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Endurance-trained sled dogs provide a unique translational model to characterize changes in hematologic and serum biochemical analytes due to the aging process. The primary objective of this study was to determine the effect of age and sex on specific hematologic and serum biochemical parameters in the endurance trained sled dog. Longitudinal and cross-sectional data were analyzed from 9,746 blood and serum samples from 4,804 dogs collected over 7 years as part of the Iditarod Trail Sled Dog Race pre-race examination program. Mixed models analysis was used for statistical analysis and P < 0.01 was considered significant. Dogs ranged from 1-12 years of age and 39% were female. Serum total calcium and phosphorus concentrations and white blood cell count decreased nonlinearly to asymptotic values by 6.6, 3.1, and 6.9 years of age, respectively, equivalent to estimated physiologic ages in human years of 44, 27, and 46 years. Serum glucose concentrations reached their lowest value at 7.8 years of age, equivalent to an estimated human physiologic age of 50 years, after which time the concentration increased. Serum globulin concentrations increased with age, but nonlinearly for females and linearly for males. Most sex-related differences were <5%; however, females had lower serum urea nitrogen (14.7%) and creatinine (7.3%) concentrations, lower serum alanine aminotransferase activity (16.6%), and higher serum total bilirubin concentration (12.8%) and platelet count (6.0%). The endurance-trained sled dog provides an excellent model to separate the physiologic effects of age from those of a sedentary lifestyle on hematologic and serum biochemical analytes., Competing Interests: The commercial company “Veterinary Specialty Center” did not contribute to the funding of this project. The author currently employed by this institution was involved in the project before employment with this institution. The funder (Iditarod Trail Sled Dog Race Committee) provided support in the form of salaries for authors [SN, TJ], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. One of the authors is employed by a commercial company “Veterinary Specialty Center” which did not play a financial, consultancy or academic role in any part of this project or the decision to publish this manuscript. As stated above, this author was involved in the project primarily before employment at this commercial company. This author did contribute some additional work on the project while being employed by the company but the work was conducted on weekends when not assigned to clinical duty therefore, her current employment does not provide a competing interest. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Chronic kidney disease (CKD) is a common renal disease in dogs and cats. Renal fibrosis is a main pathologic process leading of CKD progression. Renal biopsy is the gold standard for renal fibrosis assessment. However, it is not routinely performed in clinic due to its invasiveness. Therefore, the aim of this study was to evaluate the use of ultrasonographic strain elastography (SE), which is a non-invasive method for renal tissue stiffness determination and its association with renal function. Renal strain ratios and renal function were evaluated in 13 CKD dogs (CKDD), 38 healthy dogs (HD), 17 CKD cats (CKDC) and 26 healthy cats (HC). There were significantly lower renal cortical strain ratios than medullary strain ratios in all groups (HD; P<0.01, HC; P<0.01, CKDD and CKDC; P<0.05) and significantly lower cortical and medullary strain ratios in both CKDD and CKDC than in healthy control animals of both species (P<0.0001). In dogs, the renal cortical and medullary strain ratios significantly negatively correlated with plasma creatinine (P<0.05), blood urea nitrogen (BUN; P<0.05; P<0.01, respectively), and symmetric dimethylarginine (SDMA; P<0.01). In cats, similar correlations were found for plasma creatinine (P<0.001), BUN (P<0.05; P<0.001, respectively) and SDMA (P<0.05). SE might be a promising imaging diagnostic tool for renal-elasticity evaluation, also correlating with renal functional impairment in canine and feline CKD.

Background: Melatonin is a hormone produced and secreted primarily by the pineal gland and mainly metabolised in the liver. Increased melatonin concentrations have been reported in human cirrhosis and hepatic encephalopathy (HE), a syndrome of neurological dysfunction. The pathogenesis of canine HE is incompletely understood. Melatonin has been hypothesised as a contributor to the development of HE. The aim of this study was to investigate whether serum melatonin concentrations are increased in canine congenital portosystemic shunting (cPSS), with and without HE., Methods: Medical records were retrospectively reviewed, for which archived (-80°C) serum samples were available. A canine competitive ELISA was used to measure melatonin in two cohorts: dogs with a final diagnosis of cPSS (n=23) with and without clinical signs of HE, and healthy dogs (n=15)., Results: Melatonin concentrations were not significantly different (P=0.81) between healthy controls (median 27.2 pg/mL, range 19.8-161.5 pg/mL) and dogs with cPSS (median 25.7 pg/mL, range 18.5-244.9 pg/mL). Serum melatonin did not differ between cPSS patients with and without clinical signs of HE (P>0.99). No correlation was found between serum melatonin and blood ammonia (Spearman rank correlation coefficient, r s =-0.41, P=0.08)., Conclusion: Serum melatonin is not increased in canine cPSS with and without HE. We found no evidence that altered melatonin metabolism plays a role in the pathogenesis of cPSS-associated HE., Competing Interests: Competing interests: None declared., (© British Veterinary Association 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: The protein chromogranin A (CgA) is stored and co-released with catecholamines from the stimulated adrenal glands. Increased plasma concentrations of CgA have been shown in people with heart disease. The aim of the study was to investigate whether plasma concentrations of the CgA-derived biologically active peptides catestatin and vasostatin were associated with the severity of myxomatous mitral valve disease (MMVD) in dogs and to assess potential associations between these blood variables and dog characteristics, echocardiographic variables, heart rate (HR), blood pressure (BP) and plasma N-terminal-proBNP (NT-proBNP) concentration. Sixty-seven privately owned dogs with or without MMVD were included. The dogs underwent physical examination, blood pressure measurement, blood sample collection, and echocardiographic examination. Plasma concentrations of catestatin and vasostatin were analyzed using radioimmunoassay., Results: Catestatin concentration decreased with increasing left atrial and ventricular size (R 2  ≤ 0.09, P ≤ 0.019), and increased with increasing systolic and diastolic blood pressures (R 2  ≤ 0.08, P ≤ 0.038). Regression analyses showed no significant associations for vasostatin. No differences in plasma concentrations of catestatin or vasostatin were found between the disease severity groups used in the study., Conclusions: In the present dog population, the catestatin concentration showed weak negative associations with left atrial and ventricular sizes, both of which are known to increase with increasing severity of MMVD. Furthermore, the catestatin concentration showed weak positive associations with blood pressure.

Piroplasm species were analyzed by molecular tools in total 31 blood samples from positive dogs, previously checked by stained slides, stored until DNA extraction between 2016 to 2018 in the laboratory Clinical Analyzes in Niterói, Rio de Janeiro. The piroplasms were identified by PCR, targeting the 18S rRNA gene and sequencing. From the total number of samples only 24 (77.4%) were positive and show adequate nucleotide sequences for interpretation with identity between 93%-100% with Babesia vogeli in compared to the sequences isolated of infected dogs from other states in Brazil deposited on GenBank. Most of dogs infected with B. vogeli had anemia (62.5%) and thrombocytopenia (95.8%). The findings of this study are compatible with previous reports in the literature and highlight B. vogeli as the most incriminated species in canine piroplasmosis in Brazil, and thrombocytopenia the hematological alteration most frequently identified in this infection. It is important to note that this is the first study involving the molecular characterization of piroplasms in the metropolitan region of Rio de Janeiro, based on PCR followed by sequencing.

The ability to make an accurate prognosis, which is a prerequisite for treatment decisions, is very limited in dogs with traumatic brain injury (TBI). To determine whether serum concentrations of neuron-specific enolase (NSE) have prognostic value in dogs following TBI, we conducted a prospective, observational, controlled clinical study in an intensive care unit of a university teaching hospital. The study population comprised 24 dogs admitted to the hospital within 72 h of a known event of TBI between January 2010 and January 2015, as well as 25 control healthy shelter dogs admitted for elective neutering. Seventeen injured dogs (70%) survived to discharge, four were euthanized and three died within 48 h. Serum samples were obtained from all dogs (in injured dogs, within 72 h of TBI) and NSE concentrations were measured using enzyme-linked immonosorbent assay. Associations between NSE levels and outcome, Modified Glasgow Coma Scale, time to sampling, age or haemolysis scale were determined. Mean serum NSE concentrations were decreased in dogs with TBI compared with healthy controls (19.4 ± 4.14 ng/ml vs. 24.9 ± 4.6 ng/ml, P <0.001). No association was found between serum NSE concentrations and either survival or severity of neurological impairment. A negative correlation was found between serum NSE concentrations and time from trauma to blood collection (r = -0.50, P = 0.022). These results indicate that serum NSE concentration in dogs following TBI is not an effective marker for severity or outcome. Further studies are warranted to standardize serum NSE measurements in dogs and to determine the peak and half-life levels of this potential biomarker., Competing Interests: Conflict of Interest Statement The authors declare that there were no conflicts of interests relating to this study., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Pet-related tick-borne infections constitute an emerging problem in human and veterinary medicine worldwide. In Chile, two pathogens of the Anaplasmataceae family, Anaplasma platys and Ehrlichia canis , have been identified in recent years in dogs and vectors. This epidemiological survey aimed to determine the human and canine seroprevalence of Anaplasma spp. in urban and rural areas of different regions in Chile and to analyze the factors associated with seropositivity. Methods: We performed a cross-sectional household-based study in four regions, where healthy adults and their household dogs were included. Households were chosen by double stratified random sampling in urban areas and by convenience sampling in rural areas. Anaplasma seroreactivity was determined by a commercial microimmunofluorescence assay using Anaplasma phagocytophilum antigens. A questionnaire survey was applied to assess risk factors for Anaplasma seropositivity. Results: A total of 1105 persons and 905 dogs were included. The overall seroprevalence in humans was 9.4%, ranging from 5.6% in central Chile to 13.7% in the extreme north; in dogs the overall prevalence was 44.0% and ranged from 37.2% in the northern region to 61.1% in central Chile. Risk factors for human seropositivity were lower education and external deworming of dogs, whereas canine seropositivity was associated to urban site, mixed-breed, older animals, and tick infestation. Conclusions: This cross-sectional study suggests a broad exposure of both dogs and dog owners to Anaplasma or related agents in Chile. Further epidemiological and clinical studies are necessary to understand the complete spectrum and epidemiology of tick-borne zoonotic infections in the different ecoclimatic regions of Chile.

