Evaluation of the presence of gingivitis as confounding factor in assessing inflammatory status in serum and saliva of dogs with diabetes mellitus.

The aim of this study was to evaluate the changes in the serum and salivary inflammatory markers induced by Diabetes mellitus (DM) in dogs and to assess the possible confounding effect of gingivitis. A panel of 13 cytokines was measured in the serum and saliva of dogs diagnosed with DM and compared with healthy dogs without gingivitis (control group 1; CG1) and dogs with gingivitis but otherwise healthy (control group 2; CG2). The results of the present study showed statistically significantly higher levels of IL-8, KC-like and MCP1 in the serum of dogs with DM compared to CG1 dogs. In the case of saliva, the DM group presented statistically higher GM-CSF, IL6, IL15, and MCP1 levels compared to CG1, and lower KC-like chemokine compared to CG2. Finally, gingivitis produced changes in saliva, with salivary levels of GM-CSF, IL-6, IL-7, IL-15, IP-10, KC-like, IL-10, IL-18, MCP1, TNFα being statistically significantly higher in the saliva of CG2 dogs compared to CG1. The results of the present study indicate that dogs with DM have altered cytokine levels in serum and saliva compared to healthy dogs. In addition, this study highlights the importance of taking oral health into account when determining cytokines in dogs, as gingivitis can significantly alter their concentrations. .

Saliva Sampling Material Matters: Effects on the Results of Saliva Analysis in Pigs.

The use of saliva as a biological sample from pigs is of high practical interest because blood collection from pigs is difficult and stressful. In this study, the influence of two different materials, a cotton roll and a polypropylene sponge, in porcine saliva collection was evaluated. For this purpose, the effect of the material used for sampling was evaluated in a panel of 13 analytes, including those related to stress (cortisol and oxytocin), inflammation and immunity (adenosine deaminase, haptoglobin and myeloperoxidase), redox homeostasis (the cupric reducing ability of saliva, the ferric reducing activity of saliva, and the Trolox equivalent antioxidant capacity), and sepsis (procalcitonin), as well as other routine analytes related to metabolism and different tissues and organs, such as lactate dehydrogenase, creatine kinase, urea, and total protein concentration. The polypropylene sponge provided a higher sample volume than the cotton roll. Although the results of some salivary analytes were equivalent for both materials, other analytes, such as creatine kinase, haptoglobin and total proteins, showed significant differences depending on the material used for saliva collection. Therefore, the type of material used for salivary collection in pigs should be considered when interpreting the results of analyses of the salivary analytes.

Serum Ferritin in Obese Dogs: Changes and Comparison with Other Analytes.

Canine obesity is the most common nutritional disorder and is associated with decreased quality of life and longevity as well as comorbidities including cardiorespiratory, endocrine, oncologic, or orthopaedic disorders. Ferritin is a major acute-phase protein in dogs, increasing during inflammation; however, it could also be affected by other conditions, including trauma, iron metabolism dysregulations, neoplasia, or hypoxia. Higher ferritin levels have been reported in obese humans, but ferritin has not been explored in canine obesity. To evaluate the possible changes in serum ferritin in canine obesity, ferritin levels from lean/normal weight (CG, n = 55) and overweight/obese dogs (OG, n = 37) were measured, together with complete hemogram and biochemical analyses. Statistically significant higher ferritin levels (1.2-fold) were found in OG (median, (interquartile range), 204 (166-227.5) µg/L) in comparison to CG animals (172 (137-210) µg/L)), with median levels of ferritin in OG dogs above the reference range for healthy animals in our laboratory (60-190 µg/L). In addition, statistically significant higher mean corpuscular volume (MCV), mean cell haemoglobin concentration (MCHC), total proteins, globulins, haptoglobin, total ferric fixation capacity (TIBC), alkaline phosphatase (ALP), butyrylcholinesterase (BChE), triglycerides, and calcium were observed in OG in comparison to CG. The higher levels in ferritin, together with higher TBIC, haematocrit, and MCV, could indicate tissue hypoxia in obese dogs.

Acute Phase Proteins in Dogs with Natural Infection by Trypanosoma cruzi.

