Isolation, identification and pathogenicity of Vibrio harveyi, the causal agent of skin ulcer disease in juvenile hybrid groupers Epinephelus fuscoguttatus × Epinephelus lanceolatus.

The hybrid grouper, Epinephelus fuscoguttatus (♀) × Epinephelus lanceolatus (♂), is a newly bred cultivated marine fish species of high economic value. However, a skin ulcer disease with high mortality has occurred, and the responsible pathogen remains unknown. In this study, we summarized the epidemic status and external signs of this disease. We screened potential pathogens and finally isolated one bacterial strain ML01 from affected fish. We subjected healthy juvenile hybrid groupers to bacterial challenge tests with the isolate by immersion, immersion after dermal abrasion and intraperitoneal injection, respectively. Within 14 days post-infection, the isolate ML01 caused mass mortality of juveniles infected via immersion after dermal abrasion or intraperitoneal injection. Diseased juveniles displayed obvious signs of skin ulcers. The median lethal dose of ML01 by intraperitoneal injection was 1.10 × 105 colony-forming units. ML01 was identified as Vibrio harveyi by bacterial morphology, analytical profile index identification, 16S rDNA sequencing and multilocus sequence analysis. Antibiotic susceptibility tests showed that ML01 was sensitive to ceftriaxone, doxycycline and minocycline. The results of this study suggest that V. harveyi is the causal agent of skin ulcer disease in juvenile hybrid groupers, thus providing a basis for effective control and prevention of this disease.

Detection of type III secretion gene as an indicator for pathogenic Edwardsiella tarda.

AIMS: To differentiate pathogenic and nonpathogenic Edwardsiella tarda strains based on the detection of type III secretion system (T3SS) gene using polymerase chain reaction (PCR). METHODS AND RESULTS: Primers were designed to amplify Edw. tarda T3SS component gene esaV, catalase gene katB, haemolysin gene hlyA and 16S rRNA gene as an internal positive control. Genomic DNAs were extracted using a commercial isolation kit from 36 Edw. tarda strains consisting of 18 pathogenic and 18 nonpathogenic strains, and 50 ng of each DNA was used as the template for PCR amplification. PCR was performed with a thermocycler (TaKaRa TP600) in a 25-µl volume. Products of esaV were detected in all pathogenic strains, but not in nonpathogenic strains; katB was detected in all pathogenic strains and one of nonpathogenic strains; hlyA was not detected in any strains. CONCLUSIONS: The detection of esaV gene can be used for the assessment of pathogenic Edw. tarda strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The strategy using T3SS gene as the virulence indicator provides a useful tool for the clinical assessment of pathogenic Edw. tarda strains and prediction of edwardsiellosis risk in fish culture environments.

Screening of genes expressed in vivo after infection by Vibrio anguillarum M3.

AIMS: Genes uniquely expressed in vivo may contribute to the overall pathogenicity of an organism and are likely to serve as potential targets for the development of new vaccine. This study aims to screen the genes expressed in vivo after Vibrio anguillarum infection by in vivo-induced antigen technology (IVIAT). METHODS AND RESULTS: The convalescent-phase sera were obtained from turbot (Scophthalmus maximus) survived after infection by the virulent V. anguillarum M3. The pooled sera were thoroughly adsorbed with M3 cells and Escherichia coli BL21 (DE3) cells. A genomic expression library of M3 was constructed and screened for the identification of immunogenic proteins by colony immunoblot analysis with the adsorbed sera. After three rounds of screening, 19 putative in vivo-induced (ivi) genes were obtained. These ivi genes were catalogued into four functional groups: regulator/signalling, metabolism, biological process and hypothetical proteins. Three ivi genes were insertion-mutated, and the growth and 50% lethal dose (LD(50) ) of these mutants were evaluated. CONCLUSIONS: The identification of ivi genes in V. anguillarum M3 sheds light on understanding the bacterial pathogenesis and provides novel targets for the development of new vaccines and diagnostic reagents. SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this is the first report describing in vivo-expressed genes of V. anguillarum using IVIAT. The screened ivi genes in this study could be new virulent factors and targets for the development of vaccine, which may have implications for the development of diagnostic regents.

[Preparation, characterization and evaluation of cellulose tris (3,5-dimethylphencarbamate) chiral stationary phase].