Introduction: This study describes presentation, cardiovascular abnormalities, etiology, and outcome of canine myocarditis in geographic areas not endemic for Trypanosoma or Leishmania., Animals: Sixty-four (presumed antemortem diagnosis) and 137 (postmortem diagnosis only) client-owned dogs at two tertiary care facilities were included., Materials and Methods: Medical records of dogs with clinical or histopathological diagnosis of myocarditis were reviewed retrospectively., Results: Common examination findings in dogs with a presumed antemortem diagnosis included fever (21%) and heart murmur (19%). Median cardiac troponin I was 12.2 ng/mL (range: 0.2-808.0 ng/mL), and troponin exceeded 1.0 ng/mL in 26 of 29 (90%) dogs. Ventricular ectopy was the most common arrhythmia (54%), whereas decreased left ventricular systolic function was the most common echocardiographic abnormality (56%). An infectious etiology was diagnosed in 35 of 64 (55%) dogs. Confirmed infectious etiologies included bacterial sepsis (n = 9) or extension of endocarditis (3), toxoplasmosis or neosporosis (3), parvovirus (2), and one case each of bartonellosis, trypanosomiasis, leptospirosis, and dirofilariasis. Median survival time was 4 days (range: 0-828 days) for all dogs vs. 82 days for dogs who survived at least 2 weeks after diagnosis. Presence of pericardial effusion or azotemia was a significant predictor of non-survival. The most common inflammatory infiltrate on histopathology was neutrophilic (47%), and 20 of 137 (14.5%) dogs had concurrent bacterial endocarditis on postmortem., Conclusions: Bacterial infection was the most common confirmed etiology of myocarditis in this study. Prognosis for canine myocarditis is guarded and similar to that reported for infective endocarditis. Criteria for the antemortem diagnosis of canine myocarditis are suggested., Competing Interests: Conflicts of Interest Statement The authors declare no conflict of interest related to this study., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Visceral leishmaniasis (VL) is a major neglected disease, potentially fatal, whose control is still impaired by inefficient and/or expensive treatment and diagnostic methods. The most promising approach for VL diagnosis uses serological assays with recombinant proteins, since they are more efficient and easier to perform. Tests developed for the human form of the disease, however, have not been shown to be efficient for its diagnosis in the canine host, the major reservoir for the American VL., Methodology/principal Findings: Here, we describe a systematic approach aimed at the production of a new chimeric protein potentially able to be used for both human and canine VL diagnosis and based both on in silico gene design and experimental data. Starting from the previous identification of Leishmania infantum recombinant antigens efficient for the diagnosis of either human or canine VL, three of the best performing antigens were selected (Lci2, Lci3 and Lci12). After a preliminary evaluation validating the chimeric approach, DNA fragments encoding predicted antigenic regions from each protein, enriched with repeats, were joined in various combinations to generate a total of seventeen chimeric genes optimized for prokaryotic expression. These were assessed for optimal expression and purification yield, with four chimeric proteins being efficiently produced. Their diagnostic potential was then evaluated through ELISA assays with sera from VL afflicted humans and dogs. After two rounds of gene design, the results showed high levels of sensitivity for the best chimeric protein, named Q5, in humans (82%) and dogs (100%) with 100% specificity in comparison with healthy controls. A single non-specific reaction was seen with serum from individuals with tegumentary leishmaniasis., Conclusion: The newly described chimeric protein is potentially useful for the detection of both humans and dogs afflicted with VL, with its use in rapid tests necessary for validation as a new diagnostic tool., Competing Interests: We have read the journal's policy and the authors of this manuscript have the following competing interests: pending patent application (BR 10 2019 003639 7) jointly submitted by the institution to which the following authors OPDMN, FBM, WJTS, ALCN, DHCT, AMR and MBN are affiliated and/or from which the authors may benefit. Title: CHIMERIC PROTEIN, ITS METHOD FOR PRODUCTION AND USE, AS WELL AS NUCLEIC ACID MOLECULE, CASSETTE OF EXPRESSION, VECTOR OF EXPRESSION, CELL HOST, COMPOSITION FOR DIAGNOSIS OF LEISHMANIASIS, DIAGNOSIS KIT AND IN VITRO DIAGNOSIS METHOD FOR LEISHMANIASIS.

Spinal cord injury (SCI) involves nerve damage and often leads to motor, sensory and autonomic dysfunctions. In the present study, we have designed a clinical protocol to assess the feasibility of systemic delivery of allogenic canine bone marrow tissue-derived mesenchymal stem cell conditioned medium (BMMSC CM) to dogs with SCI. Four client-owned dogs with chronic SCI lasting more than six months underwent neurological and clinical evaluation, MRI imaging and blood tests before being enrolled in this study. All dogs received four intravenous infusions with canine allogenic BMMSC CM within one month. Between the infusions the dogs received comprehensive physiotherapy, which continued for three additional months. No adverse effects or complications were observed during the one, three and six months follow-up periods. Neither blood chemistry panel nor hematology profile showed any significant changes. All dogs were clinically improved as assessed using Olby locomotor scales after one, three and six months of BMMSC CM treatment. Furthermore, goniometric measurements revealed partial improvement in the range of joint motion. Bladder function improved in two disabled dogs. We conclude that multiple delivery of allogenic cell-derived conditioned medium to dogs with chronic SCI is feasible, and it might be clinically beneficial in combination with physiotherapy.

Background: As a co-receptor for fibroblast growth factor 23, klotho plays a pivotal role in phosphate metabolism. The kidney is known to be the main source of soluble alpha-klotho and the principal regulator of its concentration. Previous studies in human participants showed that the concentration of soluble alpha-klotho in serum and urine decreased in chronic kidney disease (CKD) patients. However, no previous study has assessed soluble alpha-klotho levels in dogs. This study aimed to measure serum and urinary alpha-klotho levels in CKD dogs and identify their associations with International Renal Interest Society (IRIS) CKD stages and other parameters known to be associated with CKD., Results: Serum and urinary alpha klotho concentrations were measured by a commercially available canine-specific sandwich enzyme-linked immunosorbent assay kit and compared between groups by a nonparametric Kruskal-Wallis test. Spearman's correlation coefficient was used to evaluate the relationships between variables. A stepwise multiple regression analysis was performed to estimate the effects of independent predictors on klotho concentrations. The urine klotho-to-creatinine ratio (UrKl/Cr) was significantly lower in stage 3 dogs than the control group and was significantly lower in dogs with stage 3 and 4 CKD than in those with stage 1 and 2 disease. UrKl/Cr was negatively correlated with serum symmetric dimethylarginine (sSDMA), blood urea nitrogen (BUN), creatinine, and phosphorus concentration. Serum alpha-klotho concentration in dogs with stages 2 and 3 CKD was significantly lower than those in the control group. There was no significant correlation between serum alpha-klotho and BUN, creatinine, and phosphorus concentrations. No statistically significant differences were observed in UrKl/Cr and serum alpha-klotho concentration between groups based on sex, age, urine protein-to-creatinine ratio (UPC), or blood pressure., Conclusions: UrKl/Cr decreased in dogs with advanced CKD, and it was negatively correlated with sSDMA, BUN, creatinine, and phosphorus concentrations. Thus, klotho is associated with CKD and its clinical consequences, including CKD-mineral bone disorder, in dogs. Although serum klotho concentration was negatively correlated with sSDMA levels, it was not apparently related to IRIS CKD stage or other parameters known to be associated with CKD.

West Nile virus (WNV) is a flavivirus transmitted by mosquitoes. Birds are the reservoir for the virus; humans, horses and other mammals are dead-end hosts. Infections caused by WNV in humans can vary from asymptomatic infections to West Nile fever (WNF) or West Nile neuroinvasive disease (WNND). In 1995, a serosurvey was performed in Slovenia on forest workers, and WNV specific IgG antibodies were confirmed in 6.8% of the screened samples, indicating that WNV is circulating in Slovenia. No human disease cases were detected in Slovenia until 2013, when the first case of WNV infection was confirmed in a retrospective study in a 79-year old man with meningitis. In 2018, three patients with WNND were confirmed by laboratory tests, with detection of IgM antibodies in the cerebrospinal fluid of the patients. In one of the patients, WNV RNA was detected in the urine sample. In 2017, 2018 and 2019, a mosquito study was performed in Slovenia. Mosquitoes were sampled on 14 control locations and 35 additional locations in 2019. No WNV was detected in mosquitoes in 2017 and 2019, but we confirmed the virus in a pool of Culex sp. mosquitoes in 2018. The virus was successfully isolated, and complete genome sequence was acquired. The whole genome of the WNV was also sequenced from the patient's urine sample. The whole genome sequences of the WNV virus detected in Slovenian patient and mosquito indicate the virus most likely spread from the north, because of the geographic proximity and because the sequences cluster with the Austrian and Hungarian sequences. A sentinel study was performed on dog sera samples, and we were able to confirm IgG antibodies in 1.8% and 4.3% of the samples in 2017 and 2018, respectively. Though Slovenia is not a highly endemic country for WNV, we have established that the virus circulates in Slovenia.

