Morphologic, cytochemical, and ultrastructural features of gray eosinophils in nine cats.

BACKGROUND: Gray eosinophils, resembling those in sighthound dog breeds, have not previously been reported in cats. OBJECTIVES: We aimed to provide a morphologic, cytochemical, and ultrastructural description of gray eosinophils in cats. METHODS: Blood films examined as part of routine hematology profiles in cats from May 2015 to July 2018 were evaluated for the presence of gray eosinophils. When identified with modified Wright stain, cells were morphologically assessed and additionally stained with Diff-Quik, ALP, Luna, and Luxol fast blue stains and compared with feline controls. Two cases were prepared for transmission electron microscopy (TEM) and compared with a feline control. RESULTS: Gray eosinophils were identified in 9 of 2641 cats during the study period. Compared with typical feline eosinophils, these cells contained abundant round granules instead of the characteristic rod-shaped specific granules. These granules lacked the characteristic intense pink/red staining with Romanowsky stains and did not stain with ALP, Luna, or Luxol fast blue stains. On TEM, the classical electron-dense core of these granules was replaced by a core with fragmented or amorphous internal material. Typical eosinophils were not identified in any cat in which gray eosinophils were identified. CONCLUSIONS: The distinct morphologic, cytochemical, and ultrastructural changes in gray feline eosinophils might be associated with a reduction or lack of major basic protein (MBP) in specific granule cores. Similar to canine gray eosinophils, accurate recognition of these cells is essential to prevent their misclassification as toxic neutrophils.

The cytologic and histologic diagnosis of ureteral fibroepithelial polyp in a dog.

A 6-year-old, intact male, brindled, 30-Lb English Bulldog presented to the Purdue University Veterinary Teaching Hospital with a recurrent history of hematuria, periuria, and lethargy that responded temporarily to antibiotic therapy. The work-up included a complete blood count, serum biochemical profile, complete urinalysis, diagnostic imaging (abdominal radiographs and ultrasound with contrast urography), and exploratory laparotomy. The diagnostic imaging findings and subsequent exploratory revealed a unilateral, intraluminal, right-sided, 3-cm ureteral mass extending from the proximal ureter into the renal pelvis. Subsequently, a unilateral right-sided ureteronephrectomy followed by biopsy with cytopathology/cytology (impression smears) and histopathology of the ureteral mass was performed. The cytopathologic interpretation was benign mesenchymal proliferation with mildly atypical urothelial cells. The association of this mass with vascular tissue and a benign nuclear appearance on cytology is similar to reports of fibroepithelial polyps (FEPs) and myxomatous tumors. Histopathology diagnosed the mass as an FEP. Cytopathology proved useful in the presumptive diagnosis of this benign urothelial polyp. To the authors' knowledge, this is the first report using cytopathology to depict and characterize FEPs in veterinary and human medical literature.

Newly recognized Anaplasma sp. in erythrocytes from Gopher Tortoises (Gopherus polyphemus).

BACKGROUND: In 2015, a previously unrecognized intracytoplasmic erythrocytic inclusion was discovered in anemic wild-caught adult gopher tortoises (Gopherus polyphemus). Subsequently, molecular diagnostics revealed this inclusion to be a novel Anaplasma sp. OBJECTIVES: The goal of this study was to morphologically characterize these erythrocytic inclusions by light and transmission electron microscopy (TEM). METHODS: Blood samples were taken from two car-injured wild-caught gopher tortoises for the preparation of Wright-Giemsa stained smears and TEM specimens. CBC data were serially performed and morphologically examined during treatment periods. RESULTS: Studies revealed a moderate to severe anemia with moderate regeneration as indicated by polychromasia and the presence of immature erythroid precursors. In addition, on light microscopy, one to two variably-sized round basophilic stippled paracentral erythrocytic inclusions were present per cell in both animals and involved 10%-25% of erythrocytes. TEM identified the intraerythrocytic inclusions as discrete membrane-bound cytoplasmic vacuoles (morulae) containing membrane-bound bacterial subunits that were of variable size, shape, and electron density. Serial hematologic data indicated complete remission of the infection in response to a single long-term course of doxycycline. CONCLUSIONS: The presence of a regenerative anemia in gopher tortoises from Florida revealed a newly recognized bacterial species that has morphologic characteristics similar to members of the genus Anaplasma.

