Identification of novel putative virulence factors, adhesin AIDA and type VI secretion system, in atypical strains of fish pathogenic Edwardsiella tarda by genomic subtractive hybridization

Edwardsiella tarda, which is known to be the causative agent of edwardsiellosis in freshwater and marine fish, has two motility phenotypes. Typical strains exhibiting motility are isolated mainly from freshwater fish and Japanese flounder. Atypical strains exhibiting non-motility are isolated mainly from marine fish, with the exception of Japanese flounder. Subtractive hybridization was performed to identify genomic differences between these two phenotypes. Two fragments which showed homology to potential virulence factors were isolated from atypical strains: the autotransporter adhesin AIDA and a component of T6SS. We analysed DNA sequences of about 5 kbp containing these fragments and identified two partial ORF, and ORF encoding for other components of T6SS. The predicted amino acid sequences showed remarkably low homology to components of T6SS reported in the typical E. tarda strain PPD130/91. Furthermore, the organization of these ORF was different from the gene cluster of the typical E. tarda strain. AIDA and T6SS may therefore be associated with different pathogenicity in typical and atypical E. tarda hosts.     

Intraspecific genetic variability of Edwardsiella tarda strains from cultured turbot

Edwardsiella tarda is an enterobacterial fish pathogen that causes mortality in various fish species worldwide. In this study, we analyzed the intraspecific variability in a collection of E. tarda strains isolated from turbot. To do this we employed 4 polymerase chain reaction (PCR)-based methods: (1) random amplified polymorphic DNA (RAPD), (2) enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR), (3) repetitive extragenic palindromic-PCR (REP-PCR) and (4) BOX-PCR. E. tarda isolates from different hosts were also included for comparison. E. tarda strains from turbot showed high molecular homogeneity when RAPD (primers P3 and P6), ERIC-PCR and BOX-PCR were employed. However, with regard to the REP-PCR and RAPD (primers P4 and P5) techniques, different genetic groups could be established within these isolates using either technique. The 2 RAPD types presented an 85% similarity, while those obtained with REP-PCR showed 74% similarity. Based on the results obtained, although a high genetic homogeneity was found in turbot isolates, the RAPD test (with primers P4 and P5) and REP-PCR were capable of discrimination within these strains, and they are therefore considered the most appropriate typing methods for studies of edwardsiellosis in turbot.     

Generation of two auxotrophic genes knock-out Edwardsiella tarda and assessment of its potential as a combined vaccine in olive flounder (Paralichthys olivaceus)

Two auxotrophic genes that play essential roles in bacterial cell wall biosynthesis--alanine racemase (alr) gene and aspartate semialdehyde dehydrogenase (asd) gene--knock-out Edwardsiella tarda (Δalr Δasd E. tarda) was generated by the allelic exchange method to develop a combined vaccine system. Green fluorescent protein (GFP) was used as a model foreign protein, and was expressed by transformation of the mutant E. tarda with antibiotic resistant gene-free plasmids harboring cassettes for GFP and asd expression (pG02-ASD-EtPR-GFP). In vitro growth of the mutant E. tarda was similar to wild-type E. tarda when D-alanine and diaminopimelic acid (DAP) were supplemented to growth medium. However, without d-alanine and/or DAP supplementation, the mutant showed very limited growth. The Δalr Δasd E. tarda transformed with pG02-ASD-EtPR-GFP showed a similar growth pattern of wild-type E. tarda when D-alanine was supplemented in the medium, and the expression of GFP could be observed even with naked eyes. The virulence of the auxotrophic mutant E. tarda was decreased, which was demonstrated by approximately 10? fold increase of LD?? dose compared to wild-type E. tarda. To assess vaccine potential of the present combined vaccine system, olive flounder (Paralichthys olivaceus) were immunized with the GFP expressing mutant E. tarda, and analyzed protection efficacy against E. tarda challenge and antibody titers against E. tarda and GFP. Groups of fish immunized with 10? CFU of the Δalr Δasd E. tarda harboring pG02-ASD-EtPR-GFP showed no mortality, which was irrespective to boost immunization. The cumulative mortality rates of fish immunized with 10? or 10? CFU of the mutant bacteria were lowered by a boost immunization. Fish immunized with the mutant E. tarda at doses of 10?-10? CFU/fish showed significantly higher serum agglutination activities against formalin-killed E. tarda than PBS-injected control fish. Furthermore, fish immunized with 10?-10? CFU/fish of the mutant E. tarda showed significantly higher ELISA titer against GFP antigen than fish in other groups. These results indicate that the present double auxotrophic genes knock-out E. tarda coupled with a heterologous antigen expression has a great strategic potential to be used as combined vaccines against various fish diseases.     

