Percutaneous malathion absorption by anuran skin in flow-through diffusion cells.

There is increased concern about the sublethal effects of organophosphorous (OP) compounds on human and animal health, including the potential role of OP compounds in the global decline of amphibian populations. Malathion is one of the most widely used OP pesticides with numerous agricultural and therapeutic applications, and exposure to environmentally applied malathion can lead to adverse systemic effects in anurans. Cutaneous absorption is considered a potentially important route of environmental exposure to OP compounds for amphibians, especially in aquatic environments. One in vitro system commonly used to determine the absorption kinetics of xenobiotics across the skin is the two-compartment Teflon flow-through diffusion cell system. To establish cutaneous absorption kinetics of malathion, six full thickness skin samples taken from both the dorsal and ventral surfaces of each of three bullfrogs (Rana catesbeiana) and three marine toads (Bufo marinus) were placed into two-compartment Teflon flow-through diffusion cells perfused with modified amphibian Ringer's solution. A 26µg/cm(2) dose of malathion-2,3-(14)C diluted in 100% ethanol was applied to each sample (0.44-0.45µCi). Perfusate was collected at intervals over a 6h period and analyzed for (14)C in a scintillation counter. At the end of 6h, surface swabs, tape strips, biopsy punches of the dosed area of skin, and peripheral samples were oxidized and analyzed for residue effects. Malathion absorption was greater across the ventral skin compared to dorsal skin in both bullfrogs and marine toads.

Retrospective study of proliferative papillary vulvitis in Florida panthers.

Proliferative, papillary vulvitis was identified in 16 of 34 (47%) free-ranging and captive female Florida panthers (Puma concolor coryi) monitored over a period from 1983-98. Gross lesions were characterized by extensive papilliferous proliferation in the mucosa of the vestibulum vaginae. Within lesions, the mean length and width of vestibular papillae were 1.07 +/- 0.39 mm (CV = 36%) and 0.55 +/- 0.11 mm (CV = 20%) respectively. Histologically, three to 12 layers of non-cornified stratified squamous epithelium with various degrees of basal cell spongiosis and rete ridge formation covered fibrous papillae. Mixed leukocytic mucosal inflammation also was observed. Infectious organisms were not observed, and immunohistochemical testing for the presence of papillomavirus antigens in specimens from seven panthers was negative. Lesions in nearly all of the panthers were first observed during a six-year period (1986-92), with one each in 1983, 1996 and 1998. There were no significant differences between the number of females having litters, the number of litters between age-matched and interval-matched females, and the interval between litters among lesions positive and lesion negative females over the 15 yr period. The severity of lesions did not appear to differ between parous and nulliparous free-ranging lesion-positive females. The cause of proliferative vulvitis remains unknown. However, the lesion did not appear to have a significant effect on reproduction.

Antimicrobial peptide defenses against pathogens associated with global amphibian declines.

Global declines of amphibian populations are a source of great concern. Several pathogens that can infect the skin have been implicated in the declines. The pathogen most frequently associated with recent die-offs is a chytrid fungus, Batrachochytrium dendrobatidis. A second fungus, Basidiobolus ranarum, was isolated from declining populations of Wyoming toads. A third pathogen, Aeromonas hydrophila, is an opportunistic bacterium found in healthy frogs, but capable of inducing disease. Among the immune defense mechanisms used by amphibians is the production of antimicrobial peptides in granular glands in the skin. These packets of natural antibiotics can be emptied onto the skin when the amphibian is injured. To determine whether antimicrobial skin peptides defend against these amphibian pathogens, six peptides (magainin I, magainin II, PGLa, CPF, ranalexin, and dermaseptin), from three species, and representing three structurally different families of peptides, were tested in growth inhibition assays. We show here that the peptides can kill or inhibit growth of both fungi but not Aeromonas. Although each peptide varied in its effectiveness, at least one from each species was effective against both fungi at a concentration of about 10-20 microM. This is the first direct evidence that antimicrobial peptides in the skin can operate as a first line of defense against the organisms associated with global amphibian declines. It suggests that this innate defense mechanism may play a role in preventing or limiting infection by these organisms.