Grey eosinophils in a Miniature Schnauzer with a poorly differentiated mast cell tumor.

A 10-year-old female spayed Miniature Schnauzer was presented for investigation of an intra-nasal mass. The mass was diagnosed by histopathologic examination as an undifferentiated round cell neoplasm with an infiltrate of segmented leukocytes, interpreted as neutrophilic inflammation. The mass was treated with palliative radiation and systemic chemotherapy due to the presence of regional lymph node metastasis. During subsequent monitoring over several months, the peripheral leukocyte concentration was repeatedly within reference intervals to slightly increased with low numbers of toxic neutrophils. Four months after the initial diagnosis, there was a significant leukocytosis of 66 100 cells/µL, and 39 700 cells/µL of the leukocytes had variably mature, lobulated, and hypolobulated nuclei, and grey cytoplasm with clear vacuoles, resembling grey eosinophils. To further characterize these cells, peripheral blood smears from the patient and a canine control with eosinophilia were stained for alkaline phosphatase (AP), peroxidase, and esterase activities, and with Luxol fast blue (LFB). Histopathologic sections of the nasal mass were stained with LFB and immunohistochemically for tryptase. On blood smears, the cytoplasm of the suspected grey eosinophils stained for AP and granules stained with LFB confirmed that there was an eosinophilic lineage. Peroxidase staining was weak, and esterase staining was absent. On histopathologic sections from the nasal mass, the segmented leukocytes contained LFB-staining granules, indicating an eosinophilic infiltrate was present. Neoplastic cells expressed tryptase, which confirms a mast cell lineage. Our findings suggest that grey eosinophils might be under-recognized and interpreted incorrectly as toxic neutrophils. This report expands the canine breeds in which these eosinophils have been identified.

Optimized immunocytochemistry using leukocyte and tissue markers on Romanowsky-stained slides from dogs and cats.

BACKGROUND: Romanowsky staining is often the initial method used to stain hematologic and cytologic materials. While immunocytochemistry (ICC) is a well-established method on air-dried smears, there are rare veterinary reports of ICC involving Romanowsky-stained slides. OBJECTIVES: This study aimed to compare immunoreactivity of unstained vs Romanowsky-stained specimens, evaluate reactions over time, and assess ICC associations with confirmatory tests of 50 lymphoma cases. Another goal aimed to optimize manual ICC protocols with cellular and tissue immunomarkers to detect CD3ε, CD20, Pax5, MHCII, lysozyme, MUM1, vimentin, cytokeratin, and Melan-A antigens on Romanowsky-stained specimens. MATERIALS AND METHODS: Cytologic specimens from cases of lymphoid and nonlymphoid neoplasms were stained with a methanolic Romanowsky method. Additional unstained slides from these cases were used for comparison with the stained materials. Antigen retrieval involved a citrate buffer pH6 or Tris/EDTA pH9 at 95°C for 25 minutes in a decloaking chamber. Immunocytochemistry used known positive and secondary antibody-only negative cytologic controls. Immunoreactivity of unstained and prestained lymphoma slides was graded by the intensity and percent of stained cells. Signal grading was monitored over time for diagnostic differences. RESULTS: Unstained and Romanowsky-stained slides had similar membrane/cytoplasm graded reactions, but unstained slides produced stronger signals. Romanowsky-stained blood films from B-cell and T-cell leukemias showed minimal loss of signal when monitored over 20 weeks. Signal differences did not change the diagnosis. There was a significant association between ICC and confirmatory tests. Optimization involved antibody dilution and antigen retrieval methodology for each antibody tested. CONCLUSIONS: Immunocytochemistry of Romanowsky-stained material can be successfully performed using antibodies against CD3ε, CD20, cytokeratin, lysozyme, Melan-A, MHCII, MUM1, Pax5, and vimentin.

Pelger-Huët anomaly in a cat.

