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Department of Animal Science, University of California, Davis, CA and * Ventria Bioscience, Incorporated, Sacramento, CA
2To whom correspondence should be addressed. E-mail: kcklasing{at}ucdavis.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • antibiotics • lactoferrin • lysozyme • broilers • growth • intestinal morphology
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) due to the emergence in food animals of zoonotic microorganisms that are resistant to antibiotics (2
,3
). This may result in a decrease in the therapeutic effectiveness of antibiotics used to treat a variety of bacterial infections in humans. This threat to human health has prompted several European countries to ban their use, and in the United States, alternatives to antibiotics are currently being encouraged (3
).
One strategy for replacing antibiotics in animal diets is to employ antibacterial molecules normally found along the digestive tract. Lactoferrin and lysozyme are present in mucosal secretions and in milk where they provide defense against bacteria along epithelial surfaces (4
–6
). Both of these molecules are highly resistant to hydrolysis by acids and proteases and to digestion in the gastrointestinal tract (7
–10
).
Lysozyme is a 1,4-ß-acetylmuramidase that hydrolyzes the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine (4
). Hydrolysis products include murimyl dipeptide, a potent adjuvant capable of enhancing immunoglobulin A (IgA)3
secretion, macrophage activation and rapid clearance of bacterial pathogens in vivo (11
). Lysozyme is also capable of binding to the lipid A portion of bacterial endotoxin (12
). Lysozyme-lipid A binding results in a conformational change that keeps endotoxin from interacting with macrophage receptors and dampens the release of the proinflammatory cytokines interleukin-1 (IL-1), interleukin-6 (IL-6) and tumor necrosis factor-
(TNF-) (13
–15
).
Lactoferrin is a cationic protein that has bacteriostatic and bactericidal effects that contribute to both systemic and mucosal immune defense. (16
) Lactoferrin is a scavenger of free iron and acts to deprive microorganisms of the essential nutrient (17
). Bactericidal activity stems from its ability to destabilize the outer membrane of gram-negative bacteria through the liberation of lipopolysaccharides (LPS) from their cell walls (18
). Lactoferrin is also capable of reducing LPS-induced proinflammatory cytokine release by monocytes, as well as blocking the LPS priming of neutrophils for superoxide production (19
).
Lysozyme and lactoferrin are efficacious for resistance to infectious diseases in experimental animals and humans after administration by oral, intravenous, intraperitoneal and topical routes. For example, intraperitoneal administration decreases the pathology resulting from a Klebsiella pneumoniae infection in mice (20
). In rainbow trout, lysozyme injections decrease mortality from a challenge with Aeromonas salmonicida by more than threefold (21
) and oral administration decreased mortality from infectious pancreatic necrosis virus by twofold (21
). Oral administration of egg white lysozyme is now used clinically in human medicine for the therapy of inflammatory diseases of respiratory and digestive epithelia (22
).
Antibiotics enable their growth-promoting effects, at least in part, by reducing stress caused by microbial challenges as indicated by lowered plasma IL-1 and corticosteroid levels (23
). Therefore, other strategies that reduce bacterial challenges or decrease proinflammatory cytokines might be efficacious. The purpose of these experiments was to determine the efficacy of rice genetically produced to express human lactoferrin (LF) or lysozyme (LZ) to serve as a substitute for antibiotics in poultry diets. Expression of recombinant proteins in grains such as rice has the advantage over other systems, e.g., Aspergillus, Saccharomyces or tobacco, of eliminating the need to purify the transgenic protein from the producing organism before feeding.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Male Cobb broiler chicks (1 d old; Foster Farms, Delhi, CA) were raised in Petersime brooder batteries (Petersime Incubator, Gettysburg, OH) located in an environmentally controlled room (25°C) with 24 h light. Chicks were provided water and commercial chick starter for ad libitum consumption. The batteries had not been cleaned after their previous use to provide a level of sanitation conducive to an antibiotic response. When the chicks were 3 d old, experimental chicks were selected for uniform body weight from a twofold larger population and randomly assigned to dietary treatments. Chicks had ad libitum access to both feed and water and were exposed to a 24-h light cycle. All experiments and procedures were approved by the Campus Animal Care and Use Committee.
