Identification of Shewanella baltica as the most important H2S-producing species during iced storage of Danish marine fish

Shewanella putrefaciens has been considered the main spoilage bacteria of low-temperature stored marine seafood. However, psychrotropic Shewanella have been reclassified during recent years, and the purpose of the present study was to determine whether any of the new Shewanella species are important in fish spoilage. More than 500 H2S-producing strains were isolated from iced stored marine fish (cod, plaice, and flounder) caught in the Baltic Sea during winter or summer time. All strains were identified as Shewanella species by phenotypic tests. Different Shewanella species were present on newly caught fish. During the warm summer months the mesophilic human pathogenic S. algae dominated the H2S-producing bacterial population. After iced storage, a shift in the Shewanella species was found, and most of the H2S-producing strains were identified as S. baltica. The 16S rRNA gene sequence analysis confirmed the identification of these two major groups. Several isolates could only be identified to the genus Shewanella level and were separated into two subgroups with low (44%) and high (47%) G+C mol%. The low G+C% group was isolated during winter months, whereas the high G+C% group was isolated on fish caught during summer and only during the first few days of iced storage. Phenotypically, these strains were different from the type strains of S. putrefaciens, S. oneidensis, S. colwelliana, and S. affinis, but the high G+C% group clustered close to S. colwelliana by 16S rRNA gene sequence comparison. The low G+C% group may constitute a new species. S. baltica, and the low G+C% group of Shewanella spp. strains grew well in cod juice at 0 degrees C, but three high G+C Shewanella spp. were unable to grow at 0 degrees C. In conclusion, the spoilage reactions of iced Danish marine fish remain unchanged (i.e., trimethylamine-N-oxide reduction and H2S production); however, the main H2S-producing organism was identified as S. baltica.     

Development of a simple and rapid fluorogenic procedure for identification of vibrionaceae family members

We describe a simple colony overlay procedure for peptidases (COPP) for the rapid fluorogenic detection and quantification of Vibrionaceae from seawater, shellfish, sewage, and clinical samples. The assay detects phosphoglucose isomerase with a lysyl aminopeptidase activity that is produced by Vibrionaceae family members. Overnight cultures are overlaid for 10 min with membranes containing a synthetic substrate, and the membranes are examined for fluorescent foci under UV illumination. Fluorescent foci were produced by all the Vibrionaceae tested, including Vibrio spp., Aeromonas spp., and Plesiomonas spp. Fluorescence was not produced by non-Vibrionaceae pathogens. Vibrio cholerae strains O1, O139, O22, and O155 were strongly positive. Seawater and oysters were assayed, and 87 of 93 (93.5%) of the positive isolates were identified biochemically as Vibrionaceae, principally Vibrio vulnificus, Vibrio parahaemolyticus, Aeromonas hydrophila, Photobacterium damselae, and Shewanella putrefaciens. None of 50 nonfluorescent isolates were Vibrionaceae. No Vibrionaceae were detected in soil, and only A. hydrophila was detected in sewage. The COPP technique may be particularly valuable in environmental and food-testing laboratories and for monitoring water quality in the aquaculture industry.     

Characterization of the 23S and 5S rRNA genes and 23S-5S intergenic spacer region (ITS-2) of Photobacterium damselae

The 23S ribosomal RNA (rRNA) gene has been sequenced in strains of the fish pathogens Photobacterium damselae subsp. damselae (ATCC 33539) and subsp. piscicida (ATCC 29690), showing that 3 nucleotide positions are clearly different between subspecies. In addition, the 5S rRNA gene plus the intergenic spacer region between the 23S and 5S rRNA genes (ITS-2) were amplified, cloned and sequenced for the 2 reference strains as well as the field isolates RG91 (subsp. damselae) and DI21 (subsp. piscicida). A 100% similarity was found for the consensus 5S rRNA gene sequence in the 2 subspecies, although some microheterogeneity was detected as inter-cistronic variability within the same chromosome. Sequence analysis of the spacer region between the 23S and 5S rRNA genes revealed 2 conserved and 3 variable nucleotide sequence blocks, and 4 different modular organizations were found. The ITS-2 spacer region exhibited both inter-subspecies and intercistronic polymorphism, with a mosaic-like structure. The EMBL accession numbers for the 23S, 5S and ITS-2 sequences are: P. damselae subsp. piscicida 5S gene (AJ274379), P. damselae subsp. damselae 23S gene (Y18520), subsp. piscicida 23S gene (Y17901), P. damselae subsp. piscicida ITS-2 (AJ250695, AJ250696), P. damselae subsp. damselae ITS-2 (AJ250697, AJ250698).     

