Optical coherence tomography for surgical margin evaluation of excised canine cutaneous and subcutaneous tumours.

Currently, intraoperative tumour margin imaging is not routinely utilized in veterinary medicine. Optical coherence tomography (OCT) allows for real-time assessment of tissue morphology of 1-2 mm depth. The aims of this study were (1) to compare the histologic and OCT features of excised canine skin and subcutaneous specimens, and (2) to determine the diagnostic accuracy of OCT for surgical margin evaluation. The authors hypothesized that OCT imaging would correlate well with histopathology and that OCT would be sensitive for detection of incomplete margins. Eighty dogs were prospectively enrolled. Tumours were excised, and the surgical margins were imaged using a spectral domain OCT system. The tumour type and completeness of excision were determined by histopathology. Nine blinded observers received training in OCT image interpretation and were then given a set of OCT images and videos. The observers assigned each image/video a grade from 1 (no tumour) to 4 (tumour) and the results were compared to histopathology. The overall median sensitivity and specificity of OCT imaging for detection of incomplete margins were 86.7% and 84.6%, respectively. A potential limitation is that observers had varied experience with OCT image interpretation, ranging from no prior experience to participating in a previous OCT project. OCT is sensitive for detection of incomplete margins and could be a promising real-time surgical margin imaging modality. Further study is needed to evaluate intraoperative applications of OCT and its impact on tumour recurrence and long-term outcome.

Pathology in Practice.

In collaboration with the American College of Veterinary Pathologists.

Treatment of indolent cutaneous T-cell lymphoma with hypofractionated radiation therapy in three cats.

BACKGROUND: Feline indolent cutaneous T-cell lymphoma (ICL) is an uncommon neoplastic disease. There is currently no consensus on treatment recommendations for ICL. OBJECTIVE: To report the clinical outcome of three cats with ICL treated with hypofractionated electron-beam radiotherapy (RT). ANIMALS: Three privately owned cats with ICL. MATERIALS AND METHODS: Medical records and client surveys were reviewed. A diagnosis of probable ICL was based on history, clinical presentation and histopathological findings, and confirmed using CD3 immunohistochemical analysis and PCR for antigen receptor gene rearrangement (PARR). All cats were treated with hypofractionated RT (four fractions of 8 Gy). RESULTS: All cats presented with skin lesions characterised by erythema and alopecia that were refractory to previous treatment with systemic glucocorticoids. Before hypofractionated RT treatment, lesions were histologically described as having diffuse infiltration of the dermis with CD3+ T cells. Molecular clonality analysis revealed clonal T-cell receptor gamma gene rearrangement. After RT, two cats showed histological improvement defined by decreased infiltration of lymphocytes, with cellular infiltrate present only in the deeper dermis; one cat had near complete histological resolution of lesions with only minimal residual lymphocytes. One cat was determined to have a complete clinical response while the other showed partial responses. No acute adverse effects of radiation were observed; chronic effects included leukotrichia, partial alopecia and mild fibrosis. All clients reported improvement in quality of life for their cats. CONCLUSIONS AND CLINICAL IMPORTANCE: Clinical and histological improvement in these cats suggests that hypofractionated RT can be a useful treatment modality for cats with ICL.

Is low-level laser therapy useful as an adjunctive treatment for canine acral lick dermatitis? A randomized, double-blinded, sham-controlled study.

