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.     

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.     

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.     

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.     

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.     

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.     

A DNA-based assay identifies Batrachochytrium dendrobatidis in amphibians

Chytridiomycosis caused by Batrachochytrium dendrobatidis (Chytridiomycota) has been implicated in declines of amphibian populations on four continents. We have developed a sensitive and specific polymerase chain reaction-based assay to detect this pathogen. We isolated B. dendrobatidis from captive and wild amphibians collected across North America and sequenced the internal transcribed spacer regions of the rDNA cassette of multiple isolates. We identified two primers (Bd1a and Bd2a) that are specific to B. dendrobatidis under amplification conditions described in this study. DNA amplification with Bd1a/Bd2a primers produced a fragment of approximately 300 bp from B. dendrobatidis DNA but not from DNA of other species of chytrids or common soil fungi. The assay detected 10 zoospores or 10 pg of DNA from B. dendrobatidis and detected infections in skin samples from a tiger salamander (Ambystoma tigrinum), boreal toads (Bufo boreas), Wyoming toads (Bufo baxteri), and smooth-sided toads (Bufo guttatus). This assay required only small samples of skin and can be used to process a large number of samples.     

Reliability of non-lethal surveillance methods for detecting ranavirus infection

Ranaviruses have been identified as the etiologic agent in many amphibian die-offs across the globe. Polymerase chain reaction (PCR) is commonly used to detect ranavirus infection in amphibian hosts, but the test results may vary between tissue samples obtained by lethal and non-lethal procedures. Testing liver samples for infection is a common lethal sampling technique to estimate ranavirus prevalence because the pathogen often targets this organ and the liver is easy to identify and collect. However, tail clips or swabs may be more practicable for ranavirus surveillance programs compared with collecting and euthanizing animals, especially for uncommon species. Using PCR results from liver samples for comparison, we defined false-positive test results as occurrences when a non-lethal technique indicated positive but the liver sample was negative. Similarly, we defined false-negative test results as occurrences when a non-lethal technique was negative but the liver sample was positive. Using these decision rules, we estimated false-negative and false-positive rates for tail clips and swabs. Our study was conducted in a controlled facility using American bullfrog Lithobates catesbeianus tadpoles; false-positive and false-negative rates were estimated after different periods of time following exposure to ranavirus. False-negative and false-positive rates were 20 and 6%, respectively, for tail samples, and 22 and 12%, respectively, for swabs. False-negative rates were constant over time, but false-positive rates decreased with post-exposure duration. Our results suggest that non-lethal sampling techniques can be useful for ranavirus surveillance, although the prevalence of infection may be underestimated when compared to results obtained with liver samples.     

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 μg/L) and chlorpyrifos (0, 2, 20, 200 μg/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 an Ambystoma tigrinum–Ambystoma tigrinum virus (ATV) model system in the laboratory to examine disease transmission dynamics. Susceptible A. tigrinum larvae 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<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 of A. tigrinum as 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 fungus Batrachochytrium dendrobatidis.     

Prevalence of scrapie infection in Great Britain: interpreting the results of the 1997-1998 abattoir survey

An accurate estimate of the prevalence of scrapie infection in the Great Britain (GB) sheep flock is essential when assessing any potential risk to human health through exposure to sheep transmissible spongiform encephalopathies (TSEs). One method for assessing the prevalence is to sample sheep intended for human consumption using a diagnostic test capable of detecting infected animals prior to the onset of clinical signs. An abattoir survey conducted in Great Britain in 1997-1998 tested brain samples from 2809 apparently healthy sheep of which none was found to be positive for scrapie by histopathology or immunohistochemistry (IHC) although 10 were positive for scrapie-associated fibrils (SAF). Subsequently, the tonsils from a subset of the animals sampled were examined using IHC, one of which tested positive. To interpret these results we use a likelihood-based approach, which accounts for the variation in the prevalence of infection with age and test sensitivity and specificity with stage of infection. Combining the results for all of the diagnostic tests yields an estimate of the prevalence of scrapie infection in the GB sheep flock of 0.22% (95% confidence interval: 0.01-0.97%). Moreover, our analysis suggests that all of the diagnostic tests used are very specific (greater than 99%). Indeed, only SAF detection yields a specificity estimate of less than 100%, which helps to account for the high number of samples found to be positive for SAF.     

