Ambystoma Tigrinum Virus Research Articles

Age- and dose-dependent susceptibility of axolotls (Ambystoma mexicanum) by bath exposure to Ambystoma tigrinum virus (ATV)

Ranaviruses are large, dsDNA viruses that have significant ecological and economic impact on cold-blooded vertebrates. However, our understanding of the viral proteins and subsequent host immune response(s) that impact susceptibility to infection and disease is not clear. The ranavirus Ambystoma tigrinum virus (ATV), originally isolated from the Sonoran tiger salamander (Ambystoma mavortium stebbinsi), is highly pathogenic at low doses of ATV at all tiger salamander life stages and this model has been used to explore the host-pathogen interactions of ATV infection. However, inconsistencies in the availability of laboratory reared larval tiger salamanders required us to look at the well characterized axolotl (A. mexicanum) as a model for ATV infection. Data obtained from five infection experiments over different developmental timepoints suggest that axolotls are susceptible to ATV in an age- and dose-dependent manner. These data support the use of the ATV-axolotl model to further explore the host-pathogen interactions of ranavirus infections.

Biomarkers of the influence of pyrethroids and neonicotinoids on amphibian larvae

Currently, most amphibian populations in the world exist under the influence of numerous stress factors. Among them, the main factors that affect almost all terrestrial animals, namely, the fragmentation of habitats, environmental pollution and anthropic transformation of landscapes. Moreover, those factors are joined by negative causes that affect only amphibians – specific viral and fungal infections: ranaviruses (Ambystoma tigrinum virus (ATV), Bohle iridovirus (BIV), and frog virus 3) (also dangerous for some reptiles) and chytrid fungi (Batrachochytrium dendrobatidis and B. salamandrivorans). All these factors are one of the main reasons for the current global decline of amphibian populations in the world. In today's world, agricultural chemicals are one of the most important in terms of toxicity, environmental emissions and total area of impact. Among them, a significant proportion are formed by synthetic insecticides, which include pyrethroids and neonicotinoids. Pollution by these substances has a negative impact on amphibian populations, despite the relatively short period of their life in the environment. The vast majority of studies on the effects of pyrethroid and neonicotinoid insecticides were made in laboratory experiments with the larval stages of tailless amphibians. Tadpoles are easy to get in sufficient quantities and kept in the laboratory. Cypermethrin reduces the viability of tadpoles and causes precocious metamorphosis of survived larvae. However, in a combination with other pesticides, it delays metamorphosis. In addition, embryos were more resistant to pyrethroids than tadpoles. Pyrethroid pesticides cause spasms indicating adverse neurological effects. Formation of oral apparatus abnormalities in tadpoles, anisochromasia and increasing number of immature erythrocytes are also caused by pyrethroids. Neonicotinoids show similar effects. Under the action of imidacloprid the erythrocytes are also disturbed that is showed up in the DNA damage and micronuclei formation. Neurological disorders are manifested in the ability to perceive or respond to a predator, disorientation, erratic movement and loss of balance. Pyrethroids and neonicotinoids have been shown cause a variety of disorders: increase mortality and reduce survival of tadpoles; have a teratogenic effect and affect the metamorphosis and morphological parameters of amphibians; change many biochemical parameters that characterize the protein metabolism and oxidative stress; have genotoxic effects and affect the state of the nervous system and animal behaviour. Most of these parameters are proposed for use as biomarkers of pesticide intoxication.

Genome Sequences of Ambystoma Tigrinum Virus Recovered during a Mass Die-off of Western Tiger Salamanders in Alberta, Canada.

Complete genome sequences of six Ambystoma tigrinum viruses (ATV) were determined directly from tail clips of western tiger salamanders (Ambystoma mavortium) from 2013 (high-mortality year) and 2014 (low-mortality year) in Alberta, Canada. The genome lengths ranged from 106,258 to 106,915 bp and contained 108 open reading frames encoding predicted proteins larger than 50 amino acids.

Co-exposure to multiple ranavirus types enhances viral infectivity and replication in a larval amphibian system.

