Veterinary Immunology and Immunopathology 152 (2013) 333–340

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Veterinary Immunology and Immunopathology

journal homepage: www.elsevier.com/locate/vetimm

Research paper

The potential for enhancement of immunity in cats by dietary

supplementation
K.J. Rutherfurd-Markwick a,∗ , W.H. Hendriks b,c , P.C.H. Morel d , D.G. Thomas d
a
Institute of Food, Nutrition & Human Health, Massey University, Albany, New Zealand
b
Animal Nutrition Group, Department of Animal Science, Wageningen University, Wageningen, The Netherlands
c
Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands
d
Institute of Food, Nutrition & Human Health, Massey University, Palmerston North, New Zealand

a r t i c l e i n f o a b s t r a c t

Article history: This study was conducted to examine the potential benefits of dietary supplementation on
Received 16 October 2012 the feline immune system. Forty three cats (8 or 9 per group) were fed a low protein control
Received in revised form
diet (22.7% DM basis), the same diet supplemented with yeast-derived nucleotides, salmon
19 December 2012
oil or l-arginine or a commercial moist high protein diet (53.0% DM basis) for a period
Accepted 10 January 2013
of five weeks. The low protein diets were formulated using a commercial moist diet base
with added fat and starch and fed ad libitum, along with water. Specific immune assays
Keywords:
Feline showed that supplementation with arginine caused a significant enhancement of lym-
Cat phocyte proliferative responses to the T-cell mitogen PHA after 35 days (P = 0.018), while
Immune function supplementation with either nucleotides or salmon oil resulted in significant enhancement
Dietary supplementation after both 14 (P = 0.0048, P < 0.0001 respectively) and 35 days (both P < 0.0001). Dietary
supplementation with arginine, nucleotides or salmon oil each led to significant increases
in blood leucocyte phagocytic activity after both 14 (P = 0.0003, P = 0.0077, P < 0.0001 respec-
tively) and 35 days (P < 0.0001). This indicates that a number of dietary ingredients have
the ability to modulate the immune system of healthy cats possibly resulting in a greater
ability to fight infection and disease.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction Dietary components can impact the health of com-

The increased importance of functional foods to
improve and maintain human health over the past few
decades has led to an increasing interest in the use of
dietary components. With the increasing humanisation of
pets, this interest has been utilised by pet food manufactur-
ers to find alternative and more consumer friendly methods
of maintaining and optimising companion animal health.
∗ Corresponding author at: Institute of Food, Nutrition & Human Health,
Massey University Albany, Private Bag 102 904, North Shore City 0745,
New Zealand. Tel.: +64 9 414 0800.
E-mail address: K.J.Rutherfurd@massey.ac.nz
(K.J. Rutherfurd-Markwick).
panion animals in two ways, firstly by supplying the
necessary nutrients required to maintain bodily func-
tions, and secondly by acting as bioactive molecules
which for example can influence digestive, gut-related
or cognitive functions, antioxidant status, or modulate
the immune system. Recent evidence suggests that while
healthy pets fed a good quality complete and balanced
diet are unlikely to experience nutritional deficiencies, the
macro-addition of certain dietary ingredients can signifi-
cantly enhance immunity, while excessive intakes of other
nutrients, can result in immunodeficiency and disease
(Bradley and Xu, 1996; Lessard et al., 1997; Chandra, 1997;
Field et al., 2002; Schwartz et al., 2012; de Heredia et al.,
2012). Therefore, while pets may be adequately nourished,
their immune systems may not be operating at optimum
levels.

