key: cord-0042074-2onyhbhn
authors: nan
title: European Society of Veterinary Clinical Pathology (ESVCP)/ European College of Veterinary Clinical Pathology (ECVCP) 15th Annual Congress
date: 2013-11-06
journal: Vet Clin Pathol
DOI: 10.1111/vcp.12091
sha: 704b233cf4785a732bd8dfeb556729bc6e098109
doc_id: 42074
cord_uid: 2onyhbhn

nan

The identification of regenerative anemia (RA) relies on the quantification of reticulocytes. Textbooks of veterinary hematology recommend to use absolute reticulocytes counts (Ret#) rather than relative counts (Ret%) or the reticulocyte production index (RPI). However, there are no studies on the diagnostic accuracy of reticulocyte variables in cats. Therefore, in this study we assessed the accuracy of Ret%, Ret# and RPI for the diagnosis of RA in cats. Data from cats with confirmed regenerative or non regenerative anemia (NRA), and from healthy cats were selected from the databases of our institutions (appr. 900 hematologic records). Sensitivity, specificity and likelihood ratio (LR+) were calculated using the cut-offs established by a receiver operating characteristic (ROC) curve. In a retrospective search 26 controls and 105 anemic cats were identified. Eight cats had RA due to hemolysis (n = 6) or chronic hemorrhage (n = 2), and 97 cats had NRA due to FIP (n = 25), renal failure (n = 17), leukemia (n = 16), other tumors (n = 15), FIV and/or FELV infection (n = 13), very recent hemorrhage (n = 6), or systemic inflammation (n = 5). Ret#, Ret% and RPI were significantly higher (P < .001) in RA cats (150.0 AE 120.0 x 10³/lL; 4.8 AE 3.2%; 0.9 AE 0.7) compared to controls (7.8 AE 13.5 x 10³/lL; 0.1 AE 0.2%; 0.0 AE 0.0) and NRA cats (19.2 AE 44.2 x 10³/lL; 0.6 AE 1.3%; 0.1 AE 0.2). Ret% and RPI were significantly higher in NRA cats than in controls. The area under the ROC curves was 97% (P < .001) for all variables. The cut-offs determined by ROC curves for Ret#, Ret% and RPI were 51.0 x 10³/lL, 1.3% and 0.3, respectively, the sensitivity 100%, the specificity 94. 3, 93.5, and 95.1%, respectively, and LR+ is 17.6, 15.4 and 20.5, respectively. These data suggests that the reticulocyte variables are comparable in discriminating feline RA from NRA, with RPI having slightly higher sensitivity and LR+ than Ret% and Ret#. Delta WBC (DWBC, ratio between the cell counts in the DIFF and BASO channel of the laser counter Sysmex XT2000iV) has been reported to be high in cats with feline infectious peritonitis (FIP). The aim of this study was to assess the diagnostic accuracy of the DWBC for FIP and to define the best cutoff value to diagnose FIP. From 56 effusions, 25 were from cats with FIP, and 31 from cats with other diseases (19 tumors; 8 inflammation; 4 cardiogen), diagnosed by cytology, necropsy or serum protein electrophoresis and a 1 -acid glycoprotein, analyzed within 12 hours from collection. WBC differential (DIFF) and basophil (BASO) counts and DWBC were recorded. Results of the 2 groups were statistically compared and sensitivity, specificity and positive likelihood ratio (LR+) were calculated. A ROC curve was designed and the ideal cut-off value determined. The DWBC was significantly higher (P < .001) in FIP (mean AE SD: 12.5 AE 11.2; median: 8.16; min-max: 0.5-36.4) than in non-FIP cats (1.1 AE 0.4; 1.0; 0.5-2.5). The BASO and DIFF counts (cells x 10 3 /lL) were significantly lower (P < .001 and P < .05) in FIP cats (BASO = 0.5 AE 1.1; 0.2; 0.0-5.3; DIFF = 4.5 AE 7.4; 1.3; 0.1-26.3) than in non-FIP cats (BASO = 43.5 AE 127.0; 10.1; 0.0-707.9 DIFF = 52.6 AE 164.6; 9.1; 0.1-34.6). All FIP cats had a DWBC > 3.0, except 2 cats, which had "atypical" FIP (one with a severe hepatic failure and one with a pericardial effusion). All non-FIP cats had a DWBC < 3.0. The area under the ROC curve was 0.95 (P < .001). The best cut-off was 1.7 (Sens: 92%; Spec: 93.5%; LR+: 14.3) . The specificity increased to 100% using a cut-off of 2.5. This study confirms the high diagnostic accuracy of the DWBC for FIP. This is due to clot formation in the BASO reagent, resulting in cell entrapment, similar to the Rivalta test reaction that has a high diagnostic accuracy for FIP. The HemoFAXS (TissueGnostic, Vienna, Austria) is an automated digital human WBC differentiation system, available with a canine classifier for enumerating canine WBC. In this study the canine WBC differentiation of the HemoFAXS classifier was evaluated and compared to 2 reference methods. Total and differential WBC counts were determined with the Sysmex-XT-2000iV in 43 canine EDTA-blood samples. Blood smears were also prepared with an automated slide maker for a 200-cell manual WBC differential and for differentiation with the HemoFAXS instrument (150 events). The HemoFAXS preclassified WBC were evaluated by a clinical pathologist for approval or reclassification. The reclassified results of the HemoFAXS and results of the 2 reference methods were used to calculate biases with 95% limits of agreement using Bland-Altman difference plots. Precision of the HemoFAXS was assessed by re-analyzing 16 times the same slide. On average 88.5% (mean) of all events were correctly classified by the HemoFAXS. Neutrophils were underestimated by the HemoFAXS compared to the manual method and the automated WBC count (mean bias Sysmex-HemoFAXS: -2.843 x10 3 /ll [-6.980 to 1.293]; manual-HemoFAXS: -3.412 x10 3 /ll [-7 .921 to 1.097]). Based on the standard monolayer settings, lymphocyte counts of the HemoFAXS showed a strong positive bias resulting in clinically relevant false positive lymphocytosis (mean bias Sysmex-HemoFAXS: 2.412 x10 3 /ll [-0.912 to 5.736 ]; manual-HemoFAXS: 2.781 x10 3 /ll [-0.831 to 6.392]). Eosinophil counts of the HemoFAXS showed only a very small bias and excellent agreement with both reference methods (mean bias Sysmex-HemoFAXS: -0.087 x10 3 /ll [-0.554 to 0.381]; manual-HemoFAXS: -0.046 x10 3 /ll [-0.547 to 0.454] ). For monocyte counts the mean bias was only slightly positive, however with wide 95% limits of agreement (mean bias Sysmex-HemoFAXS: 0.531 x10 3 /ll [-1.316 variations were lower with the HemoFAXS compared to the manual method. The software of the HemoFAXS is easy to use and pictures are of good quality. The WBC-selection process needs to be improved to reduce observed clinically relevant biases compared to manual and automated WBC differential counts. Cell cannibalism generally refers to the engulfment of cells by other cells, usually non-professional phagocytes, but its biological significance is still largely unknown. It should be differentiated from emperipolesis and overlapping of inflammatory cells with other cells. Xeno-cannibalism is the phagocytosis of non-self cells, with the leukocyte as the most often non-self phagocytized cell. Cannibal tumor cells do not distinguish or select inflammatory and sibling neoplastic cells as they have been observed engulfing even latex beads at in vitro experiments. Human studies have demonstrated cell cannibalism by neoplastic cells. Cannibalistic cells were seen more commonly in malignant fluids from cavitary effusions and urine than benign ones. They also were found in 100% of metastatic melanoma that did not present this feature at the primary site and in multiple malignancies that presented metastasis at diagnosis. Cell cannibalism is proposed as another mechanism of tumor immune escape and also representing a potential nutrient supply in an unfavorable microenvironment under conditions such as hypoxia and acidity. The actin-linker protein ezrin has been shown to be involved in the metastatic potential and the regulation of phagocytic activity of some tumors cells in human, murine and canine patients. Treatment with specific ezrin inhibitors can affect phagocytic activity (eg melanoma cells). We present two cases where cannibalism was suspected in cytological and histological examinations. Case 1 was a 9-year-old entire female American Staffordshire terrier with a metastatic squamous cell carcinoma in submandibular lymph node originated in tonsil. Large numbers of neutrophils where observed accumulated in the cytoplasm of malignant epithelial cells of lymph node. Case 2 was a 5-year-old intact female mixed breed dog with pulmonary diffuse metastatic mammary gland carcinoma. Cannibalistic cells with a crescent-shaped nucleus engulfing another cell with a round-to-oval faded nucleus were observed in histologic sections of primary and metastatic tumor tissue. In conclusion, cell cannibalism may be more frequent than reported and has clinical relevance. Future studies in veterinary patients about cell cannibalism and about ezrin as drug target against cancer are warranted. Early recognition and differentiation of systemic versus local inflammation in horses is important in order to provide appropriate treatment and prevent progression to more serious, potentially fatal conditions. The measurement of various inflammatory markers to detect inflammatory conditions is well established. The aim of this retrospective study was to investigate which of these markers are most suitable to differentiate systemic from local inflammation. The inclusion criteria were equine patients over one year of age, where total WBC and neutrophil count, myeloperoxidase index (MPXI), fibrinogen, serum amyloid A (SAA) and iron were all measured in one sample. Patients were assigned to groups with systemic inflammation (SI), local inflammation (LI) or non-inflammatory conditions/ healthy (NI) based on clinical records. Of the 143 horses included, 24 had SI, 96 had LI and 23 had NI. Data for each analyte were compared using a Kruskal-Wallis test with a Bonferroni correction (P < .05). There were no significant differences between groups for the WBC and neutrophil count or MPXI. Significant differences were found for fibrinogen and SAA between all 3 groups (Fibrinogen: median and interquartile range: SI 228 mg/dL (169-332), LI 175 mg/dL (138-227), NI 154 mg/dL , SAA (SI 1515 mg/L (302-3977), LI 280 mg/L (7-1495), NI 8 mg/L (4-9)), and for iron between the SI and LI groups (SI 61 lg/dL (44-80), LI 121 lg/dL (69-166)) and SI and NI groups ). Fibrinogen concentrations were above the reference interval (RI, 150-220 mg/dL) in 54% of patients in the SI group, in contrast to 27% in the LI group and 4% in the NI group. 96% of SI horses had increased SAA (RI <10 mg/L) versus 69% of LI and 17% of NI horses. Iron concentrations below the RI (80-240 lg/ dL) were seen in 75% of SI horses, 32% of LI horses and 4.5% of NI horses. In conclusion, markedly increased SAA together with decreased iron and increased fibrinogen is a strong indicator for equine SI. Reactive oxygen species are produced in metabolic and physiological processes, but when it exceeds the antioxidant capacity of cellular and extracellular fluids, damage and oxidation of cell membranes and tissues occur. Oxidant molecules are produced endogenously and but also originate from the outer environment. Total antioxidant capacity (TAC) is considered as a marker and total quantifier of antioxidants found in biological fluids, verifying its capacity of protecting the membrane and other cellular components against oxidative damage, and total oxidant status (TOS) is the measurement of different oxidant species in an organism. Paraoxonase-1 (PON1) is an anti-oxidative enzyme in serum primarily associated with high-density lipoproteins. The purpose of this study was to validate spectrophotometric assays for measuring serum TAC, TOS and PON1 in horses. Serum samples from horses obtained for diagnostic purposes were included in this study. The analytical validation included intra-and interassay coefficient of variation (CV) and accuracy (linearity and recovery) studies. All analyses were performed using an automated clinical chemistry analyzer. The assay for TAC Vet Clin Pathol 42/4 (2014) E20-E48 Ó2013 American Society for Veterinary Clinical Pathology E28 measurement showed intra-and inter-assay CVs < 7% and 5%, respectively. Serial dilution of serum samples resulted in linear regression equations with correlation coefficients close to 1 (r = 0.884 and 0.911). Recovery ranged between 98.9% and 102.2%. Intra-and inter-assay CVs for TOS measurements were < 10% and 8%, respectively. Serial dilution of serum samples resulted in linear regression equations with correlation coefficients close to 1 (r = 0.999 and 0.992). Recovery ranged between 92.8% and 98.3%. Intra-and inter-assay CVs for PON1 measurements were < 6% and 8%, respectively. Serial dilution of serum samples resulted in linear regression equations with correlation coefficients close to 1 (r = 0.997 and 0.990). Recovery ranged between 97.2% and 102.3%. In The quantification of circulating inflammatory biomarkers is relevant in the assessment of canine SIRS. The aim of this prospective study was to investigate the role of selected cytokines (IL6, IL8, IL10, IL15, MCP-1, and GM-CSF) during SIRS in dogs and their potential to differentiate sepsis from SIRS of noninfectious origin. Furthermore, the correlation between cytokines and other clinical and clinicopathologic variables was investigated. Twenty dogs admitted to a Veterinary Teaching Hospital with a diagnosis of SIRS related to 3 different diseases (parvovirus infection, n = 8; septic peritonitis, n = 7; acute pancreatitis; n = 5) were included and assigned to survivor (n = 15) and non-survivor (n = 5) groups. Ten blood donor dogs were included as controls. Blood was collected upon admission and immediately processed at +4°C and stored at -80°C. Clinicopathologic variables including C-reactive protein (CRP) were also assessed. Illness severity was calculated by the acute patient physiologic and laboratory evaluation fast score (APPLE). Cytokines were assayed by a multiparametric xMAP Luminex technology. Serum CRP was assayed using an immunoturbidimetric method validated for dogs. Results were analyzed by a non-parametric statistical analysis. SIRS dogs had significantly higher serum CRP compared to controls (P < .01). IL6 concentrations were significantly different between SIRS and controls dogs (P = .04). There was a significant positive correlation between IL6 concentrations in SIRS dogs with APPLE (r = 0.54, P = .02) and AST (r = 0.47, P = .04), and a significant inverse correlation with base excess (BE, r = -0.73, P = .01), serum iron (r = -0.5, P = .03) and glucose (r = -0.48, P = .04). When comparing groups for different diseases, circulating IL6 levels were significantly higher in dogs with parvovirus infection and septic peritonitis compared to dogs with pancreatitis (P = .04). Albumin, antithrombin activity, APPLE, BE, glucose, IL10, lactate, and total protein were all significantly different between survivors and non-survivors (P < .05). Preliminary results of this study indicate the potential prognostic significance of IL6 and IL10 in dogs with SIRS. Some bronchoalveolar lavage fluid (BALF) leukocytes (ie, neutrophils, mast cells and eosinophils) are used to characterize inflammatory airway disease (IAD) in horses. Different cytokines represent specific pathophysiologic events such as acute inflammation and T-helper pathways. The purpose of this study was to evaluate BALF concentration of several cytokines from controls and horses with subclinical IAD. A total of 138 Standardbred racehorses (4.7 AE 1.6 years old) in active training and racing were included in the study. BALF was recovered using videoendoscopy from both left and right lung. Classification of the BALF (IAD vs control) was based on the differential cell count performed on 300 leukocytes. Controls were defined as BALF with ≤ 5% neutrophils, < 2% mast cells and < 1% eosinophils. IAD was defined as BALF with any combination of increased proportions for these 3 cell types. BALF with evidence of exerciseinduced pulmonary hemorrhage (EIPH, ie, hemosiderophage: alveolar macrophage ratio > 20%) were excluded (n = 63). The cytokines TNFa, IFNc, IL-4 and IL-17 were measured in BALF using equine specific ELISA assays. A total of 213 BALFs were analyzed (184 IAD and 29 controls). Compared to controls, the IAD group had significantly elevated BALF concentrations of TNFa (P = .001) and IFNc (P = .016). When considering the different cytologic profiles, the IAD group with only neutrophilic subtype (n = 49) had significantly higher levels of TNFa (P < 0.05) compared to controls, while other subtypes did not. In conclusion, different BALF cytokine levels were associated with both subclinical IAD and specific cell types, especially a neutrophilic subtype. Based on these results, no particular T-cell cytokine response could be demonstrated. High mobility group box 1 (HMGB1) is a nuclear protein which is released from cells during cell necrosis or actively as a late cytokine. Gastric necrosis is one of the major complication and negative prognostic factor in dogs with gastric dilatationvolvulus syndrome (GDV). The aim of this study was to determinate diagnostic value of HMGB1 for detection of gastric necrosis in dogs with GDV. Fifty-four dogs with GDV were enrolled in this prospective study. Blood was collected before surgery from jugular vein and HMGB1 was detected using ELISA method (HMGB1, IBL International GmbH, Germany) from serum. Sixteen dogs had macroscopically evident gastric Vet Clin Pathol 42/4 (2014) E20-E48 Ó2013 American Society for Veterinary Clinical Pathology E29 necrosis and underwent partial gastrectomy or euthanasia. Thirty-eight dogs did not have gastric necrosis and were discharged from the hospital after successful treatment. Dogs with gastric necrosis had significantly higher HMGB1 levels (median 9.4 ng/ml, min-max 1.1-36.1 ng/ml) in comparison to those who had intact gastric wall (4.7 ng/ml, detection limit-16.5 ng/ ml, P < .001). A receiver operator curve (ROC) was constructed and the optimal cut-off for detection of gastric necrosis was set > 6.1 ng/ml with 75% sensitivity and 78% specificity. The area under the ROC curve was 0.795 (95% confidence interval: 0.662-0.893, P = .0001). In conclusion, HMGB1 is a useful marker of gastric necrosis in dogs with gastric dilatation and volvulus. This study was supported by grant no. IGA VFU 24/2011/ FVL and ESVCP 2012 Small Research Grant Award. Myxoma viruses (Leporipoxvirus myxomatosis, MYXV) belong to the genus Leporipoxvirus within the subfamily Chordopoxvirinae and are one of the major viral pathogens for rabbits worldwide. In the evolutionary hosts (North American brush rabbit, Sylvilagus californicus, South American tapeti, Sylvilagus brasiliensis) MYXV causes benign tumors, but in European rabbits (Oryctolagus cuniculus) the virus induces myxomatosis, a highly lethal systemic infection. Transmission to domestic and wildlife rabbits happens via flies, mosquitoes and other blood sucking arthropods. Vaccination with homologue live vaccine strains induces a constant immunity, persisting for at least 12 months. Attenuated MYXV strains differ from virulent field strains by genomic mutations and/or deletions. In the past, Shope fibroma virus (SFV), another Leporipoxvirus member with high antigenic relationship to MYXV, was also used as heterologous but less efficient vaccine. The potential spontaneous antigenic drift of MYXV field strains, affects efficiency of vaccine strains and detection systems. Myxoma viruses possess a linear double-stranded DNA with a genome size of approximately160 kb. In the present study the genomes of 6 MYXV-strains (4 field strains, one each vaccine and challenge strain) were completely sequenced and compared with published genome sequences of the MYXV reference strain Lausanne, the MYXV vaccine strain SG33 and the SFV reference strain Kasza and a quantitative TaqMan-based real-time-PCR (qPCR) was developed. This PCR allowed the differentiation between MYXV vaccine and field strains, and SFV. A primer pair amplifying a 170 bp PCR fragment allowed differentiation of MYXV from SFV. This fragment corresponds to the MYXV gene M071L, the homologue of the H3L gene in vaccinia viruses. It encodes an immunedominant surface protein. MAV primer pairs were synthesized, amplifying a unique 134 bp fragment of the M148R gene for the detection of the vaccine strain. The analytic sensitivity of the established qPCR was determined by reference to the standard curves. Analytic specificity was tested on the basis of a collection of different MYXV reference strains, vaccine strains and current field isolates. Poxviruses belonging to other genera (OPXV, PPXV, AVPV) were used as negative controls and showed any signals. Canine distemper (CD) is a highly contagious and frequently lethal disease in dogs caused by the Canine distemper virus (CDV). The aim of this study was to evaluate the diagnostic performance of direct immunofluorescent assay to detect CDV antigen in conjunctival scrapings comparing results with an established RT-PCR detection assay. Samples were collected from 23 dogs presenting central nervous system signs compatible with CD. Exfoliated epithelial cells were collected from the right and left conjunctiva of each animal using sterile cotton tipped swabs. The swabs were rubbed robustly back and forth in the lower conjunctival sac. A standard diameter area was applied with one of the swabs on a slide, was fixed in ethanol and stored frozen. Anti-CDV polyclonal antiserum conjugated to fluorescein isothiocyanate was used for staining and the sample was evaluated in a fluorescent microscope. Both swabs of each dog were immersed in sterile saline in sterile plastic tubes and stored at -20 C. After the manual stirring of swabs, the saline containing the eluted exfoliating cells was transferred into sterile vials pending extraction. The viral RNA was isolated from the supernatants using the QIAamp viral RNA Mini Kit (Qiagen, Germany). Reverse transcription and amplification were performed in a continuous RT-PCR method using the QIAGEN OneStep RT-PCR Kit (Qiagen, Germany) and primers targeting the large polymerase gene. Of the 23 dogs, 7 were positive in PCR and 5 of them in the direct immunofluorescent assay, while 16 dogs were negative in both methods. A good agreement was observed between the 2 methods with a K-value of 0.777 (95% CI: 0.488-1.065).