Rice.
Two transgenic rice strains were produced to express either lactoferrin or lysozyme (24
). Briefly, rice callus from the rice strain Taipei 309 was transformed with plasmids carrying genes for LF and LZ under the control of rice glutelin 1 gene promoter. Transgenic plants were screened for a high level of expression of both recombinant proteins. The selected lines, 159–53 and 164–12, were propagated to produce sufficient amount of rice seed for these experiments. LF and LZ rice expressed 4.0 and 2.5 g/kg of recombinant protein as determined by ELISA. Taipei 309, which is the conventional rice (CONV) that served as the host for transgenic plant production, served as a control. All rice was dehusked to yield brown rice and then ground using a comminuting machine (Fitzpatrick, Chicago, IL).
Diets.
Corn-soy-rice basal diets were formulated to meet or exceed the nutrient needs of young growing broiler chicks suggested by the NRC (25
). All experimental diets were formulated to contain the same amount of rice by substituting transgenic rice for CONV rice (Tables 1
and 2
). A range of levels of each test rice was chosen for study to determine a minimally efficacious level. The 10.0% LZ diet used in Experiment 1 was analyzed to contain 176 mg/kg lysozyme. After 6 mo of storage at room temperature, it contained 152 mg/kg lysozyme, indicating that this protein was stable to storage.
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In this experiment, 300 3-d old chicks were randomly assigned to 1 of 10 dietary treatments (Table 2)
. Each dietary treatment consisted of six replicates, with five chicks per replicate. Chick and feeder weights were determined on d 1 and 17.
Experiment 2.
A second experiment was designed to confirm the results of the first experiment using twice the number of replicates per treatment to examine more subtle effects of treatments; 360 3-d old chicks were randomly assigned to one of 5 dietary treatments (Table 2)
with 12 replicates per treatment and 6 chicks per replicate (42 chicks per treatment). Chick and feeder weights were determined on d 1 and 19.
Histology.
For both Experiments 1 and 2, intestinal samples were taken on the last day of the experiment. Sections (2.5 cm) from one bird per replicate were obtained from the duodenum at the apex of the pancreas, the jejunum at a position midway between Meckel’s diverticulum and the entrance of the bile ducts, the ileum at a position midway between Meckel’s diverticulum and the ileum-cecal junction, and the ceca at a point midway along its length (Experiment 2 only). Samples were flushed with saline, fixed in 10% buffered formalin (pH 7.0), embedded with paraffin, thin-sectioned and stained with hematoxylin-eosin (IDEXX Veterinary Services, Sacramento, CA). For enumeration of intraepithelial and lamina propria leukocytes, sections were fixed in acetone, redried, incubated with mouse anti-chicken CD45 monoclonal antibody (Southern Biochemical Associates, Birmingham, AL) for 1 h and then rinsed in PBS. Sections were incubated with rabbit anti-mouse Ig tagged with peroxidase with 5 g/L bovine serum albumin for 1 h and rinsed. Peroxidase activity was developed by incubating sections with 0.01% H2O2 and 3,3'-diamino-benzidine-tetrahydrachloride. The slides were counterstained with hematoxylin-eosin. The number of leukocytes in 10 villi per section and the number of leukocytes in the lamina propria underneath and within these 10 villi were enumerated. Cells with endogenous peroxidase activity (primarily heterophils) were also enumerated as described by Vervelde and Jeurissen (26
). For each intestinal sample, villi height, villi width, crypt depth, lamina propria thickness, number of lamina propria leukocytes and number of intraepithelial leukocytes were estimated using Image-Pro-Plus software (Media Cybernetics, Silver Spring, MD).
Statistical analysis.