0165-2427/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.vetimm.2013.01.007
334 K.J. Rutherfurd-Markwick et al. / Veterinary Immunology and Immunopathology 152 (2013) 333–340
Information on the potential benefits of feed ingredi-
ents and pro-nutrients in companion animals is scarce,
especially in the scientific literature. Metabolic differences
between species may mean that there is the potential for
dietary ingredients and supplements to have species spe-
cific effects. As such, caution should be taken to ensure that
data is not extrapolated from one species to another (NRC,
2008), and it is important that information on the efficacy
and safety of putative nutraceuticals is determined on the
individual animal species.
The cat is widely recognised as having a unique and
highly specialised metabolism (National Research Council
(NRC) 2006, 2008). Arginine, is considered a conditionally
essential amino acid in most mammals, but is essential
in the diet of the carnivorous cat due to its inability to
synthesise citrulline as a result of the low activity of two
enzymes in the arginine synthesis pathway; 1 -pyrroline-
5-carboxylate synthase and ornithine aminotransferase
(Morris and Rogers, 1978a, 1978b; Rogers and Phang,
1985). The essentiality of arginine is further demonstrated
by the fact that a single arginine free meal fed to cats can
result in death within 2 h due to hyperammonemia (Morris
and Rogers, 1978b).
Arginine plays a key role in both innate and acquired
immunity in other species, with a deficiency of arginine in
the diet leading to impaired NO synthesis and immune sup-
pression (Li et al., 2007). A number of in vitro and in vivo
studies carried out primarily in rodents and humans have
demonstrated that arginine is able to stimulate the immune
system (enhancing T-lymphocyte proliferation, increasing
IL-2 production, improving cytotoxicity of macrophages,
NK cells and T lymphocytes), accelerate wound healing and
reduce morbidity and mortality (Li et al., 2007; Wu et al.,
2009).
Lipid metabolism in cats also differs from that of other
mammalian species, with the cat having limited liver 6
desaturase activity, resulting in a restricted ability to con-
vert linoleic acid to arachidonic acid and ␣-linolenic acid to
eicosapentaenoic (EPA) and docosahexaenoic acid (DHA) in
that tissue (NRC, 2006). It does appear that the cat has suffi-
cient 6 desaturase activity in tissues such as the brain and
retina to meet tissue requirements for DHA (Filburn and
Griffin, 2005), and it has been suggested that cats may use
an alternate pathway via 8 and 5 desaturase to synthe-
sise fatty acids in the n-6 family (Morris, 2004). In cats, EPA
and DHA are of great scientific interest due to their appar-
ent ability to decrease the risk of cardiovascular disease,
lower blood pressure, decrease risk of certain cancers and
modulate immune function (Chew et al., 2000; Plantinga
and Beynen, 2003; Park et al., 2011).
Dietary supplementation with fish oil (a rich source of
EPA, DHA and other n-3 fatty acids) is thought to result in
altered platelet function in a number of species including
cats (Saker et al., 1998). Decreased concentrations of pro-
inflammatory prostaglandin E2 , and increased lymphocyte
proliferation and modulation of ␥-IFN production (Trebble
et al., 2003) have also been reported. However, conflicting
results have been obtained, and some studies have shown
that supplementation with n-3 PUFAs results in immuno-
suppression (Chew et al., 2000; Calder et al., 2006). A study
in cats showed consumption of fatty acids derived from fish
oil at an n-6:n-3 ratio of 5:1 resulted in decreased lympho-
cyte proliferation to pokeweed mitogen and a reduction
in the numbers of a subpopulation of B cells (Chew et al.,
2000).
Nucleotides have been commercially used in human
infant formulas for many years, as a way of improving gas-
tric development and early immune function, and there are
data reporting reduced digestive disorders post-weaning
in other species, including pigs (Helembai et al., 2006;
Ruihua et al., 2006). Nucleotide supplementation has been
shown to enhance a range of immune functions in humans
(Martinez-Augustin et al., 1997; Gil, 2002) and mice
(Navarro et al., 1996; Jyonouchi et al., 2003). Nucleotide-
free diets have been shown to impair cell-mediated and
humoral immune responses; which include decreased
macrophage and natural killer (NK) cell activity, delayed
type hypersensitivity responses, cytokine levels, lower
antibody production, all of which may lead to an increased
susceptibility to infection (Gil, 2002; Sanchez-Pozo and Gil,
2002). These changes can be reversed by supplementation
with nucleotides (Gil, 2002; Navarro et al., 1996). A number
of animal studies suggest that nucleotide supplemen-
tation of diets can affect immune function by enhancing
mitogen-induced lymphocyte proliferation, interleukin-2
production, cell-mediated immunity, and enterocyte and
lymphocyte maturation and improving resistance to infec-
tion (Gil, 2002; Jyonouchi et al., 2003; Yamauchi et al.,
2002).
The objective of the current study was to examine the
effects of feeding either nucleotides (in the form of an
enriched preparation derived from yeast), salmon oil or
arginine on the immune status of adult cats to ascertain
if the immunomodulatory effects reported in other species
also occur in cats.
2. Materials and methods
2.1. Animals
Forty three adult domestic short-haired cats (Felis
catus), 32 castrated males and 11 intact females aged
between 2.0 and 11.0 years (average 5.0 years) and weigh-
ing 2.2–5.9 kg from the Centre for Feline Nutrition, Massey
University (Palmerston North, New Zealand) were used in
this study as experimental animals. Cats were housed (8
or 9 cats/pen; 1.4 m × 4.4 m × 2.4 m) in a natural light and
temperature environment with access to shelter. All cats
had been vaccinated against feline rhinotracheitis, cali-
civirus and panleukopenia using a modified live vaccine
(Felocell CVR, Norden Laboratories, München, Germany)
as kittens, and received a booster vaccination every three
years. Feline leukaemia and feline immunodeficiency virus
have not been detected in the colony since its establish-
ment in 1992. The study was approved by and conformed
to the requirements of Massey University Animal Ethics
Committee (Anonymous, 2003).
2.2. Experimental design and diets
The cats were divided into 5 groups of 8 or 9 animals
(43 in total), comprising mixed groups of castrated males
 K.J. Rutherfurd-Markwick et al. / Veterinary Immunology and Immunopathology 152 (2013) 333–340 335

