Serum antibody responses to vaccinal antigens in lean and obese geriatric dogs

Abstract

The immune responses in control dogs [1 to 4 years of age, body condition score (BCS): 4 to 5 out of 9] were compared to those of aging dogs (based on breed and body size) either categorized as lean (BCS: 4 to 5 out of 9) or obese (BCS: 8 to 9 out of 9). Of interest were the serum titers to the following common agents found in vaccines, canine parainfluenza virus (CPIV), canine parvovirus (CPV), canine distemper virus (CDV), canine respiratory coronavirus (CRCoV), and Bordetella bronchiseptica. There were no statistical differences in the antibodies to CPIV, B. bronchispetica, and CRCoV, among the age/weight categories, nor among the age/weight categories and the time, in days, between the date of sample collection and the date of the last recorded vaccination for CPIV, B. bronchiseptica, CPV, and CDV. For CPV, the control dogs had significantly (P < 0.002) higher serum neutralization (SN) titers than the lean geriatric dogs and the obese geriatric dogs. For CDV SN titers, the only statistically significant (P = 0.01) difference was that the control dogs had higher SN titers than the lean geriatric dogs.

Introduction

One of the often under-acknowledged benefits of routine vaccination and control of infectious disease in human and veterinary medicine is not dying early in life from vaccine-preventable diseases. An attendant downside of age is senescence, or decay of bodily function. Immunosenescence is one of the least well-examined and understood areas of veterinary geriatric medicine. Although data are somewhat conflicting depending on how various parameters are measured, a consensus, based on studies conducted primarily in Labrador retrievers, is that there is an age-related decline in absolute numbers of lymphocytes, decreased CD4/CD8 T-cell ratio, and decreased proliferative responses to mitogens comparing “old” versus “young” dogs; these are changes that probably have more of an impact on cell-mediated responses than antibody responses (1,2). Another taken-for-granted feature of modern life in the developed world is access to a high plane of nutrition, or at least access to a lot of food, and its resultant downside, obesity, with its attendant disease-promoting effects in both humans and their pets (3). In contrast, available data indicate that diet (caloric) restriction can prolong the lifespan of dogs, an effect that was associated with averting some of the age-related changes in the immune system (3). The objective of this cross-sectional study was to add to the small database on immune responses in aging dogs, focusing on serological responses to antigens in commonly used vaccines in “lean” versus “obese” geriatric client-owned dogs.

Materials and methods

Twenty-eight lean geriatric (LG), 28 obese geriatric (OG) dogs, and 37 lean control dogs were subjects in a study that examined the effects of aging and weight on respiratory physiology that was conducted in accordance with the guidelines of the Canadian Council on Animal care. Classification of “geriatric” was based on whether each dog fell within or exceeded the geriatric age range for their body weight (4). This was done to account for the fact that large and giant breeds of dogs on average have shorter lifespans compared to small dogs (5). Geriatric dogs were ≥ 11 y of age and ≤ 10 kg, ≥ 10 y and 10 to 22 kg, ≥ 8 y and 22.1 to 40 kg, or ≥ 7.5 y and > 40 kg. For the purposes of analysis small dogs were defined as those weighing < 15 kg, while medium and large breed dogs were defined as weighing 15 to 30 kg, and > 30 kg, respectively. Geriatric dogs were divided into lean geriatric [body condition score (BCS): 4 to 5 out of 9] and obese geriatric (BCS: 8 to 9 out of 9) groups (6). For comparison, a group of client-owned, young, adult control dogs between 1 to 4 y of age that had a BCS of 4 or 5 out of 9 were recruited. An attempt was made to match the groups for gender and breed-size distribution; however, due to subject availability there was a slight overrepresentation of small-sized female dogs in both the lean geriatric and obese geriatric groups and a slight overrepresentation of medium-sized dogs in the control group.

