J. Comp. Path. 2010, Vol.

142, S85eS90

Available online at www.sciencedirect.com

www.elsevier.com/locate/jcpa

The Effect of Age on the Immune Response of

Horses to Vaccination
T. L. Muirhead*,, J. T. McClure*,, J. J. Wichtel*,, H. Stryhn*,,
R. J. F. Markham*,, D. McFarlane and D. P. Lunnx
* Department of Health Management, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, Prince
Edward Island, Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward
Island, Charlottetown, Prince Edward Island, Canada, Center for Veterinary Health Sciences, Oklahoma State University,
Stillwater, OK and x Department of Clinical Sciences, Colorado State University, Fort Collins, CO, USA

Summary
Few studies have investigated immunosenescence in the horse, but it is accepted that the primary and secondary (anamnestic) immune responses may differ between aged and younger horses. The aim of the present study
was to determine whether aged horses have a protective immune response post-vaccination. Thirty-four aged
healthy horses ($20 years) and 29 younger adult horses (4e12 years) of various breeds were vaccinated with
commercially produced killed rabies and inuenza vaccines. Rabies serum neutralizing antibody titres and
equine inuenza virus specic antibody subclasses (immunoglobulin IgGa and IgGb) and single radial
haemolysis titres were determined. Healthy aged horses mounted a primary immune response to rabies vaccine
that was similar to that of younger adult horses. However, aged horses had a signicantly reduced anamnestic
response to inuenza vaccination in comparison with the younger adult horses, even though the pre-vaccination antibody titres of aged horses were higher. Rabies antibody titres in both groups declined signicantly by 6
months post-vaccination. Serum concentrations of selenium (Se) and vitamin E were measured to test for potential confounding effects. Signicant numbers of horses had suboptimal serum Se concentrations, but Se status had no signicant impact on antibody production after vaccination.
2009 Elsevier Ltd. All rights reserved.
Keywords: geriatric; horse; immunosenescence; vaccination

Introduction
Immunosenescence refers to alterations in the
immune system associated with ageing that may
subsequently lead to an increased susceptibility to
infectious, autoimmune and neoplastic diseases. It is
estimated that geriatric horses (i.e. $20 years of
age) account for approximately 15% of the equine
population in North America (Malinowski et al.,
1997). There is an ever-increasing demand in private
practices and referral centres to provide high quality
care for these animals. These older horses experience
many changes that may be related to their level of
Correspondence to: T. L. Muirhead (e-mail: tmuirhead@upei.ca).
0021-9975/$ - see front matter
doi:10.1016/j.jcpa.2009.10.010

nutrition, their environment and/or an underlying

subclinical disease. Thus, it may be difcult to separate normal age-related changes from changes related
to the presence of disease. Many aged horses experience declining body condition, muscle tone and general well-being. It is not known whether these
changes contribute to decreased immune function or
are the result of declining immune function (Horohov
et al., 1999). A better understanding of immunosenescence in ageing horses is needed. There is evidence for
an age-related alteration in the ability of horses to
respond to antigens, as has been previously described
in man and mice.
Few studies have examined the effects of age on
the innate, cell-mediated and humoral components
2009 Elsevier Ltd. All rights reserved.

S86

T.L. Muirhead et al.