A 14-year-old, spayed female Domestic Shorthair cat was referred to the Purdue University Veterinary Teaching Hospital (PUVTH) for iodine 131 treatment of hyperthyroidism. Upon arrival, a biochemistry profile and a CBC were performed. Approximately 50% of the neutrophils and all the eosinophils observed were hyposegmented with a mature, condensed chromatin pattern. Nuclei had a band to "dumbbell" shape, and rarely a round shape, suggesting a Pelger-Huët anomaly or a pseudo Pelger-Huët. Based on both a negative FeLV and FIV tests, the absence of any clinical signs to support an inflammatory process, and the persistence of this granulocytic morphology 6 months after its previous admission to the PUVTH, a diagnosis of Pelger-Huët anomaly was established in this cat.

Multiple joint metastasis of a transitional cell carcinoma in a dog.

An 8-year-old castrated male hound mix was referred to the Purdue University Veterinary Teaching Hospital for severe lameness, pollakiuria, and dyschezia. On presentation, the dog was nonweight bearing on the right rear limb and the right carpus was diffusely swollen. Synovial fluid analysis from the right carpus revealed a population of epithelial cells displaying marked anisocytosis, anisokaryosis, multinucleation, and prominent, variably sized nucleoli. A metastatic carcinoma with presumed prostatic or urothelial origin was diagnosed based on cytomorphology. Subsequent cytologic evaluation of peripheral lymph nodes revealed the presence of a similar neoplastic population. The dog was euthanized and synovial fluid from both stifle joints, as well as impression smears of the prostate gland, were collected. Carcinoma cells were identified in each stifle joint and in the prostate gland. Immunocytochemistry was performed on synovial fluid smears from 2 of the joints (right stifle and right carpus) and on impression smears of the prostate gland. The neoplastic population in the joints and prostate gland showed strong immunoreactivity to uroplakin III, a urothelial marker, indicating metastasis of a transitional cell carcinoma to multiple joints. In addition, evidence for epithelial to mesenchymal transition was identified using cytokeratin, an epithelial marker, and vimentin, a mesenchymal marker. A necropsy was performed and histopathology confirmed the presence of metastatic transitional cell carcinoma in various tissues. This case illustrates the importance of considering metastatic disease when a patient is presented with severe lameness and joint pain, and the clinical utility of synovial fluid cytology for diagnosis of metastasis in these cases.

Cytochemical and immunocytochemical characterization of blood cells and immunohistochemical analysis of spleen cells from 2 species of frog, Rana (Aquarana) catesbeiana and Xenopus laevis.

BACKGROUND: Mechanisms of amphibian diseases are not characterized as well as those in domestic mammalian species. Antemortem laboratory testing is limited in frogs, presenting a diagnostic challenge to zoos, laboratories, and exotic veterinarians. OBJECTIVE: This study aimed to characterize blood cells and splenic cells from 2 anuran species based on characteristics identified by Wright staining, cytochemical staining, and immunochemical analysis and on histologic examination of spleens. METHODS: Blood specimens and spleens were obtained from 2 species of frog, the American bullfrog (Rana [Aquarana] catesbeiana) and the African clawed frog (Xenopus laevis). Blood smears were evaluated after Wright staining and cytochemical staining for α-naphthyl butyrate esterase (NBE), chloroacetate esterase (CAE), myeloperoxidase (PER), Sudan black B (SBB), and leukocyte alkaline phosphatase (LAP) reactions and for immunoreactivity for antibodies against CD3ε, CD79a, and BLA.36 antigens. Histologic sections of spleen were evaluated after staining with H&E and for immunoreactivity for CD3ε, CD79a, and BLA.36 antigens. RESULTS: In bullfrogs, neutrophils, eosinophils, and monocytes were positive for some or all of the following: NBE, CAE, PER, and SBB; lymphocytes occasionally were positive for CAE. In clawed frogs, neutrophils, basophils, and monocytes were positive for some or all of the following: NBE, CAE, PER, and SBB; eosinophils occasionally were positive for CAE and PER, and lymphocytes were negative for all cytochemical stains. LAP was not a useful marker for any leukocyte type. In both species, peripheral blood lymphocytes were strongly immunoreactive for CD3ε, CD79a, and BLA.36. In splenic tissue, histologic patterns varied and there was diffuse immunoreactivity for CD79a and BLA.36 with focal reactivity for CD3ε, but with different distribution patterns in each species. CONCLUSION: Cytochemical and immunochemical analysis of cells may be helpful in identification and characterization of amphibian blood cells and splenic cells for evaluation of the health of these animals.

