In all cases, saliva was obtained prior to blood to avoid any possible influence of stress associated with blood collection on the saliva results. Saliva was collected using Salivette tubes (Sarstedt, Aktiengesellschaft & Co. D-51588 Nümbrecht, Germany) containing a sponge (Esponja Marina, La Griega E. Koronis, Madrid, Spain) instead of a cotton swab. The animals were allowed to chew the sponge until thoroughly moist with the help of a flexible thin metal rod. Then, the sponge was placed into the Salivette tube. Venous blood was obtained from venipuncture of the jugular (dogs, horses, and pigs) or caudal (cows) veins, using tubes without additive (BD Vacutainer, Franklin Lakes, NJ, USA) and allowed to clot. All samples were kept in ice until arrival at the laboratory for processing (less than 2 hours).
Once at the laboratory, all saliva samples were visually checked, and those suspected of blood contamination (reddish coloration) were excluded. The saliva samples were centrifuged (Universal 320R, Hettichzentrifugen, Tuttlingen, Germany) at 3000 x g and 4º C for 10 min. The supernatant was collected in Eppendorf tubes of 1.5mL, and the sediment discarded. Blood tubes were centrifuged similarly to those of saliva, and serum was aliquoted in Eppendorf. Saliva and serum specimens were stored at -80ºC until analysis.
ADA was analyzed with a commercially available spectrophotometric automated assay (Adenosine Deaminase assay kit, Diazyme Laboratories, Poway, CA, USA). The methodology’s principle can be summarized as follows: 1) Reagent 1 containing 4-aminoantipyrine (4-AA), purine nucleoside phosphorylase (PNP), xanthine oxidase (XOD) and peroxidase in Tris-HCl buffer (pH 8.0) is pipetted to the reaction cuvette with the simple. 2) Then, the Reagent 2 containing the substrate adenosine and N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (EHSTP) in Tris-HCl buffer (pH 4.0) is added. The substrate adenosine is deaminated to inosine by ADA present in the sample. PNP then converts inosine to hypoxanthine, which is then transformed into uric acid and hydrogen peroxide (H2O2) by XOD. The amount of H2O2 produced in the reaction is proportional to the ADA activity in the sample. It is quantified by reaction with EHSTP and 4-AA in the presence of peroxidase, leading to a quinine dye kinetically monitored at a 550 nm wavelength . This method was adapted to an automated analyzer (Olympus AU400, Olympus Diagnostica GmbH, Ennis, Ireland) following the manufacturer’s protocol. Since some species salivary ADA activity was low, sample volume was increased to allow determinations. The lower limit of detection (LLOD) of this method, established in a previous study, was 0.07 IU/L .
For isoenzyme determinations, the specific ADA1 inhibitor EHNA (Merck KGaA, Darmstadt, Germany) was used. At a proper concentration, EHNA inhibits ADA1 isoenzyme, whereas ADA2 remains unaffected . Therefore, tADA and ADA2 isoenzyme can be determined when samples are analyzed in the absence and presence of EHNA, respectively. The isoenzyme ADA1 is calculated from the difference between both measurements.
Optimization of EHNA concentration for ADA2 measurement in serum and saliva in different species
To determine the appropriate concentration of EHNA that should be used in each species for total inhibition of ADA1 isoenzyme, the following samples were used:
Dogs: Samples were obtained from five healthy Beagle dogs (Canis lupus familiaris). All dogs were neutered males, 3.5 ± 0.8 years old, and 26.0 ± 7.1 Kg body weight. The animals were located in the Experimental Farm of the University of Murcia (Murcia, Spain). The animals did not show any sign after clinical examination and routine haematological or biochemistry analyses. Serum C-reactive protein (CRP) concentration was measured as previously described  and used as a marker of systemic inflammation, giving all animals values <10µg/mL .
Horses: Samples were obtained from five healthy horses (Equus caballus), one stallion and four geldings, a mean age 10.0 ± 5.1 years old, with body condition score (BCS) 3.4 ± 0.5, including three Spanish horses, one Spanish Arabian and one Warmblood. Horses showed no clinical signs of pain or discomfort after a physical examination and did not show any haematological or biochemistry alteration. The serum levels of the acute phase protein serum amyloid A (SAA) were measured as described . They was used as a marker of acute systemic inflammation, giving all animals values <2.3µg/mL.
