Neutrophil-Extracellular Traps, Cell-Free DNA, and Immunothrombosis in Companion Animals: A Review.

Immunothrombosis is a potentially beneficial physiological process that aids innate immunity and host defense against pathogen invasion. However, this process can also be damaging when it occurs to excess or in critical blood vessels. Formation of extracellular traps by leukocytes, particularly neutrophils, is central to our understanding of immunothrombosis. In addition to degranulation and phagocytosis, extracellular traps are the third mechanism by which neutrophils combat potential pathogens. These traps consist of extracellular DNA decorated with bactericidal cellular proteins, including elastase, myeloperoxidase, and cathepsins. Neutrophils can release these structures as part of a controlled cell-death process or via a process termed vital NETosis that enables the cells to extrude DNA but remain viable. There is accumulating evidence that NETosis occurs in companion animals, including dogs, horses, and cats, and that it actively contributes to pathogenesis. Numerous studies have been published detailing various methods for identification and quantification of extracellular trap formation, including cell-free DNA, measurements of histones and proteins such as high-mobility group box-1, and techniques involving microscopy and flow cytometry. Here, we outline the present understanding of these phenomena and the mechanisms of extracellular trap formation. We critically review the data regarding measurement of NETosis in companion animals, summarize the existing literature on NETosis in veterinary species, and speculate on what therapeutic options these insights might present to clinicians in the future.

C1 inhibitor in canine intravascular hemolysis (C1INCH): study protocol for a randomized controlled trial.

BACKGROUND: Immune-mediated hemolytic anemia (IMHA) is a common disease that affects all breeds of dogs and is associated with significant morbidity and mortality. Intravascular hemolysis of erythrocytes in IMHA is caused by complement activation and is often fatal. No current treatments target complement activation in canine IMHA. Human C1 esterase (C1-INH) reduces canine complement-mediated hemolysis in vitro, and a recent pharmacokinetic analysis of an FDA licensed formulation of C1-INH in dogs confirmed that a 50 IU/kg dose of C1-INH is safe to administer to dogs, and effectively inhibits canine complement mediated hemolysis ex-vivo. The C1INCH randomized controlled trial will evaluate the efficacy of this drug in dogs with intravascular hemolysis. METHODS: We will conduct a multicenter, placebo-controlled double-blind randomized clinical trial of C1-INH in dogs with intravascular hemolysis due to IMHA. We will randomize 18 dogs to receive three doses of intravenous C1-INH or saline in 24 h. Immunosuppressive and antithrombotic therapies will be standardized. Primary outcome measures will be changes in plasma free hemoglobin, serum concentrations of LDH, bilirubin, and haptoglobin. Using patient samples, we will evaluate complement activation in canine IMHA using a novel C5b-9 ELISA assay, flow cytometric detection of C3b on RBC, and by measurement of residual plasma complement activity. Secondary outcome measures will be survival to hospital discharge, duration of hospitalization, number and volume of red blood cell transfusions, and rescue therapy requirements. We will monitor dogs for adverse drug reactions. Sample size was estimated from pilot data on LDH and hemolysis index (HI) in dogs with IMHA. To detect 2-way differences between the upper and lower 50% of the LDH and HI values of equivalent size with 80% power at P < 0.05 will require 9 dogs in each arm. DISCUSSION: We anticipate that IV administration of C1-INH will significantly inhibit complement mediated hemolysis in dogs with intravascular IMHA, as determined by blood biomarker measurements (decreased plasma hemoglobin, LDH and bilirubin, increased haptoglobin). We expect this will translate into significant reductions in transfusion requirements and duration of hospitalization. TRIAL REGISTRATION: This trial has been prospectively registered with the AVMA registry (AAHSD005025).

Plasma procalcitonin concentrations predict organ dysfunction and outcome in dogs with sepsis.

