Vet Pathol 44:823–830 (2007)

Immunohistochemical Expression of Vascular Endothelial Growth

Factor and Vascular Endothelial Growth Factor Receptor Associated
with Tumor Cell Proliferation in Canine Cutaneous Squamous Cell
Carcinomas and Trichoepitheliomas
A. N. AL-DISSI, D. M. HAINES, B. SINGH, AND B. A. KIDNEY
Departments of Veterinary Pathology (ANA, BAK), Veterinary Microbiology (DMH), and Veterinary
Biomedical Sciences (BS), Western College of Veterinary Medicine, University of Saskatchewan,
Saskatoon, Saskatchewan, Canada

Abstract. The expression of 5 markers associated with angiogenesis was studied in canine squamous
cell carcinomas (SCCs) (n 5 19) and canine trichoepitheliomas (TCPs) (n 5 24). SCCs were assigned
histologic grades, and tissue sections from both tumor types were immunohistochemially stained for the
expression of vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor-2
(VEGFR-2), as well as intratumoral microvessel density (iMVD), tumor proliferation index (PI), and
tumor apoptotic index (AI), using antibodies against VEGF, VEGFR-2, von Willebrand’s factor, Ki-67
antigen, and the terminal deoxynucleotidyl transferase-mediated 29-deoxyuridine 59-triphosphate end-
labeling method (TUNEL), respectively. VEGF and VEGFR-2 were detected in 17/19 (89.4%) and 19/19
(100%) SCCs and in 17/24 (70.8%) and 20/24 (83.3%) TCPs, respectively. In SCCs, there was substantial
correlation between histologic grade and PI (r 5 0.51); and moderate correlation between VEGF and
histologic grade (r 5 0.43), VEGFR-2 and histologic grade (r 5 0.47), VEGF and PI (r 5 0.47), and
VEGFR-2 and PI (r 5 0.47) (Spearman rank correlation coefficient). In TCPs, there was substantial
correlation between VEGF and PI (r 5 0.51) and a moderate correlation between VEGFR-2 and iMVD
(r 5 0.36). The median iMVD of SCCs (15.5) was significantly higher than the median iMVD of TCPs
(9.05) (P value , .05). It was concluded that VEGF and VEGFR-2 may promote tumor cell
proliferation in TCPs and SCCs. An autocrine pathway for VEGF probably operates in canine SCCs
and TCPs, as VEGF and VEGFR-2 expression was found in most tumors and was associated with
evidence for tumor cell proliferation.

Key words: Apoptotic index; canine; immunohistochemistry; microvessel density; proliferation

index; squamous cell carcinoma; trichoepithelioma; vascular endothelial growth factor; vascular
endothelial growth factor receptor-2.

Angiogenesis, the formation of microvascula- of VEGF to VEGFR-2.9,13 VEGF and VEGFR-2

ture, is crucial for the growth and metastasis of
malignant tumors.12 Angiogenesis is promoted by
factors produced by tumor cells or their surround-
ing stroma.21 Among these factors, vascular endo-
thelial growth factor (VEGF, also known as
VEGF-A) is thought to play a key role in tumor
angiogenesis.19 Four VEGF isoforms exist as a re-
sult of alternative patterns of splicing: VEGF 121,
VEGF165, VEGF189, and VEGF206.20 VEGF
mediates angiogenesis by binding to 2 receptors
on endothelial cells known as vascular endothelial
growth factor receptor-1 (VEGFR-1) and vascular
endothelial growth factor receptor-2 (VEGFR-2).10
While both receptors bind to VEGF with high
affinity,5,39 only VEGFR-2 is capable of mediating
angiogenesis.9 VEGFR-1 is thought to play a neg-
ative regulatory role by decreasing the availability
823
may be responsible for increased tumor cell pro-
liferation.7,23,29 The simultaneous expression of
VEGF and VEGFR-2 on tumor cells may indicate
that VEGF has an autocrine function.6 VEGF may
also decrease tumor apoptosis.22 The increased
expression of VEGF has been correlated with the
histologic grade of malignancy in many human
tumors.8,40 Moreover, the relationship between the
degree of angiogenesis, often expressed as intratu-
moral microvessel density (iMVD), and many
parameters, including tumor proliferation index
(PI), an expression of tumor cell proliferation, and
tumor apoptotic index (AI), an expression of tumor
cell death by apoptosis, has been extensively
studied in human cancer.22
In the veterinary literature, the association
among VEGF, VEGFR-2, iMVD, PI, and AI has
824 Al-Dissi, Haines, Singh, and Kidney Vet Pathol 44:6, 2007

