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.

Identification of Cytauxzoon felis antigens via protein microarray and assessment of expression library immunization against cytauxzoonosis.

BACKGROUND: Cytauxzoonosis is a disease of felids in North America caused by the tick-transmitted apicomplexan parasite Cytauxzoon felis. Cytauxzoonosis is particularly virulent for domestic cats, but no vaccine currently exists. The parasite cannot be cultivated in vitro, presenting a significant limitation for vaccine development. METHODS: Recent sequencing of the C. felis genome has identified over 4300 putative protein-encoding genes. From this pool we constructed a protein microarray containing 673 putative C. felis proteins. This microarray was probed with sera from C. felis-infected and naïve cats to identify differentially reactive antigens which were incorporated into two expression library vaccines, one polyvalent and one monovalent. We assessed the efficacy of these vaccines to prevent of infection and/or disease in a tick-challenge model. RESULTS: Probing of the protein microarray resulted in identification of 30 differentially reactive C. felis antigens that were incorporated into the two expression library vaccines. However, expression library immunization failed to prevent infection or disease in cats challenged with C. felis. CONCLUSIONS: Protein microarray facilitated high-throughput identification of novel antigens, substantially increasing the pool of characterized C. felis antigens. These antigens should be considered for development of C. felis vaccines, diagnostics, and therapeutics.

Rapid High-Resolution Melt Analysis of Cytauxzoon felis Cytochrome b To Aid in the Prognosis of Cytauxzoonosis.

Cytauxzoon felis is a virulent, tick-transmitted, protozoan parasite that infects felines. Cytauxzoonosis was previously thought to be uniformly fatal in domestic cats. Treatment combining atovaquone and azithromycin (A&A) has been associated with survival rates of over 60%. Atovaquone, a ubiquinone analogue, targets C. felis cytochrome b (cytb), of which 30 unique genotypes have been identified. The C. felis cytb genotype cytb1 is associated with increased survival rates in cats treated with A&A. The purpose of this study was to design a PCR panel that could distinguish C. felis cytb1 from other cytochrome b genotypes. Primer pairs were designed to span five different nucleotide positions at which single-nucleotide polymorphisms in the C. felis cytb gene had been identified. Through the use of high-resolution melt analysis, this panel was predicted to distinguish cytb1 from other cytb genotypes. Assays were validated using samples from 69 cats with cytauxzoonosis for which the C. felis cytb genotypes had been characterized previously. The PCR panel identified C. felis cytb1 with 100% sensitivity and 98.2% specificity. High-resolution melt analysis can rapidly provide prognostic information for clients considering A&A treatment in cats with cytauxzoonosis.

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.

Pharmacogenomics of Cytauxzoon felis cytochrome b: implications for atovaquone and azithromycin therapy in domestic cats with cytauxzoonosis.

Cytauxzoon felis, an emerging virulent protozoan parasite that infects domestic cats, is treated with atovaquone and azithromycin (A&A). Atovaquone targets parasite cytochrome b. We characterized the C. felis cytochrome b gene (cytb) in cats with cytauxzoonosis and found a cytb genotype that was associated with survival in A&A-treated cats.

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.

Characterization of the intrarenal renin-angiotensin system in cats with naturally occurring chronic kidney disease.

BACKGROUND: The role of the renin-angiotensin-aldosterone system in cats with chronic kidney disease (CKD) is incompletely understood. OBJECTIVE: To characterize components of the intrarenal renin-angiotensin system (RAS) in cats with CKD. ANIMALS: Eleven cats with naturally occurring CKD (CKD group) and 8 healthy control cats. METHODS: Renal tissue samples were evaluated by reverse-transcription polymerase chain reaction for renin, angiotensinogen, angiotensin-converting enzyme (ACE), and angiotensin II type 1 receptor transcript levels, and by liquid chromatography-mass spectrometry for quantification of angiotensin I, II, III, and IV concentrations. Linear mixed models were used to compare gene transcript levels and concentrations of angiotensin peptides between groups. RESULTS: Cats of the CKD group were significantly older (P < .001) and more likely to be neutered (P = .007) than healthy control cats. Kidneys from cats with CKD had significantly higher transcript levels of angiotensinogen (P < .001) and lower transcript levels of ACE (P < .001) than those from control cats. Renal angiotensin I concentrations were increased in CKD compared with control kidneys (P = .001). No other significant differences in renal transcript levels or angiotensin peptide concentrations were noted between groups. CONCLUSION AND CLINICAL IMPORTANCE: The intrarenal RAS might be activated in cats with CKD. Small sample size and differences in age, neuter status, and dietary sodium intake between groups might have limited the ability to identify a significant difference in concentration of renal angiotensin II.