BACKGROUND: Procalcitonin (PCT) is a valuable prognostic biomarker in human sepsis that is predictive of organ dysfunction, septic shock and mortality. Data on PCT in dogs is limited. This study aimed to investigate the prognostic value of baseline and serial PCT measurements in dogs with sepsis and to determine the association between PCT and sepsis severity and the presence of organ dysfunction. PCT concentrations were measured in citrated plasma samples collected from 53 dogs with sepsis at the time of admission (T0, n = 53) and at 24 h (T1, n = 35) and 48 h (T2, n = 30) post-admission using a commercial ELISA. Dogs were classified by sepsis severity (sepsis without organ dysfunction; severe sepsis; septic shock) and outcome (survivors; non-survivors). Organ dysfunctions were recorded at T0 and during hospitalization, and the APPLEfast score calculated at T0. Healthy dogs (n = 12) were used as controls. RESULTS: There were 18 septic dogs without organ dysfunction, 24 dogs with severe sepsis and 11 with septic shock. Baseline PCT concentrations were significantly greater in dogs with sepsis compared to healthy controls (P < 0.0001), and in dogs with septic shock compared to dogs without cardiovascular compromise (P = 0.01). Baseline PCT was significantly correlated with organ dysfunction (P = 0.003). Declining PCT concentrations were documented in survivors at T1 and T2 compared to PCT at T0 (P = 0.0006), and PCT clearance at 24 h was significantly higher in survivors (n = 38) compared to non-survivors (n = 15) (P = 0.037). Canine APPLEfast score was not predictive of sepsis severity, the development of MODS or outcome. CONCLUSION: In dogs with sepsis, PCT concentrations at hospital admissions are predictive of organ dysfunction and septic shock. Serial procalcitonin monitoring may offer valuable prognostic information in canine sepsis, wherein early decreases in PCT concentrations are associated with survival.

Syntaxin 8 regulates platelet dense granule secretion, aggregation, and thrombus stability.

Platelet secretion not only drives thrombosis and hemostasis, but also mediates a variety of other physiological and pathological processes. The ubiquitous SNARE machinery and a number of accessory proteins have been implicated in regulating secretion in platelet. Although several platelet SNAREs have been identified, further members of the SNARE family may be needed to fine-tune platelet secretion. In this study we identified expression of the t-SNARE syntaxin 8 (STX8) (Qc SNARE) in mouse and human platelets. In mouse studies, whereas STX8 was not essential for α-granule or lysosome secretion, Stx8(-/-) platelets showed a significant defect in dense granule secretion in response to thrombin and CRP. This was most pronounced at intermediate concentrations of agonists. They also showed an aggregation defect that could be rescued with exogenous ADP and increased embolization in Stx8(-/-) mice in vivo consistent with an important autocrine and paracrine role for ADP in aggregation and thrombus stabilization. STX8 therefore specifically contributes to dense granule secretion and represents another member of a growing family of genes that play distinct roles in regulating granule release from platelets and thus platelet function in thrombosis and hemostasis.

Evaluation of a DLA-79 allele associated with multiple immune-mediated diseases in dogs.

Immune-mediated diseases are common and life-threatening disorders in dogs. Many canine immune-mediated diseases have strong breed predispositions and are believed to be inherited. However, the genetic mutations that cause these diseases are mostly unknown. As many immune-mediated diseases in humans share polymorphisms among a common set of genes, we conducted a candidate gene study of 15 of these genes across four immune-mediated diseases (immune-mediated hemolytic anemia, immune-mediated thrombocytopenia, immune-mediated polyarthritis (IMPA), and atopic dermatitis) in 195 affected and 206 unaffected dogs to assess whether causative or predictive polymorphisms might exist in similar genes in dogs. We demonstrate a strong association (Fisher's exact p = 0.0004 for allelic association, p = 0.0035 for genotypic association) between two polymorphic positions (10 bp apart) in exon 2 of one allele in DLA-79, DLA-79*001:02, and multiple immune-mediated diseases. The frequency of this allele was significantly higher in dogs with immune-mediated disease than in control dogs (0.21 vs. 0.12) and ranged from 0.28 in dogs with IMPA to 0.15 in dogs with atopic dermatitis. This allele has two non-synonymous substitutions (compared with the reference allele, DLA-79*001:01), resulting in F33L and N37D amino acid changes. These mutations occur in the peptide-binding pocket of the protein, and based upon our computational modeling studies, are likely to affect critical interactions with the peptide N-terminus. Further studies are warranted to confirm these findings more broadly and to determine the specific mechanism by which the identified variants alter canine immune system function.

Platelet Rho GTPases-a focus on novel players, roles and relationships.