Acute phase proteins have been used as tools for the diagnosis, monitoring, and prognosis of several diseases in domestic animals. However, the dynamics of these proteins in infection by Trypanosoma cruzi, the causative agent of Chagas disease in dogs, is still unknown. The aim of this study was to determine concentrations of acute phase proteins (C-reactive protein, haptoglobin, ferritin and paraoxonase-1) in dogs in a coastal town of Ecuador, with natural Trypanosoma cruzi infection with or without seroreactivity of Ehrlichia canis, Ehrlichia ewingii, Anaplasma phagocytophilum, Anaplasma platys, Borrelia burgdorferi and Dirofilaria immitis. For the detection of Trypanosoma cruzi serum antibodies, two different antigen-based enzyme-linked immunosorbent assay tests were implemented. For the detection of seroreactivity of Ehrlichia canis, Ehrlichia ewingii, Anaplasma phagocytophilum, Anaplasma platys, Borrelia burgdorferi and Dirofilaria immitis, an IDEXX SNAP® 4Dx® test was used. To determine the concentration of C-reactive protein and ferritin, an immunoturbidimetric assay was used; haptoglobin concentration was measured using a commercial colorimetric method validated in dogs; a spectrophotometric method was used to determine the serum concentration of paraoxonase-1. Results showed a reduction in the serum levels of paraoxonase-1 in Trypanosoma cruzi-seroreactive dogs, either with or without seroreactivity to other vector-borne diseases. A serum ferritin increment was observed in Trypanosoma cruzi-seroreactive dogs with seroreactivity to any other vector-borne diseases. Our findings suggest that paraoxonase-1 levels are reduced in Trypanosoma cruzi-seroreactive dogs without evident clinical signs of Chagas disease, despite their seroreactivity to the other vector-borne diseases studied. These findings could indicate an oxidative stress response in Trypanosoma cruzi-seroreactive dogs with no evident signs of inflammation.

Divergences between serum C-reactive protein and ferritin concentrations in canine pyometra.

The main aim of this report was to investigate and compare the response of serum C-reactive protein (CRP) and ferritin, two positive acute phase proteins (APPs) which usually show an increase in inflammatory processes, in dogs with pyometra. For this purpose, two different studies were made. In the first one , both proteins were measured together in an APPs profile in 25 dogs with pyometra, 25 dogs with pancreatitis (as an example of a positive inflammatory control group), and in 25 healthy dogs. In the second study, to advance the knowledge of the changes and evolution of serum ferritin and CRP in dogs with pyometra after treatment, the concentrations of both APPs were analyzed in 30 dogs with pyometra at diagnosis and after ovariohysterectomy and in 10 clinically healthy female dogs before and after elective spaying. In both studies, bitches with pyometra showed significant increases in serum CRP, indicating an inflammatory condition, but not in serum ferritin despite being a moderate positive APP. This divergence between the dynamics of these APPs could be a useful tool for the suspicion of cases of canine pyometra.

Evaluation of a Comprehensive Profile of Salivary Analytes for the Diagnosis of the Equine Gastric Ulcer Syndrome.

In this report, the measurement of salivary biomarkers as an aid for diagnosis of equine gastric ulcer syndrome (EGUS) was studied. A comprehensive panel of 23 salivary analytes was measured in the saliva of horses affected by EGUS and compared to healthy animals and horses with other diseases clinically similar to EGUS but with a negative diagnosis at gastroscopic examination. A total of 147 horses were included in the study and divided into heathy population (n = 12), the EGUS group (n = 110), and the group of horses with other diseases (n = 25). From the 23 analytes studied, 17 showed increased values in EGUS horses when compared to healthy ones, and uric acid, triglycerides, and calcium were significantly increased in horses with EGUS compared to the group of other diseases. The receiver operating characteristic curve analyses showed a modest but significant discriminatory power of those three analytes to identify EGUS from other diseases with similar symptoms. The discriminatory power enhanced when the results of the three analytes were combined. In conclusion, the results showed that selected salivary analytes could have potential use as biomarkers in horses with EGUS.

Changes in a Comprehensive Profile of Saliva Analytes in Fattening Pigs during a Complete Productive Cycle: A Longitudinal Study.