Overall, the results indicated that the direct immunofluorescent assay is a specific (specificity: 100% and NPV: 100%, 95% CI: 47.95-100.00%) and relatively sensitive assay (sensitivity: 71.43% PPV: 88.89%, 95% CI: 65.25-98.30%) which can be used for conjunctival scrapings, a noninvasive method that could represent a good option for the ante-mortem diagnosis of canine distemper. Pancreatitis, myocardial damage and euthyroid sick syndrome have all been observed in dogs with parvoviral enteritis, but their respective association with clinical outcome has not been sufficiently studied. The study reported here is part of a larger study evaluating the efficacy of oseltamivir in parvoviral enteritis. Specific aims were to determine a) serial serum concentrations of canine pancreatic lipase immunoreactivity (cPLI as measured by Spec cPL), troponin I (TnI), total T4, fT4 and TSH concentrations in hospitalized dogs with parvoviral enteritis receiving either oseltamivir or placebo, and b) possible associations between those variables and clinical outcome. A total of 29 dogs were included in the study, based on positive canine parvovirus (CPV) fecal antigen testing, compatible clinical signs, no previous drug administration and signed owner consent. All dogs were hospitalized for at least 5 days and received standard treatment. The study population was divided to a treatment group (n = 16, dogs receiving oseltamivir, 2 mg/kg q12 h p.o. for 5 days) and a placebo group (n = 13). Serum samples were obtained on admission and every 48 hours during the first 5 days of hospitalization. Serum concentrations of Spec cPL, TnI, total T4, fT4 and TSH were measured in all samples at the Gastrointestinal Laboratory at Texas A&M University using assays previously validated for use in dogs. Serum Spec cPL, TnI, TT4, fT4 and TSH concentrations increased significantly throughout the study period. Spec cPL was significantly higher and TT4 was significantly lower in the placebo group, compared to the treatment group. However, the proportion of dogs with serum Spec cPL concentrations diagnostic for pancreatitis (> 400 lg/L) was comparable between the 2 groups and not associated with thyroid hormone changes. None of the parameters had prognostic value regarding the outcome. Serum Spec cPL was not associated with TnI concentrations. In conclusion, serum concentrations of Spec cPL, TnI, thyroid hormone and TSH increased significantly during the first 5 days in dogs with CPV, but were not associated with a specific clinical outcome. Paraoxonase 1 (PON1) is a negative acute phase protein that circulates in blood bound to high density lipoproteins (HDLs) and exerts an antioxidant function. During systemic inflammation, the Reactive Oxygen Species (ROS) produced against pathogens leads to lipid peroxidation converting HDL to LDL. Consequently, PON1 serum activity decreases due to an increased consumption and detachment from HDL. The aim of this study was to assess the antioxidant role of PON1 towards HDL, using naturally infected dogs with babesiosis, a disease associated with changes of serum HDL concentrations, before and after treatment.

