Dr. C.M. Grieshop is with the department of animal sciences at the University of Illinois, Urbana-Champaign. She presented this paper at Alltech's 19th annual international symposium held May 12-14 in Lexington, Ky.
One goal of companion animal nutrition is optimization of the immune system. For many years, researchers have been aware of the interaction between nutrition and health status. Much of the early work in this area focused on the effects of nutrient deficiencies on the immune system, but more recent work has shifted toward enhancement of immune status, particularly at times in the animal's life when it is susceptible to an immune challenge.
Before any discussion can be made on the effects of nutrition on the immune system, a basic understanding of the physiology of the system is needed.
The immune system is a complex array of cells, tissues and signaling agents that work toward the common goal of protecting the body against foreign substances. An animal's immune system can be divided into two interactive components -- innate immunity and adaptive immunity.
Innate (also called native or natural) immunity includes physical barriers (skin and gastrointestinal tract), nonspecific phagocytic cells (neutrophils and macrophages), natural killer cells, blood proteins (complement system and inflammation mediators) and regulatory cytokines (Abbas, 2000). Components of innate immunity are present prior to exposure to the antigen and are capable of rapidly reacting to general structures that are common to groups of microbes.
In contrast to innate immunity, adaptive (also called acquired or specific) immunity is highly specific and increases in magnitude and defense capabilities with successive exposures to a particular macromolecule (Abbas, 2000). The adaptive immune response can be divided into humoral and cell-mediated immunity, which act differently to eliminate different types of microbial challenges from the body.
Humoral immunity is mediated by specific antibodies produced by B-lymphocytes in response to specific antigens. Humoral immunity is the primary defense against extracellular microbes and their toxins (Abbas, 2000). In contrast, cell-mediated immunity is mediated by T-lymphocytes, which act upon intracellular microbes inaccessible to circulating antibodies (Abbas, 2000).
Adaptive immunity is capable of distinguishing and reacting to subtle differences among antigens. After an initial exposure, adaptive immunity memory will result in a response to subsequent exposures to the same antigen that not only is more rapid, but is also of increased magnitude (Kuby, 1994). Innate and adaptive immunity do not act discretely; rather both are a part of a complex system and must act cooperatively for optimal protection from foreign invaders.
Interactions between the various cells of the immune system are mediated by cytokines, which are proteins secreted during an immune response. Cytokines are secreted either locally or systemically in response to an antigenic stimulus. Cytokines can act on different cells, tissues or organs in an endocrine, paracrine or autocrine fashion (Hall, 1998).
The gut-associated lymphoid tissue (GALT) is composed of cells residing in the lamina propria region of the gut, interspaced between epithelial cells (intraepithelial lymphocytes), and in organized lymphatic tissue (Peyer's patches and mesenteric lymph nodes). Since the GALT spans the entire intestine, it is the largest immune organ of the body (Jalkanen, 1990).
The mucosal epithelium is a barrier between the internal and external environments and, therefore, is a first line of defense against antigenic invaders. The mucosal immune system is specialized to produce large quantities of immunoglobulin A (IgA). This is the only class of antibody that is efficiently secreted through the epithelial cells into the lumen of the gastrointestinal tract (Abbas, 2000).
There are certain times in an animal's life that it is particularly susceptible to an immune challenge. Two of these critical times are immediately postweaning and late in life. Environmental inadequacies, dietary changes and numerous age-associated changes in the immune system all make these time periods particularly challenging to the animal.
In general, normal immune responses increase during fetal and early neonatal periods, reach their maximum after puberty and then decrease markedly with age (Schultz, l984). Both puppies and kittens are able to mount an immune response at birth although their immune systems are not fully mature (Felsburg, l998a; Levy and Tompkins, 1998). Unfortunately, only a small amount of immunoglobulin G (IgG; 5-10%) is transferred from dam to offspring in utero in dogs and cats (Tizard, 1987). It is, therefore, critical that these animals receive adequate amounts of colostrum and milk. Both canine and feline colostrum are rich in IgG (500-2,200 mg/dl and 4,400 mg/dl, respectively) and also contain IgA (canine, 150-340 mg/dl; feline, 340 mg/dl) and immunoglobulin M (IgM) (canine, 70-370 mg/dl; feline, 58 mg/dl) (Felsburg, 1998b; Pu and Yamamoto, 1998).