Sarcocystis neurona, a coccidian parasite shed by opossums (Didelphis spp.) in the Americas, is the major cause of equine protozoal myeloencephalitis (EPM) and induces disease in other domestic and wild animal species, including domestic dogs. Sarcocystis cruzi, despite its low pathogenicity for cattle (intermediate hosts), is worldwide distributed and uses mostly dogs as definitive hosts. The aims of this study were to test serological reactivities of dog sera to S. neurona and S. cruzi antigens, and to investigate potential serological cross-reactivity to these parasites. Sera from 353 Brazilian dogs were obtained from rural areas in the municipality of Ilhéus, Bahia, and examined by immunofluorescent antibody tests (IFAT). Antigens used in serological reactions consisted of S. neurona merozoites from a North American strain (SN138), and bradyzoites of S. cruzi obtained from Brazilian bovine hearts, with parasite species identity confirmed by PCR and sequencing of the 18S gene of the rDNA. Seropositivity to S. neurona and to S. cruzi were detected in 3.39% (12/353) and 4.81% (17/353) of the dogs, respectively. Ten canine sera reacted solely to S. neurona and 15 serum samples reacted only to S. cruzi. Two serum samples were simultaneously positive for both parasites. Sera from 14 dogs that tested positive by IFAT (9 for S. neurona and 3 for S. cruzi) and from two dogs that were negative by IFAT for the two parasites, were examined by Western blot using S. neurona as antigen; these sera reacted to a great number of protein bands, including antigens on the 16 and 30 KDa positions, which encompass immunodominant antigens for S. neurona in horses. Western blot did not show any specific pattern for S. neurona infection/exposure using canine sera. Dogs act as definitive hosts for several Sarcocystis spp. that infect farm animals, including horses, sheep, goats, water buffaloes and pigs, and for this reason, should contain antibodies to a broad repertoire of Sarcocystis spp. antigens. In conclusion, low percentages of dogs from rural areas of Ilhéus, Bahia, were reactive to both S. neurona and S. cruzi antigens. It is possible that other Sarcocystis species, besides S. neurona and S. cruzi, might have contributed for the seropositivity observed in this study. IFAT was more specific than Western blot to differentiate canine serological reactions to S. neurona and S. cruzi antigens., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Soluble interleukin-2 receptor (sIL-2r) is released directly from the surface of lymphocytes expressing interleukin-2 receptor alpha chain (CD25), and its serum concentration has been found to reflect the prognosis of human lymphoproliferative malignancies. In this study, we demonstrated the presence of sIL-2r in canine serum and developed a specific sandwich enzyme-linked immunosorbent assay (ELISA) to quantify the concentration of canine serum sIL-2r. In the immunoprecipitation (IP) assay, CD25 protein weighing approximately 45 kDa was detected in canine serum, smaller than the membrane-bound CD25 (approximately 55 kDa). To measure the concentration of serum sIL-2r in dogs, an ELISA system was developed. Serum sIL-2r levels were significantly higher in dogs with multicentric high-grade B-cell lymphoma before therapy than that in healthy dogs. Serum sIL-2r concentration was also found to be elevated in a proportion of dogs with other types of lymphoma. Changes in serum sIL-2r levels generally paralleled the changes in mass and lymph node size in dogs with high-grade B-cell lymphoma. This study demonstrated that serum sIL-2r level would be a marker to monitor tumour growth and regression in canine lymphoma., Competing Interests: Declaration of Competing Interest There is no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Homocysteine (HCY) was evaluated in healthy and chronic enteropathic dogs, however no studies on dogs with immunosuppressant-responsive enteropathy are available., Objectives: The aim was to evaluate serum HCY concentrations and its prognostic role in dogs with immunosuppressant-responsive enteropathy compared to healthy dogs., Methods: Serum HCY concentration was statistically compared between 24 healthy dogs and 29 dogs with immunosuppressant-responsive enteropathy. Correlation analyses between serum total protein, albumin (ALB), C-reactive protein (CRP), folate and cobalamin, and serum HCY concentration were performed in immunosuppressant-responsive enteropathic dogs., Results: The associations between serum HCY concentration and clinical, histological, endoscopic scores and follow-up were evaluated. Mean serum HCY concentration was higher in immunosuppressant-responsive enteropathic dogs compared to control dogs (30.22 ± 8.67 μmol/L vs. 5.26 ± 2.78 μmol/L; p < 0.0001). No association between serum HCY concentration and total protein, ALB, CRP, folate concentration as well as, clinical score, histological and endoscopic scores was found. A negative correlation between serum HCY concentration and cobalamin was noted ( p = 0.0025, r = -0.54). No significant difference in HCY was found between responsive and non-responsive dogs or between survivors and non-survivors., Conclusions: Although, serum HCY concentration was higher in immunosuppressant-responsive enteropathy, its prognostic value remains unclear. However, further prospective, large-scale studies are warranted to better investigate the possible prognostic role of HCY in immunosuppressant-responsive enteropathic dogs., Competing Interests: The authors declare no conflicts of interest. No third-party funding or support was received in association with the present study or the writing or publication of the manuscript., (© 2020 The Korean Society of Veterinary Science.)

Critical illness due to sepsis is a major global health concern associated with a high burden of mortality and cost. Glucocorticoid dysregulation in human sepsis is associated with poorer outcomes. This study examines glucocorticoid metabolism in septic canine patients to delineate elements of cellular dysregulation in common with critically ill humans and explore potential differences. This was a prospective case-control study conducted in the veterinary specialist critical care departments of two University teaching hospitals. Critically ill canine patients with naturally occurring sepsis or septic shock were compared with an in-hospital control population. Serum total, bound, and free cortisol concentrations were increased in septic shock (P < 0.001), and higher bound cortisol was associated with nonsurvival (P = 0.026). Urinary Gas Chromatography-Tandem Mass Spectrometry was performed to assess urinary glucocorticoid metabolites and estimate intracellular glucocorticoid metabolism. Decreased renal 11β-hydroxysteroid dehydrogenase 2 (11βHSD2) activity inferred from increased urinary cortisol-to-cortisone ratio was observed in critically ill dogs (P < 0.001). Decreased 11βHSD2 activity (P = 0.019) and increased A-ring reduction of cortisone (P = 0.001) were associated with nonsurvival within the critically ill dogs. Intriguingly, two dogs were identified with low circulating total cortisol (<2 mg/dL) associated with increased A-ring reduction of cortisol, not previously described. Investigation of spontaneous canine sepsis and septic shock reveals dysregulation of cortisol to cortisone conversion similar to that observed in human patients, but with differences in A-ring reduction compared with those reported in humans. In addition, two dogs with high levels of cortisol inactivation associated with low circulating cortisol concentrations were identified., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Age is a primary risk factor for multiple comorbidities including neurodegenerative diseases. Pet dogs and humans represent two populations that have experienced a significant increase in average life expectancy over the last century. A higher prevalence of age-related neurodegenerative diseases has been observed across both species, and human diseases, such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS), have canine analogs, canine cognitive dysfunction (CCD), and degenerative myelopathy (DM) respectively. In humans, protein biomarkers have proved useful in the prediction and diagnosis of neurodegeneration. Molecular signatures of many proteins are highly conserved across species. In this study, we explored the potential of the neuronal cytoskeletal protein neurofilament light chain (NfL) as a biomarker of neuro-aging in dogs using an ultrasensitive single-molecule array assay to measure plasma concentrations. Healthy dogs of different ages and dogs affected with CCD and DM were evaluated. The mean plasma NfL concentrations in the different age groups of the healthy population were as follows: 4.55 ± 1.70 pg/mL in puppy/junior group (0.43-2 years), 13.51 ± 6.8 pg/mL in adult/mature group (2.1-9 years), and 47.1 ± 12.68 pg/mL in geriatric/senior group (9.3-14.5 years). Concentrations in dogs with DM (7.5-12.6 years) and CCD (11.0-15.6 years) were 84.17 ± 53.57 pg/mL and 100.73 ± 83.72 pg/mL, respectively. Plasma NfL increases in an age-dependent manner and is significantly elevated in dogs diagnosed with neurodegenerative disease. This work identified plasma NfL as a key clinical index of neuro-aging and neurodegeneration in pet dogs. Our findings mirror recent reports from human neurodegenerative diseases.

