Immune-mediated hematological disease in dogs is associated with alterations of the fecal microbiota: a pilot study.

BACKGROUND: The dog is the most popular companion animal and is a valuable large animal model for several human diseases. Canine immune-mediated hematological diseases, including immune-mediated hemolytic anemia (IMHA) and immune thrombocytopenia (ITP), share many features in common with autoimmune hematological diseases of humans. The gut microbiome has been linked to systemic illness, but few studies have evaluated its association with immune-mediated hematological disease. To address this knowledge gap, 16S rRNA gene sequencing was used to profile the fecal microbiota of dogs with spontaneous IMHA and ITP at presentation and following successful treatment. In total, 21 affected and 13 healthy control dogs were included in the study. RESULTS: IMHA/ITP is associated with remodeling of fecal microbiota, marked by decreased relative abundance of the spirochete Treponema spp., increased relative abundance of the pathobionts Clostridium septicum and Escherichia coli, and increased overall microbial diversity. Logistic regression analysis demonstrated that Treponema spp. were associated with decreased risk of IMHA/ITP (odds ratio [OR] 0.24-0.34), while Ruminococcaceae UCG-009 and Christensenellaceae R-7 group were associated with increased risk of disease (OR = 6.84 [95% CI 2-32.74] and 8.36 [95% CI 1.85-71.88] respectively). CONCLUSIONS: This study demonstrates an association of immune-mediated hematological diseases in dogs with fecal dysbiosis, and points to specific bacterial genera as biomarkers of disease. Microbes identified as positive or negative risk factors for IMHA/ITP represent an area for future research as potential targets for new diagnostic assays and/or therapeutic applications.

In vitro Inhibition of Canine Complement-Mediated Hemolysis.

BACKGROUND: Immune-mediated hemolytic anemia (IMHA) is the most common hematologic immune-mediated disease in dogs. Complement fixation on erythrocytes causes hemolysis. Complement inhibition decreases hemolysis in people with the hemolytic disease and also may prove effective in treating IMHA in dogs. HYPOTHESIS/OBJECTIVES: Evaluate the in vitro efficacy of 2 complement inhibitors used in humans against canine complement. METHODS: The inhibitory activity of the C3-inhibitor compstatin and recombinant human C1-esterase inhibitor (C1-INH) was evaluated using an in vitro hemolytic assay and spectrophotometric measurement of released hemoglobin. Dose-response curves for each inhibitor were generated. RESULTS: Compstatin decreased approximately 50% of canine complement-mediated hemolysis in initial experiments. This inhibition largely was lost when a new lot of drug was purchased. C1-INH showed a dose-dependent inhibition. The highest concentration of C1-INH tested (500 µg/mL) decreased >80% of canine complement-mediated hemolysis, and the lowest concentration tested (31.25 µg/mL) decreased hemolysis >60%. CONCLUSIONS AND CLINICAL IMPORTANCE: Human C1-INH is a robust inhibitor of canine complement-mediated hemolysis, whereas compstatin was minimally and variably effective. Human C1-INH may substantially decrease complement-mediated hemolysis in dogs with IMHA and warrants further investigation.

Predicting Outcome in dogs with Primary Immune-Mediated Hemolytic Anemia: Results of a Multicenter Case Registry.