Multiplex PCR assay for ureC and 16S rRNA genes clearly discriminates between both subspecies of Photobacterium damselae

A multiplex-PCR approach, employing 2 primer pairs directed to internal regions of the 16S rRNA and ureC genes, was utilized to analyze a collection of Photobacterium damselae strains, including 25 isolates of subspecies piscicida and 15 isolates of subspecies damselae. With this procedure, all the P. damselae subsp. damselae strains yielded 2 amplification products, one of 267 bp and the other of 448 bp, corresponding to internal fragments of the 16S rRNA and ureC genes, respectively. However, P. damselae subsp. piscicida isolates only showed the PCR product of 267 bp (16S rRNA fragment), indicating the absence of the urease gene in its genome. We have constructed a DNA probe directed to an internal region of the ureC gene, and corroborated by dot blot hybridization that the P. damselae subsp. piscicida lacks this gene, whereas it is present in the subspecies damselae. This constitutes the first successful discrimination between both subspecies using a PCR procedure, which could become a useful tool for diagnosis of pasteurellosis in the field. In addition, since these 2 subspecies have been shown to share nearly the same rrn operon sequence, our results provided evidence that one of the steps in the P. damselae speciation proccess included gain/loss events associated with the ure operon.     

Rapid identification of marine bioluminescent bacteria by amplified 16S ribosomal RNA gene restriction analysis

To rapidly identify natural isolates of marine bioluminescent bacteria, we developed amplified ribosomal DNA restriction analysis (ARDRA) methods. ARDRA, which is based on the restriction patterns of 16S rRNA gene digested with five enzymes (EcoRI, DdeI, HhaI, HinfI, RsaI), clearly distinguished the 14 species of marine bioluminescent bacteria currently known, which belong to the genera Vibrio, Photobacterium, and Shewanella. When we applied ARDRA to 129 natural isolates from two cruises in Sagami Bay, Japan, 127 were grouped into six ARDRA types with distinctive restriction patterns; these isolates represented the bioluminescent species, P. angustum, P. leiognathi, P. phosphoreum, S. woodyi, V. fischeri, and V. harveyi. The other two isolates showing unexpected ARDRA patterns turned out to have 16S rRNA gene sequences similar to P. leiognathi and P. phosphoreum. Nevertheless, ARDRA provides a simple and fairly robust means for rapid identification of the natural isolates of marine bioluminescent bacteria, and is therefore useful in studying their diversity.     

Differentiation of Shewanella putrefaciens and Shewanella alga on the basis of whole-cell protein profiles, ribotyping, phenotypic characterization, and 16S rRNA gene sequence analysis.

Seventy-six presumed Shewanella putrefaciens isolates from fish, oil drillings, and clinical specimens, the type strain of Shewanella putrefaciens (ATCC 8071), the type strain of Shewanella alga (IAM 14159), and the type strain of Shewanella hanedai (ATCC 33224) were compared by several typing methods. Numerical analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell protein and ribotyping patterns showed that the strains were separated into two distinct clusters with 56% +/- 10% and 40% +/- 14% similarity for whole-cell protein profiling and ribotyping, respectively. One cluster consisted of 26 isolates with 52 to 55 mol% G + C and included 15 human isolates, mostly clinical specimens, 8 isolates from marine waters, and the type strain of S. alga. This homogeneous cluster of mesophilic, halotolerant strains was by all analyses identical to the recently defined species S. alga (U. Simidu et al., Int. J. Syst. Bacteriol, 40:331-336, 1990). Fifty-two typically psychrotolerant strains formed the other, more heterogeneous major cluster, with 43 to 47 mol% G + C. The type strain of S. putrefaciens was included in this group. The two groups were confirmed by 16S rRNA gene sequence analysis. It is concluded that the isolates must be considered two different species, S. alga and S. putrefaciens, and that most mesophilic isolates formerly identified as S. putrefaciens belong to S. alga. The ecological role and potential pathogenicity of S. alga can be evaluated only if the organism is correctly identified.     

