Interleukin-10 inhibits lipopolysaccharide-induced upregulation of tissue factor in canine peripheral blood monocytes

The potentially fatal hemostatic disorder of disseminated intravascular coagulation (DIC) is initiated in bacterial sepsis by lipopolysaccharide (LPS)-induced tissue factor (TF) expression on monocytes. Interleukin-10 (IL-10) is a potent inhibitory cytokine that downregulates monocyte inflammatory and procoagulant responses. We hypothesized that canine recombinant IL-10 (rIL-10) would inhibit LPS-induced TF upregulation on canine monocytes in a dose-dependent manner. Canine peripheral blood mononuclear cells (PBMC), obtained by double-density gradient centrifugation, and monocytes, purified from PBMC by immunomagnetic bead separation with an anti-canine CD14 antibody (Ab), were stimulated in suspension with LPS (0.1-1000ng/mL) for various times. Recombinant IL-10 (10-5000pg/mL) was added with LPS or up to 2h later. Tissue factor procoagulant activity was measured by cleavage of a chromogenic substrate by activated Factor X generated by the TF-factor VII complex. We found that rIL-10, when given concurrently or 1h after LPS, strongly inhibited LPS-induced TF procoagulant activity in canine PBMC and monocytes. This inhibition was dose-dependent and blocked by an anti-canine IL-10 Ab. Our results indicate that rIL-10 effectively inhibits LPS-induced TF upregulation in canine monocytes and could potentially be useful in limiting the development of DIC in dogs with endotoxemia.     

Prognostic factors for mortality and thromboembolism in canine immune-mediated hemolytic anemia: a retrospective study of 72 dogs

Medical records of 72 dogs diagnosed with immune-mediated hemolytic anemia (IMHA) were reviewed to find risk factors for the disease, for mortality, and for thromboembolism. Coagulation data of 32 patients were evaluated for mortality or thromboembolism risk factors. Cocker Spaniels were at increased risk for IMHA (P = .012). Timing of vaccination was not associated with development of IMHA. PCV ranged from 5 to 33%, with a mean of 16 +/- 5%. Autoagglutination was present in 42% of the dogs. Platelet counts (n = 60) varied from 3,000 to 793,000/microL (mean, 160,117 +/- 133,571; median, 144,000). Thrombocytopenia (platelet count, <200,000/microL) was present in 70% of the dogs, with severe thrombocytopenia (platelet count, <50,000/microL) being present in 22%. One-step prothrombin time (OSPT) was prolonged in 28% of the dogs tested, and activated partial thromboplastin time (APTT) was prolonged in 47% of the dogs tested. Fibrin(ogen) degradation products (FDPs) were detected in 16 of 28 dogs tested (57%). Disseminated intravascular coagulation (DIC) was diagnosed in 10 of 31 (32%) dogs and was suspected in 8 dogs. Thromboemboli were found in 20 of 25 dogs given postmortem examinations. Mortality rate was 58%. Thrombocytopenia (P = .008) and serum bilirubin concentration of >5 mg/dL (P = .015) were risk factors for mortality, and hypoalbuminemia approached significance (P = .053). Severe thrombocytopenia (P = .046), serum bilirubin concentration of >5 mg/dL (P = .038), and hypoalbuminemia (P = .016) were risk factors for thromboembolism. On evaluation of continuous data, decreased platelet count (P = .057), increased bilirubin (P = .062), and decreased albumin (P = .054) approached significance for decreased survival. A higher risk for thrombosis was found with increased alkaline phosphatase (ALKP) (P = .042), increased bilirubin (P = .047), and decreased albumin (P = .012).     

Case-control study of blood type, breed, sex, and bacteremia in dogs with immune-mediated hemolytic anemia

