Matthew D. Rosenbaum

Biology and Diseases of Amphibians

Amphibians are commonly used in research, and are unique in that they typically represent a transition from early aquatic life forms to terrestrial adults. There are over 6200 species of living amphibians, with tremendous variation in morphology and behavior. This chapter presents an overview of amphibian biology, husbandry and diseases, with emphasis on species most commonly used in research, including Xenopus laevis, axolotls, leopard and bullfrogs, and dendrobatid frogs.

The extracellular domain of myelin oligodendrocyte glycoprotein elicits atypical experimental autoimmune encephalomyelitis in rat and Macaque species

Atypical models of experimental autoimmune encephalomyelitis (EAE) are advantageous in that the heterogeneity of clinical signs appears more reflective of those in multiple sclerosis (MS). Conversely, models of classical EAE feature stereotypic progression of an ascending flaccid paralysis that is not a characteristic of MS. The study of atypical EAE however has been limited due to the relative lack of suitable models that feature reliable disease incidence and severity, excepting mice deficient in gamma-interferon signaling pathways. In this study, atypical EAE was induced in Lewis rats, and a related approach was effective for induction of an unusual neurologic syndrome in a cynomolgus macaque. Lewis rats were immunized with the rat immunoglobulin variable (IgV)-related extracellular domain of myelin oligodendrocyte glycoprotein (IgV-MOG) in complete Freund’s adjuvant (CFA) followed by one or more injections of rat IgV-MOG in incomplete Freund’s adjuvant (IFA). The resulting disease was marked by torticollis, unilateral rigid paralysis, forelimb weakness, and high titers of anti-MOG antibody against conformational epitopes of MOG, as well as other signs of atypical EAE. A similar strategy elicited a distinct atypical form of EAE in a cynomolgus macaque. By day 36 in the monkey, titers of IgG against conformational epitopes of extracellular MOG were evident, and on day 201, the macaque had an abrupt onset of an unusual form of EAE that included a pronounced arousal-dependent, transient myotonia. The disease persisted for 6–7 weeks and was marked by a gradual, consistent improvement and an eventual full recovery without recurrence. These data indicate that one or more boosters of IgV-MOG in IFA represent a key variable for induction of atypical or unusual forms of EAE in rat and Macaca species. These studies also reveal a close correlation between humoral immunity against conformational epitopes of MOG, extended confluent demyelinating plaques in spinal cord and brainstem, and atypical disease induction.

Trypsin and rosmarinic acid reduce the toxicity of Micrurus fulvius venom in mice

Abstract Context. Antivenom is expensive and not always available, so alternative treatments are being investigated. Objective. The efficacy of trypsin or rosmarinic acid (RA) in treating Micrurus fulvius in a murine model is determined. Materials and methods. Design: randomized controlled blinded study. Subjects: Fifty mice (20–30 g). Study groups: Intraperitoneal injections of: 1) 2 mg/kg M. fulvius venom (approximately twice the LD50 for mice; n = 10); 2) 2 mg/kg M. fulvius venom incubated in vitro for 1 h prior to injection with RA at a 1:10 ratio (n = 17); 3) 2 mg/kg M. fulvius venom incubated in vitro for 1 h prior to injection with 1 mg of trypsin (n = 17); 4)1 mg trypsin IP without venom (n = 3); and 5) RA IP without venom (n = 3). Main outcome: time to toxicity (respiratory distress (< 25 breaths/min.), loss of spontaneous locomotor activity, or inability to upright self). Statistical analysis: Time to toxicity using Tukey–Kramer HSD; Survival to 4, 6, and 12 h using Chi-square analysis. Results. Onset of toxicity: venom + saline, 120.3 + 64.4 min; venom + rosmarinic acid, 238.1 ± 139.2 min (p = 0.15 relative to venom + saline); venom + trypsin, 319.7 + 201.0 min (p = 0.007 relative to venom + saline). Venom + trypsin but not venom + RA survival to 4 h was significant compared to venom + saline (p = 0.023). Two mice in the venom + trypsin group and one mouse in the venom + RA group survived to 12 h. Mice receiving trypsin without venom or RA without venom survived to 12 h without toxicity. Discussion. This work suggests that trypsin and RA may have efficacy in treatment M. fulvius envenomation. Conclusion. In vitro neutralization of M. Fulvius venom by trypsin justifies progressing to an in vivo model in future studies.