BACKGROUND: Conventional therapy for canine acral lick dermatitis (ALD) consists of systemic antibiotics and anti-anxiety medications. Low-level laser therapy (LLLT) is a noninvasive therapy used to treat inflammatory and painful conditions. HYPOTHESIS/OBJECTIVES: The primary objective was to determine whether LLLT with conventional therapy would be beneficial as an adjunct treatment for ALD. We hypothesized that LLLT and conventional therapy combined would result in a greater reduction in licking Visual Analog Score (LVAS) compared to conventional therapy alone. Secondary objectives were to assess change in lesion/ulcer size, thickness and hair growth. ANIMALS: Thirteen dogs with a skin lesion consistent with ALD. METHODS AND MATERIALS: Dogs were randomly assigned to two groups. All dogs received systemic antibiotics and trazodone. The treatment group (TG) received LLLT by laser (130 mW, 2 min) with blue and red light-emitting diodes (LEDs), while the control group (CG) had sham therapy (laser/LEDs off). Treatments were administered three times weekly for two weeks, then twice weekly for two weeks for a total of 10 visits. Descriptive statistics were performed (mean, median); primary and secondary objectives were assessed with nonparametric ANOVA (Kruskal-Wallis test), with significance set at P < 0.05. RESULTS: Thirteen dogs (six CG, seven TG) were enrolled. There were no significant differences in median LVAS, lesion/ulcer size or thickness of the ALD lesion between TG and CG. There was a significantly greater increase (24%) in hair growth in TG (P = 0.0081) compared to CG. CONCLUSIONS AND CLINICAL RELEVANCE: Treatment of ALD requires multimodal therapy. Although combining LLLT with conventional therapy did not result in a significantly greater reduction in LVAS, there was a significant increase in hair growth compared to conventional therapy alone.

Antibody-Mediated Protection against Staphylococcus aureus Dermonecrosis: Synergy of Toxin Neutralization and Neutrophil Recruitment.

The staphylococcal α-hemolysin is critical for the pathogenesis of Staphylococcus aureus skin and soft tissue infection. Vaccine and infection-elicited α-hemolysin-specific antibodies protect against S. aureus‒induced dermonecrosis, a key feature of skin and soft tissue infection. Many interactions between α-hemolysin and host cells have been identified that promote tissue damage and modulate immune responses, but the mechanisms by which protective adaptive responses cross talk with innate responses at the tissue level are not clear. Using an established mouse model of skin and soft tissue infection and a newly developed histopathologic scoring system, we observed pathologic correlates early after infection, predicting protection against dermonecrosis by anti-α-hemolysin antibody. Protection was characterized by robust neutrophilic inflammation and compartmentalization of bacteria into discrete abscesses, which led to the attenuation of dermonecrosis and enhancement of bacterial clearance later in the infection. The ultimate outcome of infection was driven by the recruitment of neutrophils within the first day after infection but not later. Antibody-mediated protection was dependent on toxin neutralization rather than on enhanced opsonophagocytic killing by neutrophils or protection against toxin-mediated neutrophil lysis. Together, these findings advance our understanding of the mechanisms by which the early synergism between antibody-mediated toxin neutralization and tissue-specific neutrophilic inflammation preserve tissue integrity during infection.

Acquired left-to-right shunting through a valve-incompetent foramen ovale in a cat with hypertrophic cardiomyopathy and congestive heart failure.

CASE SUMMARY: An 8-year-old spayed female domestic shorthair cat was presented for a recheck evaluation of hypertrophic cardiomyopathy and chronic kidney disease. Three years prior to presentation, the patient was diagnosed with obstructive hypertrophic cardiomyopathy and started on atenolol. The left ventricular outflow tract obstruction subsequently resolved. Biochemical analysis a week prior to presentation demonstrated severe azotemia. Transthoracic echocardiograph revealed pericardial effusion, pleural effusion, severe left ventricular concentric hypertrophy, severe left atrial enlargement and continuous left-to-right flow through the interatrial septum near the fossa ovalis. The patient was euthanized owing to poor prognosis, and gross examination at necropsy revealed a valve-incompetent patent foramen ovale secondary to severe left atrial dilation. RELEVANCE AND NOVEL INFORMATION: To our knowledge, this is the first report of an acquired left-to-right shunt through a valve-incompetent foramen ovale in a cat with hypertrophic cardiomyopathy. Severe left atrial dilation was suspected to cause interatrial shunting through the valve-incompetent foramen ovale, and this finding may be relevant to echocardiographic evaluations in other cats.