OBJECTIVE: To determine whether blood type, breed, or sex were risk factors for immune-mediated hemolytic anemia (IMHA) in dogs and whether bacteremia was common in dogs with IMHA. DESIGN: Case-control study. ANIMALS: 33 dogs with IMHA, 1,014 dogs without IMHA for which blood type (dog erythrocyte antigens 1.1, 1.2, 3, 4, 5, and 7) was known, 15,668 dogs without IMHA for which breed was known, and 15,589 dogs without IMHA for which sex was known. PROCEDURE: Blood type, breed, and sex distribution of dogs with IMHA were compared with data for control dogs with Fisher exact tests and by calculating odds ratios (ORs). Results of bacterial culture of blood samples were documented for dogs with IMHA, when available. RESULTS: Dog erythrocyte antigen 7 was associated with a significant protective effect (OR, 0.1) in Cocker Spaniels with IMHA (n = 10), compared with control dogs. Cocker Spaniels, Bichon Frise, Miniature Pinschers, Rough-coated Collies, and Finnish Spitz had a significantly increased risk of IMHA, as did female dogs (OR, 2.1). Blood samples from 12 dogs with IMHA were submitted for bacterial culture, and none had bacteremia. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that blood type, breed, and sex may play a role in IMHA in dogs.     

Vaccine-Associated Immune-Mediated Hemolytic Anemia in the Dog

Vaccination has been incriminated as a trigger of immune-mediated hemolytic anemia (IMHA) in dogs and in people, but evidence to support this association is lacking. In a controlled retrospective study, idiopathic IMHA was identified in 58 dogs over a 27–month period. When compared with a randomly selected control group of 70 dogs (presented for reasons other than IMHA) over the same period, the distribution of cases versus time since vaccination was different (P < .05). Fifteen of the dogs (26%) had been vaccinated within 1 month (mean, 13 days; median, 14 days; range, 1 to 27 days) of developing IMHA (P < .0001), whereas in the control group no marked increase in frequency of presentation was seen in the first month after vaccination. The dogs with IMHA were divided into 2 groups based on time since vaccination: the vaccine IMHA group included dogs vaccinated within 1 month of developing IMHA; the nonvaccine IMHA group included dogs that developed IMHA more than 1 month after vaccination. The recently vaccinated dogs with IMHA (vaccine IMHA group) had significantly lower platelet counts (P < .05) and a trend towards increased prevalence of intravascular hemolysis and autoagglutination when compared with the nonvaccine IMHA group. Similar mortality rates were seen in the vaccine IMHA group (60%) and the nonvaccine IMHA group (44%), with the majority of fatalities (>75%) occurring in the first 3 weeks after presentation. Persistent autoagglutination was a negative prognostic indicator for survival in both groups (P < .05). Presence of icterus and hyperbilirubinemia were negative prognostic indicators for survival in the nonvaccine IMHA group (P < .0001 and P < .01, respectively) but not in the vaccine IMHA group. In the recently vaccinated dogs, combination vaccines from various manufacturers against canine distemper, adenovirus type 2, leptospirosis, parainfluenza, and parvovirus (DHLPP) were involved in each case. Vaccines against rabies virus, Bordetella spp, coronavirus, and Lyme Borrelia were administered concomitantly to some dogs. This study provides the first clinical evidence for a temporal relationship of vaccine-associated IMHA in the dog.     

Evaluation of point-of-care tests for diagnosis of disseminated intravascular coagulation in dogs admitted to an intensive care unit.

OBJECTIVE: To evaluate the accuracy of point-of-care tests for the diagnosis of disseminated intravascular coagulation (DIC) in dogs and assess the correlation and agreement of results between point-of-care and laboratory tests in the evaluation of hemostatic function. DESIGN: Prospective case series. ANIMALS: 59 critically ill dogs (affected dogs) with clinical signs of diseases known to predispose to DIC and 52 clinically normal dogs. PROCEDURES: Accuracy of the point-of-care tests (activated clotting time [ACT], estimated platelet count and number of schizocytes from a blood smear, plasma total solids [TS] concentration, and the protamine sulfate test) was evaluated, using receiver operating characteristic curves and likelihood ratios. A strategy, using likelihood ratios to calculate a posttest probability of DIC, was tested with 65% used as a threshold for initiation of treatment. Results of laboratory tests (coagulogram and plasma antithrombin III activity) were used as the standard for comparison in each dog. RESULTS: ACT and estimated platelet count provided the best accuracy for detection of DIC. The plasma TS concentration, schizocyte number, and protamine sulfate test had poor accuracy. The strategy using post-test probability of DIC identified 12 of 16 affected dogs that had DIC. Estimated platelet count was correlated and had acceptable clinical agreement with automated platelet count (r = 0.70). The plasma TS (r = 0.28) concentration and serum albumin (r = 0.63) concentration were not accurate predictors of plasma antithrombin III activity. The ACT did not correlate with activated partial thromboplastin time (r = 0.28). CONCLUSIONS AND CLINICAL RELEVANCE: Strategic use of likelihood ratios from point-of-care tests can assist clinicians in making treatment decisions for dogs suspected to have DIC when immediate laboratory support is unavailable.     

