von Willebrand's disease in Dobermann dogs in Australia

SUMMARY Over a 5-year period (1988–92), von Willebrand factor antigen (vWf:Ag) concentrations were determined on plasma samples from 614 Dobermanns. The vWf:Ag concentration was < 50 canine units (CU)/dL in 373 dogs (61%); these dogs were classified as carriers of the von Willebrand's disease (vWD) gene. In order to identify which dogs were at risk of haemorrhage due to vWD, we determined a cut-off vWf:Ag concentration below which dogs could be considered at risk. This cut-off was chosen in order to minimise the number of dogs genuinely at risk of haemorrhage, being wrongly classified as not at risk. This was done without sacrificing the specificity of the cut-off to any great extent. A vWf:Ag concentration of < 36 CU/dL was empirically chosen as the optimum cut-off concentration. In 282 dogs (76% of the carriers), the vWf:Ag concentration was below this cut-off and these dogs were, thus, classified as being at risk of haemorrhage due to vWD. Haemorrhage attributable to vWD was seen in 107 dogs (29% of the carriers, or 17% of all the dogs). Haemorrhage mostly followed trauma or surgery, but spontaneous genitourinary and gastrointestinal haemorrhages were also frequent. Of these dogs, 92 were of known age, with a median of 3 years, and 102 were of known sex, with 61% being female. In 89 dogs in which the severity of haemorrhage was subjectively assessed, mild and moderate bleeding occurred with similar frequency (48% and 43%, respectively). There were 8 cases of severe haemorrhage, with two deaths. The likelihood of haemorrhage was related to the vWf:Ag concentration: only 8% of 91 dogs with concentrations between 36 and 49 CU/dL had haemorrhage attributable to vWD, but 36% with concentrations < 36 CU/dL did so. Furthermore, dogs with haemorrhage attributable to vWD had significantly (P < 0.001) lower vWf:Ag concentrations (median 12 CU/dL, n = 107) than dogs with no such history (median 30 CU/dL, n = 132). The data indicate that vWD is a significant problem in the Dobermann breed in Australia and we accordingly recommend that steps be taken to reduce its prevalence, such as the establishment of a national testing scheme to determine the vWD status of all dogs used for breeding.     

Type III von Willebrand's disease in Dutch kooiker dogs

Summary

Type III von Willebrand's disease (vWD) was diagnosed in 38 Dutch kooiker dogs. Ten male and 9 female probands had been referred independently of each other to the Utrecht University Clinic for Companion Animals because of a moderate to severe bleeding tendency. Screening of 717 Dutch kooiker dogs, including 356 puppies, detected vWD in another 19 dogs. Diagnosis was based on non‐detectable amounts (< 1.6%) of von Willebrand factor antigen (vWF:Ag) in plasma by ELISA. Capillary bleeding time (CBT) was prolonged (> 10 min) and polybrene cofactor activity (vWF:PbCo) was not detectable in 11 dogs tested. No distinguishable protein bands were detected by multimer analysis. As in Scottish terriers with type III vWD, factor VIII clotting activity (F_VIII:C) in affected Dutch kooiker dogs was decreased but considerably less than in humans with type III vWD. A recessive mode of inheritance was indicated by the normal or subnormal but measurable amounts of vWF:Ag in the plasma of eight pairs of parents of affected dogs. The F₁ offspring resulting from the experimental mating of two affected dogs consisted of three affected males and four affected females. In 39 obligatory carriers vWF:Ag ranged from 30% to 114% with median and mean vWF values of 64% and 64.2%, respectively, and was subnormal (< 50%) in only 9 animals.     

Plasma von Willebrand factor-collagen binding activity in normal dogs and in dogs with von Willebrand's disease.

A sensitive enzyme-linked immunosorbent assay was used for the simultaneous assessment of the amount of von Willebrand factor (vWF) in canine plasma and its ability to bind to canine collagen in vitro. In 60 normal dogs, there was close correlation between the concentration of vWF and its activity as determined by vWF-collagen binding. In 14 dogs with type I expressions of von Willebrand's disease, the ratio of vWF antigen to collagen binding activity was normal or only slightly increased. In 7 dogs with type II expressions of the disease, this ratio was consistently elevated suggesting a significant functional deficiency of the protein. Plasma from 3 dogs with type III von Willebrand's disease had little collagen binding activity because of the severe quantitative deficiency of the protein. The described assay permits the rapid assessment of both the quantity and quality of vWF in a dog. This information is necessary for the detection and characterization of canine von Willebrand's disease, particularly the type II expressions, which cannot be diagnosed by quantitative vWF assays alone.     