Rho GTPases are critical for platelet function. Although the roles of RhoA, Rac and Cdc42 are characterized, platelets express other Rho GTPases, whose activities are less well understood. This review summarizes our understanding of the roles of platelet Rho GTPases and focuses particularly on the functions of Rif and RhoG. In human platelets, Rif interacts with cytoskeleton regulators including formins mDia1 and mDia3, whereas RhoG binds SNARE-complex proteins and cytoskeletal regulators ELMO and DOCK1. Knockout mouse studies suggest that Rif plays no critical functions in platelets, likely due to functional overlap with other Rho GTPases. In contrast, RhoG is essential for normal granule secretion downstream of the collagen receptor GPVI. The central defect in RhoG-/- platelets is reduced dense granule secretion, which impedes integrin activation and aggregation and limits platelet recruitment to growing thrombi under shear, translating into reduced thrombus formation in vivo. Potential avenues for future work on Rho GTPases in platelets are also highlighted, including identification of the key regulator for platelet filopodia formation and investigation of the role of the many Rho GTPase regulators in platelet function in both health and disease.

RhoG protein regulates platelet granule secretion and thrombus formation in mice.

Rho GTPases such as Rac, RhoA, and Cdc42 are vital for normal platelet function, but the role of RhoG in platelets has not been studied. In other cells, RhoG orchestrates processes integral to platelet function, including actin cytoskeletal rearrangement and membrane trafficking. We therefore hypothesized that RhoG would play a critical role in platelets. Here, we show that RhoG is expressed in human and mouse platelets and is activated by both collagen-related peptide (CRP) and thrombin stimulation. We used RhoG(-/-) mice to study the function of RhoG in platelets. Integrin activation and aggregation were reduced in RhoG(-/-) platelets stimulated by CRP, but responses to thrombin were normal. The central defect in RhoG(-/-) platelets was reduced secretion from α-granules, dense granules, and lysosomes following CRP stimulation. The integrin activation and aggregation defects could be rescued by ADP co-stimulation, indicating that they are a consequence of diminished dense granule secretion. Defective dense granule secretion in RhoG(-/-) platelets limited recruitment of additional platelets to growing thrombi in flowing blood in vitro and translated into reduced thrombus formation in vivo. Interestingly, tail bleeding times were normal in RhoG(-/-) mice, suggesting that the functions of RhoG in platelets are particularly relevant to thrombotic disorders.

Effect of institutional antimicrobial stewardship guidelines on prescription of critically important antimicrobials for dogs and cats.

BACKGROUND: Veterinary hospital antimicrobial stewardship (AMS) guidelines might help combat antimicrobial resistance (AMR). OBJECTIVES: Determine the conditions and types of infection for which antimicrobial drugs (AMDs) deemed critically important (CIA) by the World Health Organization (WHO) were prescribed and assess the effect of hospital AMS guidelines on adherence to International Society for Companion Animal Infectious Diseases published guidelines for the treatment of superficial bacterial folliculitis, respiratory tract disease and urinary tract infection in these cases. ANIMALS: Dogs and cats managed at an academic veterinary hospital from 1/21 to 6/21 and 9/21 to 6/22. METHODS: Prescriptions of cephalosporins (third or fourth generation), glycopeptides, macrolides/ketolides, polymyxins, and quinolones were identified. Data on culture and susceptibility (C/S) testing and previous AMD exposure were collected. Frequencies were compared between time periods using Fisher's exact test with Bonferroni corrections. RESULTS: In animals prescribed ≥1 WHO-CIA AMD, fluoroquinolones were the most frequently prescribed WHO-CIA class in dogs (567/1724, 32.9%) and cats (192/450, 42.7%). No animals were prescribed carbapenems, dihydrofolate reductase inhibitors/sulfonamides, or polymyxins. No cats were prescribed aminoglycosides or amphenicols. Institutional guidelines were followed in 57.8% (324/561) cases. The most frequent causes of nonadherence were failure to perform C/S testing 46.0% (109/237) and unnecessary use of a higher-tier AMD 43.0% (102/237). Bacterial C/S testing was more frequently performed after AMS guideline institution (59.7% vs. 46.8%, P = 0.0006). CONCLUSIONS AND CLINICAL IMPORTANCE: Adherence to published guidelines remained poor despite an increase in C/S testing. There were no changes in the frequencies of confirmed infections, positive cultures or AMD resistance between time periods.

Plasma concentration of thrombopoietin in dogs with immune thrombocytopenia.