Pigs: Samples were collected from five apparently healthy growing pigs (Sus scrofa domesticus), Large White x Large White males with 2-3 months-old in the last phase of fattening, housed in the Experimental Farm of the University of Murcia (Murcia, Spain). The animals did not show any sign after clinical examination, haematological or biochemistry analyses. Serum CRP concentration, measured as previously described , was used as a marker of systemic inflammation. All animals gave values <20µg/mL.
Cows: Samples were obtained from five Holstein dairy cows (Bos taurus), lactation 3.5 ± 1.0, mean age 5.3 ± 1.4 years old, days in milk 234.8 ± 9.4, from a commercial dairy herd located in the southeast of Spain. The animals were healthy at physical examination and did not show any haematological or biochemistry alteration. The acute phase protein haptoglobin (Hp) measured as previously described  was used as an indicator of inflammation, giving all animals values <20mg/L .
Each saliva and serum sample was separated into five aliquots. Then, EHNA was added to the saliva and serum samples at increasing concentrations, whereas an equal volume of diluent was added to one aliquot that was used as control. The concentration of EHNA by which ADA1 activity was wholly inhibited varied between the studied species and was determined on both 1) the concentration was able to decrease tADA activity, and 2) a no further significant reduction in the enzymatic activity was obtained with a higher concentration.
Development and validation of an automated assay for ADA2 isoenzyme measurement
An automated assay for the measurement of the ADA2 isoenzyme was developed: the adequate inhibitory concentration of EHNA previously calculated for each species was added to the reagent 1. In each species, a similar volume of samples obtained from 10 different animals (five with low and five with high ADA2 activity) were mixed to prepare two pools of serum and two pools of saliva with different ADA2 activity. Intra-assay imprecision and linearity under dilution were evaluated in serum and saliva samples from the various species by calculating the intra-assay coefficient of variation (CV) and linear regression coefficient, following previously published protocols [26, 32].
Comparison between the automated and manual measurement of ADA2 activity
The results obtained with the automated procedure were compared with those obtained after the manual addition of the inhibitor. For this approach, the pig was selected as a model because of its high activity in saliva samples. For this purpose, serum and saliva samples with low (N = 15) and high (N = 18) ADA2 activity were obtained. ADA2 isoenzyme was analyzed manually adding EHNA to the samples and automatically by adding EHNA to the reagent 1. Such concentrations in both procedures the same final concentration of EHNA in the reaction mixture was achieved.
For this purpose, tADA and its isoenzymes were measured using the fully automated method in the following samples:
Dog: 20 samples were included and divided into two groups: 1) 10 samples were collected from healthy client-owned dogs belonging to the staff of the Animal Medicine and Surgery Department of the University of Murcia. They were 3.9 ± 1.5 years old, with BCS 4.0 ± 1.0, and included three Retrievers, three mixed-breed dogs, and one of the following breeds: Beagle, French bulldog, Scottish terrier, and Brie shepherd. All were neutered males apparently healthy after physical and haematological examinations. 2) 10 samples were collected from client-owned dogs admitted to the Veterinary Teaching Hospital of the University of Murcia and diagnosed with naturally occurred Leishmania infantum infection based on clinical signs and test results. The group with leishmaniosis included three mixed breed dogs and one of the following breeds: Retriever, French bulldog, Collie, Beagle, Irish setter, German shepherd and Rottweiler. There were five males and five females, with 3.0 ± 1.0 years-old, and BCS 2.7 ± 0.5. The clinical signs described in the 10 dogs with leishmaniosis included lymphadenopathy, and anaemia (1/10), skin lesions and uveitis (2/10), weight loss and hypoalbuminaemia (3/10), and hyperglobulinaemia (6/10). The diagnoses were based on positive polymerase chain reaction (PCR) and serology results. The concentrations of the acute phase protein ferritin in serum, a biomarker of systemic inflammation in this disease, were analyzed in healthy and diseased animals as previously described . All healthy individuals had values <190µg/L.
