Evaluation of immunologic parameters in canine glioma patients treated with an oncolytic herpes virus.

Aim: To molecularly characterize the tumor microenvironment and evaluate immunologic parameters in canine glioma patients before and after treatment with oncolytic human IL-12-expressing herpes simplex virus (M032) and in treatment naïve canine gliomas. Methods: We assessed pet dogs with sporadically occurring gliomas enrolled in Stage 1 of a veterinary clinical trial that was designed to establish the safety of intratumoral oncoviral therapy with M032, a genetically modified oncolytic herpes simplex virus. Specimens from dogs in the trial and dogs not enrolled in the trial were evaluated with immunohistochemistry, NanoString, Luminex cytokine profiling, and multi-parameter flow cytometry. Results: Treatment-naive canine glioma microenvironment had enrichment of Iba1 positive macrophages and minimal numbers of T and B cells, consistent with previous studies identifying these tumors as immunologically "cold". NanoString mRNA profiling revealed enrichment for tumor intrinsic pathways consistent with suppression of tumor-specific immunity and support of tumor progression. Oncolytic viral treatment induced an intratumoral mRNA transcription signature of tumor-specific immune responses in 83% (5/6) of canine glioma patients. Changes included mRNA signatures corresponding with interferon signaling, lymphoid and myeloid cell activation, recruitment, and T and B cell immunity. Multiplexed protein analysis identified a subset of oligodendroglioma subjects with increased concentrations of IL-2, IL-7, IL-6, IL-10, IL-15, TNFα, GM-CSF between 14 and 28 days after treatment, with evidence of CD4+ T cell activation and modulation of IL-4 and IFNγ production in CD4+ and CD8+ T cells isolated from peripheral blood. Conclusion: These findings indicate that M032 modulates the tumor-immune microenvironment in the canine glioma model.

Correction: Two stable variants of Burkholderia pseudomallei strain MSHR5848 express broadly divergent in vitro phenotypes associated with their virulence differences.

[This corrects the article DOI: 10.1371/journal.pone.0171363.].

Draft Genome Sequences of Eight Crimean-Congo Hemorrhagic Fever Virus Strains.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a geographically widespread RNA virus with a high degree of genomic diversity that complicates sequence-based diagnostics. Here, we sequenced eight CCHFV strains for improved assay design and deposition into FDA-ARGOS, the FDA's pathogen database for development and verification of next generation sequencing assays.

Two stable variants of Burkholderia pseudomallei strain MSHR5848 express broadly divergent in vitro phenotypes associated with their virulence differences.

Burkholderia pseudomallei (Bp), the agent of melioidosis, causes disease ranging from acute and rapidly fatal to protracted and chronic. Bp is highly infectious by aerosol, can cause severe disease with nonspecific symptoms, and is naturally resistant to multiple antibiotics. However, no vaccine exists. Unlike many Bp strains, which exhibit random variability in traits such as colony morphology, Bp strain MSHR5848 exhibited two distinct and relatively stable colony morphologies on sheep blood agar plates: a smooth, glossy, pale yellow colony and a flat, rough, white colony. Passage of the two variants, designated "Smooth" and "Rough", under standard laboratory conditions produced cultures composed of > 99.9% of the single corresponding type; however, both could switch to the other type at different frequencies when incubated in certain nutritionally stringent or stressful growth conditions. These MSHR5848 derivatives were extensively characterized to identify variant-associated differences. Microscopic and colony morphology differences on six differential media were observed and only the Rough variant metabolized sugars in selective agar. Antimicrobial susceptibilities and lipopolysaccharide (LPS) features were characterized and phenotype microarray profiles revealed distinct metabolic and susceptibility disparities between the variants. Results using the phenotype microarray system narrowed the 1,920 substrates to a subset which differentiated the two variants. Smooth grew more rapidly in vitro than Rough, yet the latter exhibited a nearly 10-fold lower lethal dose for mice than Smooth. Finally, the Smooth variant was phagocytosed and replicated to a greater extent and was more cytotoxic than Rough in macrophages. In contrast, multiple locus sequence type (MLST) analysis, ribotyping, and whole genome sequence analysis demonstrated the variants' genetic conservation; only a single consistent genetic difference between the two was identified for further study. These distinct differences shown by two variants of a Bp strain will be leveraged to better understand the mechanism of Bp phenotypic variability and to possibly identify in vitro markers of infection.

In vitro anti-tubulin effects of mebendazole and fenbendazole on canine glioma cells.

Benzimidazole anthelmintics have reported anti-neoplastic effects both in vitro and in vivo. The purpose of this study was to evaluate the in vitro chemosensitivity of three canine glioma cell lines to mebendazole and fenbendazole. The mean inhibitory concentration (IC50 ) (±SD) obtained from performing the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay after treating J3T, G06-A, and SDT-3G cells for 72 h with mebendazole were 0.030 ± 0.003, 0.080 ± 0.015 and 0.030 ± 0.006 µM respectively, while those for fenbendazole were 0.550 ± 0.015, 1.530 ± 0.159 and 0.690 ± 0.095 µM; treatment of primary canine fibroblasts for 72 h at IC50 showed no significant effect. Immunofluorescence studies showed disruption of tubulin after treatment. Mebendazole and fenbendazole are cytotoxic in canine glioma cell lines in vitro and may be good candidates for treatment of canine gliomas. Further in vivo studies are required.