Anti-Endothelial Cell Antibodies in Dogs with Immune-Mediated Hemolytic Anemia and Other Diseases Associated with High Risk of Thromboembolism

Background: Dogs with immune-mediated hemolytic anemia (IMHA) and certain inflammatory diseases are at high risk of developing thromboembolic disease. The presence of anti-endothelial cell autoantibodies (AECA) has been associated with an increased risk of thromboembolism in humans.
Hypothesis: AECA will be detected more often in dogs at risk of thromboembolism than in healthy control animals or dogs with diseases not associated with a higher risk of thromboembolism.
Animals: Ninety-one sick dogs and 22 healthy control dogs.
Methods: Retrospective case-controlled study. Serum was screened for the presence of AECA. Dogs were identified for the study based on the risk of thromboembolism as determined by clinical impression and the underlying disease process. Flow cytometry and normal canine endothelial cells were used to screen serum samples from sick and healthy control dogs for the presence of AECA. In addition, serum from dogs with confirmed thromboemboli was also screened for the presence of AECA by immunohistochemistry.
Results: AECA were detected in 2/91 sick dogs, both with infectious diseases, but were not found in healthy dogs. Anti-endothelial antibodies were not detected in 21 dogs with IMHA and 20 dogs with systemic inflammatory response syndrome, sepsis, or both.
Conclusions: We conclude that AECA are rarely detectable in dogs considered at high risk of thromboembolism. These findings suggest that AECA may not play an important role in the pathogenesis of thromboembolism in dogs with IMHA and other inflammatory diseases.     

The use of the rapid osmotic fragility test as an additional test to diagnose canine immune-mediated haemolytic anaemia

Background

Diagnosing canine immune-mediated haemolytic anaemia (IMHA) is often challenging because all currently available tests have their limitations. Dogs with IMHA often have an increased erythrocyte osmotic fragility (OF), a characteristic that is sometimes used in the diagnosis of IMHA. Since the classic osmotic fragility test (COFT) is time-consuming and requires specialized equipment, an easy and less labour-intensive rapid osmotic fragility test (ROFT) has been used in some countries, but its diagnostic value has not yet been investigated.This study aimed to evaluate erythrocyte osmotic fragility in dogs with and without IMHA, to compare results of the classic (COFT) and rapid (ROFT) test and to assess the value of the ROFT as diagnostic test for canine IMHA.Nineteen dogs with IMHA (group 1a), 21 anaemic dogs without IMHA (group 1b), 8 dogs with microcytosis (group 2), 13 hyperlipemic dogs (group 3), 10 dogs with lymphoma (group 4), 8 dogs with an infection (group 5) and 13 healthy dogs (group 6) were included.In all dogs, blood smear examination, in-saline auto-agglutination test, Coombs’ test, COFT and ROFT were performed. In the COFT, OF5, OF50 and OF90 were defined as the NaCl concentrations at which respectively 5, 50 and 90% of erythrocytes were haemolysed.

Results

Compared with healthy dogs, OF5 and OF50 were significantly higher in group 1a (P < 0.001) and OF5 was significantly higher in group 3 (P = 0.0266). The ROFT was positive in 17 dogs with IMHA, 10 hyperlipemic dogs, one anaemic dog without IMHA and one healthy dog.

Conclusions

Osmotic fragility was increased in the majority of dogs with IMHA and in dogs with hyperlipidemia, but not in dogs with microcytosis, lymphoma or an infection. Although more detailed information was obtained about the osmotic fragility by using the COFT, the COFT and ROFT gave similar results. The ROFT does not require specialized equipment, is rapid and easy to perform and can be used easily in daily practice. Although, the ROFT cannot replace other diagnostic tests, it may be a valuable additional tool to diagnose canine IMHA.     