Platelet number and function in response to a single intravenous dose of vincristine.

BACKGROUND: Vincristine might increase circulating platelet numbers but the functional capacity of these newly released platelets is unknown. OBJECTIVE: To evaluate and compare the functionality of mature and immature (reticulated) platelets after a single intravenous dose of vincristine in dogs. ANIMALS: Ten healthy purpose-bred dogs. METHODS: Dogs prospectively received a single IV injection of 0.02 mg/kg vincristine or 0.9% saline. Before and after treatment on days 3, 5, and 7, platelets (resting and after thrombin stimulation) were assessed by flow cytometric determination of P-selectin (CD62P) expression. Reticulated platelets were distinguished using thiazole orange (TO) staining. RESULTS: Relative to saline, vincristine administration increased platelet count from day 0 to day 7 (225 ± 58 to 273 ± 65 × 103 /µL, vs 299 ± 76.4 to 214 ± 20 × 103 /µL, P = .01) and increased percentage of reticulated platelets from day 0 to day 5 (3.9 ± 1.5% to 6.1 ± 1.6%, P = .02). On all days, reticulated platelets had greater resting expression of CD62P than did mature platelets (49.6 ± 4% vs 10.2 ± 1%, P ≤ .001). Across all days, CD62P expression by reticulated platelets in the vincristine and saline-treated groups was not different when unstimulated (P = .7) or after thrombin stimulation (P = .33). CONCLUSIONS AND CLINICAL IMPORTANCE: Reticulated platelets released in response to vincristine administration function similarly to mature platelets.

Analysis of genes associated with proinflammatory and profibrotic pathways upregulated in ischemia-induced chronic kidney disease in cats.

OBJECTIVE: To use RNA sequencing (RNAseq) to characterize renal transcriptional activities of genes associated with proinflammatory and profibrotic pathways in ischemia-induced chronic kidney disease (CKD) in cats. SAMPLES: Banked renal tissues from 6 cats with experimentally induced CKD (renal ischemia [RI] group) and 9 healthy cats (control group). PROCEDURES: Transcriptome analysis with RNAseq, followed by gene ontology and cluster analyses, were performed on banked tissue samples of the right kidneys (control kidneys) from cats in the control group and of both kidneys from cats in the RI group, in which unilateral (right) RI had been induced 6 months before the cats were euthanized and the ischemic kidneys (IKs) and contralateral nonischemic kidneys (CNIKs) were harvested. Results for the IKs, CNIKs, and control kidneys were compared to identify potential differentially expressed genes and overrepresented proinflammatory and profibrotic pathways. RESULTS: Genes from the gene ontology pathways of collagen binding (eg, transforming growth factor-ß1), metalloendopeptidase activity (eg, metalloproteinase [MMP]-7, MMP-9, MMP-11, MMP-13, MMP-16, MMP-23B, and MMP-28), chemokine activity, and T-cell migration were overrepresented as upregulated in tissue samples of the IKs versus control kidneys. Genes associated with the extracellular matrix (eg, TIMP-1, fibulin-1, secreted phosphoprotein-1, matrix Gla protein, and connective tissue growth factor) were upregulated in tissue samples from both the IKs and CNIKs, compared with tissues from the control kidneys. CONCLUSIONS AND CLINICAL RELEVANCE: Unilateral ischemic injury differentially altered gene expression in both kidneys, compared with control kidneys. Fibulin-1, secreted phosphoprotein-1, and matrix Gla protein may be candidate biomarkers of active kidney injury 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.

