Molecular identification of haemoparasites in animals using blood lysate PCR: a quick and inexpensive alternative to purified whole genomic DNA.

Haemoparasitic diseases constitute a significant constraint to economic livestock farming. Diagnostic techniques that are inexpensive, rapid, reliable, and precise are crucial for the management of diseases. In this context, PCR assays are very valuable yet expensive since the samples must be processed before being included in the PCR reaction. Accordingly, the goal of the current study was to lower the PCR costs without jeopardizing the assay's sensitivity and specificity. For that purpose, the alkaline solution was optimized for low cost and quick DNA extraction (blood lysate), and PCR reagents were modified for optimum reaction. In comparison to purified whole blood genomic DNA, the currently developed and optimized blood lysate method was found to be 95.5% less expensive, as well as being equally sensitive and specific for the molecular detection (PCR) of haemoparasites like Babesia, Theileria, Trypanosoma and rickettsiales in cattle, buffaloes, horses, and dogs. The assay was also demonstrated to be quick, less likely to cross-contaminate, and appropriate for use in laboratories with limited resources. Therefore, the currently developed and optimized blood lysate method could serve as a viable alternative to purified whole blood genomic DNA for molecular detection (PCR) of haemoparasites in animals particularly in resource-limited settings.

DR-70 (fibrinogen-fibrin degradation products) as a prognostic biomarker in dogs with neoplasms.

Fibrinogen-fibrin degradation products (DR-70) are derived from tumor cells or metastases. Our previous study reported the diagnostic values in dogs with tumors, but no research has yet to be conducted to establish DR-70 as a prognostic marker. Herein, we investigated changes in DR-70 concentrations and disease courses in dogs with tumors. Overall survival time (OST) analysis was performed in 195 dogs with tumors, stratified with a recommended cut-off (1.514 µg/mL). Continual DR-70 measurements were performed during the medical interventions of 27 dogs with neoplasms. Clinical conditions and medical records were retrospectively reviewed. According to a cut-off value, dogs with plasma DR-70 concentrations above 1.514 µg/mL had shorter survival rates than those with concentrations below this threshold. In cases with complete or partial remission in response to treatment, the DR-70 concentration was decreased compared with that at the first visit, whereas it was increased in patients with disease progression. Our study suggested that changes in DR-70 concentration can be used as a prognostic biomarker for canine neoplasms. Furthermore, increased plasma DR-70 levels might be associated with shorter survival, and DR-70 concentrations may reflect responses to medical intervention.

Strain-level variations of Dirofilaria immitis microfilariae in two biochemical assays.

BACKGROUND: The increase in reports of resistance to macrocyclic lactones in the canine heartworm, Dirofilaria immitis is alarming. While DNA based tests have been well-validated, they can be expensive. In a previous study, we showed that two biochemical tests adapted to a 96- well plate format and read in a spectrophotometer could detect differences among lab validated D. immitis isolates. The two tests- Resazurin reduction and Hoechst 33342 efflux-detect metabolism and P-glycoprotein activity respectively in microfilariae isolated from infected dog blood. METHODS: Our objective was to optimize the two assays further by testing various assay parameters in D. immitis isolates not tested previously. We tested microfilarial seeding density, incubation time and the effect of in vitro treatment with ivermectin and doxycycline in five other D. immitis isolates-JYD-34, Big Head, Berkeley, Georgia III and LOL. All assays were performed in 3 technical replicates and 2-4 biological replicates. To understand the molecular basis of the assays, we also performed qPCR for selected drug metabolism and elimination associated genes of the ABC transporter and cytochrome P450 gene families. RESULTS: Metabolism and ABC transporter activity as detected by these assays varied between strains. Anthelmintic status (resistant or susceptible) did not correlate with metabolism or P-gp efflux. Basal transcriptional variations were found between strains in ABC transporter and cytochrome P450 genes. CONCLUSIONS: These assays provide a greater understanding of the biochemical variation among isolates of D. immitis, which can be exploited in the future to develop in vitro diagnostic tests capable of differentiating susceptible and resistant isolates.

