Formation of viable but non-culturable state (VBNC) of Aeromonas hydrophila and its virulence in goldfish, Carassius auratus

In this study we investigated the viable but non-culturable (VBNC) state of Aeromonas hydrophila and its virulence in goldfish. Aeromonas hydrophila cultured in a 0.35% NaCl solution at pH 7.5 and at 25 °C for 50 days showed the VBNC state. In the VBNC state we were unable to detect viable bacteria by the plate count method but we did find 10⁴ cells/ml by the direct viable count microscopical method after staining with fluorescein diacetate and ethidium bromide. The virulence comparison in goldfish showed that bacteria cultured at 25 °C for 1 day in a 0.35% NaCl solution were more virulent than bacteria cultured for 28 days. VBNC bacteria showed lower virulence in goldfish compared to 28-day-cultured bacteria by intraperitoneal injection.The results from the study suggest that A. hydrophila can remain in the aquatic environment for prolonged periods in the VBNC state but those cells are not pathogenic to goldfish.     

The effect of temperature on Aeromonas hydrophila infection in goldfish, Carassius auratus

The effect of temperature on Aeromonas hydrophila infection in goldfish, Carassius auratus , was studied using A. hydrophila strain A-3500. After comparison of four different infection methods, subcutaneous injection was selected. Different test temperatures were also tested and higher mortality was observed at 17 and 25°C during a 15-day period. SDS-PAGE analysis of outer membrane proteins prepared from A. hydrophila cultured at 10, 17, 25 and 32°C in formulated salt water showed different protein profiles. For example, a 40-kDa band was found only at 17 and 25°C. Phagocytic rates of A. hydrophila by goldfish macrophages at 10, 17, 25 and 32°C were 20.46 ± 2.07, 16.15 ± 1.39, 15.94 ± 1.85 and 22.22 ± 2.49%, respectively. The results indicated that temperature affects both the cell membrane structure of A. hydrophila and phagocytic activity of goldfish macrophages, resulting in varying fish mortality when infected at different temperatures.     

Francisella tularensis does not manifest virulence in viable but non-culturable state

Francisella tularensis is a small Gram-negative bacterium that causes tularemia in animals and man. The disease can be transmitted by handling of infected animals, by contaminated dust, by insect vectors, or by drinking contaminated water. In the present study cells of F. tularensis were subjected to extended storage in cold water devoid of carbon sources. Total cell counts remained constant throughout a 70-day period and beyond, while plate counts decreased to an undetectable level after 70 days. Attempts to resuscitate the cells were unsuccessful. Quantitative PCR targeting the 16S rDNA of F. tularensis showed an increase in variability after 25 days and the signal was lost after 45 days. Metabolic activity, measured by accumulation of rhodamine 123, declined to approximately 35% after a 140-day period. Analyses of substrate responsiveness of cells stored for 140 days in cold water showed that approximately 30% of the population increased in size after incubation in rich medium in the presence of nalidixic acid. Approximately 10(5) of these cells were injected intraperitoneally into mice. No signs or symptoms of tularemia were observed during 3 weeks. In addition, there was no evidence of stimulation of lymphocytes with F. tularensis as recall antigen. In conclusion, viable but non-culturable cells of F. tularensis are avirulent in mice, giving new insight into the ecological niche of this bacterium.     

微生物VBNC状态形成及复苏机制

99%以上的微生物因处于活的但非可培养(viable but non-culturable,VBNC)状态而无法分离培养。复苏促进因子(resuscitation-promoting factors,Rpfs)是培养获取VBNC菌的最重要突破。结合课题组近十余年从环境功能视角利用Rpf复苏培养VBNC菌的研究,本文在阐述微生物VBNC状态的形成及复苏进展的基础上,从VBNC菌形成及复苏过程出发,探究"探索因子"与群体感应的内在关系。并总结了课题组利用Rpf所复苏培养的具有潜在环境功能的VBNC菌种。本论文将为揭示微生物VBNC状态的形成及复苏机制提供新的思路,并为认识和重新评价Rpf法复苏培养VBNC菌在污染环境微生物修复中的作用提供理论依据。     

Immunoreactive antigens of the outer membrane protein of Aeromonas hydrophila, isolated from goldfish, Carassius auratus (Linn.)