Table 1
Ingredient composition of the five experimental diets.

Ingredient Diet (g/kg as-is)

HP LP LP + Arg LP + oil LP + N

Canned dieta 1000.0 796.4 796.4 796.4 796.4

Pregelatinized starchb – 134.5 127.7 134.5 101.1
Bleached lardc – 58.9 58.9 28.2 58.9
l-Arginined – – 6.8 – –
Salmon oile – – – 30.7 –
Nucleotide preparationf – – – – 33.4
Vitamin–mineral premixg – 10.2 10.2 10.2 10.2
Calculated metabolizable energyh
kJ/g dry matter 19.2 20.0 20.4 22.0 20.0
Kcal/g dry matter 4.58 4.78 4.87 5.27 4.78

HP: high-protein; LP: low-protein; LP + Arg: LP + arginine; LP + oil: LP + salmon oil; LP + N: LP + nucleotides.
a
Jellimeat (Heinz-Wattie’s Ltd., Hastings, New Zealand) Ingredient list: meat by-products and meat derived from lamb, beef, chicken and mutton,
vegetable protein, gelling agents, Ca supplement, phosphate, colouring and essential vitamins. Analysed composition (g/kg dry matter): crude protein:
530; crude fat: 313; ash: 95; arginine: 27.6; histidine: 14.8; isoleucine: 18.7; leucine: 42.3; lysine: 35.7; methionine: 9.6; cystine: 5.2; phenylalanine: 25.7;
tyrosine: 17.6; threonine: 22.9; tryptophan: 7.6; valine: 26.6; taurine: 1.56. Water content: 818 g/kg as-is.
b
Pregelatinized Starch (National Starch & Chemical, Auckland, New Zealand).
c
Bleached Lard (Farmland Foods Ltd., Wanganui, New Zealand).
d
l-Arginine (free base) (Ajinimoto, Japan).
e
Salmon Oil (New Zealand King Salmon Ltd., Nelson, New Zealand).
f
Nucleotide mixture (Nupro® , Alltech Inc., Nicholasville, KY, USA). Analysed composition (g/kg dry matter): crude protein: 505; total nucleic acids: 54;
crude fat: 2; ash: 82; arginine: 18.8; histidine: 9.7; isoleucine: 19.4,leucine: 36.0, lysine: 27.3; methionine: 7.4; cystine: 5.1; phenylalanine: 18.7; tyrosine:
15.0; threonine: 19.4; tryptophan: 4.9; valine: 24.6; taurine: 0.90. Water content: 60 g/kg as-is.
g
Provided to the diet (per kgas-is): Ca: 1.7 g; P: 1.3 g; K: 0.7 g; choline: 0.5 g; Cl: 0.2 g; Na: 0.1 g; Mg: 0.1 g; taurine: 122 mg; vitamin E: 27 mg; inositol:
45 mg; Fe: 16 mg; Zn: 15 mg; niacin: 12 mg; Cu: 1.6 mg; vitamin A: 1.5 mg; Mn: 1.5 mg; pantothenic acid: 1.4 mg; thiamine: 1.2 mg; pyridoxine: 1.2 mg;
riboflavin: 1.0 mg; I: 0.20 mg; folic acid: 0.20 mg;Co: 0.10 mg; Cr: 0.08 mg; vitamin K3 : 0.04 mg; Se: 0.03 mg; vitamin B12 : 40.8 ␮g; biotin: 20.4 ␮g; vitamin
D3 : 5.1 ␮g.
h
NRC (2006).