To this aim, serum from 30 dogs infected by Babesia canis were sampled at admission (day 0) prior to a single treatment with imidocarb dipropionate, and on days 1 and 6. A group of 15 control dogs was also included in the study to assess the actual presence of altered HDL and PON1 at day 0. PON1 and HDL were measured on all the samples using methods validated for canine serum. Compared with controls, serum concentrations of HDL and PON1 activity were significantly lower at day 0. The concentration of HDL significantly increased on days 1 and 6 compared with day 0 (P < .05 for both), and without significant differences between days 1 and 6. PON1 activity did not differ between day 0 and day 1, but significantly increased on day 6 compared with day 0 and day 1 (P < .001 for both). These results support the hypothesis that PON1 exerts an antioxidant role towards HDL also in dogs. The low serum concentration of HDL at admission is consistent with lipid peroxidation. The earlier increase of HDL (day 1) compared to PON1 (day 6) is probably dependent on increased consumption of PON1 on day 1 to reduce the oxidized lipids and to quickly restore the concentration of HDL. Following activation, platelets express activation markers on their membranes such as P-selectin. The expression promotes adhesion to leukocytes which mediates the adhesion of neutrophils and monocytes to the endothelium, and upregulates the pro-inflammatory function of neutrophils. Platelet-leukocyte heteroaggregates have been reported in people and dogs with thrombotic diseases. The purpose of this study was to investigate the occurrence of platelet activation and platelet-leukocyte heteroaggregation in canine babesiosis. EDTA blood was collected at presentation prior to any treatment, platelet concentration determined and the sample processed within one hour for flow cytometry. Leukocytes and platelets were separately harvested, washed with phosphate-buffered saline, and incubated with PE-CD61 and FITC-CD62P for determination of activated platelets, and FITC-CD62P and APC-CD14 for determination of plateletleukocyte heteroaggregates. Flow cytometric analysis was performed on a FACSCalibur (Becton Dickinson). Platelets were identified using forward/sideward scatter and CD61 positivity, and platelet activation was assessed based on CD62 expression. Platelet-leukocyte heteroaggregates were determined using CD14 to identify monocytes and sideward scatter to identify neutrophils. Of 34 Babesia-infected and 14 control dogs 27 Babesia-infected dogs survived and 7 died (mortality rate 26%). Compared to controls, the Babesia-infected dogs had significantly lower platelet counts (P < .001), significantly higher circulating platelet activity (P = .026), and platelet-monocyte heteroaggregation (P = .027). Survivors had significantly higher circulating platelet activity (P = .02) and platelet-monocyte heteroaggregation (P = .013), but no significant difference in Vet Clin Pathol 42/4 (2014) E20-E48 Ó2013 American Society for Veterinary Clinical Pathology E31 platelet-neutrophil heteroaggregation (P = .074) compared to controls. In conclusion, Babesia-infected dogs, specifically surviving dogs, had significantly up-regulated platelet activation and platelet-monocyte heteroaggregation. Dying dogs showed no difference compared to the controls, except for platelet count. Further studies and correlations with other markers of inflammation such as IL-6 and cortisol are needed to investigate these findings. Canine Leishmaniasis (CanL) is a systemic disease requiring lifelong treatment. Prognosis appears dependent on renal function; however studies on survival time and prognostic factors are limited. The aim of our study was to identify clinicopathologic variables with prognostic relevance in dogs affected by CanL and treated in a clinical setting. Dogs diagnosed by an immunofluorescence antibody titer (IFAT≥1:320) and/or direct detection of the parasite were selected from the medical records of a 12-year period. Clinical and clinicopathologic data collected upon admission were compared between survivors and non-survivors using non-parametric statistics. Survival and outcome predictors were studied with Kaplan-Meier and Cox-proportional hazard models (360 and 720 days endpoints). A total of 167 dogs (114 males, 5 neutered, 53 females, 14 spayed) met the inclusion criteria (median age 5 years, range 0,5-14). Median survival time was 720 days (range: 160-4045) for survivors and 103 days (range: 1-987) for non-survivors (P < .01). A 360 and 720 day follow-up was available for 76 and 55 patients, respectively. At both endpoints, increased death risk was associated with higher creatinine (Crea), urea, phosphorus (P), urine protein and albumin to creatinine ratios (UPC; UAC), and with lower serum albumin (Alb), WBC count, HCT and urine specific gravity (USG). In the multivariate regression model, the death risk at 360-days endpoint was associated with Crea (OR 20.40; 95%CI 2.58-161.05, p = .004), urea (OR 0.93; 95%CI 0.88-0.98, p = .011) and Alb (OR 0.22; 95%CI 0.06-0.73, P = .014), at 720-days with USG (OR 0.88; 95%CI 0.81-0.96, P = .005). For both endpoints, survival was significantly shorter in dogs with albuminuria (UAC > 0,7), proteinuria (UPC > 2), inadequate urine concentration (USG < 1030), azotemia (Crea > 1.35 mg/dl), hyperphosphatemia (P > 4.5 mg/dl), hypoalbuminemia (Alb < 2 g/dl) and anemia (HCT < 36%). IFAT and serum total protein were not associated with death risk and survival time. In conclusion, renal involvement significantly worsened prognosis and shortened survival time in a population of dogs affected by CanL during a long term follow-up. Relapsing fever (RF) is an acute human infectious disease caused by arthropod-borne spirochetes of the genus Borrelia. The disease is characterized by recurrent episodes of fever which often concur with spirochetemia. The RF borrelioses are grouped into 2 forms: louse-borne epidemic RF caused by B recurrentis, and tick-borne endemic RF transmitted by Argasid soft ticks and caused by several Borrelia spp. including B crocidurae, B coriaceae, B duttoni, B hermsii, B hispanica and B persica. Human infection with B persica is transmitted by the soft tick Ornithodoros tholozani and has been reported in Iran, Israel, Egypt, India, Central Asian republics and western China. O tholozani feeds on warm-blooded animals and commonly lives in caves, ruins, rock crevices and man-made shelters where livestock is housed. B persica RF has not been reported to infect dogs and cats previously. During 2003-2013, 5 cats and 5 dogs from Israel were detected with RF spirochetemia based on blood smear microscopy. Of these, infection was verified by PCR and sequencing of the 16S rRNA gene as caused by B persica in 4 cats and 4 dogs. The main clinical findings in cats included lethargy, anorexia and anemia in all cats (HCT 10.9-22.3%; median 17%), and thrombocytopenia in 4/5 cats. All dogs were extremely lethargic, 4/5 were febrile, 3/5 were anorectic, 5/5 were anemic (HCT 17-34%; median 27.1%) and 4/5 were thrombocytopenic. Three dogs were co-infected with babesiosis. The survival rate of both dogs and cats was 80% following treatment with antibiotics and disappearance of spirochetemia. One cat and one dog died one day after the initiation of antibiotic treatment. This is the first report of Borrelia persica infection in pet cats and dogs. Infection is associated with anemia and thrombocytopenia. RF was recorded only in infected dogs. Dogs and cats may be involved in the transmission of infection to humans via soft ticks, or serve as sentinels for human infection. The aim of this study was to determine plasma protein concentrations of the Red-tailed Amazon parrot (Amazona brasiliensis: Linnaeus, 1758) by sodium dodecyl sulphatepolyacrylamide (SDS-PAGE). Blood samples from 8 juvenile and 8 adult healthy birds in captivity were collected from the jugular vein. The plasma protein profile was determined by SDS-PAGE gel electrophoresis after staining with 0.2% Coomassie Brilliant Blue. Concentrations of the protein fractions were determined using a digital densitometer (9301PC Shimadzu, Tokyo, Japan). Proteins were identified using reference markers (Sigma Chemical Co., St. Louis, MO). In addition, the purified proteins (Sigma Chemical Co., St. Louis, MO) albumin, haptoglobin, ceruloplasmin and transferrin were also used. Identified plasma protein fractions included ceruloplasmin, transferrin, albumin, non-identified PM of 45 kDa (similar to mammal haptoglobin), a-1-acid-glycoprotein, IgA, IgG heavy and light chains and a non-identified PM of 23 kDa. These results may be useful to interpret alterations in the proteinogram of this bird species, considered and endangered species endemic to a narrow coastal plain area in southeastern Brazil. Cardiac troponin I (cTnI) correlates with severity of myocardial injury. Its serial assessment could provide information about progression of chronic heart failure (CHF). Non-specific inflammation may also occur with CHF and its assessment in dogs by C-reactive protein (CRP), haptoglobin (Hp) and ceruloplasmin (Cp) may provide valuable information. The purpose of this study was to determine whether serial serum cTnI, CRP, Hp and Cp concentrations differed among various stages of heart failure due to mitral valve disease (MVD). A group of 46 dogs diagnosed with MVD was prospectively recruited and allocated into 3 groups: I -asymptomatic disease (n = 21); II -mild to moderate CHF (n = 11); III advanced CHF (n = 14), based on the classification scheme according to the International Small Animal Cardiac Health Council guidelines. cTnI, CRP, Cp and Hp were evaluated in all dogs at admission. During a 4-month follow up period cTnI and CRP were measured bimonthly. At study start, Cp group values were indistinguishable (0.0058 AE 0.0023, group I), (0.0062, AE 0.0019, group II) and (0.0056, AE 0.0011, group III). Hp means were statistically different between groups III (7.2793, AE 4.7250) and I (3.9605, AE 1.8491) but there was no difference between groups III and II (4.4800, AE 2.5037, n = 11) and groups II and I. In the longitudinal study, there were significant negative effects of time on both logarithmic transformation of the initial CRP (b = -0.07, SE = 0.03, P = 0.003) and cTnI (b = -0.06, SE = 0.02, P = .009) variables. Post-hoc analysis using Tukey's HSD procedure to control the family-wise error rate indicated a significant difference between groups I and III (P = .004), and a marginally significant difference between groups II and III (P = .070) for CRP. Post-hoc analysis revealed that there were significant differences (P < .001) for all pair-wise comparisons of groups for cTnI. Finally, there was a significant main effect of cTnI (b = 3.50, SE = 0.91, P < .001) on CRP, indicating a positive correlation. In conclusion, long term monitoring of MVD could benefit from the serial measurements of cTnI and CRP. Dogs with pyometra have increased serum C-reactive protein (CRP) concentrations. After successful treatment of pyometra CRP concentrations are expected to decrease. The treatment of choice for pyometra is ovariohysterectomy, a surgical procedure that in itself causes tissue trauma and increased CRP concentrations. Previous studies in dogs showed that mean CRP concentrations 24 hours post ovariohysterectomy were increased compared to pre-operative levels. The objective of this study was to compare pre-and post-operative CRP concentrations in pyometra dogs undergoing ovariohysterectomy. In addition, the association of pre-operative CRP concentrations, or the difference between pre-and post-operative CRP concentrations, with outcome measured as days of post-operative hospitalization was investigated. CRP was measured with a previously validated canine-specific immunoturbidometric method (Gentian Canine CRP) in 20 pyometra dogs immediately prior and 24 AE 1 hour after ovariohysterectomy. The median (range) CRP concentrations pre-and post-operatively were 242 mg/l (<7-519 mg/l) and 180 mg/l (88-476 mg/l), respectively. The decreased CRP concentrations the day after surgery was opposite to previous reports. CRP concentrations decreased in 14 of the 20 dogs, suggesting that the removal of the inflamed uterus had a larger impact on CRP concentration than the additional tissue trauma caused by surgery. Six dogs had increased CRP concentrations the day after surgery compared to pre-operative levels. This increase was most pronounced in dogs that had low or only mildly elevated CRP concentrations pre-operatively. The number of post-operative days of hospitalization varied between 1 and 6 days. Dogs with only 1 day of post-operative hospitalization had a lower median CRP concentration pre-operatively ((n = 7, 166 mg/l) compared to dogs with a post-operative hospitalization of ≥ 2 days (n = 13, 250 mg/l). The change of CRP concentration between the pre-and post-operative measurements did not seem to be associated with days of hospitalization, although results should be interpreted with caution due to the low number of animals included in this pilot study. We previously demonstrated (Vet Clin Path 41:E10-11, 2012) that DGGR lipase enzyme-assay was a strong candidate for inexpensive, high sensitivity/specificity detection of pancreatitis in dogs. High assay efficacy is attributed to refined choice of substrate and inclusion of co-lipase and bile acids inhibitory of other lipases. We established DGGR lipase reference intervals for cats and dogs, and determined its sensitivity/specificity for pancreatitis, by comparison with tissue-and species-specific, quantitative immunoassay (PL). Amylase and a point-of-care, qualitative, "snap"-test immunoassay for canine pancreatic lipase were included for comparison. In 77 healthy dogs (no significant anemia, inflammation, liver or kidney disease, nor any clinical signs of pancreatitis such as vomiting or colic) mean AE sd was 15 AE 4 U/L (range 9-25), and for 68 cats meanAESD 28 AE 17 U/L (range 8-81) for DGGR lipase. Respective values for amylase were 960 AE 212 U/L in 78 dogs (range 403-1330) and 735 AE 225 U/L in 68 cats (range 255-1190). Cut-offs for positive tests were 5.3/200 ug/L for the immunoassay, 25/80 U/L lipase and 1400/1200 U/L amylase for cats/ dogs. Concordance of DGGR lipase and PL was 96% for 67 dogs (67% PL positive) and 97% for 31 cats (36% PL positive) for lipase but only 75% for 67 dogs and 71% for 31 cats for amylase. DGGR was 90% concordant with the snap-test, which, compared to the DGGR test had lower sensitivity (86% vs 96%) and specificity (83% vs 95%). Amylase had low concordance with PL because of poor sensitivity despite high specificity (58 / 85% in dogs, 40 / 80% in cats, respectively). We conclude that in cats and dogs a) amylase has low sensitivity but moderate specificity for pancreatitis, b) DGGR lipase activities are lower than other lipase assays, attributable to its high tissue specificity, c) DGGR lipase is > 95% concordant with PL, and d) DGGR lipase is a highly cost-effective biomarker of pancreatitis. Proteinuria is considered a renal dysfunction marker in dogs, and urine dipstick test and urinary protein to creatinine ratio (UPC) are commonly used to diagnose and monitor proteinuric patients. In people, the urinary albumin to creatinine ratio (UAC) represents the gold standard for early renal disease detection. The aim of this study was to evaluate urine dipstick test and UPC for early detection of albuminuric dogs. A total of 868 canine urine samples without active sediment and macroscopic hematuria were collected from January 2002 to December 2012 in our teaching hospital. Urinalysis, UPC and UAC were available for 550 samples, and UPC and UAC for 868 specimens. Considering UAC (immunoturbidimetric assay) as the reference method, the diagnostic accuracy of dipstick test and UPC (pyrogallol red-molybdate) was evaluated. A UAC reference interval (RI, 0-0.024) was determined for 60 healthy dogs. Data were categorized using different cut-offs for urine specific gravity (USG, 1012 or 1030), dipstick proteinuria (30 or 100 mg/dl), UPC (0.2) and UAC (0.024). Diagnostic agreement (Cohen's k coefficient) and Spearman's correlation between dipstick, UPC and UAC were evaluated. The diagnostic accuracy was estimated with the area under the receiver operator curve (ROC-AUC) analysis. Significance level was set at p < .05. Independently of urine concentration, diagnostic agreement between dipstick and UPC or UAC was substantial (k = 0.62 and k = 0.61, respectively; P < .001) with positive dipstick results ≥30 mg/dl, and was fair (k = 0.27 and k = 0.26, respectively; P < .001) with positive dipstick results ≥100 mg/dl. Diagnostic accuracy of dipstick compared to UPC and UAC was very good (AUC 0.84 and 0.84, respectively; P < .001) and negative dipstick results presented 100% sensitivity. UPC and UAC were highly correlated (r = 0,90; P < .01). Comparing UPC with UAC, diagnostic accuracy was excellent (AUC 0.94; p < .001), with maximum sensitivity and specificity for UPC ≥ 0.3. Positive dipstick results (≥ 30 mg/dl) probably detect albuminuria outside the RI, independent of USG. The urine cortisol to creatinine ratio (UCCR) is the screening test of choice for canine hyperadrenocorticism (HAC). But UCCR has low specificity. Functional tests like the low-dose dexamethasone suppression test (LDDST) or ACTH stimulation test (ACTH stim) should be performed if the UCCR is above the physiologic cutoff. The aims of this study were to investigate the sensitivity and specificity of the UCCR cutoff value (19x10 -6 in the authors' laboratory), and to calculate a receiver-operator curve (ROC) to assess the diagnostic efficacy of UCCR. Results from 601 dogs from 2005-2013 with UCCR and LDDST (524 dogs), or UCCR and ACTH stim (77 dogs) were evaluated. A HAC diagnosis was based on LDDST/8 h post-suppression value > 40 lmol/L, or 1 hour ACTH stim value > 440 lmol/L. Cortisol was measured by a chemiluminometric immunoassay. In the UCCR LDDST group 510/524 (97.3%) had an increased UCCR (median 51.6, range 19-1162.3) and 208/524 dogs (39.4%) had a positive LDDST result. In the UCCR ACTH stim group 73/77 dogs (95.8%) had an increased UCCR (median 59, range 22-777) and 26/77 (33.7%) dogs had a positive ACTH stim test. The calculated sensitivity of the UCCR using the laboratory cutoff in the LDDST group was 98%, specificity 3%. In the ACTH stim group sensitivity was 96% and specificity 5%. In the population of dogs examined the positive predictive value (PPV) for UCCR in the LDDST group was 40%, negative predictive value (NPV) 78%; with a positive likelihood ratio LR + of 1.01 and negative LR of 0.66. The area under the ROC curve was 0.77. The findings of this retrospective study confirm that UCCR is a good screening test for HAC. A positive UCCR should always be followed by a more specific test and a diagnosis of HAC should be based on the results of this as well as the pertinent clinical signs. The objective of this prospective study was to investigate effects of temperature and duration of storage on canine urine SDS-AGE. Urines with non-active sediment of 20 proteinuric (UPC > 0.5) and 20 non proteinuric (UPC ≤ 0.2) dogs were centrifuged, and aliquots were stored at room temperature (RT), 4°C, -20°C and -80°C for up to 90 days. Urine SDS-AGE was performed and results were visually interpreted by the same operator by comparison with molecular weight markers. There was no change up to 5 days of storage at 4°C and at RT, and up to 90 days at -80°C. At -20°C, from 15 days of storage, appearance or strengthening of a 150 kDa band or of a > 150 kDa band was Vet Clin Pathol 42/4 (2014) E20-E48 Ó2013 American Society for Veterinary Clinical Pathology E34 seen in 10 and 3 proteinuric dogs, respectively. Additionally a faint band > 150 kDa appeared in 12 healthy dogs. Moreover gel staining faded over time, therefore immediate analysis or digital recording are recommended. Urine SDS-AGE can securely be performed in specimens stored at RT or 4°C for up to 5 days. When a longer storage is needed, storage at À80°C is recommended. Thromboelastography (TEG) is typically performed within 2 h, no later than 8 h post sampling, making this an in-house or point-of-care test. The aim of this study was to evaluate the reliability of TEG with citrated blood samples from dairy cows and pet dogs tested at extended time points as if submitted by first opinion veterinarians. Twenty-eight samples from 27 Holstein Friesian cows (Cambridge, UK) and 16 citrated venous whole blood samples from 16 pet dogs (Bad Kissingen, Germany) were analyzed with kaolin-activated TEG. Sample aliquots stored at room temperature (18°C) were measured between 2 and 100 h post sampling. One mL of citrated whole blood was placed in a silicon coated vial containing kaolin. Pins and cups were placed in the TEG 5000 analyzer sample holder (37°C) and filled with 20 lL 0.2M calcium chloride. Then 340 lL kaolin-activated citrated whole blood was added. A comparison with a preliminary reference interval (RI) based on 62 healthy dairy Holstein Friesian cows tested within 2 h of collection showed several samples would have caused a different diagnosis. This translates to 25% of the samples being diagnosed incorrectly. With restriction to < 36 h post sampling this would be reduced to 1/10 (2/21) of the samples. Only 1/16 would be significantly changed with samples <24 h old. Sixteen canine samples were run at 2 h and between 21 and 69 h post sampling. One sample was run at 2, 24 and 47 h. Visual inspection of the TEG graphs at 2 h versus <24 h showed good agreement, and at 2 h versus <48 h acceptable agreement. Assessment of clinical significance of the differences showed that a sample run 47 h post sampling would have resulted in a different clinical interpretation in 4/5 parameters being altered by the time lag. Therefore only 1/17 runs would have had clinical significance, avoidable by analysis within 36 h. In conclusion, TEG appears stable in cows and dogs up to 36 h post sampling. The aim of this study was to evaluate the use of a human automated adapted homogenous enzyme immunoassay (Siemens Dimension T4) for the determination of total thyroxin (TT4) in canine and feline serum. An acceptance limit of total allowable error of 20%, maximum analytical imprecision of 9% and maximum bias of 6% based on canine biological variation were used. Reference intervals (RIs) used in the authors' laboratory are 17-50 nmol/L for dogs and 13-50 nmol/L for cats. The clinical decision limit for hypothyroidism in dogs is based on a TT4 < 17 nmol/L (together with an increased TSH) and the decision limit for hyperthyroidism in cats is a TT4 > 50 nmol/L. The method was linear from 50 nmol/L to a maximum tested level of 125 nmol/L; below 50 nmol/L the standard error exceeded the acceptance limits. Intra-and interassay precision was 14% at 17 nmol/L to 2.8% at 127 nmol/L. Method comparison was performed using a chemiluminescent assay (Siemens Immulite KT4) as the gold standard. Pearson correlation coefficients were 0.90 for dogs and 0.98 for cats. Regression analysis for dogs yielded a significant constant negative bias of 5.4 nmol/L. In cats there was a proportional bias for values above 100 nmol/L. Bias was 12% at the clinical decision limit of 50 nmol/L. Total error was 40% at 17 nmol/L and 24% at 50 nmol/L in dogs and 30% at 13 nmol/L and 26% at 50 nmol/L in cats. These findings show that this method is not acceptable for the measurement of TT4 in dogs due to lack of linearity and high imprecision in the clinically important lower range. In cats, although precision at the upper decision limit is acceptable, the bias and resulting total error are concerning. The use of human TT4 assays for animals should be carefully evaluated, as human RIs for TT4 are higher than for dogs and cats. Human assays may lack precision and analytical sensitivity in the low range clinically important for dogs. In veterinary medicine there is an increasing interest in the application of proteomic techniques to investigate protein patterns in healthy and diseased animals; however, data on urine proteome are still limited. The aims of this study were to identify a urine protein profile in healthy cats and to compare it with those obtained in patients affected by chronic kidney disease (CKD). Urine samples were collected by cystocentesis from 23 healthy and 18 CKD cats. Urinalysis was performed and urine protein to creatinine ratio (UPC) was calculated. Urine proteins were separated by SDS-PAGE on 4-12% gels, 20 different protein bands were cut, reduced, alkylated and digested by trypsin before ESI-Q-TOF mass spectrometry analysis; protein identification was performed using MASCOT science search engine. CKD cats had significantly higher UPC (median 0.9, P < .01) and significantly lower urine specific gravity (USG, median 1.014, P < .01) than healthy cats (UPC 0.1; USG 1.070). SDS-PAGE allowed visualization of qualitative and quantitative differences in protein profiles. CKD cats had significantly less bands (P < .01), particular at molecular weight (MW) > 100 kDa (P < .01). Eleven different proteins were identified by mass spectrometry in cat urine. In CKD a2-macroglobulin (168 kDa) and uromodulin (90 kDa) disappeared, while albumin (64 kDa), retinol-binding protein (RBP, 22 kDa) and cystatin (16 kDa) were enhanced. Cauxin (carboxylesterase 5A, 61 kDa), transferrin (80 kDa), angiotensin-converting enzyme 2 (93 kDa), inter-a-trypsin inhibitor heavy chain H4 (103 kDa), albumin-(55 kDa) and cauxin-(40 kDa) fragments, haptoglobin (45 kDa) and immunoglobulin light chains (24 kDa) were also identified. In conclusion, proteomic techniques were successfully used to investigate urine proteins, revealing different patterns between healthy and CKD cats; some of these proteins could be considered as promising biomarkers of chronic renal damage in feline patients. Cancer is a major cause of morbidity and mortality within the canine population and the most common canine hematopoietic neoplasm is lymphoma. Canine lymphoma represents a model for human non-Hodgkin lymphoma due to histological similarities in many respects. Recent data have expanded the concept that inflammation is a critical component of tumor initiation and progression. Cellular adhesion molecules have also been implicated in tumor progression and metastasis. The aim of this study was to evaluate inflammatory biomarker levels in canine lymphoma. Two groups of 15 dogs presenting with cytological lymphoma diagnosis and 15 healthy dogs were included in this study. Serum high sensitivity C reactive protein (hsCRP), IL-6, high mobility group box-1 proteins (HMGB-1), soluble intercellular adhesive molecule-1 (sICAM-1), plasminogen activator inhibitor-1 (PAI-1) and soluble urokinase receptor of plasminogen activator (suPAR) were determined (canine kits obtained from Biotang, Biotang Source International, Camarillo, USA). Dogs with lymphoma had significantly higher levels of hsCRP, ICAM-1 and IL-6 compared to healthy dogs, suggesting a proinflammatory status in dogs with lymphoma. Inflammation is known to be involved in the initiation and progression of cancer. The chronic, low-grade inflammation associated with cancer is reflected by elevated levels of hsCRP as a result of chronic stimulation with proinflammatory cytokine IL-6. The adhesion molecules are intimately involved in inflammatory reactions, but more recently a role in tumor progression has been postulated. Increased ICAM-1 expression may play a role in tumor cell adhesion to the vascular endothelium, promoting extravasation of cells and the development of metastases. Further studies are warranted to determine whether measurements of these inflammatory biomarkers are suitable in the diagnosis and monitoring of canine lymphoma. A 5-month-old male Weimaraner dog was referred to San Marco Veterinary Clinic with a 5 day history of depression, anorexia, reluctance to move and intermittent fever. On clinical examination, the dog, regularly vaccinated with a heptavalent vaccine, showed lumbar pain, hind limb stiffness, bilateral conjunctivitis and mild prescapular and submandibular lymphadenomegaly. Hematologic analysis showed mild non-regenerative normocytic normochromic anemia, monocytosis and thrombocytopenia. Blood smear evaluation revealed oval to round medium to dark purplish red cytoplasmic inclusion bodies mostly in lymphocytes but also in neutrophils and monocytes. Biochemical analysis was unremarkable with the only exception of C-reactive Protein (0.81 mg/dL (RI 0.01-0.22)), haptoglobin (206 mg/dL (RI 1-96)) and IgG (92 mg/dL (RI 323-659)). Cytologic examination of fine needle capillary sampling (FNCS) of the prescapular lymph nodes, synovial fluid of the stifle and cerebrospinal fluid (CSF) were performed. Lymph node cytology revealed a polymorphous lymphoid population, mostly consisting of mature or maturing lymphocytes, compatible with a moderate reactive lymphoid hyperplasia. Many lymphoid cells had cytoplasmic inclusions (similar ones were found in blood cells). CSF and synovial fluid evaluation revealed a mild mononuclear pleocytosis and a moderate degenerative arthropathy, respectively. No cytoplasmic inclusions were detected. The dog was positive for Canine Distemper Virus (CDV) rt-PCR tests on blood, lymph node and CSF. Indirect immunoperoxidase and immunofluorescence on air dried lymph node FNCS smears showed focal cytoplasmic brown staining and discrete cytoplasmic green fluorescence, respectively, indicative of the presence of the CDV antigen. CDV inclusion bodies in blood and lymph node cells are often few in number and may be difficult to detect. The unusual feature of this case was that the animal had been vaccinated against CDV infection. The very low IgG levels were suggestive of a possible breed related immunodeficiency state as previously reported in Weimaraner dogs, thus compromising the vaccine prophylaxis. Measurements of serum Cardiac Troponin (cTnI) concentration in dogs were assessed as marker for myocardial damage and as prognostic indicator of future cardiovascular disease. Currently, various cTnI assays are available producing different results and, consequently, using different clinical cut-off values. Therefore, cTnI values obtained from different assays cannot be interchanged nor can clinical cut-offs. A comparison study using 2 methods to assess cTnI values was performed (Fluorescence Enzyme Immuno Assay, FEIA-TB, Tosoh Bioscience; Chemiluminescence assay, Chem, DPC Immulite). Reference intervals (RI) for FEIA were calculated for a group of healthy dogs. In addition, a group of dogs affected by mitral valve disease (MVD) and cardio vascular disease (CVD), staged according to ACVIM Consensus Statement (JVIM, 2009) were studied to predict or monitor myocardial damage. Reference intervals (RI) for cTnI Chem were established at a referral laboratory. A total of 47 serum samples collected from healthy dogs (assessed by physical exam and CBC, biochemistry, and urinalysis) were used to calculate cTnI FEIA-TB RI. These samples were also tested with cTnI Vet Clin Pathol 42/4 (2014) E20-E48 Ó2013 American Society for Veterinary Clinical Pathology E36 Chem. A total of 67 samples collected from MVD-affected dogs (n = 44 stage B, n = 13 stage C, n = 10 stage D) were tested both with cTnI FEIA-TB and cTnI Chem. Statistical analysis was arranged for data collected (RI; correlation, r and regression r 2 CVD-stages dogs). RI for cTnI FEIA-TB was 0.0-0.09 ng/mL, for cTnI Chem 0.05-0.24 ng/mL. There were 7/44 B-stage dogs with cTnI out of RI for FEIA-TB and 8/44 for Chem (r = 0.85; r 2 = 0.99), 9/13 C-stage dogs out of RI for FEIA-TB and 10/13 for Chem (r = 0.97; r 2 = 0.99), and 7/20 D-stage dogs out of RI were 7/10 for FEIA-TB and 9/10 for Chem (r = 0.95; r 2 = 0.80). RI of cTnI with FEIA-TB in healthy dogs are lower compared to Chem, but they match for identification of dogs with myocardial damage. The important role of cytology as a minimally invasive diagnostic tool in veterinary medicine is well established. Cytologic examinations of young cats and dogs (< 1 year old) have been seldom reported so far. This study summarizes cases collected between October 2012 and May 2013. A total of 91 cases were classified using cytologic criteria. In puppies, 73 cytologic specimens originated from skin and subcutaneous masses (41/56.2%), lymph nodes (10/13.7%), peripheral blood (6/8.2%), cavitary effusions (3/4.1%), synovial fluid (3/4.1%), bone marrow aspiration (3/ 4.1%), bronchoalveolar liquid (2/2.7%), cerebrospinal fluid (2/ 2.7%), bone lesion (1/1.4%), nasal swab (1/1.4%), and vaginal smear (1/1.4%). In kittens, 18 cytologic specimens originated from cavitary effusions (8/44.4%), skin and subcutaneous masses (3/16.6%), bone marrow aspiration (2/11.1%), bronchoalveolar liquid (2/11.1%), conjunctival swab (1/5.6%), intestinal masses (1/5.6%), and lymph nodes (1/5.6%). This study highlights the prevalence in puppies of skin and subcutaneous mass and lymph node fine needle aspirations, the most frequent one being histiocytoma, and neuthrophilic and eosinophilic adenitis in the lymph node group. In kittens the most frequent cytologic analyses concerned cavitary effusions, and the principal diagnoses were protein rich transudates and exudates, compatible with FIP/ Coronavirus infection. In people the presence of nucleated erythrocytes (nRBCs) on blood smears can be associated with increased mortality. We frequently observe nRBCs on smears of dogs undergoing chemotherapy. So far, no information concerning the significance nRBCs is available. The aim of this study was to assess the frequency of nRBCs in tumor bearing dogs treated with different drugs, and the association with the clinical status. A total of 485 smears from 71 dogs treated for lymphoma (LYMPH, n = 41), carcinoma (CARC, n = 18), and mast cell tumor (MCT, n = 13) were examined by 2 observers. Total RBC counts and the presence of anisocytosis, polychromasia and Howell-Jolly bodies, as well as information on tumor types and therapy were recorded. In 29.2% of the dogs >1% of nRBCs were counted (LYMPH = 29.9%; CARC = 33,3%; MCT = 19.6%). In 7.6% of the dogs, >5% of nRBCs were counted, with a higher proportion in LYMPH (9.4%) than in CARC (1.5%) and MCT (3.6%). Drugs that frequently induced the presence of nRBCs were actinomycin, cytarabine, and dexamethasone (77.8% of treated dogs had >1% of nRBC; 22.2% had >5% of nRBC), Vincristine (38.1%; 20.3%), Mitoxantrone (35.7%; 7.1%) and Doxorubicin (25.0%; 8.3%) in dogs with LYMPH, carboplatin (37.0%; 2.2%) in dogs with CARC, and Vinblastine (25.0%; 5.0%) in dogs with MCT. High absolute nRBC counts (>1 x 10³/lL) were detected only in dogs with LYMPH (2.9%) or MCT (1.8%). The only drugs that induced an increased nRBC count were vincristine (9.5% of treated dogs), doxorubicin (3.3%), and vinblastine (2.5%). No association with clinical signs, anemia or RBC regeneration was found. These results suggest that vincristine, vinblastine and doxorubicine may induce the release of nRBCs from the bone marrow. However, the percentages of nRBCs may frequently increase on blood smears also using other drugs, likely relative to the decreased WBC count of treated dogs. Dipyrone (metamizole) is classified as NSAID. Analgesic action of dipyrone has already been confirmed in dogs, but potential effects on hemostasis have not been reported yet. Our study compared the effects of dipyrone, meloxicam and a combination of the 2 on hemostasis measured as buccal mucosal bleeding time (BMBT) and platelet aggregation in whole blood (PA). Six dogs were distributed in 4 groups using an aleatory crossed distribution: control (n = 4, GC, NaCl 0.9%), dipyrone (n = 3, GD, 25 mg/kg), meloxicam (n = 4, GM, 0.2 mg/kg) and dipyrone/ meloxicam (n = 6, GDM, 25 mg/kg e 0.2 mg/kg, respectively). A central venous catheter (18G) was placed in the jugular vein to administer the medications and collect blood for analysis. BMBT was measured before, and 1, 3 and 5 h after treatment. Blood samples were collected in sodium citrate 3.2% before, and 1, 2, 3, 5 and 8 h after a single treatment. PA was measured using collagen (3.2 lg) as agonist and the variable determined were amplitude (ohm), lag time (seconds) and area under curve (AUC). The results showed an increase in lag time and decrease of amplitude and AUC after one hour dipyrone administration (GD). There were no differences in BMBTs. A single dipyrone administration appeared to compromise aggregation for a short time, which was prolonged in combination with meloxicam. This emphasizes the importance of a careful choice of anti-inflammatory and/or analgesic treatment for dogs scheduled for surgery. Financial support: grant # 2012/09020-1, São Paulo Research Foundation (FAPESP) The purpose of the study was to define the influence of repeated therapeutic dosages of the low molecular weight heparin (LMWH) dalteparin on hemostatic parameters in cats. 6 healthy domestic shorthair cats received a total of 13 injections of dalteparin (75 anti-Xa IU/ kg SC) at 6 h intervals. Blood samples were collected before and 2 h after the first and second injection on days 1, 2, and 4 (ie, injections 1, 2, 5, 6, 13, and 14). Determined variables included anti-activated coagulation factor X (anti-Xa) activity, APTT, thrombin time (TT), thromboelastometry (TEM), antithrombin activity, HCT, and platelet counts. Anti-Xa activity was measured using a commercial chromogenic substrate assay. Both APTT and TT were measured using an automated coagulometer, the APTT was measured with 2 different commercial reagents, TT was measured with 2 different final concentrations of bovine thrombin (1 and 2 IU/ml). TEM was measured with the ROTEM delta analyzer, with and without kaolin activation. Ratio values were calculated (measured/baseline) and Spearman's rank correlation coefficient was calculated for the relationship between anti-Xa activity and ratio values of different tests. The treatment protocol led to reproducible peak anti-Xa activities in cats which were within the target range recommended for thrombosis treatment in people (0.5-1.0 IU/mL). Peak anti-Xa levels had only minimal effects on coagulation variables. The effect of LMWH on non-activated and kaolin activated ROTEM was more pronounced, where median ratio values at peak anti-Xa activities reached 2.87 and 2.39, but inter-individual variation was substantial. There was no significant correlation between the anti-Xa activity and CT ratios of the ROTEM analyzer, or between the anti-Xa activity and ratio values of any of the coagulation times. In conclusion, as in other species none of the coagulation assays seems suitable to monitor LMWH treatment in cats, including TEM. Viscoelastic examination techniques of hemostasis such as rotation thromboelastometry are a useful tool to detect and monitor hemostatic disorders and to monitor anticoagulant therapies. The aims of this study were to assess precision of measurements of feline citrated blood using the ROTEM delta analyzer and to establish reference intervals (RI). Intra-assayvariability was evaluated by analyzing blood samples of 2 cats in quadruplicate. RIs (2.5%-and 97.5% quantiles) were established based on 55 clinically healthy European Shorthair cats of different gender. Statistical comparisons were performed between different age groups (6-12 months [n = 8]; >12-48 months [n = 18]; >48-96 months [n = 14]; >96 months [n = 15]). Only cats with normal hematologic, biochemical, and routine hemostatic tests (PT, APTT, thrombin time) were included. Analyses with the ROTEM delta analyzer were performed with and without activation with different reagents (Extem, Intem, kaolin). For the majority of variables coefficients of variation were < 10%. The activating reagent containing tissue factor (Extem) produced the shortest clotting times (CT, RI 44.0-98.7 s; Intem: 107-255 s) and highest maximum lysis, whereas only minor differences were found between different activating reagents for the maximum clot firmness (eg, Extem: 53.1-77.7 mm; Intem: 49.5-82.0 mm). RIs of many studied variables revealed a wide inter-individual variation, for example the clot formation time (eg, Extem: 34.0-92.7 s; Intem: 107-255 s), maximum clot elasticity, and lysis parameters. Kruskal-Wallis-test revealed only sporadic differences between individual age groups. Cats aged 6 -12 months had shorter CT compared to older animals with or without kaolin activation. In conclusion, thrombelastometric analysis of feline blood using the ROTEM analyzer showed acceptable reproducibility. The heterogeneous composition of animals may have contributed to the partially wide physiological variation. The determination of population-based reference intervals (RI) according to published guidelines is difficult in wildlife and exotic species, where often only low numbers of healthy individuals are available. As an alternative subject-based RIs can be determined from a single animal or a small group, based on the measurement of biological variation including intra-individual (CV I ) and interindividual (CV G ) variation. CV I can be used to calculate the reference change value (RCV), defined as the difference between 2 consecutive test results in an individual which is statistically significant in a given proportion of similar individuals and may be significant even though both values lie within population-based RIs. The aim of this study was to calculate RCVs for hematology and clinical chemistry analytes for 4 healthy adult female African Elephants living in captivity at the Sch€ onbrunn Zoo in Vienna, Austria.

