Diagnostic and hematologic features of probable essential thrombocythemia in two dogs

The clinical and hematologic features of two cases of probable essential thrombocythemia in the dog are described. Both dogs presented with hepatosplenomegaly, severe nonregenerative anemia, neutrophilia and Thrombocytosis. Mean platelet volume and percentages of large platelets were markedly increased in both dogs. Platelet aggregation studies demonstrated hyperaggregability in one dog; platelets from the other dog aggregated spontaneously, precluding further investigation. Cytologic and histologic examination of bone marrow showed pronounced megakaryocytic hyperplasia, with erythroid hypoplasia and relative myeloid hyperplasia. Megakaryocyte morphology was abnormal, with increased numbers of small mononuclear and binucleate cells. Normal to increased hemosiderin stores suggested that apparent macrocytosis in one dog, rather than being due to iron deficiency, resulted from the hematology analyzer counting large platelets as small red blood cells. Megakaryocytic infiltration of the spleen was evident in both dogs. The hematologic findings in dogs with essential thrombocythemia can mimic those associated with iron deficiency anemia, such that diagnostic investigations should be aimed at ruling out chronic blood loss and other causes of reactive Thrombocytosis.     

Essential thrombocythemia in a dog

A two-year old male Welsh Corgi was referred for persistent thrombocytosis and occasional seizure. Hematological findings indicated marked thrombocytosis, eosinophilia, basophilia and moderate anemia. Bone marrow examination revealed marked megakaryocytic hyperplasia with morphologic abnormality. A diagnosis of essential thrombocythemia was made and the treatment was initiated with combination chemotherapy and maintained by prednisolone and busulfan. The dog successfully achieved complete remission on 100 days after initial presentation and has been good in health without chemotherapy since then.     

Generalized calcinosis cutis associated with probable leptospirosis in a dog

A 6.5-year-old male German Shepherd acutely developed renal and hepatic disease. Serology revealed high concentrations of antibodies against Leptospira copenhageni, and a presumptive diagnosis of leptospirosis was made. The dog was successfully treated with antibiotics and supportive care over a 12-day period. Sixty-two days after the initial presentation, alopecia predominantly involving the dorsum and perineal areas developed. The skin lesions expanded over a 20-day period. Histology revealed generalized calcinosis cutis with follicular atrophy. An injection of 0.01 mg kg-1 dexamethasone suppressed serum cortisol concentrations. No treatment was given and lesions resolved over the following 30 days. This is the third case of generalized calcinosis cutis that has developed in an adult dog after severe systemic disease. Both previous cases developed calcinosis cutis in association with blastomycosis. To the authors' knowledge, this is the first report of generalized calcinosis cutis in an adult dog in association with a presumptive bacterial infection.     

Thiazole orange positive platelets in a dog with Evans' syndrome

We examined transition for the percentage of reticulated platelets (RP%) and platelet count in a canine case of Evans' syndrome. The result demonstrated that measurement of the RP% can be useful in evaluating platelet production in the bone marrow and response to treatment.     

External auditory canal atresia of probable congenital origin in a dog

A nine-month-old Labrador retriever was referred to the Clinica Veterinaria Privata San Marco because of frequent headshaking and downward turning of the right ear. Clinical examination revealed that there was no external acoustic meatus in the right ear. Computed tomography confirmed that the vertical part of the right auditory canal ended blindly, providing a diagnosis of external auditory canal atresia. Cytological examination and culture of fluid from the canal and the bulla revealed only aseptic cerumen; for this reason, it was assumed that the dog was probably affected by a congenital developmental deformity of the external auditory canal. Reconstructive surgery was performed using a "pull-through" technique. Four months after surgery the cosmetic and functional results were satisfactory.     

Scanning electron microscopic studies of megakaryocytes and platelet formation in the dog and rat

Megakaryocyte morphology and platelet formation in canine and murine bone marrows were studied by scanning electron microscopy. In situ-fixed bone marrow preparations and cell suspensions of bone marrow provided complementary information for the 2 species (dogs and rats). Cylindrical processes (proplatelets) of variable length and thickness, originating from the megakaryocyte surface, were in the larger marrow sinusoids and the central vein. Regional constrictions along the length of proplatelets, particularly near their apical region, and the presence of fragments of such processes supported the concept of platelet formation through segmentation of proplatelets. Megakaryocytes presented varied morphology. Surface features resembling platelets were observed on megakaryocytes, indicating that platelets may have been released through surface budding. In conclusion, megakaryocytes formed long proplatelet processes that actively migrated to venous sinusoids to release platelets by fragmentation. Scanning electron microscopy analysis revealed a complex and variable megakaryocyte surface topography. The platelet-like structures on megakaryocyte surfaces may represent platelet release by a budding mechanism. The similarity between murine platelet release and canine platelet release demonstrates that data from rodent models may be applicable to nonrodents.     

