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Mass die-offs of amphibian populations pose a challenging problem for conservation biologists. Ranaviruses often cause systemic infections in amphibians and, in North America, are especially virulent and lethal to larvae and metamorphs. In this paper we describe a novel ranavirus isolate as well as the first recorded occurrence of ranavirus in the southern High Plains of Texas and in associated populations of the spotted chorus frog Pseudacris clarkii. The breeding sites were playas, that is, wetlands that fill via isolated thunderstorms that can occur infrequently; thus, not every playa has water or breeding amphibians annually. We did not detect ranavirus in sympatric anurans, but other reports document ranaviruses in Pseudacrisspp. elsewhere. The occurrence of multiple isolates of ranavirus in a number of Pseudacrisspecies suggests that this genus of frogs is highly susceptible to ranaviruses and may experience exceptionally high mortality rates from infection. Thus, the virus may contribute to substantial seasonal population declines and low seasonal recruitment, with negative impacts on populations of breeding adults in successive years.

A newly developed polymerase chain reaction (PCR)-based method to rapidly and specifically detect Geomyces destructanson the wings of infected bats from small quantities (1–2 mg) of tissue is described in the current study (methods for culturing and isolating G. destructansfrom bat skin are also described). The lower limits of detection for PCR were 5 fg of purified fungal DNA or 100 conidia per 2 mg of wing tissue. By using histology as the standard, the PCR had a diagnostic specificity of 100% and a diagnostic sensitivity of 96%, whereas the diagnostic sensitivity of culture techniques was only 54%. The accuracy and fast turnaround time of PCR provides field biologists with valuable information on infection status more rapidly than traditional methods, and the small amount of tissue required for the test would allow diagnosis of white-nose syndrome in live animals.

White-nose syndrome (WNS) is a cutaneous fungal disease of hibernating bats associated with a novel Geomycessp. fungus. Currently, confirmation of WNS requires histopathologic examination. Invasion of living tissue distinguishes this fungal infection from those caused by conventional transmissible dermatophytes. Although fungal hyphae penetrate the connective tissue of glabrous skin and muzzle, there is typically no cellular inflammatory response in hibernating bats. Preferred tissue samples to diagnose this fungal infection are rostral muzzle with nose and wing membrane fixed in 10% neutral buffered formalin. To optimize detection, the muzzle is trimmed longitudinally, the wing membrane is rolled, and multiple cross-sections are embedded to increase the surface area examined. Periodic acid–Schiff stain is essential to discriminate the nonpigmented fungal hyphae and conidia. Fungal hyphae form cup-like epidermal erosions and ulcers in the wing membrane and pinna with involvement of underlying connective tissue. In addition, fungal hyphae are present in hair follicles and in sebaceous and apocrine glands of the muzzle with invasion of tissue surrounding adnexa. Fungal hyphae in tissues are branching and septate, but the diameter and shape of the hyphae may vary from parallel walls measuring 2 μm in diameter to irregular walls measuring 3–5 μm in diameter. When present on short aerial hyphae, curved conidia are approximately 2.5 μm wide and 7.5 μm in curved length. Conidia have a more deeply basophilic center, and one or both ends are usually blunt. Although WNS is a disease of hibernating bats, severe wing damage due to fungal hyphae may be seen in bats that have recently emerged from hibernation. These recently emerged bats also have a robust suppurative inflammatory response.

A total of 44 amphibian mortality events and 20 morbidity events were reviewed retrospectively. The most common cause of amphibian mortality events was infection by ranaviruses (Family: Iridoviridae). Ranavirus epizootics have abrupt onset and affect late-stage larvae and recent metamorphs. Mortality events due to ranavirus infections affected only widespread and abundant amphibian species, and there was a clear association with high population densities. Chytrid fungal infections accounted for seven mortality events in postmetamorphic anurans only. Chytrid epizootics are insidious and easily overlooked in the field. While both ranavirus and chytrid fungal epizootics were associated with > 90 mortality rates at affected sites, only the chytrid fungal infections were linked to multiple amphibian population declines. Three primitive fungal organisms in the newly erected clade, Mesomycetozoa, caused morbidities and mortalities in anurans and salamanders.