Characterization of primary and memory CD8 T-cell responses against ranavirus (FV3) in Xenopus laevis

In mammals, resistance to primary and secondary viral infections critically involves major histocompatibility complex class I-restricted cytotoxic CD8+ T lymphocytes (CTLs). Although many gene homologues involved in CTL function have been identified in all vertebrate classes, antiviral CTL responses have been poorly characterized for ectothermic vertebrates. Because of the threat of emerging wildlife viral diseases to global biodiversity, fundamental research on comparative viral immunity has become crucial. Ranaviruses (family Iridoviridae) are double-stranded DNA viruses possibly implicated in the worldwide decline of amphibian populations. We used the frog Xenopus laevis as a model to evaluate adaptive immune responses to the ranavirus frog virus 3 (FV3). FV3 infects the kidneys of adults but is cleared within 4 weeks, with faster clearance upon secondary infections. In vivo depletion of CD8+ T cells markedly decreases the survival of adults after viral infection. To further investigate the involvement of anti-FV3 CD8+ T-cell effectors in host resistance in vivo, we determined the proliferation kinetics of CD8+ T cells in the spleen by bromodeoxyuridine incorporation and their infiltration of kidneys by immunohistology. Upon primary infection, CD8+ T cells significantly proliferate in the spleen and accumulate in infected kidneys from day 6 onward, in parallel with virus clearance. Earlier proliferation and infiltration associated with faster viral clearance were observed during a secondary infection. These results provide in vivo evidence of protective antigen-dependent CD8+ T-cell proliferation, recognition, and memory in fighting a natural pathogen in Xenopus.     

Immune monitoring in xenotransplantation: the multiparameter flow cytometric mixed lymphocyte culture assay

Xenotransplantation requires monitoring of complex cellular interactions in vitro. A tool to monitor cell proliferation in detail would be instrumental in understanding these cellular interactions in heterogeneous xenogeneic lymphocyte cultures and in patients after xenotransplantation. To accomplish this, we used a fluorescent cell proliferation marker, 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE), in combination with flow cytometry. CFSE, a green fluorescent molecule, binds covalently to intracellular macromolecules. Each cell division reduces the fluorescent intensity per cell by half and shows a characteristic multipeak pattern in flow cytometric analysis. For this study, human lymphocytes were labeled with CFSE and cultured in the presence of irradiated porcine lymphocytes. Cell proliferation was detected in CFSE-labeled lymphocytes in both a single and a multiparameter flow cytometry setting. Concurrently, tritiated ((3)H) thymidine incorporation, a common method to measure gross cell proliferation, was assessed. The kinetics of CFSE-labeled cell proliferation correlated with (3)H-thymidine incorporation in that both methods showed a lag phase for days 1-3 and a log phase for days 4-7. Multiparameter flow cytometric monitoring of mixed lymphocyte cultures allowed phenotyping and assessment of viability of proliferating populations in heterogeneous xenogeneic stimulated human lymphocyte cultures and complemented the classical (3)H-thymidine incorporation assay. The use of this technique will allow a wide array of immunologic parameters to be measured in a heterogeneous xenogeneic mixed lymphocyte culture. The information gained from these assays is essential to understanding the biological significance of xenogeneic cellular interaction and for monitoring the immune status of the xenotransplanted patient.     

Patterns of cellular proliferation and migration in the developing tectum mesencephali of the frog Rana temporaria and the salamander Pleurodeles waltl

The development of the tectum mesencephali was studied in the frog Rana temporaria and the salamander Pleurodeles waltl by means of nuclear staining and by labeling of cells with bromodeoxyuridine (BrdU). The general spatial and temporal pattern of cell proliferation and cell migration is the same in both species, despite drastic differences in overall tectal morphology. However, the salamander species differs from the frog species by (1) a generally lower cell proliferation rate, (2) a reduction in the activity of the lateral proliferation zone, and (3) a reduction in the formation of superficial cellular layers. Because point (3) affects processes that occur late in ontogeny, our experiments provide evidence that the simple morphology of the tectum of Pleurodeles waltl, compared with the multilayered tectum of Rana, is a consequence of a paedomorphic alteration of the ancestral developmental pattern of the amphibian tectum.     

