Consumption of milk from transgenic goats expressing human ...

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Elizabeth A. MagaEmail author; Richard L. Walker; Gary B. Anderson; James D. Murray ... Cite this article as: Maga, E.A., Walker, R.L., Anderson, G.B. et al.
Transgenic Res (2006) 15:515–519 DOI 10.1007/s11248-006-0014-3

SHORT COMMUNICATION

Consumption of milk from transgenic goats expressing human lysozyme in the mammary gland results in the modulation of intestinal microflora Elizabeth A. Maga Æ Richard L. Walker Æ Gary B. Anderson Æ James D. Murray

Received: 21 October 2005 / Accepted: 9 March 2006 Ó Springer Science+Business Media B.V. 2006

Abstract Lysozyme is a key antimicrobial component of human milk that has several health-promoting functions including the development of a healthy intestinal tract. However, levels of lysozyme in the milk of dairy animals are negligible. We have generated transgenic dairy goats that express human lysozyme (HLZ) in their milk in an attempt to deliver the benefits of human milk in a continual fashion. To test the feasibility of this transgenic approach to achieve a biological impact at the level of the intestine, feeding trials were conducted in two animal models. Pasteurized milk from HLZ transgenic animals was fed to both kid goats (ruminant model) and young pigs (human model), and the numbers of total coliforms and Escherichia coli present in the small intestine were determined. Data from this proof-of-principle study demonstrate that milk from transgenic animals was capable of modulating the

E. A. Maga (&) Æ G. B. Anderson Æ J. D. Murray Department of Animal Science, University of California, One Shields Avenue, Davis, CA 95616, USA e-mail: [email protected] R. L. Walker Department of California Animal Health and Food Safety Laboratory, University of California, Davis, CA, USA J. D. Murray Department of Population Health and Reproduction, University of California, Davis, CA, USA

bacterial population of the gut in both animal models. Pigs that consumed pasteurized milk from HLZ transgenic goats had fewer numbers of coliforms and E. coli in their intestine than did those receiving milk from non-transgenic control animals. The opposite effect was seen in goats. Milk from these transgenic animals not only represent one of the first transgenic food products with the potential of benefiting human health, but are also a unique model to study the development and role of intestinal microflora on health, well-being and resistance to disease. Keywords Lysozyme Æ Microflora Æ Transgenic Æ Intestine Æ Milk

Introduction Human milk provides the newborn with all the nutrients required to promote the health and growth of the newborn in a nutritious fashion. For example, human milk supplies lysozyme and lactoferrin, antimicrobial factors that enhance intestinal and systemic immunological functions (Levy 1998; Lonnerdal 2003). However, lactation and this supply of beneficial factors are not permanent. In contrast, the milk of dairy animals is easily and continually obtained but contains these protective factors at levels of magnitude lower than human milk. For instance, lysozyme is naturally present in human milk at concentrations

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1600–3000 times greater than in livestock milk (Chandan et al. 1968). Lysozyme is a ubiquitous antimicrobial protein found in the tears, saliva and milk of all mammals (Jolles and Jolles 1984). Lysozyme specifically cleaves the glycosidic linkage between the C-1 of N-acetylmuramic acid and the C-4 of N-acetylglucosamine of the peptidoglycan component of bacterial cell walls (Phillips 1966), thus causing leakage of the cell’s interior components or lysis. Lysozyme is one of the main non-specific immunological components present in milk and contributes in several ways to the health and well-being of breast-fed infants including defense against infection by pathogenic organisms, the stimulation of a beneficial gut microflora and development and maturation of the intestinal tract (Lonnerdal 2003). Results from many studies suggest that the impact of human milk on the gastrointestinal tract has effects during breast-feeding and beyond (Goldman 2000). Based on the concept that health and well-being are, in part, determined by diet, livestock milk is not a perfect substitute for human milk. The use of biotechnology to genetically engineer the milk of dairy animals to produce naturally-occurring human milk antibacterial compounds is a potentially powerful way of delivering a sustainable health benefit to humans by offering a supplemental and continuous supply of protective, humanized milk. As a means to introduce the beneficial properties of human milk into lactating dairy animals, we generated transgenic goats, as a model for the dairy cow, that express one of these protective components, HLZ, in the mammary gland under control of the bovine as1-casein gene (Maga et al. 2003, 2006). Here we demonstrate in two proof-of-principle trials that, when consumed by animal models, pasteurized milk from transgenic HLZ goats was capable of affecting intestinal microflora.