of the equine immune system. The innate immune

system of the horse appears to remain intact with
age. Horohov et al. (1999) investigated the effects of
exercise on the immune response of young and old
horses and demonstrated that aged horses (mean
age 25 years) had lymphokine-activated killer cell
activity equivalent to that of younger animals
(mean age 7.5 years). This nding suggests that these
killer cells are able to provide an adequate rst line
of defence when antigen is encountered. Circulating
monocyte and granulocyte counts in young and aged
horses are also reported to be similar (Guirnalda
et al., 2001). These ndings are in concordance
with those of human studies.
Horses, like other species, experience thymic involution and have similar modications in circulating
T-cell subpopulations (Perryman et al., 1988). T cells
from elderly horses show decreased proliferation on
exposure to a mitogen when compared with those
from younger horses (Horohov et al., 1999, 2002;
Adams et al., 2008). In one study, T-cell proliferation
remained depressed after supplementation with
recombinant interleukin (IL)-2, suggesting that
decreased proliferation cannot be solely attributed
to decreased expression of IL-2. This suggests that
an age-associated alteration in the T-cell IL-2 signalling pathway occurs, probably involving the IL-2 receptor (Horohov et al., 2002). McFarlane et al. (2001)
investigated age-related quantitative alterations in
lymphocyte subsets and found that healthy geriatric
horses had decreased absolute total peripheral blood
lymphocyte counts, including decreased CD4+ and
CD8+ T cells, as compared with young horses. There
was also a signicant decline in the percentage of
CD8+ cells, resulting in an increased CD4+ to
CD8+ cell ratio in the aged horses. Increased
CD4+ to CD8+ cell ratio has been associated with
non-specic inammation or immunodeciency in
other species (McFarlane et al., 2001). Another study
reported an increase in the expression of class II molecules of the major histocompatibility complex
(MHC) on the surface of T lymphocytes in adult
horses as compared with neonatal foals, suggesting
a shift toward a larger memory T-cell population
with advancing age (Lunn et al., 1993).
Studies of aged horses have also documented
changes in the humoral immune response. Aged
horses have a signicant decline in numbers of peripheral B lymphocytes as compared with younger horses.
Measurements of serum immunoglobulin IgG,
IgG(T), IgM and IgA, however, were not signicantly different between the age groups (McFarlane
et al., 2001). Two studies have investigated the specic
antibody response of aged horses to vaccines and
pathogen challenge and have shown a blunted

response when compared with younger horses (Goto

et al., 1993; Horohov et al., 1999).
The aim of the present study was to evaluate the effect of age on the specic systemic antibody response
following rabies and inuenza vaccination in 29
healthy adult (4e12 years) and 34 healthy aged
($20 years) horses of various breeds. Horses involved
in this study were from Prince Edward Island,
Canada, which is free of rabies; thus rabies vaccination is not routinely performed. Enrolled horses had
no history of previous rabies vaccination, so the use
of this vaccine was a means of assessing the primary
immune response. Since inuenza is endemic in the
horse population, inuenza vaccination was a means
of assessing an anamnestic (secondary) immune response. Other ancillary tests such as a physical examination, complete blood count, serum biochemical
panel, serum selenium (Se) and vitamin E concentrations, and serum thyroid hormones were also performed in order to determine the health status of
each horse. Serum Se concentrations were measured
due to the fact that Se deciency has been documented in the Atlantic provinces of Canada and
known to inversely affect the immune system. Vitamin E also is known to have an effect on the immune
response and it is found in adequate amounts in pasture at early stages of growth; however, this vitamin
deteriorates over time when these grasses mature or
are stored as hay or silage. Thus vitamin E intake in
forage-fed livestock is likely to uctuate throughout
the year, being lowest during the winter months
when livestock are fed conserved forages only. Both essential dietary nutrients were measured to determine
whether they had an effect on the specic rabies and
inuenza titres from each age group.
All horses were initially vaccinated with both killed
rabies and inuenza vaccines. Horses in each age
group then received either rabies or inuenza booster
vaccine 4 weeks after the initial vaccination based on
an initial random coin toss, followed by alternating
allocation. Serum samples were taken at 0, 4, 8 and
24 weeks for evaluation of the immune response and
Se status. Rabies serum neutralizing antibody titres
and equine inuenza virus specic antibody of different classes and subclasses (IgGa, IgGb, IgG[T] and
IgA), as well as single radial haemolysis (SRH) titres,
were determined. General linear models were generated for the change in titres from baseline levels. Factors tested in the model included: age, breed, initial
inuenza antibody titre, booster, sex, initial Se and
vitamin E status, serum T3, serum T4 and a-melanocyte stimulating hormone (a-MSH) concentrations.
The following factors were signicant and retained
in the nal models: age, initial inuenza antibody
titres and booster (Muirhead et al., 2008).