Multiple pathogens commonly co-occur in animal populations, yet few studies demonstrate how co-exposure of individual hosts scales up to affect transmission. Although viruses in the genus Ranavirus are globally widespread, and multiple virus species or strains likely co-occur in nature, no studies have examined how co-exposure affects infection dynamics in larval amphibians. We exposed individual northern red-legged frog Rana aurora larvae to 2 species of ranavirus, namely Ambystoma tigrinum virus (ATV), frog virus 3 (FV3), or an FV3-like strain isolated from a frog-culturing facility in Georgia, USA (RCV-Z2). We compared single-virus to pairwise co-exposures while experimentally accounting for dosage. Co-exposure to ATV and FV3-like strains resulted in almost twice as many infected individuals compared to single-virus exposures, suggesting an effect of co-exposure on viral infectivity. The viral load in infected individuals exposed to ATV and FV3 was also higher than the single-dose FV3 treatment, suggesting an effect of co-exposure on viral replication. In a follow-up experiment, we examined how the co-occurrence of ATV and FV3 affected epizootics in mesocosm populations of larval western chorus frogs Pseudacris triseriata. Although ATV did not generally establish within host populations (<4% prevalence), when ATV and FV3 were both present, this co-exposure resulted in a larger epizootic of FV3. Our results emphasize the importance of multi-pathogen interactions in epizootic dynamics and have management implications for natural and commercial amphibian populations.

DETECTION OF COPATHOGENS IN FREE-RANGING EASTERN BOX TURTLES ( TERRAPENE CAROLINA CAROLINA) IN ILLINOIS AND TENNESSEE.

Conservation efforts are investigating the impact of diseases within a species of interest, including prevalence and transmission and morbidity and mortality rates. However, the majority of these studies focus solely on the characteristics of a single pathogen. Recently, the role of copathogens has been reported to impact disease susceptibility and mortality. To that effect, a survey was conducted including 318 eastern box turtles ( Terrapene carolina carolina) from populations in Illinois and Tennessee in 2014 and 2015. Blood samples and oral swabs were collected for quantitative polymerase chain reaction (qPCR) of 15 different pathogens performed in a multiplex format using Fluidigm array technology. Four pathogens were found with varying qPCR prevalence: ranavirus (FV3; n = 2, 0.6%), Terrapene herpesvirus 1 (TerHV1; n = 129, 40.7%), box turtle Mycoplasma sp. (BT Myco; n = 14, 4.6%), and box turtle adenovirus (BT Adv1; n = 18, 11%). Thirteen pathogens were not identified in any sample, including Mycoplasma agassizii, M. testudineum, Salmonella enteriditis, S. typhmirium, Borrelia burgdorferi, Anaplasma phagocyophilum, tortoise intranuclear coccidia, Ambystoma tigrinum virus, Bohle iridovirus, Epizootic hematopoietic necrosis virus, and testudinid herpesvirus 2. Copathogen occurrence was rare but was observed in eight individuals with TerHV1-BT Myco detection and two animals with TerHV1-Adv1. Significant differences were observed in pathogen detection across season (TerHV1, BT Adv1, BT Myco, and TerHV1-Myco) and year (TerHV1, BT Adv1, and TerHV1-Myco). The results of this survey highlight that a single pathogen model may not adequately explain pathogen dynamics and that conservation efforts need to be aimed at detecting multiple pathogens in order to fully characterize population health.

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.

Pathogen richness and abundance predict patterns of adaptive major histocompatibility complex variation in insular amphibians

The identification of the factors responsible for genetic variation and differentiation at adaptive loci can provide important insights into the evolutionary process and is crucial for the effective management of threatened species. We studied the impact of environmental viral richness and abundance on functional diversity and differentiation of the MHC class Ia locus in populations of the black-spotted pond frog (Pelophylax nigromaculatus), an IUCN-listed species, on 24 land-bridge islands of the Zhoushan Archipelago and three nearby mainland sites. We found a high proportion of private MHC alleles in mainland and insular populations, corresponding to 32 distinct functional supertypes, and strong positive selection on MHC antigen-binding sites in all populations. Viral pathogen diversity and abundance were reduced at island sites relative to the mainland, and islands housed distinctive viral communities. Standardized MHC diversity at island sites exceeded that found at neutral microsatellites, and the representation of key functional supertypes was positively correlated with the abundance of specific viruses in the environment (Frog virus 3 and Ambystoma tigrinum virus). Theseresults indicate that pathogen-driven diversifying selection can play an important role in maintaining functionally important MHC variation following island isolation, highlighting the importance of considering functionally important genetic variation and host-pathogen associations in conservation planning and management.