How accurate are NT-proBNP, ANP, and cTnI levels in diagnosing dogs with myxomatous mitral valve disease?

Background: Myxomatous mitral valve disease (MMVD) is prevalent in dogs. Specialized diagnostics (radiography and echocardiography) may be unavailable in some veterinary settings. Cardiac biomarkers offer potential alternatives. Aim: This study evaluated the diagnostic value of N-terminal fragments of pro-brain natriuretic peptides (NT-proBNPs), atrial natriuretic peptides (ANPs), and cardiac troponin I (cTnI) levels in dogs with MMVD. Methods: 69 dogs with MMVD (asymptomatic and symptomatic) and 19 healthy controls were assessed. Biomarker levels were measured using commercial kit rapid tests. Results: Our results showed that the median NT-proBNP level in the symptomatic group was higher than those in the asymptomatic (p < 0.001) and control (p < 0.001) groups. Moreover, the median NT-proBNP level in the asymptomatic group was higher than that in the control group (p < 0.001). The cTnI level in the control group was lower than those in the asymptomatic (p = 0.039) and symptomatic (p = 0.001) groups. No statistically significant difference in the cTnI level was noted between the asymptomatic and symptomatic groups. The best cutoff value of the NT-proBNP level to differentiate the normal controls from dogs with MMVD with or without congestive heart failure was > 505.65 pmol/l [sensitivity, 76.8%; specificity, 89.5%; and area under the curve (AUC), 0.862]. The suggested cutoff value of the NT-proBNP level to differentiate symptomatic MMVD from asymptomatic MMVD was >787.65 pmol/l (sensitivity, 78.38%; specificity, 72.55%; and AUC, 0.792). Conclusion: NT-proBNP and cTnI may serve as point-of-care tests for dyspneic dogs, aiding MMVD assessment where specialized diagnostics are limited.

Vitamin D status in female dogs with mammary gland tumors.

BACKGROUND: Little information exists about vitamin D status in bitches with mammary tumors. OBJECTIVES: To determine whether low plasma vitamin D concentrations are found in bitches with mammary tumors. ANIMALS: Eighty-five client-owned bitches with mammary tumors (n = 21 benign, n = 64 malignant) and 39 age-matched healthy bitches. METHODS: Case-control study. Plasma ionized and total calcium, phosphorus, magnesium, urea, creatinine, albumin, total proteins, alanine aminotransferase, alkaline phosphatase, parathyroid hormone (PTH), calcitriol (1,25-dihydroxyvitamin D), and 25-hydroxyvitamin D concentrations were measured in all bitches at the time of clinical diagnosis and before any treatments. Statistical analysis was performed to compare variables among groups (control, benign, and malignant). RESULTS: No significant differences were found when plasma 25-hydroxyvitamin D concentrations in bitches with malignant (148.9 [59.9] ng/mL) and benign mammary tumors (150.1 [122.3] ng/mL) were compared with control group (129.9 [54.5] ng/mL). Parathyroid hormone was significantly higher in bitches with malignant (19.9 [20.5] pg/mL), and benign mammary tumors (14.6 [14.9] pg/mL) compared with control group (7.5 [7.5] pg/mL; P < .01). Only the presence of mammary tumors (P < .01) and age (P = .04; adjusted R2 = .22) was significant in predicting PTH. CONCLUSIONS: Bitches with mammary tumors do not have low 25-hydroxyvitamin D concentrations thus vitamin D supplementation is unlikely to be useful for prevention of mammary tumors in bitches.

Can neutrophil-to-lymphocyte and platelet-to-lymphocyte ratios be used as markers for hypercortisolism in dogs?