Canine von Willebrand's disease. A heterogeneous group of bleeding disorders

The term "von Willebrand's disease," refers to a group of inherited bleeding disorders, all of which are caused by a deficiency of the multimeric plasma glycoprotein, von Willebrand factor. The various forms of canine von Willebrand's disease can be categorized into one of three major types: in type I canine von Willebrand's disease, all sizes of von Willebrand factor multimers can be detected in the plasma; in type II canine von Willebrand's disease, only the smaller von Willebrand factor multimers are found in the plasma (larger multimers are absent); and in type III canine von Willebrand's disease, von Willebrand factor is completely absent from the plasma or present in only trace amounts. Von Willebrand's disease is common in dogs, but some forms of the disease are so mild that they are of questionable clinical significance.     

Factor XII deficiency and von Willebrand's disease in a family of miniature poodle dogs

The simultaneous occurrence of factor XII deficiency and von Willebrand's disease (VWD) is described in a family of Miniature Poodles affected concurrently with a familial non-spherocytic hemolytic anemia. Although there was a dominant distribution of factor XII deficiency in this family of dogs, only the dogs suffering from non-spherocytic hemolytic anemia had concurrent VWD gene expression. Neither the factor XII deficient dogs nor the VWD carrier dogs displayed bleeding tendencies.     

Desmopressin enhances the binding of plasma von Willebrand factor to collagen in plasmas from normal dogs and dogs with type I von Willebrand's disease.

A new in vitro von Willebrand factor-collagen binding activity (vWF:CBA) assay was used to assess qualitative changes in vWF in normal dogs and dogs with Type I von Willebrand's disease (vWD) following treatment with desmopressin acetate (DDAVP). Although DDAVP induced increases in vWF antigen concentrations at 1 hour postinfusion in both normal and vWD dogs (57% and 60% increases, respectively), there were disproportionately greater increases in vWF:CBA (96% and 103% increases). These results support the hypothesis that the enhanced hemostatic activity induced by DDAVP is, at least in part, due to the selective release of more functionally active vWF multimers. The assay, as described, provides a convenient means of simultaneously assessing vWF quantity and function before and after DDAVP administration.     

A von Willebrand's factor genomic nucleotide variant and polymerase chain reaction diagnostic test associated with inheritable type-2 von Willebrand's disease in a line of german shorthaired pointer dogs

Heritable, type-2 von Willebrand's disease (vWD) was studied in a line of German Shorthaired Pointers (GSPs) in which some members had a nucleotide variant in exon 28 of the von Willebrand factor (VWF) gene. A polymerase chain reaction (PCR) diagnostic test for the nucleotide variant was developed to establish the disorder's mode of inheritance and to eliminate it from the line. Thirty-six of the 49 GSPs in the line, 14 unrelated GSP controls, and 71 unrelated dogs of various breeds were tested for the presence of the variant nucleotide. All the dogs with a vWF antigen deficiency (<70% of normal) were either homozygous or heterozygous for the nucleotide variant. The variant was not located in any tested dog in the line or outside of the line with a vWF antigen value greater than 68%. Of the GSPs in the line tested, two were homozygous for the variant, 15 were heterozygous, and 19 were variant free. The collective evidence of this and other studies is consistent with the variant nucleotide being the cause of the type-2 vWD in this line of GSPs and German Wirehaired Pointers. The PCR diagnostic test for the variant nucleotide was successfully used to select and produce progeny that were variant free and vWD free. This test should be effective in the subsequent elimination of this same variant from other lines of dogs.     