Objective: To evaluate the relationship between azotemia and plasma lactate concentration in hypotensive dogs and cats presented to an emergency department., Design: Retrospective case-control study., Setting: University veterinary teaching hospital., Animals: The electronic medical record database was searched for dogs and cats presented to the emergency department that had severe azotemia (creatinine ≥ 443.1 μmol/L [5 mg/dL]), hypotension (systolic blood pressure ≤ 90 mm Hg), and a plasma lactate measurement within 2 h of each another. Non-azotemic, normotensive dogs and cats; non-azotemic, hypotensive dogs and cats; and azotemic, normotensive dogs and cats that presented to the emergency department were used as control populations., Interventions: None., Measurements and Main Results: Severely azotemic, hypotensive dogs (n = 10) and cats (n = 63) had a lower plasma lactate than non-azotemic, hypotensive dogs and cats (P = 0.031 and P < 0.001, respectively). Median plasma lactate concentrations in hypotensive dogs (1.75 mmol/L) and cats (1.90 mmol/L) with severe azotemia were within reference intervals., Conclusions: Hypotensive dogs and cats with severe azotemia have decreased plasma lactate concentrations as compared to hypotensive, non-azotemic dogs and cats. The median plasma lactate in azotemic, hypotensive dogs and cats was within reference intervals. This may be due to either decreased cellular production of lactate or increased excretion of lactate. Further research is needed to determine which of these mechanisms is responsible and the clinical significance of plasma lactate concentrations in azotemic, hypotensive dogs and cats., (© Veterinary Emergency and Critical Care Society 2020.)

The anti-Müllerian hormone (AMH) is a glycoprotein secreted by Sertoli cells in males and granulosa cells in females. It has first been determined in blood serum of dogs and cats by Place et al. in 2011 with the use of a human-based ELISA test. Meanwhile, different immunoassays have been validated for AMH determination in animals and a variety of studies have demonstrated the clinical significance of AMH. This review summarizes the current knowledge about AMH in dogs and cats and describes future opportunities for its diagnostic use., (© 2020 Blackwell Verlag GmbH.)

Background: Bartonella spp. comprise a genus of bacteria that frequently cause persistent, often subclinical infection. Although many Bartonella spp. have been implicated in a variety of clinical presentations, Bartonella rochalimae has yet to be documented in association with a clinical presentation other than infectious endocarditis (IE) in dogs., Objectives: To document a spectrum of clinical presentations accompanied by mild hematological abnormalities in B rochalimae-infected dogs from the United States., Animals: Eight dogs with documented B rochalimae infection., Methods: Retrospective 10-year study of B rochalimae naturally infected dogs. Clinical and clinicopathologic data, including medical history, CBC, serum biochemistry panel, urinalysis, echocardiogram, and comprehensive vector-borne disease diagnostic panel results, were reviewed., Results: Eight dogs were diagnosed with B rochalimae via polymerase chain reaction (PCR) amplification. Five dogs were diagnosed with IE. Three dogs, PCR positive for B rochalimae, were diagnosed with seizures or antibiotic responsive lameness or during routine screening of a military working dog., Conclusions: This case series provides support for an association between B rochalimae and IE and provides documentation of dogs infected with B rochalimae with other clinical diagnoses., (© 2020 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Background: Dogs with hypoadrenocorticism (HA) frequently show signs of gastrointestinal disease (SGD). The prevalence of dogs presented for chronic SGD with HA is unknown., Objectives: The aims of this study were to determine the prevalence of HA in dogs with chronic SGD and to identify clinical and laboratory variables for HA in this population., Animals: One hundred fifty-one dogs with chronic SGD., Methods: In this multicentered prevalence study a standardized workup was performed in prospectively enrolled dogs with SGD > 3 weeks duration. Basal serum cortisol concentration was measured in every dog with ACTH stimulation test (ACTHST) if basal serum cortisol concentration was <3 μg/dL., Results: Basal serum cortisol concentration was <3 μg/dL in 80/151 (53%) dogs, <2 μg/dL in 42/151 (28%) dogs, and < 1 μg/dL in 9/151 (6%) dogs. In 6/151 dogs HA was diagnosed based on ACTHST (stimulated serum cortisol concentration < 2 μg/dL), a prevalence of 4%. There was no difference in history, physical examination, and laboratory variables between dogs with HA and those with other causes of chronic SGD. In 4/6 dogs with HA, there was melena or hematochezia indicating gastrointestinal blood loss. Hyperkalemia, hyponatremia, or both was not observed in any dog., Conclusion and Clinical Importance: The prevalence of HA among dogs with chronic SGD is higher than in the general population. Based on these results, testing adrenal function should be performed as a standard screening test in dogs with chronic SGD to differentiate between HA and chronic enteropathies., (© 2020 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

The present study describes the serum biochemical alterations and histopathological abnormalities in the liver tissue of dogs with liver disease. A survey of hepatic lesions was conducted using ultrasound-guided percutaneous needle biopsies. The hematologic and biochemical changes in dogs with liver lesions were recorded. Chronic hepatitis was the most common liver histopathologic finding (37.9%). Other common findings included liver fibrosis (19.5%), vacuolar hepatopathy (10.3%), cholangiohepatitis (9.2%), hepatocellular carcinoma (4.6%), cholangitis (3.4%), cholangiocarcinoma (2.3%), and congestive hepatopathy (2.3%). Greater than 2-fold elevation of serum alanine aminotransferase (ALT) was a useful indicator, with a sensitivity of 40% to 65% for diagnosing all liver pathologies. Greater than 2-fold elevation of serum alkaline phosphatase (ALP) without significant elevation of ALT was useful for diagnosing liver diseases affected by inflammatory or regressive changes (sensitivity of 40% to 50%). Elevation of ALT, ALP, or a combination of ALT and ALP had a high sensitivity of up to 90% for identifying dogs with liver pathology. Hepatic injury and cholestasis enzymes should be interpreted together with patient history, clinical signs, and liver ultrasonographic appearance before performing a liver biopsy., (Copyright and/or publishing rights held by the Canadian Veterinary Medical Association.)

Background: Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are allelic X-linked recessive muscle diseases caused by mutations in the DMD gene, with DMD being the more severe form. We have recently shown that increased plasma low-density lipoprotein-associated cholesterol causes severe muscle wasting in the mdx mouse, a mild DMD model, which suggested that plasma lipids may play a critical role in DMD. We have also observed that loss of dystrophin in mice causes unexpected elevations in plasma lipoprotein levels., Objective: The objectives of the study were to determine whether patients with DMD and BMD also present with clinically relevant plasma lipoprotein abnormalities and to mitigate the presence of confounders (medications and lifestyle) by analyzing the plasma from patients with DMD/BMD and unmedicated dogs with DMD, the most relevant model of DMD., Methods: Levels of low-density lipoprotein-associated cholesterol, high-density lipoprotein cholesterol, and triglycerides were analyzed in patients with DMD and BMD and female carriers. Samples from unmedicated, ambulatory dogs with DMD, unaffected carriers, and normal controls were also analyzed., Results: We report that 97% and 64% of all pediatric patients with DMD (33 of 36) and BMD (6 of 11) are dyslipidemic, along with an unusually high incidence in adult patients with BMD. All dogs with DMD showed plasma lipid abnormalities that progressively worsened with age. Most strikingly, unaffected carrier dogs also showed plasma lipid abnormalities similar to affected dogs with DMD. Dyslipidemia is likely not secondary to liver damage as unaffected carriers showed no plasma aminotransferase elevation., Conclusions: The high incidence of plasma lipid abnormalities in dystrophin-deficient plasma may depict a new type of genetic dyslipidemia. Abnormal lipid levels in dystrophinopathic samples in the absence of muscle damage suggest a primary state of dyslipidemia. Whether dyslipidemia plays a causal role in patients with DMD warrants further investigation, which could lead to new diagnostic and therapeutic options., (Copyright © 2020 National Lipid Association. Published by Elsevier Inc. All rights reserved.)

Objective: To describe a serious adverse event of gastrointestinal obstruction requiring surgery following routine administration of multiple doses of activated charcoal (AC) granules, which were prescribed for carprofen toxicosis., Case Summary: A 2-year-old female neutered Airedale Terrier presented for ingestion of 207 mg/kg of carprofen. Decontamination was initiated with apomorphine to induce emesis. Along with additional supportive care, the dog received an initial dose of 75 mL of AC suspension containing sorbitol by mouth (15.6 g of AC, or 0.6 g/kg), followed by 50 g of AC granules every 8 hours for 4 additional doses. While hospitalized, the dog experienced clinical signs, including vomiting and black diarrhea, as well as bloodwork changes including mild to moderate elevations in kidney and liver enzymes. Given clinical improvement after 72 hours of hospitalization, the patient was discharged for monitoring and ongoing care at home. Two days later, the patient presented again for nausea, dark diarrhea with frank blood, and panting. Abdominal ultrasound showed findings suspicious for partially obstructive foreign material or atypical impacted fecal material partially occluding the distal ileum. Despite medical management overnight, recheck ultrasound the following day demonstrated persistent obstruction with ileal foreign material. Exploratory laparotomy and enterotomy revealed moderate distension and obstruction of the distal ileum with black granular foreign material consistent with charcoal granules. The patient remained in hospital for supportive care for 4 days following the procedure, and all clinical signs were resolved at the time of discharge., New or Unique Information Provided: This report documents a serious adverse event of gastrointestinal obstruction associated with routine multidose AC administration, which has been occasionally reported in people but not in dogs. The potential for this complication should be taken into account when prescribing multiple doses of AC granules., (© Veterinary Emergency and Critical Care Society 2020.)