Age and Equine Vaccination

Secondary Immune Response

Response to inuenza vaccination was used to evaluate the secondary or anamnestic immune response.
All horses had evidence of previous exposure to the
inuenza antigen as evidenced by detectable serum
IgGa, IgGb and SRH anti-inuenza titres prior to
vaccination. Initial IgGa and IgGb titres were significantly higher in aged horses compared with the
younger animals (P 0.004 and P 0.0027, respectively; Muirhead et al., 2008). This nding was consistent with the results of another study, which
investigated the efcacy of commercial inuenza vaccines in horses and also demonstrated that younger
horses had lower antibody titres in comparison with
older animals (Morley et al., 1999). These data suggest that older horses may be better protected against
infection. High initial anti-inuenza titres in the older
horses may reect a greater number of lifetime exposures to inuenza virus. Alternatively, this observation may be a consequence of an age-related switch
to a predominantly Th2 response. As people age,
the Th1 cell-mediated immune response declines
and the Th2 humoral response predominates (Haynes
et al., 1999, 2004; Haynes, 2005).
The younger horses had a signicantly greater increase in IgGa and IgGb post-vaccination compared
with aged horses. Four weeks post-initial vaccination
there was a signicant anti-inuenza antibody
response to the initial vaccination in animals of both
age groups for IgGa and IgGb (P # 0.001). The
younger horses had a signicantly greater increase
in anti-inuenza IgGa titres for the entire sampling
period (P # 0.001) compared with the aged horses.
The impact of initial titre was less pronounced for
older horses compared with the younger horses.
Similarly, the increase in anti-inuenza IgGb titres
post-vaccination in the younger animals was greater
compared with that of the aged horses at 4
(P 0.079), 8 (P 0.041) and 24 weeks (P 0.010)
post-initial vaccination. One possible explanation
for the more pronounced change in inuenza antibody titres in younger horses is that these animals
started with lower pre-vaccination titres, which allowed for a greater antibody response post-vaccination. However, for IgGa and IgGb, both age and
initial titre remained signicant predictors of change
in magnitude of inuenza antibody titre response,
even with the pre-vaccination age group interaction
included in the model (Muirhead et al., 2008). These
two factors were each signicant predictors of antibody response. While advanced age favours a humoral
response, humoral function does decline, resulting in
a dampened antibody response when challenged
(Haynes et al., 1999, 2004; Haynes, 2005). Similarly,

S87

Horohov et al. (1999) reported that aged horses had

a 10-fold lower anti-inuenza antibody response
than younger animals. This is consistent with the
blunted anti-inuenza antibody response observed
in the aged horses in the present study.
The SRH titres post-vaccination followed a similar
trend to IgGa and IgGb, but this did not reach statistical signicance (Muirhead et al., 2008). For equine
inuenza, SRH titres correlate well with antibody
concentrations specic for virus neutralization and
protection (Mumford et al., 1983, 1988, 1994; Morley
et al., 1995). The SRH titre corresponding to protection varies among studies. One study found that a previral challenge SRH level of $74 mm2 correlated
with protection against disease and $100 mm2 significantly reduced the time of virus shedding (Mumford
et al., 1988). In a similar study, all of the ponies that
generated SRH titres $154 mm2 following vaccination were protected against subsequent infection (no
virus cultured), while ponies that generated titres
$85 mm2 were protected against clinical disease
(Mumford et al., 1994). Mumford and Wood (1992)
showed a 90% protection against inuenza infection
at a SRH titre of $165 mm2. In the present study
the younger horses had a pre-vaccination mean
SRH titre of 31 mm2, while the aged horses had
a mean SRH titre of 89 mm2. At 24 weeks post-vaccination, 82% of the horses in our study maintained
SRH titres that would be considered protective of
clinical disease (>85 mm2). Thus, the age group differences noted in this study in the response to inuenza vaccination may not be clinically signicant.