Identification of essential and non-essential genes in Ambystoma tigrinum virus

Members of the genus Ranavirus (family Iridoviridae) are large double-stranded (ds) DNA viruses that are found world-wide infecting fish, amphibian and reptile ectothermic hosts. Ranavirus genomes range from 105 to 155kbp in length and they are predicted to encode around 90–125 genes. Currently, our knowledge of the function of ∼50% of these genes is known or inferred based on homology to orthologous genes characterized in other systems; however, the function of the remaining open reading frames (ORFS) is unknown. Therefore, in order to begin to uncover the function of unknown ORFs in ranaviruses we developed a standardized approach to generate a recombination cassette for any ORF in Ambystoma tigrinum virus (ATV). Our standardized approach quickly and efficiently assembles recombination cassettes and recombinant ATV. We have used this approach to identify two essential, one semi-essential and two non-essential genes in ATV.

Comparative Genomics of an Emerging Amphibian Virus.

Ranaviruses, a genus of the Iridoviridae, are large double-stranded DNA viruses that infect cold-blooded vertebrates worldwide. Ranaviruses have caused severe epizootics in commercial frog and fish populations, and are currently classified as notifiable pathogens in international trade. Previous work shows that a ranavirus that infects tiger salamanders throughout Western North America (Ambystoma tigrinum virus, or ATV) is in high prevalence among salamanders in the fishing bait trade. Bait ATV strains have elevated virulence and are transported long distances by humans, providing widespread opportunities for pathogen pollution. We sequenced the genomes of 15 strains of ATV collected from tiger salamanders across western North America and performed phylogenetic and population genomic analyses and tests for recombination. We find that ATV forms a monophyletic clade within the rest of the Ranaviruses and that it likely emerged within the last several thousand years, before human activities influenced its spread. We also identify several genes under strong positive selection, some of which appear to be involved in viral virulence and/or host immune evasion. In addition, we provide support for the pathogen pollution hypothesis with evidence of recombination among ATV strains, and potential bait-endemic strain recombination.

LOW DETECTION OF RANAVIRUS DNA IN WILD POSTMETAMORPHIC GREEN FROGS, RANA (LITHOBATES) CLAMITANS, DESPITE PREVIOUS OR CONCURRENT TADPOLE MORTALITY

Ranavirus (Iridoviridae) infection is a significant cause of mortality in amphibians. Detection of infected individuals, particularly carriers, is necessary to prevent and control outbreaks. Recently, the use of toe clips to detect ranavirus infection through PCR was proposed as an alternative to the more frequently used lethal liver sampling in green frogs (Rana [Lithobates] clamitans). We attempted reevaluate the use of toe clips, evaluate the potential use of blood onto filter paper and hepatic fine needle aspirates (FNAs) as further alternatives, and explore the adequacy of using green frogs as a target-sampling species when searching for ranavirus infection in the wild. Samples were obtained from 190 postmetamorphic (≥1-yr-old) green frogs from five ponds on Prince Edward Island (PEI), Canada. Three of the ponds had contemporary or recent tadpole mortalities due to Frog Virus 3 (FV3) ranavirus. PCR testing for ranavirus DNA was performed on 190 toe clips, 188 blood samples, 72 hepatic FNAs, and 72 liver tissue samples. Only two frogs were ranavirus-positive: liver and toe clip were positive in one, liver only was positive in the other; all blood and FNAs, including those from the two positive frogs, were negative. Results did not yield a definitive answer on the efficacy of testing each type of sample, but resemble what is found in salamanders infected with Ambystoma tigrinum (rana)virus. Findings indicate a low prevalence of FV3 in postmetamorphic green frogs on PEI (≤2.78%) and suggest that green frogs are poor reservoirs (carriers) for the virus.