Amphibian Pathogen Batrachochytrium dendrobatidis Is Inhibited by the Cutaneous Bacteria of Amphibian Species

Population declines of amphibian species in many parts of the world are associated with a lethal fungal pathogen, Batrachochytrium dendrobatidis. Using laboratory challenge assays, we describe the inhibition of B. dendrobatidis by members of eight genera of bacteria isolated from the skin of two amphibian species that exhibit parental care behavior (Plethodon cinereus and Hemidactylium scutatum). We found that members of three genera of bacteria isolated from the skins of the salamander P. cinereus and members of seven genera isolated from the salamander H. scutatum inhibited the growth of B. dendrobatidis. Understanding how B. dendrobatidis interacts with an ecological community of cutaneous flora may be important in explaining and preventing amphibian population declines.     

Activation markers and cell proliferation as indicators of toxicity: A flow cytometric approach

Cell proliferation is an attractive endpoint inin vitro toxicity assays, since nearly any kind of damage in a cell may result in altered cell proliferation. In toxicological applications, liquid scintillation counting, measuring radioactivity from tritiated thymidine, has been the traditional way to estimate cell proliferation. An alternative approach is the measurement of BrdU incorporation by flow cytometry. Before the actual DNA synthesis starts, several proteins are expressed on the cell surface, as well as intracellularly. Among the markers on the cell surface CD69, CD25, and CD71 are sequentially expressed on human lymphocytes after a mitogenic stimulation. The aim of this study was to evaluate information obtained by analysis of expression of activation markers on cell surfaces in lymphocyte subsets and to compare it with data from cell proliferation studies performed by liquid scintillation counting and BrdU flow cytometry. The experiments were performed with phytohemagglutinin-stimulated human lymphocytes exposed to ochratoxin A and cyclosporin A. While ochratoxin A-treated cultures showed a steep inhibition with increasing concentration, the cyclosporin A treatment gave an inhibition curve with a less steep slope. Activation marker studies showed that the effect of treatment with both of the toxins was more pronounced on the late markers CD25 and CD71, while CD69 had the advantage that significant effects could be detected as early as 6 h after ochratoxin A treatment. Cyclosporin A treatment induced only minor alterations in CD69 expression. Certain differences in expression of activation markers between CD4⁺ and CD8⁺ subsets were found both in ochratoxin A- and cyclosporin A-treated cultures. A stimulating effect was found in cell cultures exposed to the lowest concentration of ochratoxin A on CD69 and CD25 expression. Signs of an increase in frequencies of proliferating cells measured with the BrdU flow cytometry method were also seen. This increase could not be detected with liquid scintillation counting. No other differences between the liquid scintillation counting and BrdU flow cytometry measurements of proliferation were obtained. We conclude that studies of activation marker expression by the flow cytometric approach used in this report are useful complements to traditional measurements of cell proliferation as they yield subsetspecific information about cellular processes which precede proliferation of lymphocytes.Abbreviations A pulse area - BrdU bromodeoxyuridine - CD cluster of differentiation - FBS fetal bovine serum - FITC fluorescein isothiocyanate - FL fluorescence - FSC forward light scatter - H pulse height - PBS phosphate-buffered saline - PI propidium iodide - R-PE R-phycoerythrin - RPMI Roswell Park Memorial Institute - SEM standard error of the mean - SSC orthogonal light scatter - W pulse width     

Phylogeny, Life History, and Ecology Contribute to Differences in Amphibian Susceptibility to Ranaviruses