C‐reactive protein concentration in dogs with primary immune‐mediated hemolytic anemia

Background: Canine primary immune-mediated hemolytic anemia (IMHA) is associated with a high-mortality rate. C-reactive protein (CRP) is the most important acute-phase protein in dogs and may have value as a marker of prognosis or response to treatment in IMHA. Objective: The objectives of this study were to evaluate serum CRP concentration in dogs with primary IMHA at presentation and during treatment, to assess potential differences based on survival time, and to compare CRP with other laboratory parameters of inflammation and prognosis. Methods: Inclusion criteria for primary IMHA were anemia (PCV<0.30 L/L), a positive Coombs' test or persistent autoagglutination of erythrocytes, and the exclusion of underlying diseases by other diagnostic tests. Dogs were divided into 2 groups based on survival: dogs that were still alive 14 days after start of treatment (group 1) and dogs that died or were euthanized before day 14 (group 2). Serum CRP concentration, a CBC, and a biochemistry profile were performed on days 0, 3, 8, and 14. Serum CRP also was determined in 25 clinically healthy dogs. Results: CRP concentration in the 25 clinically healthy dogs ranged from 0–8.9 μg/mL (median 2.2 μg/mL). Thirty dogs were diagnosed with primary IMHA, 24 in group 1 and 6 in group 2. On day 0, CRP concentration in dogs in both groups (median 224 μg/mL) was increased above the reference interval. In group 1 dogs, median CRP concentration was 242 μg/mL on day 0, 69 μg/mL on day 3, 35 μg/mL on day 8, and 2 μg/mL on day 14. In group 2 dogs, median CRP concentration was 194 μg/mL on day 0, 119 μg/mL on day 3, and 41 μg/mL on day 8; only 1 dog in group 2 survived to day 8. There was a significant correlation between CRP and total WBC concentrations on days 0 and 3 (r=−.598, P=.003). Conclusions: Serum CRP concentration was markedly increased in dogs with primary IMHA. CRP concentration did not differ based on patient survival, but might be a marker for long-term monitoring of these patients.     

Detection of activated platelets in dogs with primary immune-mediated hemolytic anemia

Thromboembolism is a major cause of morbidity and mortality in dogs with immune-mediated hemolytic anemia (IMHA). To the authors' knowledge, the role of platelets in thromboembolic events associated with IMHA has not been extensively investigated. In the study reported here, we evaluated cell membrane expression of P-selectin with flow cytometry to determine whether platelets circulate in an activated state in association with primary IMHA. Median P-selectin expression for 20 dogs with primary IMHA was 8.1-fold greater, compared with values for 20 healthy dogs. Fifteen of 20 dogs (75%) with IMHA had P-selectin median fluorescence intensity (MFI) values that exceeded the reference interval for healthy dogs. Additionally, P-selectin MFI after activation of platelets with phorbol myristate acetate was 2.1-fold greater for dogs with IMHA than for healthy control dogs. Despite treatment of all dogs with immunosuppressive therapy and 18 dogs with subcutaneously administered low-dose unfractionated heparin, 7 dogs developed clinical signs consistent with thromboembolism. These data provide support for the hypothesis that platelets circulate in an activated state in many dogs with IMHA.     

Use of human intravenous immunoglobulin in dogs with primary immune mediated hemolytic anemia

Immune mediated hemolytic anemia (IMHA) in dogs is a severe disease with a high mortality rate. As human immunoglobulin (HIG) was reported to be beneficial for the treatment of IMHA in dogs we examined the influence of HIG on the course of the disease in our dogs with IMHA. Of 22 dogs with primary IMHA 9 dogs received in addition to routine immunosuppressive therapy HIG at a dose of 0.19 to 0.68 g/kg (median 0.35 g/kg), 13 dogs did not receive HIG (-HIG group). Both groups were similar in terms of age, weight, the presence of autoagglutination, spherocytosis, positive Coombs' test, icterus and pigmenturia. The lowest hematocrit measured during the disease was significantly lower in the +HIG group compared to the -HIG group and dogs in the +HIG group received significantly more transfusions than those of the -HIG group. This is an indication for more severe disease signs of the +HIG group dogs. Although mortality during hospitalization and the time from hospital admission to release or death was not significantly different between the two groups, we interpret this similar course of the IMHA despite more severe signs of the +HIG group dogs as a potential positive effect of the HIG therapy.     