and intact females. All cats were allowed to adapt to a low
protein diet (LP, Table 1, 22.7% crude protein DM basis) for
one week prior to the commencement of the trial. The low
protein diet was made by adding additional fat (Bleached
lard), pregelatinized starch, minerals and vitamins (listed
in Table 1) to a highly palatable moist commercial diet
(which had passed a minimum feeding protocol for proving
an adult maintenance claim for a cat food (AAFCO, 2012)),
in order to reduce the protein content. In this trial the indi-
vidual animals were considered as the experimental unit
as the effect of social hierarchy was expected to be negligi-
ble. Previous studies have shown that there was no effect
of pen or gender on the measured immune parameters
(Rutherfurd-Markwick et al., 2008).
After the adaptation period, group 1 remained on the
LP diet (control group, n = 8), group 2 was fed a moist com-
mercial high protein (53.0% protein DM basis, n-6:n-3 ratio
of 4.77:1) diet as a positive control (HP, n = 9), group 3 to
5 was fed the LP diet supplemented with either salmon
oil (LP + oil, 9% DM, n-6:n-3 ratio of 1.77:1, n = 9), free l-
arginine (LP + Arg, 1.9% DM, n = 8,) or nucleotides (LP + N,
9.25% DM, n = 9) (Table 1) for a period of 35 days. The cats
had access to food and water ad libitum during both the
acclimatisation and test period. Fresh food was provided
daily, and the amount of food consumed by each group was
calculated on a daily basis by collection of food remains.
The cats were weighed before the seven day acclimatisa-
tion period, immediately prior to commencing the trial, and
then weekly for the duration of the trial. Weekly feed intake
was expressed as g dry matter per kg body weight. A hep-
arinized blood sample (3 ml) was obtained from the cats by
jugular venipuncture, on days 0, 14 and 35 of the trial for
assessment of immune parameters. Plasma was collected
by centrifugation of the blood remaining after completion
of the other assays.
2.3. Lymphocyte proliferation assay
A whole blood cell proliferation assay was modified
from previously published methods (Hiraga et al., 1981;
Weiss, 1992; Saker et al., 2001). Lithium heparin-treated
peripheral whole blood was diluted 1:4 in complete
RPMI-1640 medium (supplemented with 10% foetal calf
serum, 2 mM l-glutamine, 100 U/ml penicillin, 100 ␮g/ml
streptomycin sulphate and 50 ␮M 2-mercaptoethanol (all
reagents from Gibco, Poole, UK). One hundred ␮l of the
diluted blood was added to four wells of a 96-well,
flat-bottomed tissue culture plate (Greiner Labortechnik,
Frickenhausen, Germany) and cultured in the presence of
either 5 ␮g/ml Concanavalin A (Con A, Sigma, St. Louis,
MO, USA), 1:49 diluted Phytohaemagglutanin (PHA, Gibco,
Poole, UK), or complete RPMI-1640 media in place of the
mitogen in the control wells. The cells were cultured for
48 h at 37 ◦ C in a 5% humidity, CO2 -air atmosphere, before
being pulsed for 18 h with 0.5 ␮Ci methyl-3 H-thymidine
(Amersham Biosciences, Amersham, UK) per well.
Each plate was harvested onto a 96-well glass fibre
mat using a cell harvester (Tomtek, Orange, CT, USA) and
counted using a Wallac-Trilux 1450 Microbeta liquid scin-
tillation and luminescence counter (Boston, MA, USA).
Stimulation index was calculated as counts per minute
(CPM) in wells with mitogen divided by CPM in wells with-
out mitogen.
336 K.J. Rutherfurd-Markwick et al. / Veterinary Immunology and Immunopathology 152 (2013) 333–340
2.4. Analysis of lymphocyte subsets
Flow cytometric analysis was used to determine the
level of expression of CD4+ (T-helper cells), CD8+ (cyto-
toxic T-cells), B cells (CD21+ ) and CD11b+ (granulocytes)
antigens on peripheral blood leucocytes and granulocytes
from whole blood. Antibodies were either feline-specific
(CD4, CD8) or canine-specific (B cells, CD11b) (Serotec Inc.,
Raleigh, NC, USA). Canine-specific antibodies have been
shown to cross react with the relevant feline antigen by
the supplier. Immunolabelling was performed according
to the method of Gill et al. (2000), and samples were
analysed using a FACSCalibur flow cytometer (Becton Dick-
inson Instruments, Cambridge, MA, USA). Negative controls
were also run. For each sample, data were collected for
10,000 gated events.
2.5. Phagocytosis
Assessment of the phagocytic capacity of peripheral
blood leucocytes by flow cytometry was based on the
method of Rutherfurd-Markwick et al. (2005). Briefly,
5 ␮l of FITC-labelled Escherichia coli bacteria (1 × 109 /ml)
(Molecular Probes Incorporated Eugene, OR, USA) was
mixed with 100 ␮l of whole blood and incubated for
