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Pathogens can cause serious declines in host species, and knowing where pathogens associated with host declines occur facilitates understanding host-pathogen ecology. Suspected drivers of global amphibian declines include infectious diseases, with 2 pathogens in particular, Batrachochytrium dendrobatidis (Bd) and ranaviruses, causing concern. We explored the host range and geographic distribution of Bd and ranaviruses in the Taiga Plains ecoregion of the Northwest Territories, Canada, in 2007 and2008. Both pathogens were detected, greatly extending their known geographic distributions. Ranaviruses were widespread geographically, but found only in wood frogs. In contrast, Bd was found at a single site, but was detected in all 3 species of amphibians in the survey area (wood frogs, boreal chorus frogs, western toads). The presence of Bd in the Northwest Territories is not congruent with predicted distributions based on niche models, even though findings from other studies at northern latitudes are consistent with those same models. Unexpectedly, we also found evidence that swabs routinely used to collect samples for Bd screening detected fewer infections than toe clips. Our use and handling of the swabs was consistent with other studies, and the cause of the apparent lack of integrity of swabs is unknown. The ranaviruses detected in our study were confirmed to be Frog Virus 3 by sequence analysis of a diagnostic 500 bp region of the major capsid protein gene. It is unknown whether Bd or ranaviruses are recent arrivals to the Canadian north. However, the genetic analyses required to answer that question can inform larger debates about the origin of Bd in North America as well as the potential effects of climate change and industrial development on the distributions of these important amphibian pathogens. Dis Aquat Org 92: [231][232][233][234][235][236][237][238][239][240] 2010

The fungal pathogen Batrachochytrium dendrobatidis threatens amphibian populations around the world. The ability to detect this pathogen on infected animals and in the environment is critical for understanding and controlling this pandemic. We tested several advances in quantitative PCR (qPCR) techniques to detect B. dendrobatidis DNA. We used a fast PCR thermocycler and enzymes that reduced the volume and the duration of the reaction. We also compared a conventional TaqMan minor groove binding (MGB) probe to an identical locked nucleic acid (LNA) counterpart. The fast qPCR reaction had a high degree of sensitivity to B. dendrobatidis DNA. The LNA probe was effective for detecting B. dendrobatidis DNA and produced results similar to those of the MGB probe. The modifications that we tested can improve the cost, time efficiency and specificity of quantitative PCR as a tool for detecting pathogen DNA. KEY WORDS: Batrachochytrium dendrobatidis · Quantitative PCR · Locked nucleic acids · Minor groove binding probe · Amphibian disease Resale or republication not permitted without written consent of the publisherDis Aquat Org 97: 249-253, 2012 250 cost, time efficiency, and specificity of the technique.Recent advances in qPCR have broadened the capability and accessibility of this approach to pathogen detection. They include faster polymerase enzymes, lower cost instrumentation with increased temperature ramp speeds and lower reaction volume requirements, and development of different types of fluorescent probes for detection of target sequences.First, we took advantage of new fast qPCR technology. The cost of fast quantitative machines is much lower than earlier instrument models, although the cost of fast reagents is slightly higher than their conventional counterparts. The greater detection sensitivity of the fast machines and the development of enzymes that can withstand faster temperature changes have reduced PCR reaction volumes and total cycle times from approximately 25 µl and 2 h, to 10 µl and 30 min, effectively lowering the actual cost per reaction. Yoder & Fishel (2008) showed that, in many cases, fast qPCR technology is compatible with established detection protocols. In the present study, we attempted to reduce the reaction volume and cycle times of 25 µl and 75 s used by Boyle et al. (2004) to 10 µl and 21 s.Finally, we tested different qPCR probe chemistries to assess the relative effectiveness and benefit of each. We compared the efficacy of the TaqMan (Applied Biosystems) minor groove binder (MGB) probe used by Boyle et al. (2004) to more recently developed locked nucleic acid (LNA) probe chemistry (Vester & Wengel 2004). Detection of genomic regions of interest requires sequence-specific primers that define the target area, while higher levels of specificity require the use of fluorescentlabeled probes that hybridize to a region between the primers and differentiate closely related sequences. The challenge of designing compatible primers and probes that satisfy reaction stringency requir...

Since host defenses to infectious disease are often costly, one would expect hosts to use their defenses only when the threat of infection is high. Southern leopard frogs (Rana sphenocephala) at Ellenton Bay in South Carolina (USA) have an extended breeding season and their eggs are exposed to a wide range of temperatures depending on the time of year when they are laid. Adults aggregate their egg masses in cold temperatures, but separate them in warm temperatures. The spatial aggregation of egg masses may insulate eggs from cold temperatures, but may also affect the transmission of pathogens between the eggs. I examined the effects of temperature, pathogens, and spatial distribution on the survival of R. sphenocephala in the egg stage. Field observations found that temperature had little effect on the number of infected eggs within egg masses, but that egg masses in colder water were more likely to be aggregated together. In a controlled laboratory experiment, the presence of aquatic oomycetes led to higher mortality in cold temperatures than they did at warmer temperatures. Infectious disease may be a selective force that favors R. sphenocephala adults that aggregate their offspring when the threat of disease is high. Host aggregation can reduce the risk of infection when pathogens have a slow generation time that hinders them from responding to an abundance of hosts.