BACKGROUND: Immune thrombocytopenia (ITP) is a common cause of severe thrombocytopenia in dogs. The pathogenesis of nonassociative, primary ITP (pITP) appears complex, with ill-defined thrombopoietic response. OBJECTIVES: Develop an immunoassay to measure plasma canine thrombopoietin (TPO) concentration and characterize TPO concentrations in dogs with pITP. ANIMALS: Forty-one healthy dogs, 8 dogs in an induced ITP model (3 control, 5 ITP), and 58 pITP dogs. METHODS: Recombinant canine TPO (rcTPO) was purchased and its identity confirmed by mass spectrometry. Monoclonal antibodies were raised to rcTPO and used to configure a sandwich ELISA using streptavidin-biotin detection. Assay performance, coefficients of variability, and healthy dog plasma TPO reference interval (RI) were determined, followed by assay of ITP samples. RESULTS: Assay dynamic range was 15 pg/mL (lower limit of detection) to 1000 pg/mL TPO, with limit of quantitation of 62 pg/mL. Plasma TPO RI was 0 to 158 pg/mL, with plasma TPO <62 pg/mL for 35/41 healthy dogs. All dogs with induced ITP developed marked increases in plasma TPO concentration. Peak values ranged from 515 to >6000 pg/mL. In contrast, only 2/58 pITP dogs had TPO values above RI. CONCLUSIONS AND CLINICAL IMPORTANCE: Plasma TPO concentration is paradoxically low at diagnosis for most dogs with pITP. This finding suggests that ineffective thrombopoiesis contributes to thrombocytopenia in pITP dogs and supports evaluating TPO receptor agonist treatment as used for pITP in humans. The TPO assay provides a new tool to study thrombopoiesis in pITP and other thrombocytopenic syndromes in dogs.

A prospective cohort study to identify clinical diagnostic and prognostic markers of primary immune thrombocytopenia in dogs.

BACKGROUND: Primary immune thrombocytopenia (pITP) in dogs presents a diagnostic challenge, and clinical markers of severity are lacking. OBJECTIVES: Identify clinicopathologic features that differentiate pITP from secondary ITP (sITP) and markers related to bleeding severity, transfusion, and survival of dogs with pITP. ANIMALS: Ninety-eight thrombocytopenic dogs (58 pITP and 40 sITP). METHODS: Client-owned dogs with platelet counts <50 000/µL were enrolled in a prospective, multi-institution cohort study. History and treatment information, through a maximum of 7 days, was recorded on standard data forms. Bleeding severity was scored daily using a bleeding assessment tool (DOGiBAT). At-admission blood samples were collected for CBC, biochemistry, C-reactive protein concentration, and coagulation panels, and to measure platelet surface-associated immunoglobulin G (PSAIg) and expression of platelet membrane proteins and phospholipids. Dogs with evidence of coincident disease were classified as sITP. RESULTS: No definitive pITP diagnostic test was found. However, pITP cases were characterized by lower platelet counts, D dimer concentrations, and platelet membrane protein expression than sITP cases. Differentiation between pITP and sITP was further enhanced using logistic regression modeling combining patient sex, coagulation profile, platelet count, D dimer, and PSAIg. A second model of pITP severity indicated that low hematocrit and high BUN concentration were associated with non-survival. Low hematocrit at admission, but not platelet count or DOGiBAT score, was associated with transfusion. CONCLUSIONS AND CLINICAL IMPORTANCE: Pending validation studies, models constructed from at-admission clinicopathologic findings may improve differentiation of pITP from sITP and identify the most severe pITP cases at the time of presentation.

Defining sepsis in small animals.

OBJECTIVE: To discuss the definitions of sepsis in human and veterinary medicine. DESIGN: International, multicenter position statement on the need for consensus definitions of sepsis in veterinary medicine. SETTING: Veterinary private practice and university teaching hospitals. ANIMALS: Dogs and cats. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Sepsis is a life-threatening condition associated with the body's response to an infection. In human medicine, sepsis has been defined by consensus on 3 occasions, most recently in 2016. In veterinary medicine, there is little uniformity in how sepsis is defined and no consensus on how to identify it clinically. Most publications rely on modified criteria derived from the 1991 and 2001 human consensus definitions. There is a divergence between the human and veterinary descriptions of sepsis and no consensus on how to diagnose the syndrome. This impedes research, hampers the translation of pathophysiology insights to the clinic, and limits our abilities to optimize patient care. It may be time to formally define sepsis in veterinary medicine to help the field move forward. In this narrative review, we present a synopsis of prior attempts to define sepsis in human and veterinary medicine, discuss developments in our understanding, and highlight some criticisms and shortcomings of existing schemes. CONCLUSIONS: This review is intended to serve as the foundation of current efforts to establish a consensus definition for sepsis in small animals and ultimately generate evidence-based criteria for its recognition in veterinary clinical practice.