Isolation of an unusual strain of Edwardsiella tarda from turbot and establish a PCR detection technique with the gyrB gene

Aims:  The aim of this study was to report an unusual Edwardsiella tarda and develop an effective method to identify this bacterium.
Methods and Results:  During the spring and summer of 2006, an epizootic occurred among cultured turbot ( Scophthalmus maximus ) in Qingdao, China. A gram-negative, rod-shaped bacterium (designated as LTB-4) was isolated from the infected fish, and was proved to be virulent to turbot. Based on the 16S rDNA sequencing and phenotypic tests, the bacterial pathogen was identified as E. tarda. Unlike those commonly described E. tarda strains, no flagellate was observed. Partial gyrB genes were amplified from E. tarda using the universal primers of gyrB genes and sequenced. The polymerase chain reaction (PCR) primers for the gyrB gene were designed specific to E. tarda . It revealed positive amplification of the gyrB fragment in E. tarda , whereas other bacterial species were negative. In addition, the technique enabled the recognition of E. tarda from diseased fish.
Conclusions:  The isolate was identified as E. tarda without flagellate and an effective method was developed to identify E. tarda based on using the gyrB gene as a taxonomic marker.
Significance and Impact of the Study:  The unusual E. tarda was first reported in China and the PCR allowed the rapid and sensitive detection of E. tarda.     

Outer membrane vesicles as a candidate vaccine against edwardsiellosis

Infection with Edwardsiella tarda, a gram-negative bacterium, causes high morbidity and mortality in both marine and freshwater fish. Outer membrane vesicles (OMVs) released from gram-negative bacteria are known to play important roles in bacterial pathogenesis and host immune responses, but no such roles for E. tarda OMVs have yet been described. In the present study, we investigated the proteomic composition of OMVs and the immunostimulatory effect of OMVs in a natural host, as well as the efficacy of OMVs when used as a vaccine against E. tarda infection. A total of 74 proteins, from diverse subcellular fractions, were identified in OMVs. These included a variety of important virulence factors, such as hemolysin, OmpA, porin, GAPDH, EseB, EseC, EseD, EvpC, EvpP, lipoprotein, flagellin, and fimbrial protein. When OMVs were administrated to olive flounder, significant induction of mRNAs encoding IL-1β, IL-6, TNFα, and IFNγ was observed, compared with the levels seen in fish injected with formalin-killed E. tarda. In a vaccine trial, olive flounder given OMVs were more effectively protected (p<0.0001) than were control fish. Investigation of OMVs may be useful not only for understanding the pathogenesis of E. tarda but also in development of an effective vaccine against edwardsiellosis.     

Isolation and characterization of Edwardsiella tarda from fall chinook salmon (Oncorhynchus tshawytscha).

A new bacterial pathogen of chinook salmon (oncorhynchus tshawytscha) was isolated from fish in Oregon's Rogue River. The bacteria are biochemically and serologically related to strains of Edwardsiella tarda. Initially isolated from chinook salmon, the bacteria were also pathogenic for steelhead and rainbow trout (Salmo gairdneri), and channel catfish (Ictalurus punctatus). The 50% lethal doses for chinook salmon, steelhead trout, and channel catfish injected intraperitoneally and maintained in 18 degrees C water were 4.1 x 10(6), 5.6 x 10(6), and 4.0 x 10(5) respectively. When chinook salmon and rainbow trout were injected intraperitoneally and held in 12 degrees C water, the mean lethal doses were 6.4 x 10(7) and 1.7 x 10(6), respectively. The invasiveness of the organism was low in steelhead trout exposed to the bacteria by the waterborne route. The optimum growth temperature of the bacteria in brain heart infusion broth was approximately 35 degrees C. The guanine plus cytosine content of DNA obtained from E. tarda isolated from salmon was 59 mol%.     