BACKGROUND: Outcome prediction in dogs with immune-mediated hemolytic anemia (IMHA) is challenging and few prognostic indicators have been consistently identified. OBJECTIVES: An online case registry was initiated to: prospectively survey canine IMHA presentation and management in the British Isles; evaluate 2 previously reported illness severity scores, Canine Hemolytic Anemia Score (CHAOS) and Tokyo and to identify independent prognostic markers. ANIMALS: Data from 276 dogs with primary IMHA across 10 referral centers were collected between 2008 and 2012. METHODS: Outcome prediction by previously reported illness-severity scores was tested using univariate logistic regression. Independent predictors of death in hospital or by 30-days after admission were identified using multivariable logistic regression. RESULTS: Purebreds represented 89.1% dogs (n = 246). Immunosuppressive medications were administered to 88.4% dogs (n = 244), 76.1% (n = 210) received antithrombotics and 74.3% (n = 205) received packed red blood cells. Seventy-four per cent of dogs (n = 205) were discharged from hospital and 67.7% (n = 187) were alive 30-days after admission. Two dogs were lost to follow-up at 30-days. In univariate analyses CHAOS was associated with death in hospital and death within 30-days. Tokyo score was not associated with either outcome measure. A model containing SIRS-classification, ASA classification, ALT, bilirubin, urea and creatinine predicting outcome at discharge was accurate in 82% of cases. ASA classification, bilirubin, urea and creatinine were independently associated with death in hospital or by 30-days. CONCLUSIONS AND CLINICAL IMPORTANCE: Markers of kidney function, bilirubin concentration and ASA classification are independently associated with outcome in dogs with IMHA. Validation of this score in an unrelated population is now warranted.

Leukemia-associated Rho guanine-nucleotide exchange factor is not critical for RhoA regulation, yet is important for platelet activation and thrombosis in mice.

BACKGROUND: RhoA is an important regulator of platelet responses downstream of Gα13 , yet we still know little about its regulation in platelets. Leukemia-associated Rho guanine-nucleotide exchange factor (GEF [LARG]), a RhoA GEF, is highly expressed in platelets and may constitute a major upstream activator of RhoA. To this end, it is important to determine the role of LARG in platelet function and thrombosis. METHODS AND RESULTS: Using a platelet-specific gene knockout, we show that the absence of LARG results in a marked reduction in aggregation and dense-granule secretion in response to the thromboxane mimetic U46619 and proteinase-activated receptor 4-activating peptide, AYPGKF, but not to adenosine diphosphate. In a ferric chloride thrombosis model in vivo, this translated into a defect, under mild injury conditions. Importantly, agonist-induced RhoA activation was not affected by the absence of LARG, although basal activity was reduced, suggesting that LARG may play a housekeeper role in regulating constitutive RhoA activity. CONCLUSIONS: LARG plays an important role in platelet function and thrombosis in vivo. However, although LARG may have a role in regulating the resting activation state of RhoA, its role in regulating platelet function may principally be through RhoA-independent pathways, possibly through other Rho family members.

Comparison of computed tomography pulmonary angiography and point-of-care tests for pulmonary thromboembolism diagnosis in dogs.

OBJECTIVES: To evaluate the feasibility of CT pulmonary angiography for identification of naturally occurring pulmonary thromboembolism in dogs using predefined diagnostic criteria and to assess the ability of echocardiography, cardiac troponins, D-dimers and kaolin-activated thromboelastography to predict the presence of pulmonary thromboembolism in dogs. METHODS: Twelve dogs with immune-mediated haemolytic anaemia and evidence of respiratory distress were prospectively evaluated. Dogs were sedated immediately before CT pulmonary angiography using intravenous butorphanol. Spiral CT pulmonary angiography was performed with a 16 detector-row CT scanner using a pressure injector to infuse contrast media through peripheral intravenous catheters. Pulmonary thromboembolism was diagnosed using predefined criteria. Contemporaneous tests included echocardiography, arterial blood gas analysis, kaolin-activated thromboelastography, D-dimers and cardiac troponins. RESULTS: Based on predefined criteria, four dogs were classified as pulmonary thromboembolism positive, three dogs were suspected to have pulmonary thromboembolism and the remaining five dogs had negative scans. The four dogs identified with pulmonary thromboembolism all had discrete filling defects in main or lobar pulmonary arteries. None of the contemporaneous tests was discriminant for pulmonary thromboembolism diagnosis, although the small sample size was limiting. CLINICAL SIGNIFICANCE: CT pulmonary angiography can be successfully performed in dogs under sedation, even in at-risk patients with respiratory distress and can both confirm and rule out pulmonary thromboembolism in dogs.

