With the increase in the population of geriatric dogs, understanding how ageing influences immune parameters is important. Similar to human subjects and other animal models, the study of age-related alterations in the immune system aims to develop interventions to modulate and ameliorate these changes, which promotes the well-being and longevity of dogs. This is important because ageing may leave the individual more susceptible to infections and cancers and compromise the quality of their life and lifespan(Reference Castle1).
IgA is the most abundant class of antibody in mucous membranes, where it represents an essential factor in the protection against infectious agents, allergens and foreign proteins. In the intestine, it is mainly produced in Peyer's patches(Reference Nishiyama, Sugimoto and Ikeda2), and IgA secretion is used as an important indicator of the mucosal immunity status(Reference Norris and Gershwin3). Among the factors that can influence IgA secretion, age, breed and diet were studied. When supplementing foods with specific substances, ileal or faecal IgA has been used to assess the dietary immunomodulatory effect(Reference Swanson, Grieshop and Flickinger4, Reference Verlinden, Hesta and Hermans5).
Mucosal immunity development has been studied in some animal species. Studies in human subjects indicate that IgA synthesis does not occur during fetal life(Reference Takemura and Eishi6), and that significant salivary IgA levels are only found after 4–6 weeks of age, and these levels continue to increase up to 18 months of age(Reference Ogra7). Younger children have few IgA+ cells in the intestine, and as they grow older, the number of these cells increase(Reference Rognum, Thrane and Stoltenberg8), which may explain the increase in IgA over time. Most of the studies about IgA production in dogs have used only animals over 2 years of age and measured serum and salivary IgA(Reference Blount, Pritchard and Heaton9, Reference HogenEsch, Thompson and Dunham10). Only one study has evaluated IgA content in nasal secretions of puppies from birth to 6 weeks of age, and observed that IgA concentrations decrease markedly during the first 2 weeks after birth, after which it remained relatively constant(Reference Schafer-Somi, Bar-Schadler and Aurich11).
Studies regarding immunosenescence in dogs have reported an age-related decrease in the proliferative response of blood mononuclear cells to mitogens, a decline in the number of peripheral blood lymphocytes, B-cells and T-cells, and a decreased ratio of CD4+:CD8+cells. Phenotypic alterations are accompanied by functional changes, such as a reduced ability to respond to stimulation by non-specific mitogens, relative change in the balance of T-helper 1 v. T-helper 1 CD4+T-cell activity and a reduced delayed-type hypersensitivity response to mitogens(Reference Day12). These changes in peripheral blood lymphocytes also seem to occur within the intestinal lamina propria of ageing dogs with reduced T-cell numbers and a lower proliferative activity of intestinal cell populations(Reference Kleinschmidt, Meneses and Nolte13). However, this aspect has not been thoroughly studied in dogs. Alterations in IgA secretion related to age are not clear because aged mice and human subjects exhibit either increased or unchanged mucosal IgA concentrations(Reference Fujihashi and McGhee14); in dogs, studies have described that both serum and salivary IgA may increase with age(Reference Blount, Pritchard and Heaton9, Reference HogenEsch, Thompson and Dunham10). Considering this, we compared faecal IgA concentration in puppies, mature and geriatric dogs.
Materials and methods
A total of twenty-four beagle dogs were divided into three age groups of eight dogs each: puppies (5 months old; 5·2 (sem 0·4) kg body weight, four females and four males, from three different litters); mature (4·6 (sem 0·5) years old; 11·36 (sem 0·55) kg body weight, eight males) and senior (10·6 (sem 0·5) years old; 11·49 (sem 0·95) kg body weight, five females and three males). All dogs were considered healthy after clinical and haematological examinations. Dogs were fed standard extruded kibble diets with similar compositions (24 % of crude protein; 14 % of acid-hydrolysed fat; 2 % of crude fibre and 8 % of ash on DM basis; composed by poultry meal, rice, maize, poultry fat, wheat bran, minerals and vitamins, and free of ingredients that may modulate gut immune status.
The study was conducted at the Laboratory of Research on Nutrition and Nutritional Diseases of Dogs and Cats, São Paulo State University, Jaboticabal, Brazil. All procedures were approved by the Ethics and Animal Welfare Committee of the Faculty of Agrarian and Veterinary Sciences, São Paulo State University according to the Brazilian animal protection law (protocol no. 021619/09).
For all dogs, fresh faecal samples (collected no later than 10 min after eliminated) were collected during three consecutive days and immediately frozen ( − 20°C). The samples were then thawed, pooled by dog and submitted for saline extraction, as described previously(Reference Peters, Calvert and Hall15). Extraction buffer (0·01 m-PBS, pH 7·4, 0·5 % Tween (Sigma-Aldrich, Poole, Dorest, UK) and 0·05 % sodium azide) was added to each tube at a ratio of 10 ml of buffer to 1 g (wet weight) of faeces. After homogenisation and centrifugation, the supernatant was transferred to a sterile eppendorf tube containing 20 μl of a protease inhibitor cocktail (Sigma-Aldrich). The samples were centrifuged at 15 000 g for 15 min at 5°C, and the supernatants were transferred to clean eppendorf tubes and stored at − 20°C until analysis.
The quantification of IgA was performed using the ELISA kit for canine IgA determination (Bethyl Laboratories, Montgomery, TX, USA). Optical density was read at 450 nm with a Microplate Reader (MRX TC Plus, Dynex Technology, Chantilly, VA, USA). To calculate the IgA concentration, the optical density of the samples was compared with the optical density of a standard with a known concentration of IgA. The standard canine IgA sample was provided in the kit, and seven dilutions of the standard were made in order to develop a regression curve between optical density and IgA amount. All samples were tested in duplicate, and results are expressed as mean values.