A comprehensive panel of 29 salivary analytes was measured in fattening pigs to evaluate its possible changes along their productive cycle. The identification of those changes would allow a better interpretation of the results according to the productive phase of the animal. Saliva samples were obtained from 49 Large-White pigs (24 females, 25 males) in suckling phase, at the beginning and the end of the nursery phase, and at the beginning and the end of the growing phase. Several analytes changed according to the phase of the productive cycle, with most of the analytes showing higher values at lactation and at the beginning of nursery. Additionally, differences were seen due to sex. When possible relations between performance parameters and analytes were evaluated, significant positive but weak relationships were found between weight at birth and salivary γ-glutamyl transferase, and between back-fat thickness and salivary lactate dehydrogenase. In conclusion, differences in the values of salivary analytes can be found in fattening pigs depending on the productive phase and sex of the animals.

Improved Joint Health Following Oral Administration of Glycosaminoglycans with Native Type II Collagen in a Rabbit Model of Osteoarthritis.

A prospective, experimental, randomized, double blinded study was designed to evaluate the effects of glycosaminoglycans, with or without native type II collagen (NC), in an osteoarthritis model induced by cranial cruciate ligament transection. The following compounds were tested: chondroitin sulfate (CS), glucosamine hydrochloride (GlHCl), hyaluronic acid (HA) and NC. Fifty-four female 12-week-old New Zealand rabbits were classified into three groups: CTR (control-no treatment), CGH (CS + GlHCl + HA) and CGH-NC (CS + GlHCl + HA + NC). Each group was subdivided into three subgroups according to survival times of 24, 56 and 84 days. Over time, all rabbits developed degenerative changes associated with osteoarthritis. CGH-NC showed significantly improved values on macroscopic evaluation, compared to CTR and CGH. Microscopically, significantly better results were seen with CGH and CGH-NC, compared to CTR, and synovial membrane values were significantly better with CGH-NC compared to CGH. A significant improvement in magnetic resonance imaging biomarkers was also observed with CGH-NC in cartilage transversal relaxation time (T2) and subchondral bone D2D fractal dimension in the lateral condyle. In conclusion, our results show beneficial effects on joint health of CGH and CGH-NC and also supports that adding NC to CGH results in even greater efficacy.

Response to Treatment with Melatonin and Clonazepam versus Placebo in Patients with Burning Mouth Syndrome.

Objective: to evaluate the efficacy of melatonin and clonazepam versus placebo in patients with burning mouth syndrome (BMS). Methods: a prospective double-blind study was carried out in patients with BMS and randomized to three groups: melatonin (1 mg once a day), clonazepam (0.5 mg/twice a day), or a placebo once a day, for 8 weeks. The clinical changes were evaluated, including xerostomia, the Oral Health Impact Profile 14 (OHIP-14) score, Pittsburg Sleep Quality Index, and the Hospital Anxiety and Depression Scale (HADS). Oxygen saturation and heart rate were recorded, with an analysis of salivary biomarkers in the forms of oxytocin, ferritin, adenosine deaminase (ADA), total proteins, and alpha-amylase. Results: a total of 64 patients were analyzed. A significant decrease in burning sensation was recorded with melatonin (7.8 ± 1.54 pre-treatment, 5.78 ± 2.54 post-treatment; p < 0.001) and clonazepam (8.75 ± 1.2 pre-treatment, 5.5 ± 3.6 post-treatment (p < 0.01). With regard to quality of life (OHIP-14), significant improvements were observed before and after the administration of melatonin (p < 0.001) and clonazepam (p = 0.001). On the other hand, with regard to the changes in salivary biomarkers following treatment, negative correlations were found between oxytocin and drainage (r = −0.410; p = 0.009) and between the HADS-D score and ferritin (r = −0.312; p = 0.05). While salivary amylase showed positive correlation with heart rate (r = 0.346; p = 0.029) and oxygen saturation (r = 0.419; p = 0.007). Conclusions: melatonin and clonazepam were shown to be effective at reducing the burning sensation and improving quality of life. Both drugs were found to be safe, with no major adverse effects in patients with BMS. Melatonin may be regarded as an alternative treatment for patients with BMS, though further studies are needed to confirm its effectiveness.