Research that identifies the potential host range of generalist pathogens as well as variation in host susceptibility is critical for understanding and predicting the dynamics of infectious diseases within ecological communities. Ranaviruses have been linked to amphibian die-off events worldwide with the greatest number of reported mortality events occurring in the United States. While reports of ranavirus-associated mortality events continue to accumulate, few data exist comparing the relative susceptibility of different species. Using a series of laboratory exposure experiments and comparative phylogenetics, we compared the susceptibilities of 19 amphibian species from two salamander families and five anurans families for two ranavirus isolates: frog virus 3 (FV3) and an FV3-like isolate from an American bullfrog culture facility. We discovered that ranaviruses were capable of infecting 17 of the 19 larval amphibian species tested with mortality ranging from 0 to 100%. Phylogenetic comparative methods demonstrated that species within the anuran family Ranidae were generally more susceptible to ranavirus infection compared to species from the other five families. We also found that susceptibility to infection was associated with species that breed in semi-permanent ponds, develop rapidly as larvae, and have limited range sizes. Collectively, these results suggest that phylogeny, life history characteristics, and habitat associations of amphibians have the potential to impact susceptibility to ranaviruses.     

Birth,survival and differentiation of neurons in an adult crustacean brain

Life‐long neurogenesis is a characteristic feature of many vertebrate and invertebrate species. In decapod crustaceans, new neurons are added throughout life to two cell clusters containing local (cluster 9) and projection (cluster 10) interneurons in the olfactory pathway. Adult‐born neurons in clusters 9 and 10 in crayfish have the anatomical properties and chemistry of mature neurons by 6 months after birth. Here we use 5‐bromo‐2′‐deoxyuridine (BrdU) incorporation to pulse label mitotically active cells in these cell clusters, followed by a survival time of up to 8 months, during which crayfish (Cherax destructor) were sacrificed at intervals and the numbers of BrdU‐labeled cells quantified. We find a decrease in the numbers of BrdU‐labeled cells in cell cluster 10 between the first and second weeks following BrdU exposure, suggesting a period of cell death shortly after proliferation. Additional delayed cell divisions in both cell clusters are indicated by increases in labeled cells long after the BrdU clearing time. The differentiation time of these cells into neurons was defined by detection of the first immunoreactivity for the transmitter SIFamide in cluster 10 BrdU‐labeled cells, which begins at 4 weeks after BrdU labeling; the numbers of SIFamide‐labeled cells continues to increase over the following month. Experiments testing whether proliferation and survival of Cluster 10 cells are influenced by locomotor activity provided no evidence of a correlation between activity levels and cell proliferation, but suggest a strong influence of locomotor activity on cell survival. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 602–615, 2014     

Ty1-copia group retrotransposon sequences in amphibia and reptilia

We have isolated sequences belonging to Tyl-copia group retrotransposons from the genomes of an amphibian (Pyxicephalus adspersa) and three reptiles (Conolophus subscristatus, Amblyrynchus cristatus and Pytas mucosus). Two different seqences were found in the amphibian (Tpa1 and Tpa2). Each is present in several copies per genome and absent from the genomes of two other amphibian species. The C. subcristatus sequence Tcs1 is present in multiple copies in both its host genome (Galapagos land iguana) and the genome of the related Galapagos marine iguana (A. cristatus). There is little or no polymorphism in Tcs1 insertions between different individual animals, suggesting that this sequence is not transposing rapidly in either iguana genome. The P. mucosus sequence Tpm1 shows a discontinuous distribution in snake species, suggesting that it has either been lost from many lineages during vertical germline transmission or has been transferred horizontally in some snake species. Phylogenetic comparisons of all these sequences with each other and with other members of this retrotransposon group from other animals and plants show that sequences within a particular vertebrate species are most closely related to each other, consistent with a vertical transmission model for their evolution.     

Effect of selenium in recovery of immunity damaged by H2O2 and60Co radiation

Con A stimulated lymphocytes proliferation was measured as [³H]thymidine incorporation and IgG was quantified by single radial immunodiffusion to study recovering or protecting effect of selenium (Se) on immunity attacked by exogenous active oxygen species, H₂O₂ and⁶⁰Co-radiation, respectively. Lipid peroxidation was also determined to observe the relation between antioxidation ability and protecting ability of Se. It was found that H₂O₂ injured lymphocytes immunocompetence deeply and⁶⁰Co-radiation decreased immune response capacity greatly, but that administration of Se counteracts this damage. The antioxidative ability of Se was correlated with its protecting ability.     