Changes in neutrophil-to-lymphocite ratio (NLR) and platelet-to-lymphocyte ratio (PLR) have been identified in dogs with hypercortisolism (HC), but, no studies have investigated the changes in these inflammatory biomarkers as cost-effective and available parameters for the diagnosis and management of HC. This study was performed to evaluate whether NLR and PLR could be used as biomarkers for the diagnosis and treatment response in dogs with HC. This retrospective study included 67 dogs with HC, 58 dogs with non-adrenal illness (NAI), and 39 healthy dogs. NLR and PLR were compared among the three groups. Cut-off values of NLR and PLR for HC screening and percent change in biomarkers for assessing treatment response were evaluated. In addition, the NLR and PLR were compared before and after trilostane treatment. NLR and PLR were significantly higher in the HC group than in the NAI and healthy groups. The NLR cut-off value of 4.227 had a sensitivity of 67.16% and specificity of 65.52%, and the PLR cut-off value of 285.0 had a sensitivity of 56.72% and specificity of 70.69% for differentiating between dogs with HC and those with NAI, respectively. Furthermore, a significant decline in NLR was observed after treatment in the well-controlled HC group. The cutoff value of percent change in NLR to identify well-controlled HC was -7.570%; sensitivity and specificity were 100% and 63.64%, respectively. Therefore, NLR and PLR might be used cautiously as supportive biomarkers for HC diagnosis, and NLR could be a potential monitoring tool in assessing the treatment response of HC in dogs.

Detection and dynamics of tumor necrosis factor alpha in the diagnosis and treatment of canine heartworm disease.

The aim of this study was to determine the concentration of TNF-alpha (TNF-α) in dogs naturally infected with Dirofilaria immitis (D. immitis) and to assess whether there are any changes in TNF-α concentration and their dependence during therapy for heartworm disease (HWD). For this study, 14 client-owned dogs with HWD were selected. Clinical and parasitological examinations (modified Knott test for circulating microfilariae and SNAP Test IDEXX for circulating D. immitis antigen) had been used for diagnosing D. immitis and HWD. All dogs were treated with an alternative therapy for HWD (oral doxycycline 10 mg/kg b.w., once daily for 6 weeks, then alternately 4 weeks without and 2 weeks with the medication, and oral ivermectin 6-14 µg/kg b.w., every 2 weeks). The dogs blood sera at the moment of HWD diagnosis, during and at the end of therapy were frozen for further quantifying of TNF-α (Canine TNF-alpha ELISA kit, Thermo scientific). At the moment of HWD diagnosis TNF-α was detected in 9 dogs (7.21±12.44 pg/ml). Concentration of TNF-α was not significantly change during the therapy, neither related to the level of D. immitis antigen nor to antigen level changes. The alternative therapy for HWD has no influence on TNF-α concentration dynamics.

A comparative analysis of canine pancreatic lipase tests for diagnosing pancreatitis in dogs.

IMPORTANCE: Early diagnosis of canine pancreatitis is challenging due to non-specific clinical signs. Currently, abdominal ultrasonography and measurement of canine pancreatic lipase (cPL) have been employed for the diagnosis of pancreatitis. OBJECTIVE: Many qualitative and quantitative commercial cPL tests have been developed and used in veterinary clinics. This study aimed to compare three different methodologies SNAP cPL, Spec cPL, and Vcheck cPL tests to assess the concordance of these assays. METHODS: Fifty serum samples were collected from 36 dogs with or without pancreatitis and subjected to SNAP cPL, Spec cPL, and Vcheck cPL tests. Agreement and correlation coefficients were calculated between the test results, and correlations were determined during the management of the patients. RESULTS: The results of the three cPL assays were strongly correlated in 47/50 serum samples (94%). Cohen's kappa analysis between the Spec cPL and Vcheck cPL showed near perfect agreement (κ = 0.960, p < 0.001), SNAP cPL and Vcheck cPL (κ = 0.920, p < 0.001), and Spec cPL and SNAP cPL (κ = 0.880, p < 0.001). The correlation coefficients (r) between data from Spec cPL and Vcheck cPL tests was calculated by Spearman's correlation test (r = 0.958, p < 0.001). Furthermore, the patterns of change in serum cPL concentrations determined using Spec cPL and Vcheck cPL were significantly consistent during the monitoring period in 11 patients. CONCLUSIONS AND RELEVANCE: Our data illustrated that Spec cPL and Vcheck cPL tests are compatible for clinical use in the diagnosis and monitoring of canine pancreatitis.