Cyclophosphamide exerts no beneficial effect over prednisone alone in the initial treatment of acute immune-mediated hemolytic anemia in dogs: a randomized controlled clinical trial

Cyclophosphamide is commonly used with prednisone in the initial treatment of severe idiopathic immune-mediated hemolytic anemia (IMHA) in dogs because retrospective reports suggest its benefit. This randomized controlled prospective clinicaltrial evaluated whether combined cyclophosphamide and prednisone therapy is more efficacious than prednisone therapy alone in the initial treatment of IMHA. Eighteen dogs with acute, severe idiopathic IMHA were randomly assigned to 1 of 2 treatment groups. The P group received prednisone therapy alone (1-2 mg/kg PO q12h), and the PC group received prednisone (1-2 mg/kg PO q12h) and cyclophosphamide (50 mg/m2 PO q24h for 4 consecutive days a week) for 4 weeks. The mortality rate in the P group was 20% (2 of 10), and in the PC group, the mortality rate was 38% (3 of 8). There was no difference in sequential CBC evaluations between the 2 groups. However, whereas dogs in the P group showed increases in reticulocyte count, reticulocytosis was suppressed in dogs in the PC group during the 1st week of therapy. Spherocytosis resolved more quickly in the P group (day 21) than in the PC group (day 28), but the time taken to achieve a negative Coombs' test result was comparable between groups. No difference was observed in the volume of packed red blood cells (pRBCs) given per transfusion between treatment groups, but more dogs in the PC group required a 2nd transfusion. The results of this limited study suggest that cyclophosphamide plus prednisone has no benefit over prednisone alone in the initial treatment of acute, severe idiopathic IMHA indogs.     

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.     

Haemoplasma infection is not a common cause of canine immune-mediated haemolytic anaemia in the UK

Objective : The aim of this study was to investigate whether the two canine haemoplasma species, Mycoplasma haemocanis and “Candidatus Mycoplasma haematoparvum,” are commonly associated with immune-mediated haemolytic anaemia (IMHA) in UK dogs. Methods : Three groups of dogs were recruited to the study: anaemic dogs with primary IMHA (n=37); anaemic dogs not meeting the inclusion criteria for primary IMHA (n=77) and non-anaemic dogs (n=113). DNA was extracted from 100 μl of blood and subjected to real-time quantitative polymerase chain reaction (qPCR) assays for both species of Mycoplasma. Each assay incorporated co-amplification of canine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an endogenous internal control. Results : Canine GAPDH was successfully amplified by qPCR from all 227 canine blood samples but none contained M. haemocanis or “Candidatus M. haematoparvum” DNA. Clinical Significance : Haemoplasma infection is uncommon in dogs in the UK and no evidence was found that these organisms act as triggers for IMHA.     

Serum and plasma latex agglutination tests for detection of fibrin(ogen) degradation products in clinically ill dogs

Abstract: An increased concentration of fibrin(ogen) degradation products (FDPs) commonly is used in conjunction with other hemostatic test abnormalities to identify patients with disseminated intravascular coagulation (DIC). Positive FDP results, however, have been observed in dogs without clinical evidence of DIC. The purpose of this study was to evaluate FDP concentrations in a group of clinically ill dogs with a variety of disorders. Dogs included in the study had the following hemostatic parameters evaluated: prothrombin time, activated partial thromboplastin time, fibrinogen concentration, platelet count, and FDP concentration. Two rapid latex agglutination methods were compared for detecting FDP in serum samples (Thrombo-Wellcotest, International Murex Technologies Corp) and plasma samples (FDP Plasma, American Bioproducts Inc). Results of the serum FDP method were positive in 8% (4/50) of the dogs tested: 3 with DIC and 1 with immune-mediated hemolytic anemia and liver disease. Results of the plasma FDP test were positive in 60% (30/50) of the animals tested: 6 with DIC, 3 with confirmed thrombosis, and 21 with a variety of conditions, including neoplasia, immune-mediated hemolytic anemia, pancreatitis, gastric dilatation-volvulus, heat stroke, severe trauma, sepsis, protein-losing nephropathy, liver disease, hyperadrenocorticism, and chronic heart failure. Because the plasma FDP test was positive more frequently than the serum FDP test in ill dogs, it may be more sensitive for the detection of canine FDP.     