Pharmacokinetics and Tolerability of the Novel Non-immunosuppressive Fingolimod Derivative, OSU-2S, in Dogs and Comparisons with Data in Mice and Rats.

In this study, we characterized the pharmacokinetics of OSU-2S, a fingolimod-derived, non-immunosuppressive phosphatase activator, in mice, rats, and dogs, as well as tolerability and food effects in dogs. Across all species tested, plasma protein binding for OSU-2S was > 99.5%, and metabolic stability and hepatic intrinsic clearance were in the moderate range. OSU-2S did not significantly modulate CYP enzyme activity up until 50 µM, and Caco-2 data suggested low permeability with active efflux at 2 µM. Apparent oral bioavailability in mice was 16% and 69% at 10 and 50 mg/kg, respectively. In rats, bioavailability was 24%, 35%, and 28% at 10, 30, and 100 mg/kg, respectively, while brain/plasma ratio was 36 at 6-h post-dose at 30 mg/kg. In dogs, OSU-2S was well tolerated with oral capsule bioavailability of 27.5%. Plasma OSU-2S exposures increased proportionally over a 2.5-20 mg/kg dose range. After 4 weeks of 3 times weekly, oral administration (20 mg/kg), plasma AUClast (26.1 µM*h), and Cmax (0.899 µM) were nearly 2-fold greater than those after 1 week of dosing, and no food effects were observed. The elimination half-life (29.7 h), clearance (22.9 mL/min/kg), and plasma concentrations of repeated oral doses support a 3-times weekly dosing schedule in dogs. No significant CBC, serum biochemical, or histopathological changes were observed. OSU-2S has favorable oral PK properties similar to fingolimod in rodents and dogs and is well tolerated in healthy animals. This work supports establishing trials of OSU-2S efficacy in dogs with spontaneous tumors to guide its clinical development as a cancer therapeutic for human patients.

Evaluation of microwave ablation for local treatment of dogs with distal radial osteosarcoma: A pilot study.

OBJECTIVE: To evaluate the feasibility and safety of microwave ablation (MWA) as a modality to induce tumor necrosis within distal radial osteosarcoma (OSA). STUDY DESIGN: Pilot study. ANIMALS: Six client-owned dogs with distal radius OSA confirmed by cytological examination. METHODS: Dogs underwent computed tomography for surgical planning before general anesthesia for fluoroscopy-guided ablation. Computed tomography was repeated 48 hours after MWA, before amputation. The ablated tumor was evaluated with histopathology. RESULTS: Six dogs underwent MWA of distal radius OSA. A lower power setting (30 W) was selected for the first two dogs to avoid collateral soft tissue damage. The power was increased to 75 W for the last four dogs. The temperature was maintained between 45°C and 55°C (113 °F-131 °F) at the bone/soft tissue interface. Tumor necrosis varied between 30% and 90% (median, 55%) according to histopathology. No intraoperative or periprocedural complications were observed. CONCLUSION: Microwave ablation induced variable tumor necrosis and did not induce immediate postablation complications in these six dogs with distal radius OSA. CLINICAL SIGNIFICANCE: These results justify further evaluation of MWA as a potential modality to treat primary bone lesions in dogs.

Continuous Microevolution Accelerates Disease Progression during Sequential Episodes of Infection.

Bacteria adapt to dynamic changes in the host during chronic and recurrent infections. Bacterial microevolution is one type of adaptation that imparts a selective advantage. We hypothesize that recurrent episodes of disease promote microevolution through genetic mutations that modulate disease severity. We use a pre-clinical model of otitis media (OM) to determine the potential role for microevolution of nontypeable Haemophilus influenzae (NTHI) during sequential episodes of disease. Whole genome sequencing reveals microevolution of hemoglobin binding and lipooligosaccharide (LOS) biosynthesis genes, suggesting that adaptation of these systems is critical for infection. These OM-adapted strains promote increased biofilm formation, inflammation, stromal fibrosis, and an increased propensity to form intracellular bacterial communities (IBCs). Remarkably, IBCs remain for at least one month following clinical resolution of infection, suggesting an intracellular reservoir as a nidus for recurrent OM. Additional approaches for therapeutic design tailored to combat this burdensome disease will arise from these studies.