Influence of platelet count,acetylsalicylic acid,von Willebrand's disease,coagulopathies, and haematocrit on results obtained using a platelet function analyser in dogs

The platelet function analyser PFA-100 aspirates blood in vitro from a sample reservoir in disposable test cartridges through a microscopic aperture cut into a biologically active membrane at the end of a capillary. In different cartridges the membrane is coated with collagen and adenosine diphosphate (ADP) or collagen and epinephrine (adrenaline) inducing a platelet plug and closure of the aperture. The closure time and total volume of blood flow through the capillary until closure of its aperture were measured. The correlation between platelet count in samples of thrombocytopenic dogs and results of the collagen/ADP cartridge (closure time: r(S)=-0.579; total volume: r(S)=-0.549) was closer than between platelet count and capillary bleeding time. No significant correlation was observed between platelet count and the results obtained with the collagen/epinephrine cartridge. In addition, a higher sensitivity was obtained for the collagen/ADP cartridge. Injection of acetylsalicylic acid into healthy dogs significantly increased closure time and total volume of both types of cartridges (P<0.01). Two dogs with von Willebrand's disease had abnormal values. In contrast, coagulopathies did not significantly influence the results of the platelet function analyser (P>0.05). Despite adequate sensitivity of measurements using the collagen/ADP cartridge to assess quantitative and qualitative platelet disorders in dogs, the influence of haematocrit (P<0.0001) will limit the clinical application of the analyser.     

Inheritance of von Willebrand's disease in a colony of Doberman Pinschers

OBJECTIVE: To determine the mode of inheritance of von Willebrand's disease (vWD) and perform linkage analysis between vWD and coat color or narcolepsy in a colony of Doberman Pinschers. ANIMALS: 159 Doberman Pinschers. PROCEDURE: von Willebrand factor antigen (vWF:Ag) concentration was measured by use of ELISA, and results were used to classify dogs as having low (< 20%), intermediate (20 to 65%), or high (> 65%) vWF:Ag concentration, compared with results of analysis of standard pooled plasma. Buccal bleeding time was measured, and mode of inheritance of vWD was assessed by pedigree analysis. RESULTS: von Willebrand's disease was transmitted as a single autosomal gene defect. Results suggested that 27.04% of dogs were homozygous for vWD, 62.26% were heterozygous, and 10.69% did not have the defect. Most homozygous and some heterozygous dogs had prolonged bleeding times. Dogs with diluted coat colors (blue and fawn) were significantly overrepresented in the homozygous group, compared with black and red dogs, but a significant link between vWD and coat color was not detected. CONCLUSIONS AND CLINICAL RELEVANCE: von Willebrand's disease is transmitted as an autosomal dominant trait with variable penetrance; most dogs in this colony (89.3%) were carriers of vWD. Homozygosity for vWD is not likely to be lethal. Some heterozygous dogs have prolonged bleeding times. An association between diluted coat colors and vWD may exist.     

Comparison of P-wave dispersion in healthy dogs,dogs with chronic valvular disease and dogs with disturbances of supraventricular conduction

Background

P-wave dispersion (P_d) is a new ECG index used in human cardiology and veterinary medicine. It is defined as the difference between the maximum and the minimum P-wave duration recorded from multiple different ECG leads. So far no studies were performed assessing the importance of P-wave dispersion in dogs.

Methods

The current study was aimed at determining proper value of P_d in healthy dogs (group I), dogs with chronic valvular disease (group II) and dogs with disturbances of supraventricular conduction (group III). The tests were carried out in 53 healthy dogs, 23 dogs with chronic valvular disease and 12 dogs with disturbances of supraventricular conduction of various breeds, sexes and body weight from 1,5 to 80 kg, aged between 0,5 and 17 years, submitted to the ECG examination. ECG was acquired in dogs in a standing position with BTL SD-8 electrocardiographic device and analyzed once the recording was enlarged. P-wave duration was calculated in 9 ECG leads (I, II, III, aVR, aVL, aVF, V1, V2, V4) from 5 cardiac cycles.

Results

The proper P-wave dispersion in healthy dogs was determined at up to 24 ms. P-wave dispersion was statistically significant increased (p < 0.01) in dogs with chronic valvular disease and dogs with disturbances of supraventricular conduction. In dogs with the atrial enlargement the P-wave dispersion is also higher than in healthy dogs, although no significant correlation between the size of left atria and Pd was noticed (p = 0.1, r = 0,17).

Conclusions

The P-wave dispersion is a constant index in healthy dogs, that is why it can be used for evaluating P wave change in dogs with chronic valvular disease and in dogs with disturbances of supraventricular conduction.     