16S rRNA gene sequence analysis of Photobacterium damselae and nested PCR method for rapid detection of the causative agent of fish pasteurellosis.

The causative agent of fish pasteurellosis, the organism formerly known as Pasteurella piscicida, has been reclassified as Photobacterium damselae subsp. piscicida on the basis of 16S rRNA gene sequence comparisons and chromosomal DNA-DNA hybridization data; thus, this organism belongs to the same species as Photobacterium damselae subsp. damselae (formerly Vibrio damselae). Since reassignment of P. damselae subsp. piscicida was based on only two strains, one objective of the present work was to confirm the taxonomic position of this fish pathogen by sequencing the 16S rRNA genes of 26 strains having different geographic and host origins. In addition, a nested PCR protocol for detection of P. damselae based on 16S rRNA was developed. This PCR protocol was validated by testing 35 target and 24 nontarget pure cultures, and the detection limits obtained ranged from 1 pg to 10 fg of DNA (200 to 20 cells). A similar level of sensitivity was observed when the PCR protocol was applied to fish tissues spiked with bacteria. The PCR approach described in this paper allows detection of the pathogen in mixed plate cultures obtained from asymptomatic fish suspected to be carriers of P. damselae subsp. piscicida, in which growth of this bacterium cannot be visualized. Our results indicate that the selective primers which we designed represent a powerful tool for sensitive and specific detection of fish pasteurellosis.     

Numerical taxonomy of Vibrionaceae isolated from cultured amberjack (Seriola dumerili) and surrounding water

A numerical taxonomic study was performed on 148 isolates of Gram-negative, heterotrophic, facultative anaerobic bacteria isolated from amberjack (Seriola dumerili) and its surrounding culture water. The study included 30 type and reference strains belonging to genera Vibrio, Listonella, and Photobacterium. The strains were characterized by 109 morphological, biochemical, physiological, and nutritional tests. Cluster analysis of similarity matrices obtained with S(SM) and S(J) coefficients was carried out. UPGMA (unweighted pair group mathematical average) analysis defined 11 phena at S(SM) values > or = 86%. Nine phena were identified as Vibrio alginolyticus, V. fischeri, V. harveyi, V. carchariae, V. mediterranei, V. splendidus, V. furnissii, V. parahaemolyticus, and Photobacterium damselae subsp. damselae. The two latter comprised strains isolated from diseased fish.     

宁海地区香鱼弧菌病病原菌鉴定

摘要：【目的】香鱼弧菌病对中国沿海地区的香鱼养殖业造成了巨大的危害,然而,病原不明导致了防治上的许多问题。本文鉴定了引起宁海地区香鱼爆发性弧菌病的病原。【方法】采用TCBS平板分离优势菌;采用回归感染试验确认病原菌,采用改进的寇氏法计算LD50;采用形态学观察、生理生化特征测定、细菌特异性引物PCR扩增检测及细菌16S rRNA和金属蛋白酶（MP）基因序列分析鉴定细菌;采用药敏实验测定它对部分抗生素的敏感性。【结果】分离并鉴定优势菌株ayu-H080701为宁海地区香鱼弧菌病的病原菌,它对香鱼的半致死量为1.2×104 CFU。形态学观察和生理生化特征测定表明,ayu-H080701与鳗利斯顿氏菌最为接近。PCR扩增检测表明,细菌16S rRNA 基因通用引物和鳗利斯顿氏菌MP基因特异引物均能扩增到预期大小的特异性条带。ayu-H080701与鳗利斯顿氏菌16S rRNA基因核苷酸序列同源性最高,为99.4%～99.5%,与同属的海弧菌和美人鱼发光杆菌分别为94.3%和91.9%;ayu-H080701与鳗利斯顿氏菌MP氨基酸序列同源性高达97.6%～98.8 %,与其它弧菌科成员则低于75.6 %,系统进化树分析也揭示ayu-H080701与鳗利斯顿氏菌进化相关性最高。【结论】引起宁海地区香鱼弧菌病的菌株ayu-H080701被鉴定为鳗利斯顿氏菌。     