Fig. 1. Skin; canine squamous cell carcinoma, grade 1. Note the prominent keratin pearl formation (arrows).
HE. Bar 5 250 mm.
Fig. 2. Skin; canine squamous cell carcinoma, grade 2. Note the arrangement of some tumor cells into small
nests (arrows), fewer keratin pearls, and more pleomorphic nuclei than in grade 1. HE. Bar 5 50 mm.
Fig. 3. Skin; canine squamous cell carcinoma, grade 3. Note the arrangement of cells in small groups and the
marked mitotic activity (arrows). HE. Bar 5 25 mm.
Vet Pathol 44:6, 2007 Angiogenesis in Squamous Cell Carcinoma and Trichoepithelioma 825

been evaluated in a few canine tumors.26–28,32–36
Therefore, the role of VEGF and VEGFR-2 in
angiogenesis and tumor cell proliferation in canine
cancer is still unclear, and studying the relationship
between these markers may aid in understanding
the role VEGF and VEGFR-2 play in cancer
progression in dogs.
The purpose of this study was to evaluate and
compare the expression of these markers in
a malignant and a benign canine skin tumor type,
cutaneous squamous cell carcinoma (SCC) and
trichoepithelioma (TCP), respectively. Both tumor
types are common in dogs and arise from
squamous epithelium.
Materials and Methods
Tissue selection and histologic grading
Nineteen and 24 tissue sections of cutaneous SCC and
benign TCP, respectively, which were submitted be-
tween 1999 and 2004 to Prairie Diagnostic Services at
the Western College of Veterinary Medicine, Saskatoon,
Saskatchewan, Canada, were selected if at least 1 cm3
of tissue was available for evaluation. SCC tissues
were given histologic grades from 1 to 3, according to
previously established criteria.15 Briefly, well-differenti-
ated tumors were given a grade of 1 (Fig. 1) if they had
abundant eosinophilic cytoplasm, prominent intercellu-
lar bridges, and many concentric laminated masses of
keratin (keratin pearls). Nuclear pleomorphism and
mitotic activity were minimal. Moderately differentiated
tumors of grades 2 and 3 in the reported classification
scheme were grouped together and given a grade of 2.
Tumor cells in this category had less eosinophilic
cytoplasm, more pleomorphic nuclei, more numerous
mitotic figures with fewer keratin pearls, and more
prominent dermal invasion (Fig. 2). Grade 3 was given
to poorly differentiated tumors that showed little
squamous differentiation, marked mitotic activity, and
deep infiltration. Tumor cells had amphophilic cyto-
plasm and often appeared as single cells or in small
groups of cells (Fig. 3). Histologic sections from each
tumor were graded independently by 2 of the authors
(ANA and BAK). Tumors receiving different histologic
grades were re-evaluated, and a consensus was estab-
lished. The histologic features of TCPs were previously
described.14
Immunohistochemical staining for VEGF, VEGFR-2, von
Willebrand’s factor, and Ki-67
An avidin-biotin-complex (ABC) immunohistochem-
ical staining method17 was used on formalin-fixed
paraffin-embedded tissues. From each paraffin block,
consecutive 5-mm tissue sections were cut, mounted, and
dried on glass slides. Tissues were deparaffinized in
Citrosolv (Fisher Scientific, Ottawa, Ontario, Canada),
followed by dehydration in graded alcohol solutions.
Endogenous peroxidase activity was blocked using 3%
hydrogen peroxide in absolute methanol for 12 minutes
at room temperature. Antigen retrieval was achieved by
steaming the tissues in a vegetable steamer for 30 min-
utes using 0.01 M sodium citrate solution at a pH of 6.8.
Nonspecific protein binding was saturated using 4%
horse serum (Invitrogen, Burlington, Ontario, Canada)
in phosphate buffer saline (Invitrogen) for 20 minutes at
42uC, except when staining for von Willebrand’s factor
(vWF), where 4% goat serum was used. Primary
antibodies consisted of the following: monoclonal
mouse anti-human VEGF121, 1/20 (NeoMarkers, Fre-
mont, CA); monoclonal mouse anti-human VEGFR-2,
1/1,000 (Santa Cruz Biotechnology, Santa Cruz, CA);
polyclonal rabbit anti-human vWF, 1/2000 (DakoCyto-
mation, Mississauga, Ontario, Canada); and prediluted
monoclonal mouse anti-human Ki-67 (Zymed Labora-
tories Inc., San Francisco, CA). The diluted primary
antibodies were applied to tissue sections, and the slides
incubated 14–18 hours at 4uC. A biotinylated polyclonal
secondary horse anti-mouse antiserum (Vector Labora-
tories, Burlington, Ontario, Canada) was applied to
tissues for VEGF, VEGFR-2, and Ki-67 staining at
a dilution of 1/400 for 20 minutes at 42uC. A 1/400
dilution of biotinylated polyclonal sheep anti-rabbit
secondary antiserum was used to stain tissues for vWF.
ABC reagent prepared according to the manufacturer’s
directions (Vectastain Elite ABC kit, Vector Laborato-
ries) was applied for 35 minutes at 42uC. Color de-
velopment was achieved by applying a 3-39-diamino-
benzidine-tetrahydrochloride reagent (Sigma, Oakville,
Ontario, Canada) for 5 minutes. Tissues were counter-
stained with hematoxylin (Sigma), rehydrated in graded
alcohol, and mounted with coverslips.
When staining tissues for VEGF and VEGFR-2, we
also stained serial sections of tissues with omission of the
primary antibody and with an isotype-matched irrele-
vant antibody (NeoMarkers). Tissue from a canine

