In vitro susceptibility of canine corneal bacterial pathogens to three cross-linking protocols.

OBJECTIVE: To assess in vitro antibacterial efficacy of three cross-linking (XL) protocols on bacteria associated with canine ulcerative keratitis. METHODS: Three XL protocols: UVA 3 mW/cm2 for 60 min, UVA 3 mW/cm2 for 30 min, and UVA 30 mW/cm2 for 3 min with and without application of riboflavin and a riboflavin-only protocol were performed in vitro on the four most common bacterial genera isolated from cases of canine ulcerative keratitis treated at Dick White Referrals, UK. Zones of bacterial growth inhibition (GIZ) associated with treatment were measured and compared. RESULTS: The four most common isolates were Pseudomonas aeruginosa (PA) (48/140, 34.3%), Streptococcus spp. (32/140, 22.9%), Staphylococcus spp. (24/140, 17.1%) and Escherichia coli (EC) (11/140, 7.9%). PA, EC, Streptococcus canis (SC), and Staphylococcus pseudintermedius (SP), isolated from canine corneas, were selected for testing. EC and SC demonstrated growth inhibition following all UVA/riboflavin protocols. PA and SP only displayed growth inhibition following the 60 min UVA/riboflavin protocol. GIZ areas for 60 min UVA/riboflavin protocols were significantly greater than 30 and 3 min UVA/riboflavin protocols (p < .01) and there was no significant difference between 30 and 3 min UVA/riboflavin protocols. In respect to GIZ areas, EC was significantly more susceptible to XL than SP (p = <.01). CONCLUSIONS: All UVA/riboflavin XL protocols caused growth inhibition of EC and SC in vitro. PA and SP did not show clear growth inhibition in vitro following exposure to XL protocol settings of UVA 3 mW/cm2 for 30 min and UVA 30 mW/cm2 for 3 min.

Case Report: Diffuse Lumbar Hyperostosis Causing Vertebral Canal Stenosis in a Dog With Concurrent Multicentric T-Cell Lymphoma.

A 4-year-old female spayed Bullmastiff-cross presented with a 24-h history of progressive paraparesis. Neurological examination was consistent with L4-S3 myelopathy. On magnetic resonance imaging (MRI), all vertebrae showed homogenously increased short tau inversion recovery (STIR) signal with strong contrast enhancement. The vertebral canal was concentrically narrowed along the length of the L5 vertebra secondary to bony proliferation of the vertebral pedicles, dorsal lamina, and vertebral body. Cytological analysis of the peripheral lymph nodes and subsequent flow cytometry was consistent with T-cell lymphoma. The dog was euthanised due to poor prognosis. Necropsy confirmed the presence of stage V multicentric T-cell lymphoma, as well as diffuse hyperostosis of the vertebral bodies. This is the first report of presumed paraneoplastic lumbar skeletal hyperostosis.

Comparison of corneal culture results obtained using the ESwab™ and Amies charcoal swab in canine and feline patients.

OBJECTIVE: The objective of the study was to compare corneal culture results using the ESwab™ and Amies charcoal swab. ANIMALS STUDIED: One hundred fourteen canine and fifteen feline eyes. PROCEDURES: Retrospective analysis of Dick White Referrals bacterial and fungal corneal culture data was conducted. Results were included from canine and feline patients, which presented with suspected infectious keratitis that had samples taken using an Amies charcoal swab followed by an ESwab™ in the same eye. In respect to positive and negative cultures, a McNemar test was conducted comparing instances of disagreement between swab types, and the Kappa coefficient (κ) was calculated to assess the level of agreement between swab types. RESULTS: The ESwab™ produced more positive corneal cultures (71/129 [55.0%]) than the Amies charcoal swab (41/129 [31.8%]). 2/129 eyes produced positive fungal cultures. Considering 37/129 eyes in which both swab types detected a positive corneal culture, the same bacterial species were cultured from each swab type in 34/37 (91.9%) eyes, and an additional bacterial species was cultured by the ESwab™ in 3/37 (8.1%) eyes. In 34/38 (89.5%), instances of disagreement between swab types, the ESwab™ showed a positive culture, and the Amies charcoal swab showed a negative culture from the same eye, and this difference was significant (p < 0.0001). There was a moderate level of agreement between results from both swab types (κ = 0.432). CONCLUSIONS: ESwab™ sampling alone may be superior to Amies charcoal swabs for detecting presence of bacteria in suspected infectious keratitis in cats and dogs.

