Evaluación de adenosina desaminasa en saliva y suero, y α-amilasa salival, en piometra canina en el momento del diagnóstico y después de la ovariohisterectomía

The Veterinary Journal 236 (2018) 102–110
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The Veterinary Journal
journal homepage: www.elsevier.com/locate/tvjl
Original Article
Evaluation of adenosine deaminase in saliva and serum, and salivary
a-amylase, in canine pyometra at diagnosis and after
ovariohysterectomy
F. Teclesa , D. Escribanob , M.D. Contreras-Aguilara , C.P. Rubioa , M. Szczubiałc, J.J. Ceróna ,
R. Da˛browskic,* , A. Tvarijonaviciutea
a
Interdisciplinary Laboratory of Clinical Analysis of the University of Murcia (Interlab-UMU), Regional Campus of International Excellence ‘Campus Mare
Nostrum’, University of Murcia, Campus de Espinardo s/n, 30100 Espinardo, Murcia, Spain
b
Department of Animal and Food Science, Faculty of Veterinary Medicine, Universitat Autonoma de Barcelona, 08193 Bellaterra, Spain
c
Department and Clinic of Animal Reproduction, Faculty of Veterinary Medicine, University of Life Sciences in Lublin, 20-612 Lublin, Poland
A R T I C L E I N F O
Article history:
Accepted 25 April 2018
Keywords:
a-Amylase
Adenosine deaminase
Bitch
Pyometra
Saliva
A B S T R A C T
An assay for adenosine deaminase (ADA) was validated in serum and saliva in dogs. Changes in ADA and
salivary a-amylase activities were analysed in 26 bitches diagnosed with pyometra and compared with
activities in 19 healthy bitches. All animals were classified according to the American Society of
Anaesthesiologists (ASA) scoring for physical status. In the validation study, the ADA assay had an
imprecision < 12% and determination coefficients > 0.90 in linearity under dilution experiments, with
recoveries of 99.2–114.4%. On the day of presentation, salivary ADA activity was significantly higher in
dogs with pyometra than in healthy dogs (median values 7.1 IU/L vs. 0.8 IU/L, respectively; P < 0.01). ADA
had a moderate positive correlation with leucocyte and band neutrophil counts, haptoglobin, salivary
a-amylase and ASA score, and a low positive correlation with C-reactive protein. There were no
significant differences in salivary a-amylase activity between dogs with pyometra and healthy dogs
(57.3 IU/L vs. 27.4 IU/L, respectively). Salivary a-amylase had a low correlation with ASA grade, and
leucocyte and band neutrophil counts. In 7/26 bitches with pyometra that were sampled 3 and 10 days
after ovariohysterectomy, there were no significant changes in a-amylase or ADA activities. These results
indicate that ADA activity is increased in the saliva of bitches with pyometra, probably related to systemic
inflammation.
© 2018 Elsevier Ltd. All rights reserved.
Introduction
Pyometra is relatively common in intact bitches and its
diagnosis is aided by ultrasonography (Bigliardi et al., 2004),
along with measurement of acute phase proteins and leucocyte
(white blood cell, WBC) counts; assessment of these parameters
during recovery may be helpful for monitoring systemic inflammation and for detecting early complications (Da˛browski et al.,
2009; Jitpean et al., 2014). Saliva can be used for evaluation of
markers of inflammation and stress, and can be collected relatively
non-invasively and with minimal stress in veterinary species,
including dogs, for the evaluation of markers of inflammation and
stress (Vincent and Michell, 1992; Parra et al., 2005).
* Corresponding author.
E-mail address: roman.dabrowski@up.lublin.pl (R. Da˛browski).
https://doi.org/10.1016/j.tvjl.2018.04.018
1090-0233/© 2018 Elsevier Ltd. All rights reserved.
Adenosine deaminase (ADA; Enzyme Commission number
3.5.4.4) is an enzyme that catalyses the irreversible conversion of
adenosine and deoxyadenosine to inosine and deoxyinosine,
respectively. This enzyme has been proposed in human beings
as a biomarker of inflammation (Mishra et al., 1994). ADA activity is
increased in saliva in human beings with squamous cell carcinoma
of the tongue (Rai et al., 2011). In experimental infection of dogs
with Ehrlichia canis, serum ADA activity decreased at 12 days, then
increased at 30 days after infection (Da Silva et al., 2013). ADA
activity is decreased in bitches with mammary tumours compared
to unaffected dogs (Machado et al., 2015) and in dogs with
leishmaniasis compared to uninfected dogs (Tonin et al., 2016).
