D. Earl Green1, DVM, ACVM; Sanford H. Feldman2, DVM, PhD, DACLAM; J. Wimsatt2,3, DVM, PhD
Multiple un-explained mass mortality events in free-living larval anurans of the genus Rana have occurred between September 1999 and March 2002. Mortality rates in many instances exceeded 90% of tadpoles, resulting in negligible recruitment that year. However, adult frogs appeared unaffected and successful breeding occurred in subsequent years. A seasonal appearance of the organism was clearly associated with the presence of 1–4 mo-old tadpoles, with outbreaks beginning December to March in southern states and April to July in central and northern states. Die-offs occurred in the following states: Florida, Mississippi, Virginia, New Hampshire and Minnesota. Sporadic infections that were not known to be associated with die-offs also occurred in Alaska, Minnesota, Virginia and North Carolina. Infected ranid tadpoles included wood frogs (Rana sylvatica), Southern leopard frogs (R. sphenocephala),mink frogs (R. septentrionalis), green frogs (R. clamitans), Gopher frogs (R. silvosa) and bullfrogs (R. catesbeiana). Gross pathologic findings are characteristic: mild to massive enlargement of the liver, spleen, pronephroi and mesonephroi; affected organs are homogenously white. Histopathologically and cytologically, these four organs and others have massive infiltrates of spheric, darkly basophilic organisms morphologically reminiscent of primitive protozoa of the newly erected clade, Mesomycetozoa (e.g., Dermocystidium, Dermosporidium, rosette agent and Ichthyophonus).
DNA was isolated from tadpole livers from several outbreaks. A"Perkinsus-like" generic PCR was developed by sequence alignment of several organisms. The PCR amplifies a 280-nucleotide region of the 18S ribosomal RNA gene. Sequences of 18S rRNA aligned were identified from the NIH GenBank, and included Ichthyophonus irregularis (Accession AF232303), Ichthyophonus hoeferi, (Accession U25637), Amoebidium parasiticus (Y191555), Pseudoperkinsus tapetus (AF192386), Dermocystidium salmonis (U21337) and Rhinosporidium seeberi (AFA399715). Ribosomal RNA 18S sequences from Xenopus laevis (XO4025) and Bufo valliceps (M59386) were used to identify regions to be avoided in primer design. Sequence alignment was performed using PILEUP in the GCG Wisconsin Analysis Package (Oxford Medical, Oxford, UK). PCR products obtained from affected liver tissues were subcloned into pCR4 TOPO (Invitrogen Co, Carlsbad, CA) and sequenced (Davis Sequencing, U.C. Davis) in both forward and reverse directions. Next, a new PCR was designed that was specific to the Perkinsus-like parasite under examination, which amplified a 238-nucleotide region. The NIH GenBank was searched using the BLAST (the Basic Alignment Sequence Tool) routine, using consensus sequences of PCR product obtained from twelve clones as a reference. BLAST identified closest phylogenetic relatives whose 18S rRNA sequences were truncated for use in construction of the most parsimonious relationship. Tree construction was performed using the PHYLIP software including DNApars, Seqboot and Consense.
The PCR-amplified sequence from amphibian livers was most closely related to organisms in the genus Perkinsus, a group of well-known marine shellfish pathogens (e.g., P. marinus, etc.). Blinded specific PCR testing was performed on liver samples previously characterized as positive or negative for the agent based on light microscopy. All samples from outbreaks were identified by PCR, whereas amphibian tissues containing Ichthyophonus (diagnosed by histology) were consistently negative.
Our results suggest a close relationship between the larval anuran die-offs, light microscopic findings of massive systematic tissue invasion, and the presence of DNA sequences consistent with a new Perkinsus-like agent. Our inability to culture the agent (at this time) precludes the fulfilment of Koch's postulates. Further work is warranted to establish the geographic distribution, host range, life cycle, and the relationship (if any) of this new agent to marine invertebrate pathogens. A definitive role of this emerging new agent in periodic mass mortality events of wild anuran population declines in the United States is suspected.
The authors would like to thank Nick Douris for technical assistance. Partial grant support was provided by USGS to JW and SHF.