Idiopathic immune-mediated hemolytic anemia: treatment outcome and prognostic factors in 149 dogs

BACKGROUND: Canine idiopathic immune-mediated hemolytic anemia (IMHA) is associated with a high mortality, especially in the 1st 2 weeks after diagnosis despite treatment. OBJECTIVES: To determine treatment outcome and identify prognostic variables in order to define areas of future research. ANIMALS: One hundred forty-nine dogs with hematocrit <30% and either a positive Coombs' test or spherocytosis and with no evidence of disease that can trigger IMHA were included. METHODS: Retrospective cohort study. All dogs were treated with prednisolone and azathioprine according to a standard protocol. Survival analysis was performed by the Kaplan-Meier method. Variables recorded at the time of diagnosis were tested as possible prognostic variables in a univariate and multivariate Cox proportional hazard model. RESULTS: The main predictors for mortality in dogs with idiopathic IMHA are the presence of increased plasma urea concentration, bands, thrombocytopenia, and petechiae at the time of diagnosis. The estimated Kaplan-Meier half-year survival was 72.6% (95% confidence interval [CI]: 64.9-81.3%). Mortality occurred mostly within the 1st 2 weeks. Cox proportional hazards analysis indicated that increased plasma urea concentration, icterus, and petechiae were the major independent predictors of mortality in the 1st 2 weeks. In most dogs that survived IMHA, a 3-month protocol of azathioprine with prednisolone maintained clinical remission. The estimated half-year survival for dogs that survived the 1st 2 weeks was 92.5% (95% CI: 86-99.3%). CONCLUSIONS AND CLINICAL IMPORTANCE: If the dogs survived IMHA, a 3-month protocol of prednisolone and azathioprine was effective with regard to survival and clinical outcome. Future research should be directed at identifying whether thrombotic tendency in dogs with IMHA is the main contributor to the development of increased plasma urea concentration, icterus, thrombocytopenia, and petechiae.     

Clinical presentation of 26 anaplasma phagocytophilum-seropositive dogs residing in an endemic area

Anaplasma (A.) phagocytophilum, the etiological agent of canine granulocytic anaplasmosis, is capable of inciting moderate to severe clinical disease in a variety of mammals and is endemic in the upper midwest. The purpose of this study was fourfold: to describe the range of clinical signs in dogs seropositive to A. phagocytophilum; to examine the prevalence of immune-mediated hemolytic anemia (IMHA) in this population; to evaluate whether specific clinical signs were associated with coexposure to Borrelia (B.) burgdorferi in actively infected dogs; and to determine whether clinical response to doxycycline was complete in treated dogs. Medical records of dogs seropositive for A. phagocytophilum were reviewed retrospectively. Peripheral blood smears were also reviewed retrospectively for granulocytic Anaplasma morulae. Lethargy (81%), inappetence (58%), and lameness (50%) were the most common clinical signs, followed by fever (46%). Thrombocytopenia was the most common laboratory abnormality, and IMHA was diagnosed in three dogs. Dogs that were thrombocytopenic and had antibodies to both A. phagocytophilum and B. burgdorferi had a median platelet count of 51,000/μL (range 20,000 to 171,000/μL), which was significantly lower than the count in dogs with antibodies only to A. phagocytophilum (P=0.04). Some dogs had an apparent relapse of clinical signs after an appropriate course of doxycycline. Testing for A. phagocytophilum by polymerase chain reaction, serum antibody assays, and/or blood smear evaluation should be considered in dogs with IMHA, cough, or epistaxis and that reside in A. phagocytophilum-endemic areas. If moderate to severe thrombocytopenia is present, testing for concurrent B. burgdorferi infection may be warranted.     

The utility of screening diagnostic tests in identifying associative immune-mediated haemolytic anaemia in dogs