As the maternal supply of immunoglobulins declines, neonatal levels increase. In puppies, serum IgM and IgG concentrations reach adult levels by 4-8 and 16 weeks of age, respectively. As with other species, serum IgA does not reach adult levels until approximately one year of age (Felsburg, 1998a). In kittens at 90 days of age, serum immunoglobulin values are 80% (IgG), 7% (IgA) and 100% (IgM) those of adult cats (Yamada et al., l991).
Animals are particularly susceptible to an immune challenge during their senior years as well. As companion animal diets improve and advances are made in veterinary health care, life expectancy is increased. Although determining the specific chronological age at which a dog or cat can be considered physiologically "old" is difficult, the following ages have been established based on the age at which the animal exhibits diseases associated with aging: small dogs, 11.5 years; medium dogs, 11.0 years; large dogs, 9.0 years; giant dogs, 7.5 years, and cats, 12 years (Hayek, l998).
Unfortunately, the geriatric pet population tends to have multiple health problems, such as a less desirable intestinal microbial balance and diminished immune capacity (Kearns et al., 1998; Shultz, 1984). Numerous physiological changes occur with increasing age that reduce the immune capacity of the animal. The lymph nodes, Peyer's patches, tonsils and thymus all involute with age. Phagocytosis and chemotaxis are less efficient and, in general, there is a reduction in the animal's immunological competence, in spite of a normal number of cells (Mosier, 1989).
The bone marrow and spleen also show physiological changes with age. Old animals may require twice as long to restore red blood cells after bleeding compared to young or mature animals. The leukocyte response is reduced, and most dogs over nine years of age have a lower number of antibody-forming cells and specific IgG antibodies compared to young dogs (Mosier, l989).
A recent longitudinal study with German Shepherds reported numerous changes in immunological parameters with age. Absolute numbers of white blood cells (10.68 ;R 2.34 x 109 cells/L versus 8.63 ;R 2.65 x 109 cells) and peripheral blood lymphocytes (2.97 ;R 0.94 x 109 cells/L versus 2.08 ;R 0.75 x 109 cells) both were higher in young (2-4 years) versus old (8-13 years) dogs (Strasser et al., 1999).
One of the most dramatic changes that occur with age in the dog is a decrease in lymphoproliferative response. Lymphoproliferative responses of both young (mean 2.4 years) and middle-aged (mean 5.8 years) Labrador Retrievers to concanavalin A, phytohemagglutinin, pokeweed mitogen and staphylococcal enterotoxin B are higher than those of old dogs (mean 9.1 years; Greeley et al., 1996). In addition, an age-related decline in absolute numbers of lymphocytes, T-cells, CD4-cells and CD8-cells is observed, and the distribution of lymphocyte subsets shifts with age such that the percentages of B-cells decline while those of T-cells increase (Greeley et al., 2001).
Hayek (1998) observed similar age-associated declines in the ability of lymphocytes from young and old Fox Terriers and Labrador Retrievers to respond to different mitogens. In this research, the age-associated decreases in lymphocyte response to concanavalin A, phytohemagglutinin and pokeweed mitogen ranged from -216 to -292% in Fox Terriers and -102 to -114% in Labrador Retrievers. From the information presented, it is evident that there is an age-related decline in immunity of companion animals. This decline could increase the susceptibility to, and severity of, an antigen challenge in senior pets.
Nutrition x immunity
Many nutrients have been implicated in either enhancement or suppression of the immune system. Early studies on the interaction of nutrition and immunity focused on the effects of protein-energy deprivation. Adequate supply of these nutrients continues to be of critical importance for maintenance of a healthy immune system throughout the life of companion animals.
