2011 (SBTE) 25th Annual Meeting Proceedings - International ...

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E.A. Maga & J.D. Mur urray. 2011. Genetic engineering of livestock to improve human health: The human lysozyme transgenic goat model. Acta Scientiae Veterinariae. 39(Suppl 1): s295 - s300. animals significantly slowed the growth of S. aureus, E. coli and P. fragi as demonstrated by an overall lower mean number of colony forming units (CFU)/mL after incubation than milk from non-transgenic controls. The growth of a lactic acid bacteria (L. lactis) was not affected by the presence of HLZ in milk. Milk from HLZ transgenic animals was also capable of slowing the growth of bacteria in vivo. Milk from HLZ transgenic animals was found to have a different bacterial population corresponding to a longer shelf life [20]. Fewer bacteria grew in milk of transgenic animals and milk survived at room temperature for longer periods than control milk before bacterial growth occurred. The differential growth of bacteria in milk from transgenic animals indicates that HLZ expressed in milk is able to act in an antimicrobial fashion to alter the growth of bacteria in vivo. V. IMPACT OF CONSUMING HLZ TRANSGENIC GOAT MILK As the efficacy of HLZ in milk was confirmed as described above, the biological action of HLZ milk at the level of the intestine after consumption was evaluated by assessing the growth of coliform bacteria in the small intestine in two animal models, the goat and the pig. Pigs represent a monogastric animal with a digestive tract similar to humans. The use of pigs as a relevant human medical model is well documented [33] as pigs are frequently used in cardiovascular and nutritional research. Due to the antimicrobial properties of HLZ and evidence that natural components of milk result in different intestinal microbiota and overall intestinal development, it is our hypothesis that the consumption of milk containing active HLZ will impact intestinal microbiota, and thus intestinal health, and resistance to intestinal illness. The first part of this hypothesis was shown to be correct as pasteurized milk from HLZ transgenic animals was capable of modulating intestinal bacteria in both ruminant and non-ruminant animal models [21]. In the more humanrelevant model, weanling pigs receiving pasteurized milk from HLZ transgenic animals for 16 days had significantly lower numbers of coliforms and E. coli in their intestine than did pigs fed milk from non-transgenic control animals [21]. These data indicate that HLZ expressed in the milk of dairy animals can indeed be biologically active in the intestine and modulate gut microbiota, much like human milk. Further work in pigs confirmed the second part of our hypothesis by demonstrating that consumption of pasteurized milk from HLZ transgenic goats resulted in beneficial changes in gut histology and protection against intestinal infection [3]. Pigs fed HLZ milk had fewer numbers of coliforms and E. coli in both the duodeneum and ileum than did pigs fed milk from non-transgenic controls, repeating the findings of our first study. Animals receiving HLZ milk had significantly wider villi in the duodenum indicating a healthier gut with increased absorptive area. The number of intraepithelial lymphyocytes per micron of villi height was significantly decreased in the duodenum of HLZ-fed animals, an additional indicator of increased gastrointestinal tract health. Animals fed HLZ milk for a period of 4 weeks and then challenged with a porcine-specific enteropathogenic Escherichia coli (EPEC) had significantly lower levels of coliforms and E. coli in their ileum than did those receiving milk from non-transgenic control animals, indicating a protective effect of HLZ milk against EPEC infection. In addition, standard CBC analysis indicated that no allergic response was occurring upon consumption of HLZ milk. Furthermore, there was no significant difference in the expression of key proinflammatory cytokines (TNF-α and IL-8) in intestinal tissue of pigs consuming HLZ or control milk, indicating that an inflammatory response is not induced upon consumption of HLZ milk [6]. Serum from non-challenged pigs was also subjected to a metabolite profiling analysis [4]. A total of 234 metabolites were quantified (178 known, 56 unknown) with levels of 18 known metabolites and 4 unknown metabolites being significantly different in pigs reared on HLZ milk compared to pigs that received control milk. These differences could be broken down into effects of bacteria, increased growth, healthier gastrointestinal tract, and modulation of the immune system with the direction of changes indicative of a healthier gut. In addition, consumption of HLZ milk significantly increased the expression of the anti-inflammatory cytokine TGF-â1 in the small intestine [6], again indicative of a healthier gut. Taken together, these data strongly suggest that milk from HLZ transgenic goats can be used to improve human health by fighting the high childhood mortality rates associated with common diarrheal illnesses. VI. CONCLUSIONS Data collected over the years on most applications of transgenic animals for agriculture indicate that the implementation of GE animals can indeed have a positive impact on animal productivity and sustainability as well as animal and human health. s298