Studies on the association of malondialdehyde as a biomarker for oxidative stress and degree of malignancy in dogs with mammary adenocarcinomas.

BACKGROUND: Mammary adenocarcinomas are one of the most common tumour diseases in bitches. The relationship between oxidative stress and the degree of malignancy of the tumour has not been sufficiently researched in veterinary medicine. OBJECTIVES: The main objective was to investigate the potential role of MDA as a practice-relevant biomarker for the assessment of systemic oxidative stress and to determine whether this parameter can indicate the malignancy grade of a mammary adenocarcinoma. METHODS: In the present pilot study, MDA plasma concentrations were analysed in 55 bitches with (n = 28) and without (n027) malignant adenocarcinomas of the mammary gland using two different measurement methods and the relationship to tumour size was investigated. RESULTS: The mean MDA concentration measured by enzyme-linked immunosorbent assay (ELISA) was 289 ng/mL (range 365-634 ng/mL) in dogs with grade 1 adenocarcinoma (n = 13), 288.5 ng/mL (range 85-752 ng/mL) in dogs with grade 2 adenocarcinoma (n = 10), 332 ng/mL (range 239-947 ng/mL) in dogs with grade 3 (n = 5) adenocarcinoma and 293 ng/mL (range 175-549 ng/mL) in dogs without a mammary tumour (n = 27). When MDA was measured by HPLC, the average MDA concentration in the study group (n = 11) was 0.24 µmol/L (range 0.16-0.37) and that of the control group (n = 15) was 0.27 µmol/L (range 0.16-1.62). Thus, there were no significant differences between the study group with malignant adenocarcinomas and the control group in both examination methods (p > 0.05). Furthermore, there was no correlation between the MDA concentrations and the approximate volume of the mammary tumour. CONCLUSION: The results highlight the challenges of providing a prognosis for the malignancy of a mammary adenocarcinoma based on MDA concentrations in plasma using ELISA or HPLC. As a result, histopathological examination remains the gold standard for diagnosing and differentiating adenocarcinomas of the mammary gland.

Detection of rat lungworm (Angiostrongylus cantonensis) infection by real-time PCR from the peripheral blood of animals: a preliminary study.

Rat lungworm disease or neuroangiostrongyliasis is a cerebral parasitic infection that affects humans and animals alike. Its clinical signs and symptoms can range from mild self-resolving to serious life-threatening conditions. Studies suggest therapeutic interventions during the early stages of infection to be more effective than in later stages. However, early diagnosis of infection is usually problematic without the knowledge of exposure and/or detection of the parasite's DNA or antibody against the parasite in the cerebrospinal fluid. This requires a lumbar puncture, which is an invasive procedure that generally requires hospitalization. This study evaluates an affordable and less invasive alternative to detect parasitic DNA by PCR from the peripheral blood of potentially infected animals. Blood samples from 58 animals (55 dogs and 3 cats) with clinical suspicion of infection were submitted to our lab between February 2019 and August 2022 by local, licensed veterinarians. DNA was extracted from whole blood, plasma, serum, and/or packed cells using the Qiagen DNeasy Blood & Tissue Kit as per the manufacturer's protocol. All 58 animals were tested by real-time PCR using the AcanITS1 assay and 32 of these animals (31dogs; 1 cat) were also tested using the AcanR3990 assay. The PCR results for both assays were classified into strongly positive > positive > weakly positive > negative, and equivocal for ambiguous results, based on the strength of the signal. The percent infection detected using the AcanITS1 and AcanR3990 assays was 12.72% (7/55) and 20.68% (6/29), respectively. The overall percent infection detected was 34.37% (11/32), with only two animals testing positive by both assays. The three cats involved in this study tested negative by both assays. These results are promising and warrant further investigations to increase sensitivity including variables that might affect detection in the blood, such as parasite load, and laboratory methodologies.

Longitudinal analysis of echocardiographic and cardiac biomarker variables in dogs with atrial fibrillation: The optimal rate control in dogs with atrial fibrillation II study.