MOOSE ( ALCES ALCES) MORTALITY ASSOCIATED WITH CAPRINE HERPESVIRUS 2 (CPHV-2) IN A ZOOLOGICAL COLLECTION.

Malignant catarrhal fever (MCF) can affect both domestic and wild artiodactyls. In a zoological setting, in which subclinical carriers and susceptible species are often housed in close proximity, the disease can prove fatal. This report describes a case of goat-associated MCF in a captive moose ( Alces alces). The diagnosis was confirmed by histopathology, which showed lymphocytic vasculitis in the brain and panuveitis, and by detection of caprine herpesvirus 2 DNA in tissues. Identical viral DNA sequences amplified from the clinically affected moose and from domestic, petting goats ( Capra aegagrus hircus) housed in the zoo suggest that the goats were the source for the virus transmutation. This is the first report, to our knowledge, of confirmed goat-associated MCF in any moose in North America and of the surveillance measures and procedures put in place to prevent additional spread of the disease.

Pulmonary capillary hemangiomatosis and hypertrophic cardiomyopathy in a Persian cat.

Pulmonary capillary hemangiomatosis (PCH) and pulmonary veno-occlusive disease (PVOD) are rare causes of primary pulmonary hypertension in humans, and, in 2016, were reported in dogs. A 1-y-old, neutered male Persian cat was presented for autopsy after sudden death several hours after grooming. Grossly, the lungs were mottled red-to-pink, contained rubbery-to-firm nodular foci, and there was moderate-to-marked left-sided cardiomegaly and left atrial dilation, consistent with hypertrophic cardiomyopathy. Microscopically, there was multifocal to regionally extensive capillary proliferation within pulmonary alveolar septa and around respiratory bronchioles, with nodular aggregates of densely arranged capillaries that replaced pulmonary alveolar spaces. Rare occlusive venous remodeling was identified in Verhoeff-van Gieson-stained sections. The gross and microscopic changes were consistent with PCH with rare features of PVOD. Hypertrophic cardiomyopathy was interpreted as potentially contributing to the cause of death, but unrelated to the pulmonary vascular proliferation.

Unsupervised learning techniques reveal heterogeneity in memory CD8+ T cell differentiation following acute, chronic and latent viral infections.

CD8+ T lymphocytes are critical for the control of gammaherpesvirus latency. To determine how memory CD8+ T cells generated during latency differ from those primed during acute or chronic viral infection, we adoptively transferred naive P14 CD8+ T cells into uninfected recipients, and examined surface proteins, cytokines and transcription factors following infection with the Armstrong (acute) or Clone 13 (chronic) strains of lymphocytic choriomeningitis virus (LCMV), or murine gammaherpesvirus 68 (MHV68) expressing the LCMV epitope DbGP33-41. By performing k-means clustering and generating self organizing maps (SOM), we observed increased short-lived effector-like, CD27lo CD62Llo and Bcl-6lo CD8+ T cells following latent infection. In addition, we found that memory CD8+ T cells from latent primed mice underwent less expansion following adoptive transfer and antigen rechallenge. Data from cluster models were combined and visualized by principal component analysis (PCA) demonstrating memory CD8+ T cells from latent infection occupy an intermediate differentiation space.

C4 Deficiency is a predisposing factor for Streptococcus pneumoniae-induced autoantibody production.