Therefore, the role of ADA as a marker of inflammation in dogs is
still to be fully elucidated.
Dogs may perceive different degrees of stress when they face
new situations, such as a visit to a veterinary clinic or being
hospitalised. This induced stress reaction can involve the
F. Tecles et al. / The Veterinary Journal 236 (2018) 102–110
sympathetic-adrenomedullary (SAM) system and the hypothalamic-pituitary-adrenal (HPA) axis. Salivary chromogranin A and
a-amylase (Enzyme Commission number 3.2.1.1) are considered to
be markers of activation of the SAM system in the dog, whereas
cortisol concentrations increase in dogs after activation of the HPA
(Contreras-Aguilar et al., 2017a; Srithunyarat et al., 2017). Serum
cortisol concentrations are increased in bitches with pyometra and
decrease after treatment (Reinoldes, 2010).
The aims of this study were to evaluate salivary ADA as a possible
indicator of inflammation in bitches with pyometra, as well as to
evaluate salivary a-amylase as a marker of activation of the SAM
system. To achieve these aims, a commercial assay for ADA was
validated in canine serum and saliva, and salivary ADA and a-amylase
were evaluated, in dogs with pyometra and unaffected dogs. Changes
in these analytes after treatment were assessed to determine their
possible usefulness for monitoring responses to treatment.
Materials and methods
Animals
Bitches undergoing ovariohysterectomy at the Department and Clinic of Animal
Reproduction, University of Life Sciences, Lublin, Poland, from November 2016 to May
2017 were included in this study. The first group comprised unaffected bitches that
were classified as healthy after complete physical examination, haematology and
biochemistry. The second group comprised bitches diagnosed with pyometra after
clinical examination and additional testing, including haematology and biochemistry.
In most cases the clinical examination revealed pyrexia, polydipsia, polyuria, anorexia,
apathy and abnormal colour of mucous membranes. Vaginal discharge was observed
in all diseased animals and there were abundant degenerate neutrophils with
intracellular bacteria on cytological examination of vaginal smears. Findings on
abdominal ultrasound in all affected bitches were consistent with pyometra.
Bitches with pyometra were treated by ovariohysterectomy after premedication
with 30 mg/kg medetomidine (Domitor, Pfizer) intramuscularly, 0.2 mg/kg butorphanol (Torbugesic, Zoetis) intravenously and 2 mg/kg ketamine (VetaKetam, VetAgro)
intravenously. Anaesthesia was maintained with 2% isoflurane (Iso-Vet, Chanelle).
Cephalexim (Cefalexim 18%, ScanVet) was administered for 5 days post-surgery at
10 mg/kg subcutaneously. Pyometra was confirmed by histopathological examination
of the uterus and ovaries by veterinary pathologists in the Department of Pathological
Anatomy, University of Life Sciences, Lublin, Poland. Uterine pus from all affected
bitches was submitted for bacteriology. Bitches with pyometra were classified
according to their physical status using a scoring system adapted from the American
Society of Anaesthesiologists (ASA grade; Table 1) (Thurmon et al., 1996).
Collection of samples
Saliva was collected using Salivette tubes (Sarstedt) containing a sponge. Each
dog was allowed to chew the sponge until it was thoroughly moist, then the sponges
were placed in the tubes and kept refrigerated during transportation to the
laboratory. At the laboratory, the tubes were centrifuged at 3000 g and 4 C for
10 min. Blood samples were obtained by cephalic vein venepuncture using 21 G
needles and 5 mL syringes.