Quantifying lymphocyte kinetics in vivo using carboxyfluorescein diacetate succinimidyl ester (CFSE)

The cytoplasmic dye carboxyfluorescein diacetate succinimidyl ester (CFSE) is used to quantify cell kinetics. It is particularly important in studies of lymphocyte homeostasis where its labelling of cells irrespective of their stage in the cell cycle makes it preferable to deuterated glucose and BrdU, which only label dividing cells and thus produce unrepresentative results. In the past, experiments have been limited by the need to obtain a clear separation of CFSE peaks forcing scientists to adopt a strategy of in vitro labelling of cells followed by their injection into the host. Here we develop a framework for analysis of in vivo CFSE labelling data. This enables us to estimate the rate of proliferation and death of lymphocytes in situ, and thus represents a considerable advance over current procedures. We illustrate this approach using in vivo CFSE labelling of B lymphocytes in sheep.     

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.     

Silence of ClC-3 chloride channel inhibits cell proliferation and the cell cycle via G/S phase arrest in rat basilar arterial smooth muscle cells

Abstract. Objectives: Previously, we have found that the ClC‐3 chloride channel is involved in endothelin‐1 (ET‐1)‐induced rat aortic smooth muscle cell proliferation. The present study was to investigate the role of ClC‐3 in cell cycle progression/distribution and the underlying mechanisms of proliferation. Materials and methods: Small interference RNA (siRNA) is used to silence ClC‐3 expression. Cell proliferation, cell cycle distribution and protein expression were measured or detected with cell counting, bromodeoxyuridine (BrdU) incorporation, Western blot and flow cytometric assays respectively. Results: ET‐1‐induced rat basilar vascular smooth muscle cell (BASMC) proliferation was parallel to a significant increase in endogenous expression of ClC‐3 protein. Silence of ClC‐3 by siRNA inhibited expression of ClC‐3 protein, prevented an increase in BrdU incorporation and cell number induced by ET‐1. Silence of ClC‐3 also caused cell cycle arrest in G₀/G₁ phase and prevented the cells’ progression from G₁ to S phase. Knockdown of ClC‐3 potently inhibited cyclin D1 and cyclin E expression and increased cyclin‐dependent kinase inhibitors (CDKIs) p27^KIP and p21^CIP expression. Furthermore, ClC‐3 knockdown significantly attenuated phosphorylation of Akt and glycogen synthase kinase‐3β (GSK‐3β) induced by ET‐1. Conclusion: Silence of ClC‐3 protein effectively suppressed phosphorylation of the Akt/GSK‐3β signal pathway, resulting in down‐regulation of cyclin D1 and cyclin E, and up‐regulation of p27^KIP and p21^CIP. In these BASMCs, integrated effects lead to cell cycle G₁/S arrest and inhibition of cell proliferation.     

Immunotoxicity Biomarkers in Fish: Development,Validation and Application for Field Studies and Risk Assessment

Imunocompetence is usually monitored using a tiered approach that is based upon several parameters including immunopathology, immune function, and host resistance. Through the efforts of numerous investigations, well-characterized immune assays validated in rodents for their sensitivity and reproducibility in assessing xenobiotic-induced immunotoxicity are currently available. Recently, many of these same endpoints have been utilized in non-mammalian species as indicators to predict chemical-induced immunotoxicity. In this laboratory, immune assays that measure immunopathology, antibody-forming cell response to T-dependent antigens, lymphocyte proliferation, macrophage function, antioxidant activity, and host resistance against infectious bacteria have been employed successfully to assess metal-, pesticide-, aromatic hydrocarbon-, and mixture-induced immunotoxicity in laboratory-reared Japanese medaka (Oryzias latipes). These same assays have also proven successful in feral fish populations for predicting risk(s) associated with habitation in contaminated aquatic environments. For example, smallmouth bass (Micropterus dolomieu) collected from a polychlorinated biphenyl-contaminated site had reduced phagocyte function, oxyradical production, and antioxidant levels (compared to reference fish), while circulating leukocyte profiles and lymphocyte proliferation by splenic T-cells were altered in organochlorine-exposed walleye (Stizostedium vitreum vitreum). Results of the aforementioned studies demonstrate that immune assays developed and validated in a laboratory fish model can be successfully applied to feral fish populations to predict the toxicological hazards associated with exposure to immunomodulating aquatic pollutants.     