Background: Cognitive dysfunction syndrome (CDS) is a common progressive neurodegenerative disease that is poorly defined. Specific multitargeted protocols do not exist for setting the diagnosis and the prognosis of the syndrome., Hypothesis/objectives: To quantify Aβ42 and Aβ40 peptides in blood and cerebrospinal fluid (CSF) and to investigate their contribution to CCDS., Animals: A total of 61 dogs from a hospital population., Methods: Case-control study. Six young (YG: 0-4 years old), 8 middle-aged (4-8 years old), 17 cognitively unimpaired and aged (CU: 8-20 years old), and 30 cognitively impaired and aged (CI: 8-17 years). From the CI group, 10 dogs exhibited mild impairment (CI-MCI) and 20 exhibited severe impairment (CI-SCI). Cognitive status was assessed using a validated owner-based questionnaire. Direct and indirect Aβ markers were determined in plasma fractions (total-TP, free-FP, bound to plasma components-CP) and CSF using commercial ELISA assays (AΒtest, Araclon Biotech)., Results: TPAβ42/40 facilitated discrimination between CI-MCI and CU aged dogs with area under curve ≥ 0.79. CSFAβ42 levels were higher (P = .09) in CU (1.25 ± 0.28 ng/mL) than in MCI (1.04 ± 0.32 ng/mL) dogs. CSF Aβ42 levels were correlated with the CP fragment (CPAβ40: P = .02, CPAβ42: P = .02). CPAβ42 was higher in the CI-MCI (23.03 ± 11.79 pg/μL) group compared to the other aged dogs (CU: 10.42 ± 7.18 pg/μL, P = .02, SCI: 11.40 ± 12.98 pg/μL, P = .26)., Conclusion and Clinical Importance: The Aβ should be determined in all of the 3 plasma fractions (TP, FP, CP). In the clinical approach, TPAβ42/40 could be used as an efficient preselection tool for the aged canine population targeting dogs with mild cognitive impairment., (© 2020 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Chronic kidney disease is a common disease in dogs, and factors such as serum concentrations of creatinine, albumin, and phosphorus at the moment of diagnosis may influence the survival of these patients. The present retrospective study aimed to evaluate the relationship between survival in dogs with chronic kidney disease and laboratory parameters (creatinine, phosphorus, albumin, and hematocrit) and nutritional parameters (body condition score, muscle mass score, type of food, appetite and feeding method). A total of 116 dogs with chronic kidney disease stages 2 to 4 were included, and survival was calculated considering the time between diagnosis and death. Survival curves were configurated by Kaplan-Meier analysis and a comparison between survival curves was performed by the log-rank test. Factors related to survival were disease stage (p<0.0001), serum phosphorus concentration (p = 0.0005), hematocrit (0.0001), body condition score (p = 0.0391), muscle mass score (p = 0.0002), type of food (p = 0.0009), feeding method (p<0.0001) and appetite (p = 0.0007). Based on data obtained in this study, it is possible to conclude that early diagnosis, as well as nutritional evaluation and renal diet intake, are determinant strategies to increase survival in dogs with chronic kidney disease., Competing Interests: The authors have declared that no competing interests exist.

The purpose of this study was to do a serological survey on three rickettsial species: Rickettsia rickettsii and Rickettsia parkeri, two species of the spotted fever group (SFG) that are considered to be great importance for public health; and Rickettsia bellii, a species of unknown pathogenicity that infects a variety of human-biting ticks. Serum samples from 273 dogs were tested using the indirect immunofluorescence assay (IFA). A total of 52 samples (19.04%) were seropositive for at least one of the three Rickettsia spp. antigens. Thirty-eight (73.07%), twelve (23.07%) and one (1.92%) of these dogs showed homologous reactions to R. bellii, R. rickettsii and R. parkeri, respectively. Our results showed that the seroprevalence of Rickettsia spp. was relatively low. However, the positive serological tests indicated that these dogs had become infected by these agents at some point in their lives. Lastly, our study adds to the previous knowledge on the epidemiology of rickettsiosis in the state of Goiás by doing the first record of detection of anti-R. rickettsii, R. parkeri and R. bellii antibodies by IFA among dogs, thus indicating that these agents may be circulating in the dog population analyzed.

Non-neoplastic bone marrow disorders such as non-regenerative immune-mediated anemia, pure red cell aplasia, and myelodysplastic syndrome are major causes of non-regenerative anemia in dogs. However, there has been no study on the clinical and clinicopathological features of canine non-neoplastic bone marrow disorders in Japan. Hence, we first investigated the breed disposition of non-neoplastic bone marrow disorders that induce anemia as a retrospective study and found that Miniature Dachshund (MD) was a predisposed breed. Based on this finding, we investigated the clinical and clinicopathological features of non-neoplastic bone marrow disorders in MDs as a preliminary retrospective study, and we compared them between immunosuppressive treatment-responsive and -resistant MDs. We found that treatment-resistant MDs showed thrombocytosis and increased frequencies of dysplastic features in the peripheral blood. These results indicate that bone marrow disorders in treatment-resistant MDs might manifest distinct features compared with those in treatment-sensitive MDs, and sensitivity to immunosuppressive treatments could be predicted based on thrombocytosis and dysplastic features in the peripheral blood. Further studies that examine aberrations in the genome are needed to elucidate the pathophysiology of bone marrow disorders in MDs.

Human patients with inflammatory bowel disease may have poor prognosis with hypozincemia. However, there are limited data on zinc concentrations in the blood of dogs with lymphocytic-plasmacytic enteritis (LPE). The purpose of this study was to investigate the serum zinc concentration in dogs with LPE and its influence on disease severity and prognosis. Thirty-five dogs with LPE were recruited. Serum zinc concentration was measured using atomic absorption spectrometry. Hypozincemia was observed in 18/35 (51%) dogs with LPE. Serum zinc concentration was inversely correlated with histological and clinical severities. Overall survivals were significantly shorter in dogs with hypozincemia than in those without it. These findings suggest that serum zinc concentration is a useful biomarker for LPE severity and prognosis in dogs.

Myocardial energy deprivation plays a causal role in the development of heart failure. A cardiac protection blend (CPB) of nutrients including medium chain triglycerides, fish oil and other key nutrients was developed to slow the progression of canine myxomatous mitral valve disease (MMVD). A six-month dietary intervention demonstrated efficacy of CPB in slowing MMVD progression. Untargeted metabolomic analysis of serum from these dogs identified 102 differential metabolites (adjusted P < 0.05). The ratios of omega-6 to omega-3 fatty acid (FA) changed from 2.41 and 1.46 in control and CPB groups at baseline to 4.30 and 0.46 at 6 months respectively. A 2.7-fold increase of α-aminobutyrate, a myocardial modulator of glutathione homeostasis, was found in CPB dogs compared to 1.3-fold increase in control dogs. Arginine and citrulline, precursors of nitric oxide biosynthesis, were both increased 2-fold; caprate, a medium chain FA, was increased 3-fold; and deoxycarnitine, precursor of carnitine biosynthesis, was increased 2.5-fold in CPB dogs. Margarate and methylpalmitate decreased in response to CPB, a potential benefit in MMVD dogs as positive correlations were found between changes in both these FAs and left atrial diameter (r = 0.69, r = 0.87 respectively, adjusted P < 0.05). Sphingomyelins with very long chain saturated FAs associated with decreased risk of heart failure in humans were increased in MMVD dogs fed the CPB diet. Our data supports the hypothesis that CPB improves FA utilization and energetics, reduces oxidative stress and inflammation in MMVD dogs. More studies are needed to understand the roles of specific metabolites in MMVD., Competing Interests: QL is a current employee and DPL and JEB are paid consultants of the Nestlé Purina PetCare Company. This does not alter our adherence to the PLOS ONE policies on sharing data and materials.

Background: Adiponectin (ADPN) is an adipocytokine with insulin-sensitizing, vascular-protective, and anti-inflammatory properties for which concentration changes occur in response to inflammation. Little is known about the regulation of ADPN and the impact of this adipocytokine in septic dogs., Objective: We aimed to assess the diagnostic and prognostic value of ADPN vs other traditional acute-phase proteins (APPs), such as albumin (ALB), haptoglobin (HPT), fibrinogen (FBG), ferritin (FRT), and C-reactive protein (CRP) in dogs with naturally acquired sepsis., Methods: This prospective observational study included 20 dogs with sepsis, 27 with low-grade systemic inflammation (LGSI), and 18 clinically healthy dogs as controls. For method analyses, plasma samples were obtained from all dogs on admission and then every 24-48 hours until discharge or death in the septic group., Results: Septic dogs had lower ADPN (2.4 ± 0.46 vs 4.5 ± 0.41mg/L, P < .001) dand ALB (17 ± 1 vs 22 ± 0.8g/L, P = .002), and tended to have higher CRP (87 ± 4.8 vs 73 ± 4.1mg/L, P < .079) concentrations than dogs with LGSI on admission. Only ADPN and ALB were able to successfully discriminate animals with LGSI from those presenting with sepsis with areas under the curve (AUCs) for the receiver operating characteristic (ROC) curves of 0.811 and 0.789, respectively. In the septic group, ADPN concentration did not differ between survivors and non-survivors, either on admission or at discharge or death., Conclusions: Although plasma ADPN can be used as a reliable negative APP in dogs with sepsis, further studies are warranted to confirm the usefulness of this biomarker in terms of disease progression and recovery., (© 2020 American Society for Veterinary Clinical Pathology.)

