Tiger Frog Research Articles

Phenotypic characterization and phylogenetic analysis of a virulent Bacillus cereus strain from the tiger frog, Hoplobatrachus rugulosus Wiegmann.

Bacillus cereus is a common opportunistic human pathogen well known as a food contaminant. In recent years, tiger frog farming has been undertaken in China and many other countries to meet human food habits. However, there is no definitive data to indicate the contamination of potentially pathogenic B. cereus strains to fresh cultured tiger frogs. In this study, a virulent B. cereus strain W10 with hemolytic activity was first isolated from the liver of cultured tiger frogs. It was initially identified based on the spore-forming characterization and phenotypic characterization using API identification kits and its taxonomic position, was further determined by nucleotide blast search in NCBI website and phylogenetic analysis. The constructed phylogenetic tree using neighbor-joining method further showed that strain W10 was closely related to B. cereus strain F46 (GenBank accession no. EF203906), which was isolated from the contaminated food. To further confirm the virulence and potential danger of strain W10, its LD50 value was tested to be 5.22×104 cfu/g, which indicated that strain W10 had strong potential virulence to healthy tiger frogs. Pathological changes were observed in liver and body surface of the challenged tiger frogs. Thus, as a potential pathogen of a zoonotic disease, the distribution of the virulent B. cereus strain, may be a threat to the safety of tiger frog food and more importance should be attached to the virulent B. cereus strain in tiger frog farming. Key words: Tiger frog, Bacillus cereus, phenotypic characterization, phylogenetic analysis.

Development and evaluation of a loop-mediated isothermal amplification assay for rapid detection of lymphocystis disease virus

A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of lymphocystis disease virus (LCDV). A set of five specific primers, two inner and two outer primers and a loop primer, were designed on the basis of the major capsid protein gene of LCDV. The reaction time and temperatures were optimized for 60 min at 63 °C, respectively. LAMP amplification products were detected by a ladder-like appearance on agarose gel electrophoresis or a naked-eye inspection of a color change in the reaction tube by addition of SYBR Green I. The assay was specific for LCDV, and there was no cross-reactivity with white spot syndrome virus (WSSV) or six other Iridoviridae viruses (epizootic hematopoietic necrosis virus, EHNV; tiger frog virus, TFV; Bohle iridovirus, BIV; soft-shelled turtle iridovirus, STIV; infectious spleen and kidney necrosis virus, ISKNV; red sea bream iridovirus, RSIV). The detection limit of the LAMP assay was 15 fg, which was similar to that of real-time quantitative polymerase chain reaction (PCR) and 10-fold higher than the conventional PCR. The LAMP assay was evaluated using 109 clinical samples, and the results indicated the suitability and simplicity of the test as a rapid, field diagnostic tool for detection of LCDV. The LCDV LAMP assay has potential for early diagnosis of LCDV infection.

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.

Tiger frog virus can infect zebrafish cells for studying up- or down-regulated genes by proteomics approach

Tiger frog virus (TFV), a member of the iridovirus family, causes high mortality of cultured tiger frog tadpoles in southern China. To better understand TFV infection and its interaction with host cells, zebrafish embryonic fibroblast (ZF4) cells, a stable polyploid cell line with most clear genetic map, was used for our present study. Our results showed that TFV caused typical lytic plaque forming cytopathic effect (CPE) and that various stages of viral proliferation were observed using electron microscopy and indirect immunofluorescence assay. Two-dimensional electrophoresis also showed that some cellular proteins were differentially expressed in the ZF4 cells infected with TFV. A total of 10 proteins were identified using the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) technique, including 7 that were up-regulated and 3 that were down-regulated after infection. Among the 10 identified proteins, alterations in Hsp90 and α-tubulin expression were further confirmed by Western blot analysis. Furthermore, reorganization of microtubules was also observed in TFV-infected cells and can probably be attributed to the overexpression of translationally controlled tumor protein. The present study is the first attempt to reveal cellular responses to TFV infection by proteomics. The results suggest that the ZF4 cell line could be used as a model to study TFV infection.

