Evaluation of coagulation and platelet activation state and function in heartworm-infected dogs.

BACKGROUND: Enhanced platelet responses have been demonstrated in heartworm-infected (HWI) dogs; however, the cause and clinical implications of altered platelet function have not been fully elucidated. OBJECTIVE: This study evaluated platelet function in HWI dogs. METHODS: Anticoagulated whole blood collected from eight HWI and eight uninfected dogs was evaluated using turbidometric platelet aggregometry, a platelet function analyzer (PFA-100), a total thrombus analysis system (T-TAS), tissue factor-activated and tissue plasminogen activator modified thromboelastography (TF- and tPA-TEG), CBC, von Willebrand Factor activity, and fibrinogen concentrations. Platelet activation state and the presence of reticulated platelets were assessed via flow cytometric expression of P-selection (CD-62P) and thiazole orange staining. RESULTS: Platelet aggregation responses to adenosine diphosphate (ADP, 10 µM) or collagen (20 µg/mL), PFA-100 closure times, and T-TAS occlusion times did not differ between groups. TEG values TF-R, tPA-R, TF-K, and TF-LY60 were decreased (P = .025, P = .047, P = .038, P = .025) and TF-MA, tPA-MA, TF-G, tPA-G and TF-alpha angle were increased (P < .04) in HWI dogs. HWI dogs had higher fibrinogen concentrations (465.6 ± 161 mg/dL vs 284.5 ± 38 mg/dL, P = .008) and eosinophil counts (0.686 ± 0.27 × 103/µL vs 0.267 ± 0.20 × 103/µL, P = .003). There was no difference in hematocrit, activation state, or percent of reticulated platelets. Non-activated reticulated platelets exhibited higher CD62P expression compared with mature platelets. CONCLUSIONS: Chronic canine heartworm disease was accompanied by hypercoagulability, hyperfibrinogenemia, and decreased fibrinolysis. Enhanced platelet activation was not identified in this group of HWI dogs.

Prognostic significance of CD25 expression in dogs with a noninvasive diagnosis of B-cell lymphoma treated with CHOP chemotherapy.

Prior studies have identified high CD25 expression in canine diffuse large B-cell lymphoma as a negative prognostic indicator. The objective of this retrospective study was to evaluate CD25 expression as a prognostic indicator in dogs with B-cell lymphoma (BCL) diagnosed with commonly used noninvasive diagnostics (cytology and flow cytometry [FC]) and treated with CHOP chemotherapy. Lymph node aspirates from 57 dogs with cytologic diagnosis of lymphoma composed of intermediate to large lymphocytes were analysed with FC. Percentage of neoplastic B-cells expressing CD25 and median fluorescence intensity (MFI) of CD25 were measured. Relationships of CD25 percent positivity and MFI to progression free survival (PFS) and survival time were evaluated. Median survival time (MST) of all dogs was 272 days (95% CI, 196-348 days) and median PFS was 196 days (95% CI, 172-220 days). Higher percentage of B-cells positive for CD25 was associated with decreased risk of death in multivariable analysis (p = .02). Dogs with higher CD25 positivity had longer MST and PFS than dogs with lower CD25 positivity (318 days versus 176 days and 212 days versus 148 days, respectively), but these differences were not significant. CD25 MFI was not significantly associated with outcome. Based on the results of this study, the association of CD25 expression and prognosis in dogs with BCL diagnosed using noninvasive methods should be interpreted with caution. Further evaluation, with studies that include histopathologic differentiation of lymphoma subtypes, is needed.

Evaluation of profibrotic gene transcription in renal tissues from cats with naturally occurring chronic kidney disease.