Construction of a safe, stable, efficacious vaccine against Photobacterium damselae ssp. piscicida

Vaccination with bacterial auxotrophs, particularly those with an interruption in the common pathway of aromatic amino-acid biosynthesis, known as the shikimate pathway, has been shown to be effective in the prevention of a variety of bacterial diseases. In order to evaluate this approach to vaccine development in the important marine pathogen Photobacterium damselae subsp. piscicida, the aroA gene of the shikimate pathway was identified from a P. damselae subsp. piscicida genomic library by complementation in an aroA mutant of Escherichia coli. The complementing plasmid was isolated and the nucleotide sequence of the P. damselae subsp. piscicida genomic insert was determined. Subsequent analysis of the DNA-sequence data demonstrated that the identified plasmid contained 3464 bp of P. damselae subsp. piscicida DNA, including the complete aroA gene. The sequence data was used to delete a 144 bp MscI fragment, and the kanamycin resistance gene (kan) from transposon Tn903 was ligated into the MscI site. This delta(aro)A::kan construct was sub-cloned into a suicide plasmid and transferred to a wild-type P. damselae subsp. piscicida by conjugation and allelic exchange. One selected mutant, LSU-P2, was confirmed phenotypically to require supplementation with aromatic metabolites for growth in minimal media, and was confirmed genotypically by PCR and DNA sequencing. Further, LSU-P2 was demonstrated to be avirulent in hybrid striped bass and to provide significant protection against disease following challenge with the wild-type strain.     

Photobacterium damselae ssp. damselae associated with diseased black tiger shrimp Penaeus monodon Fabricius in India

AIM: To characterize and identify Photobacterium damselae ssp. damselae present in black gill diseased Penaeus monodon collected from east coast of India. METHODS AND RESULTS: Photobacterium damselae ssp. damselae was isolated from hepatopancreas, muscles and gills by using the thiosulfate citrate bile salts sucrose agar supplemented with 1.5% NaCl (TCBS-1) medium. A total of 32 Ph. damselae ssp. damselae isolates were studied together with two reference strains. The biochemical tests and analysis of ureC and 16S rRNA genes confirmed the phenotypic characterization of the isolates as Ph damselae ssp. damselae. Experimental infection studies revealed that the LD50 values of P. monodon and P. indicus ranged from 2x10(3) to 5x10(5) CFU per shrimp and from 4x10(2) to 2x10(4) CFU per shrimp, respectively. CONCLUSIONS: Photobacterium damselae ssp. damselae was found in the internal organs of P. monodon and it showed pathogenic to shrimp. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study on the Ph. damselae ssp. damselae present in the black gill diseased P. monodon in India and therefore might serve as a basis for future studies and diagnosis purpose to shrimp culturists.     

Amplified fragment length polymorphism (AFLP) and biochemical typing of Photobacterium damselae subsp. damselae