The study aimed to (1) define the proportion of dogs with immune-mediated haemolytic anaemia (IMHA) that have associative and non-associative disease and (2) evaluate the utility of screening diagnostic tests in identifying potential triggers of associative IMHA. Medical records of 78 dogs diagnosed with IMHA at a specialist hospital in Sydney from July 2008 to August 2017 were reviewed. The original diagnosis was revised according to published guidelines (Garden et al., 2019) as either diagnostic, supportive or suspicious for IMHA. Associative IMHA was confirmed if immunosuppressive therapy was discontinued within six weeks of effective treatment of a potential trigger. Associative IMHA was considered possible when a potential trigger was identified but its significance could not be confirmed. Associative IMHA was confirmed (3) or suspected (7) in 10 dogs (13%, confidence interval [CI] 7.1%–22%), with 68 cases presumed to be non-associative. Associative IMHA was present in 3/29 (10.3%) of dogs with criteria diagnostic for IMHA, 4/42 (9.5%) of dogs with criteria supportive for IMHA and 3/7 (42.9%) of dogs with criteria suspicious for IMHA. Abdominal ultrasound was performed in 68 dogs and identified possible triggers in five (7.3%, CI 3.2% to 16%). Thoracic radiographs were performed in 70 dogs but did not identify any potential triggers (0%, CI 0% to 5.2%). Urine culture was performed in 22 dogs and was positive in three (14%, CI 4.7% to 33.3%). Routine screening tests, particularly thoracic radiographs, have a low yield in identifying potential triggers of associative IMHA, but are more likely to be useful in dogs fulfilling less stringent diagnostic criteria of IMHA.     

Treatment of Immune‐Mediated Hemolytic Anemia with Individually Adjusted Heparin Dosing in Dogs

Background: A major cause of death in dogs with immune‐mediated hemolytic anemia (IMHA) is thromboembolism. Previous studies suggest unfractionated heparin (UH) is not effective in preventing thromboembolism in IMHA; however, subtherapeutic dosing could explain the seeming lack of efficacy. Hypothesis: Providing therapeutic plasma concentration of UH by individually adjusting doses based on antifactor Xa activity would improve survival in IMHA. Animals: Fifteen dogs with primary IMHA. Methods: Randomized, prospective, controlled clinical trial. Dogs received standardized therapy for IMHA and either constant dose (CD) (150 U/kg SC) (n = 7) or individually adjusted dose (IAD) (n = 8) UH, monitored via an anti‐Xa chromogenic assay, adjusted according to a nomogram. UH was administered every 6 hours until day 7, and every 8 hours thereafter. UH dose was adjusted daily in IAD dogs until day 7, weekly until day 28, then tapered over 1 week. Dogs were monitored for 180 days. Results: At day 180, 7 dogs in the IAD group and 1 in the CD group were alive (P= .01). Median survival time for the IAD group was >180 days, and 68 days for the CD group. Thromboembolic events occurred in 5 dogs in the CD group and 2 dogs in the IAD group. Doses of UH between 150 and 566 U/kg achieved therapeutic anti‐Xa activity (0.35–0.7 U/mL). Conclusions and Clinical Importance: This study suggests that IAD UH therapy using anti‐Xa monitoring reduced case fatality rate in dogs with IMHA when compared with dogs receiving fixed low dose UH therapy.     

Perinuclear antineutrophil cytoplasmic autoantibodies in dogs infected with various vector-borne pathogens and in dogs with immune-mediated hemolytic anemia

Objective-To determine the prevalence of perinuclear antineutrophil cytoplasmic autoantibodies (pANCA) in dogs with confirmed or suspected immune-mediated hemolytic anemia (IMHA) or dogs infected with various vector-borne pathogens, including Rickettsia rickettsii, Bartonella henselae, Bartonella vinsonii subsp berkhoffii, Ehrlichia canis, Borrelia burgdorferi, and Leishmania infantum. Animals-55 dogs with confirmed or suspected IMHA, 140 dogs seroreactive for vector-borne pathogens, and 62 healthy dogs and dogs seronegative for vector-borne pathogens. Procedures-Samples were allocated to subgroups on the basis of the health status of the dogs and the degree of seroreactivity against various vector-borne pathogens. Serum samples were tested retrospectively via indirect immunofluorescence assay to determine pANCA status. Results-26 of 55 (47%) dogs with confirmed or suspected IMHA and 67 of 140 (48%) dogs seroreactive for vector-borne pathogens had positive results when tested for pANCA. Serum samples with the highest antibody concentrations against L infantum antigen had the highest proportion (28/43 [65%]) that were positive for pANCA. One of 20 (5%) dogs seronegative for tick-borne pathogens and 8 of 22 (36%) dogs seronegative for L infantum had positive results for pANCA. One of 20 (5%) healthy dogs had serum antibodies against pANCA. Conclusions and Clinical Relevance-pANCA were detected in a high percentage of dogs with IMHA and vector-borne infectious diseases. Therefore, pANCA may be a relatively nonspecific marker for dogs with inflammatory bowel disease, although they could represent a biomarker for immune-mediated diseases and infections.