BACKGROUND: Increased gene transcription of hypoxia-induced mediators of fibrosis in renal tissue has been identified in experimentally induced, ischemic chronic kidney disease (CKD). OBJECTIVE: To characterize hypoxia-induced profibrotic pathways in naturally occurring CKD in cats. ANIMALS: Twelve client-owned cats with CKD and 8 healthy control cats. METHODS: In this prospective, cross-sectional study, bilateral renal tissue samples were assessed histologically for inflammation, tubular atrophy, and fibrosis, and by reverse transcription-quantitative PCR for characterization of transcript levels of hypoxia-inducible factor-1α (HIF1A), matrix metalloproteinases-2 (MMP2), -7 (MMP7), and -9 (MMP9), tissue inhibitor of metalloproteinase-1 (TIMP1), transforming growth factor-ß1 (TGFB1), and vascular endothelial growth factor-A (VEGFA). Linear mixed models were used to compare gene transcription between diseased and healthy kidneys, and to examine the association between transcript levels and serum creatinine concentration for all cats, and between transcript levels and histologic scores of diseased kidneys. RESULTS: Kidneys from cats with CKD had significantly higher transcript levels of HIF1A, MMP2, MMP7, MMP9, TIMP1, and TGFB1 (all P < .001), and lower levels of VEGFA (P = .006) than those from control cats. Transcript levels of MMP7 (P = .05) and TIMP1 (P = .005) were positively associated with serum creatinine in cats with CKD, but not in control cats. In diseased kidneys, transcript levels of MMP2 (P = .002), MMP7 (P = .02), and TIMP1 (P = .02) were positively, whereas those of VEGFA (P = .003) were negatively, associated with histologic score severity. CONCLUSION AND CLINICAL SIGNIFICANCE: Evaluation of the expression of the corresponding proteins in larger populations could identify therapeutic targets and/or biomarkers of tubulointerstitial fibrosis in cats.

Multicenter flow cytometry proficiency testing of canine blood and lymph node samples.

BACKGROUND: Flow cytometry (FC) is used increasingly in veterinary medicine for further characterization of hematolymphoid cells. Guidelines for optimizing assay performance and interpretation of results are limited, and concordance of results across laboratories is unknown. OBJECTIVES: This study aimed to determine inter-investigator agreement on the interpretation of FC results from split samples analyzed in different laboratories using various protocols, cytometers, and software; and on the interpretation of archived FC standard (FCS) data files contributed by the different investigators. METHODS: This was a multicenter observational cross-sectional study. Anticoagulated blood or lymph node aspirate samples from nine client-owned dogs were aliquoted and shipped to participating laboratories. Samples were analyzed with individual laboratory-developed protocols. In addition, FCS files from a set of separate samples from 11 client-owned dogs were analyzed by participating investigators. A person not associated with the study tabulated the results and interpretations. Agreement of interpretations was assessed with Fleiss' kappa statistic. RESULTS: Prolonged transit times affected sample quality for some laboratories. Overall agreement among investigators regarding the FC sample interpretation was strong (κ = 0.86 ± 0.19, P < .001), and for specific categories, ranged from moderate to perfect. Agreement of the lymphoproliferation or other leukocyte sample category from the analysis of the FCS files was weak (κ = 0.58 ± 0.05, P < .001). CONCLUSIONS: Lymphoproliferations were readily identified by FC, but identification of the categories of hematolymphoid neoplasia in fresh samples or archived files was variable. There is a need for a more standardized approach to maximize the enormous potential of FC in veterinary medicine.

Profibrotic gene transcription in renal tissues from cats with ischemia-induced chronic kidney disease.

OBJECTIVE: To characterize transcription of profibrotic mediators in renal tissues of cats with ischemia-induced chronic kidney disease (CKD). SAMPLE: Banked renal tissues from 6 cats with experimentally induced CKD (RI group) and 8 healthy control cats. PROCEDURES: For cats of the RI group, both kidneys were harvested 6 months after ischemia was induced for 90 minutes in 1 kidney. For control cats, the right kidney was evaluated. All kidney specimens were histologically examined for fibrosis, inflammation, and tubular atrophy. Renal tissue homogenates underwent reverse transcription quantitative PCR assay evaluation to characterize gene transcription of hypoxia-inducible factor-1α (HIF-1α), matrix metalloproteinase (MMP)-2, MMP-7, MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), transforming growth factor-ß1, and vascular endothelial growth factor A. Gene transcription and histologic lesions were compared among ischemic and contralateral kidneys of the RI group and control kidneys. RESULTS: Ischemic kidneys had greater transcript levels of MMP-7, MMP-9, and transforming growth factor-ß1 relative to control kidneys and of MMP-2 relative to contralateral kidneys. Transcription of TIMP-1 was upregulated and that of vascular endothelial growth factor A was downregulated in ischemic and contralateral kidneys relative to control kidneys. Transcription of HIF-1α did not differ among kidney groups. For ischemic kidneys, there were strong positive correlations between transcription of HIF-1α, MMP-2, MMP-7, and TIMP-1 and severity of fibrosis. CONCLUSIONS AND CLINICAL RELEVANCE: Transcription of genes involved in profibrotic pathways remained altered in both kidneys 6 months after transient renal ischemia. This suggested that a single unilateral renal insult can have lasting effects on both kidneys.