Quantitation of reticulated platelets in healthy dogs and in nonthrombocytopenic dogs with clinical disease

Background — Thrombocytopenia is a common disorder in dogs and development of an objective diagnostic assay to measure platelets newly released from bone marrow into the blood would provide a noninvasive way to predict megakaryocytopoiesis. Reticulated platelets are newly released platelets with increased concentrations of RNA that can be detected by flow cytometric analysis of blood stained with thiazole orange (TO).
Objectives — The goals of this study were to establish a reproducible method to quantitate reticulated platelets in dogs, to establish a reference interval for reticulated platelet percentages in healthy dogs, and to determine whether the percentage of reticulated platelets was nonspecifically increased in nonthrombocytopenic dogs with clinical disease.
Methods — Blood samples were obtained from healthy dogs and from nonthrombocytopenic dogs presented for a variety of disorders. An aliquot of whole blood was stained with TO and a phycoerythrin-labeled monoclonal antibody to platelet CD61, then analyzed by flow cytometry.
Results — The coefficients of variation were 7.8% to 15.6% (intra-assay precision) and 6.1% to 19.5% (interassay precision). Overnight storage for 18 to 26 hours, under variable conditions, resulted in an increase in the percentage of platelets staining with TO. The reference interval for reticulated platelets in the healthy control group was 0–4.3% (0–12,095/μL). No significant differences were found in the mean percentage of reticulated platelets or absolute concentration of reticulated platelets between control and affected dogs.
Conclusions — These studies demonstrate a reliable, noninvasive diagnostic assay for measurement of reticulated platelets in whole blood and provide a baseline for assessment of the clinical utility of the assay.     

Indocyanine green disposition in healthy dogs and dogs with mild, moderate, or severe dimethylnitrosamine-induced hepatic disease.

Disposition kinetics of indocyanine green (ICG) were used to evaluate hepatic function in healthy Beagles (group 1; n = 6) and Beagles with progressive hepatic disease induced by oral administration of dimethylnitrosamine, a hepatospecific toxin. Three classes of hepatic disease were defined by histologic features: mild (group 2; n = 5), moderate (group 3; n = 6), and severe (group 4; n = 5). Disposition of ICG was studied 3 weeks following the last dose of toxin. A rapid IV injection of 0.5 mg of ICG/kg was administered and serum samples were obtained at certain intervals during 60-minute periods. Serum ICG was analyzed by use of visible spectrophotometry. Disposition kinetics were determined from serum ICG concentrations vs 15- and 60-minute time curves and compared between one another and among groups. Data based on 60-minute time curves were not significantly different from those based on 15-minute curves. Area under the curve for ICG was greatest in group 3. Clearance of ICG was decreased and mean resident time was increased in groups 3 and 4, compared with those in groups 1 and 2. When disposition data (60 minutes) were normalized for differences in hepatic weight among dogs, group-3 mean resident time was significantly greater than that of group 4. This study supports the diagnostic benefits of using ICG disposition kinetics as a method of evaluating hepatic function in dogs with progressive liver disease.     

Plasma and platelet serotonin concentrations in healthy dogs and dogs with myxomatous mitral valve disease

ObjectivesSerotonin has been implicated in canine myxomatous mitral valve disease (MMVD); however, the sources of serotonin have not been fully elucidated. This study compared the concentration of serotonin in plasma and platelets of normal healthy small breed dogs with predisposition to MMVD and dogs with naturally occurring MMVD.Animals43 small-breed client-owned dogs with an approximate weight of <10 kg and age of 6 years or above were divided into 2 groups: a healthy control group (n = 20) and a group with echocardiographic evidence of MMVD (n = 23).Methods5 ml samples of blood were collected. Plasma and platelets were separated by centrifugation and assayed for serotonin measured by enzyme linked immunosorbent assay (ELISA).ResultsMedian plasma serotonin concentration was not significantly different (p = 0.3630) between normal healthy dogs (3.7 ng/ml) and dogs with MMVD (4.3 ng/ml). Males had higher plasma serotonin concentration than females (4.7 and 2.9 ng/ml respectively, p = 0.0043). Platelet serotonin concentration was not different between healthy dogs and dogs with MMVD (128.6 ng/10⁹ platelets and 176.6 ng/10⁹ platelets respectively, p = 0.4575). Age, echocardiographic indices and platelet count showed no correlation with plasma or platelet serotonin concentration.ConclusionsCirculating plasma serotonin is unlikely a major source of serotonin signaling in canine MMVD. Platelets could be a source of serotonin in canine MMVD through platelet adhesion to the mitral valve; however, the amount of serotonin stored in platelets of healthy dogs and dogs with MMVD is not different.