Characterization of the Plesiomonas shigelloides genes encoding the heme iron utilization system

Plesiomonas shigelloides is a gram-negative pathogen which can utilize heme as an iron source. In previous work, P. shigelloides genes which permitted heme iron utilization in a laboratory strain of Escherichia coli were isolated. In the present study, the cloned P. shigelloides sequences were found to encode ten potential heme utilization proteins: HugA, the putative heme receptor; TonB and ExbBD; HugB, the putative periplasmic binding protein; HugCD, the putative inner membrane permease; and the proteins HugW, HugX, and HugZ. Three of the genes, hugA, hugZ, and tonB, contain a Fur box in their putative promoters, indicating that the genes may be iron regulated. When the P. shigelloides genes were tested in E. coli K-12 or in a heme iron utilization mutant of P. shigelloides, hugA, the TonB system genes, and hugW, hugX, or hugZ were required for heme iron utilization. When the genes were tested in a hemA entB mutant of E. coli, hugWXZ were not required for utilization of heme as a porphyrin source, but their absence resulted in heme toxicity when the strains were grown in media containing heme as an iron source. hugA could replace the Vibrio cholerae hutA in a heme iron utilization assay, and V. cholerae hutA could complement a P. shigelloides heme utilization mutant, suggesting that HugA is the heme receptor. Our analyses of the TonB system of P. shigelloides indicated that it could function in tonB mutants of both E. coli and V. cholerae and that it was similar to the V. cholerae TonB1 system in the amino acid sequence of the proteins and in the ability of the system to function in high-salt medium.     

Effect of oxytetracycline-medicated feed on antibiotic resistance of gram-negative bacteria in catfish ponds.

The effect of oxytetracycline-medicated feeds on antibiotic resistance in gram-negative bacteria from fish intestines and water in catfish ponds was investigated. In experiments in the fall and spring, using ponds with no previous history of antibiotic usage, percentages of tetracycline-resistant bacteria in catfish intestines obtained from medicated ponds increased significantly after 10 days of treatment. In the fall, resistance of the intestinal and aquatic bacteria returned to pretreatment levels within 21 days after treatment. In the spring, resistance declined after treatment but remained higher than pretreatment levels for at least 21 days in intestinal bacteria and for 5 months in aquatic bacteria. Plesiomonas shigelloides, Aeromonas hydrophila, and Citrobacter freundii were isolated frequently in both spring and fall; Escherichia coli, Klebsiella pneumoniae, Edwardsiella tarda, and Enterobacter spp. were isolated primarily in the spring. Oxytetracycline treatment did not affect the distribution of bacterial species in the fall but may have accelerated a shift toward greater prevalence of members of the family Enterobacteriaceae in the spring. Multiple antibiotic resistance did not appear to be elicited by oxytetracycline treatment.     

Distribution of Plesiomonas shigelloides in various components of pond ecosystems in Dhaka, Bangladesh.

Plesiomonas shigelloides is considered to be a waterborne agent of human gastroenteritis. An ecological study was carried out in five ponds in Dhaka city over a period of one year to elucidate the distribution and seasonality of this organism in various components of pond ecosystems. Samples were collected from hydrophytes, water, phytoplankton and sediment every 15 days over 12 months and cultured for P. shigelloides. P. shigelloides was isolated from a total of 120 samples including 25 (20.8%), 16 (13.3%), 22 (18.3%) and 35 (29.2%) of hydrophytes, water, phytoplankton and sediment samples, respectively. Distinct seasonal patterns of isolation of P. shigelloides were observed in the four components with two distinct peaks. The highest peaks were observed in hydrophytes and water samples in May and in phytoplankton and sediment in November. P. shigelloides was isolated from all components from all ponds during the study period. These results suggest that P. shigelloides is an autochthonous member in the freshwater pond ecosystems in Dhaka, Bangladesh.     