Background: Flow cytometry (FC) is used increasingly in veterinary medicine for further characterization of hematolymphoid cells. Guidelines for optimizing assay performance and interpretation of results are limited, and concordance of results across laboratories is unknown., Objectives: This study aimed to determine inter-investigator agreement on the interpretation of FC results from split samples analyzed in different laboratories using various protocols, cytometers, and software; and on the interpretation of archived FC standard (FCS) data files contributed by the different investigators., Methods: This was a multicenter observational cross-sectional study. Anticoagulated blood or lymph node aspirate samples from nine client-owned dogs were aliquoted and shipped to participating laboratories. Samples were analyzed with individual laboratory-developed protocols. In addition, FCS files from a set of separate samples from 11 client-owned dogs were analyzed by participating investigators. A person not associated with the study tabulated the results and interpretations. Agreement of interpretations was assessed with Fleiss' kappa statistic., Results: Prolonged transit times affected sample quality for some laboratories. Overall agreement among investigators regarding the FC sample interpretation was strong (κ = 0.86 ± 0.19, P < .001), and for specific categories, ranged from moderate to perfect. Agreement of the lymphoproliferation or other leukocyte sample category from the analysis of the FCS files was weak (κ = 0.58 ± 0.05, P < .001)., Conclusions: Lymphoproliferations were readily identified by FC, but identification of the categories of hematolymphoid neoplasia in fresh samples or archived files was variable. There is a need for a more standardized approach to maximize the enormous potential of FC in veterinary medicine., (© 2020 American Society for Veterinary Clinical Pathology.)

Whole transferrin receptor (TfR) is present in reticulocyte exosomes. Soluble transferrin receptor (sTfR) is cleaved from whole TfR in human plasma, with the remnant cytoplasmic domain (cTfR) remaining membrane associated. In humans, sTfR is a biomarker that can detect iron deficiency in the presence of inflammatory disease. This condition is still a diagnostic dilemma in veterinary species. We aimed to (1) confirm the presence of exosomes and exosome-associated TfR in the serum of dogs, cats, and horses; and (2) to assess and compare the proportion of cTfR to total (cTfR + whole) in exosomal membranes of healthy and diseased dogs and cats and in healthy horses to indirectly predict their anticipated levels of circulating sTfR. We used discarded serum and whole blood samples from canine and feline patients, separated into healthy and diseased groups based on the health status of each patient, and healthy equine participants from a previous study. Ultracentrifugation, followed in some experiments by OptiPrep discontinuous density gradient fractionation, was used to isolate exosomes. Exosomes and associated TfR were identified using TEM and Western blot for TfR, respectively. Densitometry tracings of Western blots of serum exosomes were used to measure the proportion of cTfR to total TfR. Extracellular vesicles compatible with exosomes were successfully isolated and expressed TfR. The proportion of cTfR in dogs was greater than 50%, indicating that a majority of the whole TfR was cleaved to produce sTfR (and remnant cTfR). There was significant interindividual variation and no significant difference between healthy and diseased animals. The proportion of cTfR in cats was very low at 11%, indicating that very little sTfR was likely produced. There was a small yet significant difference between healthy and diseased cats. Healthy horses do not appear to cleave exosome-associated TfR. Diagnosis of iron deficiency in the presence of inflammatory disease remains a challenge in veterinary medicine. Our results indicate that TfR is poorly or unpredictably cleaved in veterinary species, revealing that there are species differences in exosomal TfR handling. These data suggest that development of an assay for the detection and quantification of sTfR in the species investigated may not be warranted., (Copyright © 2020 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

The aim of this study was to evaluate the intestinal and cardiac biomarkers in the determination of intestinal and cardiac damage in dogs with parvoviral enteritis. The material of this study consisted of 10 healthy dogs (control group) and 30 dogs with parvoviral enteritis (experimental group) admitted to the Department of Internal Medicine, Faculty of Veterinary Medicine, Selcuk University.Serum samples were extracted from the collected blood samples taken from vena cephalicavenipuncture for analysis of blood gases, haemogram and to measure the levels of intestinal-fatty acid-binding protein (I-FABP), trefoil factor 3 (TFF-3), claudin-3 (CLDN-3), heart-type fatty acid-binding protein (H-FABP), cardiac troponin I (cTnI), and creatine kinase-myocardial band (CK-MB) by enzyme linked immunosorbent assay (ELISA) test kits. Statistically significant decreases in the blood gas hydrogen ion concentration (pH), partial pressure of oxygen (pO2), sodium (Na), bicarbonate (HCO3), and oxygen saturation (SatO2) levels and significant increase in the levels of I-FABP, TFF-3, CK-MB, cTnI and also in the haemogram, a decrease in leukocyte (WBC) level and an increase in platelet (THR) level were detected in parvoviral dogs compared to the control group (p⟨0.05). Also ROC analysis revealed on 0th hour for the utility of I-FABP and on 48th hour for TFF-3 in differentiating in the experimental group between the survivor and non-survivor dogs. Other intestinal-related biomarker (CLDN-3) and none of the cardiac-related biomarkers (H-FABP, CK-MB and cTnI) are not high enough for prediction of mortality.In conclusion, it was determined that I-FABP and TFF-3 for the intestinal injury and morta-lity prediction, and CK-MB and cTnI for the cardiac injury were useful and reliable biomarkers to determine the damage caused by parvovirus in dogs., (Copyright© by the Polish Academy of Sciences.)

Background: The Mindray BC-5000Vet hematology analyzer is a flow cytometry-based automated hematology analyzer that generates a complete blood count with a five-part white blood cell (WBC) differential count., Objectives: We aimed to validate reliability results of the Mindray BC-5000Vet for use in dog and cat blood., Methods: Imprecision was performed using the manufacturer's quality control material at low, normal, and high levels. Blood sample results of healthy and ill dogs and cats were studied for comparability between manual methods and the Mindray BC-5000Vet analyzer. Forty dogs and 40 cats were included in the study., Results: Precision for red blood cell (RBC) parameters was excellent, with a coefficient of variation within-run (%CV w ) and between-run (%CV b ) of <2%. WBC count and differential count showed %CV w and %CV b <8%; however, %CV w and %CV b of low-level control material gave >9% eosinophils. The correlation between the BC-5000 Vet and manual methods in normal and abnormal canine and feline blood samples showed excellent correlations for the RBC counts, hemoglobin concentrations, hematocrits, and WBC counts (r > .93). The differential WBC analysis of canine blood showed good correlation (r = .80-.92). Feline blood samples showed excellent correlations for neutrophils and lymphocytes, with a good correlation for monocytes and eosinophils., Conclusions: The Mindray BC-5000Vet hematology analyzer proved a suitable instrument for routine analysis in dogs and cats with various hematologic abnormalities., (© 2020 American Society for Veterinary Clinical Pathology.)

Gastric dilation-volvulus (GDV) syndrome in dogs is associated with complex metabolic, acid-base, and electrolyte abnormalities. The aim of this study was to evaluate previously analyzed factors (lactate and BE) in combination with other acid-base parameters (pH, pCO 2 , bicarbonate, base excess [BE], anion gap [AG], and strong ion difference) and electrolyte concentrations and to evaluate their association with the incidence of gastric necrosis and outcome in dogs with GDV. A retrospective study in 75 dogs with gastric dilation-volvulus syndrome, University veterinary teaching hospital. Medical records were reviewed including signalment, history, initial plasma lactate, acid-base parameters, and electrolyte concentrations, surgical findings and outcome. The overall mortality was 18.7%. In dogs with gastric necrosis, higher initial plasma lactate (median 5.84 vs. 3.36 mmol/L) and AG (20.7 vs. 16.55 mmol/L) and lower pH (7.29 vs. 7.36), bicarbonate (18.7 vs. 22.9 mmol/L), and BE concentration (-8.1 vs. -1.85 mmol/L) were found compared to dogs without gastric necrosis. Anorganic phosphorus was the only electrolyte investigated for which a significant difference was noted between dogs with and without gastric necrosis (1.93 vs. 1.39 mmol/L). The initial plasma lactate concentration (3.36 mmol/L vs. 9.68 mmol/L) and AG (16.8 vs. 20.95 mmol/L) were lower in survivors than nonsurvivors. Survivors had higher pH (7.35 vs. 7.27), bicarbonate concentrations (22.9 vs. 17.35 mmol/L), and BE (-1.9 vs. -9.55 mmol/L) compared to nonsurvivors. Anorganic phosphorus was ultimately the only electrolyte with a significant difference between survivors and nonsurvivors (1.4 vs. 1.84 mmol/L). A multivariate logistic regression model of combination lactate, pH, bicarbonate, BE, AG, and anorganic phosphorus identified pH ≤7.331 and bicarbonate as factors independently associated with gastric necrosis. Similarly, pH ˃7.331, bicarbonate and anorganic phosphorus were independently associated with outcome. Higher initial plasma lactate, AG and anorganic phosphorus levels, and lower pH, BE and bicarbonate concentrations were found in GDV dogs with gastric necrosis. Similarly, initially higher plasma lactate, AG and anorganic phosphorus concentrations, and lower pH, BE and bicarbonate were found in GDV dogs who required euthanasia or who died after surgery. Of these parameters, pH and bicarbonate were strongly and independently associated with gastric necrosis, and pH, bicarbonate and phosphorus were independently associated with outcome., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: In human medicine, extremely high CRP (C-reactive protein) concentrations > 100 mg/l are indicators of bacterial infection and the need of antibiotic treatment. Similar decision limits for septic pneumonia are recommended for dogs but have not yet been evaluated for other organ systems. The aim of the retrospective study was to investigate the prevalence and evaluate dogs with CRP concentrations > 100 mg/l regarding the underlying etiology, the affected organ system and the prognostic significance., Results: Prevalence of CRP > 100 mg/l was investigated in dogs presented between 2014 and 2015 and was 12%. For evaluation of etiology and organ systems, dogs with CRP > 100 mg/l presented between 2014 and 2016 were enrolled. Dogs were classified into 4 main disease categories, i.e. inflammatory, neoplastic, tissue damage or "diverse". Diseases were assigned to the affected organ system. If an organ classification was not possible, dogs were classified as "multiple". 147 dogs with CRP 101-368 mg/l were included and classified into disease categories: 86/147 (59%) with inflammatory etiology (among these, 23/86 non-infectious, 44/86 infectious (33/44 bacterial), 19/86 inflammation non-classifiable), 31/147 (21%) tissue damage, 17/147 (12%) neoplastic (all malignant) and 13/147 (9%) diverse diseases. The affected organ systems included 57/147 (39%) multiple, 30/147 (20%) trauma, 21/147 (14%) gastrointestinal tract, 10/147 (7%) musculoskeletal system, 8/147 (5%) respiratory tract, 7/147 (5%) urinary/reproductive tract, 6/147 (4%) skin/subcutis/ear, 6/147 (4%) central/peripheral nervous system and 2/147 (1%) heart. The disease group (p = 0.081) or organ system (p = 0.17) did not have an impact on CRP. Based on CRP, a detection of bacterial infection was not possible. The prognostic significance was investigated by determining the 3-months survival and hospitalization rate in a subgroup with known outcome. The 3-months survival rate was 46/73 (63%) while the majority 66/73 (90%) of patients was hospitalized., Conclusions: CRP concentrations > 100 mg/l are occasionally seen in a clinic population. They indicate a severe systemic disease of various etiologies with guarded prognosis. Extremely high CRP concentrations do not allow a conclusion of the underlying etiology or an identification of bacterial inflammation.