Blood samples were collected for hematology and clinical chemistry analyses under standardized conditions from each individual elephant 7 times over a period of 9 months (June 2011-March 2012). The CV I was generated for total WBC count (10,3%), RBC count (9.4%), HGB concentration (7.3%), HCT (7.4%), MVC (3.3%) and MCHC (2.1%), urea (19.4%), creatinine (10.9%), total protein (6.1%), bilirubin (22.8%), AST (17.3%), calcium (7.0%), phosphorus (15.8%), sodium (2.3%) and potassium (23.6%). The analytical variation for each analyte (CV A ) was calculated from internal laboratory quality control data for the period in question and was used together with the CV I to calculate the RCVs: WBC 29.2%, RBC 26.4%, Hb 20.5%, Hct 20.8%, MCV 9.5%, MCHC 6.6%, urea 56.2%, creatinine 36.6%, total protein 17.8%, bilirubin 68.8%, AST 52.6%, calcium 23.8%, phosphorus 44.4%, sodium 8.5% and potassium 65.7%. CV I for hematologic and clinical chemistry variables in these elephants were comparable to dogs but greater than in people. The RCVs generated for these elephants can potentially be Vet Clin Pathol 42/4 (2014) E20-E48 Ó2013 American Society for Veterinary Clinical Pathology E38 used to detect clinically relevant changes in hematology and clinical chemistry analytes in these individuals. Although some concerns exist on the practical utility of total nucleated cell counts (TNCCs) on bronchoalveolar lavage (BAL) fluids, the TNCCs is considered a useful tool to estimate the quality of the sample, based on the assumption that only when samples have a sufficient number of cells cytology may provide reliable information. No information about the possible presence of artifacts in automated counts on BAL fluids from dogs is available. The aim of this study was to assess the frequency of enumeration errors on BAL fluids using the laser counter Sysmex XT2000iV. A total of 60 BAL fluids collected from dogs with respiratory disorders using standard endoscopic techniques were analyzed. Counts generated in DIFF and BASO channels and DWBC were recorded. The DIFF channel counts the cells stained with a fluorescent dye that specifically binds to nucleic acids; the BASO channel counts the cellular residues produced after contact with an acidic reagent that collapses the nucleated cells. DWBC is the ratio between DIFF and BASO counts, which should be close to 1.00 without artifacts. In our caseload, only 4 samples (6.7%) had a DWBC of 1.00 AE 0.05, probably not affected by artifacts. Conversely, 56 samples (93.3%) had an abnormal DWBC. Specifically, 42 samples (70%) had a DWBC < 1.00 (mean AE SD=0.57 AE 0.24; median=0.57; min-max=0.12-0.94), likely due to the presence of non-cellular particles (eg, fragments of mucus clots, cellular debris), resulting in higher BASO compared with DIFF counts. On the contrary, 13 samples (21.7%) had a DWBC higher than 1.00 (2,15 AE 1,74; 1,32; 1,06-6,17), due to mucous clots in BASO reagent entrapping the cells, resulting in falsely low BASO counts, or DNA positive non-cellular particles (eg bacterial aggregates), falsely elevating the DIFF count. In conclusion, this study demonstrated that the automated TNCCs on canine BAL fluids may suffer from pre-analytical errors resulting in inaccurate results. Cytologic examination of BAL fluids is required to verify automated counts and potential artifacts. Most veterinary students are not familiar with the preanalytical phase and its requirements. The purpose of the study was to provide a summary of preanalytical conditions collected from original articles, and to provide the corresponding articles in a data base to users. Original articles on domestic, laboratory and wild animals were identified from authors' files and various literature surveys. Main information summarized in an Excel spreadsheet and copies of articles as PDFs are made available on the veterinary school Moodle internet teaching platform. It is open to any user at the following address: http://svmoodle.envt.fr/course/view. php?id=11&edit=1&sesskey=fU56XipJUN