A portion of each blood sample was transferred into tubes (Medlab Products)
containing ethylenediaminetetraacetic acid (K3-EDTA) for haematology. The
remainder of each blood sample was drawn into Vakuette silicone test tubes
(Greiner Labortechnik) and allowed to clot for 30 min, then the tubes were
centrifuged (15 min) at 3000 g to obtain serum. Seven out of the 26 bitches with
pyometra were sampled after treatment for both serum and saliva. In these animals,
samples were collected before surgery (T0), at 3 days (T1) and at 10 days (T2) after
surgery. The research protocols used in the current study were approved by the
University of Lublin institutional animal care and ethics committee (approval
number 27/2015, date of approval 18th May 2015).
103
Analytical measurements
Complete blood cell counts were performed with a haematology analyser (Scil Vet
Plus+ Horiba ABX) and leukocyte differential counts were performed manually by
examination of routinely stained blood smears. Routine biochemistry included testing
for alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin, urea and
creatinine (MindrayBS-130 W; Shenzhen Mindray Bio-medical Electronics Company)
These analyses were performed in the Department and Clinic of Animal Reproduction,
Faculty of Veterinary Medicine, University of Life Sciences, Lublin, Poland.
Serum and saliva samples were frozen at 76 C (Polar 110 H, Angelantoni
Industries) until transported to the Interdisciplinary Laboratory of the University of
Murcia, Spain (Interlab-UMU) for further analyses. Samples were shipped on dry ice
and the time elapses between sampling and shipping was < 6 months. Serum Creactive protein (CRP) concentrations were measured using a human immunoturbidimetric test (CRP OSR 6147 Olympus Life and Material Science) previously
validated in dogs (Martínez-Subiela and Cerón, 2005). Serum haptoglobin (Hp)
concentration was measured using a commercial colorimetric method (Tridelta
Phase Range Haptoglobin Kit, Tridelta Development), previously validated in the
authors’ laboratory for canine samples (Martínez-Subiela and Cerón, 2005). These
analytes were measured using a chemistry analyser (Olympus AU2700, Olympus
Diagnostica). Serum cortisol concentrations were determined using a competitive
chemiluminescence immunoassay (Immulite, Diagnostic Products Corporation)
previously validated for use in dogs (Singh et al., 1997).
Total ADA activity was measured in serum and saliva samples using a
spectrophotometric automated method (Adenosine Deaminase Assay Kit, Diazyme
Laboratories) validated at Interlab-UMU (see Appendix: Supplementary material).
Salivary a-amylase activity was measured using a commercially available method
(a-Amylase, Beckman Coulter) previously validated for canine saliva at InterlabUMU (Contreras-Aguilar et al., 2017b). Total protein concentrations in saliva
samples were determined using a colorimetric method (Protein in Urine and CSF,
Spinreact) to evaluate the influence of saliva concentration on the results. ADA and
a-amylase activities are expressed as IU/g of protein. These analytes were measured
in an automated clinical chemistry analyser (Olympus AU2700).
Statistical analysis
Means, standard deviations, coefficients of variation (CVs) and regression
analyses were determined. Data obtained from healthy and diseased animals were
evaluated for normality of distribution, using Shapiro–Wilk/Kolmogorov–Smirnov
tests. Multivariate linear regression was performed to assess whether age could be a
significant predictor for the analytical parameters. To assess any difference between
groups, normally distributed data were checked using a two-tailed t test. The
Mann–Whitney U was used for data that was not normally distributed. For the
seven bitches in which follow-up was recorded, changes were assessed with a oneway analysis of variance of repeated measures, followed by Tukey’s post-hoc test for
normal data, and Friedman’s test followed by Dunn’s multiple comparisons test in
the case of non-normally distributed data. Effect size was assessed by Cohen’s d and
f coefficients: d > 0.8 and f > 0.4 were considered to be large size effects (Cohen,
1988). Correlations of salivary ADA and a-amylase activities with ASA scores,
inflammatory markers (WBC and band counts, CRP, Hp and rectal temperature) and
stress biomarkers (serum cortisol) were studied by Spearman correlation assays
and linear regression plots were constructed. Data analyses were performed using
Excel 2000 (Microsoft Corporation), GraphPad Prism version 5 for Windows
(GraphPad Software) and SPSS Statistics version 24 (IBM Corporation). Values of
P < 0.05 were considered to be statistically significant.