Hematology, leukocyte cytochemical analysis, plasma biochemistry, and plasma electrophoresis of wild-caught and captive-bred Gila monsters (Heloderma suspectum).

BACKGROUND: The Gila monster (Heloderma suspectum), one of several venomous lizard species in the world, is found within the United States and Mexico and is recognized as an iconic symbol of the American Southwest. Thus, Gila monsters are of growing interest in the captive reptile trade and within zoological and educational institutions. OBJECTIVES: The aims of this study were to determine results for CBCs, describe cytochemical reactions in WBCs, and obtain plasma biochemical and protein electrophoresis results from wild-caught and captive-bred H. suspectum. METHODS: Ventral tail vein blood samples were obtained from 16 captive (14 wild-caught and 2 captive-bred) Gila monsters. CBCs, RBC morphometric analysis, plasma biochemical analysis, and protein electrophoresis were performed. Leukocytes were stained for peroxidase, Sudan black B (SBB), chloroacetate esterase, napthyl butyrate esterase, and leukocyte alkaline phosphatase, and blood smears were examined for the presence of hemoparasites. RESULTS: The median (range) PCV was 37% (22-50%) and WBC count was 4.6 × 10(3) /µL (3.3-6.4 × 10(3) /µL) with approximately 50% heterophils and fewer lymphocytes, basophils, azurophils, and monocytes in decreasing order. Cytochemical reactions were unique among reptiles with strong staining for peroxidase and SBB in monocytes/azurophils. Biochemical results were similar to those of earlier reports with slight increases in uric acid and urea concentrations. Plasma electrophoretic results indicated that albumin was approximately equal to the combined globulin fractions. CONCLUSIONS: Results of blood analysis in healthy wild-caught and captive-bred H. suspectum may be useful for monitoring health status in this species.

Sarcocystis sp. encephalomyelitis in a cat.

A 5-month-old male neutered domestic shorthair cat was evaluated for spinal pain, ataxia, and anisocoria. Neuroanatomic localization indicated diffuse or multifocal central nervous system disease. On cerebrospinal fluid analysis, neutrophilic pleocytosis and intracellular protozoal merozoites were observed. The merozoites were oval, 2-4 microm in width and 4-6 microm in length, and had linear arrays of nuclear material concentrated at one pole. Serum was positive for Sarcocystis sp. antibodies and negative for Toxoplasma gondii antibodies. The organism was determined to be either Sarcocystis neurona or Sarcocystis dasypi based on sequence analysis of the internal transcribed spacer 1 ribosomal RNA genomic region. Clinical disease resolved following treatment with 3 different protocols for protozoal infection. This case is the first to demonstrate the antemortem diagnosis and survival of a domestic cat with Sarcocystis sp.-associated encephalomyelitis. Clinicians and cytopathologists should include Sarcocystis sp. as a differential for feline inflammatory central nervous system disease characterized by neutrophilic pleocytosis.

Effect of furosemide and furosemide-carbazochrome combination on exercise-induced pulmonary hemorrhage in Standardbred racehorses.

The objective was to quantify the effect of furosemide and carbazochrome on exercise-induced pulmonary hemorrhage (EIPH) in Standardbred horses using red blood cell count and hemoglobin concentration in bronchoalveolar lavage (BAL) fluid. Six healthy Standardbred horses with prior evidence of EIPH performed a standardized treadmill test 4 h after administration of placebo, furosemide, or furosemide-carbazochrome combination. Red blood cell (RBC) counts and hemoglobin concentrations were determined on the BAL fluid. The RBC count in BAL ranges were (2903-26,025 cells/microL), (45-24,060 cells/microL), and (905-3045 cells/microL) for placebo, furosemide, and furosemide-carbazochrome, respectively. Hemoglobin concentration ranges were (0.03-0.59 mg/mL), (0.01-0.55 mg/mL), and (0.007-0.16 mg/mL) for placebo, furosemide, and furosemide-carbazochrome groups, respectively. No significant differences were detected among treatments. However, there was great variability among horses, suggesting that a larger sample size or better selection of horses was needed.