Fig. 4. Skin; canine squamous cell carcinoma. Immunohistochemical staining of endothelial cells for von
Willebrand factor antigen. Note the density of labeled vessels (arrows). ABC diaminobenzidine method,
hematoxylin counterstain, Bar 5 50 mm.
Fig. 5. Skin; canine squamous cell carcinoma, grade 2. Immunohistochemical staining of tumor cells for VEGF
antigen. Note the granular staining in the cytoplasm of tumor cells (arrows). ABC diaminobenzidine method,
hematoxylin counterstain, Bar 5 50 mm.
Fig. 6. Skin; canine squamous cell carcinoma, grade 2 (same tumor as shown in Fig. 5). Immunohistochemical
staining of tumor cells for VEGFR-2 antigen. Note the granular staining in the cytoplasm of tumor cells (arrows).
ABC diaminobenzidine method, hematoxylin counterstain, Bar 5 50 mm.
826 Al-Dissi, Haines, Singh, and Kidney
splenic hemangiosarcoma was used as a positive control
for VEGF. Tissue from a normal canine lymph node
was used a positive control for Ki-67. Normal vessels in
the surrounding tissue of each tumor were used as
a positive control for vWF.
Terminal deoxynucleotidyl transferase-mediated
29-deoxyuridine 59-triphosphate end-labeling assay for
apoptosis
Apoptosis was detected using the ApopTag Peroxi-
dase in Situ detection kit (Chemicon International Inc.,
Temecula, CA). Briefly, 5-mm tissue sections were
deparaffinized and rehydrated as described above.
Protein digestion was achieved using 20 mg/ml pro-
teinase K solution (Invitrogen/VWR, Saskatoon, Sas-
katchewan, Canada) for 15 minutes at room tempera-
ture. Endogenous peroxidase activity was blocked using
3.0% hydrogen peroxide. An equilibration buffer
supplied with the kit was applied for 15 seconds,
followed by working-strength terminal deoxynucleotidyl
transferase enzyme for 1 hour at 37uC. The slides were
covered with an antidigoxigenin conjugate and incubat-
ed in a humidified chamber for 30 minutes at room
temperature. Color development and counterstaining
were as described above for immunohistochemical
stains.
Immunohistochemical scoring for VEGF and VEGFR-2
In each of the SCC sections, the percentage of
positive tumor cells and the darkness of the brown
staining were estimated. A score ranging from 0 to 4 was
assigned to each tumor. SCCs were given a score of 0 if
they showed no staining, a score of 1 if less than 50% of
cells were positive with pale staining, a score of 2 if less
than 50% of cells were positive with dark staining,
a score of 3 if more than 50% of cells were positive with
pale staining, and finally a score of 4 if more than 50%
of cells were positive with dark staining. TCPs were
given a score of 0 or 1 if they were negative or positive,
respectively. All cases were evaluated independently by
2 of the authors (ANA and BAK). In instances of
discordant scores, a score was established by consensus.
Microvessel density, Ki-67, and apoptosis evaluation
iMVD was evaluated with antiserum detecting vWF
(Fig. 4), using the ‘‘hot-spot method.’’18 Slides were
examined at low power (403) to identify 10 areas with
high vessel density followed by re-examination of each
area at high power (4003) and counting of all positively