Results
Clinical findings
The healthy group included 18 bitches 0.5–6 years of age (mean
2.8 years), body weight 5.0–42 kg (mean 24.2 kg), body condition
score (BCS) 2.0–4.0 (mean 3.2 out of 5) and rectal temperature
Table 1
Classification of physical status using a modification of the American Society of Anaesthesiologists (ASA) grade (Thurmon et al., 1996).
Category
I
II
III
IV
V
Physical status
Normal healthy animal
Animal with mild systemic disease
Examples of conditions in this category
No discernible disease; animals entered for ovariohysterectomy or castration
Skin tumour; fracture without shock; uncomplicated hernia; cryptorchidectomy; localised
infection; compensated cardiac disease
Animal with severe systemic disease
Pyrexia; dehydration; anaemia; cachexia; moderate hypovolaemia
Animal with severe systemic disease that is a constant Uraemia; toxaemia; severe dehydration and hypovolaemia; anaemia; cardiac decompensation;
thread to life
emaciation; high fever
Moribund animal not expected to survive 1 day with or Extreme shock and dehydration; terminal malignancy or infection; severe trauma
without operation
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F. Tecles et al. / The Veterinary Journal 236 (2018) 102–110
37.1–38.7 C (mean 38.0 C) (Table 2). Seven healthy bitches were
of mixed breed; the remainder consisted of six German shepherds,
two Labrador retrievers, and one of each of Dachshund, Bull terrier
and Jack Russell terrier. All healthy bitches had an ASA score of 1.0.
The pyometra group was composed of 25 bitches 3–15 years of age
(mean 8.7 years; P < 0.001 with respect to the healthy group), body
weight 3–42 kg (mean 21.7 kg), body condition score 2.0–4.0
(mean 3.1) and rectal temperature 37.0–40.1 C (mean 38.7 C;
P < 0.01 with respect to the healthy group). Twelve bitches with
pyometra were of mixed breed; the remainder consisted of six
German shepherds and one of each of Maltese, French bulldog,
Golden retriever, Yorkshire terrier, Pekinese, Dachshund and
Husky. The median ASA score of bitches with pyometra was 4.0,
which was significantly higher (P < 0.001) than healthy bitches.
Escherichia coli was recovered from uterine pus from all bitches
with pyometra.
Haematology, serum biochemistry and acute phase proteins
Results of the different analytes in blood, serum and saliva in
bitches with pyometra and healthy bitches are shown in Table 2.
There were no statistically significant differences in red blood cell
parameters between the two groups. In bitches with pyometra,
there were significantly higher WBC counts (P < 0.001), band
neutrophil counts (P < 0.001), and serum ALT (P < 0.01), bilirubin
(P < 0.05), urea (P < 0.001), creatinine (P < 0.01), CRP (P < 0.001),
Table 2
Haematology, serum and saliva biochemistry in healthy bitches (n = 18) and bitches
with pyometra (n = 25) at the day of presentation; results are given in mean
(standard deviation) or median (25th percentile–75th percentile), respectively
when data followed normal or non-normal distribution.
