Jiang R, L？nnerdal B (2012) Biochemical and molecular impacts of lactoferrin on small intestinal growth and development during early life. Biochem Cell Biol. 90: 476–484.
[39]
Koppelmann T, Pollak Y, Mogilner J, Bejar J, Coran AG, et al. (2012) Reversal of severe methotrexate-induced intestinal damage using enteral n-3 fatty acids. Br J Nutr. 28: 1–10.