Validation of a spectrophotometric method for GGT measurement in canine urine and determination of the urine GGT-to-creatinine ratio reference interval and biological variation in 41 healthy dogs.

The urine gamma-glutamyl transferase (GGT)-to-creatinine ratio has been used to monitor patients at risk of acute renal injury. We validated the spectrophotometric quantification of GGT in urine in a commercial biochemistry analyzer. The assay was precise, accurate, and linear. Intra-assay precision was 3.59% in 4 samples, with GGT concentrations of 47-195 U/L. Inter-assay precision in 3 samples with activities of 11-51 U/L was 7.74%. Accuracy was 97.3%, with an absolute bias of 2.7 U/L. Urine GGT was unaffected by hematuria, hemoglobinuria, or bacteriuria. Urine GGT was stable at 20°C and 4°C for up to 3 d. Storage by freezing at -20°C resulted in a significant reduction in enzyme activity. A pH outside the range of 6.5-8 resulted in reduced GGT activity. The biological variation of urine GGT-to-creatinine ratio provided an index of individuality of 1.6, indicating that a population-based reference interval (RI) can be used. The reference change value was calculated, and an increase in consecutive measurements >43% is required to be regarded as significant. The urine GGT-to-creatinine ratio RI obtained in a population of 41 healthy dogs was 8.5-28.5 U/g.

Indolent, T-cell, large granular lymphocytic leukaemia in a dog presenting with severe neutropenia and an absence of lymphocytosis.

In humans, large granular lymphocytic leukaemia (LGLL) is a low-grade, indolent lymphoproliferative disorder of large granular lymphocytes (LGL) associated with autoimmune disorders; including rheumatoid arthritis and single or multiple cytopenias; particularly neutropenia. Therapy largely centres around immunosuppression which aims to resolve the immune-mediated secondary pathology, often without eradicating the neoplastic clone. The most effective agents appear to be cyclophosphamide, cyclosporine and methotrexate. This case report describes the presentation, diagnostics, therapeutic approach and outcome of a 6 year-old Golden Retriever presenting with severe neutropenia. Chlorambucil, prednisolone and cyclosporine failed to improve the neutropenia but subsequent cyclophosphamide resulted in a sustained albeit temporary improvement in neutrophil count and the ability to withdraw prophylactic antibacterials. This case closely mirrors the diagnostics and therapeutic response in human LGLL.

Comparison of quantitative immunoturbidimetric and semiquantitative latex-agglutination assays for D-dimer measurement in canine plasma.

BACKGROUND: D-dimer measurement in dogs is considered the most reliable test for detecting disseminated intravascular coagulation or thromboembolism. OBJECTIVES: The purposes of this study were to compare 2 D-dimer assays, a quantitative immunoturbidimetric and a semiquantitative latex agglutination assay, and to assess the effect of hemolysis and storage conditions on D-dimer concentration using the quantitative assay. METHODS: The immunoturbidimetric assay was validated using canine citrated plasma samples containing different concentrations of D-dimer. The effect of storage at various temperatures and times was assessed. Hemolysis was produced by adding lysed RBCs to the samples for a final hemoglobin concentration of 0.35 g/dL. RESULTS: For clinically relevant values (>250 microg/L), intra-assay and interassay coefficients of variation were 6.8% and 7.2%. The assay was linear (r(2)=1.00), and the tests had good agreement (kappa=0.685, P<.001). Storage at 4 degrees C and -20 degrees C and hemolysis had no significant effect on D-dimer concentrations. In hemolyzed samples stored at room temperature for > or =48 hours, fine clots were noted and often resulted in falsely increased D-dimer concentrations. CONCLUSIONS: Our findings suggest that the immunoturbidimetric assay validated in this study is reliable and accurate for the measurement of D-dimer in canine plasma. Samples can be stored for up to 1 month at -20 degrees C and moderate hemolysis does not significantly affect the D-dimer concentration in frozen or refrigerated samples.

RAS, FLT3, and C-KIT mutations in immunophenotyped canine leukemias.