AIMS: The aim of the present study was to characterize subspecifically Photobacterium damselae subsp. damselae strains isolated from cultured Sparus aurata and Dicentrarchus labrax by means of phenotypic and molecular typing techniques (amplified fragment length polymorphism, AFLP). METHODS AND RESULTS: Seventy-one strains of P. damselae subsp. damselae were isolated from 38 cultured fishes at different fish farms located on the Mediterranean coast near Valencia, Spain. Most fish studied were asymptomatic and some were recovered during infectious outbreaks. Phenotypic characterization revealed a considerable degree of variability within the subspecies, including some characters, such as production of urease, which are used to differentiate P. damselae subsp. damselae from P. damselae subsp. piscicida. Genetic characterization was conducted on a selection of 33 strains, including two reference strains. Dice coefficient (Sd) and the unweighted pair group method with average linkage (UPGMA) were used for numerical analysis of banding patterns. AFLP type was defined on the basis of 100% similarity in the dendrogram obtained, yielding 24 distinct AFLP profiles. At 70% similarity, 13 clusters were defined, thus confirming the great variability observed for the phenotypic traits. CONCLUSIONS: The AFLP variability shown by the isolates was high enough to discriminate between different strains which colonize the same fish. However, closely related AFLP types were usually derived from strains isolated at the same fish farm, indicating an epidemiological relationship. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has confirmed that the AFLP technique allows discrimination of individual strains within P. damselae subsp. damselae for epidemiological studies, and that this subspecies exhibits greater variability than that described for subspecies piscicida.     

Effect of salinity on the immune response of tiger shrimp Penaeus monodon and its susceptibility to Photobacterium damselae subsp. damselae

Addition of NaCl at 2.5% to tryptic soy broth (TSB) significantly increased the growth of Photobacterium damselae subsp. damselae. Tiger shrimp Penaeus monodon held in 25 per thousand seawater were injected with P. damsela subsp. damselae grown in TSB containing NaCl at 0.5%, 1.5%, 2.5% and 3.5% at a dose of 8.48 x 10(4)colony-forming units (cfu)shrimp(-1). Over 24-96 h, the cumulative mortality was significantly higher for the shrimp challenged with P. damselae subsp. damselae grown in 2.5% NaCl than those grown in 0.5%, 1.5% and 3.5% NaCl. In another experiment, P. monodon held in 25 per thousand were injected with TSB-grown P. damselae subsp. damselae (8.48 x 10(4)cfushrimp(-1)), and then transferred to 5 per thousand, 15 per thousand, 25 per thousand (control) and 35 per thousand. After 96 h, the mortality was highest for the P. damselae subsp. damselae-injected shrimp held in 5 per thousand, and the lowest for the P. damselae subsp. damselae-injected shrimp held in 25 per thousand. In a separate experiment, P. monodon held in 25 per thousand and then transferred to 5 per thousand, 15 per thousand, 25 per thousand (control) and 35 per thousand were examined for immune parameters, and phagocytic activity and clearance efficiency of P. damselae subsp. damselae after 12-96 h. The THC, hyaline cell, phenoloxidase activity, respiratory burst, superoxide dismutase (SOD) activity, phagocytic activity and clearance efficiency decreased significantly for the shrimp held in 5 per thousand, 15 per thousand and 35 per thousand after 12h. It is concluded that tiger shrimp P. monodon transferred from 25 per thousand to low salinity levels (5 per thousand and 15 per thousand) and high salinity (35 per thousand) had reduced immune ability and decreased resistance against P. damselae subsp. damselae infection.     

Occurrence of both subspecies of Photobacterium damselae in mullets collected in the river Magra (Italy)

The natural reservoirs and biological characteristics of pathogenic populations of both subspecies of Photobacterium damselae in aquatic habitants remain unclear because of difficulties in obtaining pathogenic strains from the environment. In the present study, we assessed the occurrence of Photobacterium damselae subsp. piscicida and Photobacterium damselae subsp. damselae , considered to be the causative agent of past epizootic outbreaks in mullets collected in the river Magra, Italy. Two hundred and seventy-eight mullets were collected during a period of two years (2008-2009) and analyzed using multiplex PCR. During this period, 57% of fishes were positive for Photobacterium damselae subsp. piscicida and 37% for Photobacterium damselae subsp. damselae, with an higher presence in summer months although none of PCR-positive mullets showed clinical signs of disease. Our results indicate that the two micro-organisms are widespread in the population of mullets studied, and this could be a possible cause for outbreaks in favourable environmental conditions.     