Ranavirosis in Invasive Bullfrogs, Belgium

To the Editor: Massive global declines in amphibians have been attributed to various causes, including infectious diseases such as chytridiomycosis and ranavirosis. Chytridiomycosis and ranaviral disease are international notifiable diseases because they have been listed by the World Organisation for Animal Health in its Animal Health Code. Ranavirosis is caused by icosahedral cytoplasmic DNA viruses that belong to the family Iridoviridae, in particular by 4 species of Ranavirus: Frog Virus 3 (FV3), Bohle iridovirus, Ambystoma tigrinum virus, and a possible species Rana catesbeiana virus Z. In Europe, FV3 has been identified in several outbreaks of ranavirosis, characterized by mass deaths, notably in green frogs (Pelophylax sp.) in Denmark, Croatia, and the Netherlands (1,2); Rana temporaria and Bufo bufo in the United Kingdom (3,4); and Alytes obstetricans and Ichthyosaura alpestris in Spain (5). The invasive exotic bullfrog (Lithobates catesbeianus) has been introduced in several European countries and has established large breeding populations in France, Italy, Germany, Greece, and Belgium (6). In addition to their direct effect on native amphibians through competition and predation, bullfrogs are thought to be carriers of chytridiomycosis (7,8) and, possibly, ranaviruses. Although mass deaths of L. catesbeianus tadpoles has been reported in aquaculture facilities, L. catesbeianus tadpoles are generally considered a subclinical reservoir of ranaviruses in the United States (9). To assess the role of bullfrogs as carriers of ranaviruses in Europe, we collected 400 clinically healthy tadpoles of L. catesbeianus from 3 invasive bullfrog populations at Hoogstraten, Belgium (51°47′Ν, 4°75′E) during May–June 2010. All larvae were euthanized as part of an invasive species eradication project and stored at –20°C until further use. At necropsy, liver tissues were collected, and DNA was extracted by using the Genomic DNA Mini Kit (BIOLINE, London, UK). PCR to detect ranavirus was performed as described by Mao et al. (10). Three samples showed positive results with this PCR. These samples were sequenced by using primers M4 and M5 described by Mao et al. (10) and blasted in GenBank. A 100% homology with the common midwife toad (A. obstetricans) ranavirus partial major capsid protein gene (GenBank accession no. {type:entrez-nucleotide,attrs:{text:FM213466.1,term_id:209869735,term_text:FM213466.1}}FM213466.1) was found (5). Despite the low prevalence of Ranavirus infection (0.75%) in the bullfrog tadpoles examined, this study shows that invasive bullfrogs, a known reservoir of chytridiomycosis, are also a likely carrier of ranaviral disease in Europe.

Innate Immune Evasion Mediated by the Ambystoma tigrinum Virus Eukaryotic Translation Initiation Factor 2α Homologue

Ranaviruses (family Iridoviridae, genus Ranavirus) are large, double-stranded DNA (dsDNA) viruses whose replication is restricted to ectothermic vertebrates. Many highly pathogenic members of the genus Ranavirus encode a homologue of the eukaryotic translation initiation factor 2α (eIF2α). Data in a heterologous vaccinia virus system suggest that the Ambystoma tigrinum virus (ATV) eIF2α homologue (vIF2αH; open reading frame [ORF] 57R) is involved in evading the host innate immune response by degrading the interferon-inducible, dsRNA-activated protein kinase, PKR. To test this hypothesis directly, the ATV vIF2αH gene (ORF 57R) was deleted by homologous recombination, and a selectable marker was inserted in its place. The ATVΔ57R virus has a small plaque phenotype and is 8-fold more sensitive to interferon than wild-type ATV (wtATV). Infection of fish cells with the ATVΔ57R virus leads to eIF2α phosphorylation, in contrast to infection with wtATV, which actively inhibits eIF2α phosphorylation. The inability of ATVΔ57R to prevent phosphorylation of eIF2α correlates with degradation of fish PKZ, an interferon-inducible enzyme that is closely related to mammalian PKR. In addition, salamanders infected with ATVΔ57R displayed an increased time to death compared to that of wtATV-infected salamanders. Therefore, in a biologically relevant system, the ATV vIF2αH gene acts as an innate immune evasion factor, thereby enhancing virus pathogenesis.

OA021-01. Construction and characterization of replication competent attenuated NYVAC-based vectors as potential HIV vaccines

To decrease virulence, we deleted the E3L gene, which is required for interferon-resistance and virulence, and replaced it with a gene from Ambystoma tigrinum virus (ATV, the new virus is NYVAC-C+12-ATV), which restores a single round of replication. Results In vitro characterization of the constructs demonstrates restoration of replication in primary and human cell lines. NYVAC-C+12-ATV leads to induction of pro-inflammatory signal transduction pathways. Pathogenicity studies in newborn mice demonstrate attenuation of 3–5 logs when compared to wt vaccinia virus or to NYCBH.

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.

Spatial and temporal patterns of Ambystoma tigrinum virus (ATV) prevalence in tiger salamanders Ambystoma tigrinum nebulosum

Amphibian ranaviruses have been documented as causes of mass mortality in amphibian populations throughout the world. The temporal and spatial dynamics of ranavirus infections when epidemics are not apparent remains unclear. To address this question, we collected tissue samples from 2003 to 2006 in 4 geographically separated tiger salamander Ambystoma tigrinum nebulosum host populations on the Kaibab Plateau in northern Arizona. We tested for Ambystoma tigrinum virus (ATV), a lethal ranavirus of tiger salamanders, calculated ATV prevalence for each sampling date, and examined temporal and spatial patterns by quantifying the annual level of ATV synchrony among populations using the intraclass correlation coefficient. Salamander populations were commonly infected with ATV. We observed no morbidity or mortality in these populations even as ATV prevalence values varied from 0 to 57%. Infection prevalence across the landscape was more similar within a given year than between years. There was no statistically significant spatial pattern in prevalence across the landscape. Our findings highlight the need to explore new hypotheses regarding the population level impact of these pathogens on amphibian communities.