Cytologic Criteria for Mast Cell Tumor Grading in Dogs With Evaluation of Clinical Outcome.

A 2-tiered histologic grading scheme for canine cutaneous mast cell tumors (MCTs) is based on morphologic characteristics of neoplastic cells, including karyomegaly, multinucleation, nuclear pleomorphism, and mitotic figures. Aspirates from MCTs may provide the same information more quickly, inexpensively, and less invasively. This study used these criteria to develop a cytologic grading scheme for canine MCTs to predict outcome. Three anatomic pathologists graded histologic samples from 152 canine MCTs. Three clinical pathologists evaluated aspirates from these masses using similar criteria. A cytologic grading scheme was created based on correlation with histologic grade and evaluated with a kappa statistic. Survival was evaluated with Kaplan-Meier survival curves. Cox proportional hazards regression was used to estimate hazard ratios for tumor grades and individual grading components. Simple logistic regression tested for relationships between risk factors and mortality. The cytologic grading scheme that best correlated with histology (kappa = 0.725 ± 0.085) classified a tumor as high grade if it was poorly granulated or had at least 2 of 4 findings: mitotic figures, binucleated or multinucleated cells, nuclear pleomorphism, or >50% anisokaryosis. The cytologic grading scheme had 88% sensitivity and 94% specificity relative to histologic grading. Dogs with histologic and cytologic high grade MCTs were 39 times and 25 times more likely to die within the 2-year follow-up period, respectively, than dogs with low grade MCTs. High tumor grade was associated with increased probability of additional tumors or tumor regrowth. This study concluded that cytologic grade is a useful predictor for treatment planning and prognostication.

A Novel Inherited Cerebellar Abiotrophy in a Cohort of Related Goats.

Cerebellar abiotrophies, also known as cerebellar ataxias, are characterized by premature post-natal degeneration of cerebellar neurons. This report describes the clinical, magnetic resonance imaging (MRI), gross, histopathological and immunohistochemical features of a novel inherited cerebellar abiotrophy in a cohort of three closely related mixed-breed goats (Capra aegagrus hircus) in the southeastern USA. The animals all presented with early juvenile-onset ataxia, hypermetria, wide-based stance, head tremors and nystagmus. On MRI and at gross examination, there was moderate thinning of the cerebellar vermis and sharpening of the folia. Histologically, the vermis, paravermis and flocculonodular lobe had moderate to severe segmental loss of Purkinje cells with sparing of the hemispheres and secondary loss of granule cells and astrogliosis. Heritable cerebellar ataxias have been reported in many domestic animal species, but not, to the authors' knowledge, as a heritable condition in goats.

Nasal tumor with widespread cutaneous metastases in a Golden Retriever.

An intact, 8-year-old, male Golden Retriever dog was presented for evaluation of a nasal mass and approximately 30 firm, raised, variably ulcerated dermal and subcutaneous masses. Histopathology of both nasal and multiple skin masses revealed multiple nonencapsulated, infiltrative masses comprising clusters, anastomosing trabeculae, and packets of neoplastic, round to ovoid, hyperchromatic cells with marked nuclear molding. Surrounding the neoplastic cells was a marked stromal response in which many of the spindle-shaped cells expressed muscle-specific actin and had ultrastructural features consistent with myofibroblasts. A literature search indicates that this is the first report in a peer-reviewed journal of cutaneous metastasis of a nasal neuroendocrine tumor in any domestic animal species.

Vesicular and ulcerative dermatopathy resembling superficial necrolytic dermatitis in captive black rhinoceroses (Diceros bicornis).

The histopathology, clinical presentation, and epidemiology of a cutaneous and oral mucosal disease affecting 40 black rhinoceroses (Diceros bicornis) at 21 zoological parks (50% of the captive US population) were investigated. Twenty-seven biopsies were examined from recent lesions, and clinical information was available from 127 episodes. The cutaneous lesions began as plaques that progressed to vesicles, bullae, or ulcers. Lesions waxed and waned in individual cases. Lesions were predominantly bilaterally symmetrical, affecting pressure points, coronary bands, tips of the ears and tail, and along the lateral body wall and dorsum. Oral lesions were first noticed as ulcers and were present on the lateral margins of the tongue, palate, and mucocutaneous junctions of the lips. All recent lesions had similar histopathologic findings of prominent acanthosis, hydropic degeneration of keratinocytes in the stratum spinosum, spongiosis, intraepithelial vesicles, and parakeratosis without dermal inflammation. Chronic lesions were ulcerated. No pathogens were identified by culture or electron microscopy. Most episodes coincided with stress events (transportation, sudden cold temperatures, intraspecific harassment, estrus, advanced pregnancy) or concurrent diseases (toxic hepatopathy, hemolytic anemia, respiratory or urinary tract infections). Affected rhinoceroses usually were lethargic and had weight loss. Affected rhinoceroses also had lower hematocrit, serum albumin, and cholesterol values than captive healthy or wild rhinoceroses. The clinical patterns and histopathologic findings are similar to those of superficial necrolytic dermatitis in dogs and necrolytic migratory erythema in humans. The high prevalence of this skin disease in captive black rhinoceroses under many circumstances suggests that their epidermis is acutely sensitive to any disruption of metabolic homeostasis. We propose that metabolic changes secondary to a stress response from maladaptation or nutritional inadequacy of captive diets may contribute to the development of this disease in rhinoceroses without hepatopathies.