Ultrastructural morphology of platelets and fibrin networks of lactating and non-pregnant rabbits

Platelets and fibrin play an important role in the coagulation process where they are involved in the maintenance of haemostasis. Fibrin dysfunction is associated with the development of vascular complications, while proneness to the formation of tight and rigid fibrin networks is independently associated with thrombotic disease. Rabbits have long been used successfully as animal models, and are often the species of choice for models of antithrombotic efficacy. It was previously shown that rabbit and human platelet and fibrin morphology are very similar in ultrastructure and fibrin fibre thickness. It was also previously reported that the thin minor fibres forms a thick fine network cover over the major fibres during pregnancy. According to research, white blood cell counts also changes during pregnancy and stays changed for up to 6 weeks post-partum; where the number of neutrophils increased, and the number of lymphocytes, basophils and eosinophils decreased. Here, we show that the same ultrastructure and white blood cell count changes occur in lactating rabbits (4 weeks post-partum). We therefore suggest that a rabbit morphology model studying platelet and fibrin morphology can be used successfully, either to study the effect of pharmaceutical products to be used during lactation and pregnancy in humans, or used in veterinary research. Furthermore, the effects of pharmaceutical products on immunology and white blood cell counts can possibly also be used successfully.     

Measurement of thrombopoietic activity through the quantification of megakaryocytes in bone marrow cytology and reticulated platelets

Reticulated platelets are considered as marker for bone marrow thrombopoiesis. The aim of the study was evaluate the role of reticulated platelets as markers of thrombopoiesis in dogs. Reticulated platelets analysis by flow cytometry and megakaryocyte quantification by bone marrow cytology were determined in 29 healthy adult dogs (control group), 14 dogs with thrombocytopenia without megakaryocytic hypoplasia (group A) and 14 dogs with thrombocytopenia which presented megakaryocytic hypoplasia (group B), detected by bone marrow aspiration cytology. Blood samples were collected and the platelet rich plasma was obtained for reticulated platelets quantification in flow cytometry. Megakaryocytes were quantified in aspiration cytology by two techniques in marrow particles, and correlated to reticulated platelets counts. There are no differences between megakaryocyte quantification. Although there is no correlation between reticulated platelet values and megakaryocyte in bone marrow cytology, the interpretation of reticulated platelet values can be based both on absolute or relative corrected values.     

Flow cytometric detection of activated platelets in the dog

Background: Platelet activation appears to play a role in a variety of canine thrombotic disorders. At present, tests for the detection of activated platelets are not used routinely in veterinary clinical laboratories. Objective: The purpose of this study was to develop a clinically applicable method to detect activated canine platelets. Methods: A flow cytometric assay was developed to detect activated platelets, platelet aggregates, and platelet microparticles in the dog. Blood was collected from healthy dogs using EDTA or sodium citrate as the anticoagulant, and platelet‐rich plasma was prepared. Platelets were activated by adding phorbol myristate acetate. In some experiments, platelets were fixed by incubation with 0.5% paraformaldehyde. In other experiments, platelets were stored for 4 or 24 hours at 4°C before analysis. Activated platelets were detected by measuring surface expression of P‐selectin and by determining the percentages of platelet aggregates and microparticles using forward‐angle vs side‐angle light scatter plots. Results were analyzed by using 2‐way ANOVA and the SchefféF‐test. Results: Platelets collected in EDTA had minimal expression of P‐selectin, whereas platelets collected in sodium citrate had greater median fluorescence intensity. Fixation with 0.5% paraformaldehyde before labeling platelets with anti‐P‐selectin did not affect antibody binding or the percentages of platelet aggregates and microparticles. Storage of platelet‐rich plasma at 4°C for 4 hours did not affect antibody binding or the percentages of platelet aggregates or microparticles. Activation of platelets ex vivo by addition of 10 ng/mL phorbol myristate acetate resulted in a large increase in expression of P‐selectin but only slight increases in platelet aggregates and microparticles. Conclusion: Determination of platelet P‐selectin expression and percentages of platelet aggregates and platelet microparticles may provide a clinically applicable means for detection of activated platelets in dogs. The capacity to use EDTA‐anticoagulated blood samples and to fix platelets for evaluation at a later time makes the test attractive as a routine diagnostic tool.     

