Influence of temperature on Ranavirus infection in larval salamanders Ambystoma tigrinum

Abstract

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 ...

Antimicrobial Peptide Defenses in the Salamander, Ambystoma Tigrinum, Against Emerging Amphibian Pathogens

“…Our initial hypothesis, that the markedly different responses of A. tigrinum to ATV observed at three environmentally relevant temperatures (Rojas et al, 2005) is due to differences in production of antimicrobial peptides, was therefore not supported. Rather, our results suggest that the observed differences in susceptibility and time to death at the three experimental temperatures may largely be due to differences in immune functions other than skin peptides.…”

“…Ambient temperature has a strong effect on susceptibility of A. tigrinum to ATV (Rojas et al, 2005), but the influence of temperature on susceptibility to B. dendrobatidis has not been investigated. The ambient temperature at which tiger salamanders were acclimated had a notable influence on the effectiveness of skin peptides against B. dendrobatidis.…”

“…The ATV is responsible for localized die-offs of A. tigrinum across western North America . However, A. tigrinum larvae infected with ATV over a range of environmental temperatures (10 C, 18 C, and 26 C) differ markedly in their mortality and time to death (Rojas et al, 2005). At 26 C most larvae survive ATV exposure, but some retain the virus for up to 60 days.…”

“…However, at 18 C mortality is rapid and total, while at 10 C mortality is total but requires far longer to be expressed. In cell culture, ATV replicates at all three temperatures, but viral replication is faster at warmer temperatures (Rojas et al, 2005).…”

Environmental and Anthropogenic Factors Influencing Salamanders in Riparian Forests: A Review

“…In addition, temperature can play a role in disease ecology and infections. For instance, temperature has a strong influence on percent mortality and time to death of salamanders exposed to the Ambystoma tigrinum virus [79].…”