Introduction: A surge in Food and Drug Administration (FDA) consumer complaints identified concerns that legume-rich, grain-free diets were associated with nutritionally-mediated dilated cardiomyopathy (DCM). Golden retrievers represent the most reported breed affected by this condition and previous studies documented the disease is responsive to dietary change and taurine supplementation. Although dietary findings across cases are compelling, prospective studies with control groups are lacking. The role of diet in developing taurine deficiency and echocardiographic changes consistent with DCM in healthy dogs is unknown., Objectives: We hypothesized that golden retrievers eating non-traditional diets are at a higher risk of having taurine deficiency and nutritionally-mediated DCM compared with those eating traditional commercial diets. We aimed to compare taurine concentrations and echocardiographic indices of systolic function between golden retrievers in each diet group and elucidate associations between diet and these variables. Additionally, we aimed to generate breed-specific reference intervals for whole blood and plasma taurine concentrations., Animals: 86 golden retrievers., Methods: Golden retrievers eating traditional or non-traditional diets were evaluated and diet history, taurine concentrations and echocardiographic data were collected. Dietary features, taurine concentrations and echocardiographic findings were compared between diet groups. Relative risks were calculated for the likelihood of echocardiographic abnormalities and taurine deficiency in each diet group. Breed-specific reference intervals were constructed for taurine concentrations in dogs from the traditional diet group., Results: Golden retrievers eating non-traditional diets had significantly lower taurine concentrations and more frequent systolic dysfunction. Breed specific reference intervals are higher than previously reported across breeds., Conclusions: Non-traditional diets, which were typically grain-free and contained legumes in this study, were significantly associated with and have increased relative risk for the identification of taurine deficiency and echocardiographic abnormalities consistent with nutritionally-mediated DCM. These findings were identifiable in the absence of clinical signs and support the findings of multiple previous studies and the ongoing FDA investigation., Competing Interests: A.J. Fascetti is the Scientific Director of and J. Yu is employed by the Amino Acid Laboratory at the University of California, Davis that provides amino acid analysis on a fee for service basis. This did not influence the collection or interpretation of results in this study. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Despite vulnerability and unsanitary conditions of animal hoarding may predispose environmental contamination and spread of vectors and pathogens, no study to date has focused on their impact on public health and zoonotic diseases. Accordingly, this study aimed to assess the seroprevalence of anti-Toxoplasma gondii antibodies and associated factors in individuals with animal hoarding disorder (AHD) and their dogs in Curitiba, Southern Brazil. Blood samples were obtained from 264 dogs (21 households) and 19 individuals with AHD (11 households). Their blood was tested by indirect fluorescent antibody test (IFAT). Overall, anti-Toxoplasma gondii seropositivity was found in 21/264 dogs (7.95%; 95% CI: 4.69-11.22) with titers ranging from 16 to 4096, and in 7/19 individuals with AHD (36.84%; CI: 15.15-58.53) with titers ranging from 16 to 64. Serological analysis for anti-T. gondii antibodies were considered positive in at least one individual or dog in 9/11 (81.82%; 95% CI: 59.03-100.00) cases that were thoroughly assessed. Surprisingly, the seropositivity of individuals with AHD and their dogs was among the lowest reportedly observed in human and dog populations of Brazil. There was no significant association between positive owners and positive dogs or the presence of cats in the household. Regard epidemiological variables, a significant association was found between dog's seropositivity and the type of dog food. To the authors' knowledge, the present study represents the first investigation of T. gondii seroprevalence in individuals with hoarding disorder and their dogs. In conclusion, despite low sanitary conditions, anti-Toxoplasma gondii antibodies frequency in individuals with AHD and their dogs are lower than the general population likely due to low protozoan load in such isolated households., Competing Interests: The authors have declared that no competing interests exist.

Canine leishmaniosis is an important vector-borne zoonosis caused mainly by Leishmania infantum. Diagnosis and treatment of affected individuals can be particularly complex, hindering infection control in endemic areas. Methods to prevent canine leishmaniosis include the use of topical insecticides, prophylactic immunotherapy and vaccination. Four vaccines against canine leishmaniosis have been licensed since 2004, two in Brazil (Leishmune®, the production and marketing licence of which was withdrawn in 2014, and Leish-Tec®) and two in Europe (CaniLeish® and LetiFend®). After several years of marketing, doubts remain regarding vaccine efficacy and effectiveness, potential infectiousness of vaccinated and infected animals or the interference of vaccine-induced antibodies in L. infantum serological diagnosis. This review summarises the scientific evidence for each of the vaccines commercially approved for canine leishmaniosis, while discussing possible weaknesses of these studies. Furthermore, it raises the need to address important questions related to vaccination impact in Leishmania-endemic countries and the importance of post-marketing pharmacological surveillance., (© 2020 John Wiley & Sons Ltd.)

Ehrlichiosis is a common vector-borne disease caused by Ehrlichia spp. This retrospective matched cohort study was performed to determine if dogs with Ehrlichia spp. antibodies had an increased incidence of chronic kidney disease (CKD). Exposure to Ehrlichia spp. was defined as having an Ehrlichia spp. antibody-positive result recorded at any point in their available patient history. The outcome of CKD was defined as concurrent increased symmetric dimethylarginine (>14 µg/dL) and creatinine (>1.5 mg/dL) for a minimum of 25 days with inappropriate urine specific gravity (<1.030). Patients were matched using propensity score matching to control for age, geography, and breed. A total of 22,440 patients and controls in E canis -endemic regions of the United States were used in this analysis. Contingency tables were used to compare dogs with and without exposure to Ehrlichia spp.-infected ticks and CKD outcome. The relative risk of CKD for patients exposed to ticks carrying Ehrlichia spp. was found to be 2.12 (95% confidence interval [1.35-3.15], p < 0.0006). This study identified that testing positive for Ehrlichia spp. antibodies in E canis -endemic regions is associated with higher incidence of CKD in dogs.

Cysteine proteinases are well-known virulence factors of Leishmania spp. with demonstrated actions in both experimental mouse infection and human infection. However, studies on these enzymes in canine leishmaniasis are scarce. Here, we show, for the first time, the reactivity of sera from dogs living in an endemic area to a recombinant protein from the COOH-terminal region of cysteine B protease. In this work, enzyme-linked immunosorbent assays were performed using a 14kDa rcyspep protein obtained through a pET28-a expression system in Escherichia coli. First, 96-well plates were coated with rcyspep (500ng/well) and incubated with sera from dogs (1:100). Subsequently, IgG antibody detection was performed using rabbit anti-dog IgG antibodies conjugated with peroxidase. Sera from dogs (n=114), including suspect (n=30) and positive (n=50) dogs from a leishmaniasis-endemic area and dogs from a nonendemic area, (n=34), negative for leishmaniasis, were assessed. The results showed that sera from the suspect (42%) and positive (68%) groups responded differently to the antigen titers tested above the cut-off (Optical Density=0.166). This finding suggests that the immune response detected against cyspep may be related to clinical disorders present in these animals. Collectively, the data gathered here suggest that cyspep can sensitize the immune systems of dogs from a leishmaniasis-endemic area to elicit a humoral response, an immunological parameter indicating the contribution of this protein in host-parasite interaction., (Copyright © 2020 Sociedade Brasileira de Infectologia. Published by Elsevier España, S.L.U. All rights reserved.)