Frog virus 3-like infections in aquatic amphibian communities

Frog virus 3 (FV3) and FV3-like viruses, are members of the genus Ranavirus (family Iridoviridae), and they have been associated with infectious diseases that may be contributing to amphibian population declines. We examined the mode of transmission of an FV3-like virus, and potential hosts and reservoirs of the virus in a local amphibian community. Using the polymerase chain reaction to detect infected animals, we found an FV3-like virus in south-central Ontario, Canada, amphibian communities, where it infects sympatric amphibian species, including ranid and hylid tadpoles (Rana sylvatica, Hyla versicolor, and Pseudacris spp.), larval salamanders (Ambystoma spp.), and adult eastern-spotted newts (Notophthalmus viridescens). The high prevalence of FV3-like infections in caudate larvae suggests that salamanders are likely to be both hosts and reservoirs. In laboratory FV3 challenges of R. sylvatica, the rate of infection was dependent on the amount of virus to which the animals were exposed. In addition, although vertical transmission was suspected, horizontal transmission through exposure to infected pond water is the most likely route of infection in tadpoles. Based on our observations, a simple model of FV3/FV3-like virus transmission postulates that, in aquatic amphibian communities, transmission of the virus occurs between anuran and urodele species, with ambystomatid salamanders the most likely reservoir for the ranavirus in our study.     

Studies of mechanisms of antibody formation: III. Effects of 5-bromodeoxyuridine upon normal and neoplastic B-lymphocyte function

Although 5-bromodeoxyuridine (BrdU) does not interfere with the rate of proliferation, or with total protein, DNA, or RNA synthesis in normal mouse spleen B cells exposed to specific antigenic excitation or to polyclonal activators, this agent has the capacity to block the appearance of IgM antibody-forming cells (AFC) in response to antigenic challenge in vitro with sheep red blood cells (sRBC), as well as the generation of anti-sRBC or 2,4,6-trinitrobenzene sulfonate-AFC in the course of polyclonal expansion. The effect of BrdU can be blocked competitively by a 10-fold excess of thymidine (TdR). CsCl density equilibrium analysis indicates that B cells with 55% BrdU substitution of TdR in a single DNA strand could participate in at least one additional division cycle, resulting in a double DNA strand substitution. BrdU substitution in a single DNA strand was sufficient, however, to block antibody secretion in B cells (unifilar dominance). When cells with single-strand BrdU substitution were allowed to proliferate in the presence of a 10-fold excess of TdR, the cells regained the capacity to synthesize and secrete specific antibody, demonstrating the reversibility of the inhibitory effect of BrdU upon this specialized B-cell function. Cells stimulated with LPS in the presence of BrdU exhibited higher rates of synthesis of total RNA as well as of the RNA species of the translation machinery (28, 18, and 5S and tRNA), measured by the rate of incorporation of radioactive precursors at the time of peak proliferation. Exposure to BrdU blocked the secretion of μ and light chains, although such cells had high levels of membrane IgM (mIgM) and were able to regenerate mIgM after enzymatic stripping. The regeneration of mIgM could be suppressed with α-amanitine and by actinomycin D, suggesting that the incorporation of BrdU into DNA does not affect the transcripition of nucleotide sequences for mIgM. MOPC 315 and 70 plasmocytoma cells cultured in the presence of BrdU for 60 hr regularly incorporated BrdU into one and two DNA strands. There was a 55–60% substitution of BrdU in single DNA strands. This situation produces complete inhibition of the production of IgM, μ, or light chains in normal cells. In contrast, plasmocytoma cells treated with BrdU secreted as many α, γ, and light chains as the corresponding control cells grown in the presence of TdR. This result points to possibly profound differences in the regulation of transcripition of Ig nucleotide sequences in lymphocytes which have undergone malignant transformation.     