First isolation and identification of Elizabethkingia meningoseptica from cultured tiger frog, Rana tigerina rugulosa

Elizabethkingia meningoseptica has been recognised as an occasional but serious opportunistic bacterial pathogen to human beings. Recently, it was frequently isolated from tiger frog, Rana tigerina rugulosa, with cataract disease, which is the most common disease of unknown aetiology of frogs in Hainan, China. The purpose of this study was to identify and characterise the bacterial strains isolated from the recent outbreaks of cataract disease in farmed tiger frog in Hainan, China, and to evaluate their pathogenicity to the frog and their sensitivity to 20 chemotherapeutic agents. The 16S rRNA gene sequences of strains W0701 (1478 bp), W0702 (1477 bp) and W0703 (1478 bp) showed 98.6–98.7% similarity with the sequence of E. meningoseptica type strain (ATCC 13253) and 99.9–100% similarity with that of E. meningoseptica NTU 870424-IL. Six strains (W0701–W0706) were selected to represent 24 isolates retrieved from six moribund frogs. The morphological, physiological and biochemical characteristics of the six representative isolates were consistent with those of E. meningoseptica strains. The organisms were only susceptible to vancomycin and moderately susceptible to cefoperazone among the 20 investigated chemotherapeutic agents. Virulence test with strain W0702 was conducted and pathogenicity (by intramuscular injection) was demonstrated in the tiger frog. In conclusion, 24 isolates obtained from frogs with cataract disease were the E. meningoseptica strains highly pathogenic to tiger frog, and this is the first report of E. meningoseptica as a pathogen for tiger frog.

Isolation and characterization of microsatellite loci in tiger frog (Hoplobatrachus rugulosus)

Nine novel microsatellite loci were developed from the tiger frog (Hoplobatrachus rugulosus) using enrichment methods. The number of alleles ranged from 6 to 11 with an average of 8.56 alleles per locus. The observed and expected heterozygosity ranged from 0.571 to 0.935 (average 0.796) and 0.765 to 0.888 (average 0.837) respectively. These polymorphic loci can be used in population structure, gene flow, and population differentiation.

Isolation of Laribacter hongkongensis, a novel bacterium associated with gastroenteritis, from Chinese tiger frog

Laribacter hongkongensis is a recently discovered novel bacterium associated with community-acquired gastroenteritis. Although the bacterium has been isolated from freshwater fish and natural freshwater environments, it is not known if other freshwater animals could also be a source of L. hongkongensis. In a surveillance study on freshwater food animals (other than fish) in Hong Kong, L. hongkongensis was isolated from eight of 10 Chinese tiger frogs ( Hoplobatrachus chinensis), a widespread frog species commonly consumed in China and southeast Asia. The large intestine was the site with the highest recovery rate, followed by the small intestine and stomach. None of the 30 Malaysian prawns, 20 pieces of sand shrimp, 20 Chinese mystery snails or 10 Chinese soft-shelled turtles was found to harbor the bacterium. Among the eight positive frogs, a total of 26 isolates of L. hongkongensis, confirmed by phenotypic tests and PCR, were obtained. As with human, freshwater fish and natural water isolates, a heterogeneous population of L. hongkongensis in frogs was identified by pulsed-field gel electrophoresis, with 6 different patterns among the 26 isolates and a single frog often carrying different strains. The present report represents the first to describe the isolation of L. hongkongensis from amphibians. The high isolation rate and genetic heterogeneity of L. hongkongensis among the Chinese tiger frogs suggested that these animals are also natural reservoir for the bacterium. Caution should be exercised in handling and cooking these frogs.

Development of an RT-PCR detection method for mud crab reovirus

Mud crab reovirus (MCRV) causes high mortality in the cultivated mud crab. To control better an outbreak of this virus, a rapid, specific and sensitive detection method based on RT-PCR was developed. The MCRV detection method was designed based on the one-step and two-step RT-PCR which resulted in the amplification of predicted products of 433 and 304 bp. The method is specific as no cross-reaction was observed between grass carp hemorrhage virus (GCHV), white spot syndrome virus (WSSV), tiger frog virus (TFV), infectious spleen and kidney necrosis virus (ISKNV), fish nervous necrosis virus (NNV) and the muscovy duck virus (MDRV). One-step PCR amplification could detect 10 −8 μg of purified MCRV dsRNA, while two-step PCR amplification could detect 10 −9 μg MCRV dsRNA. At an early stage of infection, MCRV could be detected in the heart, thoracic ganglion, muscle, intestine, gut, hemolymph, gonad and gill, but not in the stomach or hepatopancreas. However, in the moribund mud crabs, MCRV could be detected in all tissues examined.