With the deposition of cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC) on aminopropylated silica gel (mean particle size, 5 microns; pore size, 13 nm; surface area, 110 m2/g) by using two different methods (evaporation and precipitation), two chiral stationary phases (CSP1 and CSP2) characterized by elemental analysis and scanning electron micrography were obtained. They were also evaluated by using seven racemic compounds with n-hexane/ethanol(95/5, V/V) and n-hexane/2-propanol (90/10, V/V) as mobile phases. The results showed that the chiral stationary phase CSP1 obtained by the evaporation method had better efficiency and chiral resolution ability than CSP2 by the precipitation method.

Endogenous firing patterns of murine spiral ganglion neurons.

Current-clamp recordings with the use of the whole cell configuration of the patch-clamp technique were made from postnatal mouse spiral ganglion neurons in vitro. Cultures contained neurons that displayed monopolar, bipolar, and pseudomonopolar morphologies. Additionally, a class of neurons having exceptionally large somata was observed. Frequency histograms of the maximum number of action potentials fired from 240-ms step depolarizations showed that neurons could be classified as either slowly adapting or rapidly adapting. Most neurons (85%) were in the rapidly adapting category (58 of 68 recordings). Measurements of elementary properties were used to define the endogenous firing characteristics of both the neuron classes. Action potential number varied with step and holding potential, spike amplitude decayed during prolonged depolarizations, and spike frequency adaptation was observed in both rapidly and slowly adapting neurons. The apparent input resistance, spike amplitude decrement, and instantaneous firing frequency differed significantly between rapidly and slow adapting neurons. Inward rectification was evaluated in response to hyperpolarizing contrast current injections. Present in both electrophysiological classes, its magnitude was graded from neuron to neuron, reflecting differences in number, type, and/or voltage dependence of the underlying channels. These data suggest that spiral ganglion neurons possess intrinsic firing properties that regulate action potential number and timing, features that may be crucial to signal coding in the auditory periphery.

Heterogeneous voltage dependence of inward rectifier currents in spiral ganglion neurons.

Inward rectification was characterized in neonatal spiral ganglion neurons maintained in tissue culture. Whole cell current and voltage-clamp techniques were used to show that the hyperpolarization-activated cationic (Ih) current underlies most or all of the inward rectification demonstrated in these neurons. The average reversal potential (-41.3 mV) and cesium sensitivity were typical of that found in other neurons and cell types. What was unique about the hyperpolarization-activated currents, however, was that the half-maximal voltages (V1/2) and slope factors (k) that characterized Ih current activation were graded from neuron to neuron. Voltage-clamp recordings made with standard bath and pipette solutions revealed V1/2 values that ranged from -78.1 to -122.1 mV, with slope factors from 7.6 to 13.1. These gradations in the voltage-dependent features of the Ih current did not result from variability in the recording conditions because independently measured Na+ current-to-voltage relationships were found to be uniform (peak current at -20 mV). Moreover, the range and average V1/2 and slope values could be altered with activators [8-(4-chlorophenylthio) adenosine 3',5'-cyclic monophosphate in combination with okadaic acid] or inhibitors {N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide}of protein indicating that Ih current heterogeneity most likely resulted from differential phosphorylation.

Effects of IL-1 beta on neuronal activities in the dorsal motor nucleus of the vagus in rat brain slices.

Effects of recombinant human interleukin-1 beta (IL-1 beta) on the neuronal activities in the rat dorsal motor nucleus of the vagus (DMV) were investigated by extra- and intracellular recordings in slice preparations. Twelve (52%) of 23 spontaneously firing neurons recorded extracellularly, 7 of which were electrophysiologically identified as vagal motoneurons, were inhibited by a bath application of IL-1 beta at a dose of either 5.8 x 10(-8) or 5.8 x 10(-8) M. The duration of the responses ranged widely from about 10 min to more than 2 h. Two (9%) of the 23 neurons were excited, whereas the remaining 9 (39%) were not affected by IL-1 beta. Of 42 DMV neurons recorded intracellularly, 19 (45%) showed a hyperpolarization following an application of 5.8 x 10(-8) M IL-1 beta, which still persisted in a TTX-containing solution. Two (5%) displayed depolarization and 21 (50%) were unaffected. The hyperpolarization in 16 of the 19 neurons (84%) ranged from -5 to -10 mV and lasted for more than 30 min without changing the input resistance. The IL-1 beta-induced hyperpolarization was completely blocked by concurrent perfusion with sodium salicylate. The remaining three neurons showed a short-lasting (5-14 min) hyperpolarization (ranging from -6 to -15 mV) with a decrease in the input resistance. These findings indicate that IL-1 beta mainly inhibits the vagal motoneurons in the DMV, at least partly through prostaglandin synthesis. This provides a mechanism that could account for the central action of IL-1 beta on visceral processes such as the inhibition of gastric acid secretion.