Canine parainfluenza virus (CPIV)-specific antibodies were measured using an indirect enzyme-linked immunosorbent assay (ELISA) as previously reported (7). Briefly, 96-well plates were coated overnight with CPIV antigen or cellular extract control antigen. Non-specific binding sites were blocked with 0.2% gelatin. Control and test serum samples were diluted at 1:50 in phosphate-buffered saline (PBS) containing 0.05% Tween 20 and 0.2% gelatin and tested in duplicate wells of CPIV and CPIV control antigens. CPIV-specific IgG was detected using an HRP sheep anti-dog IgG conjugate at 1:5000. Antibodies to CRCoV were similarly measured using an indirect ELISA as previously described (7). Bordetella bronchiseptica-specific antibodies were detected by an indirect ELISA as previously reported (7). Briefly, 96-well plates were coated overnight with sonicated B. bronchiseptica antigen. Non-specific binding sites were blocked with 1% gelatin. Control and test serum samples were diluted at 1:50 in PBS containing 0.05% Tween 20 and 0.2% gelatin and tested in duplicate wells of B. bronchiseptica antigen. Bordetella bronchiseptica-specific IgG was detected using HRP sheep anti-dog IgG conjugate at 1:6000. Canine distemper virus (CDV)-specific antibodies were measured by endpoint titration in a standard previously described virus microneutralization assay (8) using a concentration of 100 tissue culture infective dose (TCID₅₀) CDV stock virus. Canine parvovirus (CPV)-specific antibodies were measured in a standard previously described hemagglutination inhibition test (HAI) (9). Five serial 2-fold dilutions of dog sera, beginning at 1:80, were tested with 8 HA units of CPV. Endpoint titers were determined after the addition of 0.5% pig erythrocytes. A titer of ≥ 1:32 in the CDV seroneutralization assay and a titer of ≥ 1:80 in the CPV HAI test have been considered indicative of protection (9–11), although there are virtually no population-based case-control studies to validate these “cutoff ” values.

For all ELISA tests optical density (OD) values for test samples were expressed as ELISA units that were calculated as follows:

$$100\times [\text{sample\hspace{0.17em}OD}-\text{blank\hspace{0.17em}}(\text{buffer\hspace{0.17em}only\hspace{0.17em}OD})]/[\text{positive\hspace{0.17em}standard\hspace{0.17em}OD}-\text{blank\hspace{0.17em}}(\text{buffer\hspace{0.17em}only})\hspace{0.17em}\text{OD}]$$

Statistical analyses were performed using commercially available software programs (SPSS 20.0 for Windows; SPSS, Chicago, Illinois, USA). Descriptive analyses were completed and variables were recoded as necessary for statistical modeling.

Neither the immune response data nor the time between sample collection and previous vaccination data were normally distributed; therefore, non-parametric Kruskal-Wallis tests were used for all analyses. Pairwise comparisons were made for each age/weight category, i.e., control, lean geriatric and lean obese and the test variable of interest. The test variable for CPIV, B. bronchispetica and CRCoV was the respective IgG ELISA units for each organism and for CDV and CPV it was the serum neutralization (SN) titers and HAI titers, respectively. If dogs were not vaccinated for any of CPIV, B. bronchiseptica, CDV, or CPV they were excluded from any analysis observing that organism. All dogs were included in the CRCoV analysis since there is no vaccine for CRCoV at this time. For the age/weight variable the group identified as the control group (BCS: 4 or 5 out of 9, age between 1 and 4 y) was the primary reference group in all analyses, but comparisons between lean and obese geriatric dogs were also performed. A Bonferroni correction was applied due to multiple pairwise comparisons and therefore results were considered statistically significant when P ≤ 0.02.

Results

Canine parainfluenza virus (CPIV)

Out of the 93 enrolled dogs, 66 had vaccination dates for CPIV recorded. These included 29 controls, 21 lean geriatric dogs, and 16 obese geriatric dogs. CPIV IgG ELISA titers were not statistically (P = 0.16) different between the control dogs (median: 27, range: 2 to 101) and the lean geriatric dogs (median: 28, range: 3 to 142) or between the control dogs and the obese geriatric dogs (P = 0.78, median: 18, range: 5 to 106). No statistical difference (P = 0.49) was noted between the control dogs (median: 279, range: 0 to 1458) and the lean geriatric dogs (median: 334, range: 13 to 1922) or between the control dogs and the obese geriatric dogs (P = 0.23, median: 158, range: 0 to 2008) for time in days between sample collection and last vaccination. There was also no statistically significant difference between CPIV IgG ELISA titers (P = 0.21) or time between sample collection and vaccination in days (P = 0.08) between the lean and obese geriatric dogs.