30 min at 37 ◦ C. Immediately after incubation, the cells
were fixed with para-formaldehyde, and the erythrocytes
lysed by the addition of 1 ml of ice-cold water. Follow-
ing centrifugation (2700 × g) the pellet was re-suspended
in 500 ␮l of phosphate buffered saline, and 50 ␮l of 4%
Trypan blue added to quench extraneous fluorescence.
Two types of negative controls were prepared, both by
incubating and fixing the blood samples in the absence
of the FITC-labelled E. Coli; one set then had the FITC-
labelled E. coli added following the fixation and prior to the
lysing step, while the other did not. The phagocytic activity
was determined using a FACSCalibur flow cytometer (Bec-
ton Dickinson Instruments, Cambridge, MA, USA), and is
expressed as the percentage of gated blood leukocytes that
were phagocytically active. Each sample was processed
individually.
2.6. Statistical analysis
Immune parameters were analysed by repeat measure
analysis, with diet and week and their interaction fitted as
fixed effects, cat within diet as a random effect, and age
as a covariate. No differences between sex were observed
and this effect was not included in the model (Proc Mixed,
SAS). A linear model with diet and week as fix effect was
fitted to the weekly feed intake data (Proc GLM, SAS).
Differences with P < 0.05 were considered as achieving sta-
tistical significance. All values are expressed as means ± SE.
Two dietary treatments group included 8 cats (LP and
LP + Arg) and three included 9 cats (HP, LP + N, LP + oil).
During some weeks individual cat observations were miss-
ing: 1 observation for B-cell, 4 observations for ConA and
2 observations for PHA. The different number of obser-
vations per diet × week resulted as expected, in different
SE.
Fig. 1. Effect of dietary supplementation with arginine, salmon oil
or nucleotides on feline lymphocyte proliferative responses to Phyto-
haemagglutanin (PHA). Stimulation index was calculated as counts per
minute (CPM) in wells with mitogen divided by CPM in wells with-
out mitogen. HP: high-protein; LP: low-protein; LP + Arg: LP + arginine;
LP + oil: LP + salmon oil; LP + N: LP + nucleotides. Values with different
superscript letters within treatment group (a, b, c) and treatment period
(x, y, z) are significantly different (P < 0.05). Results are mean ± SE.
3. Results
3.1. Body weight and feed intake
All cats completed and remained healthy throughout
the trial. There was no significant difference in body weight
between groups at the start or at any time during the trial
(results not shown). Over the period of the trial, there was
no significant difference in average energy intake between
the groups. The LP + oil group had the highest daily energy
intake (85.67 ± 1.42 kcal/kg BW), and the LP + Arg group,
had the lowest energy intake (82.54 ± 1.42 kcal/kg BW).
3.2. Effect of dietary supplementation on lymphocyte
proliferative responses
To evaluate the effect of dietary supplementation on
T-cell function in cats, proliferative responses of periph-
eral blood leucocytes to the T-cells mitogens: Con A and
PHA were measured in vitro at day 0, 14 and 35. The
results (Table 2) showed that there were no diet, week of
diet × week interaction effects on proliferative responses
to Con A stimulated peripheral blood leucocytes. However,
there were significant effects of dietary supplementation
on lymphocyte proliferative responses to PHA (Fig. 1).
The group fed the LP diet (negative control) showed no
change in lymphocyte proliferative responses over the
35 day study. Compared to the day 0 values, the HP
group (positive control) and the LP + Arg group showed
significant increases (P = 0.003, P = 0.018 respectively) in
lymphocyte proliferative responses to PHA after 35 days
of supplementation. Both the LP + N and LP + oil groups
showed significant time dependent increases in prolife-
rative responses to PHA after 14 (P = 0.0048, P = 0.0001
respectively) and 35 days (both P < 0.0001) of feeding com-
pared to day 0.
 K.J. Rutherfurd-Markwick et al. / Veterinary Immunology and Immunopathology 152 (2013) 333–340
Table 2
Effect of dietary supplementation with arginine, salmon oil or nucleotides on feline lymphocyte proliferative responses to Concanavalin A (Con A).
Stimulation index was calculated as counts per minute (CPM) in wells with mitogen divided by CPM in wells without mitogen.
Mitogen Day Diet
HP LP LP + Arg
n=9 n=8 n=8
Con A 0 37.78 ± 6.29 20.99 ± 6.32 24.53 ± 6.17
14 36.24 ± 6.29 20.68 ± 6.32 24.43 ± 6.40
35 32.87 ± 6.29 21.93 ± 6.57 23.82 ± 6.17