BACKGROUND: Rate control (RC; meanHRHolter ≤ 125 bpm) increases survival in dogs with atrial fibrillation (AF). The mechanisms remain unclear. HYPOTHESIS/OBJECTIVES: Investigate echocardiographic and biomarker differences between RC and non-RC (NRC) dogs. Determine if changes post-anti-arrhythmic drugs (AAD) predict successful RC in subsequent Holter monitoring. Evaluate if early vs late RC affects survival. ANIMALS: Fifty-two dogs with AF. METHODS: Holter-derived mean heart rate, echocardiographic and biomarker variables from dogs receiving AAD were analyzed prospectively at each re-evaluation and grouped into RC or NRC. The primary endpoint was successful RC. Between group comparisons of absolute values, magnitude of change from admission to re-evaluations and end of study were performed using Mann-Whitney tests or unpaired t-tests. Logistic regression explored variables associated with inability to achieve RC at subsequent visits. Kaplan-Meier survival analysis was used to compare survival time of early vs late RC. RESULTS: At visit 2, 11/52 dogs were RC; at visit 3, 14/52 were RC; and at visit 4, 4/52 were RC. At the end of study, 25/52 remained NRC. At visit 2, both groups had increased cardiac dimensions, but NRC dogs had larger dimensions; biomarkers did not differ. At the end of study, RC showed decreased cardiac dimensions and end-terminal pro-brain natriuretic peptide (NT-proBNP) compared with NRC. No variables were useful at predicting RC success in subsequent visits. Survival analysis found no differences between early vs late RC. CONCLUSIONS AND CLINICAL IMPORTANCE: The RC dogs had decreased cardiac dimensions and NT-proBNP, suggesting HR-mediated reverse-remodeling might benefit survival, even with delayed RC achievement. Pursuit of RC is crucial despite initial failures.

Factors influencing serum concentrations of levetiracetam in dogs with epilepsy.

BACKGROUND: Factors affecting serum concentrations of levetiracetam in dogs are unknown and could affect the efficacy of levetiracetam in controlling seizures in dogs with epilepsy. HYPOTHESIS/OBJECTIVES: Higher PO doses of levetiracetam will be needed in dogs to achieve serum concentrations shown to be effective in humans. Determine factors that could influence serum levetiracetam concentrations and justify dose adjustment in some epileptic dogs. ANIMALS: Sixty-nine client-owned dogs with epilepsy treated with levetiracetam alone or in combination, based on 127 trough serum concentration measurements of levetiracetam. METHODS: Retrospective cohort study. Linear mixed models were used to assess the effect of patient signalment and concurrent drug administration on serum concentrations of levetiracetam and the effect of serum concentration of levetiracetam on seizure frequency reduction. RESULTS: The PO dose of levetiracetam significantly explained changes in serum levetiracetam concentration, and this causal link was stronger with monotherapy (R2 = 0.59, P < .001). Phenobarbital significantly decreased serum levetiracetam concentration in a dose dependent manner (R2 = 0.30, P = .003). Based on our model, a levetiracetam dosage of 99-216 mg/kg/day is necessary to obtain a serum levetiracetam concentration of 20 µg/mL when used alone or concurrently with 7 mg/kg/day of phenobarbital. No other factors were found to influence serum levetiracetam concentrations. No therapeutic range could be identified. CONCLUSION AND CLINICAL IMPORTANCE: Our data suggest that a dosage of 99-216 mg/kg/day of levetiracetam is needed to achieve a serum concentration known to be therapeutically effective in humans, especially when administered concomitantly with phenobarbital.

Evaluation of the relationship between plasma glucagon-like peptide-2 and gastrointestinal dysbiosis in canine chronic enteropathies.

