Depauperate major histocompatibility complex variation in the endangered reticulated flatwoods salamander (Ambystoma bishopi).

Reticulated flatwoods salamander (Ambystoma bishopi) populations began decreasing dramatically in the 1900s. Contemporary populations are small, isolated, and may be susceptible to inbreeding and reduced adaptive potential because of low genetic variation. Genetic variation at immune genes is especially important as it influences disease susceptibility and adaptation to emerging infectious pathogens, a central conservation concern for declining amphibians. We collected samples from across the extant range of this salamander to examine genetic variation at major histocompatibility complex (MHC) class Iα and IIß exons as well as the mitochondrial control region. We screened tail or toe tissue for ranavirus, a pathogen associated with amphibian declines worldwide. Overall, we found low MHC variation when compared to other amphibian species and did not detect ranavirus at any site. MHC class Iα sequencing revealed only three alleles with a nucleotide diversity of 0.001, while MHC class IIß had five alleles with a with nucleotide diversity of 0.004. However, unique variation still exists across this species' range with private alleles at three sites. Unlike MHC diversity, mitochondrial variation was comparable to levels estimated for other amphibians with nine haplotypes observed, including one haplotype shared across all sites. We hypothesize that a combination of a historic disease outbreak and a population bottleneck may have contributed to low MHC diversity while maintaining higher levels of mitochondrial DNA variation. Ultimately, MHC data indicated that the reticulated flatwoods salamander may be at an elevated risk from infectious diseases due to low levels of immunogenetic variation necessary to combat novel pathogens.

ESTIMATING OCCURRENCE, PREVALENCE, AND DETECTION OF AMPHIBIAN PATHOGENS: INSIGHTS FROM OCCUPANCY MODELS.

Understanding the distribution of pathogens across landscapes and their prevalence within host populations is a common aim of wildlife managers. Despite the need for unbiased estimates of pathogen occurrence and prevalence for planning effective management interventions, many researchers fail to account for imperfect pathogen detection. Instead raw data are often reported, which may lead to ineffective, or even detrimental, management actions. We illustrate the utility of occupancy models for generating unbiased estimates of disease parameters by 1) providing a written tutorial describing how to fit these models in Program PRESENCE and 2) presenting a case study with the pathogen ranavirus. We analyzed ranavirus detection data from a wildlife refuge (Maryland, US) using occupancy modeling, which yields unbiased estimates of pathogen occurrence and prevalence. We found ranavirus prevalence was underestimated by up to 30% if imperfect pathogen detection was ignored. The unbiased estimate of ranavirus prevalence in larval wood frog (Lithobates sylvaticus; 0.73) populations was higher than in larval spotted salamander (Ambystoma maculatum; 0.56) populations. In addition, the odds of detecting ranavirus in tail samples were 6.7 times higher than detecting ranavirus in liver samples. Therefore, tail samples presented a nonlethal sampling method for ranavirus that may be able to detect early (nonsystemic) infections.

Times from Infection to Disease-Induced Death and their Influence on Final Population Sizes After Epidemic Outbreaks.

For epidemic models, it is shown that fatal infectious diseases cannot drive the host population into extinction if the incidence function is upper density-dependent. This finding holds even if a latency period is included and the time from infection to disease-induced death has an arbitrary length distribution. However, if the incidence function is also lower density-dependent, very infectious diseases can lead to a drastic decline of the host population. Further, the final population size after an epidemic outbreak can possibly be substantially affected by the infection-age distribution of the initial infectives if the life expectations of infected individuals are an unbounded function of infection age (time since infection). This is the case for lognormal distributions, which fit data from infection experiments involving tiger salamander larvae and ranavirus better than gamma distributions and Weibull distributions.

Characterization of a PKR inhibitor from the pathogenic ranavirus, Ambystoma tigrinum virus, using a heterologous vaccinia virus system.