The summary table and library of articles is continuously increasing as new information and references are included. It allows finding information on technical and biological factors of preanalytical variability of hematologic, biochemical and cytologic analytes in domestic, laboratory, exotic, and wild animals. The main difficulty is the identification of relevant sources and the retrieval of original articles, some of which are "old" and not available from websites. As this tool is currently under development, it has not yet been tested with students but should also allow them to have a critical vision of "big tables" which are oversimplifications of original scientific publications. Moreover, no selection is made on the scientific quality of the articles, which should be evaluated by the students as part of their training. In conclusion, this new tool should allow students and other interested individuals to improve their knowledge of the preanalytical phase, thus improving the efficient use of the clinical pathology laboratory. The serum concentration of high density lipoproteins (HDLs) is affected by oxidative phenomena that characterize inflammation. The aims of this study were to assess the concentration of HDLs in dogs with leishmaniasis, and to investigate the correlation between HDL, C-reactive protein (CRP), globulin fractions, and activity of the antioxidant enzyme paraoxonase (PON1). The serum concentration of HDLs was also measured during treatment to assess whether it may be a good marker to monitor response to therapy. HDLs were measured in serum from 10 leishmaniotic dogs (7 classified as sick and 3 as severely sick according to the staging system of the Canine Leishmaniasis Working Group), at admission and during the follow up (3, 7, 14, 21 , and 28 days after the beginning of treatment with antimonials and allopurinol). Concentrations of total cholesterol, PON1, CRP and globulin fractions were also measured. At admission, the concentration of HDLs did not differ between sick and severely sick dogs. The correlation between HDL, PON1, CRP, and globulin fractions was assessed on the whole set of data. There was a negative correlation (P < .05, r = -0.42) between total cholesterol and c-globulins, and positive correlations between HDL and PON1 (P = .002; r = 0.55), and HDLs and A/G ratio (P = .019, r = 0.44). There was a negative correlation between HDLs and CRP (r = -0.37, P =,058). HDLs increased in sequential samples collected during the follow up after 14 and 28 days (P = .033 and P = .024). These results support an influence of c-globulins on lipid metabolism as reported in people, and suggest that inflammation is associated with a decreased HDLs concentration, likely due to the oxidative phenomena that decrease PON1 activity. Moreover, these results suggest that HDLs may be useful to assess decreased oxidation/inflammation after successful treatments. In conclusion, HDLs may serve as an additional inflammation and oxidation marker, including dogs with leishmaniasis. Blood lactate measurement is a useful prognostic tool in human intensive care. In dogs prognostic value of lactate measurement has been evaluated in gastric dilatation and volvulus, immune mediated anemia, trauma and pyometra. Our goal was to evaluate the prognostic value of blood lactate and lactate clearance in dogs suffering from various types of diseases in a critical care setting. A total of 26 dogs with ASAP score ≥ 3 admitted at the Intensive Care Unit of the Internal Medicine Department were included in the study. Clinical examination, CBC, lactate, glucose, electrolytes, acid-base examination (venous pH, pCO 2 , pO 2 , HCO 3 , BE), anion gap, ALT, total protein, albumin, urea, and creatinine were measured prior to therapy. Venous blood lactate was measured with a hand-held lactate analyzer (Lactate SCOUT, Radiometer Copenhagen) at admission. If at admission blood lactate was > 2.5 mmol/l, it was repeated 6 and 16 h later, and lactate clearance was calculated. Disease groups included anemia, hepatopathy, nephropathy, encephalopathy, multiorgan failure (MOF), neoplastic disease, and pulmonary disease. Dogs were also evaluated according to systemic inflammatory response criteria (SIRS). Survival time was followed up to 2 weeks after hospitalization. Hyperlactemia (HL) was present in 32% (9/27) of all cases. Survival was 63% (4/7) in dogs with HL and 37% (7/19 cases) in normolactemic dogs (P = .31). Dogs with anemia, MOF and pulmonary disease had higher lactate levels than other groups. Dogs with SIRS had higher lactate levels compared to dogs without SIRS (2.96 vs 2.08 mmol/l) and SIRS was associated with significantly higher mortality (P < .001). Lactate clearance in survivors compared to non survivors was 25.2 vs 29.7 mmol/l (P = .74) at 6 hours and 11.7 vs 50.4 mmol/l (P = .002) at 16 hours after admission. In conclusion, SIRS criteria were a better prognostic factor of mortality than admission blood lactate level in dogs suffering from various diseases in a critically ill setting. However, increased lactate clearance after therapy was strongly associated with better survival. Most dogs with leishmaniasis due to Leishmania infantum are seropositive. Accurate rapid in-clinic serologic tests may permit immediate confirmation of the diagnosis and implementation of therapeutic measures. The aim of this study was to evaluate the diagnostic accuracy of 2 commercial, rapid in-clinic serologic tests for the detection of anti-Leishmania antibodies in sera of dogs, the Snap Canine Leishmania Antibody Test kit and the Immuno-Run Antibody Detection kit, using immunofluorescent antibody test (IFAT) as the gold standard. A total of 109 sera collected from 65 seropositive and 44 seronegative dogs were used. The sensitivities of the Snap Canine Leishmania Antibody Test kit and of the ImmunoRun Antibody Detection kit were 89.23% (95% CI: 79.05-95.54%) and 86.15% (95% CI: 75.33-93.45%), respectively, and the specificity of both tests was 100%. A good agreement between each of the rapid in-clinic serologic tests and IFAT and between the 2 rapid in-clinic serologic tests was found. Both rapid in-clinic serologic tests showed an adequate diagnostic accuracy and can be used for the fast detection of antibodies against L infantum in dogs. Paraoxonase 1 (PON1) is an esterase synthesized in the liver and secreted to blood, where it circulates associated with high-density lipoprotein cholesterol (HDL-C). Its primary physiologic role is the protection of low-density lipoprotein cholesterol (LDL-C) and HDL-C from oxidative modifications through enzymatic hydroxylation of oxidized phospholipids. In human medicine a decrease in PON1 activity was described in obesity being associated with oxidative stress. However, in dogs no such studies have been reported. The purpose of the study was to evaluate PON1 activity in obese dogs before and after a weight loss program. 35 dogs were included in the study. These dogs participated in a study on the effects of weight loss on inflammatory biomarkers. Obesity (body fat mass >35%) of all dogs was confirmed by dual-energy X-ray absorptiometry. All dogs underwent a weight loss program. PON1 and HDL-C were measured before and after weight loss. Median (range) PON1 activity in dogs before was 2.7 IU/L (0.9 -3.8 IU/L) and decreased to 2.2 IU/L (0.2 -3.4) after weight loss (P < .001), in contrast to observations in human medicine. This divergence could be explained by different metabolism of HDL-C in these species, as human obesity is associated with reduced levels of HDL-C, while in canine obesity HDL-C is increased. This hypothesis is supported by the observed strong correlation between PON1 and HDL-C (r = 0.622; P < .001). In conclusion, in this study canine obesity was associated with increased levels of PON1 possibly because of the increased circulating levels of HDL-C.

