Many equids are vaccinated against equine influenza annually as it causes a highly contagious respiratory infection. In horses, both age and adiposity contribute to increased levels of inflammatory markers, which could affect the response to vaccination. In humans, a chronic inflammatory state associated with obesity can impair response to vaccination or infection (Sheridan et al., 2012). The objective of this study was to determine which factors would influence the response to the equine influenza vaccination.
Material and methods
Surplus to diagnostic requirement serum samples were obtained from 55 donkeys that had recently received a booster dose of a commercially-available inactivated virus equine influenza vaccine. Antibody levels against a component strain of the vaccine (influenza A/equine/Newmarket/2/93 [H3N8]) were measured using the single radial haemolysis assay. An equine-specific ELISA (R&D Systems) was used to measure serum tumour necrosis factor alpha (TNF-α). High molecular weight (HMW) adiponectin was measured using a human ELISA kit (Millipore) previously shown to be relevant for the horse (Woolridge et al., 2012). Non-esterified fatty acid (NEFA) levels were measured using a kit from Randox with some modifications to the manufacturer’s instructions. Additional data including age, weight, body condition score (BCS), and total cholesterol and triglyceride serum levels were kindly provided by the Donkey Sanctuary. Univariate analysis was conducted using Pearson correlation for normally distributed variables and Spearman’s rank correlation for variables that were not normally distributed. To evaluate the effect of gender, comparisons were made using a t-test for normally-distributed variables or a Mann-Whitney test for variables not normally distributed. Significant differences were determined at the level of p<0.05.
In this study, the factor with the greatest influence on the response to vaccination was gender, with females having significantly higher antibody levels than males (Table 1). In addition, the mean age of the female donkeys was significantly greater than that of the male donkeys. There was no correlation between BCS or weight and antibody levels, but NEFA levels were negatively correlated with antibody levels (p=0.044). Associations between NEFA and age, weight and days since vaccination also reached statistical significance. Triglyceride levels were also positively correlated with days since vaccination although antibody levels were not. Positive correlations were seen between serum adiponectin and age, and triglyceride and cholesterol levels.
Table 1. Influence of gender on variables measured
Variable Mean ±SD (Range) p-value
Males (n=28) Females (n=27)
Age (years) 19.2 ±11.8 25.0 ±6.4 .028
Antibody (area of lysis - mm2) 178.9 ±39.3 208.9 ±42.3 .009
Negative correlations were expected between antibody levels and both age and BCS, but were not seen. This may have been confounded by the narrow distribution of BCS in the study population (the majority had a BCS score of 2.5–3.5 on a scale of 1–5) and the overwhelming influence of gender with female donkeys having a higher antibody response despite a greater mean age. It has been demonstrated in human subjects that influenza vaccination can cause alterations to the lipid profile (Tsai et al., 2005). The correlation between NEFA and serum antibody levels warrants further investigation as does the finding that gender has a significant impact on response to equine influenza vaccination in donkeys.
Acknowledgments The authors gratefully acknowledge The Donkey Sanctuary for providing surplus to diagnostic requirement serum samples and data, Waltham Centre for Pet Nutrition for funding the study and Dr Marnie Brennan for assistance with the statistical analysis.
P. A. Sheridan, H. A. Paich, J. Handy, E. A. Karlsson, M. G. Hudgens, A. B. Sammon, L. A. Holland, S. Weir, T. L. Noah, M. A. Beck. 2012. Obesity is associated with impaired immune response to influenza vaccination in humans. International Journal of Obesity, 36, 1072-1077.
M. Y. Tsai, N. Q. Hanson, R. J. Straka, T. R. Hoke, J. M. Ordovas, J. M. Peacock, V. L. Arends, D. K. Arnett. 2005. Effect of influenza vaccine on markers of inflammation and lipid profile. The Journal of Laboratory and Clinical Medicine, 145, 323-327.
A. A. Wooldridge, H. G. Edwards, E. P. Plaisance, R. Applegate, D. R. Taylor, J. Taintor, Q. Zhong, R. L. Judd. 2012. Evaluation of high-molecular weight adiponectin in horses. American Journal of Veterinary Research, 73, 1230-40.