ACVIM consensus statement on the diagnosis of immune thrombocytopenia in dogs and cats.

Immune thrombocytopenia (ITP) is the most common acquired primary hemostatic disorder in dogs. Immune thrombocytopenia less commonly affects cats but is an important cause of mortality and treatment-associated morbidity in both species. Immune thrombocytopenia remains a diagnosis of exclusion for which diagnostic guidelines are lacking. Primary, or non-associative, ITP refers to autoimmune platelet destruction. Secondary, or associative, ITP arises in response to an underlying disease trigger. However, evidence for which comorbidities serve as ITP triggers has not been systematically evaluated. To identify key diagnostic steps for ITP and important comorbidities associated with secondary ITP, we developed 12 Population Evaluation/Exposure Comparison Outcome (PECO) format questions. These questions were addressed by evidence evaluators utilizing a literature pool of 287 articles identified by the panelists using a structured search strategy. Evidence evaluators, using panel-designed templates and data extraction tools, summarized evidence and created guideline recommendations that then were integrated by diagnosis and comorbidity domain chairs. The revised PECO responses underwent a Delphi survey process to reach consensus on final guidelines. A combination of panel expertise and PECO responses were employed to develop algorithms for diagnosis of ITP in dogs and cats, which also underwent 4 iterations of Delphi review. Comorbidity evidence evaluators employed an integrated measure of evidence (IME) tool to determine evidence quality for each comorbidity; IME values combined with evidence summaries for each comorbidity were integrated to develop ITP screening recommendations, which also were subjected to Delphi review. Commentary was solicited from multiple relevant professional organizations before finalizing the consensus. The final consensus statement provides clinical guidelines for the diagnosis of, and underlying disease screening for, ITP in dogs and cats. The systematic consensus process identified numerous knowledge gaps that should guide future studies. This statement is a companion manuscript to the ACVIM Consensus Statement on the Treatment of Immune Thrombocytopenia.

ACVIM consensus statement on the treatment of immune thrombocytopenia in dogs and cats.

Management of immune thrombocytopenia (ITP) in dogs and cats is evolving, but there are no evidence-based guidelines to assist clinicians with treatment decisions. Likewise, the overall goals for treatment of ITP have not been established. Immunosuppressive doses of glucocorticoids are the first line treatment, but optimal treatment regimens beyond glucocorticoids remain uncertain. Additional options include secondary immunosuppressive drugs such as azathioprine, modified cyclosporine, and mycophenolate mofetil, usually selected based on clinician preference. Vincristine, human IV immunoglobulin (hIVIg), and transfusion of platelet or red blood cell-containing products are often used in more severe cases. Splenectomy and thrombopoietin receptor agonists are usually reserved for refractory cases, but when and in which patient these modalities should be employed is under debate. To develop evidence-based guidelines for individualized treatment of ITP patients, we asked 20 Population Intervention Comparison Outcome (PICO) format questions. These were addressed by 17 evidence evaluators using a literature pool of 288 articles identified by a structured search strategy. Evidence evaluators, using panel-designed templates and data extraction tools, summarized evidence and created guideline recommendations. These were integrated by treatment domain chairs and then refined by iterative Delphi survey review to reach consensus on the final guidelines. In addition, 19 non-PICO questions covering scenarios in which evidence was lacking or of low quality were answered by expert opinion using iterative Delphi surveys with panelist integration and refinement. Commentary was solicited from multiple relevant professional organizations before finalizing the consensus. The rigorous consensus process identified few comparative treatment studies, highlighting many areas of ITP treatment requiring additional studies. This statement is a companion manuscript to the ACVIM Consensus Statement on the Diagnosis of Immune Thrombocytopenia in Dogs and Cats.

Increased thrombin activatable fibrinolysis inhibitor activity is associated with hypofibrinolysis in dogs with sepsis.