Relationship between separation time of plasma from heparinized whole blood on plasma biochemical analytes of loggerhead sea turtles (Caretta caretta).

Concentrations and activities of selected biochemicals of loggerhead sea turtles (Caretta caretta) were determined for plasma that was separated from whole blood samples that were kept up to 96 hr post collection (PC) in a refrigerator. Blood samples collected from seven juvenile captive loggerhead sea turtles were added to tubes containing lithium heparin and were placed on ice. Equal amounts of anticoagulated whole blood from the lithium heparin tubes were then aliquoted into plastic tubes and stored as whole blood under refrigeration until they were centrifuged at 0, 4, 24, 48, and 96 hr PC. Plasma was removed and the analytes that were measured were alkaline phosphatase (ALP), aspartate aminotransferase (AST), gamma glutamyl transferase (GGT), creatine kinase (CK), sodium, chloride, potassium, magnesium, calcium, phosphorus, cholesterol, glucose, urea nitrogen, uric acid, total protein, albumin, and globulin. Compared with values at 0 time, the only analyte to be significantly different at 24 hr PC was GGT (activity decreased by 25%). Compared with values at 0 time, significant differences at 96 hr PC were only seen in AST (2% increase), GGT (25% decrease), glucose (7% decrease), and uric acid (25% increase). Although a statistically significant difference was found in concentrations of phosphorus and cholesterol over time by repeated measures analysis of variance (ANOVA), the follow-up multiple comparison procedure could not define the specific time points at which significant differences occurred. For all other analytes, significant differences over the time course of the study were not found. In these instances, the power of the ANOVA was sufficient (> or = 0.80) to detect any arithmetic differences of a clinically relevant magnitude. Although plasma should be separated from the cellular component of blood as soon as possible PC, in a field situation in which a centrifuge is unavailable, samples can be stored in a portable cooler up to 24 hr without appreciable change in select biochemical analytes.

Results of diagnostic ophthalmic testing in healthy guinea pigs.

OBJECTIVE-To report values for tear production, central corneal touch threshold (CTT), and intraocular pressure (IOP) in healthy guinea pigs and determine results of aerobic bacterial culture and cytologic examination of conjunctival swab specimens. DESIGN-Cross-sectional study. ANIMALS-31 Healthy guinea pigs (62 eyes) of various ages and breeds. PROCEDURES-Tear production was measured by the phenol red thread tear test (PRT) and Schirmer tear test (STT) before and after topical anesthetic application, CTT was measured with an esthesiometer, and IOP was measured by applanation tonometry. RESULTS-Combining data from all eyes, mean +/- SD PRT values before and after topical anesthetic administration were 21.26 +/- 4.19 mm/15 s and 22.47 +/- 3.31 mm/15 s, respectively, and mean IOP was 18.27 +/- 4.55 mm Hg. Median STT values before and after topical anesthetic administration were 3 mm/min (range, 0 to 12 mm/min) and 4 mm/min (range, 0 to 11 mm/min), respectively, and median CTT was 2.0 cm (range, 0.5 to 3.0 cm). Values did not differ between eyes for any test, but significant differences were identified for PRT values between males and females and between values obtained before and after topical anesthetic administration. Common bacterial isolates included Corynebacterium spp, Streptococcus spp, and Staphylococcus spp. Cytologic examination of conjunctival swab specimens revealed mainly basal epithelial cells; lymphocytes were common. CONCLUSIONS AND CLINICAL RELEVANCE-Results provided information on values for PRT, STT, CTT, and IOP in healthy guinea pigs and on expected findings for aerobic bacterial culture and cytologic examination of conjunctival swab specimens.