Healthy
Pyometra
Cohen’s d
Clinical data
Age (years)
Body weight (kg)
BCS
ASA
Temperature ( C)
2.0 (1.8–5.0)
24.2 (11.7)
3.0 (3.0–4.0)
1.0 (1.0–1.0)
38.0 (0.4)
8.0 (7.0–11.0)c
21.7 (11.9)
3.0 (3.0–3.0)
4.0 (3.5–4.0)c
38.7 (1.0)b
2.13
0.21
0.21
5.84
0.84
Haematology
RBC (1012/L)
HGB (g/dL)
HCT (%)
WBC (109/L)
Bands (109/L)
PLT (109/L)
6.2 (1.1)
15.2 (2.5)
41.5 (3.8)
11.4 (2.2)
0.2 (0.1)
280.5 (62.4)
5.6 (1.2)
14.4 (2.5)
40.5 (7.7)
31.0 (13.0)c
1.5 (1.0)c
318.6 (125.6)
0.56
0.33
0.16
1.94
1.83
0.37
Serum biochemistry
ALT (IU/L)
AST (IU/L)
Creatinine (mmol/L)
Urea (mmol/L)
Bilirubin (mmol/L)
CRP (nmol/L)
Hp (g/L)
Cortisol (nmol/L)
ADA (IU/L)
40.0 (34.7–42.5)
52.5 (13.8)
106.0 (24.2)
10.8 (10.2–13.8)
1.1 (0.5)
28.1 (23.8–41.9)
1.9 (0.5–3.2)
107.9 (80.9–140.5)
4.2 (2.7–7.1)
64.0 (39.3–89.5)b
60.3 (35.6)
162.7 (65.2)b
22.1 (16.1–28.0)c
1.7 (1.0)a
850.5 (372.4–930.5)c
4.8 (4.4–5.0)c
193.8 (146.0–288.3)c
5.6 (2.9–7.9)
0.67
0.27
0.84
0.57
0.72
2.21
1.84
1.04
0.06
Saliva biochemistry
ADA (IU/L)
ADA (IU/g)
a-Amylase (IU/L)
a-Amylase (IU/g)
0.8 (0.1–3.2)
0.4 (0.0–1.1)
26.5 (17.9–49.4)
14.7 (6.6–21.3)
7.1 (2.2–15.5)c
1.9 (0.9–3.0)b
57.3 (19.9–111.9)
17.0 (9.4–30.2)
0.62
0.35
0.07
0.03
BCS, body condition score; ASA grade, American Society of Anaesthesiologists score;
RBC, red blood cells count; HGB, haemoglobin concentration; HCT, haematocrit;
WBC, white blood cells count; PLT, platelets count; ALT, alanine aminotransferase;
AST, aspartate aminotransferase; CRP, C-reactive protein; Hp, haptoglobin; ADA,
adenosine deaminase.
a
P < 0.05.
b
P < 0.01.
c
P < 0.001.
Hp (P < 0.001) and cortisol concentrations than in healthy bitches.
Since differences in age were statistically different between
affected and unaffected bitches groups, a multivariate linear
regression was performed to assess whether age might have
influenced analytical results. Multivariate analysis showed that age
was not a significant predictor for any of the studied analytical
parameters (Pillai–Bartlett trace P = 0.338).
Serum and salivary adenosine deaminase and salivary a-amylase
There was no significant difference in serum ADA activity
between groups. Salivary ADA activity was significantly higher in
bitches with pyometra than in healthy bitches (P < 0.01), whereas
there was no significant difference in salivary a-amylase between
the two groups. The correlations of salivary ADA and a-amylase
activities with haematology, serum biochemistry, rectal temperature and ASA scale are shown in Figs. 1 and 2. Salivary ADA activity
expressed in IU/L had a high positive correlation with activity
expressed in IU/g of protein; therefore, only ADA activity in IU/L
was used for the remainder of the correlations. ADA had a
moderate positive correlation with WBC and band neutrophil
counts, Hp concentration, salivary a-amylase activity and ASA
score, but a low positive correlation with CRP. There was no
correlation between salivary and serum ADA activities. Salivary
a-amylase activity expressed in IU/L had a high positive correlation
with activity expressed in IU/g of protein; therefore, only
a-amylase in IU/L was used for the rest of the correlations. A
low positive correlation was observed between salivary a-amylase
activity and WBC and band neutrophil counts, as well as ASA grade.
Follow-up of bitches with pyometra after ovariohysterectomy
Follow-up data for seven bitches with pyometra after ovariohysterectomy are shown in Table 3. ASA grade was significantly
decreased (P < 0.01) at T2 compared to T0. Rectal temperature was
significantly decreased at T1 (P < 0.05) and T2 (P < 0.01) when
compared to T0. Haematology results showed only a significant
decrease in WBC and band neutrophil counts at T2 compared to T0
(P < 0.05). There were significant decreases in urea (P < 0.001) and
CRP concentrations (P < 0.05) in bitches with pyometra after
ovariohysterectomy. No significant changes was observed in serum
and salivary ADA activity, and salivary a-amylase activity, after
ovariohysterectomy.
