Parasites play a prominent role in the ecology, evolution, and more recently, conservation of many organisms. For example, emerging infectious diseases, including a group of lethal ranaviruses, are associated with the declines and extinctions of amphibians around the world. An increasingly important basic and applied question is: what controls parasite virulence? We used a dose-response experiment with three laboratory-bred clutches of tiger salamander larvae (Ambystoma tigrinum) to test how the size of inoculum and host genetic factors influence the dynamics and outcome of ranavirus infections. We found that infection rates increased with dose and were strongly affected by clutch identity and host life history stage. Case mortality increased with dose of inoculum, but was unaffected by host characteristics. Average survival time decreased with dose and differed among clutches, but this was largely due to differences in the time to onset of symptoms. Overall, our results suggest that dose of inoculum and host characteristics (life history stage and genetic background) influence the establishment and early virus replication, and therefore the virulence of ranavirus infections.

Temperature strongly influenced percent mortality and time to death of salamanders exposed to the Ambystoma tigrinum virus (iridovirus) (ATV). Most salamanders survived when exposed at 26°C, whereas all died at 18°C and nearly all died at 10°C. Some asymptomatic salamanders that survived 60 d at 10 or 26°C were found to be carrying virus. Polymerase chain reaction (PCR) confirmed the presence of virus in ATV-exposed salamanders but was found to be less sensitive than cell culture in detecting ATV at low concentrations. PCR products were 100% identical to ATV in the major capsid protein sequence. Virus titer was higher in salamanders held at 10°C than at 18°C but little virus, if any, was present in the small number of salamanders that died at 26°C. These results may help explain periodic viral epizootics in field populations of A. tigrinum where water temperatures fluctuate widely. KEY WORDS: Epizootic · Cell culture · Iridovirus · Mortality Resale or republication not permitted without written consent of the publisherDis Aquat Org 63: [95][96][97][98][99][100] 2005 MATERIALS AND METHODSGeneral methods. ATV was cultured using epithelioma papilloma cyprini (EPC) cells (Fijan et al. 1983). Plaque and TCID 50 (tissue culture infectious dose 50 ; Reed & Muench 1938) assays employing EPC cells were used to estimate the concentration of virus used in each experiment. Three to 4 mo old Ambystoma tigrinum nebulosum larvae originated from stock held in the laboratory for at least 2 generations. No mortality due to possible virus infection was observed in the rearing facility during this period. During the experiments, each salamander was held individually in 300 ml of water in a Zip Lock ® (S. C. Johnson) container and water was changed weekly. Salamanders were fed brine shrimp Artemia sp. 3 times a week and observed daily. Salamanders that died were stored at -70°C. At the end of each experiment, body wall samples were taken from all dead larvae and tail clips from surviving salamanders for virus detection. Each sample was placed in a Stomacher ® (Seward) bag containing 2 ml of Eagle's Minimum Essential Medium (MEM) + 2% Fetal Bovine Serum (FBS) + penicillin-streptomycin-neomycin (PSN; Sigma) + diatomaceous earth powder, and homogenized in a Stomacher device. The homogenate was centrifuged at 9000 × g for 10 min and the supernatant was stored at -80°C. Supernatant (100 µl) was inoculated onto EPC cells in each well of 12-or 24-well plates, the preparation was rocked for 1 h and then 1 ml of MEM + 10% FBS + PSN was added. Cells were incubated at 22°C under 5% CO 2 and observed for 2 wk for cytopathic effect (CPE). Samples showing no CPE were passed through 2 further sets of EPC cells to confirm the presence or absence of virus.To determine the multiplication of virus at the 3 experimental temperatures, replicate EPC cultures in 24-well plates were inoculated with dilutions of ATV and held at 10, 18 or 26°C for 10 to 16 d. Development of CPE was observed and TCID 50 was calculated during incubation.In order ...

Batrachochytrium dendrobatidis (Bd) infection on post-metamorphic frogs and salamanders is commonly diagnosed using polymerase chain reaction (PCR) of skin scrapings taken with mildly abrasive swabs. The technique is sensitive, non-lethal, and repeatable for live animals. Tadpoles are generally not sampled by swabbing but are usually killed and their mouthparts excised to test for the pathogen. We evaluated a technique for non-lethal Bd diagnosis using quantitative PCR (qPCR) on swabs scraped over the mouthparts of live tadpoles. The sensitivity of non-lethal (swabbing) and lethal (removal of mouthparts) sampling was assessed using 150 Bd-infected Rana subaquavocalis tadpoles. Swabbing was consistently less sensitive than lethal sampling, but still detected Bd. Experimental Bd prevalence was 41.1% when estimated by destructively sampling mouthparts and 4.7 to 36.6% (mean = 21.4%) when estimated with swabs. Detection rates from swabbing varied with investigator and time since infection. The likelihood of detecting Bd-infected tadpoles was similar regardless of size and developmental stage. Swabbing mouthparts of live tadpoles is a feasible and effective survey technique for Bd, but, because it is less sensitive, more tadpoles must be sampled to estimate prevalence at a confidence level comparable to destructive sampling. KEY WORDS: Batrachochytrium dendrobatidis · Tadpole · Detection · PCR · Technique · Chytridiomycosis Resale or republication not permitted without written consent of the publisherDis Aquat Org 72: [77][78][79][80][81][82][83][84][85] 2006 et al. 2004, Knapp & Morgan 2006, and are killed for histological diagnosis. This limits studies of amphibian disease, particularly those that span life-history stages.The polymerase chain reaction (PCR) is the basis for a highly sensitive, non-destructive diagnostic technique for sampling post-metamorphic amphibians. An animal's skin is scraped or swabbed lightly with a finely pointed, slightly abrasive object, such as a wooden toothpick or cotton surgical swab, to remove the outermost skin cells, which are then screened for Bd DNA using real-time Taqman PCR assays (Boyle et al. 2004). Researchers studying chytridiomycosis in amphibians have used the swabbing technique for about 4 yr, and it is a generally accepted protocol used by several laboratories (Briggs & Vredenberg 2004, Livo 2004, Kriger et al. 2006. We evaluate the sensitivity and feasibility of swabbing tadpole mouthparts for Bd diagnosis as a non-lethal alternative to histology. The project arose from a desire to learn more about the rare Ramsey Canyon leopard frog Rana subaquavocalis in southeastern Arizona, where Bd has been detected at several sites and tadpoles often remain at sites after frogs have become scarce or absent (K. J. Field pers. obs.). Wildlife managers are rightly reluctant to test for Bd by killing large numbers of tadpoles in small, vulnerable populations. Our question was: Could we detect Bd reliably and non-lethally in tadpoles using quantitative PCR? MATERIALS AND...