Ambystoma tigrinum virus (ATV) (family Iridoviridae, genus Ranavirus) was isolated from diseased tiger salamanders (Ambystoma tigrinum stebbinsi) from the San Rafael Valley in southern Arizona, USA in 1996. Genomic sequencing of ATV, as well as other members of the genus, identified an open reading frame that has homology to the eukaryotic translation initiation factor, eIF2α (ATV eIF2α homologue, vIF2αH). Therefore, we asked if the ATV vIF2αH could also inhibit PKR. To test this hypothesis, the ATV vIF2αH was cloned into vaccinia virus (VACV) in place of the well-characterized VACV PKR inhibitor, E3L. Recombinant VACV expressing ATV vIF2αH partially rescued deletion of the VACV E3L gene. Rescue coincided with rapid degradation of PKR in infected cells. These data suggest that the salamander virus, ATV, contains a novel gene that may counteract host defenses, and this gene product may be involved in the presentation of disease caused by this environmentally important pathogen.

The Ambystoma tigrinum virus (ATV) RNase III gene can modulate host PKR activation and interferon production.

The iridovirus RNase III gene is one of 26 conserved core genes among the family Iridoviridae. Initial studies suggest this viral protein functions to suppress RNA interference pathways that may attack viral RNA during infection. Therefore, to determine if the Ambystoma tigrinum virus (ATV) RNase III-like gene (ORF 25R) can modulate the host innate immune response fish and human cells ectopically expressing 25R were treated with polyI:C and monitored for interferon synthesis and phosphorylation of eIF2α and PKR. We found a decrease in cellular IFN production and modulation of the PKR pathway. In addition, ATV deleted of the RNase III gene (ATVΔ25R) shows reduced pathogenicity in tiger salamanders. Collectively our data suggest that the ATV 25R protein is a pathogenesis factor that may function to help evade the host's immune response by masking activators of the IFN pathway.

User-friendly Taqman probe coupled-insulated isothermal PCR (iiPCR) for rapid detection of emerging Ambystoma tigrinum virus (ATV) in western tiger salamanders (Ambystoma mavortium) on a compact, portable instrument.

Portable user-friendly diagnostic tests can benefit detection and surveillance of wildlife diseases. Here, the performance of a compact POCKIT™ Nucleic Acid Analyzer for detection of Ambystoma tigrinum virus (ATV), an emerging Iridovirus that is associated with high host mortality in the western tiger salamander (Ambystoma mavortium) in North America was assessed. Tissue samples from 188 larval tiger salamanders collected from sites in Alberta, Canada were tested by both iiPCR and by conventional PCR. Results of the two assays showed 96.3% agreement. All 176 samples that tested positive by conventional PCR were also positive by iiPCR, while 12 of the samples that were negative by conventional PCR were positive by iiPCR. Comparison of the limits of detection of the two assays shows that the iiPCR assay was more sensitive than conventional PCR and had a LOD95 of 20 copies per reaction. The instrument automatically analyzes and displays results within 40min following nucleic acid extraction. The novel technology could enhance detection of, and response to, wildlife pathogens, particularly those that occur sporadically, cause rapid outbreaks, and/or occur within isolated geographical regions.

Combined effects of virus, pesticide, and predator cue on the larval tiger salamander (Ambystoma tigrinum).

Emerging diseases and environmental contamination are two of the leading hypotheses for global amphibian declines. Yet few studies have examined the influence of contaminants on disease susceptibility, and even fewer have incorporated the role of natural stressors such as predation. We performed a factorial study investigating the interaction of the insecticide carbaryl, dragonfly predator cue, and the emerging pathogen Ambystoma tigrinum virus (ATV) on fitness correlates and disease susceptibility in tiger salamander larvae. Four week old larvae were exposed for 22 days in a 2 (0, 500 µg/l carbaryl) × 2 (control, predator cue water) × 2 (0, 1 × 10(4) pfu ATV) factorial designed laboratory study. Results show significant impacts to survival of larvae for both virus and predator cue treatments, as well as an interactive effect between the two, in which predator cue strongly exacerbated disease-driven mortality. There was a clear pattern of reduced survival with the addition of stressors, with those where all three stressors were present exhibiting the worst effects (a decrease in survival from 93 to 60%). On those that survived, we also detected several sub-lethal impacts in mass, SVL, and development. Predator cue and pesticide treatments significantly reduced both SVL and mass. Virus and predator treatments significantly slowed development. Stressors also exhibited opposing effects on activity. Predator cue caused a significant reduction in activity, whereas virus caused a significant increase in activity over time. These results highlight the importance of examining combined natural and introduced stressors to understand potential impacts on amphibian species. Such stressors may contribute to the emergence of ATV in particular regions, raising concerns about the influence of pesticides on disease emergence in general.