Correlations between analytes obtained during the follow up of
bitches with pyometra after ovariohysterectomy are shown in
Figs. 3 and 4. Since both salivary ADA and a-amylase expressed in
IU/L had very high and high correlations, respectively, with
activities expressed in IU/g of protein, only activities expressed in
IU/L were used for the rest of the correlations. Salivary ADA activity
had a moderate positive correlation with WBC and band neutrophil
counts, as well as a low positive correlation with ASA grade. There
was no correlation between salivary and serum ADA activities.
There was no correlation was observed between salivary a-amylase activity and other analytes, ASA grade or rectal temperature.
WBC and band neutrophil counts had a high positive correlation
with ASA score (Spearman correlation coefficients 0.848 and 0.801,
respectively; P < 0.001). ASA score had a moderate positive
correlation with CRP concentration (Spearman correlation coefficient 0.569; P < 0.01) and rectal temperature (Spearman correlation coefficient 0.547; P < 0.05).
Discussion
In this study, an inflammatory response was observed in bitches
with pyometra at the time of presentation, since inflammatory
markers, such as WBC and band neutrophil counts, and CRP and Hp
F. Tecles et al. / The Veterinary Journal 236 (2018) 102–110
105
Fig. 1. Linear regression plots and Spearman correlation coefficients for bitches with pyometra obtained at the day of presentation between salivary adenosine deaminase
(ADA) expressed in IU/L and salivary ADA (expressed in IU/g of protein), serum ADA, American Society of Anaesthesiologists (ASA) score, rectal temperature, white blood cell
(WBC) and band neutrophil counts, serum C-reactive protein (CRP), serum haptoglobin (Hp), serum cortisol and salivary a-amylase (a-A).
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F. Tecles et al. / The Veterinary Journal 236 (2018) 102–110
Fig. 2. Linear regression plots and Spearman correlation coefficients for bitches with pyometra obtained at the day of presentation between salivary a-amylase (a-A)
expressed in IU/L and salivary a-A (expressed in IU/g of protein), American Society of Anaesthesiologists (ASA) score, rectal temperature, white blood cell (WBC) and band
neutrophil counts, serum C-reactive protein (CRP), serum haptoglobin (Hp) and serum cortisol.
concentrations were increased. All cases were uncomplicated, with
no evidence of anaemia, leucopaenia, vomiting, dehydration,
cachexia or systemic inflammatory response syndrome (SIRS), nor
high levels of hepatic enzymes, urea or creatinine. Inflammatory
markers decreased in monitored animals following ovariohysterectomy.
Salivary ADA activity was significantly higher in dogs with
pyometra than in healthy dogs and was significantly correlated
with the serum inflammatory biomarkers CRP, Hp and WBC count.
Since previous studies have correlated ASA grade with outcome
(Sigrist et al., 2008; Goggs et al., 2015), the relationship between
ASA and salivary parameters was studied in order to evaluate their
possible prognostic usefulness. ADA activity was correlated with
ASA grade, indicating that it could be of value as a prognostic factor,
although this should be tested in a larger population of dogs and
with a range of post-operative outcomes.
Salivary ADA activity was not correlated with serum ADA
activity. Furthermore, serum ADA activity was not correlated with
any inflammatory marker, so the role of serum ADA as
inflammatory biomarker in dogs requires further study, since it
could be unaffected, as in our study, or could increase (Da Silva
et al., 2013) or decrease (Machado et al., 2015; Tonin et al., 2016),
F. Tecles et al. / The Veterinary Journal 236 (2018) 102–110
107
Table 3
Follow up of the American Society of Anaesthesiologists (ASA) score, rectal temperature, haematology, serum and saliva biochemistry in bitches with pyometra (n = 7) at the
day of presentation (T0), 3 days after surgery (T1) and 10 days after surgery (T2); results are given as mean (standard deviation) or median (25th percentile–75th percentile),
respectively when data followed normal or non-normal distribution.
