Severe perkinsea data was based on the observation of characteristic gross and microscopic lesions and the morphological identification of Perkinsea organisms within the lesions. Necropsies and gross evaluation of carcasses were carried out under a dissecting microscope. Specimen were collected and submitted to the NWHC as part of mortality investigations and collection of specimen from apparently healthy populations as part of ongoing amphibian health monitoring.
(ii) Epidemiological information was compiled from each anuran sample, including the detection type (mortality event or health monitoring study), location (state and county), anuran species reported in the event, life-stage of species reported in the event (tadpoles or adults), tadpole life-stage of specimen submitted for examination, date of first observation of the event, estimate of the size of the event (based on total number of moribund and dead tadpoles encountered), and aetiology of the event. Anuran species and life-stage identification was confirmed at arrival by an NWHC expert herpetologist and pathologist based on external morphological features. Event information was summarized from reports submitted to the NWHC by field biologists and from diagnostic reports generated by NWHC staff pathologists and laboratory diagnosticians. As such, event summaries had variable detail. We reported event date as the month and year of the first observation and collection of specimen. Event aetiologies were those considered to significantly contribute to mortality or morbidity based on expert epidemiological and diagnostic interpretation of pathological findings, bacteriology, parasitology and virology results (i.e., pathogens found in low abundance or without evidence of significant pathological impact to the host were not reported).
(iii) Tadpole liver that had been stored at -80°C was screened for the presence of Perkinsea within the NAG01 clade. DNA was extracted from the tadpole liver using the Gentra®Puregene® Tissue Kit (Qiagen Inc., Valencia, California, USA) according to the manufacturers instructions. To amplify DNA of the 18S rRNA gene of Perkinsea-like organisms within NAG01, primers 86F-B, 300F-B, 1294 R, and 1282 R were used 7. Polymerase chain reaction was carried out using GoTaq® Flexi DNA polymerase (Promega Corporation, Madison, Wisconsin, USA). Each 25 µl reaction contained 13.375 µl nuclease-free water, 5 µl buffer, 2 µl dNTPs (2.5 mM each), 1.5 µl MgCl2 (25 mM), 1.25 µl of each primer (20 µM), 0.125 µl enzyme, and 0.5 µl undiluted or 10-fold diluted DNA template. The reaction was carried out under the following cycling conditions: 95°C for 2 min; 40 cycles of 95°C for 30 sec, 55°C for 30 sec, 72°C for 2 min; and a final extension of 72°C for 10 min. Each sample was run in four separate reactions (one for each possible primer pairing). The presence of potential Perkinsea-like DNA was assessed by the generation of an approximately 1 kb amplicon as viewed on a 1% agarose gel.
(iv) To confirm the morphology-based species identification of tadpoles, a subset (one representative of each host species from each site) of PCR amplicons representing host mitochondrial DNA (12S-16S ribosomal DNA) were sequenced. Double-stranded sequencing was conducted as described above using primers 12L1, 16H1, 12Sm, 16Sc, 16Sh, and 16Sa34,39 . In some instances, sequences could not be interpreted due to the presence of multiple overlaid peaks. This was thought to be the result of primers binding to multiple locations on the amplicon. When this occurred, smaller products were generated from the original sample by using internal sequencing primers for initial amplification (i.e., primer pairs 12L1 and 16Sh, 12L1 and 16Sa, 12Sm and 16Sa, 12Sm and 16H1, and 16Sc and 16H1) and modified cycling conditions (95°C for 5 min; 40 cycles of 95°C for 1 min, 55-60°C for 1 min, 72°C for 2 min; final extension 72°C for 10 min) and then sequencing the fragments with those same primers.