Low-cost do-it-yourself (DIY) mannequin for blood collection: A comprehensive evaluation about its use in teaching.

The use of mannequins to practice different clinical procedures in undergraduate students complies with the 3R principle (Replacement, Reduction, Refinement) without affecting professional competencies. However, commercial solutions are often expensive and therefore, not available for many schools. The main aim of this study was to describe the development and validation of an economical Do-It-Yourself mannequin for jugular blood collection from dogs. The use of the mannequin was evaluated by (1) assessment of the opinion of students and experts and (2) by conducting a pilot study where salivary biomarkers of stress were determined in students and dogs. The costs of the materials needed for the mannequin confection were less than 60 Euros and it was easily built in less than 1 h. The mannequin was very well accepted and scored by both, students and experts, being mostly liked a lot and considered it to be very useful for the practices (Scored 8, 9 or 10/10). Students that could first practice with the mannequin reported a self-perceived higher level of confidence and had lower levels of alpha-amylase in saliva after the procedure. Overall, the mannequin enables the initiation of blood sampling skills in agreement with 3R principles and is easy to perform, economically affordable and sustainable. This model could be adapted to other vein simulations and animal species, and has the potential to help students deal with stressful situations such as taking blood samples from a live animal.

Clinical leishmaniosis in a captive Eurasian otter (Lutra lutra) in Spain: a case report.

BACKGROUND: Captive and free-ranging wild mammals have been recognized as potential reservoirs of Leishmania infantum infection. The aim of this study was to describe the first clinical case of leishmaniosis in the Eurasian otter. CASE PRESENTATION: A case of clinical leishmaniosis is reported in a 4-year-old male Eurasian otter housed at a wildlife park (Murcia, South Eastern Spain). The Eurasian otter showed bilateral epistaxis, anorexia, apathy, and weight loss. A complete blood cell count and biochemical analyses revealed hyperproteinemia, hyperglobulinemia, decreases of paraoxonase-1, increases of haptoglobin and ferritin, and proteinuria. Bilateral nephropathy with hydronephrosis, mesenteric lymphadenomegaly, and ascites were also observed. L. infantum infection was confirmed by microscopy (amastigotes were detected in macrophages from spleen aspirate), molecular diagnosis (L. infantum DNA was detected by real-time polymerase chain reaction), and serology (anti-Leishmania IgG2 antibodies were detected by time-resolved immunofluorometry). The animal was treated with allopurinol for 3 months and gained weight, the epistaxis disappeared, and the ferritin concentration decreased. CONCLUSIONS: This is the first report of clinical leishmaniosis in the Eurasian otter. Our results suggest that Eurasian otters are susceptible to infection with L. infantum and can develop clinical leishmaniosis in endemic areas.

Salivary Biomarkers and Their Correlation with Pain and Stress in Patients with Burning Mouth Syndrome.

OBJECTIVE: To evaluate a panel of salivary analytes involving biomarkers of inflammation, stress, immune system and antioxidant status in patients with burning mouth syndrome (BMS) and to study their relationship with clinical variables. MATERIALS AND METHODS: A total of 51 patients with BMS and 31 controls were consecutively enrolled in the study, with the recording of oral habits, the severity of pain using a visual analogue scale (VAS), the Hospital Anxiety and Depression (HAD) score and the Oral Health Impact Profile-14 (OHIP14) score. Resting whole saliva was collected with the drainage technique, followed by the measurement of 11 biomarkers. RESULTS: The salivary flow was higher in patients with BMS. Among all the biomarkers studied, significantly higher levels of alpha-amylase, immunoglobulin A (IgA), and macrophage inflammatory protein-4 (MIP4) and lower levels of uric acid and ferric reducing activity of plasma (FRAP) were observed in the saliva of patients with BMS and the controls (p < 0.05 in all cases). Positive correlations were found between pain, oral quality of life and anxiety scores and salivary biomarkers. CONCLUSIONS: BMS is associated with changes in salivary biomarkers of inflammation, oxidative stress and stress, being related to the degree of pain and anxiety.

Salivary biomarkers in Alzheimer's disease.