A COMPARATIVE STUDY OF SEROLOGICAL TESTS FOR THE DETECTION OF CANINE ANTI-LEISHMANIA INFANTUM ANTIBODIES L. Solano-Gallego 1 , S. Villanueva-Saz 1 , M. Carbonell 1 , T. Furlanello 2 , A. Natale 3 , M. Trotta 2 . 1 Facultat de Veterin aria,Universitat Aut onoma de Barcelona UAB , Barcelona, Spain; 2 Laboratorio Privato Veterinario San Marco, Padova, Italy; 3 Istituto Zooprofilattico Sperimentale delle Venezie, Padova, Italy.

The aim of this study was to evaluate the diagnostic performance of 6 serological tests for the detection of canine antibodies against Leishmania infantum antigen. Three commercially available Vet Clin Pathol 42/4 (2014) E20-E48 Ó2013 American Society for Veterinary Clinical Pathology E40 quantitative ELISA (Leiscan, ID Screen and Leishmania 96), one qualitative test (Speed Leish K) and 2 quantitative serological inhouse techniques (ELISA and IFAT) were analyzed. Clinically sick dogs (n = 36), seropositive clinically healthy dogs (n = 18), high to low seropositive dogs (n = 53), uninfected dogs from L infantum endemic areas (n = 32), uninfected dogs from nonendemic areas (n = 50), and dogs seropositive for other pathogens (n = 14) were studied. Reference dogs were selected based on clinical history, complete physical examination, routine laboratory tests, quantitative serology and real-time PCR. Sensitivity, specificity, accuracy, area under receiver-operating curve (ROC-AUC) and Kappa index were determined. The UAB ELISA was the most sensitive test (1.000), followed by the Leiscan and ID Screen (0.953), Leishmania 96 (0.925), and IFAT (0.869), whilst Speed Leish K was the least sensitive (0.636). Maximum specificity (1.000) was obtained for all diagnostic tests except the IFAT (0.917) and Leishmania 96 (0.896). The UAB ELISA showed the greatest accuracy (1.000) followed by Leiscan test and ID Screen test (0.975) and Leishmania 96 test (0.911). IFAT (0.891) and Speed Leish K (0.808) had the lowest accuracy. UAB ELISA (1.000) had the highest ROC-AUC, followed by ID Screen (0.993), Leiscan (0.990), Leishmania 96 (0.962), IFAT (0.926) and the Speed Leish K (0.818). UAB ELISA (1.000) had the best Kappa index and Speed Leish K (0.622) the lowest result. Overall, the UAB ELISA, Leiscan and ID Screen were superior to IFAT (P < .05), and all quantitative serological tests were superior (P < .05) to the only rapid test evaluated (Speed Leish K). These findings are important for clinical practice since Speed Leish K is used to diagnose clinical leishmaniasis and for screening prior to vaccination with CaniLeish. S-phase fraction (SPF) is a good indicator of proliferative activity in neoplastic populations. In human non-Hodgkin lymphomas (NHL) it correlates with tumor grading while contrasting results have been reported concerning its prognostic significance. Little information exists in veterinary medicine. The purpose of the study was to evaluate diagnostic and prognostic significance of SPF in lymph node aspirates from canine NHL by flow cytometry. Seventy-four canine NHL were evaluated. Based on immunophenotype and cytologic features, NHL were classified according the updated Kiel classification adapted for the dog. Fine-needle aspirates from lymph nodes were labeled with propidium iodide and SPFs were determined as part of the cell cycle using Multicycle software. SPFs ranged from 0,63% to 46,36% (median 9,36%; mean 10,31%AE7,45%). A statistically significant difference was detected between high grade (HG; 10,84%) and low grade (LG; 1,94%) forms. Areas under the receiver operator curve (ROC-AUC) showed a high accuracy in discriminating HG and LG (AUC=94,8%) and reported 3,95% as the best cut-off value to detect HG (sensitivity = 85,1%, specificity = 100%). There was no significant difference between B-and T-NHL. SPF had no prognostic value for overall survival using median value, 25 th or 75 th percentile to stratify groups, both considering all subtypes together and only high grade B lymphomas. In conclusion, SPF can be used as a diagnostic tool to discriminate between HG and LG but not as prognostic marker for overall survival. Canine leishmaniasis (CanL) due to Leishmania infantum is a life threatening zoonotic disease transmitted by Phlebotomus sand flies endemic in more than 70 countries. Reticulocyte indices such as reticulocyte volume (rMCV) and reticulocyte hemoglobin content (CHr) are provided by laser-based hematology analyzers and have demonstrated their utility as early and cost-effective biomarkers of iron deficiency anemia in both human and veterinary patients. The objectives of this retrospective study were to describe reticulocyte indices (RIs) in naturally infected dogs with L infantum and to correlate these data with serology titers and major clinical findings. 30 dogs of different ages (mean 7.5 years old), breeds and reproductive status with positive serology (ELISA for anti-leishmania antibodies) were included. Most common clinical findings were recorded based on physical examination and anamnesis. Results of CBC including reticulocyte analysis (ADVIA 120) and biochemistry profile including serum protein electrophoresis and urine protein-to-creatinine ratio were collected when available. Spearman's rho was used to study lineal correlate between serology titer and main clinical signs with CBC parameters. Statistically significant (P < .05) linear correlation was observed between HCT, RBC, HGB, RDW and CHr, with serology titer. Animals with higher serology had lower values except for RDW. Only lymphadenomegaly had a statistically significant correlation with rVCM and CHr. Kruskal-Wallis test was performed to compare animals with low, medium or high serology values and RIs, and lymphadenomegaly grade and RIs. The inflammatory response present in clinical leishmaniasis is associated with anemia of chronic disease, and may be related to lymphadenomegaly, one of the most common clinical manifestation of leishmaniasis. These conditions may also affect reticulocytes variables as an indicator of erythropoiesis. Animals with higher serology and generalized lymphadenomegaly tended to have lower CHr. Delayed or ineffective RBC maturation can be induced by several mechanisms including changes in iron homeostasis and other mechanism in anemia of chronic disease. The possible value of CHr in monitoring treatment response in dogs with leishmaniasis should be confirmed by further studies. In the last decade, measurement of acute phase proteins (APPs) has been widely used for monitoring treatments and in the assessment of disease activity in inflammatory or infectious conditions. APP measurements in animal production have demonstrated to be useful in screening for ante/post mortem inspection, as well as detection of subclinical infections and evaluation of health status. Other uses for APPs include the assessment of anti-microbial or anti-inflammatory agents and vaccine development. The use of APPs as biomarkers implies that assays must be standardized. Reference APPs materials are necessary to ensure reliability and comparability of the results of chemical analysis. The aim of this study was to develop new reference materials for acute phase proteins in pig: Pig Major Acute Phase Protein (pig-MAP) and porcine CRP. Serum obtained from healthy pigs was frozen at -80°C until its processing. A pilot batch of the serum was delipidated, diafiltrated and then spiked with purified PigMAP and porcine CRP. Stability studies in this reference material were performed in order to verify that it was homogenous and stable. The method used to measure these proteins was immunoturbidimetry, and it was performed with species-specific reagents on an Olympus AU400 platform. According to the Clinical and Laboratory Standards Institute (CLSI), "individual laboratories should focus more on verifying reference intervals established elsewhere." One proposed option is the transference after method comparison, which does not require specimens collected from reference individuals. The purpose of this study was to transfer a previously published canine reference interval (RI) for D-Dimers obtained by one analyzer (STA-Compact, Diagnostica Stago) in one laboratory for use with the same analyzer and reagents in another laboratory. The first batch of analyses was performed at the Ecole Nationale V et erinaire de Toulouse on 71 canine specimens stored in a plasma bank and using a STA-Compact analyzer with the STA-Liatest D-Di kit (Diagnostica Stago). The 71 specimens were then re-frozen at -80°C and sent with dry ice to the Justus-Liebig-Universit€ at Gießen were the second batch of analyses was immediately performed using the same analyzer, reagents and quality controls. Comparison of results was based on Spearman correlation and Passing-Bablok agreement. The 71 D-Dimers results covered a wide range of concentrations, from 0.03 to 6.8 mg/L. Correlation coefficient (r) was 0.89. Passing-Bablok equation was D-Di Toulouse = 0.88•D-Di Gießen -0.02. The 95% confidence intervals (CIs) were [0.76 -0.97] and [-0.06 -0.01] for slope and intercept respectively. The published RI for D-Dimers at the Justus-Liebig-Universit€ at Gießen is [0.023 -0.654] mg/L based on 56 healthy dogs. The published RIs were converted according to Passing-Bablok equation and, taking into account the limit of quantification (manufacturer's printout), the corresponding transferred RI was [<0.27 -0.56] mg/L. Comparability of canine population was presumed to be fulfilled as canine breeds represented in Germany and France are similar. In this first step, comparability of the techniques was considered to be satisfactory: r was high and the CIs of Passing-Bablok agreement coefficients were narrow. However, the validation of this transference has not yet been performed by a test with a small number of reference individuals as recommended by CLSI. The serum concentration of acute phase proteins (APPs) increases in the presence of disease or stress, which makes APPs valuable parameters for the global assessment of animal health and welfare. APP measurements in animal production have demonstrated to be useful for the evaluation of herd health status, in the detection of subclinical infection or stress conditions causing poor productive performance, as well as for ante/post mortem inspection. APPs can be also interesting tools in studies aimed to evaluate the efficiency of antibiotic or anti-inflammatory treatments or vaccination. The APP pattern is species dependant. Pig Major acute phase protein (pig-MAP), also known as ITIH4, is one of the main APPs in pigs.