Cottontail Rabbit Papillomavirus in Langerhans Cells in Sylvilagus spp.

A wildlife sanctuary presented an adult female cottontail rabbit (Sylvilagus spp.), age unknown, to the Colorado State University Pathology service for postmortem examination. Gross examination revealed numerous pigmented wartlike lesions arising from the skin of the head surrounding the ears, eyes, nares, mouth, and dorsum. Masses were firm, friable, and easily detached from the underlying skin. Differential diagnoses included Cottontail rabbit papillomavirus, Rabbit fibroma virus, and Myxoma virus. Histological examination revealed multiple papillary masses lined by stratified squamous epithelial cells with central cores of fibrovascular connective tissue and parakeratotic hyperkeratosis. Cells of the Stratum spinosum were frequently swollen with abundant perinuclear, cytoplasmic, clearing, and occasional intranuclear basophilic, glassy, spherical inclusions up to 3 μm in diameter. The lesions were consistent with Cottontail rabbit papillomavirus infection. Papilloma virus antigens were identified by immunohistochemistry. In addition, papillomavirus particles were identified by transmission electron microscopy within Langerhans cells of the epidermis, suggesting a unique mechanism for systemic dissemination of the virus. The present case report highlights the finding of viral particles within the Langerhans cells and suggests a novel mechanism of pathogenesis.

Long-term efficacy of pressure immobilization bandages in a porcine model of coral snake envenomation

BACKGROUND Pressure immobilization bandages delay mortality for 8 hours after coral snake envenomation, but long-term efficacy has not been established. OBJECTIVE The objective of this study is to determine the long-term efficacy of pressure immobilization bandages after coral snake envenomation in the absence of antivenom therapy. METHODS A randomized, observational pilot study was conducted. Ten pigs (17.3-25.6 kg) were sedated, intubated for 5 hours, and injected subcutaneously with 10 mg of lyophilized Micrurus fulvius venom resuspended in water. Pigs were randomly assigned to a control group (no treatment) or a treatment group (compression bandage and splint) approximately 1 minute after envenomation. Bandage pressure was not controlled. Pigs were monitored daily for 21 days for signs of respiratory depression, decreased oxygen saturations, and paralysis. In case of respiratory depression, pigs were humanely euthanized and time to death recorded. Statistical analysis was performed with Fisher exact test, Mann-Whitney U test, and Kaplan-Meier survival curve as appropriate. RESULTS Median survival time of control animals was 307 minutes compared with 1172 minutes in treated animals (P = .10). Sixty percent of pigs in the treatment group survived to 24 hours vs 0% of control pigs (P = .08). Two of the treatment pigs survived to the end point of 21 days but showed necrosis of the distal lower extremity. CONCLUSIONS Long-term survival after coral snake envenomation is possible in the absence of antivenom with the use of pressure immobilization bandages. The applied pressure of the bandage is critical to allowing survival without necrosis. Future studies should be designed to accurately monitor the pressures applied.

Solitary foot mass on a Sprague-Dawley rat

1East Carolina University, Department of Comparative Medicine, Brody School of Medicine, Greenville, NC. 2Colorado State University, Department of Microbiology, Immunology, and Pathology, Fort Collins, CO. Correspondence should be addressed to M.D.R. (rosenbaumm@ecu.edu). Solitary foot mass on a Sprague-Dawley rat Matthew D. Rosenbaum, DVM, MS1, Matthew R. Feirer, DVM2, Karen Fox, DVM2 & Lon Kendall, DVM, PhD, DACLAM2