The Veterinary Journal 236 (2018) 102–110 Contents lists available at ScienceDirect 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: [email protected] (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 22.214.171.124) 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 126.96.36.199) 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 104 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). 106 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. References Bigliardi, E., Parmigiani, E., Cavirani, S., Luppi, A., Bonati, L., Corradi, A., 2004. Ultrasonography and cystic endometrial hyperplasia–pyometra-complex in the bitch. Reproduction of Domestic Animals 39, 136–140. Cohen, J., 1988. Statistical Power Analysis for the Behavioural Sciences, Second Ed. Lawrence Erlbaum Associates, New York, NY, USA. Contreras-Aguilar, M.D., Escribano, D., Martínez-Subiela, S., Martínez-Miró, S., Rubio, M., Tvarijonaviciute, A., Tecles, F., Cerón, J.J., 2017a. Inﬂuence of the way of reporting alpha-amylase values in saliva in different naturalistic situations: a pilot study. Public Library of Science One 12, e0180100. Contreras-Aguilar, M.D., Tecles, F., Martínez-Subiela, S., Escribano, D., Bernal, L.J., Cerón, J.J., 2017b. Detection and measurement of alpha-amylase in canine saliva and changes after an experimentally induced sympathetic activation. BioMed Central Veterinary Research 13, 266. Da˛browski, R., Kostro, K., Lisiecka, U., Szczubiał, M., Krakowski, L., 2009. Usefulness of C-reactive protein, serum amyloid A component, and haptoglobin determinations in bitches with pyometra for monitoring early postovariohysterectomy complications. Theriogenology 72, 471–476. Da Silva, A.S., Munhoz, T.D., Faria, J.L., Vargas-Hérnandez, G., Machado, R.Z., Luz, N.C., Moritz, C.E., Casali, E.A., Bottari, N.B., Stefani, L.M., et al., 2013. Inﬂuence of experimental canine ehrlichiosis on the E-ADA activity and purine levels in serum and possible functional correlations with pathogenesis. Veterinary Microbiology 166, 602–606. Goggs, R., Dennis, S.G., Di Bella, A., Humm, K.R., McLauchlan, G., Mooney, C., Ridyard, A., Tappin, S., Walker, D., Warman, S., et al., 2015. Predicting outcome in dogs with primary immune-mediated hemolytic anemia: results of a multicenter case registry. Journal of Veterinary Internal Medicine 29, 1603–1610. Grigoleit, J.S., Kullmann, J.S., Oberbeck, R., Schedlowski, M., Engler, H., 2013. Salivary a-amylase response to endotoxin administration in humans. Psychoneuroendocrinology 38, 1819–1823. Haririan, H., Bertl, K., Laky, M., Rausch, W.D., Böttcher, M., Matejka, M., Andrukhov, O., Rausch-Fan, X., 2012. Salivary and serum chromogranin A and a-amylase in periodontal health and disease. Journal of Periodontology 83, 1314–1321. Jitpean, S., Ström-Holst, B., Emanuelson, U., Höglund, O.V., Pettersson, A., AlnerydBull, C., Hagman, R., 2014. Outcome of pyometra in female dogs and predictors of peritonitis and prolonged postoperative hospitalization in surgically treated cases. BioMed Central Veterinary Research 10, 6. Jitpean, S., Stridsberg, M., Pettersson, A., Höglund, O.V., Holst, B.S., Hagman, R., 2015. Decreased plasma Chromogranin A361-372 (Catestatin) but not Chromogranin A17-38 (Vasostatin) in female dogs with bacterial uterine infection (pyometra). BioMed Central Veterinary Research 11, 14. Machado, V.S., Crivellenti, L.Z., Bottari, N.B., Tonin, A.A., Pelinson, L.P., BorinCrivellenti, S., Santana, A.E., Torbitz, V.D., Moresco, R.N., Duarte, T., et al., 2015. Oxidative stress and inﬂammatory response biomarkers in dogs with mammary carcinoma. Pathology, Research and Practice 211, 677–681. Martínez-Subiela, S., Cerón, J.J., 2005. Validation of commercial assays for the determination of haptoglobin, C-reactive protein and serum amyloid A in dogs. Archivos de Medicina Veterinaria 37, 61–66. Mishra, O.P., Gupta, B.L., Ali, Z., Nath, G., Chandra, L., 1994. Adenosine deaminase activity in typhoid fever. Indian Pediatrics 31, 1379–1384. Parra, M.D., Tecles, F., Martínez-Subiela, S., Cerón, J.J., 2005. C-reactive protein measurement in canine saliva. Journal of Veterinary Diagnostic Investigation 17, 139–144. Rai, B., Kaur, J., Jacobs, R., Anand, S.C., 2011. Adenosine deaminase in saliva as a diagnostic marker of squamous cell carcinoma of tongue. Clinical Oral Investigations 15, 347–349. Reinoldes, A., 2010. Avaliação dos Níveis de Glicose, Insulina, Cortisol e Glucagon em cães com Sepse Grave Submetidos ao Tratamento Intensivo. Master of Veterinary Medicine Thesis. University of São Paulo, Brazil. Sigrist, N., Mosing, M., Iff, I., Larenza, M.P., Lang, J., Spreng, D., 2008. Inﬂuence of preanaesthetic thoracic radiographs on ASA physical status classiﬁcation and anaesthetic protocols in traumatized dogs and cats. Schweizer Archiv für Tierheilkunde 150, 507–514. Singh, A.K., Jiang, Y., White, T., Spassova, D., 1997. Validation of nonradioactive chemiluminescent immunoassay methods for the analysis of thyroxine and cortisol in blood samples obtained from dogs, cats, and horses. Journal of Veterinary Diagnostic Investigation 9, 261–268. Srithunyarat, T., Hagman, R., Höglund, O.V., Stridsberg, M., Hanson, J., Lagerstedt, A. S., Pettersson, A., 2017. Catestatin, vasostatin, cortisol, and visual analog scale scoring for stress assessment in healthy dogs. Research in Veterinary Science 117, 74–80. Thurmon, J.C., Tranquilli, W.J., Benson, G.J., 1996. Lumb & Jones’ Veterinary Anesthesia, Third Ed. Hrsg. Lippincott, Williams & Wilkins, Baltimore, MD, USA 928 pp. Tonin, A.A., Calado, A.M., Bottari, N.B., Dalenogare, D., Thomé, G.R., Duarte, T., Duarte, M.M., Morsch, V.M., Schetinger, M.R., Alves, L.C., et al., 2016. Novel markers of inﬂammatory response and hepatic dysfunction in canine leishmaniasis. Comparative Immunology, Microbiology and Infectious Diseases 44, 61–64. Vincent, I.C., Michell, A.R., 1992. Comparison of cortisol concentrations in saliva and plasma of dogs. Research in Veterinary Science 53, 342–345.