HP: high-protein; LP: low-protein; LP + Arg: LP + arginine; LP + oil: LP + salmon oil; LP + N: LP + nucleotides. Results are mean ± SE.
3.3. Lymphocyte subsets
Table 3 shows the percentages of lymphocyte subsets
in the peripheral blood of control cats and cats fed the
different dietary supplements for 5 weeks. There were
no significant diet × week interactions observed over the
period of the trial. Although week effects were observed for
the expression of CD4+ and CD11b+ cell markers (Table 3).
3.4. Phagocytic function of peripheral blood leucocytes
The effect of dietary supplementation on peripheral
blood phagocytosis is shown in Fig. 2. A diet, week and a
diet x week interaction was observed. The LP group showed
no change in the level of phagocytic activity over the
duration of the trial. A time dependent increase in phago-
cytic activity in each of the other groups was observed
albeit to varying extents. The HP group showed a sig-
nificant increase (P = 0.04) in phagocytic activity after 35
days, whilst the LP + Arg, LP + N and LP + oil groups attained
significant increases after 14 days of feeding (P = 0.0003,
P = 0.0077, P < 0.0001 respectively), and a further increase
after 35 days of feeding (P < 0.0001 for all three groups).
Fig. 2. Effect of dietary supplementation with arginine, salmon oil or
nucleotides on phagocytic activity of feline peripheral blood lympho-
cytes. HP, high-protein; LP: low-protein; LP + Arg: LP + arginine; LP + oil:
LP + salmon oil; LP + N: LP + nucleotides. Values with different superscript
letters within treatment group (a, b, c) and treatment period (x, y, z) are
significantly different (P < 0.05). Results are mean ± SE.
337
P value
LP + oil LP + N Diet Week Diet × week
n=9 n=9
27.67 ± 5.80 16.83 ± 6.29 0.423 0.600 0.531
33.18 ± 5.80 25.13 ± 6.02
28.51 ± 5.80 30.54 ± 5.83
4. Discussion
Dietary supplementation did not affect body weight
(P > 0.05), or effect energy intake over the trial period in the
treatment groups. To ascertain whether it was necessary to
use a low protein (LP) diet in order to observe significant
changes in immune parameters due to dietary supplemen-
tation, or if a higher protein complete and balanced diet
alone could be used as the base diet for future studies, a
positive control diet (HP) was included in the present study,
thus allowing direct comparison with the LP diet. The HP
diet contained 53.0% crude protein (DM basis) compared
with the LP diet with 22.7% crude protein (DM basis). How-
ever, the LP diet still contained 51.7 g crude protein per
kcal ME which is higher than the minimum recommended
crude protein allowance for a maintenance diet (50.0 g/kcal
ME) as specified by the NRC (2006). The immune param-
eters measured did not change over time for cats fed the
LP diet. Although some of the parameters did change sig-
nificantly over time for cats fed the HP diet, the changes
observed were not as large as that observed for cats fed the
LP + supplement diets. It appears that there is no need to
use a diet containing close to the minimum allowance for
crude protein (LP: 22.7% crude protein DM basis) and a HP
diet (53.0% protein DM basis) would be a suitable base diet
for future work.
Lymphocyte proliferative responses to mitogens are
widely used to assess both T- and B-cell function (Gill et al.,
2000). In the present study, dietary supplementation had
no significant effects on proliferative responses to the T-cell
mitogen Con A (Table 2). However, significant effects were
seen on proliferative responses to PHA for cats fed several of
the diets. Cats fed the HP and LP + Arg diets showed signifi-
cant time dependent increases after 35 days of supplemen-
tation, while those fed the LP + N and LP + oil diets showed
increases in lymphocyte proliferative responses to PHA
after both 14 and 35 days. The results of nucleotide supple-
mentation are in agreement with work carried out in mice
and humans where dietary nucleotide supplementation
led to significant enhancement of lymphocyte proliferative
responses to T cell mitogens (Kulkarni et al., 1989; Carver
and Walker, 1995; Gil, 2002). Likewise studies in rats and
humans have shown that dietary supplementation with
arginine results in enhancement of T-lymphocyte prolif-
eration (Evoy et al., 1998; Li et al., 2007). The enhancement
of lymphocyte proliferative responses to PHA seen in the
cats fed the LP + oil diet in this study is in contrast to that
of Chew et al. (2000) who saw no significant effect of
338 K.J. Rutherfurd-Markwick et al. / Veterinary Immunology and Immunopathology 152 (2013) 333–340