Parameter
T0
T1
T2
Cohen’s f
Clinical data
ASA
Temperature ( C)
4.0 (3.0–4.0)
38.8 (0.9)
4.0 (4.0–4.0)
38.1 (0.5)a
2.0 (1.0–2.0)b
37.7 (0.5)b
1.74
0.77
Haematology
RBC (1012/L)
HGB (g/dL)
HCT (%)
WBC (109/L)
Bands (109/L)
PLT (109/L)
5.7 (1.5)
14.4 (3.8)
40.5 (10.6)
31.2 (17.4)
1.6 (1.4)
247.0 (79.5)
5.4 (1.7)
13.8 (2.8)
39.4 (9.8)
35.7 (18.3)
1.1 (0.9)
207.3 (64.1)
6.1 (0.5)
14.8 (2.9)
38.8 (7.8)
15.5 (3.2)a
0.3 (0.2)a
256.4 (87.6)
0.24
0.13
0.07
0.67
0.65
0.28
Serum biochemistry
ALT (IU/L)
AST (IU/L)
Creatinine (mmol/L)
Urea (mmol/L)
Bilirubin (mmol/L)
CRP (nmol/L)
Hp (g/L)
Cortisol (nmol/L)
ADA (IU/L)
43.0 (22.0–64.0)
43.0 (25.8–78.0)
156.0 (70.6)
23.6 (16.4–32.2)
0.5 (0.4–2.3)
876.2 (360.0–937.2)
4.9 (4.8–5.1)
251.3 (166.2–305.5)
5.2 (2.6–7.1)
25.1 (20.0–60.0)
37.0 (30.0–41.0)
103.3 (47.5)
14.3 (12.5–27.3)
0.8 (0.8–2.1)
372.4 (133.3–437.2)
5.0 (4.5–5.1)
182.7 (63.7–317.4)
7.1 (3.6–8.4)
35.0 (18.0–52.0)
36.0 (17.0–44.0)
93.2 (26.1)
12.5 (10.7–18.3)c
1.1 (0.6–1.4)
150.5 (130.5–250.5)a
5.1 (4.6–5.1)
147.4 (67.0–213.8)
7.1 (4.1–20.6)
0.20
0.26
0.57
0.14
0.14
1.15
0.20
0.45
0.34
Saliva biochemistry
ADA (IU/L)
ADA (IU/g)
a-Amylase (IU/L)
a-Amylase (IU/g)
4.9 (1.4–20.6)
1.1 (0.4–2.9)
57.3 (15.7–127.1)
8.8 (4.4–30.7)
8.2 (4.0–9.7)
1.6 (1.1–2.0)
46.3 (33.6–67.4)
10.2 (7.2–18.3)
4.6 (1.4–6.8)
0.8 (0.4–1.3)
66.3 (30.7–96.8)
8.7 (6.8–22.8)
0.39
0.39
0.43
0.37
ASA grade, American Society of Anaesthesiologists score; RBC, red blood cell count; HGB, haemoglobin concentration; HCT, haematocrit; WBC, white blood cell count; PLT,
platelets count; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CRP, C-reactive protein; Hp, haptoglobin; ADA, adenosine deaminase.
a
P < 0.05.
b
P < 0.01.
c
P < 0.001.
depending on the specific disease. One dog in the healthy group
had a very high ADA activity in saliva for an unknown reason; this
activity remained high when values were normalised by the
protein content. The possible different sources of this enzymatic
activity in saliva should be studied further to improve our
understanding of its role.
In human beings, increases in salivary ADA activity have been
associated with oral diseases, such as squamous cell carcinoma of
the tongue, increasing as the stage of the disease increases (Rai
et al., 2011). Accelerated purine metabolism and the salvage
pathway activity of purine nucleotides in cancerous human oral
tissues has been proposed as a mechanism, suggesting that ADA
could have a role in the progression and invasion of cancer (Rai
et al., 2011).
Salivary ADA did not significantly decrease after ovariohysterectomy. This may be due to a slow ADA response; further studies
monitoring animals for a longer period should be done to generate
data on the half-life of salivary ADA. Alternatively, subclinical
inflammation could maintain higher ADA activity after clinical
signs have improved or resolved. The presence of ongoing
subclinical inflammation might be indicated by persistently
elevated CRP values; 6/7 bitches still had CRP values over the
reference limit of our laboratory at T2.
Salivary a-amylase activity was not significantly different
between bitches with pyometra and healthy bitches, and there
were no significant changes after ovariohysterectomy, similar to
chromogranin A (Jitpean et al., 2015). Therefore, it is postulated
that the SAM axis is not activated in bitches with pyometra. In
contrast, bitches with pyometra may have activation of the HPA
axis, since they had higher serum cortisol concentrations than
healthy bitches, similar to a previous report (Reinoldes, 2010).