Aeromonas hydrophila causes disease under stress conditions or in concert with infection by other pathogens in goldfish. Sero-diagnostic and/or immunoprophylactic tools against Aeromonas infection in goldfish are not available so far. The present study was undertaken to fractionate and characterise the outer membrane proteins (OMP) of A. hydrophila and to identify suitable immunoreactive components. A total of 10 fractions were generated from crude OMP antigens upon gel permeation and subsequent ion-exchange chromatography. One of the fractionated antigens (GPID2), primarily a 57-kDa polypeptide, showed maximum sero-reactivity, even higher than the crude OMP. Suitability of GPID2 antigen for use in diagnostic preparations was assessed by dip-stick ELISA. In vitro goldfish lymphoproliferative ability of fractionated antigen, GPIID2 (primarily a 23-kDa polypeptide) was observed to be higher than all the fractionated antigens as well as crude OMP. It can be concluded that the 57 kDa and 23 kDa polypeptides of the OMP of A. hydrophila, possessing high immunoreactivity, should be given due attention while preparing immunodiagnostic and immunoprophylatic tools against Aeromonas infections in goldfish.     

Gordonia jinhuaensis sp. nov., a novel actinobacterium,isolated from a VBNC (viable but non-culturable) state in pharmaceutical wastewater

A Gram-stain positive, aerobic, non-motile and rod-shaped actinobacterial strain, designated as ZYR 51^T, was isolated from pharmaceutical wastewater in Jinhua city, Zhejiang province, Eastern China. Isolation was aided by using a resuscitation-promoting factor, suggesting the strain was recovered from a viable but non-culturable state. Strain ZYR 51^T was characterized by a polyphasic taxonomic approach. Growth was found to occur at 10–45 °C, pH 6.0–10.0 and 0–9 % NaCl (w/v). Based on the 16S rRNA gene sequence, phylogenetic analysis clearly demonstrated that strain ZYR 51^T belongs to the genus Gordonia and showed low level similarities (below 97 %) with all other members of this genus. The strain was found to possess meso-diaminopimelic acid (meso-DAP), along with MK-9(H₂) as the predominant menaquonine. Mycolic acids were found to be present. C_16:0 (34.9 %), 10-methyl C_18:0 (30.3 %), iso-C_18:0(8.2 %), and summed feature 3 (C_16:1 ω6c and/or C_16:1ω7c as define by MIDI; 18.8 %) were identified as the major cellular fatty acids. The polar lipid profile of strain ZYR 51^T was found to consist of diphosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannosides and some unknown lipids. The genomic DNA G+C content of strain ZYR 51^T was determined to be 67.7 mol%. The combined genotypic and phenotypic data showed that the strain represents a novel species of the genus Gordonia, for which the name Gordonia jinhuaensis sp. nov. is proposed, with the type strain is ZYR 51^T (=CGMCC 1.12827^T = NBRL B-59111^T = NBRC 110001^T).     

CadF expression in Campylobacter jejuni strains incubated under low-temperature water microcosm conditions which induce the viable but non-culturable (VBNC) state

Campylobacter jejuni is a major gastrointestinal pathogen that colonizes host mucosa via interactions with extracellular matrix proteins such as fibronectin. The aim of this work was to study in vitro the adhesive properties of C. jejuni ATCC 33291 and C. jejuni 241 strains, in both culturable and viable but non-culturable (VBNC) forms. To this end, the expression of the outer-membrane protein CadF, which mediates C. jejuni binding to fibronectin, was evaluated. VBNC bacteria were obtained after 46–48 days of incubation in freshwater at 4 °C. In both cellular forms, the expression of the cadF gene, assessed at different time points by RT-PCR, was at high levels until the third week of VBNC induction, while the intensity of the signal declined during the last stage of incubation. CadF protein expression by the two C. jejuni strains was analysed using 2-dimensional electrophoresis and mass spectrometry; the results indicated that the protein, although at low levels, is also present in the VBNC state. Adhesion assays with culturable and VBNC cells, evaluated on Caco-2 monolayers, showed that non-culturable bacteria retain their ability to adhere to intestinal cells, though at a reduced rate. Our results demonstrate that the C. jejuni VBNC population maintains an ability to adhere and this may thus have an important role in the pathogenicity of this microorganism.     