Introduction: Disorders of coagulation are well-recognized in dogs with sepsis, but data regarding fibrinolysis disorders are limited. We aimed to characterize fibrinolysis in dogs with sepsis compared to healthy controls. We hypothesized that dogs with sepsis would be hypofibrinolytic, and that hypofibrinolysis would be associated with non-survival. Methods: This was a prospective observational cohort study. We enrolled 20 client-owned dogs with sepsis admitted to the Cornell University Hospital for Animals and 20 healthy pet dogs. Coagulation and fibrinolytic pathway proteins including antiplasmin activity (AP), antithrombin activity (AT), thrombin activatable fibrinolysis inhibitor activity (TAFI), D-dimer concentration, fibrinogen concentration, and plasminogen activity were measured and compared between groups. Overall coagulation potential, overall fibrinolysis potential, and overall hemostatic potential were calculated from the curve of fibrin clot formation and lysis over time. Results: Compared to healthy controls, dogs with sepsis had lower AT (P = 0.009), higher AP (P = 0.002), higher TAFI (P = 0.0385), and higher concentrations of fibrinogen (P < 0.0001) and D-dimer (P = 0.0001). Dogs with sepsis also had greater overall coagulation potential (P = 0.003), overall hemostatic potential (P = 0.0015), and lower overall fibrinolysis potential (P = 0.0004). The extent of fibrinolysis was significantly negatively correlated with TAFI. No significant differences were observed between survivors and non-survivors. Discussion: Dogs with sepsis were hypercoagulable and hypofibrinolytic compared to healthy dogs, suggesting potential utility of thromboprophylaxis in this patient population. The association between high TAFI and low overall fibrinolysis potential might provide a potential mechanism for this hypofibrinolysis.

Apolipoprotein A1 and serum amyloid A in dogs with sepsis and septic shock.

Introduction: Apolipoprotein-A1 (Apo-A1) acts as a negative acute phase protein (APP) during inflammatory states, and has a potential prognostic value in people and dogs with sepsis. The aim of this retrospective study was to investigate the association of serum Apo-A1 concentration with disease severity, multiorgan dysfunction syndrome (MODS) and outcome in a population of dogs with sepsis, and to assess its correlation with major canine APPs. Methods: Ninety-nine dogs with uncomplicated sepsis (n = 78) or septic shock (n = 21) were included. The serum concentration of Apo-A1, C-reactive protein (CRP) and serum amyloid A (SAA) were recorded, alongside the canine acute patient physiologic and laboratory evaluation fast (APPLEfast) score and the presence of MODS. Results: Dogs with septic shock had significantly lower serum Apo-A1 concentrations (106.3 ± 22.7 mg/dl; reference interval: 123.0-142.3 mg/dl), higher APPLEfast score (30, 13-38) and greater frequency of MODS (67%) compared to those with uncomplicated sepsis (117.9 ± 19.3 mg/dl; 25, 6-33 and 8%, respectively) (P = 0.0201; P = 0.0005; P < 0.0001, respectively). Similarly, dogs with MODS had significantly lower serum Apo-A1 concentrations (104.1 ± 4.6 mg/dl) and higher APPLEfast score values (31, 13-38) compared to those without MODS (118.32 ± 2.1 mg/dl and 26, 6-33, respectively) (P = 0.0050 and P = 0.0038, respectively). Conversely, neither CRP nor SAA were different between these groups. No difference in serum APPs concentrations was detected between survivors and non-survivors. Significant negative correlations were detected between serum Apo-A1 and SAA (P = 0.0056, r = -0.277), and between serum Apo-A1 and the APPLEfast score (P = 0.0027, r = -0.3). In this population, higher values of the APPLEfast score and the presence of MODS were independently associated with a higher risk of death. Discussion: Our study shows that Apo-A1 is a useful biomarker of sepsis severity in dogs, since it is decreased in those with septic shock and MODS. Further prospective investigations are deemed to evaluate the applicability of Apo-A1 to predict sepsis course and response to treatment in septic dogs.

Prevalence of antibiotic use for dogs and cats in United States veterinary teaching hospitals, August 2020.

