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Puppy Immune System

Archive document IAMS COMPANY

Iams is since 1999 a registered trademark of The Procter & Gamble Company. This is an archive document used historically by the Iams Pet Food company or for Iams Pet Food products. Any and all mentions included herein needs to be replaced in context of the time and geography of its initial use as circumstances and products may have changed since then. Products and relative data are for US only. No assertion or further use of these documents can be made without P&G's authorization.

THE INFLUENCE OF DIET ON THE PUPPY'S DEVELOPING IMMUNE SYSTEM

Stefan P. Massimino, MS
Leighann Daristotle, DVM, PhD
Michael A. Ceddia, PhD
Michael G. Hayek, PhD
Research and Development Division
The Iams Company, Lewisburg, Ohio, USA
Presented at the Iams Breeder' Symposium, February 9, 2002

OVERVIEW OF THE IMMUNE SYSTEM

In all mammals, the body has various defenses against invading disease agents (pathogens), ranging from non-specific to specific. Non-specific physical barriers, such as skin and mucous membranes, protect against the initial entry of pathogens such as bacteria, viruses, and parasites. However, once those barriers are overcome, a functional immune system is required to mount a specific response to protect and clear the body from infection. The immune system is an intricate network of specialized and interacting tissues, organs, cells, and chemicals.

Immunity can be innate (inherent) or acquired. Puppies and other mammals including humans are born with innate immunity, which consists of both cellular and chemical defense mechanisms that rely heavily on detection of the difference between invading microorganisms and what is considered "self" or part of the individual's body. When invaders are detected, enzymes that can digest bacterial cell walls are activated, and cells that recognize these invading microorganisms and destroy them are deployed. This response is specific and does not require priming, but is slow and usually not sufficient to clear the body of a pathogen. Rather, it serves to contain the infection until the next level of defense - known as acquired immunity - develops.

Acquired immunity is a complex, sophisticated system that can rapidly develop a specific response against invading pathogens. It can be divided into the following two components: cell-mediated and humoral immunity. Cell-mediated immunity includes both T and B cells (lymphocytes) and their interactions. These blood cells recognize foreign invaders by their protein (antigen) makeup and through cell to cell interactions and cytokine release (soluble immune mediators), result in production of additional T and B cells (proliferative response). These cells are then responsible for sustaining the immune response, destroying the invading pathogen and infected cells, and terminating the immune response once the infection has been cleared. As well, some cells survive that are known as memory cells. In the event of another attack by the same pathogen, the immune system is able to respond much more rapidly and vigorously as a result of these memory cells.

Humoral immunity is also referred to as the antibodymediated immune response. When an invading pathogen has been recognized, antigen-specific B cells proliferate and are transformed into antibody-secreting cells (plasma cells). Antibodies are blood-borne immune proteins (immunoglobulins) that are able to bind specifically with infected cells, as well as free microorganisms, which lead to their destruction. There are five different immunoglobulin isotypes (IgG, IgA, IgM, IgE, and IgD). The two isotypes most common in serum are IgG and IgM. As with T cells, memory B cells remain after the infection to produce specific antibodies if the same pathogen is detected.

NEONATAL IMMUNITY

When puppies are born, they emerge from a sterile environment (the uterus) to become exposed to a host of microorganisms, all of which are potentially pathogenic. Unfortunately, the immune system is not fully functional and developed for some time after birth. As a result, newborn puppies are especially vulnerable to infection in the first few weeks of life and require immune assistance in order to survive. This assistance is provided by the mother, via transfer of immune cells and immunoglobulins through the colostrum and milk, and immediately confers some level of immune protection for the newborn. This transfer of immunity from mother to newborn is essential for the newborn's survival.