Many additional nutrients, such as vitamins, minerals and fatty acids, also have been implicated in maintenance of optimal immune capacity, but due to space limitations, this paper will focus on energy, protein and oligosaccharides only.
Effects of energy
Given the typical energy content of commercial companion animal diets, energy deficiencies are uncommon in healthy adult animals. On the contrary, up to 40% of the dogs and cats seen by veterinarians are overweight (Sunvold and Bouchard, 1998). It is commonly assumed that most senior dogs are overweight. In reality, a greater proportion of older dogs and cats are underweight than any other age group (Laflamme, 1997), possibly resulting from decreased feed intake.
Energy requirement is known to change over the life of an animal. One of the most intriguing areas being addressed in companion animal research today is the relationship between age and maintenance energy requirements. Unfortunately, many of the studies that evaluate the effect of aging on maintenance energy requirement only utilize food intake as an indicator of energy requirement, rather than monitoring changes in bodyweight and/or composition.
Kienzle and Rainbird (1991) observed a 22 and 16% reduction, respectively, in maintenance energy requirements of young (1-2 years) versus old (more than seven years) Labradors and Beagles. Similarly, reductions in senior dog energy requirements were observed by Finke (1994) and Burger (1994). Laflamme et al. (2000) also demonstrated a reduction of 25% in maintenance energy requirement of old dogs (10.9 ;R 0.7 years) compared to young dogs (2.5 ;R 0.5 years) but found that the ability to digest nutrients was unaffected by age.
Few studies have evaluated the energy requirements of aged cats. Daily energy required for cats to maintain bodyweight over a four-week period was estimated to be 20% lower in cats more than seven years of age (Perez-Camargo and Rudnick, 2002). However, a possibly confounding factor in this research was that cats more than 12 years old demonstrated a slow and progressive loss of bodyweight starting at least two years prior to death. These data suggest that assessment of energy requirement over a four-week maintenance period may not be suitable in old cats (Perez-Camargo and Rudnick, 2002).
In contrast, neither Harper (1998) nor Taylor et al. (l995) reported a significant age effect on energy intake in cats. There is no obvious reason that dogs demonstrate such a dramatic decrease in energy intake with age while cats do not experience this phenomenon, although it is thought to be related to physical activity. In contrast to dogs that experience an age-related reduction in activity, cats are relatively inactive throughout their life (Harper, l998).
Dietary energy concentration is of particular importance to an optimal immune response. In mice, natural killer cell activity, which is thought to aid in defense against tumor growth, is reduced with age (Weindruch et al., 1983). Aged mice (30-33 months) fed restricted diets (50 kcal per week) possess natural killer cell activity similar to those of young (2-3 months) mice fed either restricted or control diets (85 kcal per week). Energy restriction in mice also increases survival time 10-20% and tends to inhibit spontaneous lymphoma (Weindruch and Walford, l982).
Research also has demonstrated a benefit of caloric restriction in maintaining optimal immune responses in the senior dog. Newberne et al. (1966) evaluated the effect of overnutrition on resistance of dogs to distemper virus. Purebred Beagles (5-7 months) were fed a balanced diet containing 90-100 (high), 70-75 (moderate) or 40-45 (low) kcal/kg bodyweight per day. High caloric intake resulted in obesity in 5-6 weeks. All dogs were exposed to distemper virus after a six-week dietary adaptation period. Mortality rate of dogs fed the high-calorie diet was 87% compared to 74 and 31% for the moderate- and low-calorie groups, respectively.
Average survival time also was reduced by overfeeding (8, 10 and 14 days for dogs fed the high-, moderate- and low-energy diets, respectively). Clinical responses to the virus were most severe in the high-calorie group, and the majority of these animals developed paralytic encephalitis in 8-10 days (Newberne, 1966).