Unusual physiology of scale-less carp,Gymnocypris przewalskii,in Lake Qinghai: a high altitude alkaline saline lake

The scale-less carp (Gymnocypris przewalskii) inhabits Lake Qinghai located on the Qinghai-Tibet plateau (elevation, 3200 m) in western China. The lake waters are alkaline (pH 9.4, titratable alkalinity=30 mmol l(-1)), Mg(2+)-rich (18.7 mmol l(-1)), Ca(2+)-poor (0.30 mmol l(-1)) and saline (9 per thousand ). These fish make annual spawning migrations into freshwater rivers. We investigated the physiology of nitrogen excretion and ionoregulation of fish from the lake and river. Fish from both waters were ammonotelic, although ammonia-N excretion rates were lower in lake fish (175 vs. 344 micromol kg(-1) h(-1), P<0.05) resulting in unusually high levels of ammonia in blood plasma (2.23 vs. 0.32 mmol l(-1)), bile, liver, muscle and brain. Exposure to 0.4 mmol l(-1) total ammonia in lake water ([NH(3)]=0.16 mmol l(-1)) killed fish within 8 h. River fish survived exposure to 1.0 mmol l(-1) total ammonia in river water at pH 8.0 ([NH(3)]=0.023 mmol l(-1)) for 24 h suggesting high ammonia tolerance in lake fish. High glutamate dehydrogenase and glutamine synthetase activities in tissues probably allow the fish to alleviate ammonia toxicity by amino acid accumulation. Neither lake nor river fish relied on urea excretion to remove excess N. Urea-N excretion rates were below 20 micromol kg(-1) h(-1) for both groups, and levels of urea in plasma and tissues were moderate. When exposed to elevated ammonia, urea-N excretion increased slightly (approximately 50 micromol kg(-1) h(-1)) and liver and muscle urea levels increased in the river fish. Plasma ion levels were within the range typical of cyprinids, but river fish had significantly higher plasma [Na(+)] and [Cl(-)] and lower [K(+)] than fish from the lake. During 48-h lake-to-river water transfer, plasma Na(+) and Cl(-) levels rose significantly. Significantly higher Na(+)/K(+)-ATPase activity in the gills of river fish may be related to the higher plasma ion levels. Plasma [Mg(2+)] and [Ca(2+)] were tightly regulated despite the great differences in the lake and river water levels.     

The icthyofauna of the Mhlathuze coastal lakes: some preliminary results

The fish faunas of the four Mhlathuze coastal lakes and the lower river comprise a diverse assortment of over fifty marine, estuarine and freshwater species. Three freshwater species are endemic to KwaZulu-Natal and nine estuarine species are endemic to southern Africa. Five species are of conservation significance. Species numbers in Lakes Mzingazi and Cubhu are similar historically and both lakes served as secondary nursery habitats for estuarine associated fishes. This role has been impacted by the construction of weirs at their outlets which prevent successful recruitment of estuarine species, especially during drought years when lake water levels are low. The fish faunas of Lakes Nsezi and Mangeza are depauperate and lack marine or estuarine components. In order that these systems fulfil their potential function as secondary nursery habitats to many estuarine fish species, minimum lake water levels must be set to ensure sufficient outflows at proposed fish ladders during critical spawning and migration times.     

Protection of tilapia (Oreochromis mosambicus) from edwardsiellosis by vaccination with Edwardsiella tarda ghosts

The vaccine potential of Edwardsiella tarda ghosts produced by gene E mediated lysis was investigated using tilapia (Oreochromis mosambicus). Tilapia immunized with E. tarda ghosts (ETG) and formalin killed E. tarda (FKC) vaccines showed significantly higher serum agglutination titers than control fish. Fish immunized with ETG showed no significant differences with fish immunized with FKC in serum agglutination titers, but showed significantly higher bactericidal activity than fish immunized with FKC. Furthermore, fish immunized with ETG showed higher protection than fish immunized with FKC. As this promising type of a non-living whole cell envelope preparation seems to be favorable over conventional vaccines, we suggest E. tarda ghosts as a new vaccine candidate.     