Immunomodulatory in vitro effects of oclacitinib on canine T-cell proliferation and cytokine production.

BACKGROUND: Oclacitinib is a Janus kinase inhibitor used to control pruritus and skin lesions in canine allergic skin disease; its effect on canine T cells is not well-characterized. HYPOTHESIS/OBJECTIVES: To evaluate the impact of oclacitinib on cultured T cells using peripheral blood mononuclear cells from dogs. ANIMALS: Six bluetick coonhounds. METHODS AND MATERIALS: Lymphocyte-enriched cells were incubated with or without the T-cell mitogen concanavalin A (Con A), oclacitinib (0.5, 1 or 10 µM), ciclosporin (200 ng/mL), Con A + oclacitinib 1 µM and Con A + ciclosporin. We assessed both T-cell proliferation and the secretion of cytokines. RESULTS: Ciclosporin and oclacitinib both inhibited the spontaneous proliferation of T cells; this effect was significant only after incubation with oclacitinib at 10 µM. At this concentration, oclacitinib significantly reduced the spontaneous secretion of clonal activator cytokines [interleukin (IL)-2, IL-15], pro-inflammatory cytokines (interferon-gamma (IFN-γ), IL-18) and the regulatory cytokine IL-10; tumour necrosis factor alpha (TNF-α) and IL-6 cytokine production was mildly inhibited. After Con A stimulation, only T cells co-treated with ciclosporin achieved a significant proliferation inhibition and reduction of IL-2, IL-10, IL-15, IL-18, IFN-γ and TNF-α. Surprisingly, oclacitinib at 1 µM (337 ng/mL, corresponding to the oral dosage of 0.4-0.6 mg/kg) did not significantly affect Con A-stimulated T-cell proliferation nor cytokine production (IL-2, IL-10, IL-15, IL-18, IFN-γ and TNF-α). CONCLUSIONS: Although a limited number of dogs were investigated, these preliminary results suggest that oclacitinib appears to have immunosuppressive properties, but only at dosages above those used to treat allergic pruritus in dogs.

Extreme lymphocytosis with myelomonocytic morphology in a horse with diffuse large B-cell lymphoma.

An 11-year-old, 443-kg Haflinger mare was presented to the North Carolina State University Veterinary Teaching Hospital with a 2-week history of lethargy and a 3-day duration of anorexia, pyrexia, tachycardia, and ventral edema. Severe pitting edema, peripheral lymphadenopathy, and a caudal abdominal mass were noted on physical examination. An extreme leukocytosis (154.3 × 103 /µL) and microscopic hematologic findings suggestive of myelomonocytic leukemia were observed. Serum protein electrophoresis revealed a monoclonal gammopathy and urine protein electrophoresis revealed a monoclonal light chain proteinuria. Necropsy and histopathology confirmed widespread neoplastic infiltration in many organs with a heterogenous population of cells; there was no apparent evidence of bone marrow involvement. Immunohistochemistry confirmed presence of a majority of B cells with a limited antigen expression, admixed with a lower number of T cells. Molecular clonality analysis of IgH2, IgH3, and kappa-deleting element (KDE, B cell) on whole blood and KDE on infiltrated tissues revealed clonal rearrangements, and the KDE intron clones that amplified in blood and in infiltrated tissue were identical. In contrast, the clonality analysis of T-cell receptor γ revealed no clonality on blood cells and infiltrated tissues. In conjunction with the histopathologic changes, the lesion was interpreted to be composed of neoplastic B cells with a reactive T-cell population. Polymerase chain reaction testing for equine herpes virus 5 was negative. The final diagnosis was diffuse large B-cell lymphoma with a marked hematogenous component.

What is your diagnosis? Particulate material in peritoneal fluid from a dog.