Inflammation-Induced Reactivation of the Ranavirus Frog Virus 3 in Asymptomatic Xenopus laevis

Natural infections of ectothermic vertebrates by ranaviruses (RV, family Iridoviridae) are rapidly increasing, with an alarming expansion of RV tropism and resulting die-offs of numerous animal populations. Notably, infection studies of the amphibian Xenopus laevis with the ranavirus Frog Virus 3 (FV3) have revealed that although the adult frog immune system is efficient at controlling RV infections, residual quiescent virus can be detected in mononuclear phagocytes of otherwise asymptomatic animals following the resolution of RV infections. It is noteworthy that macrophage-lineage cells are now believed to be a critical element in the RV infection strategy. In the present work, we report that inflammation induced by peritoneal injection of heat-killed bacteria in asymptomatic frogs one month after infection with FV3 resulted in viral reactivation including detectable viral DNA and viral gene expression in otherwise asymptomatic frogs. FV3 reactivation was most prominently detected in kidneys and in peritoneal HAM56+ mononuclear phagocytes. Notably, unlike adult frogs that typically clear primary FV3 infections, a proportion of the animals succumbed to the reactivated FV3 infection, indicating that previous exposure does not provide protection against subsequent reactivation in these animals.     

Enhancement of proliferation and differentiation in bone marrow hematopoietic cells by Spirulina (Arthrospira) platensis in mice

This study evaluates whether Spirulina, including its components such as phycocyanin, enhances or sustains immune functions by promoting immune competent-cell proliferation or differentiation. The effects of Spirulina of a hot-water extract (SpHW), phycocyanin (Phyc), and cell-wall component extract (SpCW) on proliferation of bone marrow cells and induction of colony-forming activity in mice were investigated. The Spirulina extracts, SpHW, Phyc, and SpCW, enhanced proliferation of bone-marrow cells and induced colony-forming activity in the spleen-cell culture supernatant. Granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-3 (IL-3) were detected in the culture supernatant of the spleen cells stimulated with the Spirulina extracts. Bone marrow-cell colony formation in soft-agar assay was also significantly induced by the blood samples and the culture supernatants of the spleen and Peyer's patch cells of the mice which ingested Spirulina extracts orally for 5 weeks in in vivo study. Ratios of neutrophils and lymphocytes in the peripheral blood and bone marrow, consequently, increased in the mice. Spirulina may have potential therapeutic benefits for improvement of weakened immune functions caused by, for example, the use of anticancer drugs.     

Expression analysis of immune response genes in fish epithelial cells following ranavirus infection

Ranaviruses (family Iridoviridae) are a growing threat to fish and amphibian populations worldwide. The immune response to ranavirus infection has been studied in amphibians, but little is known about the responses elicited in piscine hosts. In this study, the immune response and apoptosis induced by ranaviruses were investigated in fish epithelial cells. Epithelioma papulosum cyprini (EPC) cells were infected with four different viral isolates: epizootic haematopoietic necrosis virus (EHNV), frog virus 3 (FV3), European catfish virus (ECV) and doctor fish virus (DFV). Quantitative real-time PCR (qPCR) assays were developed to measure the mRNA expression of immune response genes during ranavirus infection. The target genes included tumour necrosis factor α (TNF-α), interleukin-1β (IL-1β), β2-microglobulin (β2M), interleukin-10 (IL-10) and transforming growth factor β (TGF-β). All ranaviruses elicited changes in immune gene expression. EHNV and FV3 caused a strong pro-inflammatory response with an increase in the expression of both IL-1β and TNF-α, whereas ECV and DFV evoked transient up-regulation of regulatory cytokine TGF-β. Additionally, all viral isolates induced increased β2M expression as well as apoptosis in the EPC cells. Our results indicate that epithelial cells can serve as an in vitro model for studying the mechanisms of immune response in the piscine host in the first stages of ranavirus infection.