Primary Immune Response

A primary immune response occurs when an animal is
exposed to an antigen for the rst time. This response
involves recruitment of nave lymphocytes, which become activated, proliferate and nally create memory
T and B cells which have a role in providing protection from subsequent challenges by the same antigen
(Janeway et al., 2001). Studies in aged man and mice
reveal a shift in lymphocyte populations, with
a greater number of memory cells and fewer nave
cells in the aged subjects. This potentially could result
in a reduced capacity of the elderly to respond to
a novel pathogen compared with younger subjects
(Rocha et al., 1992; Cossarizza et al., 1997; Ginaldi
et al., 1999; Castle, 2000). Impaired primary immune
response has also been reported in aged horses
(Horohov et al., 1999, 2002). In aged horses it has
been shown that there is a shift in lymphocyte subsets
and a decrease in peripheral blood lymphocytes
including a decrease in CD4+ and CD8+ cells that
could alter immune function (McFarlane et al.,

T.L. Muirhead et al.

Serum Se and the Equine Immune Response

As previously stated, serum concentrations of Se and
vitamin E were measured in all horses in the present
study as a screening test. Studies investigating the
effects of Se on antibody production have shown minimal effects on the magnitude of the total or specic
antibody responses in a variety of domestic animals
(Baalsrud and Overnes, 1986; Knight and Tyznik,
1990; Turner and Finch, 1991; Finch and Turner,
1996). One study in ponies illustrated that Se supplementation signicantly increased haemagglutination
titres as well as IgG concentrations (Knight and

100
90

Proportion of horses (%)

2001). Assessment of primary immune response has

traditionally been performed by measuring antibody
production following vaccination using a novel antigen such as sheep red blood cells or bacteriophage l
(Bandilla et al., 1969; Peacock et al., 1973). In the present study, antibody production following exposure to
rabies antigen was used to measure primary immune
response. This was possible because the study was performed on Prince Edward Island, which is rabies free,
thus most horses are not vaccinated against rabies.
Younger horses tended to have higher median antirabies antibody titres at all time points compared with
their aged counterparts, but this did not reach statistical signicance. The anti-rabies antibody titres generated by both the younger and aged horses that
received a single dose of rabies vaccine were generally
low. At 8 weeks post-vaccination, 82% of the aged
horses and 50% of the younger horses had anti-rabies
antibody titres of <0.5 IU/ml (P 0.08). A titre of
<0.5 IU/ml in rabies vaccinated people is an indication for a booster vaccine (Health Canada, 2007).
Eighty-nine percent of horses receiving a single dose
of vaccine had anti-rabies antibody titres below
0.5 IU/ml (Fig. 1). In comparison, all but one horse
receiving a rabies booster vaccine had titres
>0.5 IU/ml at 8 weeks post-initial vaccination, although 28% of these horses had serum antibody titres
below this level at 24 weeks (Fig. 2). Currently, there
is no recommended protective titre established for
horses. The current labelled protocol for the commercial rabies vaccine used in our study is a single initial
vaccination followed by annual boosters. This recommendation was established based on an experimental
challenge in horses, but unfortunately, serum antibody titres were not monitored during this challenge
trial (personal communication, Dr. N. Plourde,
Merial Canada Inc.). The ndings of the present
study suggest that aged horses may have greater difculty maintaining an adequate antibody titre after
a single dose of rabies vaccine compared with younger
horses (Muirhead et al., 2008).

Protective titre
Unprotective titre

80
70
60
50
40
30
20
10
0
Young 8 week
(n =12)

Aged 8 week Young 24 week Aged 24 week

(n =15)
(n =12)
(n =12)

Fig. 1. Proportion (%) of horses at 8 and 24 weeks after a single

rabies vaccination that have a potentially non-protective
rabies serum antibody titre (<0.5 IU/ml).