Reductions in C4 levels may predispose individuals to infection with encapsulated bacteria as well as autoimmunity. In this study, we examined the role C4 has in protection against Streptococcus pneumoniae-induced autoimmunity. Mild respiratory infection with serotype 19F pneumococci selectively induced systemic anti-dsDNA IgA production in naive C4(-/-) mice, but not in C3(-/-) or wild-type mice. Systemic challenge with virulent serotype 3 pneumococci also induced anti-dsDNA IgA production in immune C4(-/-) mice. Remarkably, pneumococcal polysaccharide (PPS) vaccination alone induced C4(-/-) mice to produce increased anti-dsDNA IgA levels that were maintained in some mice for months. These effects were most pronounced in female C4(-/-) mice. Importantly, immunization-induced increases in anti-dsDNA IgA levels were strongly associated with increased IgA deposition in kidneys. Cross-reactivity between pneumococcal Ags and dsDNA played a partial role in the induction of anti-dsDNA IgA, but a major role for PPS-associated TLR2 agonists was also revealed. Administration of the TLR2/4 antagonist, OxPAPC, at the time of PPS immunization completely blocked the production of anti-dsDNA IgA in C4(-/-) mice without suppressing PPS-specific Ab production. The TLR2 agonist, Pam3CSK4, similarly induced anti-dsDNA IgA production in C4(-/-) mice, which OxPAPC also prevented. LPS, a TLR4 agonist, had no effect. Pam3CSK4, but not LPS, also induced dsDNA-specific IgA production by C4(-/-) splenic IgA(+) B cells in vitro, indicating that TLR2 agonists can stimulate autoantibody production via B cell-intrinsic mechanisms. Collectively, our results show an important role for C4 in suppressing autoantibody production elicited by cross-reactive Ags and TLR2 agonists associated with S. pneumoniae.

Type I interferon signaling enhances CD8+ T cell effector function and differentiation during murine gammaherpesvirus 68 infection.

UNLABELLED: CD8(+) T cell responses are critical to the control of replication and reactivation associated with gammaherpesvirus infection. Type I interferons (IFNs) have been shown to have direct and indirect roles in supporting CD8(+) T cell development and function during viral infection; however, the role of type I interferons during latent viral infection has not been examined. Mice deficient in type I IFN signaling (IFNAR1(-/-) mice) have high levels of reactivation during infection with murine gammaherpesvirus 68 (MHV68), a murine gammaherpesvirus model for Epstein-Barr virus. We hypothesized that type I IFNs function to enhance the anti-gammaherpesvirus CD8(+) T cell response. To test this, IFNAR1(-/-) mice were infected with MHV68 and the CD8(+) T cell response was analyzed. In the absence of type I IFN signaling, there was a marked increase in short-lived effector CD8(+) T cells, and MHV68-specific CD8(+) T cells had upregulated expression of PD-1 and reduced tumor necrosis factor alpha (TNF-α), gamma IFN (IFN-γ), and interleukin-2 (IL-2) production. Suppressing MHV68 replication early in infection using the antiviral cidofovir rescued CD8(+) T cell cytokine production and reduced PD-1 expression. However, suppressing high levels of reactivation in IFNAR1(-/-) mice failed to improve CD8(+) T cell cytokine production during latency. T cell-specific abrogation of type I IFN signaling showed that the effects of type I IFNs on the CD8(+) T cell response during MHV68 infection are independent of direct type I IFN signaling on T cells. Our findings support a model in which type I IFNs likely suppress MHV68 replication, thus limiting viral antigen and facilitating an effective gammaherpesvirus-directed CD8(+) T cell response. IMPORTANCE: The murine gammaherpesvirus MHV68 has both genetic and biologic homology to the human gammaherpesvirus Epstein-Barr virus (EBV), which infects over 90% of humans. Latent EBV infection and reactivation are associated with various life-threatening diseases and malignancies. Host suppression of gammaherpesvirus latency and reactivation requires both CD8(+) T cells as well as type I interferon signaling. Type I IFNs have been shown to critically support the antiviral CD8(+) T cell response in other virus models. Here, we identify an indirect role for type I IFN signaling in enhancing gammaherpesvirus-specific CD8(+) T cell cytokine production. Further, this function of type I IFN signaling can be partially rescued by suppressing viral replication during early MHV68 infection. Our data suggest that type I IFN signaling on non-T cells can enhance CD8(+) T cell function during gammaherpesvirus infection, potentially through suppression of MHV68 replication.