Serial assessment of the coagulation status of dogs with immune-mediated haemolytic anaemia using thromboelastography.

This study investigated the coagulation status of dogs with immune-mediated haemolytic anaemia (IMHA) over time. Thirty animals with primary IMHA were blood sampled on three occasions over a 5 day period and assays performed included prothrombin time, activated partial thromboplastin time, D-dimer and fibrinogen concentration, antithrombin activity and recalcified unactivated thromboelastography (TEG). Based on TEG, dogs with IMHA were significantly hypercoagulable vs. controls (P<0.001) and over the 5 day period, 3/4 of the TEG parameters reflected increased clotting kinetics (P ≤ 0.02). The 30 day survival of these patients was 80% and, at hospital admission, the TEG maximum amplitude (MA) was significantly higher in survivors than non-survivors (P=0.015). Each unit increase in MA was associated with an increased odds of 30 day survival of 1.13 (95%; CI 1.02-1.25). Based on TEG, most dogs with IMHA were hypercoagulable on admission and their clotting kinetics increased with time. Relative hypocoagulability identified by TEG at initial assessment was found to be a negative prognostic indicator.

Hypercoagulability in dogs with protein-losing enteropathy.

BACKGROUND: Dogs with protein-losing enteropathy (PLE) have previously been reported to present with thromboembolism; however, the prevalence and pathogenesis of hypercoagulability in dogs with PLE have not been investigated so far. HYPOTHESIS: Dogs with PLE are hypercoagulable compared with healthy control dogs. ANIMALS: Fifteen dogs with PLE. Thirty healthy dogs served as controls (HC). METHODS: A prospective study was performed including 15 dogs with PLE. All dogs were scored using the canine chronic enteropathy activity index (CCECAI). Thromboelastography (TEG) and other measures of coagulation were evaluated. Recalcified, unactivated TEG was performed and reaction time (R), kinetic time (K), alpha angle (α), and maximum amplitude (M(A)) values were recorded. Nine dogs were reassessed after initiation of immunosuppressive treatment. RESULTS: All dogs with PLE in the study were hypercoagulable with decreased R (PLE: median 7.8, range [2.4-11.2]; HC: 14.1 [9.1-20.3]), decreased K (PLE: 2.5 [0.8-5.2]; HC: 8.25 [4.3-13.1]), increased α (PLE: 56.7 [38.5-78.3]; HC: 25.6 [17-42.4]), and increased M(A) (PLE: 68.2 [54.1-76.7]; HC: 44.1, [33.5-49]) (all P < .001). Median antithrombin (AT) concentration was borderline low in PLE dogs; however, mean serum albumin concentration was severely decreased (mean 1.67 g/dL ± 5.1, reference range 2.8-3.5 g/dL). Despite a significant improvement in serum albumin and CCECAI, all 9 dogs with PLE were hypercoagulable at re-examination. CONCLUSIONS AND CLINICAL IMPORTANCE: The hypercoagulable state in dogs with PLE cannot be solely attributed to loss of AT. Despite good clinical response to treatment, dogs remained hypercoagulable and could therefore be predisposed to thromboembolic complications.

Use of serum concentrations of interleukin-18 and monocyte chemoattractant protein-1 as prognostic indicators in primary immune-mediated hemolytic anemia in dogs.