Tyznik, 1990). Another study concluded that horses

that received vitamin E and Se had signicantly
greater antibody response to equine inuenza virus
when compared with animals that did not (Baalsrud
and Overnes, 1986). In the present study, Se and
vitamin E status had no signicant effect on the
immune response in either group of horses. However,
approximately 89% of the horses in this study had Se
concentrations below adequate levels (<0.140 ppm;
Muirhead et al., 2008). This high proportion of horses
with an inadequate Se status in both age groups may
have reduced our ability to identify an effect of Se
deciency on the immune response. It is also possible
that the inadequate selenium levels contributed to the
poor primary immune response to a single dose of
rabies vaccine.

100

Protective titre
Unprotective titre

90
80

Proportion of horses (%)

S88

70
60
50
40
30
20
10
0
Young 8 week Aged 8 week
(n =12)
(n =15)

Young 24 week Aged 24 week

(n =12)
(n =12)

Fig. 2. Proportion (%) of horses at 8 and 24 weeks after initial

rabies vaccination with a potentially non-protective rabies
serum antibody titre (<0.5 IU/ml). These animals also
received a rabies booster vaccination 4 weeks after the
initial vaccination.

Age and Equine Vaccination

Conclusions
The results of the present study suggest that immunosenescence can play a role in the ability of aged horses
to respond to vaccination. The aged horses had
a blunted anamnestic response to inuenza vaccination, although the primary immune response to the
rabies vaccination in the aged horses was similar to
that in their younger counterparts. The clinical significance of these ndings warrants further research.

Conflict of Interest
The rst author was an invited speaker at the Merial
European Comparative Vaccinology Symposium
and received travel expenses and an honorarium for
this presentation.

Acknowledgments
Funding for this project was provided though the
generosity of the Sir James Dunn Animal Welfare
Center.

References
Adams AA, Breathnach CC, Katepalli MP, Kohler K,
Horohov DW (2008) Advanced age in horses affects
divisional history of T cells and inammatory cytokine
production. Mechanisms of Aging and Development, 129,
656e664.
Baalsrud KJ, Overnes G (1986) Inuence of vitamin E and
selenium supplement on antibody production in horses.
Equine Veterinary Journal, 18, 472e474.
Bandilla KK, McDufe FC, Gleich GJ (1969) Immunoglobulin classes of antibodies produced in the primary
and secondary responses in man. Clinical and Experimental
Immunology, 5, 627e641.
Castle SC (2000) Clinical relevance of age-related immune
dysfunction. Clinical Infectious Diseases, 31, 578e585.
Cossarizza A, Ortolani C, Monti D, Franceschi C (1997)
Cytometric analysis of immunosenescence. Cytometry,
27, 297e313.
Finch JM, Turner RJ (1996) Effects of selenium and
vitamin E on the immune responses of domestic animals.
Research in Veterinary Science, 60, 97e106.
Ginaldi L, De Martinis M, DOstilio A, Marini L,
Loreto MF et al. (1999) The immune system in the
elderly: II. Specic cellular immunity. Immunologic
Research, 20, 109e115.
Goto H, Yamamoto Y, Ohta C, Shirahata T, Higuchi T
et al. (1993) Antibody responses of Japanese horses to inuenza viruses in the past few years. Journal of Veterinary
Medical Science, 55, 33e37.
Guirnalda PD, Malinowski K, Roegner V, Horohov DW
(2001) Effects of age and recombinant equine somatotropin (eST) administration on immune function infemale horses. Journal of Animal Science, 79, 2651e2658.