Spatial and Temporal Relations between Fluvial and Allacustrine Yellowstone Cutthroat Trout, Oncorhynchus clarki bouvieri, Spawning in the Yellowstone River, Outlet Stream of Yellowstone Lake

Yellowstone cutthroat trout (YCT), Oncorhynchus clarki bouvieri, that spawn in the outlet of Yellowstone Lake show two potamodromous migration patterns, fluvial and allacustrine. The main purpose of this study was to determine whether those fluvial and allacustrine YCT represent reproductively isolated stocks. Redd surveys indicated spawning occurred during about 5 consecutive weeks between late May and mid-July 1993–1995. Lake fish (N=6), defined as radiotagged YCT that entered Yellowstone Lake after the spawning period (i.e. allacustrine pattern), were found in the river between the lake outlet (river kilometer [Rkm] 0) and Rkm 20.0 during spawning. Probable lake fish (N=28; tagged YCT that were last detected near the lake outlet) were found between Rkm 0 and Rkm 22.5 during spawning. River fish (N=4; tagged YCT that remained in the river when annual tracking concluded in fall, i.e. fluvial pattern) were found between Rkm 1.1 and Rkm 18.0 during spawning. Fidelity to spawning areas used between consecutive years was suggested by one of five lake fish and the single river fish for which data were available. Spatial overlap in spawning and a lack of temporal separation between the life-history types during spawning suggested that fluvial and allacustrine YCT were not reproductively isolated. Radiotagging, as well as visual observations made annually from boats during April and May, indicated fluvial YCT overwintered downstream from Rkm 14 and were few, probably on the order of 10% of all YCT that spawned in the Yellowstone River.     

Habitat fragmentation and extinction rates within freshwater fish communities: a faunal relaxation approach

Aim To estimate population extinction rates within freshwater fish communities since the fragmentation of palaeo‐rivers due to sea level rise at the end of the Pleistocene; to combine this information with rates estimated by other approaches (population surveys, fossil records); and to build an empirical extinction–area relationship. Location Temperate rivers from the Northern Hemisphere, with a special focus on rivers discharging into the English Channel, in north‐western France. Methods (1) French rivers. We used a faunal relaxation approach to estimate extinction rates in coastal rivers after they became isolated by the sea level rise. Tributaries within the Seine were used to build a species–area relationship for a non‐fragmented river system to predict species richness in coastal rivers before their fragmentation. (2) Other rivers. Extinction rates obtained for four other Holarctic river systems fragmented at the end of the Pleistocene, the fragmented populations of one salmonid species (Japan) and the fossil records from the Mississippi Basin were included in the study. Results (1) French rivers. Within strictly freshwater fish species, rare and/or habitat specialist species were the most affected by fragmentation. In contrast, euryhaline species were not affected. A negative relationship between extinction rate and river basin size was observed. (2) Other rivers. Our study established a common scaling relationship for freshwater fish population extinction rates that spans seven orders of magnitude in river basin size. Main conclusions This study strongly suggests that extinctions of fish populations occurred within French coastal rivers after they became isolated 8000 years ago. The patterns observed at regional and inter‐continental scales are consistent with the expectation that large populations are less prone to extinction than small ones, resulting in a strong extinction–area relationship coherent over a large spatio‐temporal scale. Our study is the first multi‐scale quantitative assessment of background extinction patterns for freshwater fishes.     

青菱湖中长春鳊生物学的研究

1983-1985年对通长江的青菱湖中长春鳊的年龄与生长、食性、繁殖及其鱼群江湖交流等生态习性进行了调查。发现相当部分标本的鳞片上有明显的幼轮标志;性成熟后,雌鱼的生长较雄鱼迅速;摄取的食物种类与湖中各种水生植物的季节性盛衰相适应,但有一定的选择性;亲鱼能在湖中成熟产卵,但鱼卵因受自然条件限制,不能发育成活。湖中鱼群的补充仍依靠数量不定的1龄幼鱼由长江干流逆水或顺水伺机窜入湖内。江湖之间闸门的启闭主要根据农田水利的需要,因此,长春鳊鱼群规律性的江湖交流仍然受到阻碍。