Effects of vasopressin and angiotensin II on neurones in the rat dorsal motor nucleus of the vagus, in vitro.

1. Extracellular recordings were made from 297 spontaneously firing neurones in the dorsal motor nucleus of the vagus (DMV) in slice preparations of rat medulla oblongata. Some of the neurones recorded were identified to be vagal motoneurones by antidromic stimulation. The cells fired with a slow irregular pattern at an average rate of 1.1 +/- 0.1 spikes/s (mean +/- S.E.M.). 2. Arginine vasopressin (AVP) was applied by perfusion in 196 of the 297 cells. Most of the neurones (190/196, 97%) were excited by 10(-6) M AVP with an increase in firing rate from the basal level of 1.1 +/- 0.1 to a maximum of 2.5 +/- 0.2 spikes/s. There was a dose-dependent relation between the concentration of AVP and the increased firing rate in all DMV neurones tested (n = 38). The threshold concentration of the peptide to produce changes in firing rate was assumed to be about 10(-10) M. The remaining six neurones were not affected by application of AVP. 3. Application of oxytocin (OXT, 10(-6) M) increased the firing rate of all thirty-eight neurones tested. The effects of AVP and OXT on all neurones examined (n = 20 and 4, respectively) still persisted after blocking the synaptic transmission in a low-Ca2+ or Ca(2+)-free-high-Mg2+ solution, indicating the direct action of both AVP and OXT on the postsynaptic membranes. 4. The AVP-induced excitatory responses were completely but reversibly blocked by the V1-type receptor antagonists, [1-(beta-mercapto-beta, beta-cyclopentamethylene-propionic acid), 2-(O-methyl)tyrosine]-arginine vasopressin (d(CH2)5Tyr(Me)AVP) (n = 5) and Phaa-D-Tyr(Et)Phe-Gln-Asn-Lys-Pro-Arg-NH2 (n = 6), whereas a selective and reversible OXT receptor antagonist, desGly-NH2d(CH2)5[Tyr-(Me)2Thr4]ornithine vasotocin, which suppressed the OXT-induced excitation, did not block the responses to AVP (n = 11). 5. Application of angiotensin II (AII, 10(-6) M) to 153 neurones increased the firing rates of 60 (39%) neurones. The firing rate was increased from the basal level of 1.0 +/- 0.1 to a maximum of 1.8 +/- 0.2 spikes/s (n = 60). The effect of AII was completely abolished by an AII receptor antagonist, [Sar1,Ile8]angiotensin II (n = 6). There was a dose dependence of the excitatory response on AII concentration in all of eleven neurones tested. The threshold concentration was assumed to be about 10(-9) M. The activity of 5 (3%) of 153 neurones was decreased, and the remaining 88 (58%) neurones were not affected by AII.(ABSTRACT TRUNCATED AT 400 WORDS)

Comparison of toxic effects of methylguanidine, guanidinosuccinic acid and creatinine in rats with adenine-induced chronic renal failure.

Methylguanidine (MG), guanidinosuccinic acid (GSA) and creatinine (Cr), which accumulate in the body in parallel with the progress of renal failure after adenine administration, were given separately to rats in order to compare their toxicities. Food containing adenine was given to rats for 24 days to induce renal failure, and then each of the test substances was administered intraperitoneally from the following day, the survival rates of the rats being subsequently determined. Administration of MG at varying doses produced a dose-dependent decrease in the survival rate, whereas the survival curves obtained for rats given GSA or Cr indicated weak toxicity. The levels of MG, GSA or Cr accumulated in the body were extraordinarily high in surviving rats after 14 days of administration of each respective compound. The toxic effects are discussed on the basis of these results.