S89

Haynes L (2005) The effect of aging on cognate function

and development of immune memory. Current Opinion
in Immunology, 17, 476e479.
Haynes L, Eaton SM, Burns EM, Rincon M, Swain SL
(2004) Inammatory cytokines overcome age-related
defects in CD4 T cell responses in vivo. Journal of
Immunology, 172, 5194e5199.
Haynes L, Linton PJ, Eaton SM, Tonkonogy SL,
Swain SL (1999) Interleukin 2, but not other common
gamma chain-binding cytokines, can reverse the defect
in generation of CD4 effector T cells from naive T cells
of aged mice. Journal of Experimental Medicine, 190,
1013e1024.
Health Canada (2007) Active immunization. In: Canadian
Immunization Guide, 7th Edit., Publishing and Depository
Services, Public Works and Government Services,
Ottawa, pp. 285e297.
Horohov DW, Dimock A, Guirnalda P, Folsom RW,
McKeever KH et al. (1999) Effect of exercise on the
immune response of young and old horses. American
Journal of Veterinary Research, 60, 643e647.
Horohov DW, Kydd JH, Hannant D (2002) The effect of
aging on T cell responses in the horse. Developmental and
Comparative Immunology, 26, 121e128.
Janeway CA, Travers P, Walport M, Shlomchik M (2001)
T cell-mediated immunity. In: Immunobiology: the Immune
System in Health and Disease, 5th Edit., Garland Publishing, New York, pp. 295e406.
Knight DA, Tyznik WJ (1990) The effect of dietary selenium on humoral immunocompetence of ponies. Journal
of Animal Science, 68, 1311e1317.
Lunn DP, Holmes MA, Duffus WP (1993) Equine T-lymphocyte MHC II expression: variation with age and
subset. Veterinary Immunology and Immunopathology, 35,
225e238.
Malinowski K, Christensen RA, Konopka A, Scanes CG,
Hafs HD (1997) Feed intake, body weight, body condition score, musculation, and immunocompetence in
aged mares given equine somatotropin. Journal of Animal
Science, 75, 755e760.
McFarlane D, Sellon DC, Gibbs SA (2001) Age-related
quantitative alterations in lymphocyte subsets and
immunoglobulin isotypes in healthy horses. American
Journal of Veterinary Research, 62, 1413e1417.
Morley PS, Hanson LK, Bogdan JR, Townsend HG,
Appleton JA et al. (1995) The relationship between single radial hemolysis, hemagglutination inhibition, and
virus neutralization assays used to detect antibodies specic for equine inuenza viruses. Veterinary Microbiology,
45, 81e92.
Morley PS, Townsend HG, Bogdan JR, Haines DM (1999)
Efcacy of a commercial vaccine for preventing disease
caused by inuenza virus infection in horses. American
Journal of Veterinary Research, 215, 61e66.
Muirhead TL, McClure JT, Wichtel JJ, Stryhn H, Frederick Markham RJ et al. (2008) The effect of age on serum
antibody titers after rabies and inuenza vaccination in
healthy horses. Journal of Veterinary Internal Medicine, 22,
654e661.

S90

T.L. Muirhead et al.

Mumford JA, Wilson H, Hannant D, Jessett DM (1994)

Antigenicity and immunogenicity of equine inuenza
vaccines containing a carbomer adjuvant. Epidemiology
and Infection, 112, 421e437.
Mumford JA, Wood J (1992) Establishing an acceptability
threshold for equine inuenza vaccines. Developments in
Biological Standardization, 79, 137e146.
Mumford JA, Wood JM, Folkers C, Schild GC (1988)
Protection against experimental infection with inuenza
virus A/equine/Miami/63 (H3N8) provided by inactivated whole virus vaccines containing homologous
virus. Epidemiology and Infection, 100, 501e510.
Mumford J, Wood JM, Scott AM, Folkers C, Schild GC
(1983) Studies with inactivated equine inuenza
vaccine. 2. Protection against experimental infection

with inuenza virus A/equine/Newmarket/79 (H3N8).

Journal of Hygiene, 90, 385e395.
Peacock DB, Jones JV, Gough M (1973) The immune
response to thetaX 174 in man. I. Primary and secondary antibody production in normal adults. Clinical and
Experimental Immunology, 13, 497e513.
Perryman LE, Wyatt CR, Magnuson NS, Mason PH
(1988) T lymphocyte development and maturation in
horses. Animal Genetics, 19, 343e348.
Rocha B, Vassalli P, Guy-Grand D (1992) The extrathymic T-cell development pathway. Immunology Today,
13, 449e454.
Turner RJ, Finch JM (1991) Selenium and the immune response. Proceedings of the Nutrition Society,
50, 275e285.