Horse: Samples from 20 horses (10 considered as healthy after physical and blood examinations, and 10 with acute abdominal pain) were included. The healthy animals were male horses admitted for castration or routine health check. They included different breeds (seven Spanish horses, one Spanish-Arabian, one Warmblood, and one crossbreed), mean age 8.0 ± 4.2 years-old, and BCS 3.5 ± 0.4. They showed no clinical signs of abdominal pain or other diseases during the physical examination, as well as haematological or biochemical abnormalities. The group of diseased animals consisted of horses with the acute abdominal disease. This group included animals with different breeds (five Spanish horses, two Warmblood horses, one Lusitanian horse, one Holsteiner, and one crossbreed) all males, mean age 11.3 ± 3.3 years-old, and BCS 3.4 ± 0.7. The diagnoses were based on clinical history, physical examination, haematology and plasma biochemistry, transabdominal ultrasonography, rectal examination, nasogastric intubation, and laparotomy findings in surgical cases. The following diagnoses were obtained: three colon impaction with large colon displacement, three stomach impaction, one nephrosplenic entrapment, one impaction of the pelvic flexure, one large colon displacement, and one enteritis. SAA was measured as a marker of acute systemic inflammation in all animals. All healthy animals showed SAA values <2.3µg/mL.
Pig: Samples from 20 animals (Large White x Large White males with 2-3 months-old in the last phase of fattening) housed in the Experimental Farm of the University of Murcia (Spain) were used. The healthy group was composed of 10 apparently healthy pigs after physical examination at the farm. The diseased group was formed of 10 lame pigs. The presence of lameness was considered based on the observation of the animals according to the scoring system published by Main et al. . An animal was considered lame when a lameness score of ≥1 was achieved. Serum CRP concentration was used in all animals as a marker of systemic inflammation. All healthy animals gave values <20µg/mL.
Cow: Samples from 10 dairy cows (seven Holstein, two Montbellier and one crossbreed), mean age 4.9 ± 1.6 years-old, parity 3.4 ± 1.6, and BCS 3.4 ± 0.7, from a commercial dairy herd located in the southeast of Spain were used. All animals were at the last phase of gestation, apparently healthy and no lameness, mastitis, metritis, ketosis, or other health issues were observed. Blood and saliva samples were obtained 13 ± 7 days before calving (Before calving) and on the day of calving (At calving), between January and February of 2019, to avoid any change in the results due to seasonal reasons. Serum Hp and the total WBC (Advia 120 haematology Analyzer, Siemens Healthcare GmbH, Erlangen, Germany) were used as indicators of inflammation. Healthy animals showed values lower than 20mg/L and 11x103/mm3 for serum Hp and WBC count, respectively [51, 54].
Data obtained from ADA measurements were analyzed for normality, giving a non-normal distribution. The changes due to the presence of EHNA at different concentrations were assessed by Friedman’s, followed by Dunn’s multiple comparison tests. ADA2 results obtained after manual and automated inhibition in the 33 samples (15 with low and 18 with high ADA2 activity) of porcine serum and saliva were compared by linear regression and Bland-Altman plot in which difference between methods was plotted against the average value. Unpaired Mann-Whitney test was used to compare tADA and isoenzymes results between healthy and diseased animals. The Wilcoxon signed-rank test was used to compare tADA and isoenzymes results between the two different measurements performed in cows. Spearman correlation coefficients (r) were calculated between ADA results and the biomarkers of inflammation. The correlations were considered according to the r value as very high (≥ 0.90), high (0.70-0.89), moderate (0.50-0.69), low (0.30-0.49), and negligible (< 0.30), following the Rule of Thumb . Data analyses were performed using Excel 2000 (Microsoft Corporation, Redmond, WA, USA) and Graph Pad Software Inc (GraphPad Prism, version 5 for Windows, Graph Pad Software Inc, San Diego, CA, USA). A P value of less than 0.05 was considered significant.