9-year-old castrated male Greyhound dog was presented for evaluation of vomiting and lethargy of 1-week duration. On physical examination, the dog was febrile and dehydrated with a tense abdomen and petechial hemorrhages. Clinicopathologic abnormalities included relative polycythemia, mild lymphopenia with reactive lymphocytes, hypoalbuminemia, hypocholesterolemia, hyperbilirubinemia, increased ALP, mild hypokalemia, hyperamylasemia, hyperlipasemia, increased D-dimer concentration, and hyperfibrinogenemia. Cytologic evaluation of peritoneal fluid revealed marked suppurative inflammation with intracellular barium sulfate particles. The day before presentation, the referring veterinarian had administered oral barium sulfate in an upper gastrointestinal contrast study. Radiographs revealed free contrast material in the peritoneal cavity, consistent with gastrointestinal perforation, and leakage of contrast material. Abdominal exploratory surgery revealed a mid-jejunal perforation and a hepatic nodule. Histopathologic diagnosis of the jejunal and liver lesions was T-cell lymphoma. The patient recovered well postoperatively and received chemotherapy for treatment of lymphoma. Most commercial barium sulfate preparations contain relatively uniform, weakly birefringent, pale yellow particles <1 microm in diameter. Because barium sulfate is found occasionally in clinical specimens, cytopathologists should be familiar with its cytologic appearance.

Gallbladder aspirate from a dog.

A 7-year-old, male, castrated, Labrador Retriever with a history of pancreatitis and inflammatory bowel disease presented for vomiting and anorexia. Serum biochemistry findings were indicative of cholestasis, hepatocellular insult, and decreased hepatic function. Ultrasound examination showed sediment and gas within the gallbladder, and a diagnosis of emphysematous cholecystitis was made. Emergency gallbladder resection was performed. Cytologic examination of bile fluid collected at surgery showed a mixed population of bacteria (bactibilia) together with fungal organisms consistent with Cyniclomyces guttulatus (previously known as Saccharomycopsis guttulatus). Similar fungal organisms were seen on a fecal smear. Bacteria cultured were normal gastrointestinal flora, supporting ascending infection; the fungal organisms were interpreted as incidental. Histopathology of the gallbladder indicated active (suppurative) and chronic (lymphocytic) cholecystitis and sections of liver tissue had evidence of chronic liver disease. A positive liver culture indicated concurrent bacterial hepatitis or cholangiohepatitis. Despite supportive care, the dog continued to decline and was euthanized 30 days later. Necropsy results confirmed end stage liver disease, but an initiating cause was not found. This case highlights the role of bactibilia in the development of acute cholecystitis and the unique cytologic appearance of C guttulatus as an incidental finding in bile fluid.

Reluctant to dive: coelomic effusion in a frog.

An adult female, albino South African Clawed frog (Xenopus laevis) from a research colony at the Biological Resources Facility of the College of Agriculture and Life Sciences at North Carolina State University (NCSU) was presented with depression, lethargy, loss of diving reflex, and a distended abdomen. Cytologic examination of coelomic effusion fluid at the NCSU veterinary teaching hospital revealed a mixed population of inflammatory cells, including heterophils and a predominance of large mononuclear cells (macrophages) that often contained intracytoplasmic, negatively-stained, rod-shaped to filamentous organisms consistent with Mycobacterium sp. Ziehl-Neelsen stain revealed bright pink to red, acid-fast organisms with a beaded appearance. Histopathologic findings in tissues obtained at necropsy included marked, multifocal to coalescing, heterophilic, granulomatous and fibrinous coelomitis as well as severe multifocal heterophilic and granulomatous hepatitis, interstitial pneumonia and sinusitis/rhinitis. Slender gram-positive, acid-fast bacterial rods were identified in sections of coelomic pleura, kidneys, nasal cavities, spleen, liver, and pulmonary interstitium, indicative of systemic mycobacteriosis. Based on mycobacterial culture, the organism was identified as M marinum complex. Mycobacteria are variably gram-positive, often acid-fast, small rods that are ubiquitous in aquatic environments. The clinical and pathologic spectrum of disease in amphibians depends on host and pathogen status. Xenopus sp and several other frogs are good models for studying the pathogenesis of M tuberculosis infection. In addition to culture, polymerase chain reaction assays may be used for definitive identification of the organisms; accurate speciation may require further genetic investigation.