OBJECTIVES: The objective of this study was to evaluate the changes that can occur in saliva components in patients with Alzheimer's disease (AD) of different severity and determine if any of these components could be a biomarker of this disease. Therefore, a panel of selected analytes related to the amyloid cascade, the immune and adrenergic systems, among others, were analyzed in the saliva of patients with Alzheimer's disease. METHODS: A total of 152 patients with AD and controls were included. The severity of the disease was established according to the Global Deterioration Scale. Unstimulated whole saliva was collected. RESULTS: Salivary amyloid-ß42 was significantly lower, and complement C4 was significantly higher in the patients with AD than in the controls (p < 0.05 in both cases). Only complement C4 maintained its significant effect in the multivariate regression analysis. However, the area under the receiver operating characteristic curve of C4 was 0.613. No changes were found in any analyte regarding the severity of the disease. CONCLUSIONS: A decrease in amyloid-ß42 and an increase in complement C4 were detected in the saliva of patients with AD, but the changes did not show a high diagnostic performance for the detection of AD and were not associated with its severity. CLINICAL RELEVANCE: Although some analytes showed significant differences in saliva in patients with AD, in our study conditions the salivary biomarkers analyzed were not of enough diagnostic utility for being used in routine.

Comparison of acute phase proteins in different clinical classification systems for canine leishmaniosis.

The main objective of the present study was to evaluate the acute phase protein (APP) concentrations that dogs naturally infected with Leishmania infantum show in different clinical stages of disease staged according to the Leishvet and CLWG classifications. In addition, these classifications are compared with the groups based on APP recommendations published recently. Medical records of 458 dogs with leishmaniosis whose samples were submitted to our laboratory for clinical pathology evaluation were reviewed and 77 cases met the inclusion criteria. All dogs were classified according to the CLWG system and the majority of the dogs (33.8%) were classified in stage D. Although some dogs (41.6%) could not be classified by the Leishvet system since it includes only dogs with clinical disease, most of the classified dogs (27.3%) were at Leishvet stage II. According to the APP classification, the majority of dogs (32.5%) were classified in stage 3a. Dogs in the more advanced stages of Leishvet and CWLG classifications had significant increases in serum ferritin and C-reactive protein (CRP) and decrease in Paraoxonase 1 (PON1). These findings indicate that APPs show more significant changes in the more advanced stages of Leishvet and CWLG classifications corresponding with more severe cases of canine leishmaniosis.

Relationship between serum anti-Leishmania antibody levels and acute phase proteins in dogs with canine leishmaniosis.

This study examined the relationship between two serologic assays which quantify anti-Leishmania antibodies (a commercial enzyme-linked immunosorbent assay (ELISA) and a time-resolved immunofluorometric assay (TR-IFMA)) and selected acute phase proteins (APPs) and analytes related to protein concentration. Data were obtained from 205 canine serum samples from different veterinary clinics located in an area in which canine leishmaniosis (CanL) is endemic. The samples were submitted to the Interdisciplinary Laboratory of Clinical Analysis (Interlab-UMU), University of Murcia, Spain, for analysis. The biochemical analytes evaluated were serum ferritin, C-reactive protein (CRP), haptoglobin, paraoxonase-1 (PON-1) and albumin as APPs and total proteins and globulins as indicative analytes of protein concentration. Samples were submitted for the initial diagnosis of CanL, or to monitor the response to treatment in patients with CanL. The evaluation of the biochemical analytes did not show differences between Leishmania-seronegative and Leishmania-seropositive dogs. However, dogs with high antibody titers showed more pronounced clinicopathological abnormalities. Both serological assays had correlations of different significance with the biochemical analytes, showing higher significant correlations with total proteins and globulins than with the rest of the analytes. When the samples submitted for diagnosis and treatment monitoring were analyzed separately, serological assays showed lower correlation in samples for treatment monitoring (r = 0.531, p < 0.0001) than in samples for diagnosis (r = 0.769, p < 0.0001). In addition, higher correlations were found between TR-IFMA and analytes such as serum ferritin and CRP in the treatment monitoring group than with the ELISA. These results may help to clarify the relationship between anti-Leishmania antibody levels and selected biochemical analytes related to inflammation and protein concentration in CanL.