Faecal DM was determined by sample drying at 105°C, and results are expressed as mg IgA per g of dry faeces. All analyses were carried out in duplicate with a CV of less than 5 %.
Statistical analysis
One-way ANOVA was used to compare variables across the three age classes. When a significant difference was identified by ANOVA (P < 0·05), Tukey's test (post hoc) was used to identify differences among groups (P < 0·05). Analysis was performed using the GraphPad Prism software (version 5.0; Graph-Pad Software, Inc., San Diego, CA, USA). All data were found to comply with ANOVA assumptions. Results are expressed as means with their standard errors.
Results
Puppies (0·32 (sem 0·05) mg IgA per g of dry faeces) presented less faecal IgA (P < 0·05) than mature dogs (2·34 (sem 0·44) mg IgA per g of dry faeces). Senior dogs (1·45 (sem 0·41) mg IgA per g of dry faeces) did not differ from either puppies or mature animals (P>0·05). A large deviation from the mean was observed for both mature and senior animals, but puppies had the most homogeneous distribution (Fig. 1).
Fig. 1 Faecal IgA concentrations (mg/g of dry faeces) of puppies (n 8; ■), mature (n 8; ▲) and senior dogs (n 8; ▾). a,b Mean values with unlike letters were significantly different by Tukey's test (P < 0·05).
Discussion
Studies of the effects of ageing on IgA secretion differ between species and authors. Results have shown that ageing can decrease, increase or have no effect on the number of mucosal IgA-producing cells(Reference Fujihashi and McGhee14, Reference Schmucker, Owen and Outenreath16). The results of faecal IgA in puppies reported in the present study agree with the data reported for serum and salivary IgA concentrations(Reference Blount, Pritchard and Heaton9, Reference HogenEsch, Thompson and Dunham10), suggesting that as the dogs mature, an increase in IgA secretion was observed. However, for senior dogs, faecal IgA concentrations exhibited a slight reduction compared with mature dogs. While this result was not significant, it differed from the data observed for salivary IgA by HogenEsch et al. (Reference HogenEsch, Thompson and Dunham10), which found higher salivary IgA concentrations in 12-year-old dogs compared with 3-year-old dogs. Interestingly, senior dogs could be separated in two groups: one group of four animals with >1·5 mg IgA per g of dry faeces and another group of four animals with < 1 mg IgA per g of dry faeces.
Currently, the study of age-related changes in mucosal or secreted IgA concentrations is more applicable than performing extrapolations from the concentrations of serum IgA because serum IgA is not a good predictor of secretory IgA content(Reference Rinkinen, Teppo and Harmoinen17). Moreover, a higher serum IgA level observed in some elderly individuals may reflect a monomeric IgA increase that does not bind to the polymeric Ig receptor and is not transported to the mucosal surface as a secretory IgA(Reference Schmucker, Owen and Outenreath16).
Mucosal IgA measurements have been used for several reasons in veterinary and human medicine. This Ig is considered a marker of severity and progression of some gastrointestinal diseases in both dogs and human subjects(Reference Peters, Calvert and Hall15). In dogs, IgA deficiency has been correlated with chronic enteropathies; especially in German shepherd dogs, the lack of IgA would predispose to small-intestinal bacterial overgrowth(Reference Batt, Barnes and Rutgers18, Reference Littler, Batt and Lloyd19). Other methods can be used to assess mucosal immunity, e.g. immunohistochemistry to characterise leucocyte subsets, antibodies and cytokine-producing cells, or mucosal cell explants to study ex vivo cytokine production. However, these methods are more invasive, requiring gut biopsies, and were not used in the present study.
The reduced faecal IgA in puppies was not reported in the consulted literature. IgA secretion in the intestine is important because it actively binds to micro-organisms, enterotoxins and other antigens to prevent adherence and subsequent penetration into the gut wall(Reference Flickinger, Grieshop and Merchen20). Dogs are considered immunologically mature immediately after birth, but the immune response matures with age, and the humoral response, especially the IgA, seems to develop later(Reference Holsapple, West and Landreth21). Understanding changes in the immune system during growth and the nutritional interventions that may beneficially modulate their function could be important to the development of better diets for young animals.
For old dogs, the reported differences on faecal IgA concentration were not related to any identifiable condition, including age, sex, body weight or body condition, which were similar between the animals. Other studies in old dogs have also found large CV for IgA, 92(Reference Blount, Pritchard and Heaton9) or 121 %(Reference HogenEsch, Thompson and Dunham10), values even greater than those found in the present study (81 %). This probably refers to the fact that dogs, as humans, do not age consistently, and chronological age does not always match physiological age(Reference Burkholder22).
Ageing is associated with a general decline in the intestinal mucosal immune response(Reference Kleinschmidt, Meneses and Nolte13), and our data suggest that some senior dogs also presented reduced faecal IgA concentrations. These alterations are linked to an increased morbidity and mortality due to infectious diseases in elderly individuals(Reference Schmucker, Owen and Outenreath16). Thus, these animals might benefit from studies that enable a better comprehension of these changes and the nutritional interventions that could ameliorate them.
Conclusions
Puppies have reduced faecal IgA concentrations in comparison with mature dogs. Senior dogs presented intermediary faecal IgA concentrations, but some animals presented very low faecal IgA levels.
Acknowledgements
L. Z. and A. C. C. conceived the present study and drafted the manuscript. L. Z. was the main executer, and C. F., M. d. O. S. G., M. M., L. T. and R. S. V. contributed to the execution of the present study. All authors contributed to the critical revision of the manuscript. The present study was supported by Mogiana Alimentos (Guabi), Campinas, Brazil and Biorigin, Lençóis Paulista, SP, Brazil. None of the authors has any conflicts of interest.
