Emerging Pathogens and a Current-Use Pesticide: Potential Impacts on Eastern Hellbenders.

Populations of the eastern hellbender Cryptobranchus alleganiensis alleganiensis have been declining for decades, and emerging pathogens and pesticides are hypothesized to be contributing factors. However, few empirical studies have attempted to test the potential effects of these factors on hellbenders. We simultaneously exposed subadult hellbenders to environmentally relevant concentrations of either Batrachochytrium dendrobatidis (Bd) or a frog virus 3-like ranavirus (RV), a combination of the pathogens, or each pathogen following exposure to a glyphosate herbicide (Roundup). Additionally, we measured the ability of the skin mucosome to inactivate Bd and RV in growth assays. We found that mucosome significantly inactivated RV by an average of 40% but had no negative effects on Bd growth. All treatments that included RV exposure experienced reduced survival compared to controls, and the combination of RV and herbicide resulted in 100% mortality. Histopathology verified RV as the cause of mortality in all RV-exposed treatments. No animals were infected with Bd or died in the Bd-only treatment. Our results suggest that RV exposure may be a significant threat to the survival of subadult hellbenders and that Roundup exposure may potentially exacerbate this threat.

Ranavirus could facilitate local extinction of rare amphibian species.

There is growing evidence that pathogens play a role in population declines and species extinctions. For small populations, disease-induced extinction may be especially probable. We estimated the susceptibility of two amphibian species of conservation concern (the dusky gopher frog [Lithobates sevosus] and boreal toad [Anaxyrus boreas boreas]) to an emerging pathogen (ranavirus) using laboratory challenge experiments, and combined these data with published demographic parameter estimates to simulate the potential effects of ranavirus exposure on extinction risk. We included effects of life stage during pathogen exposure, pathogen exposure interval, hydroperiod of breeding habitat, population carrying capacity, and immigration in simulations. We found that both species were highly susceptible to ranavirus when exposed to the pathogen in water at environmentally relevant concentrations. Dusky gopher frogs experienced 100 % mortality in four of six life stages tested. Boreal toads experienced 100 % mortality when exposed as tadpoles or metamorphs, which were the only life stages tested. Simulations showed population declines, greater extinction probability, and faster times to extinction with ranavirus exposure. These effects were more evident with more frequent pathogen exposure intervals and lower carrying capacity. Immigration at natural rates did little to mitigate effects of ranavirus exposure unless immigration occurred every 2 years. Our results demonstrate that disease-induced extinction by emerging pathogens, such as ranavirus, is possible, and that threat may be especially high for species with small population sizes. For the species in this study, conservation organizations should incorporate ranavirus surveillance into monitoring programs and devise intervention strategies in the event that disease outbreaks occur.

Water Temperature Affects Susceptibility to Ranavirus.

The occurrence of emerging infectious diseases in wildlife populations is increasing, and changes in environmental conditions have been hypothesized as a potential driver. For example, warmer ambient temperatures might favor pathogens by providing more ideal conditions for propagation or by stressing hosts. Our objective was to determine if water temperature played a role in the pathogenicity of an emerging pathogen (ranavirus) that infects ectothermic vertebrate species. We exposed larvae of four amphibian species to a Frog Virus 3 (FV3)-like ranavirus at two temperatures (10 and 25°C). We found that FV3 copies in tissues and mortality due to ranaviral disease were greater at 25°C than at 10°C for all species. In a second experiment with wood frogs (Lithobates sylvaticus), we found that a 2°C change (10 vs. 12°C) affected ranaviral disease outcomes, with greater infection and mortality at 12°C. There was evidence that 10°C stressed Cope's gray tree frog (Hyla chrysoscelis) larvae, which is a species that breeds during summer-all individuals died at this temperature, but only 10% tested positive for FV3 infection. The greater pathogenicity of FV3 at 25°C might be related to faster viral replication, which in vitro studies have reported previously. Colder temperatures also may decrease systemic infection by reducing blood circulation and the proportion of phagocytes, which are known to disseminate FV3 through the body. Collectively, our results indicate that water temperature during larval development may play a role in the emergence of ranaviruses.

High tissue burden of Toxoplasma gondii is the hallmark of acute virulence in mice.

Toxoplasma gondii virulence is commonly determined by mortality rate of infected mice. Limited data showed that virulent T. gondii strains had increased parasite growth in mice compared to that of less virulent strains. To determine if this is a common phenomenon for a variety of strains and to develop an alternative assay to test acute virulence in mice, we measured parasite burdens in experimentally infected outbred CD-1 mice for 19 T. gondii isolates, in which the virulence phenotypes had previously been determined by mortality assay. Our results showed that parasite concentrations in spleen tissues were two orders of magnitude higher in the virulent than the intermediately and non-virulent isolates at day 7 post infection. In competition assays, mice inoculated with mixed tachyzoites of virulent and intermediately virulent strains or virulent and non-virulent strains showed that the former always reached a higher concentration at day 7 post infection. In mixed infection of intermediate and non-virulent strains, both strains were detectable in mice at day 7 post infection. In conclusion, our data showed that the virulence of T. gondii can be predicted by parasite load in the spleen tissue of infected mice at 7 days post infection, providing an alternative method to determine virulence of Toxoplasma.

Differential gene expression in mice infected with distinct Toxoplasma strains.

Toxoplasma gondii is the causative agent of toxoplasmosis in human and animals. In a mouse model, T. gondii strains can be divided into three groups, including the virulent, intermediately virulent, and nonvirulent. The clonal type I, II, and III T. gondii strains belong to these three groups, respectively. To better understand the basis of virulence phenotypes, we investigated mouse gene expression responses to the infection of different T. gondii strains at day 5 after intraperitoneal inoculation with 500 tachyzoites. The transcriptomes of mouse peritoneal cells showed that 1,927, 1,573, and 1,009 transcripts were altered more than 2-fold by type I, II, and III infections, respectively, and that the majority of altered transcripts were shared. Overall transcription patterns were similar in type I and type II infections, and both had greater changes than infection with type III. Quantification of parasite burden in mouse spleens showed that the burden with type I infection was 1,000 times higher than that of type II and that the type II burden was 20 times higher than that of type III. Fluorescence-activated cell sorting revealed that type I and II infections had comparable macrophage populations, and both were higher than the population with type III infection. In addition, type I infection had a higher percentage of neutrophils than type II and III infections. Taken together, these results suggested that there is a common gene expression response to T. gondii infection in mice. This response is further modified by parasite strain-specific factors that determine their distinct virulence phenotypes.