Chronic enteropathies are a common cause of morbidity in dogs and are associated with disruption of the normal gastrointestinal mucosal barrier. The objective of this prospective study was to determine the association between measures of gastrointestinal dysbiosis and plasma concentrations of glucagon-like peptide-2, a hormone responsible for normal mucosal structure, in dogs with chronic enteropathies. Fecal 16S V4 rRNA gene sequencing and quantitative PCR via the dysbiosis index was performed on 16 healthy controls and 18 dogs with chronic enteropathy prior to and 1 month after initiation of individualized therapy. Fasting and post-prandial plasma GLP-2 concentrations were measured via ELISA in healthy dogs and chronic enteropathy dogs at both time points. Alpha and beta diversity indices, as well as bacterial population abundances were compared between groups and time-points. Principal component analysis combined with least squares regression was used to identify taxa contributing to glucagon-like peptide-2 variance among groups. While the dysbiosis index did not differ between healthy dogs and dogs with chronic enteropathy, 16S V4 genomic sequencing identified 47 operational taxonomic units that differed between the groups, all but 2 of which resolved following chronic enteropathy treatment. Principal component analysis identified 6 families and 19 genera that contributed to differences in glucagon-like peptide-2 concentrations between groups. Dysbiosis associated with chronic enteropathies in dogs may contribute to the observed lower plasma glucagon-like peptide-2 concentrations. Further research into mechanisms of microbiota impact on the enteroendocrine system is needed. Association between glucagon-like peptide-2 secretion and microbiome indices may help to guide research into future treatment strategies for dogs with chronic enteropathy.

Evaluation of clopidogrel, hypercoagulability, and platelet count in dogs undergoing splenectomy for splenic masses.

Dogs that had splenectomy are predisposed to fatal thrombotic conditions, and thrombocytosis is a risk factor for post-splenectomy hypercoagulability. However, in veterinary medicine, there are no specific therapeutic approaches for managing this hypercoagulability. This study aimed to determine the preventive effect of clopidogrel on post-operative hypercoagulability during the first 2 weeks post-splenectomy in dogs with splenic masses. This study included 12 dogs that had splenectomy. Seven dogs received no treatment (group A), and five were treated with clopidogrel (group B). Clopidogrel was loaded at 10 mg/kg on day 2 and continued at 2 mg/kg until day 14. Blood samples were collected on the day of surgery and 2, 7, and 14 days after splenectomy in both groups. In group B, thromboelastography (TEG) was performed on the same days. In group A, there was significant elevation of platelet counts on days 7 (p = 0.007) and 14 (p = 0.001) compared to day 0. In group B, the platelet counts were significantly elevated on day 7 (p = 0.032) but no significant difference was found on day 14 compared to day 0. Platelet counts on day 14 were significantly higher in group A than in group B (p = 0.03). The lower platelet counts were correlated with alterations in TEG parameters, and no significant differences were found in the K and α-angle values at all postoperative assessment points compared to day 0. Our study suggests that clopidogrel may reduce post-operative thrombocytosis and hypercoagulability in dogs that undergo splenectomy for splenic masses.

Pilot study to evaluate isolation by size of circulating tumour cells in canine oral melanoma.

Liquid biopsy for circulating tumour cell (CTC) detection is generally unexplored in veterinary medicine. Dogs with highly aggressive and heterogeneous tumours, such as oral malignant melanoma (OMM), could benefit from studies involving size-based isolation methods for CTCs, as they do not depend on specific antibodies. This pilot study aimed to detect CTCs from canine OMM using Isolation by Size of Epithelial Tumor Cells (ISET), a microfiltration methodology, followed by immunocytochemistry (ICC) with Melan-A, PNL2, and S100 antibodies. Ten canine patients diagnosed by histopathology and confirmed as OMM by immunohistochemistry were enrolled, their prognostic data was assessed, and blood samples were collected for CTC analysis. Results have shown the detection of intact cells in 9/10 patients. ICC has shown 3/9 Melan-A-positive, 3/9 PNL2-positive, and 8/9 S100-positive patients, confirming the importance of opting for a multimarker assay. A significant number of negative-stained CTCs were found, suggesting their high heterogeneity in circulation. Microemboli stained with either PNL2 or S100 were found in a patient with a high isolated cell count and advanced clinical stage. Preliminary statistical analysis shows a significant difference in CTC count between patients with and without lymph node metastasis (p < .05), which may correlate with tumour metastatic potential. However, we recommend further studies with more extensive sampling to confirm this result. This pilot study is the first report of intact CTC detection in canine OMM and the first application of ISET in veterinary medicine, opening new possibilities for liquid biopsy studies in canine OMM and other tumours.