Measurement of urea and creatinine in saliva of dogs: a pilot study.

BACKGROUND: Urea and creatinine in saliva have been reported to be possible markers of chronic kidney disease (CKD) in humans. The aim of this study was to assess if urea and creatinine could be measured in canine saliva, and to evaluate their possible changes in situations of CKD. RESULTS: The spectrophotometric assays for urea and creatinine measurements in saliva of dogs showed intra- and inter-assay imprecision lower than 12% and coefficients of correlation close to 1 in linearity under dilution tests. Healthy dogs showed median salivary concentrations of urea of 39.6 mg/dL and creatinine of 0.30 mg/dL, whereas dogs with CKD showed median salivary urea of 270.1 mg/dL and creatinine of 1.86 mg/dL. Positive high correlations were found between saliva and serum activities of the two analytes (urea, r = 0.909; P < 0.001; creatinine, r = 0.819; P < 0.001). CONCLUSIONS: Urea and creatinine concentrations can be measured in canine saliva with commercially available spectrophotometric assays. Both analytes showed higher values in saliva of dogs with CKD compared with healthy dogs and their values were highly correlated with those in serum in our study conditions.

Changes in serum anti-Leishmania antibody concentrations measured by time-resolved immunofluorometric assays in dogs with leishmaniosis after treatment.

The aim of this study was to evaluate the changes in anti-Leishmania IgG2 and IgA antibodies measured by two time-resolved immunofluorometric assays (TR-IFMAs) recently validated and by means of a commercially available ELISA test in dogs with leishmaniosis after treatment. Serum samples from 16 dogs with clinical leishmaniosis were obtained on days 0, 30 and 180 of treatment. In addition, these serological changes were compared with the clinical signs and selected analytes (total proteins, albumin, globulins and urinary protein:creatinine ratio). Concentrations of IgG2 and IgA by TR-IFMA were significantly lower on days 30 (p < 0.05) and 180 of treatment (p < 0.0001) compared to day 0 in dogs that showed a positive response to treatment. Magnitudes of decrease of IgG2 (1.66 and 20.4-fold) and IgA (1.3 and 11.43-fold) concentrations on days 30 and 180 were greater than those of the commercially available ELISA test (1.29 and 2.06-fold), and that of other analytes (total proteins: 1.11 and 1.25-fold; globulins: 1.22 and 1.74-fold; and albumin: 0.93 and 0.8-fold). This study shows that serum IgG2 and IgA anti-Leishmania antibodies measured by TR-IFMAs were useful for treatment monitoring in dogs with leishmaniosis, showing a significant reduction in antibody concentrations earlier than the commercial ELISA assay. Results suggest that the method used for antibody measurements greatly influences the results and, consequently, the usefulness for measuring anti-Leishmania antibodies to monitor the treatment of canine leishmaniosis.

Prevention of disease progression in Leishmania infantum-infected dogs with dietary nucleotides and active hexose correlated compound.

BACKGROUND: The prevalence of Leishmania infantum infection in clinically healthy dogs can be several times higher than that of clinical disease in endemic areas. Although treatment is not recommended in dogs with subclinical infection, these animals should be managed to prevent disease progression and parasite transmission to human beings or to other dogs. Dietary nucleotides and active hexose correlated compound (AHCC) have been shown to modulate the immune response. A recent study in dogs with clinical leishmaniosis receiving an initial 28-day course of methylglucamine antimoniate showed that six-month administration of a dietary supplement containing nucleotides plus AHCC achieves similar efficacy to allopurinol. Since the type of immune response plays a key role in the evolution of patients with leishmaniosis, the present study was aimed at evaluating the preventive effect of this supplement in avoiding or delaying disease progression in clinically healthy Leishmania-infected dogs. METHODS: Forty-six dogs were included in this multicenter, randomized, double-blind, placebo-controlled trial. Dogs received once-daily oral administration of a placebo or a dietary supplement containing nucleotides plus AHCC. Disease progression was monitored throughout the study in both groups. At 0, 60, 180 and 365 days of treatment, clinical signs were evaluated using a validated clinical scoring system, and several analytes were measured from blood, urine, and bone marrow samples. RESULTS: During the study, a significantly lower (P = 0.047) proportion of dogs changed their clinical status and became sick in the supplement group (3/20; 15%), compared to the placebo group (10/22; 45.5%). ELISA-determined antibody titers were significantly reduced compared to baseline at all time points with the supplement (P < 0.01), but not with the placebo. The mean clinical score of disease severity was significantly lower in the supplement group after 180 days (P = 0.014). No significant differences were observed for the other parameters. The dietary supplement was well tolerated. CONCLUSIONS: Oral administration of nucleotides plus AHCC for 365 days in clinically healthy L. infantum-infected dogs is safe, allows a significant reduction in anti-Leishmania antibodies, and leads to a lower disease progression rate, hence exerting a preventive effect.