Pathogen host switching in commercial trade with management recommendations.

Global wildlife trade exacerbates the spread of nonindigenous species. Pathogens also move with hosts through trade and often are released into naïve populations with unpredictable outcomes. Amphibians are moved commercially for pets, food, bait, and biomedicine, and are an excellent model for studying how wildlife trade relates to pathogen pollution. Ranaviruses are amphibian pathogens associated with annual population die-offs; multiple strains of tiger salamander ranaviruses move through the bait trade in the western United States. Ranaviruses infect amphibians, reptiles, and fish and are of additional concern because they can switch hosts. Tiger salamanders are used as live bait for freshwater fishing and are a potential source for ranaviruses switching hosts from amphibians to fish. We experimentally injected largemouth bass with a bait trade tiger salamander ranavirus. Largemouth bass became infected but exhibited no signs of disease or mortality. Amphibian bait ranaviruses have the potential to switch hosts to infect fish, but fish may act as dead-end hosts or nonsymptomatic carriers, potentially spreading infection as a result of trade.

Combined effects of atrazine and chlorpyrifos on susceptibility of the tiger salamander to Ambystoma tigrinum virus.

Several hypotheses have been examined as potential causes of global amphibian declines, including emerging infectious diseases and environmental contaminants. Although these factors are typically studied separately, animals are generally exposed to both stressors simultaneously. We examined the effects of the herbicide atrazine and the insecticide chlorpyrifos on the susceptibility of tiger salamander larvae, Ambystoma tigrinum, to a viral pathogen, Ambystoma tigrinum virus (ATV). Environmentally relevant concentrations of atrazine (0, 20, 200 microg/L) and chlorpyrifos (0, 2, 20, 200 microg/L) were used along with ATV in a fully factorial experimental design whereby individually housed, 4-week-old larvae were exposed for 2 weeks. Atrazine alone was not lethal to larvae, and chlorpyrifos alone was lethal only at the highest concentration. When combined with ATV, chlorpyrifos increased susceptibility to viral infection and resulted in increased larval mortality. A significant interactive effect between atrazine and ATV was detected. Atrazine treatments slightly decreased survival in virus-exposed treatments, yet slightly increased survival in the virus-free treatments. These findings corroborate earlier research on the impacts of atrazine, in particular, on disease susceptibility, but exhibit greater effects (i.e., reduced survival) when younger larvae were examined. This study is the first of its kind to demonstrate decreases in amphibian survival with the combination of pesticide and a viral disease. Further examination of these multiple stressors can provide key insights into potential significance of environmental cofactors, such as pesticides, in disease dynamics.

Mortality rates differ among amphibian populations exposed to three strains of a lethal ranavirus.

Infectious diseases are a growing threat to biodiversity, in many cases because of synergistic effects with habitat loss, environmental contamination, and climate change. Emergence of pathogens as new threats to host populations can also arise when novel combinations of hosts and pathogens are unintentionally brought together, for example, via commercial trade or wildlife relocations and reintroductions. Chytrid fungus (Batrachochytrium dendrobatidis) and amphibian ranaviruses (family Iridoviridae) are pathogens implicated in global amphibian declines. The emergence of disease associated with these pathogens appears to be at least partly related to recent translocations over large geographic distances. We experimentally examined the outcomes of novel combinations of host populations and pathogen strains using the amphibian ranavirus Ambystoma tigrinum virus (ATV) and barred tiger salamanders (Ambystoma mavortium, formerly considered part of the Ambystoma tigrinum complex). One salamander population was highly resistant to lethal infections by all ATV strains, including its own strain, and mortality rates differed among ATV strains according to salamander population. Mortality rates in novel pairings of salamander population and ATV strain were not predictable based on knowledge of mortality rates when salamander populations were exposed to their own ATV strain. The underlying cause(s) for the differences in mortality rates are unknown, but local selection pressures on salamanders, viruses, or both, across the range of this widespread host-pathogen system are a plausible hypothesis. Our study highlights the need to minimize translocations of amphibian ranaviruses, even among conspecifc host populations, and the importance of considering intraspecific variation in endeavors to manage wildlife diseases.