Gill sphaerosporosis in goldfish (Carassius auratus)

Infections caused by a Sphaerospora sp. resembling S. chinensis are reported for the first time in goldfish (Carassius auratus) from North America. The myxosporean was found in the respiratory epithelium of the gill of pond-reared fish. Spores from stained tissue sections were spherical with an equal mean length and width of 6.3 microns. Spore valves were thickened at the suture which lies in a plane perpendicular to two prominent pyriform polar capsules. The polar capsules were 4.0 x 2.8 microns in length and width. Both monosporous and disporous development within a surrounding "pseudoplasmodium" was detected. Infections caused moderate hyperplasia and occasional necrosis of the respiratory epithelial cells of the gill.     

Effect of orally administered azadirachtin on non-specific immune parameters of goldfish Carassius auratus (Linn. 1758) and resistance against Aeromonas hydrophila

Modulation of the immune responses using active bio-ingredients as a possible prophylaxis measure has been novel prospect for aquaculture. The present study evaluated the effects of azadirachtin EC 25% on non-specific immune responses in goldfish Carassius auratus and resistance against pathogenic bacteria Aeromonas hydrophila. The experimental trial for effects of azadirachtin on immuno-haematoloical parameters in goldfish was conducted by feeding the various levels of azadirachtin as control T₀ (without azadirachtin), T₁ (0.1%), T₂ (0.2%), T₃ (0.4%), T₄ (0.8%) and T₅ (1.6%) for a period of 28 days. Fishes were challenged with A. hydrophila 28 days post feeding and relative percentage survival (%) was recorded over 14 days post infection. Immuno-haematoloical (total erythrocyte count, total leukocyte count, haemoglobin, packed cell volume, NBT activity, phagocytic activity, serum lysozyme activity, myeloperoxidase activity, total immunoglobulin) and serum biochemical parameters (serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT) and blood glucose) of fishes were examined at 14 and 28 days of feedings. Fish fed with azadirachtin, showed significantly (p < 0.05) enhanced TEC, TLC, Total Ig, total protein, NBT activity, serum lysozyme activity and myeloperoxidase level in different treatment groups in comparison with control group. Similarly, SGOT, SGPT and blood glucose level were found to be significantly (p < 0.05) high but PCV and Hb did not differ significantly (p > 0.05) in the treatment groups compared to control groups. Azadirachtin at the concentration of 4 g kg^?1 showed significantly (p < 0.05) higher relative percentage survival (42.60%) when compared with the control against A. hydrophila infection. This study indicated that azadirachtin EC 25% (4 g kg^?1) showed higher NBT activity, serum lysozyme, protein profiles, leukocyte counts and resistance against A. hydrophila infection and thus, can be used as a potential immunostimulant in aquaculture.     

Starvation survival and viable but nonculturable states in Aeromonas hydrophila

The behavior of Aeromonas hydrophila stored at 4 degrees C and 25 degrees C in nutrient-poor filtered sterilized distilled water was investigated. At 4 degrees C, the A. hydrophila population declined below the detection level (0.1 cell mL(-1)) after 7 weeks, whereas the number of cells with intact membrane as determined by the LIVE/DEAD method decreased only by 1 log unit. Although, this response is reminiscent of the so-called VBNC state, the cells could not be resuscitated by an upshift to 25 degrees C. A mixture of rods with normal size and elongated cells was observed in this state. At 25 degrees C, viable cells and cells with intact membrane declined only by 0.8 log unit over the 10-week storage period, and thus A. hydrophila entered the classical starvation survival state. During this state, a mixture of rods and cocci was observed. Prestarvation at 25 degrees C for 24 h and especially 49 days delayed significantly the rate of entry into the VBNC state. However, stationary phase cells were not significantly more tolerant than exponential phase cells. No significant improvements in recovery yield were obtained on LB agar plates amended with catalase or sodium pyruvate. During cold incubation, high variability in responses was observed. Intermittent cryptic regrowth might be responsible for this variability in responses.     