Bordetella bronchiseptica

Out of the 93 enrolled dogs, 30 had vaccination dates for B. bronchiseptica recorded. These included 17 controls, 10 lean geriatric and 3 obese geriatric dogs. Bordetella bronchiseptica IgG ELISA titers were not statistically (P = 0.4) different between the control dogs (median: 80, range: 15 to 104) and the lean geriatric dogs (median: 47, range: 27 to 110). No statistical difference (P = 0.28) was noted between the controls (median: 249, range: 0 to 772) and the lean geriatric dogs (median: 331, range: 60 to 966) for time between sample collection and last vaccination. Bordetella bronchiseptica IgG ELISA titers were also not statistically (P = 0.67) different between the control dogs (median: 80, range: 15 to 104) and the obese geriatric dogs (median: 59, range: 43 to 103). No statistical difference (P = 0.21) was noted between the control dogs (median: 249, range: 0 to 772) and the obese geriatric dogs (median: 9, range: 4 to 313) for time in days between sample collection and last vaccination. There was also no statistically significant difference between B. bronchiseptica IgG ELISA titers (P = 0.31) or time between sample collection and vaccination in days (P = 0.12) between the lean and obese geriatric groups.

Canine respiratory coronavirus (CRCoV)

All 93 enrolled dogs, including 37 controls, 28 lean geriatric and 28 obese geriatric dogs, were included in this analysis. CRCoV IgG ELISA titers were not statistically (P = 0.05) different between the control dogs (median: 5, range: 0 to 36) and the lean geriatric dogs (median: 11, range: 0 to 34) or between the control dogs and the obese geriatric dogs (P = 0.60, median: 13, range: 0 to 178). There was also no statistically significant difference between CRCoV IgG ELISA titers (P = 0.54) between in the lean and obese geriatric groups of dogs.

Canine parvovirus (CPV)

Out of the 93 enrolled dogs, 66 had vaccination dates for CPV recorded. These included 29 controls, 21 lean geriatric dogs, and 16 obese geriatric dogs. Including all dogs for which sera were available, 29/30 controls, 26/28 lean geriatrics, and 28/28 obese geriatrics had CPV HAI titers ≥ 80. Canine parvovirus HAI titers were statistically (P = 0.002) higher in the controls (median: 1920, range: 60 to 1920) than in the lean geriatric group (median: 480, range: 60 to 1920). No statistical difference (P = 0.55) was noted between the control dogs (median: 279, range: 0 to 1458) and the lean geriatric dogs (median: 334, range: 13 to 1922) for time in days between sample collection and last vaccination. Canine parvovirus HAI titers were also statistically (P = 0.002) higher in the control dogs (median: 1920, range: 60 to 1920) compared to the obese geriatric dogs (median: 480, range: 60 to 1920) but no statistical difference (P = 0.10) was noted between the control dogs (median: 279, range: 0 to 1458) and the obese geriatric dogs (median: 158, range: 0 to 2008) for time in days between sample collection and last vaccination. There was also no statistically significant difference between CPV SN titers (P = 0.58) or time between sample collection and vaccination in days (P = 0.04) between in the lean and obese geriatric groups of dogs.

Canine distemper virus (CDV)