Specific dynamic action of ambystomatid salamanders and the effects of meal size, meal type, and body temperature

The past decade has witnessed a dramatic increase in studies of amphibian and reptile specific dynamic action (SDA). These studies have demonstrated that SDA, the summed energy expended on meal digestion and assimilation, is affected significantly by meal size, meal type, and body size and to some extent by body temperature. While much of this attention has been directed at anuran and reptile SDA, we investigated the effects of meal size, meal type, and body temperature on the postprandial metabolic responses and the SDA of the tiger salamander (Ambystoma tigrinum tigrinum). We also compared the SDA responses among six species of Ambystoma salamanders representing the breadth of Ambystoma phylogeny. Postprandial peaks in VO(2) and VO(2), duration of elevated metabolism, and SDA of tiger salamanders increased with the size of cricket meals (2.5%-12.5% of body mass). For A. tigrinum, as for other ectotherms, a doubling of meal size results in an approximate doubling of SDA, a function of equal increases in peak VO(2) and duration. For nine meal types of equivalent size (5% of body mass), the digestion of hard-bodied prey (crickets, superworms, mealworms, beetles) generated larger SDA responses than the digestion of soft-bodied prey (redworms, beetle larvae). Body temperature affected the profile of postprandial metabolism, increasing the peak and shortening the duration of the profile as body temperature increased. SDA was equivalent among three body temperatures (20 degrees, 25 degrees, and 30 degrees C) but decreased significantly at 15 degrees C. Comparatively, the postprandial metabolic responses and SDA of Ambystoma jeffersonianum, Ambystoma maculatum, Ambystoma opacum, Ambystoma talpoideum, Ambystoma texanum, and the conspecific Ambystoma tigrinum mavortium digesting cricket meals that were 5% of their body mass were similar (independent of body mass) to those of A. t. tigrinum. Among the six species, standard metabolic rate, peak postprandial VO(2), and SDA scaled with body mass with mass exponents of 0.72, 0.78, and 1.05, respectively.     

Acceleration of Ambystoma tigrinum metamorphosis by corticotropin-releasing hormone

Previous work of others and ours has shown that corticotropin-releasing hormone (CRH) is a positive stimulus for thyroid and interrenal hormone secretion in amphibian larvae and that activation of CRH neurons may mediate environmental effects on the timing of metamorphosis. These studies have investigated CRH actions in anurans (frogs and toads), whereas there is currently no information regarding the actions of CRH on metamorphosis of urodeles (salamanders and newts). We tested the hypothesis that CRH can accelerate metamorphosis of tiger salamander (Ambystoma tigrinum) larvae. We injected tiger salamander larvae with ovine CRH (oCRH; 1 microg/day; i.p.) and monitored effects on metamorphosis by measuring the rate of gill resorption. oCRH-injected larvae completed metamorphosis earlier than saline-injected larvae. There was no significant difference between uninjected and saline-injected larvae. Mean time to reach 50% reduction in initial gill length was 6.9 days for oCRH-injected animals, 11.9 days for saline-injected animals, and 14.1 days for uninjected controls. At the conclusion of the experiment (day 15), all oCRH-injected animals had completed metamorphosis, whereas by day 15, only 50% of saline-injected animals and 33% of uninjected animals had metamorphosed. Our results show that exogenous oCRH can accelerate metamorphosis in urodele larvae as it does in anurans. These findings suggest that the neuroendocrine mechanisms controlling metamorphosis are evolutionarily conserved across amphibian taxa.     