细菌有活力但不可培养状态及其机制研究进展

有活力但不可培养(viable but non-culturable,VBNC)状态是细菌遭遇逆境时进入的一种特殊状态,该状态下的菌体在条件适宜时可复苏并恢复其致病性,被认为是细菌躲避不良环境的一种生存策略。VBNC状态菌体对人类医学和工农业生产具有巨大的潜在威胁,开展关于VBNC状态的检测及诱导、复苏及其机制研究可为减少或避免该状态细菌的危害提供理论基础。本文简要综述了细菌VBNC状态在诱导、复苏及致病性等方面的研究进展,并结合本实验室及国内外相关团队近年来在植物病原细菌VBNC状态研究中的结果,详细总结了VBNC状态细菌的形成和复苏机制,对植物病原细菌在环境胁迫下的存活机制、病害田间初侵染来源分析及VBNC状态菌体在病害循环中的作用等相关研究具有重要参考意义。     

A BAC library for the goldfish Carassius auratus auratus (Cyprinidae, Cypriniformes)

A goldfish (Carassius auratus auratus) bacterial artificial chromosome genomic library (BAC library) was constructed from one aquarium-bred male specimen (tetraploid, 4n=100, genome size=3.52 pg/cell). The library consists of 128,352 positive clones with an average insert size of 150.4 kb, covering the genome 11-fold. All clones were spotted onto nylon filters and thus are available for screening of genomic regions of interest, such as candidate genes, gene families, or large-sized syntenic DNA regions of cyprinid species. Preliminary screens with two genes were conducted with hybridizing probes to the genes RAG1 and lgi1. RAG1 is a single-copy gene in zebrafish and is duplicated in C. a. auratus. We found a very close correlation between the number of positive BAC clones and the expected library coverage. Two copies of lgi1 were found in zebrafish. We have detected four different copies in C. a. auratus, not in the expected abundance, which indicates some variation in the coverage of the BAC library. The preliminary screens indicate that many duplicated genes that resulted from the ancient fish-specific genome duplication persist in the tetraploid goldfish genome. Hence, the BAC library will provide a useful resource for the future work on comparative genomics, polyploidy, diploidization, and evolutionary genomics in fishes.     

空肠弯曲菌活而不可培养状态的产毒素能力及毒素基因表达的检测

目的 通过诱导空肠弯曲菌进入活的不可培养(VBNC)状态建立细胞学模型,用VeroE6细胞和HeLa细胞检测空肠弯曲菌产细胞膨胀性肠毒素(cytotoxic distendingtoxin,CDT)能力的改变,并在RNA水平上进一步验证毒素的表达。方法 采用4℃冷藏及-70℃冷冻的方法诱导空肠弯曲菌进入VBNC状态,利用细胞总数计算、活细胞染色计数及可培养细胞计数间接确证VBNC状态细菌的存在,细胞对于VBNC状态下的细菌仍然敏感,并用反转录-聚合酶链反应(RT-PCR)证实空肠弯曲菌毒素基因的表达。结果 3种不同状态的细胞计数结果表明存活细胞进入了VBNC状态,细菌经4℃冷藏及-70℃冷冻后数量减少约1个数量级,在冷冻前,细菌对细胞毒性较强,72h时70%以上细胞凋亡,冷冻后的VBNC状态细菌对VeroE6和HeLa细胞的生长仍有影响,72h时约有40%细胞进入凋亡状态。结论 低温、贫养条件下空肠弯曲菌可进入VBNC状态,用常规传统的羊血平板培养基仅能使不到10%的空肠弯曲菌复苏生长,从而可以检测,在VBNC状态时,空肠弯曲菌产毒素能力改变较大,远远低于正常状态,但仍具备产毒素的能力,也从RNA水平实验得到证实。     

