Strategies to prevent, treat, and provoke Corynebacterium-associated hyperkeratosis in athymic nude mice

Abstract

Athymic nude mice infected with Corynebacterium bovis typically exhibit transient hyperkeratotic dermatitis. Our vivarium experienced an increased incidence of disease characterized by persistent skin lesions and increased mortality, leading to this study. For detection of infection, skin and buccal swab methods showed comparable sensitivities in nude mice. Various prevention, treatment, and eradication strategies were evaluated through clinical assessment, microbiology, and histopathology. In experimentally naïve athymic nude mice, a 2-wk course of prophylactic amoxicillin-containing diet (1200 ppm amoxicillin; effective dose, 200 mg/kg) was ineffective at preventing infection or disease. There was also no significant difference in disease duration or severity in athymic nude mice that received amoxicillin diet or penicillin-streptomycin topical spray (penicillin, 2500 U/mL; streptomycin, 2500 μg/mL). Prolonged treatment with 4 or 8 wk of amoxicillin diet cleared only a small number of athymic nude mice that had subclinical C. bovis infections. Antibiotic sensitivity of C. bovis isolates demonstrated a small colony isolate with less susceptibility to all antibiotics compared with a large colony isolate. Resistance did not appear to develop after prolonged treatment with amoxicillin. Provocation testing by administration of cyclophosphamide (50 mg/kg i.p. every 48 to 72 h for 90 d) to subclinically infected athymic nude mice resulted in prolonged clinical disease that waxed and waned without progression to severe disease. Our findings suggest that antibiotic prophylaxis and treatment of clinical disease in experimentally naïve mice is unrewarding, eradication of bacterial infection is difficult, and severe disease associated with C. bovis is likely multifactorial.

Figure 1.

Growth of C. bovis on Columbia agar with 5% sheep blood. The small and large colony-types are distinguishable, measuring approximately 1 mm and 2 mm, respectively, in diameter.

Figure 2.

Skin; mouse. (A) Section of lesional skin demonstrating acanthosis (white asterisks; consistent with average epidermal cell thickness of 5), orthokeratotic hyperkeratosis (red arrows), and a mild infiltrate of lymphocytes and plasma cells with fewer mast cells and neutrophils within the superficial dermis (surrounding white crosses; consistent with inflammation score of 2). Hematoxylin and eosin stain; bar, 50 μm. (B) Section from periphery of lesional skin, demonstrating relatively normal epidermal thickness (yellow asterisks) and paucity of inflammatory cells within the dermis. Hematoxylin and eosin stain; bar, 50 μm.

Figure 3.

Skin; mouse. Intracorneal clusters of gram-positive, rod-shaped bacteria with fewer gram-positive cocci (red asterisk; consistent with bacterial burden score of 3). Modified Twort stain; bar, 20 μm.

Figure 4.

Classic manifestation of Corynebacterium-associated hyperkeratosis. Experimentally naïve mice typically display mild to moderate skin lesions (Grade 2 disease shown here) with white flakes adherent over the dorsum, ventrum, and muzzle.