Neutrophil-to-lymphocyte ratio and red blood cell distribution width to platelet ratio and their relationships with inflammatory and antioxidant status in dogs with different stages of heart failure due to myxomatous mitral valve disease.

We aimed to evaluate the red blood cell distribution width-to-platelet ratio (RDW/PLT) with other complete blood cell count (CBC) indices and their correlations with serum proinflammatory cytokines, acute phase proteins (APPs), and antioxidant biomarkers in dogs at different stages of heart failure (HF). A total of 29 dogs were divided into four groups according to the ACVIM Consensus Statement: stage-A (healthy/controls, n = 8), stage-B2 (n = 6), stage-C (n = 10), and stage-D (n = 5). Seventeen CBC indices were calculated and correlated with the measurements of inflammatory, APPs, and antioxidant biomarkers, as well as selected echocardiographic variables in all dogs. At stage-C, CBC indices were evaluated 14 days after the treatment. Statistically significant changes were observed only for RDW/PLT and neutrophil-to-lymphocyte ratio (NLR) between groups. NLR increased, but RDW/PLT deceased in dogs with HF, compared to controls (P < 0.05). There were no statistically differences between pre- and post-treatment CBC indices. There were significantly positive and negative correlations between the CBC indices, serum parameters and selected echocardiographic variables in dogs with HF(P < 0.05). ROC analysis showed the best sensitivity (57% and 68%) and specificity (100% and 57%) for NLR > 5.8 and RDW/PLT ≤ 0.057 for predicting the severity of HF, respectively. Results showed that NLR and RDW/PLT may have potential for monitoring severity of the disease and the effect of treatment in dogs with HF. Imbalances between indices of circulating blood cells can contribute to immunoinflammatory and antioxidant responses in pathogenesis of canine HF, which may provide us alternative targets to develop new diagnostic and therapeutic strategies in veterinary medicine.

Factors correlating with circulating T-zone-like cells of undetermined significance (TZUS) in dogs resident in Italy.

T-zone-like cells of undetermined significance (TZUS) share the same phenotypic pattern (CD45-CD5+) with T-zone lymphoma cells and were first described a few years ago in the peripheral blood (PB) of healthy aged American Golden retrievers (GR). History of bladder and eye disease increased the odd of circulating TZUS in the American GR population. Since differences among dogs may exist according to the geographical region of origin, herein we screened 489 PB samples to assess potential factors predisposing to the presence of circulating TZUS in dogs living in Italy. Overall, TZUS were found in 174 (35.6%) samples. Among 83 clinical variables, significant associations emerged with sex, age, diagnosis of neoplasia, history of neoplasia, history of infectious or parasitic disease, history of osteoarticular disease, presence of traumatic lesions or foreign bodies, and lymphocytes count. Only age and history of neoplasia retained significance at multivariate analysis (p=0.019 and p=0.036, respectively). Thus, older age and history of neoplasia are the main factors associated with circulating TZUS in Italian dogs. Future studies should focus on elucidating the biological role of TZUS and determining reproducible criteria for their identification, distinguishing them from infiltrating TZL.

Phosphocalcic metabolism and its potential association with biomarkers of kidney disease in dogs with spontaneous hyperadrenocorticism.