In a longitudinal study that monitored Labrador Retrievers from eight weeks of age to death, diet-restricted (75% of total food consumption of pair-fed controls) dogs exhibited a slower rate of age-related decline in lymphoproliferative responses to concanavalin A, phytohemagglutinin and pokeweed mitogens. Restricted-fed dogs had lower numbers of B-cells than pair-mates, and dietary restriction slowed the rate of decline for CD4, CD8 and total T-cell numbers (Greeley et al., 2002). The authors reported that immune predictors of increased survival (high lymphoproliferative responses, high percentage and number of CD8 cells and high numbers of T-cells) were positively affected by diet restriction (Greeley et al., 2002).
Effects of protein
Dispute also exists over the changes in protein requirement that occur with aging in companion animals. Wannemacher and McCoy (1966) demonstrated that the dietary protein needed to maintain nitrogen balance and maximize the liver protein-to-DNA ratio was up to 50% higher in old beagles (12-13 years old) compared to young Beagles (1-2 years old). This contradicts the currently held belief that protein requirement decreases with age. Since many products of the immune system are proteins, such as immunoglobulins, lymphokines and bacteriocidal enzymes, it is not surprising that a protein-deficient diet impairs the immune response (Burkholder and Swecker, 1990).
The detrimental effect of severe protein depletion on the immune response is well documented in numerous species. Infant mice subjected to severe protein deprivation (28 versus 6% casein diets) at weaning (20 days of age) exhibited persistent decreases in thymus-derived lymphoid cells and diminished specific antibody responses (Jose et al., 1973).
The ability to mount a specific mucosal IgA response also is impaired by protein deficiency. This is demonstrated by rats fed a low-protein diet (3.2% casein), which subsequently had markedly impaired mucosal immune response to cholera toxoid/toxin compared to rats fed a control diet (24% protein). This impairment was rapidly reversed by refeeding with the high-protein diet (Barry and Pierce, 1979).
Protein deficiencies also influence antibody production. Rats and mice fed protein-deficient versus control diets (3 versus 16% casein in rat diets; 1 versus 16% casein in mouse diets) showed a marked reduction in the number of antibody-producing cells in response to a sheep red blood cell challenge (Mathur et al., l972).
Most commercial pet foods are formulated to provide more than the required protein levels, making severe protein deficiencies unlikely in most companion animals. Certain life stages do exist, such as the neonatal and senior periods, where food intake is decreased and mild protein deficiencies are possible.
In addition, deficiencies of specific essential amino acids also have been shown to affect the immune system of animals. Mice fed synthetic diets limited in a single essential amino acid (leucine, isoleucine, valine or lysine) for three weeks after weaning had significantly increased susceptibility to Salmonella typhimurium infection, as indicated by a higher mortality rate and spread of the bacterial cells compared to control mice (Petro and Bhattacharjee, l981).
Cats have a particularly high dietary requirement for the amino acid taurine. This is partly due to the low hepatic activity of cysteinesulfinic acid decarboxylase, which limits the synthesis of taurine from ethionine and cysteine (Schuller-Levis, l990). Healthy adult cats typically do not experience taurine deficiencies since their natural diet of meat and fish contains a high concentration of taurine (Hayes and Trautwein, 1989).
Problems may arise, though, when cats are fed commercially formulated diets based on cereals and grains or diets that are arbitrarily restricted by owners, such as vegetarian diets (Hayes and Traitwein, l989). Additional factors that may affect a cat's taurine requirement include the type of diet fed (i.e., expanded dry diets versus canned cat foods) and level of sulfur amino acids in the diet (Case et al., 2000).
Severe deficiency of dietary taurine can result in abnormalities in the immune system. Domestic female cats fed taurine-deficient diets for at least one year have significantly lower total white blood cell counts than taurine-supplemented cats. Feeding a taurine-deficient diet also results in significant leukopenia, a shift in the percentage of polymorphonuclear and mononuclear leukocytes. Polymorphonuclear cell functionality and histologic characteristics of the lymph nodes and spleen also are altered by taurine deficiencies in these cats (Schuller-Levis, 1990).