Antimicrobial peptide defenses in the salamander, Ambystoma tigrinum, against emerging amphibian pathogens.

Skin peptides were collected from living Ambystoma tigrinum larvae and adults and tested against two emerging pathogens, Batrachochytrium dendrobatidis and the Ambystoma tigrinum virus (ATV), as well as bacteria isolated from A. tigrinum. Natural mixtures of skin peptides were found to inhibit growth of B. dendrobatidis, Staphylococcus aureus, and Klebsiella sp., but activity against ATV was unpredictable. Skin peptides collected from salamanders held at three environmentally relevant temperatures differed in activity against B. dendrobatidis. Activity of the A. tigrinum skin peptides was found to be strongly influenced by pH.

Habitat fragmentation as a result of biotic and abiotic factors controls pathogen transmission throughout a host population.

1. The hypothesis that habitat fragmentation (biotic or abiotic) alters the transmission of disease within a population is explored using field data from a well-studied amphibian-pathogen system. 2. We used the Ambystoma tigrinum-A. tigrinum virus (ATV) model system to show how habitat fragmentation as a result of emergent vegetation and habitat management affects disease transmission dynamics in ponds across a landscape. 3. We quantified variation in ATV infection over time and across the landscape. ATV infection was significantly higher in ponds modified for livestock use (P = 0.032). Disease incidence decreased with increased amounts of emergent vegetation (P < 0.001). These factors appear to control disease transmission by altering the host contact rate and with it disease transmission. 4. A field experiment to test the effect of emergent vegetation on the distribution of larvae in ponds demonstrated a behavioural change in larvae found in sparsely vegetated ponds. Microhabitat choices resulted in larvae being concentrated at the pond edge resulting in a 'halo effect' in sparsely vegetated ponds, whereas larvae in heavily vegetated ponds were distributed more evenly throughout. Microhabitat choice affects the effective density that larvae experience. This 'halo effect' increases contact rates in the shallows of sparsely vegetated ponds and increases the transmission of a directly transmitted pathogen. 5. Despite recurrent epidemics of a lethal Ranavirus in tiger salamanders on the Kaibab Plateau, Arizona, USA, these populations persist. We discuss the implications of our results in the context of density-dependent transmission and homogeneous mixing, two increases key assumptions of epidemiological theory.

Transmission dynamics of the amphibian ranavirus Ambystoma tigrinum virus.

Transmission is central to pathogen fitness and strongly influences the impact of pathogens on host populations. Particularly important to transmission dynamics is the distinction between direct transmission requiring close physical contact (e.g. bumping, fighting, or coughing) and indirect transmission from environmental sources such as contaminated substrates. We present data from 4 experiments addressing the form, routes, and timing of transmission of Ambystoma tigrinum virus (ATV) among tiger salamanders Ambystoma tigrinum nebulosum. Our data suggest that ATV is efficiently transmitted by direct interactions between live animals (bumping, biting and cannibalism) as well as by necrophagy and indirectly via water and fomites. Determining which form of transmission is most important in nature is essential for understanding transmission at the population level. Our experiments also revealed an important temporal aspect of infectiousness: larval salamanders become infectious soon after exposure to ATV and their propensity to infect others increases with time. These results begin to clarify the mechanisms and dynamics of ATV transmission and lead to key questions that need to be addressed in future research.

Susceptibility of the endangered California tiger salamander, Ambystoma californiense, to ranavirus infection.

Emerging infectious diseases are implicated in the declines and extinctions of amphibians worldwide. Ranaviruses in the family Iridoviridae are a global concern and have caused amphibian die-offs in wild populations in North America, Europe, South America, and in commercial populations in Asia and South America. The movement of amphibians for bait, food, pets, and research provides a route for the introduction of ranaviruses into naive and potentially endangered species. In this report, we demonstrate that the California tiger salamander, Ambystoma californiense, is susceptible to Ambystoma tigrinum virus (ATV). This virus has not been previously reported in California tiger salamander, but observed mortality in experimentally infected animals suggests that California tiger salamander populations could be adversely affected by an ATV introduction.