The concentration of APPs in pig serum is usually measured by ELISA, a technique affected by a relatively high variability. A robust, turbidimetric method for the determination of Pig-MAP has been developed and validated. Pig-MAP was purified by affinity chromatography using a previously developed specific monoclonal antibody. The assay was developed using polyclonal antibodies raised in rabbits. The immunoturbidimetric method showed good precision (Intra assay CV < 5%; Inter-assay CV < 10%) and accuracy, with a linearity range up to 5 mg/mL. A good correlation was observed between the values obtained with the turbidimetric method and ELISA (R > 0.98). In this report, a listeria encephalitis was defined pathologically in a 3-year-old Holstein cow.

Clinical symptoms were gait disturbance, loss of appetite, one-sided facial paralysis and some decrease in swallowing reflexes. Necropsy revealed opaque meninges of the brain, and large soft areas in pons and medulla oblongata of the brainstem. Plenty of neutrophil granulocytes were seen in the cytological preparations prepared from these regions. Tissue samples taken from organs were fixed in 10% formalin and then routinely processed, including paraffin blocks in 5 lm thickness, H&E stain, and immunohistochemical (IHC) staining by the ABC method and L. monocytogenes primer. Histopathologic examinations revealed gliosis, areas with liquefaction necrosis and microabscesses containing neutrophils, gitter cells and spheroids in pons and medulla oblongata. In the meninges and perivascular areas near the micro-abscesses, a small number of neutrophils and infiltrations with lymphoid cells were seen. Positive staining was observed in the cytoplasm of the macrophages present in liquefaction necrosis and perivascular infiltration areas, confirming encephalitic listeriosis. Cows with similar neurological symptoms after feeding with silage of poor quality were reported with a similar history, as the cow being diagnosed by cytological, pathological and immunohistochemical methods and presented by this report. Cytology is commonly used as a diagnostic procedure to evaluate splenic masses in veterinary medicine. However, few studies have addressed the accuracy of cytology in the evaluation of splenic masses in a clinical setting. The purpose of this study was to compare the diagnostic accuracy of cytology, in comparison with histopathology, in a series of splenic masses. Cytologic and histopathologic specimens of 67 splenic masses from 67 dogs were retrospectively evaluated. Cytologic samples were obtained by fine needle aspirate or tissue imprint. Histopathology was evaluated from tissues collected by surgical biopsy or necropsy. A cytologic diagnosis of neoplasia was obtained in 36 cases (33 true positive cases, 3 false positive cases). Sarcoma (20/67) and lymphoma (7/ 67) were the most common tumors diagnosed by cytology. A total of 31 samples were classified as "negative for neoplasia" (12 true negative cases, 19 false negative cases) by cytology. Cytology had an overall 67.2% (45/67) agreement with histopathologic analysis. In diagnosing neoplasia, cytology had a sensitivity of 89%, a specificity of 80%, a predictive value of positive test of 91.2% and a predictive value of negative test of 38.7%. The results of this study confirmed cytology as a reliable and useful diagnostic procedure for the evaluation of splenic neoplastic lesions in canine small animal practice. The Beagle dog is commonly used as non-rodent species in preclinical toxicity studies during drug development. Because of inter-individual variation and the limited number of dogs per treatment group, it is important to establish baseline hematology and clinical chemistry variables for each animal prior to start of dosing in a non-clinical safety study. At our facility, historical control data (HCD) on clinical pathology variables are established for each species and the HCD's are being actualized at regular intervals. Starting early 2013 several hematology and clinical chemistry results during pre-dose testing showed either a shift towards the lower limits of the HCD range or fell outside the range, and this for multiple consecutive studies. A number of experimental conditions had changed in a short period of time and all possible factors were investigated. Pre-dose data from 3 different studies were analyzed and showed very similar trends for changes in urea, creatinine, total protein, albumin, ALT, RBC count, HGB concentration, and HCT levels. The findings were comparable in male and female dogs. Parameters investigated were a new clinical chemistry analyzer, different location of origin of dogs, and the age of the animals. The observed decreases in several biochemistry variables with urea being the most prominent corresponded with published data on young Beagle dogs. The dog's mean age in the investigated studies was 4.5 -5.5 months versus previously 6 -8 months. The facility's HCD data cover an age range of 5.3 -8.5 months. In conclusion, the observed alterations in clinical chemistry and hematology variables during pre-dose testing of Marshall Beagle dogs were due to the younger age of the animals at start of the study. Stratification according to age is important for establishment of HCD. Furthermore, narrow age intervals must be chosen as rapid changes occur during the first 6 months of life. This is an important factor in pediatric dog studies in drug development. Semen contamination can affect urine protein concentration in people and dogs, and retro-ejaculation is known to occur frequently in male dogs. The objective of the study was to investigate the effect of sex on the qualitative evaluation of urine protein electrophoresis in dogs. Urine specimens were collected from 20 healthy adult dogs (5 castrated males, 5 intact males, 5 neutered females, 5 intact females) with RPCU < 0.3. Urine sodium dodecyl sulfate-agarose gel electrophoresis (SDS-AGE) was performed followed by qualitative interpretation according Vet Clin Pathol 42/4 (2014) E20-E48 Ó2013 American Society for Veterinary Clinical Pathology E43 to molecular weight. 5 out of the 20 dogs had a low intensity low molecular weight band (LMWb). They all were intact males and 4/5 of them had visible spermatozoids at direct microscopic urinalysis. Results suggest that, in intact non proteinuric healthy dogs, probable urine contamination by semen, with or without microscopically visible spermatozoids, can result in the appearance of a LMWb. This band seems to reflect seminal protein contamination and not renal tubular proteins and thus warrants caution for the interpretation in intact males. More investigations are needed to confirm this hypothesis. In this study 45 female egg-laying turkeys were distributed in 3 groups, with 15 birds in each group: 1) GI control, 2) G2 vaccinated against Newcastle disease with LaSota activated strain (by ocular-nasal route), 3) G3 vaccinated against Newcastle disease with LaSota inactivated strain (subcutaneously). Turkeys were vaccinated with LaSota activated strain at 32, 40 and 48 weeks of age and with LaSota inactivated strain only at 32 weeks of age. Blood samples were collected at 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 , and 52 weeks of age. Protein serum concentrations were determined by SDS-PAGE electrophoresis. Purified chicken ovotransferrin iron-free from Sigma-Aldrich (St. Louis, MO, USA) was also used to perform protein gel electrophoresis by means of SDS-PAGE stained in Coomassie Brilliant Blue 0.2%. The standard ovotransferrin showed a molecular weight of 80kD for all the birds evaluated. Ten protein fractions were obtained: IgA, ceruloplasmin, transferrin, ovotransferrin, albumin, IgG heavy and light chains, non-identified PM 45 kDa, a-1 acid glycoprotein and a non-identified PM 23 kDa. Statistically significant differences were found for a-1 acid glycoprotein, IgG, ovotransferrin and albumin between G2 and G3 at 36, 38 and 48 weeks of age and for total serum protein and a-1 acid glycoprotein between G1 and G3 at 46 and 52 weeks of age. This study was supported by FAPESP/Brazil. An 11-year-old neutered male domestic short-hair cat presented with an invasive mass located in the perianal area that also involved the right sciatic region. On physical examination a slight exsudative discharge was seen, and the cat had been on antibiotic and anti-inflammatory therapy for approximately 2 weeks. Although the discharge disappeared, a distinct solid mass was palpable and fine needle aspiration was performed. No more clinical and laboratory results were available at that time. Cytological examination was consistent with epithelial malignancy and the most likely differential diagnosis included basal cell carcinoma, spindle cell carcinoma, anal sac gland adenocarcinoma, and apocrine gland carcinoma. On the basis of cytological results the mass was subsequently excised with wide margins and processed for histopathologic examination, which was consistent with anal sac gland carcinoma. Immunohistochemistry for CAM 5.2 supported the histological diagnosis. Anal sac gland carcinoma is a wellknown tumor in dogs, but is rarely documented in cats either for clinical and morphological features including cytologic appearance. Feline anal sac gland carcinomas tend to infiltrate the capsular fibrous tissue and surrounding soft tissue and can metastasize. Immunohistochemistry using monoclonal anticytokeratin CAM 5.2, which recognizes CK7 and CK8, has been suggested as a useful tool to further classify this type of tumor. Hypercalcemia is a well-know paraneoplastic syndrome occurring in dogs with anal sac gland carcinoma. However, little is known about the frequency of hypercalcemia and overproduction of PTHrP in cats with this tumor. In the case presented here no further details were available at that time. Clinician and pathologists should be aware of this uncommon neoplasia in cats presenting with clinical signs of anal sac disease. A 2-year-old male castrated Doberman was presented with a 2week history of progressive right hind limb lameness and a firm swelling on the right proximal tibia. A medio-lateral radiograph of the stifle revealed an osteolytic lesion of the proximal tibia, with extensive cortical and periosteal changes and soft tissue swelling. Fine needle aspirate of the lesion was performed as well as tissue biopsy. Cytologic examination revealed a spindle cell proliferation, and histological examination was supportive of hemangiosarcoma, which was confirmed by immunohistochemistry using an anti-Factor VIII antibody. The dog underwent right hindquarter limb amputation with coxo-femoral disarticulation. Histologic examination of the amputated leg revealed infiltration of the fibromuscular tissue adjacent to the stifle joint. The dog received 3 intravenous doxorubicin treatments once every 3 weeks. The owner reported that the dog was euthanized 9 months after first diagnosis. Hemangiosarcoma should be considered as a differential diagnosis for osteolytic bone lesions associated to cytological findings of spindle cell proliferation. A thorough staging and adjuvant chemotherapy should be advised in patients with monostotic disease due to the high metastatic rate. however, are different from other species. SPE RIs for dairy sheep using agarose gel as the supporting matrix has not been reported so far. The purpose of this study was to evaluate serum concentration of total protein and protein fractions (albumin and globulins) determined by agarose gel electrophoresis (AGE) in dairy ewes to establish RIs and to assess potential differences between Sarda (S) and Lacaune (L) sheep-breeds. Blood samples were collected from 175 clinically healthy multiparous mid-lactating (week 10 to 15 post-partum) ewes, including 119 L-and 56 S-sheep ranging from 2 to 6 years of age. Total protein concentration (TP) was determined by an automated biuret method. SPE was assessed by a semi-automated AGE system. The data were submitted to statistical analysis (MeanAESD, Min, Max, 99% Confidence Intervals). Data from Sarda and Lacaune sheeps were compared using the Wilcoxon-Mann-Whitney test. Significant differences (P < .05) were found between S and L sheep-breeds and separate RIs were calculated for TP (76,7-82,4 g/L; 71,9-74,4 g/L), albumin (34,0-37,9 g/L; 37,8-39,8 g/L),a1-globulin (3, 8 g/L; 3,1-3,5 g/L) a2-globulin (7,9-8,7 g/L; 7,4-7,9 g/L), b1-globulin (2,4-2,9 g/L; 1,6-2,0 g/L), b2-globulin (5,3-7,3 g/L; 3,4-3,9 g/L), c1-globulin (16,4-21,8 g/L; 12,0-13,6 g/L) and c2-globulin (3,3-4,3 g/L; 4,6-5,6 g/L) concentrations and albumin/globulin ratio (0,77-1,05; 1,11-1,23), respectively. S showed higher values for TP, albumin, a2-, b1-, b2and c1-globulins, whereas L had higher values for c2-globulin and A/G. AGE as supporting matrix for SPE in sheep gives excellent resolution and accurate results. A total of 7 protein fractions were standardized and differences between breeds were detected. RIs for an italian (Sarda) and a french (Lacaune) dairy sheep-breed are reported as an additional diagnostic aid for veterinary clinicians. Cats have naturally occurring alloantibodies, therefore they should be transfused with blood of the same blood type A, B, or AB to avoid major and minor transfusion reactions. The purpose of this study was to describe the blood type distribution in cats presented at 2 university blood banks in northern (Milan) and central (Perugia) Italy between September 2010 and June 2013 as potential blood donors, and to assess the risk of major and minor transfusion in this population. Blood typing was performed using an immunochromatographic cartridge method. A total of 357 cats belonging to 15 different breeds, 158 female (45.3%) and 191 male (54.7%), with a mean age of 3.8 years (SDAE2.9) was evaluated. Of these 90.5% (322) were blood type A, 5.6% (20) type B and 3.9% (14) type AB. The majority of the cats (195; 54.6%) were European Domestic Short Hair (EDSH), of which 92.3% (180) were type A, 5.1% (10) type B and 2.6% (5) type AB, 75 (21%) were Maine Coon (MC) cats, and 100% of these were blood type A. The estimated frequencies of transfusion reactions following an unmatched transfusion between EDSH (donors and recipients), MC (donor and recipients), EDSH donors and MC recipients and MC donors and EDSH recipients were 4.8%, 0%, 0%, and 5.1% for major reactions and 7.2%, 0%, 7.7%, 0% for minor transfusions reactions. The frequencies of the 3 feline blood types in cats in this study are comparable to those previously reported, with a predominance of blood type A. All MC cats in the study were blood type A. In a population of blood donors including EDSH and MC the risk of a transfusion reaction could be in excess of 5% if the donors and recipient are not blood typed. Blood typing and cross-matching should therefore be performed before transfusion in order to minimize the risk of transfusion reactions.

MEASUREMENT OF CEREBROSPINAL FLUID ALBU-MIN IN HEALTHY DOGS. E. Ramery 1 , M. Girod 1 , F. Allerton 1 , K. Gommeren 1 , J. DeMarchin 2 , D. Peeters 1 . 1 Faculty of Veterinary Medicine, University of Liege, Belgium; 2 Citalab, Citadelle Hospital, Liege, Belgium.