Table 3
Effect of dietary supplementation with arginine, salmon oil or nucleotides on percentage of feline peripheral blood lymphocyte subsets.

Subset Day Diet P value

HP LP LP + Arg LP + oil LP + N Diet Week Diet × week

n=9 n=8 n=8 n=9 n=9

CD 4+ cells (%) 0 36.82 ± 2.46 33.18 ± 2.43 36.71 ± 2.36 32.76 ± 2.21 32.34 ± 2.23 0.275 <0.0001 0.278
14 35.45 ± 2.46 30.79 ± 2.43 35.01 ± 2.36 28.39 ± 2.21 30.93 ± 2.23
35 33.62 ± 2.46 29.54 ± 2.43 35.27 ± 2.36 28.37 ± 2.21 31.34 ± 2.23
CD 8+ cells (%) 0 16.49 ± 1.98 14.40 ± 1.95 16.69 ± 1.89 13.53 ± 1.78 16.41 ± 1.79 0.676 0.055 0.848
14 16.43 ± 1.99 15.25 ± 1.95 17.39 ± 1.89 13.68 ± 1.78 16.87 ± 1.79
35 16.26 ± 1.98 15.37 ± 1.95 17.48 ± 1.89 14.72 ± 1.78 17.39 ± 1.79
B cells (%) 0 31.40 ± 3.56 38.35 ± 3.60 28.34 ± 3.44 40.68 ± 3.23 38.48 ± 3.25 0.052 0.218 0.074
14 30.92 ± 3.56 38.47 ± 3.54 30.71 ± 3.86 46.02 ± 3.28 36.66 ± 3.25
35 33.48 ± 3.56 37.21 ± 3.54 29.13 ± 3.44 40.66 ± 3.23 33.87 ± 3.25
CD 11b+ cells (%) 0 98.21 ± 1.37 95.40 ± 1.41 96.68 ± 1.39 96.62 ± 1.31 93.73 ± 1.31 0.314 0.023 0.337
14 95.13 ± 1.37 94.92 ± 1.41 93.92 ± 1.39 95.56 ± 1.36 94.66 ± 1.31
35 96.84 ± 1.37 94.75 ± 1.41 97.21 ± 1.39 95.86 ± 1.31 94.05 ± 1.31

HP: high-protein; LP: low-protein; LP + Arg: LP + arginine; LP + oil: LP + salmon oil; LP + N: LP + nucleotides. Results are mean ± SE.