These results clearly demonstrate significant immunological consequences to prolonged taurine deficiency in cats. Taurine is important not only for prevention of deficiency in the adult animal, but also for maintenance of the immune system in a senior animal. Taurine is capable of augmenting the proliferative responses of T-cells from both young and old mice, although the augmentation is more marked in old T-cells (Negoro and Hara, 1992).
Functional food ingredients
Recent research not only has focused on the suppression of the immune system by nutritional deficiencies but also on enhancement via nutrient supplementation. Dietary ingredients can elicit an effect on the immune system via: a localized effect on the GALT, alteration in microbial population (prebiotic effect) or a systemic effect on the immune system.
In reality, these mechanisms do not act exclusively but rather interact to cause an overall effect. Non-digestible oligosaccharides, such as fructoologosaccharides (FOS) and mannan oligosaccharides (MOS), are examples of compounds that elicit an effect on the gastrointestinal ecology and immune system when supplemented to companion animal diets.
Non-digestible oligosaccharides can affect gastrointestinal health by promoting beneficial bacterial populations (prebiotic effect) and/or directly enhancing the immune system. Fructans (including oligofructose (OF), inulin and short-chain FOS) are prebiotic oligosaccharides that aid in host resistance to pathogenic bacteria by promoting growth of beneficial bacteria.
These beneficial bacteria compete with less desirable bacteria for nutrients and binding sites and produce antimicrobial compounds against pathogenic strains of bacteria. Alterations in microbial populations, or the short-chain fatty acids that are produced as a result of oligosaccharide fermentation, can influence the immune system by inhibiting growth of harmful bacteria, restoring normal intestinal flora and acting as immunomodulators (Meyer et al., 2000).
Some oligosaccharides, such as inulin, a storage carbohydrate found in many plants and vegetables, are specifically fermented in the colon by the beneficial bacteria bifidobacterium and lactobacillus (Meyer et al., 2000). Ingestion of only 5 g per day of OF, a hydrolyzed product of inulin, by humans increased the fecal concentration of bifidobacteria after only 11 days (Rao, 2001). Feeding adult cats FOS (0.75% diet) for 12 weeks had no effect on anaerobic or aerobic duodenal fluid bacteria concentrations (Sparks et al., 1998a).
In contrast, the same feeding regimen reduced fecal concentrations of Escherichia coli (6.3 versus 7.5 log10 CFU/g) and increased fecal concentrations of bacteroides (9.5 versus 8.0 log10 CFU/g) and the beneficial lactobacilli (7.9 versus 5.7 log10 CFU/g) compared to the basal diet. FOS supplementation also tended to reduce fecal concentrations of the potentially negative bacteria Clostridium perfringens (4.9 versus 6.6 log10 CFU/g; Sparks et al., l998b). This study indicates that low levels of OF supplementation can elicit changes in the colonic microbial population in cats.
Short-chain FOS are synthesized from sucrose by bacteria. Willard et al. (1994) supplemented diets of German Shepherds affected with small intestinal bacterial overgrowth, a condition that results in increased diarrhea and weight loss, with 1% FOS. Results indicated decreased aerobes and facultative anaerobes in duodenal fluid and in the duodenal mucosa. These results signify a positive health response and support a role for FOS in the maintenance of gut health.
Swanson et al. (2002a) also reported that supplementation of adult dogs with 4 g per day FOS positively influenced indices of gut health, including increased concentrations of the beneficial bacteria bifidobacteria and lactobacilli, and decreased concentration of the potentially negative bacteria C. perfringens. Supplementation of FOS also increased fecal butyrate and lactate concentrations.
This is of particular interest since short-chain fatty acids, particularly butyrate, are the main energy source for colonocytes. FOS supplementation also decreased the fecal concentration of several putrefactive compounds (e.g., branched-chain fatty acids, phenols, indoles).