Condylomatous genital lesions in cynomolgus macaques from Mauritius.

Genital condyloma-like lesions were observed on male and female cynomolgus macaque monkeys (Macaca fascicularis) originating from the island of Mauritius. Cytobrush and/or biopsy samples were obtained from lesions of 57 affected macaques. Primary histologic features included eosinophilic, neutrophilic, and lymphoplasmacytic penile and vulvar inflammation, epidermal hyperplasia with acanthosis, and increased collagenous stroma. Polymerase chain reaction-based assays to amplify viral DNA revealed the presence of macaque lymphocryptovirus (LCV) DNA but not papillomavirus or poxvirus DNA. Subsequent DNA analyses of 3 genomic regions of LCV identified isolates associated with lesions in 19/25 (76%) biopsies and 19/57 (33%) cytology samples. Variable immunolabeling for proteins related to the human LCV Epstein Barr Virus was observed within intralesional plasma cells, stromal cells, and epithelial cells. Further work is needed to characterize the epidemiologic features of these lesions and their association with LCV infection in Mauritian-origin macaques.

An alternative pathway electrosurgical unit injury in a dog.

OBJECTIVE: To report esophageal perforation, caused by alternative current pathway from the use of a monopolar electrosurgery unit (ESU), during routine orthopedic surgery in a dog. STUDY DESIGN: Clinical report. ANIMALS: Two-year-old male Labrador retriever. METHODS: Medial meniscectomy and lateral suture stabilization were performed on a healthy Labrador retriever with a ruptured cranial cruciate ligament. Monopolar electrosurgery was used during the procedure for hemostasis and tissue dissection. Anesthetic monitoring was augmented with an esophageal electrocardiogram (ECG) probe. The day after surgery, the dog appeared dehydrated. After intravenous fluid therapy, respiratory distress was noted and thoracic radiography and contrast fluoroscopy revealed an esophageal perforation. RESULTS: Exploratory surgery was planned to repair the defect. Cardiac arrest occurred after induction. Gross necropsy findings and histopathologic examination revealed lesions consistent with thermal necrosis of the esophagus and myocardial degeneration. An internal investigation of this medical device accident revealed that multiple factors may have contributed to the injury. CONCLUSIONS: An alternative current pathway from the monopolar ESU to the esophageal ECG probe resulted in a full-thickness esophageal thermal injury and cardiac failure.

Magnetic resonance imaging of radiation-induced thymic atrophy as a model for pathologic changes in acute feline immunodeficiency virus infection.

The development of a protocol to reproducibly induce thymic atrophy, as occurs in feline immunodeficiency virus (FIV) infection and other immunosuppressive diseases, and to consistently estimate thymic volume, provides a valuable tool in the search of innovative and novel therapeutic strategies. Magnetic resonance imaging (MRI) using the short tau inversion recovery (STIR) technique, with fat suppression properties, was determined to provide an optimized means of locating, defining, and quantitatively estimating thymus volume in young cats. Thymic atrophy was induced in four, 8-10-week-old kittens with a single, directed 500 cGy dose of 6 MV X-rays from a clinical linear accelerator, and sequential MR images of the cranial mediastinum were collected at 2, 7, 14, and 21 days post irradiation (PI). Irradiation induced a severe reduction in thymic volume, which was decreased, on average, to 47% that of normal, by 7 days PI. Histopathology confirmed marked, diffuse thymic atrophy, characterized by reduced thymic volume, decreased overall cellularity, increased apoptosis, histiocytosis, and reduced distinction of the corticomedullary junction, comparable to that seen in acute FIV infection. Beginning on day 7 PI, thymic volumes rebounded slightly and continued to increase over the following 14 days, regaining 3-35% of original volume. These findings demonstrate the feasibility and advantages of using this non-invasive, in vivo imaging technique to measure and evaluate changes in thymic volume in physiologic and experimental situations. All experimental protocols in this study were approved by the Institutional Animal Care and Use Committee (IACUC) at Auburn University.