For both Experiments 1 and 2, data were analyzed for main effect of diet using the general linear model (Minitab; State College, PA). When main effects were significant (P < 0.05), differences due to dietary treatment were determined using Tukey’s means comparisons.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Chicks fed diets containing 0.1% LF, 1% LF, 5% LF, human lactoferrin, 0.2% LZ, 10% LZ, or 0.1% LF + 0.2% LZ did not differ from chicks fed CONV in any of the parameters measured, and therefore will not be mentioned further. Feed intake and body weight gain were not affected by dietary treatments (P > 0.05), and averaged 36.10 and 28.96 g/(chick · d), respectively. Chicks fed 5% LF + 10% LZ had significantly greater feed conversion compared with chicks fed CONV (Table 3)
. Chicks fed antibiotics (bacitracin + roxarsone; Antibiotics) also tended (P = 0.058) to have greater feed conversion than those fed CONV.
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. There were no significant differences in villous height, villous width or crypt depth due to dietary treatments in any intestinal segment (data not shown). Chicks fed 5% LF + 10% LZ had significantly thinner lamina propria in the duodenum compared with those fed CONV (P < 0.05). Chicks fed Antibiotics had jejuni with significantly thinner lamina propria and lower counts of lamina propria leukocytes compared with chicks fed CONV (P < 0.05). Chicks fed 5% LF + 10% LZ or Antibiotics tended (P = 0.068) to have lower counts of lamina propria leukocytes in the ileum compared with those fed CONV.
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Chicks fed the corn-soy diet, which was devoid of rice, did not differ from chicks fed CONV in any of the parameters measured, and therefore will not be mentioned further. There was no significant difference (P > 0.05) in body weight gain due to dietary treatments, which averaged 37.89 g/(chick · d). Chicks fed 5% LF + 10% LZ or 10% LZ consumed significantly less feed than those fed CONV (Table 5)
. Chicks fed either 10% LZ, 5% LF + 10% LZ, or Antibiotics had greater feed efficiency compared with chicks fed CONV (Table 5)
. As in the first experiment, there were no differences in body weight gain, food intake or feed efficiency between chicks fed LF or LZ rice and those fed Antibiotics.
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. Chicks fed CS + 10% LZ or Antibiotics had significantly thinner lamina propria in the ileum and fewer leukocytes in the ileal lamina propria compared with chicks fed CONV. There were no other significant differences due to diet in villous width, crypt depth or intraepithelial leukocytes in any intestinal segment.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Our highest level of LZ (10% of the diet) provided 0.3 g/kg lysozyme, which is similar to the highest concentrations in human milk. Inclusion of LF at 5% provided 0.125 g/kg, which is low relative to human milk. We were precluded from testing higher levels of lactoferrin because of scarcity of this strain of plant at the time of these studies.
Previous investigators demonstrated that the inclusion of antibiotics in the diet results in increased growth rate and improved feed efficiency compared with controls fed antibiotic-free diets (23
,28
–32
). In our experiments, feed efficiency tended to be improved by dietary antibiotics in the first experiment and was significantly improved in the second experiment, in which a greater number of replicates were used. We did not observe an effect of dietary antibiotics on weight gain and this could be due to lower efficacy of antibiotics in a clean environment (23
,33
,34
). Our experiments were conducted in cages, whereas much of the published work has utilized floor pens containing reused litter. Although sanitation standards were relaxed in our cage environment, cleanliness was still better than what is typically found in reused litter environments. Diets containing 5% LF + 10% LZ improved feed efficiency in both experiments and diets containing 10% LZ resulted in an improvement in the second experiment. Diets containing the highest levels of LF and LZ were at least as effective in improving feed efficiency as antibiotics.
Antibiotics are thought to improve animal productivity by lowering microbial infectious challenges along the gastrointestinal tract (23
,35
). This mode of action is supported by the observation that antibiotics are ineffective at improving animal productivity in a germfree environment (23
,31
,34
). Antibiotic-fed chicks have lower small intestinal weights than chicks fed antibiotic-free diets (36
–38
). The difference in intestinal weight is not due to alterations in fat, moisture content or length, (36
) but rather to changes in intestinal tissue layers (33
,37
,39
). Jukes et al. (37
) demonstrated that antibiotics caused significantly thinner duodenal lamina propria and cross-sectional diameter. Small intestines of chicks raised in a germfree environment also have diminished lamina propria and a greater proportion of epithelial layer compared with conventionally raised chicks (39
). We observed reduced thickness of the lamina propria and increased villi length in the small intestine of chicks fed either antibiotics or high levels of LZ and LF. Presumably the increased villi length, and consequently surface area, would facilitate better nutrient digestion and absorption, explaining the improved efficiency of feed utilization.