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.

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.

Limited genetic variability of Cytauxzoon felis apical membrane antigen-1 (ama1) from domestic cats and bobcats.

BACKGROUND: Cytauxzoon felis is a tick-transmitted apicomplexan that causes cytauxzoonosis in domestic cats (Felis catus). Even with intensive care, the mortality rate of acute cytauxzoonosis approaches 40% in domestic cats, while bobcats (Lynx rufus), the natural intermediate host of C. felis, remain clinically asymptomatic. However, multiple reports of domestic cats surviving acute disease without any treatment exist. One hypothesis for survival of these cats is infection with unique C. felis genotypes of lower pathogenicity. Prior studies have identified genetically distinct C. felis isolates containing polymorphisms within internal transcribed spacer regions (ITS) of the rRNA operon. However, these polymorphisms do not correlate with the clinical outcome of cytauxzoonosis, and so additional genetic markers are needed to test this hypothesis. We selected C. felis apical membrane antigen-1 (ama1) as a potential genetic marker of differential pathogenicity. AMA1 is a vaccine candidate for relatives of C. felis within Plasmodium spp.; however its historically high level of genetic polymorphism has resulted in escape from vaccine-induced immunity. While such diversity has hindered vaccine development, the expected polymorphism within the ama1 gene may be useful to evaluate population genetics. RESULTS: A 677 bp sequence of the C. felis ama1 gene was PCR-amplified from 84 domestic cats and 9 bobcats and demonstrated 99.9% sequence identity across all samples. A single nucleotide polymorphism (SNP) was identified in domestic cats and bobcats with evidence for co-infection with both genotypes identified in two domestic cats. The prevalence of the two genotypes varied with geographical distribution in domestic cats. Nucleotide diversity (π) and haplotype diversity (H) were calculated for C. felis ama1 and ama1 of related apicomplexans to assess genetic diversity. Based on these values (π = 0.00067 and H = 0.457), the diversity of the C. felis ama1 gene region analyzed is considerably lower than what is documented in related apicomplexans. CONCLUSIONS: In surprising contrast to related apicomplexans, our results support that the sequence of the C. felis ama1 gene is highly conserved. While lack of genetic diversity limits utility of C. felis AMA1 as a genetic marker for clinical outcome, it supports further investigation as a vaccine candidate for cytauxzoonosis.

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.

Genetic conservation of Cytauxzoon felis antigens and mRNA expression in the schizont life-stage.

Cytauxzoonosis is a highly fatal disease of domestic cats caused by the apicomplexan protozoan Cytauxzoon felis, which is most closely related to Theileria spp. The growing prevalence, high morbidity and mortality, and treatment cost of cytauxzoonosis emphasize the need for vaccine development. Traditional approaches for vaccine development, however, have been hindered by the inability to culture C. felis in vitro. Recent availability of the annotated C. felis genome combined with genome-based vaccine design and protein microarray immunoscreening allowed for high-throughput identification of C. felis antigens that could serve as vaccine candidates. This study assessed the suitability of three of these vaccine candidates (cf30, cf63, cf58) in addition to a previously reported vaccine candidate (cf76) based on two criteria: genetic conservation among diverse C. felis geographic isolates and expression in tissues containing the C. felis schizont life stage, which has been previously associated with the development of a protective immune response. A comparison of seventeen C. felis isolates across seven states demonstrated high sequence identity (99-100%) for cf30, cf63, and cf58, similar to the degree of conservation previously reported for cf76. RNAscope® in situ hybridization using acutely infected feline splenic tissue revealed robust levels of all transcripts in the schizont life stage of the parasite. These data support the suitability of these three antigens for further investigation as vaccine candidates against cytauxzoonosis.

Cerebrospinal fluid from a dog with hind limb ataxia.