The pathogenesis of increased serum phosphate concentration and proteinuria in dogs with spontaneous hyperadrenocorticism (HAC) is unclear. A potential link between proteinuria and calcium/phosphate metabolism has never been studied in dogs with HAC. The aims of the study were: (1) To evaluate calcium/phosphate metabolism in dogs with spontaneous HAC and compare to healthy dogs as well as to dogs with non-HAC illness; (2) to look for associations between markers of calcium/phosphate metabolism and biomarkers of kidney disease in dogs with HAC. Fifty-four dogs were included in the study, classified as HAC (n=27), non-HAC disease (n=17), and healthy (n=10). Serum calcium, phosphate, 25(OH)Vitamin D, 1,25(OH)2Vitamin D, plasma intact parathyroid hormone concentration (iPTH), FGF23, and urinary fractional excretion of calcium and phosphate were evaluated in all dogs at diagnosis and compared between each group. The correlation between these variables and urine protein-to-creatinine ratio (UPC) and urinary N-acetylglucosaminidase-to-creatinine ratio (uNAG/C) was evaluated in the HAC group. Medians [range] of serum phosphate concentration, urinary fractional excretion of calcium (FE(Ca)), and iPTH were significantly higher in dogs with HAC than in dogs with non-HAC illness (P<0.01) and healthy dogs (P<0.01). Increased 1,25(OH)2Vitamin D/25(OH)Vitamin D was also observed (P<0.001). In HAC group, UPC was significantly negatively correlated with 25(OH)Vitamin D (r(s): -0.54; P<0.01). Urinary NAG/C was significantly positively correlated with serum phosphate (r(s): 0.46; P=0.019). Increased serum phosphate, urinary excretion of calcium, and hyperparathyroidism were observed in dogs with HAC. Vitamin D metabolism may be shifted towards increased 1-alpha hydroxylation.

Evaluation of coagulation and platelet activation state and function in heartworm-infected dogs.

BACKGROUND: Enhanced platelet responses have been demonstrated in heartworm-infected (HWI) dogs; however, the cause and clinical implications of altered platelet function have not been fully elucidated. OBJECTIVE: This study evaluated platelet function in HWI dogs. METHODS: Anticoagulated whole blood collected from eight HWI and eight uninfected dogs was evaluated using turbidometric platelet aggregometry, a platelet function analyzer (PFA-100), a total thrombus analysis system (T-TAS), tissue factor-activated and tissue plasminogen activator modified thromboelastography (TF- and tPA-TEG), CBC, von Willebrand Factor activity, and fibrinogen concentrations. Platelet activation state and the presence of reticulated platelets were assessed via flow cytometric expression of P-selection (CD-62P) and thiazole orange staining. RESULTS: Platelet aggregation responses to adenosine diphosphate (ADP, 10 µM) or collagen (20 µg/mL), PFA-100 closure times, and T-TAS occlusion times did not differ between groups. TEG values TF-R, tPA-R, TF-K, and TF-LY60 were decreased (P = .025, P = .047, P = .038, P = .025) and TF-MA, tPA-MA, TF-G, tPA-G and TF-alpha angle were increased (P < .04) in HWI dogs. HWI dogs had higher fibrinogen concentrations (465.6 ± 161 mg/dL vs 284.5 ± 38 mg/dL, P = .008) and eosinophil counts (0.686 ± 0.27 × 103/µL vs 0.267 ± 0.20 × 103/µL, P = .003). There was no difference in hematocrit, activation state, or percent of reticulated platelets. Non-activated reticulated platelets exhibited higher CD62P expression compared with mature platelets. CONCLUSIONS: Chronic canine heartworm disease was accompanied by hypercoagulability, hyperfibrinogenemia, and decreased fibrinolysis. Enhanced platelet activation was not identified in this group of HWI dogs.

Spurious hyperbicarbonatemia and a negative anion gap in a cat and a dog with severe rhabdomyolysis.

A 3-year-old male neutered domestic shorthair cat and a 2-year-old male neutered Labrador-mix dog were separately presented to the Veterinary Medical Center for evaluation after sustaining significant muscle trauma due to a dog attack and seizure activity, respectively. In both cases, biochemical analysis was consistent with rhabdomyolysis. Additionally, a markedly increased measured serum bicarbonate concentration and negative calculated anion gap were observed. As these biochemical abnormalities were not expected and deemed incompatible with life, an interference with the analyzer measurement of bicarbonate involving marked increases in pyruvate and lactate dehydrogenase (LDH) following myocyte injury was suspected. Venous blood gas analysis calculated bicarbonate concentration and anion gap were within reference interval, while measured LDH activity was markedly increased. These findings supported an analyzer-generated interference. This is the first published report of a previously described chemistry analyzer interference of markedly increased LDH activity with serum bicarbonate concentration measurement in dogs and cats. Awareness of this interference is important, particularly in the emergency setting, as it may influence case management.