Measurement of CSF albumin aids diagnosis in human medicine but technical difficulties related to its low CSF concentration prohibit its routine use in veterinary medicine. High-resolution electrophoresis (HRE) has been described but often results in non-interpretable integration profiles preventing albumin determination. Fraction quantification using HRE may be more precise after concentration (cHRE) using a membrane microconcentrator technique but has not been evaluated in CSF with total protein levels below 20 mg/dL. An immunoturbidimetry assay (ITA) is routinely used for human CSF albumin measurement and was recently applied on canine samples with encouraging results. The purpose of this study was to compare HRE and cHRE and ITA for the measurement of albumin levels in normal canine CSF. A total of 30 CSF specimens from 15 healthy dogs were evaluated. CSF total protein was measured by the pyrogallol red method and CSF albumin was determined by HRE (n = 15), cHRE (n = 30) and ITA (n = 30). Validation of the human ITA was performed using a purified canine albumin standard. Mean CSF total protein was 17.5 (range 7-39) mg/dL. HRE integration profiles were noninterpretable in all unconcentrated specimens. However, clear distinction of the major protein fractions was achieved for all cHRE specimens. CSF albumin levels were measureable in 29/30 specimens using ITA. Excellent correlation (Pearson r = 0.92, P < .001) was found between the 2 techniques. ITA and cHRE may be used for routine measurement of CSF albumin in dogs.

and ASVCP recommendations. The purpose of the study was retrospective determination of population and individual RIs of routine hematology variables in laboratory Beagles. Data of control Beagles at the Sanofi centre in Alfortville, France (AAALAC accredited) between 2008 and 2012 were used. Beagles < 3 years old originated from Marshall Farms (USA). Analyses were performed in K2-EDTA specimens after overnight fasting with an ADVIA 2120 analyzer (Siemens, France). RIs and effects of sex, age, and weight were analyzed with Reference Value Advisor and Systat. A total of 147 females and 193 males were included. There was no effect of sex on age, mean (range) being 14.9 (9.4-34.7) months, with 91% of animals being ≤1.5 year old. Males were heavier than females (P < .001), respective medians and range being 8.9 (5.6-15.2) and 7.6 (4.1-12.8) kg. Weight increased with age (P < .018). There was an effect of: 1/age on all blood analytes except for MCV and neutrophils, 2/sex on MCV, lymphocytes, monocytes, basophils and platelets. The overall RIs were 6.0-8.310 12 /L for RBC, .5g/L for HGB, 0.399-0.547L/L for HCT, 62.4-71.0fL for MCV, 21.5-25.1pg for MCH, 330-368g/L for MCHC, 11.3-14.5 for RDW, 6.0-14.110 9 /L for WBC, 3.4-9.510 9 /L for neutrophils, 1.5-4.010 9 /L for lymphocytes, 0.2-0.910 9 /L for monocytes, 0.1-1.110 9 /L for eosinophils, and 220.4 -480610 9 /L for platelets. For all variables inter-individual (CV G ) and intra-individual (CV I ) variability were very close and indexes of individuality ranged from 0.52 to 1.63. In conclusion, the homogeneity of the genetic origin, age and regulatory housing conditions is the probable the cause of the very low inter-individual variability observed. However, these results cannot be transferred to other canine populations, including pet Beagles.

VALIDATION OF A NEW CANINE SPECIES-SPECIFIC C-REACTIVE PROTEIN ASSAY ON THE PENTRA 400. S. Klenner 1 , S. Zielinsky 2 , N. Kneier 1 , J. Klotz 1 , N. Bauer², A. Moritz 2 . 1 scil animal care company GmbH, Viernheim, Germany; 2 Justus-Liebig University of Giessen, Germany.

A new canine-specific C-reactive protein (CRP) assay including calibrators and species-specific controls is available from Gentian AS (Moss, Norway). The aim of the study was to validate this assay on the ABX Pentra 400 clinical chemistry analyzer (Horiba ABX SAS, Montpellier, France) as the use of canine-specific reagents and controls are supposed to be more precise and accurate than the previously used human-based reagents. Linearity was assessed using 7 serial dilutions of a high canine CRP serum sample. Within-run precision and between-run precision was performed using 4 different levels of canine serum pools. Interference study included evaluation of hemolysis, icterus and lipemia. Three different CRP samples were used to assess hook-effect (455 mg/L, 676 mg/L, 890 mg/L). Method comparison study included the human Randox CRP-reagent calibrated with canine specific control material using 279 canine serum samples. Quality specifications were set according to the literature and based on biologic variation. Linearity under dilution was shown from 7-281 mg/l. CV% was excellent except in very low CRP concentrations, which was acceptable as these low concentrations are without clinical relevance. Interference was not present up to a concentration of 5 g/L HGB, 800 mg/l bilirubin, and 10 g/l intralipid. Hook-effect occurred at extremely high concentrations >676 mg/l. Spearman correlation coefficient was >0.99, Bland-Altman difference plot revealed a slight bias of -1.2% with a confidence interval of -6.8% -4.4% The new assay performed in all experiments within the set quality specifications. The new Gentian CRP assay accurately and precisely measures canine CRP samples on the Pentra 400 and is an excellent alternative to human-based reagents. This study was supported by the German Federal Ministry of Education and Research (promotional reference 01QE1110). The risks associated with blood transfusion include the possibility of disease transmission, like anaplasmosis, which causes anemia, and as a consequence, the affected animals may require blood replacement. Therefore, it is important the inactivation of pathogens in blood bags collected from animals that do not have the signs of disease. The techniques of inactivation currently known are methods which have as objective to stop the pathogens from replicating their genome, for example, the treatment with riboflavin associated with ultraviolet radiation (UVR). This study aimed at evaluating the action of the treatment using riboflavin associated with UVR in an attempt to inactivate, reduce or completely eliminate Anaplasma marginale in bovine blood preserved for transfusion. A total of 8 calves were used in the study, as follows: group 1 consisted of 2 splenectomized calves which received blood with parasite without treatment; group 2 consisted of 3 healthy animals, and group 3 consisted of 3 splenectomized animals, which received blood treated with riboflavin associated with UVR. Stained blood smear (SBS), indirect immunofluorescence reaction (IIR) and quantitative polymerase chain reaction (qPCR) were performed before and after treatment, as well as inoculation in calves to evaluate the infectivity of the parasite. At time zero (T0), the mean parasite load in bags was 30.60% and after treatment, it reduced to 15.26%; IIR was positive at T0 and negative after treatment; qPCR showed lower values in blood of calves which received the treated blood. We concluded that the action of riboflavin associated with UVR inactivated A marginale, and did not cause disease in sensitive animals which had received blood with parasite treated with riboflavin associated with UVR. 

UMS 006, Laboratoire Central de Biologie M edicale

Currently available hematology reference intervals (RIs) in laboratory Beagles are from old studies and do not meet CLSI-IFCC

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Espada 1,2 , J. Martorell 1,2 . 1 Hospital Cl ınic Veterinari, 2 Depart. Medicina i Cirurgia Animal, Facultat de Veterin aria

Physical examination showed moderate abdominal distention and a firm mass in the mid abdomen. CBC was normal. Biochemistry abnormalities included hypertriglyceridemia and hypoproteinemia. Serum protein electrophoresis showed hypoalbuminemia and increased a/b-globulins. A FNA of a hypoechoic 4,8 cm abdominal mass revealed a uniform population of intermediate to large lymphocytes with multiple nucleoli and aberrant mitotic figures. Findings were compatible with lymphoma. Abdominal effusion consisted of a pinkish, milky fluid, with high cholesterol (Chol, 100 mg/dL) and triglycerides (TG, 651,1 mg/dL) concentrations, (Chol:TG ratio 0.15). Plasma Chol was 166,9 mg/dL and TG was 87,3 mg/dL TG. Total nucleated cell count (TNCC) was 57,310cells/lL, total protein concentration was 3,5 g/dL and specific gravity was 1.028. The predominant cells were nondegenerate neutrophils (62%), macrophages (25%) and lymphocytes (13%)

E20-E48 Ó2013 American Society for Veterinary Clinical Pathology E47

Different pathogenesis of DIC is expected in these diseases with different alterations in hemostatic profiles. The aim of this study was to describe and compare alterations in hemostatic profiles in dogs with GDV or H. Blood was collected from dogs with GDV (n = 12) and H (n = 6) at admission into citrate or EDTA tubes and platelet count (PLT), PT, APTT, fibrinogen (FBG), and D-dimers were measured, and thromboelastography (TEG) was performed. Median PLT was 186 vs 7410 9 /l (GDV vs H, min-max

subclinical persistence in infected animals, and as a consequence, it is likely that some hemoparasites remain viable in the stored blood. Moreover, the possibility exists of disease transmission as anaplasmosis. This study had as objective to demonstrate the importance of anaplasmosis diagnosis before blood collection for transfusion, and evaluate hematological and biochemical changes which occur in bovine blood from healthy animals compared to changes in bovine blood infected by Anaplasma marginale, when the latter was stored in plastic bags containing CDPA-1 at 4°C during 21 days. Group I consisted of plastic bags containing blood from clinically healthy bovine animals, and Group II consisted of plastic bags containing blood from bovine animals parasitized by A marginale. The exams were performed as follows: total number of RBC and WBC, HGB, globular volume, MCV, total plasma protein, fibrinogen, sodium, potassium and lactate determined each 3 days, over 21 days. For both groups, a reduction was found in mean values of RBC, HGB, globular volume, total plasma protein, WBC, and sodium; and an increase was found in MCV, potassium, and lactate. In addition, a reduction was observed in parasitemia and indirect immunofluorescence reaction. Therefore, we could conclude that before blood collection for transfusion from donor bovine animals, it is important to perform exams of parasitemia, indirect immunofluorescence reaction and PCR for diagnosis of anaplasmosis.

HAWS SYNDROME AND SUSPECTED FIP -CASE REPORT. J. Francuski, S. Ne si c, V. Krsti c, N. Andri c. Faculty of Veterinary Medicine, University of Belgrade, Serbia.A 5-month-old indoor male Russian Blue cat was presented to our clinic with a history of conjunctivitis, weight loss and antibiotic treatment over the preceding 1 month, with the symptoms of lethargy, anorexia and mucus in the stool. Bilateral protrusion of the third eyelids without miosis or mydriasis, serous conjunctivitis and ascites was detected at clinical examination. Hematology and biochemistry shows leukocytosis (22.33 x 10 9 ) with neutrophilia (19.77 x 10 9 ) hypoalbuminemia (18 g/l) and hyperglobulinemia (52 g/l) (decreased albumin-to-globulin ratio -0.35). Adult nematodes were observed in feces. Abdominal radiographs revealed peritoneal effusion but no visible changes in thorax radiographs. In the yellow, clear non viscous abdominal effusion, negative on microbiological examination, total protein content was 26 g/l, albumin 16 g/l, with total nucleated cell count (TNCC) 1500/lL and SG 1010. Cytology of abdominal effusion showed neutrophils and macrophages in a dense proteinaceous background. FIP/FIV/FeLV snap test (VetAll Laboratories, Korea) was negative. RT-PCR on FeCoV from a blood sample was also negative while competitive enzyme-linked immunosorbent assay showed high titers of antibody to Corona virus. Seven days after deworming, metronidazole therapy with infusions and vitamins, the cat died. An autopsy determined granulomatous peritonitis and lymphadenitis, hyperemia and pulmonary edema. Difficulties in definitively diagnosing FIP antemortem arise from the lack of specific clinical signs, the lack of pathognomonic biochemical abnormalities, and the low sensitivity and specificity of tests routinely used in practice. Cause of Haws syndrome is unknown but speculatively it may be associated with tapeworm infestation or virus infection. To the authors, knowledge there is no published data on PubMed about Haws syndrome and FIP infection in cats. Although the cause of Haws syndrome is unknown, it appears there is a strong association between the occurrence of ocular diseases and FIP infection. An increase in serum CRP and SAA concentration has been described after different surgical procedures in dogs. Orchiectomy is used widely to modify undesirable behavior, prevent health problems, and control pet population, thus exposing a large number of male dogs to surgery each year. The aim of this study was to investigate CRP and SAA concentrations before and after orchiectomy. Twelve clinically healthy male dogs were admitted to elective orchiectomy. Blood samples were collected from the distal cephalic vein just before the orchiectomy (base values) as well as after 24 hours (Day 1), 72 hours (Day 3) and 168 hours (Day 7) post surgery. The mean serum levels of CRP on Day 1 (68.39 AE 7.87 lg/mL) and Day 3 (29.67 AE 2.11 lg/mL) were significantly higher (P < .001 for Day 1 and P < .05 for Day 3) than the base value (4.84 AE 0.73 lg/mL). No statistical difference was found between the base value and Day 7 (7.95 AE 0.85 lg/mL). The mean serum levels of SAA on Day 1 Chyloabdomen (CHA) is a rare condition and can be primary or acquired. In cats it has been associated with neoplasia, including lymphoma, biliary cirrhosis, vitamin E deficiency-associated steatitis, hypertrophic cardiomyopathy and infectious disease. In dogs, it occurs with neoplasia, nontraumatic rupture of mesenteric lymphatic vessels, lymphatic obstruction, and inflammation or as a complication of thoracic duct ligation surgery. In people, the most common causes in adult patients are neoplasia (30% lymphoma), hepatic cirrhosis and infectious disease. Typically the predominant cells in chylous effusion are small lymphocytes, but non-degenerate neutrophils can be present due to a secondary chronic inflammatory reaction as chyle is an irritant to the serosal surface. Lymphoma is a common tumor in ferrets; its classification and clinical presentation depend on the organs affected. In this case, CHA was likely secondary to an obstruction of lymphatic drainage due to a lymphoma. To the authors' knowledge this is the first case reported in ferret.