Identification of novel biomarkers for treatment monitoring in canine leishmaniosis by high-resolution quantitative proteomic analysis.

The objective of this study was to use the Tandem Mass Tag (TMT) isobaric label-based proteomic approach, in order to identify new potential biomarkers for the treatment monitoring of canine leishmaniosis that could not be identified by the use of gel-based techniques. For this purpose serum samples were obtained from 5 clinically diseased dogs before and one month after the treatment of canine leishmaniosis. The non-depleted serum samples were subjected to reduction, alkylation and trypsin digestion, and the resulting peptides were labeled using 6-plex TMT reagents. To obtain information about protein identities and relative quantification, liquid chromatography-MS analysis of multiplexed TMT-labeled peptides was employed. This gel-free, label-based quantitative proteomic approach enabled identification of 117 canine proteins. Among these, 23 showed significant difference (p<0.05) in expression (two downregulated and 21 upregulated ranging from 1.25 to 2.5 fold change). Comparison of gel-free TMT-based quantification and a gel-based approach previously applied to the same samples resulted in the identification of some common markers (Apo-A1, vitamin D binding protein and RBP4). However, 20 additional differentially represented proteins were highlighted by the gel-free approach, 13 of which have not been previously reported in canine leishmaniosis. In conclusion, the TMT-based proteomic approach allowed identification of new serum proteins that significantly change in concentration after canine leishmaniosis treatment. These proteins are involved in various physiopathological processes such as inflammatory, coagulation or defense mechanisms, and could potentially be suitable biomarkers for treatment monitoring of this parasitic disease.

New wide dynamic range assays for quantification of anti-Leishmania IgG2 and IgA antibodies in canine serum.

The aims of this study were (1) to develop and validate time resolved-immunofluorometric assays for the detection of anti-Leishmania IgG2 and IgA antibodies in canine serum and (2) to evaluate the ability of these assays to quantify different amounts of anti-Leishmania antibodies in Leishmania-seronegative and seropositive dogs, determined by a commercial ELISA assay, and between different clinical stages according to LeishVet guidelines. The analytical validation showed that the assays had a good precision with intra- and inter-assay coefficients of variation lower than 10%. In addition, the assays allowed the quantification of very low concentration of antibodies as well as demonstrated a high level of accuracy, as determined by linearity under dilution (R2=0.99) and recovery tests (>85%). Moreover, no cross-reactions with Ehrlichia canis, Canine Parvovirus Type 2, Anaplasma phagocytophilum, Babesia canis, Dirofilaria immitis and pyometra were found. The assays were able to detect higher values of anti-Leishmania IgG2 and IgA antibodies in seropositive dogs compared with seronegative dogs (p<0.0001), although an overlap between groups existed in the case of IgA. In addition, significantly higher values for both antibodies were detected in LeishVet groups II (p<0.05) and III (p<0.01) when compared with LeishVet group I. From our study, it could be concluded that the immunofluorometric assays developed would be suitable for determination of anti-Leishmania IgG2 and IgA antibodies in serum samples with an adequate precision, analytical sensitivity and accuracy. In addition, these assays showed a wider difference in the concentration of both IgG2 and IgA antibodies between seronegative and seropositive dogs and between different clinical stages of CanL than a current commercial ELISA kit. Further studies would be recommended to evaluate the diagnostic sensitivity and specificity of these new assays as well as their application in monitoring CanL.