Identification of two novel membrane proteins from the Tiger frog virus (TFV)

The Tiger frog virus (TFV) belongs to the genus Ranavirus in the family Iridoviridae, and its genome was completely sequenced in 2002. In order to better understand the viral structure and functional genes involved in infection and virus–host interactions, two candidate genes, ORF001L and ORF020R, were selected for our study. ORF001L and ORF020R were analyzed by genomic comparison and by using the TMHMM software. Both genes were conserved in the genus Ranavirus, may encode putative membrane proteins, and were determined as late genes by temporal analysis. In order to identify whether these two proteins were structural proteins or not, ORF001L and ORF020R were cloned and expressed in the pET32a (+) vector. Antisera against the two proteins were prepared by immunization of mice with purified proteins. Western blot analysis suggested that both ORF001L and ORF020R were structural proteins. Indirect immunofluorescence assay (IFA) revealed that the subcellular location of the two proteins was confined to the cytoplasm, especially at the viral assembly site (AS). Immunogold electron microscopy (IEM) further localized these two proteins, showing that they were envelope proteins.

Evolutionary significance of oral morphology in the carnivorous tadpoles of tiger frogs, genus Hoplobatrachus (Ranidae)

We describe the larval stages of three species of the Asian-African tiger frogs Hoplobatrachus chinensis, H. occipitalis and H. tigerinus. The tadpoles of all three species are very similar, with peculiar oral features: (1) double rows of needle-like labial teeth, (2) strong emarginations on the large jaw sheaths and (3) keratinized spurs on the buccal floor. Characters 1 and 2 (and perhaps 3) are probably related to the carnivorous habits of these tadpoles. A molecular phylogeny based on 2430 base pairs of two nuclear and four mitochondrial genes corroborated monophyly of Asian and African Hoplobatrachus, and identified Euphlyctis as their sister group. Tadpoles of the latter genus lack buccal spurs and double labial tooth rows but share large jaw sheaths, the upper with a medial projection. Therefore, the common ancestor of Euphlyctis and Hoplobatrachus probably was also characterized by this state, and may have been facultatively carnivorous. Further carnivorous specializations in Hoplobatrachus could explain why tiger frogs have been so successful in populating arid environments where ponds are at high risk of desiccation. Larval morphology may prove to be the key innovation which enabled them to disperse, in the Late Cenozoic, into their current very wide distribution area in Asia and Africa. © 2004 The Linnean Society of London, Biological Journal of the Linnean Society, 2004, 81, 171–181.

Genomic sequence of a ranavirus (family Iridoviridae) associated with salamander mortalities in North America

Disease is among the suspected causes of amphibian population declines, and an iridovirus and a chytrid fungus are the primary pathogens associated with amphibian mortalities. Ambystoma tigrinum virus (ATV) and a closely related strain, Regina ranavirus (RRV), are implicated in salamander die-offs in Arizona and Canada, respectively. We report the complete sequence of the ATV genome and partial sequence of the RRV genome. Sequence analysis of the ATV/RRV genomes showed marked similarity to other ranaviruses, including tiger frog virus (TFV) and frog virus 3 (FV3), the type virus of the genus Ranavirus (family Iridoviridae), as well as more distant relationships to lymphocystis disease virus, Chilo iridescent virus, and infectious spleen and kidney necrosis virus. Putative open reading frames (ORFs) in the ATV sequence identified 24 genes that appear to control virus replication and block antiviral responses. In addition, >50 other putative genes, homologous to ORFs in other iridoviral genomes but of unknown function, were also identified. Sequence comparison performed by dot plot analysis between ATV and itself revealed a conserved 14-bp palindromic repeat within most intragenic regions. Dot plot analysis of ATV vs RRV sequences identified several polymorphisms between the two isolates. Finally, a comparison of ATV and TFV genomic sequences identified genomic rearrangements consistent with the high recombination frequency of iridoviruses. Given the adverse effects that ranavirus infections have on amphibian and fish populations, ATV/RRV sequence information will allow the design of better diagnostic probes for identifying ranavirus infections and extend our understanding of molecular events in ranavirus-infected cells.