Materials, results and discussion Two animal models, one ruminant (goats) and one monogastric (pigs), were used to asses the impact of HLZ milk consumption on the growth of intestinal microbes. The line of transgenic goats used for study was of Alpine and Toggenburg in origin and expressed active HLZ in their milk at a mean of

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270 lg/ml, approximately 68% of the level of lysozyme found in human milk (Maga et al. 2006). Milk from five individual transgenic females of the founding, F1, and F2 generations in their first (n=2), second (n=1), third (n=1) and fourth (n=1) lactations and 10 breed-, age-, parity- and stage of lactationmatched controls from the university herd was collected by machine in the morning and evening, pooled into respective containers and pasteurized to 165°F before feeding to animals. Transgenic and nontransgenic lactating does were housed in adjacent pens and treated equally. Milk from transgenic animals was monitored weekly for the presence and quantity of HLZ by western blot. Kid goats and young pigs were fed pasteurized milk from either transgenic or non-transgenic control animals, and the total number of coliforms and E. coli present in the intestine were enumerated based on the ease of analysis and importance of these organisms. For each animal model, trials were conducted contemporaneously with each treatment group housed in adjacent pens. All animals were treated identically, had equal access to ad libitum milk (goats) or lixit containers (pigs) and were cared for under AAALAC-approved conditions. The ruminant model consisted of randomly placing non-transgenic male kid goats into one of two groups at birth. Six goats received colostrum and milk from non-transgenic goats, and six received colostrum and milk from HLZ transgenic does. After colostrum administration in the first 24 h of life, all goats were hand-fed 10 oz of milk 3 times a day for the first week, then ad libitum milk via coolers for the duration of the trial (6 weeks). Weights of each animal were taken twice weekly. At the end of the feeding period each animal was euthanized and sections of the upper and lower small intestine were taken for microbial population analysis. The number of colony forming units (CFU) of total coliforms and E. coli present in a 2-g sample of material isolated from these sections were determined by Petrifilm Coliform Count Plates (3 M). Values reported are CFU/g intestinal contents and each value was log-transformed prior to statistical analysis by ANOVA. The consumption of milk from HLZ transgenic animals was capable of affecting the composition of the gut microflora in the ruminant model. Kid goats fed milk from HLZ transgenic animals had greater numbers of total coliforms (CFU/g intestinal

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contents) in the small intestine than did those fed milk from non-transgenic control animals (Table 1). Kid goats receiving milk from HLZ transgenic does had a significantly higher mean number of total coliforms in the duodenum (P =0.010) and ileum (P =0.011) than did those kids receiving milk from non-transgenic control animals. The mean number of E. coli was not significantly different between the treatment groups in either the upper (P =0.066) or lower (P =0.104) small intestine. The presence of coliforms in the small intestine of goats is usually a result of infection, as healthy goats generally contain a higher percentage of Gram-positive bacteria than Gram-negative bacteria such as coliforms and E. coli in the small intestine (Mohammed et al. 2000; Barlow et al. 2004). Here, HLZ-fed animals had higher levels of coliforms in the gut, yet showed no signs of illness or diarrhea. As lysozyme is known to be more active against Gram-

positive bacteria (Costerton et al. 1974), and milk from HLZ does was active against a Gram-positive test microorganism Micrococcus lysodeiktus (Maga et al. 2006), it is likely that the HLZ consumed was active against the predominant Gram-positive species in the gut and/or rumen, thereby reducing competition and allowing more Gram-negative coliforms to grow. Consumption of protective milk proteins such as lysozyme and lactoferrin in other species has been correlated with a growth advantage by acting at the level of the intestine. (Humphrey et al. 2002; Robblee et al. 2003). Here, the mean cumulative weight gain for animals fed HLZ milk was significantly higher during weeks 3–5 of the trial (Fig. 1). A regression analysis revealed that during this time frame, HLZfed animals gained on average 0.62 lbs/day while control-fed animals gained only 0.31 lbs/day. However, throughout the duration of the trial, overall

Table 1 Total coliform and Escherichia coli counts (CFU/g intestinal contents) in the small intestine of animal models that received milk from HLZ transgenic or non-transgenic control goats Duodenum

Goats HLZ-fed 4332 4337 4338 4339 4351 4355 Control-fed 4014 4015 4016 4017 4018b 4350b Pigs HLZ-fed 28–8 28–9 28–16 28–17 Control-fed 28–10 28–12 28–13 28–14 a

Ileum

Total coliforms

E. coli

Total coliforms

E. coli

40 2200 310 420,000 720 400