staining structures, irrespective of whether a lumen was
identified. Continuous vessels were counted as 1 vessel.
The average of the 10 fields was determined and was
expressed as count per high-power field in each tumor.
A similar method was used to evaluate sections
stained with Ki-67 monoclonal antibody to demonstrate
dividing tumor cells. The tissue sections were first
examined at low power (403) to identify 5 areas with
high numbers of positive cells. Within each area, a high-
power field (4003) was selected and 200 cells were
counted. A total of 1,000 cells were counted in 5 high-
Vet Pathol 44:6, 2007
power fields. The average number of positive cells in the
5 fields represented the PI. The AI was similarly
determined by counting 200 cells in 5 selected areas
and averaging numbers of positive cells using the
terminal deoxynucleotidyl transferase-mediated 29-
deoxyuridine 59-triphosphate end-labeling (TUNEL)
method assay in five 4003 fields.
Statistical analysis
Statistical analysis was performed using Statistix 7 for
Windows 2000 (Analytical Software, Tallahassee, FL).
The strength of the association between the 6 variables
evaluated (histologic grade, VEGF expression, VEGFR-
2 expression, iMVD, PI, and AI) was assessed by the
Spearman rank correlation coefficient. Correlation coef-
ficients were interpreted as follows: 0 $ r, no correlation;
0 , r , 0.1, trivial correlation; 0.1 # r , 0.3, slight
correlation; 0.3 # r , 0.5, moderate correlation; 0.5 # r
, 0.7, substantial or large correlation; and r $ 0.7, very
large correlation.4
The Kruskal-Wallis test was used to test for
differences in median iMVD, AI, and PI among VEGF
expression, VEGFR-2 expression, or histologic grade
group of SCCs. This test was also used to evaluate the
differences in VEGF or VEGFR-2 expression levels
among the different histologic grades of SCCs.
The Wilcoxon rank sum test was used to compare
median iMVD, PI, and AI between SCCs and TCPs.
Finally, the Wilcoxon rank sum test was also used in
TCPs to compare the median iMVD, PI, and AI
between VEGF- or VEGFR-2-positive and negative
tumor tissues. A P value calculated from Kruskal-Wallis
and Wilcoxon rank sum tests less than .05 was
considered significant.
Results
SCCs
Of the 19 SCCs, 8 (42.1%) were assigned
a histologic grade of 1; 8 (42.1%), grade 2; and 3
(15.8%), grade 3. The results for each of the
parameters evaluated are shown in Table 1. There
was a substantial correlation between histologic
grade and PI (r 5 0.51); a moderate correlation
between VEGF and histologic grade (r 5 0.43),
VEGFR-2 and histologic grade (r 5 0.47), VEGF
and PI (r 5 0.47), and VEGFR-2 and PI (r 5 0.47);
and a slight correlation between VEGF and
VEGFR-2 (r 5 0.20) (Spearman rank correlation
test). There were no significant differences in
median iMVD, PI, or AI among the different
histologic grades or among the different expression
levels of VEGF or VEGFR-2 (Kruskal-Wallis test).
TCPs
The results of testing canine TCPs are shown in
Table 2. There was a substantial correlation
between VEGF expression and PI (r = 0.51),
Vet Pathol 44:6, 2007 Angiogenesis in Squamous Cell Carcinoma and Trichoepithelioma 827