The viable but non-culturable state of wine micro-organisms during storage

Colony counting and DEFT did not give the same results when wine micro-organisms were enumerated. Both methods were used to monitor the population of acetic acid bacteria (AAB) and lactic acid bacteria (LAB) during wine storage. Results suggest that part of the populations had reached a viable but non-culturable (VBNC) state. These cells were unable to produce colonies but could hydrolyse fluorescent esters and could be counted by DEFT. For AAB, O2 deprivation quickly induced this state. Recovery from this state was very rapid as soon as O2 was available. The response was not so clear for LAB during wine storage. However, a similar state was induced by sulfiting. Moreover, filtration of wine stored in barrels and contaminated by Brettanomyces, AAB and LAB demonstrated that cell size was not homogeneous. Cells which remained in wine after several weeks could pass through a 0.45-microm membrane. However, when they re-entered a growing phase, they were again retained by membrane filtration. During and after the decline phase, wine micro-organisms might survive as smaller cells in a VBNC state.     

Gut-associated lymphoid tissue (GALT) of the goldfish,Carassius auratus

The head kidney and spleen are major sites of haemopoiesis in fish; a secondary center is found in loose connective tissue of the intestine. In this study we determined the nature of gut-associated haemopoietic tissue in the goldfish, Carassius auratus, using light and electron microscopy. This tissue is a loose stroma of reticular cells and fibers vascularized by capillaries, venules, and arterioles. The cellular population includes lymphoblasts, small and medium-sized lymphocytes, plasmocytes, macrophages, and various granulocytes. The most abundant granulocyte is the mast cell, whose large granules stain with Alcian blue and toluidine blue. Heterophils are found in the intestinal connective tissue as well as two other granulocytes: one with ovoid granules having dense parallel lamellae and another with granules containing crystalline inclusions. Immature forms of both granulocytes were also noted. Macrophages containing phagocytosed debris were often located close to the epithelium; they were observed forming clusters with lymphocytes. The epithelium contained a number of migrating leucocytes including lymphocytes and lymphoblasts, macrophages, and heterophils. Although many granulocytes were found in the connective tissue, granulopoiesis does not seem to be a major function. Gut-associated haemopoietic tissue in goldfish resembles diffuse lymphoid tissue and may be involved in intestinal immune responses.     

A new myxozoan from feral goldfish (Carassius auratus)

In February 2004, a mass die-off of common goldfish Carassius auratus L., presumptively caused by bacterial coldwater disease (Flavobacterium psychrophilum), occurred at Fern Ridge Reservoir, Oregon. A range of size classes was affected, but all mature fish were female and all fish were infected with a single myxozoan, Chloromyxum auratum n. sp. No histological changes were observed associated with the parasite. Infection was represented by mictosporic plasmodia and free-floating spores in the gall bladder. Parasite spores were nearly spherical, 13.6 microm long x 12.6 microm wide x 13.1 microm thick, and possessed 4 equal-sized polar capsules. Spores had a coglike appearance in apical view because of distinct ridges 2.1 microm high protruding from the valve cells. There were 6-9 extrasutural ridges per valve (15-20 ridges per spore), aligned along the longitudinal axis, with some branching, and convergence at both poles. Morphologically, spores identified most closely with Chloromyxum cristatum Léger, 1906; however, 18S rDNA sequence data indicated only 97.5% similarity over 2,076 bp with Chloromyxum cyprini, the only synonym of C. cristatum for which DNA data are available; additional sequence data may reveal the other synonyms to be distinct species. This is the first record of a species of Chloromyxum from goldfish.     

Temperature tolerance in the goldfish,Carassius auratus

• 1.Lower and upper temperature tolerances of 240 goldfish, Carassius auratus, were measured at constant acclimation temperatures of 5, 15, 25 and 35 °C via critical thermal methodology.
• 2.Mean critical thermal minima and maxima ranged from 0.3 to12.6 °C and 30.8 to 43.6 ° C, respectively, and were significantly linearly related to acclimation temperature. Acclimation temperature accounted for approximately 90% of the variance in temperature tolerance. Ultimate critical thermal minimum and maximum equaled 0.3 and 43.6 °C, respectively.
• 3.Integrating the temperature tolerance polygon yielded an area of temperature tolerance of 1429 °C², which is approximately 17% larger than the polygon measured via the incipient lethal temperature approach. This difference is explained by methodological differences in these two techniques to quantify temperature tolerance.