40. From vaccination to vacillation

Disease is among the suspected causes of amphibian population declines, and an iridovirus and a chytrid fungus are the primary pathogens associated with amphibian mortalities. Ambystoma tigrinum virus (ATV) and a closely related strain, Regina ranavirus (RRV), are implicated in salamander die-offs in Arizona and Canada, respectively. We report the complete sequence of the ATV genome and partial sequence of the RRV genome. Sequence analysis of the ATV/RRV genomes showed marked similarity to other ranaviruses, including tiger frog virus (TFV) and frog virus 3 (FV3), the type virus of the genus Ranavirus (family Iridoviridae), as well as more distant relationships to lymphocystis disease virus, Chilo iridescent virus, and infectious spleen and kidney necrosis virus. Putative open reading frames (ORFs) in the ATV sequence identified 24 genes that appear to control virus replication and block antiviral responses. In addition, >50 other putative genes, homologous to ORFs in other iridoviral genomes but of unknown function, were also identified. Sequence comparison performed by dot plot analysis between ATV and itself revealed a conserved 14-bp palindromic repeat within most intragenic regions. Dot plot analysis of ATV vs RRV sequences identified several polymorphisms between the two isolates. Finally, a comparison of ATV and TFV genomic sequences identified genomic rearrangements consistent with the high recombination frequency of iridoviruses. Given the adverse effects that ranavirus infections have on amphibian and fish populations, ATV/RRV sequence information will allow the design of better diagnostic probes for identifying ranavirus infections and extend our understanding of molecular events in ranavirus-infected cells.

Outbreaks of an iridovirus disease in cultured tiger frog, Rana tigrina rugulosa, in southern China

Outbreaks of an iridovirus disease in cultured tiger frog, <i>Rana tigrina rugulosa</i>, in southern China

Immunocytochemical Localization of Growth Hormone-Releasing Hormone-like Peptide in the Brain of the Tiger Frog, Rana tigrina

Using antisera directed against carp growth hormone-releasing hormone (cGHRH), we found more extensive brain distribution of GHRH-like immunoreactive (ir) neurons in the tiger frog, Rana tigrina, than reported in previous studies, which employed mammalian GHRH antibodies. In the telencephalon, GHRH-ir perikarya were present in the pallium dorsale, pallium laterale (pars dorsalis and pars ventralis), pallium mediale, nucleus entopeduncularis, amygdala pars medialis, and in the ventral portion of the lateral preoptic area. Most GHRH somata were present in the nucleus infundibularis ventralis located around the third ventricle, extending from the region posterior to the optic chiasma to the caudal end of infundibulum. In the thalamic region, GHRH-ir perikarya occurred in the area ventrolateralis thalami, the nuclei posterocentralis thalami, and the posterolateralis thalami. The ir cell bodies in the nucleus posteroventralis tegmenti mesencephali represented the caudal-most brain GHRH perikarya. Extensive GHRH-ir fibers occurred around the nonreactive cells in the ventral preoptic area and ventral area of the infundibulum. GHRH-ir fibers were present in the outer layers of the median eminence, but not in the neural lobe or pars distalis of the pituitary gland. This wider neuroanatomical distribution of GHRH-like peptide in the brain of R. tigrina should, provide the basis for future studies to establish the exact role of GHRH-like peptides in anuran brain.

Sequence Analysis of the Complete Genome of an Iridovirus Isolated from the Tiger Frog

We have isolated a tiger frog virus (TFV) from diseased tiger frogs, Rana tigrina rugulosa. The genome was a linear double-stranded DNA of 105,057 basepairs in length with a base composition of 55.01% G+C. About 105 open reading frames were identified with coding capacities for polypeptides ranging from 40 to 1294 amino acids. Computer-assisted analyses of the deduced amino acid sequences revealed that 39 of 105 putative gene products showed significant homology to functionally characterized proteins of other species in the GenBank/EMBL/DDBJ databases. These proteins included enzymes and structural proteins involved in virus replication, transcription, modification, and virus–host interaction. The deduced amino acid sequences of TFV gene products showed more than 90% identity to FV3, but a low degree of similarity among TFV, ISKNV, and LCDV-1. The results from this study indicated that TFV may belong to the genus Ranavirus of the family Iridoviridae.