BACKGROUND: Awareness of prescribing practices helps identify opportunities to improve antibiotic use (AU). OBJECTIVES: To estimate AU prevalence in dogs and cats in U.S. veterinary teaching hospitals (VTHs) and identify antibiotic drugs commonly prescribed, indications for use, and evidence of bacterial infection. ANIMALS: Medical record data were collected from dogs and cats examined at 14 VTHs. METHODS: Data were collected from VTH medical records of dogs and cats examined by primary care, urgent care, emergency and critical care, internal medicine, and surgery services on a single day during August 13-September 3, 2020. Data included signalment; clinical service; inpatient or outpatient status; clinical conditions; diagnostic tests; evidence of bacterial infection; intended reason for AU; name and route of antibiotics prescribed. RESULTS: Of 883 dogs and cats, 322 (36.5%) were prescribed at least 1 antibiotic. Among 285 antibiotics administered systemically intended for treatment of infection, 10.9% were prescribed without evidence of infection. The most common class of antibiotics presribed for systemic administration was potentiated penicillin for dogs (115/346, 33.3%) and cats (27/80, 33.8%). For dogs and cats, first-generation cephalosporins (93/346, 26.9% and 11/80, 13.8%, respectively) and fluoroquinolones (51/346, 14.7% and 19/80, 23.8%, respectively) was second or third most-prescribed. Common AU indications included skin, respiratory, and urinary conditions, and perioperative use. CONCLUSIONS AND CLINICAL IMPORTANCE: Collaborative data collection provides a sustainable methodology to generate national AU prevalence estimates and bring attention to areas requiring additional research and detailed data collection. These efforts can also identify practice improvement opportunities in settings where future veterinarians are trained.

Prospective investigation of factors associated with success on the American College of Veterinary Emergency and Critical Care certification examination (2016-2018).

OBJECTIVE: To assess the association of candidate attributes and residency training factors with success on the American College of Veterinary Emergency and Critical Care (ACVECC) board certification examination and to develop multivariable models of first-attempt success. DESIGN: Prospective survey-based study. SETTING: Post-assessment ACVECC examination candidates. ANIMALS: None. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Comprehensive surveys were distributed to ACVECC examination candidates in 2016 to 2018 after completion of their assessments, but prior to publication of examination results. Unique anonymous candidate identification numbers were used to match survey responses to outcome data from the office of the ACVECC Executive Secretary. After curation to retain only the first response from each candidate, there were 97 unique candidate responses available for analysis. Univariate analyses identified multiple factors associated with first-attempt success and multiple differences between academic and private practice residency programs. Multivariable logistic regression modeling suggested that 5 factors were independently associated with first-attempt success on the ACVECC examination, specifically younger age, more weeks of study prior to the examination, training at a facility with more ACVECC Diplomates, training at a facility with more ACVECC residents, and having no requirement to manage both Emergency Room (ER) and Critical Care (CC) cases simultaneously. CONCLUSIONS: Numerous resident and training center factors are associated with success in the ACVECC board certification examination. Residents and training centers might be able to use these data to enhance training, but caution must be exercised because these data are associative only.

Clinical features and posttreatment monitoring of dogs administered rivaroxaban (2018-2020): 19 cases.

OBJECTIVE: To describe a population of sick dogs administered rivaroxaban monitored with a rivaroxaban-calibrated anti-Xa activity assay (aXa). DESIGN: Descriptive retrospective study. SETTING: Two veterinary teaching hospitals. ANIMALS: Client-owned dogs administered rivaroxaban and monitored with aXa from January 2018 to January 2020 were eligible for study. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Medical records were reviewed and 19 dogs with a variety of underlying disease processes were identified. Rivaroxaban was administered to 12 of 19 dogs (63%) with confirmed thrombosis, 4 of 19 dogs (21%) with a strong clinical suspicion of thrombosis, and in 3 of 19 dogs (16%) with no current evidence of thrombosis. The median rivaroxaban dose administered was 0.96 mg/kg/day (0.62-1.58 mg/kg/day), with 15 of 19 dogs (79%) receiving rivaroxaban once daily. Clopidogrel was concurrently administered to 11 of 19 dogs (58%). Complete or partial thrombus resolution was identified in 5 of 12 (42%) and 3 of 12 (25%) dogs, respectively. Rivaroxaban appeared safe, with only 1 of 19 dogs (5%), concurrently administered clopidogrel, developing evidence of mild hematuria. Posttreatment monitoring revealed that 8 of 19 dogs (42%) had aXa below the target (aXa range of 150-250 ng/ml associated with effective treatment and prevention of venous thrombosis in people). The remaining 3 to 19 dogs (16%) achieved this range, and 8 of 19 dogs (42%) exceeded the range. No significant relationship between the initial rivaroxaban dose administered and the corresponding aXa result was identified. There were also no significant differences in baseline clinicopathological variables in dogs in which aXa fell within or outside this range. CONCLUSIONS: aXa was most commonly measured in dogs receiving rivaroxaban with confirmed or suspected thrombosis. Dogs in this study received a range of rivaroxaban dosages and attained variable aXa values that were not directly correlated with dosage.