OBJECTIVE: To determine the frequency of FLT3, C-KIT, and RAS mutations in canine leukemia patients. MATERIALS AND METHODS: Ethylenediamine tetra-acetic acid blood samples were recruited from dogs with suspected leukemia, categorized by quantitative and cytological evaluation and immunophenotyping. Flow cytometry was carried out using antibodies against CD3; CD3e; CD4; CD5; CD8; CD11a, b, c, and d; CD14; CD21; CD34; CD45 and 45RA; CD79a; CD90 (THY-1); major histocompatibility complex II; myeloperoxidase; MAC387; and neutrophil-specific antibody. Genomic DNA was extracted from whole blood and analyzed for mutations in N, H, and K-RAS, FLT3, and C-KIT genes by polymerase chain reaction and sequencing. RESULTS: Fifty-seven (77.0%) of 74 samples submitted from dogs with suspected leukemia had cytologically and immunophenotypically confirmed leukemia. There were 36 (63.2%) acute leukemias, 16 (28.1%) chronic, 3 (5.3%) prolymphocytic, 1 natural killer cell, and 1 chronic leukemia undergoing blast transformation. N-RAS mis-sense mutations were identified in 14 (25%) dogs with acute myeloid (AML) or lymphoid (ALL) leukemia, and also in one dog in the leukemic phase of lymphoma. Mutations in K-RAS were found in two dogs with AML. There were no H-RAS mutations. FLT3 internal tandem duplications were identified in three dogs with ALL, and a mis-sense mutation was found in one dog with ALL. C-KIT mutations were identified in three dogs with AML. Sixty-one percent of dogs with acute leukemia harbored mutations in N/K-RAS, FLT3, or C-KIT. CONCLUSION: RAS, FLT3, and C-KIT mutations, analogous to those found in human leukemia, occur commonly in acute canine leukemia.

Solitary plasmacytoma of bone in two successfully treated cats.

This is the first report of feline solitary plasmacytoma of bone. We describe the clinical, clinico-pathological, radiographic and pathological findings of two successfully treated cats with long-term follow-up. The first case presented with spinal pain and neurological deficits. Radiographs demonstrated sclerosis of lumbar vertebra L6 and a myelogram confirmed interference to flow of contrast in the L4-7 region. A biopsy of L6 revealed neoplastic plasma cell infiltration. There was no evidence of paraproteinaemia on serum protein electrophoresis. The cat underwent hypofractionated megavoltage radiotherapy. Clinical signs resolved completely and 4 years after diagnosis the cat remains well and has no electrophoretically detectable paraproteinaemia. The second case presented with neurological deficits of the tail and spinal radiographs revealed extensive osteolysis of the sacrum. A biopsy of sacral bone demonstrated neoplastic plasma cell infiltration. The animal was normoglobulinaemic. The cat improved clinically with induction chemotherapy (melphalan and methylprednisolone). The same chemotherapeutics were continued at maintenance doses for 4.3 years, at which time there was recurrence of neurological deficits and a palpable sacral mass. Cytological examination of a fine needle aspirate confirmed recurrence of plasma cell neoplasia. A low concentration monoclonal paraproteinaemia was detected. Vincristine was administered resulting in resolution of neurological deficits and a palpably smaller sacral mass. Eighteen months into vincristine therapy, there was recurrence of clinical signs and the cat was euthanased, more than 6 years after the initial diagnosis.

Myeloma-related disorders in cats commonly present as extramedullary neoplasms in contrast to myeloma in human patients: 24 cases with clinical follow-up.