Salivary a-amylase activity was correlated with salivary ADA
activity, WBC count and ASA grade in bitches with pyometra on the
day of presentation. An increase in salivary a-amylase activity has
been reported in human beings in response to endotoxin (Grigoleit
et al., 2013).
The lack of significant changes between groups and also
following ovariohysterectomy could have been influenced by the
very high inter-individual variability in salivary a-amylase values,
which had ranges of 15.6–775.3 IU/L at T0, 23.7–96.8 IU/L at T1 and
12.6–105.6 IU/L at T2. A similar high variability in a-amylase
activity has been reported previously (Contreras-Aguilar et al.,
2017b). Furthermore, an increase in salivary a-amylase activity
due to the stress associated with an unfamiliar place, such as the
veterinary hospital, could have influenced results.
Limitations of this study include: (1) the mean age of groups
was significantly different, although the statistical study indicated
that did not affect the results; (2) local inflammation, such as
gingivitis, could affect salivary results, as has been reported in
human beings (Haririan et al., 2012); (3) the data on salivary
analytes should be taken with caution since size effect was
considered as medium for salivary ADA and low for salivary
a-amylase, due to the high inter-individual variability; and (4)
evaluation of the analytes for treatment monitoring should also
include different outcomes in order to study the prognostic use of
these analytes.
The aim of this study was to explore changes in ADA and
a-amylase activities in saliva in dogs with pyometra, whereas it did
not have the purpose of evaluating saliva as an alternative to blood.
The data presented in this paper showed that there is an overlap in
these analytes between bitches with pyometra and healthy
bitches. In addition, there were no significant changes in ADA or
108
F. Tecles et al. / The Veterinary Journal 236 (2018) 102–110
Fig. 3. Linear regression plots and Spearman correlation coefficients obtained during follow up of 7 bitches with pyometra between salivary adenosine deaminase (ADA)
expressed in IU/L and salivary ADA (expressed in IU/g of protein), serum ADA, American Society of Anaesthesiologists (ASA) score, rectal temperature, white blood cell (WBC)
and band neutrophil counts, serum C-reactive protein (CRP), serum haptoglobin (Hp), serum cortisol and salivary a-amylase (a-A).
F. Tecles et al. / The Veterinary Journal 236 (2018) 102–110
109
Fig. 4. Linear regression plots and Spearman correlation coefficients obtained during follow up of 7 bitches with pyometra between salivary a-amylase (a-A) expressed in IU/
L and salivary a-A (expressed in IU/g of protein), American Society of Anaesthesiologists (ASA) score, rectal temperature, white blood cell (WBC) and band neutrophil counts,
serum C-reactive protein (CRP), serum haptoglobin (Hp) and serum cortisol.
a-amylase activities following ovariohysterectomy and these
enzymes had lower correlations with ASA score than WBC and
band neutrophil counts, CRP or rectal temperature. Therefore, CBC
counts and blood smear evaluations, as well as the analysis of
serum CRP would be preferred for the evaluation and treatment
monitoring of pyometra.
there were no significant differences in salivary a-amylase activity
between bitches with pyometra and healthy bitches. Further
studies should be undertaken in order to elucidate and refine our
knowledge about ADA and a-amylase in the saliva of dogs with
pyometra, including their possible differences in complicated and
uncomplicated pyometra, as well as in other canine-specific
diseases.
Conclusions
Conflict of interest statement
Although this was a preliminary study, with results that should
be confirmed in a larger population of dogs, it has shown that ADA
activity is increased in saliva of bitches with pyometra and that this
increase could be related to systemic inflammation. In addition,
None of the authors of this paper has a financial or personal
relationship with other people or organisations that could
inappropriately influence or bias the content of the paper.
110
F. Tecles et al. / The Veterinary Journal 236 (2018) 102–110
Acknowledgement
This study was funded by a grant from the Seneca Foundation of
Murcia Region, Spain (19894/GERM/15).
Appendix: Supplementary data
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.tvjl.2018.04.018.
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