Identifying potential biomarkers and therapeutic targets for dogs with sepsis using metabolomics and lipidomics analyses.

Sepsis is a diagnostic and therapeutic challenge and is associated with morbidity and a high risk of death. Metabolomic and lipidomic profiling in sepsis can identify alterations in metabolism and might provide useful insights into the dysregulated host response to infection, but investigations in dogs are limited. We aimed to use untargeted metabolomics and lipidomics to characterize metabolic pathways in dogs with sepsis to identify therapeutic targets and potential diagnostic and prognostic biomarkers. In this prospective observational cohort study, we examined the plasma metabolomes and lipidomes of 20 healthy control dogs and compared them with those of 21 client-owned dogs with sepsis. Patient data including signalment, physical exam findings, clinicopathologic data and clinical outcome were recorded. Metabolites were identified using an untargeted mass spectrometry approach and pathway analysis identified multiple enriched metabolic pathways including pyruvaldehyde degradation; ketone body metabolism; the glucose-alanine cycle; vitamin-K metabolism; arginine and betaine metabolism; the biosynthesis of various amino acid classes including the aromatic amino acids; branched chain amino acids; and metabolism of glutamine/glutamate and the glycerophospholipid phosphatidylethanolamine. Metabolites were identified with high discriminant abilities between groups which could serve as potential biomarkers of sepsis including 13,14-Dihydro-15-keto Prostaglandin A2; 12(13)-DiHOME (12,13-dihydroxy-9Z-octadecenoic acid); and 9-HpODE (9-Hydroxyoctadecadienoic acid). Metabolites with higher abundance in samples from nonsurvivors than survivors included 3-(2-hydroxyethyl) indole, indoxyl sulfate and xanthurenic acid. Untargeted lipidomic profiling revealed multiple sphingomyelin species (SM(d34:0)+H; SM(d36:0)+H; SM(d34:0)+HCOO; and SM(d34:1D3)+HCOO); lysophosphatidylcholine molecules (LPC(18:2)+H) and lipophosphoserine molecules (LPS(20:4)+H) that were discriminating for dogs with sepsis. These biomarkers could aid in the diagnosis of dogs with sepsis, provide prognostic information, or act as potential therapeutic targets.

Serial evaluation of thoracic radiographs and acute phase proteins in dogs with pneumonia.

BACKGROUND: Acute phase proteins (APP) may guide treatment of pneumonia in dogs but correlations with radiographic abnormalities are poorly characterized. OBJECTIVES: Develop a thoracic radiographic severity scoring system (TRSS), assess correlation of radiographic changes with APP concentrations, and compare time to APP and radiograph normalization with duration of antimicrobials treatment. ANIMALS: Sixteen client-owned dogs, 12 with aspiration pneumonia, and 4 with community-acquired pneumonia. METHODS: Concentrations of C-reactive protein (CRP), serum amyloid A (SAA), and haptoglobin were measured on days 1, 3, 7, 14, 28, and 60 and orthogonal 2-view thoracic radiographs were obtained on days 1, 7, 14, 28, and 60. Treatment was clinician-guided and blinded to APP concentrations. Radiographic severity scores were assigned by blinded, randomized retrospective review by 2 board-certified radiologists with arbitration by a third radiologist. RESULTS: Median (interquartile range [IQR]) time to normalization of CRP (7 days [7-14]) and SAA concentrations (7 days [7-14]) were shorter than antimicrobial treatment duration (17.5 days [14.5-33.5]; P = .001 and .002, respectively) and TRSS normalization (14 days [8.8-52], P = .02 and .02, respectively). The CRP and SAA concentrations were positively correlated with TRSS (CRP rs , 0.643; SAA rs , 0.634; both P < .0001). Both CRP and SAA identified normal thoracic radiographs area under the curve (AUC) 0.873 and 0.817, respectively, both P < .0001. Interobserver agreement for TRSS assignment was moderate (κ, .499; P < .0001). CONCLUSION AND CLINICAL IMPORTANCE: Concentrations of CRP and SAA normalized before radiographic resolution and before clinicians discontinued antimicrobial treatment. The CRP and SAA concentrations may guide duration of antimicrobial treatment for dogs with pneumonia.