Background: Long-term use of doxorubicin (DOX) is limited by cumulative dose-dependent cardiotoxicity., Objectives: Identify plasma extracellular vesicle (EV)-associated microRNAs (miRNAs) as a biomarker for cardiotoxicity in dogs by correlating changes with cardiac troponin I (cTnI) concentrations and, echocardiographic and histologic findings., Animals: Prospective study of 9 client-owned dogs diagnosed with sarcoma and receiving DOX single-agent chemotherapy (total of 5 DOX treatments). Dogs with clinically relevant metastatic disease, preexisting heart disease, or breeds predisposed to cardiomyopathy were excluded., Methods: Serum concentration of cTnI was monitored before each treatment and 1 month after the treatment completion. Echocardiography was performed before treatments 1, 3, 5, and 1 month after completion. The EV-miRNA was isolated and sequenced before treatments 1 and 3, and 1 month after completion., Results: Linear mixed model analysis for repeated measurements was used to evaluate the effect of DOX. The miR-107 (P = .03) and miR-146a (P = .02) were significantly downregulated whereas miR-502 (P = .02) was upregulated. Changes in miR-502 were significant before administration of the third chemotherapeutic dose. When stratifying miRNA expression for change in left ventricular ejection fraction, upregulation of miR-181d was noted (P = .01). Serum concentration of cTnI changed significantly but only 1 month after treatment completion, and concentrations correlated with left ventricular ejection fraction and left ventricular internal dimension in diastole., Conclusion and Clinical Significance: Downregulation of miR-502 was detected before significant changes in cTnI concentrations or echocardiographic parameters. Further validation using a larger sample size will be required., (© 2020 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Background: Sensitivity and specificity for commercial and in-house pancreatic lipase immunoreactivity (cPLI) assays have been reported, but repeatability under routine clinical conditions is unknown., Objectives: To determine: Coefficient of variation (CV) between replicates of a commercial assay (Spec cPL) and 2 in-house assays (VetScan cPL, Vcheck cPL) under routine conditions. Effects of sample condition or personnel on results. Potential directional bias between assays., Animals: Serum from 12 canine clinical patients., Methods: Prospective study. Serum Spec cPL, VetScan cPL, and Vcheck cPL (6 aliquots each) were measured, and CVs were calculated, effects of sample condition and personnel were assessed using a linear mixed model, and direction of bias was assessed using least square mean cPLI concentration., Results: Mean %CVs for Spec cPL, VetScan cPL, and Vcheck cPL were 5.5, 17.0, and 23.7%. Three of 6 VetScan cPL samples and 5/9 Vcheck cPL samples had an unacceptably high %CV (>20%). Transportation (Spec cPL) and sample condition or personnel (VetScan cPL, Vcheck cPL) did not affect repeatability. Least square mean cPL was higher for Spec cPL (807.9 μg/L) than for VetScan cPL (558.5 μg/L) or Vcheck cPL (399.8 μg/L)., Conclusions and Clinical Importance: For clinical use, the commercial Spec cPL has the highest repeatability, and Vcheck cPL has significantly lower repeatability. Both in-house assays evaluated may provide discrepant categorical results ("pancreatitis" versus "equivocal" versus "not pancreatitis") for the same sample. In-house pancreatic lipase concentrations may be lower than those determined by the Spec cPL assay., (© 2020 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Background: Asymmetric dimethylarginine (ADMA) has been proposed as a severity marker in humans with acute pancreatitis (AP)., Objectives: To evaluate ADMA in dogs with AP compared with healthy dogs and its association with severity of disease, mortality, and indicators of the systemic inflammatory response syndrome (SIRS), including serum C-reactive protein (CRP) concentration, WBC count, and band neutrophils., Animals: Fifty-four dogs with AP and a control group (CG) of 28 healthy dogs., Methods: Cohort study including dogs with AP diagnosed using clinical and laboratory variables, abnormal canine pancreatic lipase (cPL) concentration, and compatible abdominal ultrasound examination findings performed within 48 hours of admission. Canine AP severity (CAPS) was calculated. Serum concentration of ADMA was measured using high performance liquid chromatography. Blood donor-, staff-, and student-owned dogs were enrolled in the CG., Results: Dogs with AP had higher median admission serum ADMA concentrations compared with the CG (62 versus 48.5 μg/dL; P = .003). Dogs with CAPS ≥11 had higher serum ADMA concentrations than did dogs with CAPS <11 (92 versus 54.6 μg/dL P = .009). Univariable analysis for mortality, CAPS score, band neutrophils, CRP, and ADMA were included in multivariable logistic regression, in which only ADMA was associated with mortality (P = .02). Survivors had a significant decrease in ADMA at first reevaluation compared to admission (P = .02)., Conclusions and Clinical Importance: Because serum ADMA concentrations were higher in AP dogs compared with the CG, it may have value as a biomarker in the diagnosis of AP in dogs. In addition, because ADMA was associated with mortality, it may have prognostic value., (© 2020 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Background: The use of serological markers to diagnose inflammatory bowel disease (IBD) in humans is well-established. Because of the frequency of IBD in dogs and resources required for its diagnosis with current methods, new approaches are desired., Objective: The goal is to evaluate novel serologic markers to differentiate clinical cohorts in dogs with gastrointestinal (GI) disease and assess their potential to develop a serum-based IBD diagnostic test., Animals: Seventy dogs diagnosed with biopsy-confirmed IBD, 23 dogs with non-IBD predominantly acute GI diseases, and 58 normal dogs., Methods: Prospective control study. ELISA methods were developed to detect autoantibodies to polymorphonuclear leukocytes (APMNA) and calprotectin (ACNA), antibodies against gliadins (AGA), microbial outer membrane porin C (ACA), and flagellins (AFA) isolated from diseased dogs based on clinical and histopathological scoring., Results: IBD dogs displayed a 39%-76% prevalence of seropositivity against selected serologic markers that markedly decreased to 0%-13% in non-IBD and normal dogs. ROC analysis showed statistical significance in differentiating the cohorts, with seropositivity against OmpC being the highest single performance marker. The combination of markers such as OmpC and APMNA reached specificities of 93%-99% and 79%-98% and sensitivities of 76%-97% and 66%-86% when comparing IBD versus normal cohorts and non-IBD cohorts, respectively., Conclusion and Clinical Importance: Seropositivity of canine immunoglobulins A against selected serologic markers in dogs appears promising in the detection and differentiation of IBD versus other acute GI conditions. Among them, antibody reactivity to Escherichia coli OmpC and canine autoantibodies against polymorphonuclear leukocytes displayed the highest single marker discriminating performance., (© 2020 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Canine Leishmaniasis caused by Leishmania infantum is endemic in Mediterranean countries. The disease represents an important public health issue in Kosovo in which the autochthonous cases by humans were reported in the last years. The objective of this study was to survey the serological and clinical data of Canine Leishmaniasis in dogs in southwestern region of Kosovo. The blood samples were collected from stray dogs in four municipalities, respectively Prizren, Gjakova, Rahovec and Deçan. The total 125 samples were collected between April/September 2016. Antibodies of Leishmania infantum were detected by competitive enzyme-linked immunosorbent assay (ELISA IgG). Out of total 125 serum samples, 23 dogs (18.4%) were positive for Leishmania infantum. In three of 23 infected dogs (13%) were shown the clinical signs of Canine Leishmaniasis as skin and ocular lesions, decreased appetite, lameness, diarrhea, lethargy and progressive weight loss. The highest infection rate was detected in Prizren municipality (21.6%), followed by Gjakova (17.6%), Rahovec (17.1%) and Deçan (7.8%). The present study confirmed that the Leishmaniasis is endemic in southwestern part of Kosovo and emphasized the need to establish the surveillance and control of this disease.

Circulating tumor DNA (ctDNA), which carries tumor-specific mutations, is an emerging candidate biomarker for malignancies and for monitoring disease status in various human tumors. Recently, BRAF V595E mutation has been reported in 80% of dogs with urothelial carcinoma. This study investigates the BRAF V595E allele concentration in circulating cell-free DNA (cfDNA) and assesses the clinical significance of BRAF-mutated ctDNA levels in canines with urothelial carcinoma. A total of 15 dogs with urothelial carcinoma were included. cfDNA concentration was measured using a real-time polymerase chain reaction (PCR) of the LINE-1 gene. To measure the concentration of the mutated BRAF gene in cfDNA, allele-specific real-time PCR with a locked nucleic acid probe was performed. BRAF mutations were detected in 11 (73%) of the 15 tested tumor samples. BRAF-mutated ctDNA concentrations were significantly higher in dogs with the BRAF mutation (14.05 ± 13.51 ng/ml) than in wild-type dogs (0.21 ± 0.41 ng/ml) (p = 0.031). The amount of BRAF-mutated ctDNA in plasma increased with disease progression and responded to treatment. Our results show that BRAF-mutated ctDNA can be detected using allele-specific real-time PCR in plasma samples of canines with urothelial carcinoma with the BRAF V595E mutation. This ctDNA analysis may be a potentially useful tool for monitoring the progression of urothelial carcinoma and its response to treatment., Competing Interests: The authors have declared that no competing interests exist.