Out of the 93 enrolled dogs, 66 had vaccination dates for CDV recorded. These included 29 controls, 21 lean geriatric dogs, and 16 obese geriatric dogs. Including all dogs for which sera were available, 29/30 controls, 22/28 lean geriatrics, and 26/28 obese geriatrics, had CDV SN titers > 32. Canine distemper virus SN titers were statistically (P = 0.01) higher in the control dogs (median: 324, range: 18 to 2916) than in the lean geriatric dogs (median: 108, range: 12 to 972) but no statistical difference (P = 0.55) was noted between the control dogs (median: 279, range: 0 to 1458) and the lean geriatric dog (median: 334, range: 13 to 1922) for time in days between sample collection and last vaccination. Canine distemper virus SN titers were not statistically (P = 0.16) different between the control dogs (median = 324, range: 18 to 2916) and the obese geriatric dogs (median: 108, range: 12 to 2916). No statistical difference (P = 0.10) was noted between the control dogs (median: 279, range: 0 to 1458) and the obese geriatric dogs (median: 158, range: 0 to 2008) for time between sample collection and last vaccination in days. There was also no statistically significant difference between CDV titers (P = 0.36) or time in days between sample collection and vaccination in days (P = 0.04) between the lean and obese geriatric groups.

Discussion

As with many studies in small animal veterinary medicine, including previous studies of immunosenscence, a major limitation of this study is the relatively small number of experimental subjects and sufficient intragroup and interbreed variation to preclude the detection of differences among groups. Relatedly, interbreed differences in the effects of aging and relative body weights may have also affected the outcome in this cross-sectional study; different life expectancies and body types for different breeds render the definition of “old” and “obese” variable, respectively. In a previous study of more than 900 adult (2- to 6-year-old) client-owned dogs that had been vaccinated approximately 12 mo previously (9), anti-CPV antibody titers were significantly higher in “super light” (< 5 kg) versus “medium” (10 to 19.9 kg) and “heavy” (> 20 kg) dogs, and also significantly higher in “light” (5 to 9.9 kg) versus heavy dogs; whereas, anti-CDV titers were significantly higher in super light, light, and medium dogs compared to heavy dogs. However, the relation of these findings to BCS (or breed) was not examined. Life-long dietary restriction (25% of “normal” diet) has been shown to have life-prolonging effects on Labrador retrievers maintained under laboratory conditions, that can be associated with changes in various immunological, primarily cellular, parameters; however, the impact of restricted diet on serological responses to routine vaccination were not examined in those studies (3).

It has been reported that the percentage of CD45R+, presumably naïve, CD4 T-cells is markedly lower, while the percentage of presumed memory CD4 and CD8 T-cells expressing CD44 is increased in older dogs (2). As well, CD21+ B-cells and plasma concentrations of IgM and IgG are not different in young versus old dogs (2). Together these immunological phenomena could account for the likelihood that primary antibody responses may be reduced in older dogs, as has been documented in the case of rabies vaccines (12), but responses to “recall” antigens do not diminish significantly with age (2). This latter possibility may in part explain the lack of significant differences in responses to CPIV and B. bronchiseptica (and CRCoV) among these groups because the observed antibody responses were not likely the result of primary exposure to the antigens. In contrast, the observed differences in responses to CDV and CPV in young adults versus some geriatric dogs could be consistent with a senescence in antibody responses to vaccination.

The failure to detect an association between time since last vaccination and differences in antibody titers among all the dogs could, in part, indicate an important role for natural exposure, in addition to number and frequency of vaccinations, in maintaining serum antibody concentrations, especially to endemic respiratory pathogens. This effect could vary with the “lifestyle” of the pathogen (extensiveness of replication in vivo and resultant effect on immunological memory, transmissibility, stability in the environment) or the lifestyle of the dog (frequency of exposure to other dogs). The incidence of natural exposure is difficult to measure (7); however, in 1 study vaccinated dogs with a “high-risk” lifestyle (more frequent exposure to other dogs) maintained higher titers to the acutely infecting CPIV than did dogs with less frequent exposure (13). Similarly, the generally lower concentrations of antibodies to CRCoV (for which there is currently no vaccine) in these household dogs compared to the common seroconversion in dogs in a shelter environment reflects the effect of environmental co-factors (7). These lifestyle effects on exposure are likely to occur in the case of responses to B. bronchiseptica as well (7,14,15).

Based on these data, and data from other, primarily rodent, models, it is likely that both age and nutritional status affect the canine response to infectious diseases, including responses to commonly used vaccines. Therefore, further investigation of this complex subject in household dogs is certainly warranted, although such studies (8,9) are logistically challenging, especially with the extant limited resources available in small animal medicine. CVJ