Ancient DNA assessment of tiger salamander population in Yellowstone National Park

Recent data indicates that blotched tiger salamanders (Ambystoma tigrinum melanostictum) in northern regions of Yellowstone National Park are declining due to climate-related habitat changes. In this study, we used ancient and modern mitochondrial haplotype diversity to model the effective size of this amphibian population through recent geological time and to assess past responses to climatic changes in the region. Using subfossils collected from a cave in northern Yellowstone, we analyzed >700 base pairs of mitochondrial sequence from 16 samples ranging in age from 100 to 3300 years old and found that all shared an identical haplotype. Although mitochondrial diversity was extremely low within the living population, we still were able to detect geographic subdivision within the local area. Using serial coalescent modelling with Bayesian priors from both modern and ancient genetic data we simulated a range of probable population sizes and mutation rates through time. Our simulations suggest that regional mitochondrial diversity has remained relatively constant even through climatic fluctuations of recent millennia.     

A serological and bacteriological survey of brucellosis in wild boar (Sus scrofa) in Belgium

ABSTRACT: BACKGROUND: Brucellosis is frequently reported among wild boar populations in Europe. The aim of the study was to assess the epidemiological situation in Belgium, regarding the steady increase of wild boar populations over the last decades. Several serological tests were used and compared with culture and IS711 polymerase chain reaction (PCR), to determine the most suitable combination of diagnostic tools for conducting a successful prevalence study in wildlife. RESULTS: An indirect enzyme-linked immunosorbent assay (iELISA) was used on 1168 sera from hunter-killed wild boar sampled between 2003 and 2007 in 4 natural regions of southern Belgium. Results gave an apparent prevalence of 54.88% (95% CI 52.03-57.73). Prevalence was significantly affected by age and by the year of study, but not by sex nor by the region of sampling. The relative sensitivities of the complement fixation test (CFT), the Rose Bengal test (RBT), and the slow agglutination test (SAT) versus the iELISA differed widely between tests, reaching 62.67%, 46.68%, and 34.77%, respectively. The relative specificities of the CFT, RBT and SAT versus the iELISA were respectively 99.01%, 92.49%, and 99.1%. From seropositive animals (iELISA), 9% were positive by culture and 24% by PCR when testing spleen and/or tonsils. Sensitivity of the PCR was higher on tonsils than on spleen. All bacterial isolates were identified as Brucella suis biovar 2. CONCLUSIONS: Brucellosis is widespread among wild boar in southern Belgium, with seroprevalences having increased over ten years, and constitutes a growing risk of spillback to outdoor-farmed pig herds. The iELISA showed a better sensitivity than the CFT, RBT and SAT. Serological tests must be associated with direct diagnosis and PCR proved more sensitive than culture under wildlife sampling conditions. Spleen and tonsils are lymphoid tissues usually sampled in multi-disease monitoring programs. They remain top-grade organs for direct diagnosis of brucellosis, with a preference for tonsils.     

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.     

Innate immune evasion mediated by the Ambystoma tigrinum virus eukaryotic translation initiation factor 2alpha 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.     

Inferring population history and demography using microsatellites, mitochondrial DNA, and major histocompatibility complex (MHC) genes

Microsatellites and mitochondrial DNA (mtDNA) have traditionally been used in population genetics because of their variability and presumed neutrality, whereas genes of the major histocompatibility complex (MHC) are increasingly of interest because strong selective pressures shape their standing variation. Despite the potential for MHC genes, microsatellites, and mtDNA sequences to complement one another in deciphering population history and demography, the three are rarely used in tandem. Here we report on MHC, microsatellite, and mtDNA variability in a single large population of the eastern tiger salamander (Ambystoma tigrinum tigrinum). We use the mtDNA mismatch distribution and, on microsatellite data, the imbalance index and bottleneck tests to infer aspects of population history and demography. Haplotype and allelic variation was high at all loci surveyed, and heterozygosity was high at the nuclear loci. We find concordance among neutral molecular markers that suggests our study population originated from post-Pleistocene expansions of multiple, fragmented sources that shared few migrants. Differences in N(e) estimates derived from haploid and diploid genetic markers are potentially attributable to secondary contact among source populations that experienced rapid mtDNA divergence and comparatively low levels of nuclear DNA divergence. We find strong evidence of natural selection acting on MHC genes and estimate long-term effective population sizes (N(e)) that are very large, making small selection intensities significant evolutionary forces in this population.     