The immune system then requires time to develop to its fully functional capacity. Both the distribution of immune cell types and their responses have been reported to change as puppies and kittens grow and develop.¹ T cell populations are significantly smaller and their proliferation response to an antigen is less in puppies, compared with that in adult dogs.^2,3 By 16 weeks of age, however, puppies have lymphocyte populations that appear similar to those of healthy adult dogs.^3,4

Although our understanding of how and when the puppy immune system matures is incomplete, much of what we do know is related to the ability of the puppy to respond to vaccination.
Studies have shown that puppies are able to develop a specific immune response to vaccination, similar to the adult response in character but of lower magnitude, within the first 24 hours after birth,⁵ and even when maternal antibodies were also present.⁶ These studies demonstrated that puppies possess both a functional B cell and T cell system.

The condition of the immune responses of young puppies is an important consideration for breeders and veterinarians as they try to protect these animals against infectious disease. Vaccinations are critical to this protection. The purpose of vaccinations is to stimulate humoral (antibody) and/or cellular immune responses to an antigen and generate an appropriate immune memory so that subsequent exposure of the animal to an infectious agent will not result in disease.

Because nursing puppies are protected against disease by the antibodies they receive from their mother and because their immune responses are immature, the vaccination process is usually begun at weaning and continued until the transferred (maternal) antibodies are gone and the puppy's immune responses are more durable. In practice, this translates to administering the first set of vaccinations at 6-9 weeks of age and giving boosters every 3-4 weeks until the animal is about 16 weeks (Table). For animals that have not received colostrum or maternal milk, vaccinations can be given as early as 2-3 weeks of age and continued every 3-4 weeks until they are 16 weeks old. A notable exception is rabies vaccines, which confer longer-lasting immunity and are not given until 12 weeks or as indicated by local law. Owners and breeders should discuss vaccination strategies with their veterinarian to obtain specific recommendations for their animals.

NUTRITION AND IMMUNE FUNCTION

Interactions between nutrition and immunity have been well documented. Diets deficient in protein, energy, minerals, vitamins, and essential fatty acids have long been known to impair immunity.⁷ More recently, supplementation above and beyond the required levels has been reported to be successful in improving health and immune function in a wide range of species. One group of nutrients that has attracted special interest for enhancing immune function is known as antioxidants.

Antioxidants are thought to benefit immune function by their effects on free radicals.⁸ Free radicals (also known as reactive oxygen species) are chemically reactive compounds with an unpaired electron that are produced daily in the body as a result of aerobic (oxidative) metabolism and normal immune system functioning. If free radical accumulation is not controlled, however, it can damage healthy cells. Primary targets for damage of these reactive oxygen species are double bonds, typically found in high concentrations in polyunsaturated fatty acids in cell membranes. Immune cells are especially susceptible to free radical damage because their cell membranes contain high levels of these polyunsaturated fatty acids. The body has systems in place to combat these free radicals, including enzyme systems, endogenous factors, and antioxidants. Antioxidants that can be obtained through the diet include vitamin E, β-carotene, and lutein.

Vitamin E

Vitamin E is a term used to encompass a group of potent, chemically similar antioxidants. One form of vitamin E, a-tocopherol, is most abundant in the body,⁹ has the highest biological activity,^10,11 and reverses vitamin E-deficiency symptoms in humans.¹² In cells, vitamin E contributes to membrane stability, regulates membrane fluidity, and protects cellular components from oxidative damage. Interestingly, immune cells possess a higher vitamin E level than other cells^13,14; as previously mentioned, these cells contain a high level of fatty acids that make them more susceptible to oxidative damage.

Vitamin E supplementation has been reported to increase various measures of immune function in several species, including cats.¹⁵ Studies have demonstrated that adding vitamin E to vaccines as an adjuvant increased the vaccination titer (antibody) response in several species.^16-19 Intramuscular injection of vitamin E prior to vaccination, as well as nutritional supplementation with vitamin E, also resulted in increased titer responses.^20-23 Although supplementation with moderate levels of vitamin E can enhance vaccination responses, studies in humans have shown that over-supplementation with high levels of vitamin E was associated with lower antibody titer response to influenza vaccination.²³ Recent studies in cats conducted by The Iams Company have identified a nutrient range for vitamin E that provides immune system benefits, and this range is thought to be similar for dogs.