BACKGROUND: Myeloma-related disorders (MRD) are rare neoplasms of plasma cells. Published case reports describe a diversity of clinical presentations with confusing terminology and diagnostic criteria as a consequence of the assumption that MRD in cats are analogous to those in dogs or humans. OBJECTIVE: The aim of the study was to describe clinical, clinicopathologic and imaging findings, response to treatment, survival and possible associations with other diseases or vaccination in a large case series. A priori hypotheses were that cats with MRD commonly present with extramedullary involvement and uncommonly have radiographic bone lesions, in contrast to human patients. ANIMALS: Twenty-four cats with MRD confirmed by cytology or histopathology and immunohistochemistry. METHOD: A multicenter retrospective study was performed. RESULTS: Two types of clinical presentation were observed. The first group (n = 17) had neoplasia involving abdominal organs, bone marrow, or both. All developed systemic clinical signs and paraproteinemia. Five of 7 cats that received chemotherapy improved clinically or had decreased serum globulin concentration (median survival, 12.3 months; range, 8.5-22 months). The second group comprised 7 cats with skin masses, 2 of which were paraproteinemic and developed rapidly worsening systemic signs. In cats without systemic signs, excision of the skin masses appeared to be associated with prolonged survival (up to 2.4 years). Cats with MRD commonly presented with extramedullary involvement (67%), versus humans with MRD (5%) (P < .001), and uncommonly presented with radiographic bone lesions (8%) versus humans with MRD (80%) (P < .001). CONCLUSIONS: Radiographic bone lesions are uncommon in cats with MRD and extramedullary presentation is common, relative to human myeloma.

Identification of acute myeloid leukemia in dogs using flow cytometry with myeloperoxidase, MAC387, and a canine neutrophil-specific antibody.

BACKGROUND: Flow cytometry may be used to determine immunophenotype or lineage of leukemic cells, but few antibodies are available that are specific for cells of monocytic and granulocytic lineage. OBJECTIVE: The purpose of this study was to evaluate the flow cytometric staining patterns of 3 commercial monoclonal antibodies for monocytes and granulocytes in clinically healthy dogs and in dogs with acute myeloid leukemia (AML). METHODS: Mouse antihuman macrophage antibody (MAC387), mouse anti-human myeloperoxidase (MPO), and a canine neutrophil-specific antibody (NSA) were evaluated using flow cytometry on blood from 6 clinically healthy control dogs, and on blood (n = 7) and/or bone marrow (n = 2) from 8 dogs with AML. A diagnosis of acute leukemia was confirmed by >30% blasts in bone marrow or >30% blasts in peripheral blood, together with bi- or pancytopenia, circulating CD34-positive blast cells, and clinical signs of disease. Leukemic samples also were evaluated using a wide panel of monoclonal antibodies. RESULTS: MAC387 stained neutrophils and monocytes from control dogs, although the staining profiles for the 2 cell types differed. MPO and NSA resulted in strong positive staining of neutrophils; MPO also stained monocytes weakly. Lymphocytes did not stain with any of the antibodies. One case was classified as AML of granulocytic lineage (AML-M1), 6 cases were classified as acute monocytic leukemia (AML-M5), and 1 case was classified as acute myelomonocytic leukemia (AML-M4). Neoplastic myeloblasts in the dog with granulocytic AML were positive for MPO, NSA, MAC387, and CD4. All monoblasts from the dogs with AML-M5 were positive for CD14, 5 of 6 were positive for MAC387, and 2 were positive for MPO. NSA staining was negative in the 2 dogs with AML-M5 in which it was evaluated. In the dog with AML-M4 variable percentages of blast cells were positive for CD14, MPO, MAC387, CD4, and NSA. CONCLUSIONS: Antigens identified by antibodies to MAC387, MPO, and NSA were expressed not just by normal mature neutrophils and monocytes, but also by neoplastic myeloblasts and monoblasts. These 3 antibodies may be useful as part of a wider panel for immunophenotyping AML in dogs.

High serum troponin I concentration as a marker of severe myocardial damage in a case of suspected exertional heatstroke in a dog.

A young, overweight dog presented with sudden onset lethargy and collapse following exercise in warm environmental conditions. Investigations revealed systolic hypotension, multiform ventricular premature complexes, irregular myocardial echogenicity with poor left ventricular systolic function and a markedly elevated troponin cTnI (180ng/mL, reference range <0.3ng/mL) consistent with severe myocyte damage. Infectious causes of myocarditis were ruled out on the basis of serological and polymerase chain reaction blood tests. Exercise-induced malignant hyperthermia was excluded from the history, an exercise tolerance test and genetic testing for the RYR1 V547A mutation. The diagnosis was myocardial damage secondary to suspected exertional heatstroke, from which the dog recovered uneventfully over a number of weeks and serum troponin normalised. This is the first case report in any species including man, documenting high troponin as a marker of severe myocardial damage following suspected heatstroke.