Sublethal Effects of Nitrite on Eastern Tiger Salamander (<Emphasis Type="Italic">Ambystoma tigrinum tigrinum</Emphasis>) and Wood Frog (<Emphasis Type="Italic">Rana sylvatica</Emphasis>) Embryos and Larvae: Implications for Field Populations

Ephemeral pools, which can have high animal biomass and low dissolved oxygen, may be prone to nitrite accumulation. As such, it is important to understand how exposure to nitrite might affect development and growth of amphibians that breed in these ephemeral pools. Wood frog (Rana sylvatica) and eastern tiger salamander (Ambystoma tigrinum tigrinum) embryos and tadpoles and young larvae were exposed to elevated concentrations of nitrite derived from sodium nitrite: 0, 0.3, 0.6, 1.2, 2.1, 4.6, and 6.1 mg l⁻¹ NO₂–N. Increasing nitrite exposure slowed embryonic and larval development in both the eastern tiger salamander and the wood frog, reduced growth in tiger salamander embryos and larvae, and delayed metamorphosis in the wood frog. At concentrations less than 2 mg l⁻¹ NO₂–N nitrite delayed hatching, and at concentrations above 2 mg l⁻¹ time to hatching decreased causing more individuals to hatch at less developed stages. Nitrite also increased asynchrony in tiger salamander hatching. The sublethal effects of nitrite on amphibian development, growth and hatching could have serious repercussions on amphibian fitness in ephemeral environments. Potential increases in mortality on field populations caused by sublethal effects of nitrite are discussed.     

Evaluation of two methods for measuring nonspecific immunity in tiger salamanders (Ambystoma tigrinum)

Study of amphibian immunotoxicology is a growing area of research, but very little information is available on how environmental contaminants affect disease resistance in urodele amphibians. Urodele amphibians lack the more highly evolved aspects of the specific immune system that are present in anurans, birds, and mammals. Instead, these animals rely more heavily on innate defense mechanisms than do anurans to provide rapid, nonspecific protection from pathogens. Thus, it is prudent that immunotoxicologic research with urodele amphibians includes an evaluation of effects of contaminant exposure on nonspecific immunity. The objectives of this study were to measure the phagocytic and oxidative-burst activity of peritoneal neutrophils collected from a urodele, the tiger salamander (Ambystoma tigrinum), and to evaluate the use of these assays in immunotoxicologic research using urodele amphibians. Using tiger salamanders collected in August 2000, phagocytosis and oxidative-burst assays modified from mammalian protocols were conducted through October 2001. Results indicated that large numbers of peritoneal neutrophils for use in immunotoxicologic tests can be collected from salamanders injected with thioglycollate. Moreover, these neutrophils readily engulfed foreign material (phagocytic activity) and produced measurable amounts of hydrogen peroxide (oxidative-burst activity). Phagocytosis was effectively inhibited by incubating cells with sodium azide (P<0.001), and quantification of phagocytosis using flow cytometry was well correlated with manual counts (r=0.84, P<0.001). Dexamethasone treatment reduced phagocytic activity as measured by manual counts (P<0.02), suggesting that this test is useful for detecting alteration by immunosuppressive agents. In contrast, oxidative function was unaffected by dexamethasone treatment, and results from the oxidative-burst assay were generally less consistent than those from the phagocytosis assay. Based on these results, phagocytic activity of peritoneal neutrophils may be a useful endpoint in immunotoxicologic studies to evaluate the impact of environmental contaminants on innate defense mechanisms in urodele amphibians.