Table 1. Squamous cell carcinomas, VEGF, VEGFR-2, iMVD, PI, AI, and histologic grade.*

Tumor No. Histologic Grade VEGF VEGFR-2 iMVD PI AI

1 1 0 1 37.7 23 1
2 1 1 1 13.8 2.4 0.6
3 1 1 2 12 42 3
4 1 1 1 18.8 23.8 11.8
5 1 3 1 10.3 38.2 10
6 1 1 1 22.9 12.6 3.2
7 1 1 2 15.4 52.6 2.2
8 1 1 1 21.1 36 6
9 2 4 2 13.3 97.4 3.4
10 2 1 3 9.7 68.8 0.4
11 2 3 3 16.7 35.2 8.6
12 2 1 4 31.6 62.2 4
13 2 1 1 38.4 31.4 0.2
14 2 3 3 26.4 50.2 3.8
15 2 0 1 15.5 47 6.2
16 2 3 1 29.6 40.8 0
17 3 1 3 14.2 25.4 0
18 3 3 3 13.1 55.8 0.4
19 3 4 1 5.8 37.2 4.8
* VEGF 5 vascular endothelial growth factor, VEGFR-2 5 vascular endothelial growth factor receptor-2, iMVD 5
intratumoral microvessel density, PI 5 proliferation index, AI 5 apoptotic index.

a moderate correlation between VEGFR-2 expres-
sion and iMVD (r = 0.36), and no correlation
between AI and any other parameter (Spearman
rank correlation test). Tumors expressing VEGF
had a significantly higher median PI (56) than
tumors not expressing VEGF (43). Median iMVD
was significantly higher in tumors expressing
VEGFR-2 (10.2) than in tumors negative for
VEGFR-2 (6.1).
Comparison between SCCs and TCPs
VEGF expression (Fig. 5) was detected in 17/19
(89.4%) SCCs compared with 17/24 (70.8%) TCPs.
VEGFR-2 expression (Fig. 6) was detected in all of
the 19 SCCs (100%) compared with 20/24 (83.3%)
TCPs. Results of iMVD, PI, and AI comparisons
between the SCCs and TCPs are summarized in
Table 3. Median iMVD was significantly higher in
SCCs than in TCPs.
Discussion
The present study demonstrated for the first time
the expression of VEGFR-2 in canine SCCs,
although the protein has been detected in canine
melanomas32 and mammary tumors.33 In the
present study, VEGF was detected on 89% of
canine SCCs, while in a previous report, 100% of
canine SCCs were VEGF-positive.26 The difference
in percentages could be due to differences in the
methodology used to detect VEGF antigen, or
there may be an absence of VEGF expression in
a low number of canine SCCs. There was
a moderate correlation between the histologic
grade of SCC and both VEGF and VEGFR-2
expression; however, in agreement with the pre-
vious study,26 the present study failed to demon-
strate differences in VEGF expression that related
to histologic grade. The absence of differences in
VEGF expression (and, in the present study,
VEGFR-2) among the different histologic grades
could be due to the small sample sizes in both
studies.
Ki-67, a nuclear-associated protein expressed in
proliferating cells, has been evaluated in many
canine tumors.16,25,28,30,31,37,38,44 Ki-67 expression is
a useful prognostic indicator in canine mast cell
and mammary tumors.30,38 In canine lung cancer,
adenosquamous and squamous cell carcinomas
differed from the other histologic types in having
significantly higher proliferation indices.16 In the
present study, there was a correlation between
VEGF and VEGFR-2 expression and PI in SCCs
and between VEGF expression and PI in TCPs.
These findings suggest that VEGF may play a role
in stimulating tumor cell division. Such a role for
VEGF has been recently demonstrated in human
breast cancer, where treatment of a breast cancer
cell line with exogenous VEGF resulted in in-
creased cancer cell proliferation and increased
expression of BCL-2, an antiapoptotic protein.24
The lack of a significant difference in median PI
that related to the level of VEGF or VEGFR-2
828 Al-Dissi, Haines, Singh, and Kidney Vet Pathol 44:6, 2007