E.A. Maga & J.D. Mur urray. 2011. Genetic engineering of livestock to improve human health: The human lysozyme transgenic goat model. Acta Scientiae Veterinariae. 39(Suppl 1): s295 - s300. The HLZ transgenic goat model has produced convincing data that the adoption of GE livestock has the potential to benefit human health with little risk. Future work with this approach will revolve around translating the use of HLZ milk to treat/prevent diarrhea by developing a pig model of infection and generating lactoferrin transgenic goats. Our hope is to contribute a new solution to an old problem and prevent the suffering and deaths associated with common diarrhea in Brazil and throughout the world. REFERENCES 1 Bleck G. T., White B. R., Miller D. J & Wheeler M. B. 1998. Production of bovine α-lactalbumin in the milk of transgenic pigs. Journal of Animal Science. 76(12): 3072–3078. 2 Brophy B., Smolenski G., Wheeler T., Wells D., L’Huillier P. & Laible G. 2003. Cloned transgenic cattle produce milk with higher levels of β-casein and γ-casein. Nature Biotechnology. 21(2): 157–162. 3 Brundige D.R., Maga E.A., Klasing K.C. & Murray J.D. 2008. Lysozyme transgenic goats’ milk influences gastrointestinal morphology in young pigs. Journal of Nutrition. 138(5): 921-926. 4 Brundige D.R., Maga E.A., Klasing K.C. & Murray J.D. 2010. Consumption of pasteurized human lysozyme transgenic goats’ milk alters serum metabolite profile in young pigs. Transgenic Research. 19(4): 563-574. 5 Chandan R.C., Parry R.M. & Shahani K.M. 1968. Lysozyme, lipase, and ribonuclease in milk of various species. Journal of Dairy Science. 51(4): 606-607. 6 Cooper C.A., Brundige D.R., Reh W.A., Maga E.A. & Murray J.D. 2011. Lysozyme transgenic goats’ milk positively impacts intestinal cytokine expression and morphology. Transgenic Research. DOI 10.1007/s11248-011-9489-7. 7 Du S. J., Gong Z., Fletcher G., Shears M., King M.J., Idler D.R. & Hew C.L. 1992. Growth enhancement in transgenic Atlantic salmon by the use of an “all fish” chimeric growth hormone gene construct. Biotechnology. 10(2): 176–181. 8 Ebert K.M., Selgrath J.P., DiTullio P., Denman J., Smith T.E., Memon M.A., Schindler J.E., Monastersky G.M., Vitale J.A. & Gordon K. 1991. Transgenic production of a variant of human tissue-type plasminogen activator in goat milk: generation of transgenic goats and analysis of expression. Biotechnology. 9(9): 835-838. N 9 Edmunds T., Van Patten S.M., Pollock J., Hanson E., Bernasconi R., Higgins E., Manavalan P., Ziomek C., Meade H., McPherson J.M. & Cole E.S. 1998. Transgenically produced human antithrombin: structural and functional comparison to human plasma-derived antithrombin. Blood. 91(12): 4561-4571. 10 Goldman A.S., Garza C., Johnson C.A., Nichols B.L., & Goldblum R.M. 1982. Immunologic factors in human milk during the first year of lactation. Journal of Pediatrics. 100(4): 563-567. 11 Goldman A.S. 2007. The immune system in human milk and the developing infant. Breastfeed Medicine. 2(4): 195-204. 12 Golovan S.P., Meidinger R.G., Ajakaiye A., Cottrill M., Wiederkehr M.Z., Barney D.J., Plante C., Pollard J.W., Fan M.Z., Hayes M.A., Laursen J., Hjorth J.P., Hacker R.R., Phillips J.P. & Forsberg C.W. 2001. Pigs expressing salivary phytase produce low-phosphorus manure. Nature Biotechnology. 19(10): 741-745. 13 Hammer R.E., Pursel V.G., Rexroad Jr. C.E., Wall R.J., Bolt D.J., Ebert K.M., Palmiter R.D. & Brinster R.L. 1985. Production of transgenic rabbits, sheep and pigs by microinjection. Nature. 315(6021): 680-683. 14 Jackson K.A., Berg J.M., Murray J.D. & Maga E.A. 2010. Evaluating the fitness of human lysozyme transgenic dairy goats: Growth and reproductive traits. Transgenic Research. 19(6): 977-986. 15 Kuroiwa Y., Kasinathan P., Sathiyaseelan T., Jiao J.A., Matsushita H., Sathiyaseelan J., Wu H., Mellquist J., Hammitt M., Koster J., Kamoda S., Tachibana K., Ishida I. & Robl J.M. 2009. Antigen-specific human polyclonal antibodies from hyperimmunized cattle. Nature Biotechnology. 27(2): 173-181. 16 Lai L., Kang J.X., Li R., Wang J., Witt W.T., Yong H.Y., Hao Y., Wax D.M., Murphy C.N., Rieke A., Samuel M., Linville M.L., Korte S.W., Evans R.W., Starzl T.E., Prather R.S. & Dai Y. 2006. Generation of cloned transgenic pigs rich in omega-3 fatty acids. Nature Biotechnology. 24(4): 435-436. 17 Lonnerdal B. 2003. Nutritional and physiologic significance of human milk proteins. American Journal of Clinical Nutrition. 77(6): 1537S-1543S. 18 Maga E.A., Sargent R.G., Zeng H., Pati S., Zarling D.A., Oppenheim S.M., Collette N.M.B., Moyer A.L., Conrad-Brink J.S., Rowe J.D., BonDurant R.H., Anderson G.B. & Murray J.D. 2003. Increased efficiency of transgenic livestock production. Transgenic Research. 12(4): 485-496. s299
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