Complete sequence determination of a novel reptile iridovirus isolated from soft-shelled turtle and evolutionary analysis of Iridoviridae

BackgroundSoft-shelled turtle iridovirus (STIV) is the causative agent of severe systemic diseases in cultured soft-shelled turtles (Trionyx sinensis). To our knowledge, the only molecular information available on STIV mainly concerns the highly conserved STIV major capsid protein. The complete sequence of the STIV genome is not yet available. Therefore, determining the genome sequence of STIV and providing a detailed bioinformatic analysis of its genome content and evolution status will facilitate further understanding of the taxonomic elements of STIV and the molecular mechanisms of reptile iridovirus pathogenesis.ResultsWe determined the complete nucleotide sequence of the STIV genome using 454 Life Science sequencing technology. The STIV genome is 105 890 bp in length with a base composition of 55.1% G+C. Computer assisted analysis revealed that the STIV genome contains 105 potential open reading frames (ORFs), which encode polypeptides ranging from 40 to 1,294 amino acids and 20 microRNA candidates. Among the putative proteins, 20 share homology with the ancestral proteins of the nuclear and cytoplasmic large DNA viruses (NCLDVs). Comparative genomic analysis showed that STIV has the highest degree of sequence conservation and a colinear arrangement of genes with frog virus 3 (FV3), followed by Tiger frog virus (TFV), Ambystoma tigrinum virus (ATV), Singapore grouper iridovirus (SGIV), Grouper iridovirus (GIV) and other iridovirus isolates. Phylogenetic analysis based on conserved core genes and complete genome sequence of STIV with other virus genomes was performed. Moreover, analysis of the gene gain-and-loss events in the family Iridoviridae suggested that the genes encoded by iridoviruses have evolved for favoring adaptation to different natural host species.ConclusionThis study has provided the complete genome sequence of STIV. Phylogenetic analysis suggested that STIV and FV3 are strains of the same viral species belonging to the Ranavirus genus in the Iridoviridae family. Given virus-host co-evolution and the phylogenetic relationship among vertebrates from fish to reptiles, we propose that iridovirus might transmit between reptiles and amphibians and that STIV and FV3 are strains of the same viral species in the Ranavirus genus.

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.

Testing a key assumption of host‐pathogen theory: density and disease transmission

Conventional disease theory suggests that extinction with density‐dependent transmission is unlikely as the threshold host density (KT) is greater than zero. Extinction may result if transmission is frequency dependent or the pathogen has an environmental reservoir. Given the importance of understanding how pathogens affect species richness and diversity there are few empirical tests of these conclusions. We used anAmbystoma tigrinum–Ambystoma tigrinumvirus (ATV) model system in the laboratory to examine disease transmission dynamics. SusceptibleA. tigrinumlarvae were exposed to three different densities and proportions of infected larvae for 24 h. We then housed susceptible hosts individually for 28 days and monitored them for infection. The density of infected hosts to which susceptible hosts were exposed was the best predictor of infection (p=0.037). There was no effect of host clutch on the probability of becoming infected (p=0.67). Larvae in the highest density treatments died sooner than larvae in lower density treatments (p&lt;0.001). Asymptomatic but infected hosts shed sufficient virus into the water in a 24‐h period to infect susceptible hosts without any direct contact between individuals. ATV transmission was best described by a power function, leading to the prediction that extinction ofA. tigrinumas a result of this pathogen is unlikely. Indeed, field observations show that larval salamander populations that experience ATV‐driven epidemics may decrease, but not to extinction, and then recover. Disease is proposed as a possible explanation for the global decline of amphibians. Ranaviruses infect many amphibian populations, but based on our results may not be a general cause of declines to extinction. In contrast, frequency dependent transmission, environmental reservoirs and alternative hosts may be the most likely explanation for the enigmatic decline, at times to extinction, of some amphibian populations as a result of emerging infectious diseases, like the chytrid fungusBatrachochytrium dendrobatidis.