MOS are oligosaccharides derived from the cell wall of Saccharomyces cerevisiae. In vitro data indicate that MOS is highly fermentable by canine fecal inoculum (Swanson, unpublished data). MOS can affect the immune system by a number of mechanisms including bacterial exclusion (Spring et al., 2000), neutralization of mycotoxins (Devegowda et al., 1994) and immunostimulation. MOS exclude many pathogenic bacteria by inhibiting intestinal binding via bacterial fimbriae.
Many harmful bacterial species, such as salmonellae, possess the mannose-sensitive type 1 fimbriae that undergo agglutination by MOS in vitro (Oyofo et al., 1989a; Finucane et al., 1999) and colonize in lower concentrations in animals supplemented with MOS (Spring et al., 2000, and Oyofo et al., 1989b).
MOS also can protect the animal by neutralizing mycotoxins (Devegowda et al., l994), systemically increasing IgG (Savage et al., 1996) and increasing neutrophil activity (O'Carra, 1998). Supplementation of aflatoxin-infected broilers with S. cerevisiae culture results in alleviation of toxic effects (Devegowda et al., l994). It has also been shown that immunostimulation by MOS results in systemic increases in IgG concentrations in turkeys (Savage et al., 1996).
Swanson et al. (2002b) supplemented dogs with 2 g MOS, 2 g FOS or 2 g MOS plus 2 g FOS per day for 14 days. Dogs supplemented with MOS had an enhanced systemic immune status as evidenced by a significant increase in percent serum lymphocytes and a tendency for increased serum IgA concentrations compared to control dogs. In addition, dogs supplemented with 2 g FOS plus 2 g MOS per day had significantly greater ileal IgA concentrations than control dogs, indicating enhanced local immunity also. MOS-fed dogs had lower total fecal aerobic bacteria and tended to have greater fecal concentrations of lactobacilli.
In this study, MOS supplementation tended to decrease dry matter and organic matter digestibilities compared to control dogs. In a subsequent study, dogs supplemented with 4 g FOS plus 2 g MOS per day exhibited increased fecal bifidobacteria and fecal and ileal lactobacilli concentrations, but serum, fecal and ileal immunoglobulin concentrations were unchanged. Dogs supplemented with FOS plus MOS tended to have lower concentration of blood neutrophils and greater concentration of blood lymphocytes than unsupplemented dogs (Swanson et al., 2002c).
Zentek et al. (2002) also demonstrated that dogs supplemented with 1 g MOS/kg bodyweight had reduced apparent total tract digestibility of dry matter (82 versus 90%), crude protein (80 versus 91%) and nitrogen-free extract (83 versus 93%) and had lower fecal dry matter (32 versus 37%) compared to unsupplemented dogs. Alteration in the solubility of nutrients in the intestinal chyme and activity of the intestinal microflora were offered by the author as possible mechanisms for the reductions in nutrient digestibilities observed (Zentek et al., 2002).
O'Carra (1998) evaluated the effects of supplemental MOS on the immune system of both rats and dogs. Rats fed 2-4 g MOS/kg feed exhibited a linear increase in intestinal IgA concentrations on days 12-32. In contrast, rats fed 8-10 g MOS/kg feed had a maximum intestinal IgA concentration on day 18. For all MOS supplementation levels, pooled treatment means were significantly higher than rats fed a control diet (0% MOS).
Feeding dogs 2 g MOS/kg feed resulted in a numerical increase in number of circulating neutrophils in response to a vaccine, beginning seven days post-vaccination and continuing over the 63-day trial. In this case, neutrophil activity was used as an indicator of enhanced protection against pathogenic infection.
Individual nutrients can have profound effects on the immune systems of companion animals. This paper has reviewed only a few of the many well-established interactions that exist. Although historical research has focused on immunological defects caused by nutrient deficiencies, current research is exploring the potential for enhancement of the immune response through nutrient supplementation.
As demonstrated in this paper, there are certain times in an animal's life, such as immediately postweaning and late in life, when it is particularly susceptible to an immune challenge. A great potential for enhancement of the immune system exists during these times.
Future research will focus on elucidating the role of specific nutrients on the immune system and their potential to improve the health of companion animals throughout their life.