Out of Asia: Mitochondrial DNA Evidence for an Oriental Origin of Tiger Frogs, Genus Hoplobatrachus

Most examples of intercontinental dispersal events after the Miocene contact between Africa and Asia involve mammal lineages. Among amphibians, a number of probably related groups are known from both continents, but their phylogenies are so far largely unresolved. To test the hypothesis of Miocene dispersal against a Mesozoic vicariance scenario in the context of Gondwana fragmentation, we analyzed fragments of the mitochondrial 16S rRNA gene (572 bp) in 40 specimens of 34 species of the anuran family Ranidae. Results corroborated the monophyly of tiger frogs (genus Hoplobatrachus), a genus with representatives in Africa and Asia. The African H. occipitalis was the sister group of the Asian H. crassus, H. chinensis, and H. tigerinus. Hoplobatrachus was placed in a clade also containing the Asian genera Euphlyctis and Nannophrys. Combined analysis of sequences of 16S and 12S rRNA genes (total 903 bp) in a reduced set of taxa corroborated the monophyly of the lineage containing these three genera and identified the Asian genus Fejervarya as its possible sister group. The fact that the African H. occipitalis is nested within an otherwise exclusively Asian clade indicates its probable Oriental origin. Rough molecular clock estimates did not contradict the assumption that the dispersal event took place in the Miocene. Our data further identified a similar molecular divergence between closely related Asian and African species of Rana (belonging to the section Hylarana), indicating that Neogene intercontinental dispersal also may have taken place in this group and possibly in rhacophorid treefrogs.

Cardiovascular effects of bPTH-(1–34) and isoproterenol in the frog, Rana tigrina

1. The cardiovascular effects of bovine parathyroid hormone fragment [bPTH-(1–34)] and isoproterenol (ISO) on frog ( Rana tigrina) isolated atria and helical strips of blood vessels were examined since PTH produces a β-adrenergic-like effect in the mammal. 2. Data showed that both bPTH-(1–34) and ISO were vasorelaxant in KC1 and arginine vasoctocin (AVT) preconstricted dorsal aorta, iliac and femoral arteries. 3. They both relaxed extracellular calcium-dependent contrations. 4. There was no additive nor synergistic effect between them in AVT preconstricted strips. 5. Both bPTH-(1–34) and ISO were positively inotroplc but differed in their chronotroplc effects, being negative and positive. 6. In the tiger frog, bPTH-(1–34) shows β-adrenergic like contractile responses in both the cardiac and vascular smooth muscle as in the mammal, but not in the heart rate.

The cardiac effects of parathyroid hormone in the tiger frog, Rana tigrina

1. 1. The cardiac effects of the N-terminal (1–34) peptide fragment of bovine parathyroid hormone [bPTH-(1–34)] on isolated atria were examined in the frog, Rana tigrina. 2. 2. bPTH-(1–34) produced dose-related inotropic response but no chronotropic response. This inotropic response varied at different times of the year. 3. 3. The inotropic effect of bPTH-(1–34) was attenuated in the presence of verapamil and imidazole. 4. 4. The mechanism of action of bPTH-(1–34) is probably a stimulation of calcium influx directly or indirectly via cAMP production.

Osmotic Regulation in the Crab-Eating Frog (Rana Cancrivora)

ABSTRACT The osmotic and ionic regulatory abilities of adults of the euryhaline crab-eating frog (Rana cancrivora) have been studied. Adult frogs tolerated environmental salinities as high as 28%,, at 30°C. Tadpoles of this form tolerated salinities as high as 39 % at the same temperature. Changes in body weight of frogs following transfers to different environmental salinities indicate both that the skin of this frog is permeable to water and that these animals do not swallow large volumes of external medium, even in high salinities. Above salinities of about 9 %, plasma Δrises with increasing environmental Δ. Plasma Δ is always higher than environmental Δ. Increases in plasma concentration above fresh-water levels are due partly to increased NaCl concentration (about 40%), partly to increased urea concentration (about 60%). Urea concentrations as high as 0·48 M (2·9%) have been measured. Urinary A parallels plasma Δ, but is always lower than plasma Δ. Considerable quantities of urea are lost via the urine, even though urinary urea levels are below plasma levels. Measurements of short-circuit current indicate that active uptake by the skin of inorganic ions continues in R. cancrivora acclimatized to high salinities. R. cancrivora is no less susceptible to water loss by evaporation from the skin than are other amphibians. In preference experiments R. cancrivora chooses salinities below 18 %, but shows no strong preference for a particular salinity. Similar observations on osmoregulatory mechanisms in a close relative of R. cancrivora, the tiger frog (R. tigerina), show that the latter species is similar to ordinary fresh-water frogs. The striking physiological convergence between R. cancrivora and the elasmobranch fishes is discussed, as are various possible implications of our data regarding nitrogen metabolism in tadpoles and kidney function in adult frogs.