A 9-year-old spayed female German Shepherd dog with a history of orthopedic disease was presented to the North Carolina State University Veterinary Teaching Hospital for evaluation of recent, progressive, bilateral, hindlimb ataxia. Analysis of cisternal and lumbar cerebrospinal fluid (CSF) samples revealed normal total nucleated cell counts and a mild increase in protein concentration in the lumbar sample. In cytocentrifuged specimens of both CSF samples, aggregates of refractile, angular to irregular, pale blue to colorless, crystalline material were observed in the background. Some of the material appeared birefringent under polarized light. Differentials for the material included contrast agent, epidural anesthetics or other pharmacologic agents, or artifact introduced through sample processing, collection, or handling. Based on investigation of clinical and laboratory processes it was determined that tubes used to collect CSF in the hospital recently had been changed from additive-free glass tubes to silica-coated shatter-resistant plastic tubes (BD Vacutainer Plus serum tubes, silicone-coated, Becton Dickinson). A cytocentrifuged preparation of saline placed in a silica-coated tube contained crystalline material identical to that observed in the CSF samples; saline placed in an additive-free glass tube contained no material. In this case, we document the microscopic appearance of highly concentrated silica particles in cytocentrifuged preparations of CSF and underscore the importance of recognizing and identifying this artifact in cytologic preparations.

Deep and superficial skin scrapings from a feline immunodeficiency virus-positive cat.

An 8-year-old, neutered male, domestic shorthair cat housed at the North Carolina State University, College of Veterinary Medicine, Laboratory Animal Research facility as part of a research colony was examined because of mulifocal skin lesions. The lesions consisted of patchy alopecia with mild crusting of the periauricular region, neck, and dorsum; periauricular excoriations; marked dorsal seborrhea and scaling; and generalized erythematous papules. A moderate amount of ceruminous exudate was present in both ear canals. Results of testing for feline immunodeficiency virus (FIV) were positive. An ear swab specimen and superficial and deep skin scrapings were obtained, mounted with oil on glass slides, and coverslipped for microscopic examination. Two populations of mites were observed: a large population of slender, long (approximately 200 microm), adult mites with long, tapering abdomens that comprised two-thirds of the total body length; and a smaller population of more translucent and shorter mites (approximately 100 microm) with wide, blunt abdomens that had prominent transverse ridges. The interpretation was demodicosis, with Demodex cati and D gatoi co-infection. Histologic sections of biopsies from skin lesions on the neck, dorsum, and periauricular area contained a mild perivascular and perifollicular inflammatory infiltrate composed predominantly of histiocytes, lymphocytes, and plasma cells. Diffusely within the follicular lumina and occasionally within the superficial keratin, a myriad of Demodex organisms were observed. Intrafollicular mites were compatible in appearance with D cati whereas those in the corneal layer were suggestive of D gatoi. Demodicosis is an uncommon disease of cats, and rare cases of dual infection have been documented, occasionally in FIV-infected cats. The dual infection emphasizes the importance of doing both superficial and deep skin scrapings and of recognizing the unique microscopic features of different Demodex mites.

Diagnosis and management of pyothorax in a domestic ferret (Mustela putorius furo).

OBJECTIVE: To describe the diagnosis, management, and outcome of pyothorax in a domestic ferret (Mustela putorius furo). CASE SUMMARY: A domestic ferret was evaluated for a history of lethargy, anorexia, and pyrexia. Pleural effusion was detected with radiography and ultrasonography, and a diagnosis of pyothorax was made following cytologic evaluation of pleural fluid. Bilateral thoracostomy tubes were placed for thoracic drainage and lavage, and the ferret was treated with intravenous crystalloid fluids, antimicrobials, and analgesics. Bacterial culture of the pleural fluid yielded Fusobacterium spp. and Actinomyces hordeovulneris. This treatment protocol resulted in resolution of pyothorax, and a positive clinical outcome. NEW OR UNIQUE INFORMATION PROVIDED: This is the first reported case of successful management of pyothorax caused by Fusobacterium spp. and A. hordeovulneris in a ferret.

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.

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.