feeding fish oil to adult cats at an n-6:n-3 ratio of 5:1 after
either 6 or 12 weeks (42 or 84 days) on lymphocyte prolif-
eration to Con A or PHA. The difference in the results may
be due to either the different lengths of the trials, or the
different n-6:n-3 ratios fed. The n-6:n-3 ratio of foods con-
sumed by pet cats has been reported to range from 5:1 to
17:1, while that of feral cats is more likely to be approxi-
mately 2:1 (Plantinga et al., 2011). The n-6:n-3 ratio used
by Chew et al. (2000) is at the lower end of the pet food
range and more similar to that found in the HP diet used
in this study (n-6:n-3 ratio 4.77:1). In contrast, the n-6:n-3
ratio of the LP + oil diet used in the current study (1.77:1)
was lower and more similar to that described for feral cats.
The enhancement of lymphocyte proliferative responses to
PHA seen in the LP + oil fed group in this study may be a
consequence of the different n-6:n-3 ratio used. This is in
agreement with the suggestion that it is the total dietary
ratio of n-6:n-3 fatty acids that is important rather than
the absolute amount of n-3 given. Clearly more work is
required to determine the optimum ratio of n-6:n-3 fatty
acids for overall health in the cat, along with the effects
of feeding different n-6:n-3 ratios over varying periods of
time as it is possible that some changes may be transient.
In the present study we did not observe any changes
in the level of expression of peripheral blood leucocyte
subsets in response to dietary supplementation. This is a
similar result to that reported in elderly humans where
arginine supplementation in combination with zinc did
not affect lymphocyte population numbers (Provinciali
et al., 1998). It is also a similar result to that reported for
mice and term infants (Gil, 2002; Hawkes et al., 2006),
receiving nucleotide supplementation, but in contrast to
a study by Navarro et al. (1999), who reported a signifi-
cant increase in CD4+ cells in preterm infants fed dietary
nucleotides. The authors suggested that this difference may
be due to nucleotides exerting their action on immature
neonate lymphocytes. However, since the animals used in
the present study were all adult, it is not surprising that we
did not observe a similar effect.
Park et al. (2011) also observed no change in the level
of expression of CD5+ (Total T cells) or CD4+ cells in cats
fed a diet enriched in omega 3 fatty acids from fish oil
(n-6:n-3 ratio 5:1) for 12 weeks (but not at 6 weeks) com-
pared to control animals fed a diet with an n-6:n-3 ratio
of 20:1 when it was fed as part of a high fat diet (22% fat).
However, when the omega 3 enriched diet was consumed
as part of a lower fat diet (14%), expression of both CD5+
and CD4+ were significantly lower compared to cats fed the
control diet (14%, n-6:n-3 ratio 20:1). In contrast, the CD21+
B cell population was significantly lower in the cats fed the
high omega 3 diet, high fat diet, but remained unchanged in
the cats fed the high omega 3, lower fat, diet. Expression of
CD8+ cells did not change in cats fed the omega 3 enhanced
diet at either fat level (Park et al., 2011). An 8 week study
carried out in aged beagles, where the diet fed contained
an n-6:n-3 ratio of 1.4:1 (21.3% fat, DM basis) content also
showed a significantly lower percentage of CD4+ cells after
vaccination (Hall et al., 1999).
In the current study we did not observe any signifi-
cant changes in the level of expression of the lymphocyte
subsets that we measured. However, our dietary supple-
mentation period was only 5 weeks, compared to 8 week
and 12 week periods used by others (Hall et al., 1999; Park
et al., 2011). As previously mentioned, the n-6:n-3 ratio
used in the current study was higher that used by Park et al.
(2011), as was the fat content of the diets (HP 31% fat, LP
30% fat DM basis) and these design differences may explain
the contrasting results.
Phagocytic cells are major effectors of natural immu-
nity, playing an important role in protection against
infection. In the current study we observed a time depend-
ent enhancement of phagocyte function in adult cats fed
diets supplemented with salmon oil, nucleotides and argi-
nine, compared to the LP control-fed animals which did
not change and the HP-fed animals which exhibited a
significant increase in phagocytic activity only after 35
days feeding. The results observed in the current study
with dietary supplementation of arginine and nucleotides
in cats are similar to those observed in other species. In
studies carried out in rats fed a diet supplemented with
arginine, enhancement of phagocyte function and bacteri-
cidal activity was observed (Wang et al., 2003; Izgut-Uysal
et al., 2004). Previous studies in mice have shown that
animals on nucleotide free diets had significantly lower
 K.J. Rutherfurd-Markwick et al. / Veterinary Immunology and Immunopathology 152 (2013) 333–340
macrophage phagocytic activities than those fed a diet sup-
plemented with nucleotides (Carver, 1994; Kulkarni et al.,
1986). A study in healthy beagles reported that dietary fish
oil supplementation had no effect on bactericidal activity
of activated peripheral blood neutrophils (Hall et al., 2011).
Although we did not measure bactericidal activity, we did
observe an enhancement of phagocytic activity in the cats
fed salmon oil.
Having an optimally functioning immune system is
essential for host defence against invading pathogens and
spontaneously developing cancers. The results of this study
demonstrate that several different dietary supplements
have the ability to modulate immune responses in healthy
cats. Some of the responses seen were similar to those
described in other species, while others were not, empha-
sising that results cannot be extrapolated across species.
The enhanced phagocyte function and T-cell function seen
here may result in an increased resistance to invading
pathogens and disease and therefore benefit the animals’
overall health status. Given these results it would be
interesting to carry out supplementation at different con-
centrations in order to determine the optimal formulations
and also feeding for a longer time period to determine if
and when a plateau occurs and also to elucidate the safety
of long term feeding.
Conflicts of interest statement
The authors declare that they have no conflicts of inter-
est.
Acknowledgements
This work was funded in part from a grant from the
Foundation for Research Science and Technology, New
Zealand (Contract MAUX0201) and the Centre for Feline
Nutrition, Institute of Food, Nutrition and Human Health,
Massey University, Palmerston North, New Zealand. We
gratefully acknowledge the expert technical skills of M. C.
McGrath, K. Weidgraaf, M. Hekman and Y. H. Cottam.
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