Isolation of a rotavirus from a newborn dog with diarrhea

A rotavirus was isolated from a newborn dog that died after having clinical signs of diarrhea. Virus particles with rotaviral morphologic features were observed by transmission electron microscopy in the intestinal homogenate collected at necropsy. Cytopathic effects were observed, and rotaviral antigens were detected by indirect immunofluorescence in MA-104 monolayer cultures (a fetal rhesus macaque kidney cell) inoculated with intestinal homogenate. This rotavirus isolate, designated LSU 79C-36, may be a specific canine rotavirus or a rotavirus from another species.     

1株犬类志贺邻单胞菌的分离鉴定及序列分析

从病死犬病料中分离到1株类志贺邻单胞菌,并对该菌做了生理生化鉴定、药敏试验,致病性试验。结果表明,本菌对多种抗生素耐药,其庆大霉素耐药机制与所携带的质粒相关;本菌对小白鼠有强致病性,其LD50为1．6×10^7.4CFU。用PCR方法扩增分离菌株16SrDNA基因并测序,并将其与GenBank上其他细菌16SrDNA核苷酸序列进行同源性分析。结果表明,分离株的16SrDNA核苷酸序列与类志贺邻单胞菌（GQ359962．1）的同源性为98％,因此将该分离菌株鉴定为致病性肠球菌,命名为YN-1株（云南-1株）。     

Pleural fluid from a dog with marked eosinophilia

A 12-year-old neutered male Shar-Pei was presented to the North Carolina State University Veterinary Teaching Hospital cardiology service with a 2-week history of coughing and a 2-day history of lethargy and anorexia. Pleural effusion and a mediastinal mass were detected with thoracic radiographs. Ten mL of fluid were removed via thoracocentesis, and cytologic examination of the fluid revealed marked eosinophilic inflammation and few atypical mast cells. Mast cell neoplasia was suspected. Aspirates of the mediastinal mass, abdominal lymph nodes, and bone marrow contained similar pleomorphic mast cells and increased numbers of eosinophils. The dog was diagnosed with systemic (visceral) mastocytosis, a rare form of neoplasia in dogs, and was euthanized. These tumors carry a poor to grave prognosis and the etiology is uncertain.     

Cerebrospinal fluid from a dog with neurologic collapse

A 3-year-old Staffordshire Terrier was presented to the Texas Veterinary Medical Center with a short progressive history of anorexia, weight loss, and weakness that had progressed to ataxia and collapse with empirical treatment. The dog was tetraparetic and obtunded. Results of a complete neurologic evaluation were consistent with severe, multifocal to diffuse disease involving the forebrain, spinal cord, and brainstem. Cerebrospinal fluid, obtained via cerebellomedullary cisternal puncture, was highly cellular and contained large atypical round cells with small numbers of nondegenerate neutrophils and large mononuclear cells. Rare eosinophils and small lymphocytes were noted. The atypical round cells were approximately 15–25 μm in diameter with a single nucleus set in a small amount of cytoplasm. The nuclei were typically round to slightly ovoid; however, occasional notched, lobulated, and reniform nuclei were observed. These cells were interpreted as malignant lymphocytes. Owing to a grave prognosis, the animal was euthanized and a necropsy was performed. No gross lesions were found in the central nervous system. Multiple sections of cerebellum, medulla, and spinal cord contained a diffuse neoplastic infiltrate that was predominantly meningeal with rare superficial neuropil invasion. The neoplastic cells were arranged in sheets, cords, and rosettes. Immunohistochemical staining for vimentin, pancytokeratin, CD3, CD79a, synaptophysin, S-100, and neuronspecific enolase was negative; glial fibrillary acidic protein (GFAP) staining was equivocal. Based on histologic findings, a diagnosis of medulloblastoma was made. This case documents the rare occurrence of a canine medulloblastoma and illustrates the difficulty in distinguishing between some embryonal brain tumors and lymphoma.