β -Carotene

Another group of antioxidants that can improve immune function is the carotenoids. Carotenoids are naturally-occurring plant pigments that have been suggested to play important roles in modulating immunity and health of humans and animals.^24-27 Studies have shown that β-carotene increases the number^28,29 and function of immune cells,^29-33 as well as modulates nonspecific cellular defenses.^29,34

Iams-sponsored studies have revealed that β-carotene is effectively absorbed in dogs.^35,36 In addition, feeding 20 mg of β -carotene per day to adult Beagles increased prevailing antibody levels after 8 weeks and was also associated with an increase in the delayed-type hypersensitivity (DTH) response (a measure of cell-mediated immunity.)³⁵ In a follow-up Iams-sponsored study, dietary supplementation with β-carotene modified immune cell numbers, increased T and B cell proliferation responses, and increased DTH response in dogs.³⁷ Feeding β-carotene also has been shown to improve various measures of immune function in senior, as well as adult, dogs.^35,37

Lutein

Lutein is another carotenoid antioxidant found abundantly in plants and microorganisms, and is a major blood carotenoid in some species (humans, chickens). Like β-carotene, lutein functions as an antioxidant, protecting lipid-containing cell membranes from oxidative damage³⁸ and having a pronounced synergistic protective effect when mixed with other carotenoids.³⁹

In dogs, lutein can be absorbed from the diet and taken up in blood lymphocytes.^40,41 Lutein has been reported to increase cell-mediated immune responses, such as the delayed-type hypersensitivity and lymphocyte proliferation responses in dogs after 6 weeks of supplementation.⁴¹ In addition, humoral immunity, measured as elevated IgG levels, was also enhanced in these dogs with lutein supplementation.

ANTIOXIDANT STUDY

Antioxidant Combination Package

It has been proven that vitamin E, β-carotene, and lutein improve immune function in a variety of species, including dogs. However, their effects in puppies have not been reported. To determine the effect of these antioxidants as a combination package on immune function, 40 puppies were randomized at weaning (6 weeks of age) by litter, body weight, and gender into two treatment groups. The control group was fed a nutritionally complete diet (Eukanuba® Medium Breed Puppy Formula) and the experimental group was fed the same diet with supplemental vitamin E, lutein, and β-carotene. Puppies were fed for four months, with blood samples collected at various time points for lymphocyte proliferation determination. Puppies were vaccinated throughout the feeding period according to standard procedures. At the end of the 4-month feeding period, puppies were vaccinated again (for secondary vaccination response measurement) and blood samples were collected to measure antibody titer response. After the last sample was collected, animals were then inoculated with a novel antigen (sheep red blood cells [sRBC]); for measurement of a primary response to vaccination) and samples were collected at various time points for serum antibody titer analysis.

Results

Results from this study show that puppies fed the antioxidant-supplemented diet had significantly higher lymphocyte proliferation responses to different cell stimuli, testing both T and B cell function (Figures 1-3). The longer the puppies were fed the diets, the greater the difference between the treatment groups. Dietary antioxidant supplementation thus significantly improves both T and B cell-mediated immune function in puppies.

The antioxidant-supplemented puppies also exhibited higher antibody titer responses to distemper, parainfluenza and parvovirus vaccine at the end of the feeding period (Figure 4).

These higher antibody response levels indicate a more successful vaccination response, with concurrent lower likelihood of subsequent infection with these viruses. In addition, the antioxidant-supplemented group generated significantly higher IgM titers to sRBC inoculation (Figure 5). The IgM response is the initial response produced in humoral immunity, and as such confirms that antioxidant-supplemented puppies exhibit significantly enhanced non-specific humoral immune function.

CONCLUSION

Scientific evidence indicates that puppies can benefit from a boost in immune function. Puppies possess a lower level of immune response than adult dogs, contributing to the development of a vulnerable period that puts them at higher risk for disease and death. Previous research in dogs, as well as other species, shows that nutritional supplementation can influence immune function. This recent study shows that in neonatal dogs, dietary supplementation with antioxidants can improve both cell-mediated and humoral immune function and enhance the responses necessary to protect puppies against infectious disease.

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