Both germfree and antibiotic-fed chicks have lower counts of reticuloendothelial cells in the subepithelial tissue of their small intestines and lower ileocecal tonsil weights compared with conventionally raised or antibiotic-free chicks, respectively (31
,40
,41
). In our second experiment, we found that the diminished number of leukocytes in the lamina propria of the small intestine due to antibiotics was duplicated by 10% LZ. Fewer leukocytes suggest lower levels of microbial challenges and improved intestinal health due to these dietary treatments.
We observed effects of antibiotics and rice on the thickness of the lamina propria and numbers of lamina propria leukocytes in both experiments, yet the region of the small intestine in which they occurred differed. This variability might be explained by the difference in the age at which the birds were sampled. Alternately, the differences could be due to the microbial populations that colonized the intestines. The microbial ecology of the intestines is extremely variable over time and it is almost certain that there were differences in the communities of microbes inhabiting the intestines of the birds used in these two experiments. These differences could easily explain the variability in the intestinal regions in which we observed treatment effects.
In general, we found that the combination of 5% LF + 10% LZ was more efficacious at improving feed efficiency and histological indices of intestinal health than 10% LZ alone; 5% LF alone was without effect. These observations suggest that at the levels used, lysozyme was much more efficacious than lactoferrin and that there was synergism between these two proteins. In vitro studies utilizing a combination of lactoferrin and lysozyme on bacterial growth have demonstrated increased bacteriostatic activity compared with either lactoferrin or lysozyme alone (5
). Lactoferrin binds to the lipid A component of LPS of several serotypes of bacteria involved in septic shock, resulting in the release of LPS from the membrane and enhanced susceptibility of the bacteria to lysis by lysozyme (5
,16
).
In vitro, lactoferrin decreases the production of IL-1 and TNF- from mixed lymphocyte cultures (19
). Lysozyme decreases in vitro IL-6 production by macrophages, and in vivo production of TNF- (13
,14
). The proinflammatory cytokines IL-1, IL-6 and TNF- mediate the impaired growth rates and feed efficiency that accompany the immune response to bacteria and their LPS (42
,43
). Roura et al. (23
) demonstrated that feeding antibiotics lowers circulating bioactive IL-1 levels, which presumably permits faster growth and better feed efficiency. Whether the effects of LF and LZ were mediated by the bacteriostatic properties of lactoferrin and lysozyme, by their modulation of pro-inflammatory cytokine profiles or by a combination of both remains to be determined.
Feeding antibiotics remains efficacious at improving animal productivity after > 50 y of steady use. However, evidence suggesting the transfer of antibiotic resistance genes from commensal bacteria of poultry, pigs and cattle to human pathogens has driven a search for alternate strategies. Our experiments demonstrate the potential of rice expressing lactoferrin and lysozyme to serve as an alternative to antibiotics in broiler diets. The antibacterial properties of lysozyme and lactoferrin evolved in the presence of commensal and pathogenic microflora, and there is no indication that bacteria have become resistant to these proteins in nature. However, the development of bacterial resistance after prolonged feeding of these proteins has yet to be examined.
The approach of reinforcing the innate immune system by feeding protective proteins normally found along the intestinal epithelium may have applications beyond chickens. In particular, weanling baby pigs are very reliant on dietary antibiotics for maintenance of intestinal health and prevention of diarrhea. Because the transgenic proteins used in these experiments were of human origin, this strategy may also be relevant to the nutrition of human infants.
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FOOTNOTES |
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3 Abbreviations used: Antibiotics (bacitracin + roxarsone); CONV, conventional rice; Ig, immunoglobulin; IL, interleukin; LF, rice genetically produced to express lactoferrin; LPS, lipopolysaccharides; LZ, rice genetically produced to express lysozyme; TNF, tumor necrosis factor.
Manuscript received 13 November 2001. Initial review completed 21 December 2001. Revision accepted 7 March 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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