Effects of atrazine and iridovirus infection on survival and life-history traits of the long-toed salamander (Ambystoma macrodactylum).

Environmental contaminants and emerging infectious diseases are implicated as factors contributing to global amphibian declines. However, few studies have tested the interaction of these factors. We exposed six-week-old, larval long-toed salamanders (Ambystoma macrodactylum) to Ambystoma tigrinum virus (ATV; 0 or 10(3.5) plaque-forming units/ml) and sublethal concentrations of atrazine (0, 1.84, 18.4, and 184 microg/L) in a 4 x 2 factorial design for 30 d. We tested the effects of atrazine and virus on mass and snout-vent length (SVL) at metamorphosis and larval period as well as on rates of mortality and viral infectivity. We confirmed ATV transmission to A. macrodactylum via polymerase chain reaction, but infection rates were lower than expected, consistent with the theory predicting lower pathogen transmission to nonnative hosts. Larvae exposed to both atrazine and ATV had lower levels of mortality and ATV infectivity compared to larvae exposed to virus alone, suggesting atrazine may compromise virus efficacy. The highest atrazine level (184 microg/L) accelerated metamorphosis and reduced mass and SVL at metamorphosis significantly relative to controls. Exposure to ATV also significantly reduced SVL at metamorphosis. The present study suggests moderate concentrations of atrazine may ameliorate effects of ATV on long-toed salamanders, whereas higher concentrations initiate metamorphosis at a smaller size, with potential negative consequences to fitness.

Atrazine increases ranavirus susceptibility in the tiger salamander, Ambystoma tigrinum.

Pathogenic diseases and environmental contaminants are two of the leading hypotheses for global amphibian declines, yet few studies have examined the influence of contaminants on disease susceptibility. In this study, we examined effects of ecologically relevant doses of atrazine (0, 1.6, 16, and 160 microg/L), sodium nitrate (0, 6.8, 68 mg/L), and their interactions on susceptibility of four laboratory-bred tiger salamander families to Ambystoma tigrinum virus (ATV), a pathogen implicated in global amphibian die-offs. Salamanders were from Arizona populations where a coevolutionary history with ATV is supported, and thus cofactors rather than recent introduction may contribute to disease epizootics. Use of atrazine and nitrogenous fertilizers are ubiquitous; therefore, the impact of these cofactors on disease susceptibility is an important consideration. Atrazine and sodium nitrate significantly decreased peripheral leukocyte levels, suggesting an impact of these contaminants on the immune system. As expected from this result, atrazine significantly increased susceptibility of larvae to ATV infection. In contrast, nitrate had a marginally significant main effect and significantly decreased infection rate at the highest level. However, neither atrazine nor sodium nitrate had significant effects on viral copy number per individual. These results suggest that ecologically relevant concentrations of atrazine and nitrates have immunosuppressive effects, and atrazine may contribute to ATV epizootics, raising concerns about the influence of contaminants on diseases in general.

Influence of temperature on Ranavirus infection in larval salamanders Ambystoma tigrinum.

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 degrees C, whereas all died at 18 degrees C and nearly all died at 10 degrees C. Some asymptomatic salamanders that survived 60 d at 10 or 26 degrees 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 degrees C than at 18 degrees C but little virus, if any, was present in the small number of salamanders that died at 26 degrees C. These results may help explain periodic viral epizootics in field populations of A. tigrinum where water temperatures fluctuate widely.

Transmission of the Ambystoma tigrinum virus to alternative hosts.

Ambystoma tigrinum virus (ATV) is a lethal virus originally isolated from Sonora tiger salamanders Ambystoma tigrinum stebbinsi in the San Rafael Valley in southern Arizona. USA. ATV is implicated in several salamander epizootics. We attempted to transmit ATV experimentally to fish and amphibians by injection, water bath exposure, or feeding to test whether ATV can cause clinical signs of infection or be recovered from exposed individuals that do not show clinical signs. Cell culture and polymerase chain reaction of the viral major capsid protein gene were used for viral detection. Salamanders and newts became infected with ATV and the virus was recovered from these animals, but virus could not be recovered from any of the frogs or fish tested. These results suggest that ATV may only infect urodeles and that fish and frogs may not be susceptible to ATV infection.