Table 2. Trichoepitheliomas: VEGF, VEGFR-2, iMVD, PI, and AI.*

Tumor No. VEGF VEGFR-2 iMVD PI AI

1 0 1 22.2 22.2 16.4
2 0 1 19.1 26.2 7.8
3 0 1 7.5 15 0.4
4 0 0 6 12.2 5.2
5 0 1 8.7 39.4 2.4
6 0 1 14.4 36.8 6.2
7 0 1 9.2 61 6
8 1 1 20.6 20.6 11.2
9 1 1 8.9 56.8 0
10 1 1 7.7 25.8 1.2
11 1 1 17.3 74.8 1.8
12 1 1 8.3 47.6 0
13 1 1 14.4 52.4 1.4
14 1 1 14.2 61.4 10.2
15 1 1 11.9 37.2 5.6
16 1 1 8.1 34.6 3.4
17 1 1 23.2 87.6 4.8
18 1 0 6.1 78.8 2.4
19 1 0 6.1 64.4 0
20 1 1 5.6 61.6 4.6
21 1 1 11.2 89.8 6.4
22 1 0 14.1 50 11.6
23 1 1 8.3 41.2 3.2
24 1 1 3.5 67 1.4
* VEGF 5 vascular endothelial growth factor, VEGFR-2:5 vascular endothelial growth factor receptor-2, iMVD 5
intratumoral microvessel density, PI 5 proliferation index, AI 5 apoptotic index.

expression in canine SCCs could be due to the
small sample size. TCPs had a higher PI than
SCCs, although the difference was not statistically
significant. Although the difference in the AI is not
statistically significant between the 2 tumor types,
the trend points in the right direction to counteract
the higher PI noted in TCPs. The association of
VEGF expression and iMVD in SCCs is uncertain.
Some studies report that iMVD correlates with
VEGF expression in human esophageal SCCs,22
while others1,41 failed to find such a correlation. In
the present study, no correlation was found
between iMVD and either VEGF expression or
histologic grade. This is in contrast to other studies
of canine SCC27 that reported a significant differ-
ence in iMVD among histologic grades of tumors.
The disparity with the current study could be due
to the differences in iMVD evaluation methods.
The present study used expression of vWF to detect
endothelial cells, while the previous report utilized
expression of CD31; the latter may be a superior
marker for endothelial cells in canine tissues.11
SCCs had higher iMVD than TCPs. The higher
iMVD in malignant tumors may reflect their ability
to proliferate and metastasize by promoting angio-
genesis. The inflammatory neoangiogenesis associ-
ated with the presence of free keratin in the dermis in
SCCs could also be responsible for the high iMVD.
The finding of a significantly higher median iMVD
in TCPs expressing VEGFR-2, but not VEGF, may

Table 3. Median iMVD, PI, and AI: comparison between SCCs and TCPs.*

Median (1st, 3rd quartiles)

iMVD PI AI
{
SCCs 15.5 (13.1, 26.4) 40.8 (25.4, 55.8) 3.2 (0.4, 6.0)
TCPs 9.1 (7.6, 14.4) 48.8 (34.2, 63.7) 4.0 (1.4, 6.4)
* iMVD 5 intratumoral microvessel density, PI 5 proliferation index, AI 5 apoptotic index, SCCs 5 squamous cell
carcinomas, TCPs 5 trichoepitheliomas.
{ P value , .05 using Wilcoxon rank sum test. Median iMVD of SCCs was significantly higher than that of TCPs.
Vet Pathol 44:6, 2007 Angiogenesis in Squamous Cell Carcinoma and Trichoepithelioma
indicate that VEGFR-2 plays a role in vascular
proliferation through a pathway that is independent
of VEGF. Other members of the VEGF family (i.e.,
VEGF-C) can stimulate VEGFR-2 and may con-
tribute to angiogenesis.3 The expression of VEGF
and VEGFR-2 in TCPs was high compared with the
15% (3/20) reported in basal cell tumors.26
The detection of VEGF and VEGFR-2 in canine
SCCs and TCPs and the correlation between the
expression of these molecules and PI suggest an
autocrine function of VEGF in these tumor cells.
Autocrine function of VEGF has been documented in
human breast and pancreatic cancer.42,43 In pancreatic
cancer, VEGF treatment resulted in phosphorylation
of the mitogen-activated protein kinase and growth of
VEGFR-2 expressing pancreatic adenocarcinoma cell
lines.42 The AI did not correlate with the studied
parameters of angiogenesis.
In conclusion, VEGF and VEGFR-2 expression
was detected in neoplastic cells of TCPs and SCCs.
The expression of both molecules was associated
with increased PI, which suggests that they may
stimulate tumor cell growth through an autocrine
pathway. Since most SCCs were found to express
VEGF and VEGFR-2, these tumors may respond
to therapy targeting these molecules.
Acknowledgements
We thank Ms. B. Trask and Dr. C. Waldner for their
technical and statistical assistance, respectively. This
study was funded by a grant from the Western College
of Veterinary Medicine Companion Animal Health
Fund, Saskatoon, Saskatchewan, Canada.
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