BACKGROUND: The cytokine response in immune-mediated hemolytic anemia (IMHA) is poorly characterized and correlation with outcome is unknown. HYPOTHESIS/OBJECTIVES: To determine if cytokine activity is correlated with outcome in dogs with IMHA. ANIMALS: Twenty dogs with primary IMHA and 6 control dogs. METHODS: Prospective study on dogs with IMHA with blood sampling at admission. Serum activity of interleukin-2 (IL-2), IL-4, IL-6, IL-7, IL-8, IL-10, IL-15, IL-18, monocyte chemoattractant protein-1 (MCP-1), granulocyte-macrophage colony stimulating factor (GM-CSF), interferon-inducible protein-10, interferon-gamma, and keratinocyte chemoattractant (KC) was assessed. RESULTS: Thirty-day case fatality rate was 25% (5/20 dogs). Increased concentrations (median [range]) of IL-2 (45.5 ng/L [0;830] versus 0 ng/L [0;46.8]), IL-10 (8.2 ng/L [0;60.6] versus 0 ng/L [0;88.2]), KC (1.7 µg/L [0.3;4.7] versus 0.5 µg/L [0.2;1.1]), and MCP-1 (162 ng/L [97.6;438] versus 124 ng/L [90.2;168]) were observed in dogs with IMHA compared with controls. The cytokine profile was indicative of a mixture of pro- and anti-inflammatory cytokines of various cellular origins. Cytokines/chemokines strongly associated with macrophage/monocyte activation and recruitment were significantly increased in nonsurvivors compared with survivors; IL-15 (179 ng/L [48.0;570] versus 21.3 ng/L [0;193]), IL-18 (199 ng/L [58.7;915] versus 37.4 ng/L [0;128]), GM-CSF (134 ng/L [70.0;863] versus 57.6 ng/L [0;164]), and MCP-1 (219 ng/L [135;438] versus 159 ng/L [97.6;274]), respectively. Logistic regression suggested increased IL-18 and MCP-1 concentrations were independently associated with mortality in this population (P<.05, Wald's type 3). CONCLUSIONS AND CLINICAL IMPORTANCE: A mixed cytokine response is present in dogs with IMHA and mediators of macrophage activation and recruitment might serve as prognostic indicators.

Cavitary pulmonary lesion associated with Aspergillus fumigatus infection in a German shepherd dog.

A two-year-old female German shepherd dog was presented with chronic cough and haemoptysis. Thoracic radiographs revealed a thin-walled cavitary lesion within a consolidated left cranial lung lobe. Bronchoalveolar lavage confirmed a concurrent bacterial infection; however, despite antibiotic and anthelmintic therapy the clinical signs failed to resolve. A left cranial lung lobectomy was performed. Histopathology and fungal culture confirmed the presence of Aspergillus fumigatus. The necrotic cavity had features compatible with a bronchial origin, possibly a form of cystic bronchiectasis, arising either as a congenital anomaly or acquired secondary to infection. Surgery provided resolution of clinical signs for just over a year before the dog deteriorated again and was subsequently euthanised. Necropsy was declined by the owners. This case report presents a unique presentation in which the predominant clinical sign was coughing due to pulmonary involvement. Aspergillus fumigatus was isolated from the left cranial lung lobe.

Concurrent immune-mediated haemolytic anaemia and severe thrombocytopenia in 21 dogs.

The medical records of 21 dogs with concurrent immune-mediated haemolytic anaemia (imha) and severe thrombocytopenia (defined as an automated platelet count of less than 50x10(9)/l, confirmed by the examination of a blood smear) were reviewed. Their mean (sd) age was 5.8 (2.5) years. When compared with the 24,759 dogs in the hospital population for the same period Airedale terriers and dobermanns appeared to be over-represented with odds ratios of 22.5 (95 per cent confidence interval [ci] 5.2 to 97.9) and 7.6 (95 per cent ci 1.8 to 32.7) respectively. The median duration of the dogs' clinical signs was seven days, with a range from one to 17 days. Eleven of the dogs had a history of a tendency to bleed, and 15 had evidence of bleeding when examined. Twenty of the 21 dogs had been treated with glucocorticoids, nine with vincristine, and seven with azathioprine. Their median stay in hospital was four days, with a range from one to 17 days. The median period for which they survived after admission to hospital was five days, with a range from one to 558 days, and 16 of the 21 dogs had died or been euthanased within 30 days of their admission.