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TABLE 1. Prevalence of B. dendrobatidis infection of Blanchard’s Cricket Frog (Acris crepitans blanchardi) samples from the midwestern U.S., asdetermined by standard PCR assay.No. infected / PrevalenceState County Locality Latitude Longitude total sampled of infectionOhio Auglaize St. Marys Fish Hatchery 40.526°N 84.418°W 6 / 42 0.14Ohio Preble Woodland Trails W.A. 39.750°N 84.633°W 3 / 19 0.16Ohio Greene Caesar Creek St. Park 39.748°N 83.816°W 0 / 1 0.00Ohio Greene Fish & Game Club 39.856°N 83.943°W 2 / 9 0.11Ohio Clinton Unspecified pond 39.667°N 83.964°W 3 / 10 0.30Michigan Kalamazoo Harrison Lake 42.149°N 85.680°W 0 / 1 0.00Michigan Barry Lux Arbor Reserve 42.613°N 85.228°W 2 / 8 0.25Michigan Lenawee Ives Road gravel pit 42.180°N 84.156°W 2 / 10 0.20Michigan Washtenaw Ypsilanti 42.207°N 83.578°W 1 / 10 0.10Michigan St. Clair Port Huron 42.970°N 82.425°W 1 / 10 0.10Michigan Ottawa Grand Rapids gravel pit 42.941°N 85.820°W 1 / 9 0.11Michigan Kent Unspecified pond 42.989°N 85.509°W 2 / 6 0.33Illinois Jackson Unspecified pond 37.727°N 89.209°W 0 / 9 0.00Illinois Effingham Unspecified pond 38.995°N 88.621°W 5 / 16 0.31Illinois Will Unspecified pond 41.570°N 88.072°W 1 / 14 0.07Iowa Madison Unspecified pond 41.300°N 93.744°W 1 / 3 0.33Iowa Guthrie Unspecified pond 41.686°N 94.359°W 0 / 1 0.00Iowa Lucas Unspecified pond 41.016°N 93.115°W 0 / 8 0.00Iowa Ringgold Unspecified pond 40.741°N 94.241°W 0 / 5 0.00Kansas Jefferson Unspecified pond 39.082°N 95.546°W 0 / 5 0.00Oklahoma Ellis Packsaddle Wildlife Area 35.846°N 99.616°W 1 / 9 0.11samples were transported to the College of Wooster (Wooster,Ohio) for processing with a PCR-based assay.All DNA from tissue samples were extracted according to theanimal tissues protocol provided by the manufacturer (Qiagen,Valencia, CA; DNeasy Blood and Tissue Handbook, 07/2006).Skin swab samples were agitated for thirty seconds in a vortexand the swab was removed and squeezed along the side of thetube to remove the maximum amount of solution. An aliquot of300 µl was removed from the tube and centrifuged in a 2.5 mltube for five minutes. The supernatant was subsequently removedand the remaining pellet was used for the remainder of the extractionprocedure. Following the extraction procedure, all sampleswere concentrated to approximately 20 µl by centrifugal evaporation.Amplification of the extracted samples was completed with standardpolymerase chain reactions (PCR) using the primers reportedin Annis et al. (2004). Amplification reactions consisted of 13 µlof deionized water, 5.0 µl of template (containing approximately10–30 µg of DNA), 2.5 µl 10x PCR buf fer (Qiagen, Valencia,CA), 2.5 µl dNTPs (10 uM of each), 1.0 mM MgCl 2, 0.5 µl ofeach primer at a concentration of 50 pmol/µl, and 0.25 µl Taqpolymerase (5 units/mL) in a volume of 24 µl. The amplificationof the mixture took place according to the following steps: an initialdenaturation at 94°C for 10 minutes, followed by 30 cycles of45 seconds at 93°C and 45 seconds at 60°C, and then a final extensionat 72°C for 10 minutes to complete the amplification. Productswere then viewed on ethidium bromide stained 1.0% agarosegel dissolved in TAE (40 mM Tris [pH 8.0], 20 mM acetic acid, 1mM EDTA) alongside a 1 kb ladder. A band approximately 300base-pairs in length indicated the presence of B. dendrobatidisinfection.A positive control of B. dendrobatidis broth culture andzoospores was used to optimize and determine the sensitivity ofthe PCR assay. A dilution series of the positive control was runand successful amplification took place in 1/5, 1/10, 1/50, 1/100,and 1/500 dilutions. All PCRs were run twice for each sample.Amplification in both replicates was considered a true positivesignal of infection, while no amplification in both replicates wasconsidered a true negative signal. Templates that amplified oncewere subject to a third replicate, in which a successful amplificationwas taken as indicating infection. To avoid false-positive andfalse-negative results, negative and positive controls were usedwith all samples analyzed.Available information suggests that declines in Blanchard’scricket frogs may be moving from north to south, and inward fromboth the western and eastern range boundaries (Lannoo andGrundel 2004; Lehtinen and Skinner 2006). Based on where declineshave been reported, we used chi-squared analyses to testfor differences in infection prevalence north and south of threelatitudes (38°N, 40°N, and 42°N) and east and west of two longitudes(88°W and 90°W). Also, the difference in prevalence of infectionbetween skin swab samples and tissue samples was testedusing a chi-square test. All statistical analyses were performed usingSPSS (version 13.0, SPSS Inc., Chicago).194 Herpetological Review 39(2), 2008