Photobacterium damselae ssp. piscicida: detection by direct amplification of 16S rRNA gene sequences and genotypic variation as determined by amplified fragment length polymorphism (AFLP)

A PCR protocol for the rapid diagnosis of fish 'pasteurellosis' based on 16S rRNA gene sequences was developed. The procedure combines low annealing temperature that detects low titers of Photobacterium damselae but also related species, and high annealing temperature for the specific identification of P. damselae directly from infected fish. The PCR protocol was validated on 19 piscine isolates of P. damselae ssp. piscicida from different geographic regions (Japan, Italy, Spain, Greece and Israel), on spontaneously infected sea bream Sparus aurata and sea bass Dicentrarchus labrax, and on closely related American Type Culture Collection (ATCC) reference strains. PCR using high annealing temperature (64 degrees C) discriminated between P. damselae and closely related reference strains, including P. histaminum. Sixteen isolates of P. damselae ssp. piscicida, 2 P. damselae ssp. piscicida reference strains and 1 P. damselae ssp. damselae reference strain were subjected to Amplified Fragment Length Polymorphism (AFLP) analysis, and a similarity matrix was produced. Accordingly, the Japanese isolates of P. damselae ssp. piscicida were distinguished from the Mediterranean/European isolates at a cut-off value of 83% similarity. A further subclustering at a cut-off value of 97% allowed discrimination between the Israeli P. damselae ssp. piscicida isolates and the other Mediterranean/European isolates. The combination of PCR direct amplification and AFLP provides a 2-step procedure, where P. damselae is rapidly identified at genus level on the basis of its 16S rRNA gene sequence and then grouped into distinct clusters on the basis of AFLP polymorphisms. The first step of direct amplification is highly sensitive and has immediate practical consequences, offering fish farmers a rapid diagnosis, while the AFLP is more specific and detects intraspecific variation which, in our study, also reflected geographic correspondence. Because of its superior discriminative properties, AFLP can be an important tool for epidemiological and taxonomic studies of this highly homogeneous genus.     

Variation in 16S-23S rRNA intergenic spacer regions in Photobacterium damselae: a mosaic-like structure

Phenotypically, Photobacterium damselae subsp. piscicida and P. damselae subsp. damselae are easily distinguished. However, their 16S rRNA gene sequences are identical, and attempts to discriminate these two subspecies by molecular tools are hampered by their high level of DNA-DNA similarity. The 16S-23S rRNA internal transcribed spacers (ITS) were sequenced in two strains of Photobacterium damselae subsp. piscicida and two strains of P. damselae subsp. damselae to determine the level of molecular diversity in this DNA region. A total of 17 different ITS variants, ranging from 803 to 296 bp were found, some of which were subspecies or strain specific. The largest ITS contained four tRNA genes (tDNAs) coding for tRNA(Glu(UUC)), tRNA(Lys(UUU)), tRNA(Val(UAC)), and tRNA(Ala(GGC)). Five amplicons contained tRNA(Glu(UUC)) combined with two additional tRNA genes, including tRNA(Lys(UUU)), tRNA(Val(UAC)), or tRNA(Ala(UGC)). Five amplicons contained tRNA(Ile(GAU)) and tRNA(Ala(UGC)). Two amplicons contained tRNA(Glu(UUC)) and tRNA(Ala(UGC)). Two different isoacceptor tRNA(Ala) genes (GGC and UGC anticodons) were found. The five smallest amplicons contained no tRNA genes. The tRNA-gene combinations tRNA(Glu(UUC))-tRNA(Val(UAC))-tRNA(Ala(UGC)) and tRNA(Glu(UUC))-tRNA(Ala(UGC)) have not been previously reported in bacterial ITS regions. The number of copies of the ribosomal operon (rrn) in the P. damselae chromosome ranged from at least 9 to 12. For ITS variants coexisting in two strains of different subspecies or in strains of the same subspecies, nucleotide substitution percentages ranged from 0 to 2%. The main source of variation between ITS variants was due to different combinations of DNA sequence blocks, constituting a mosaic-like structure.     

VHH/TLH溶血素基因在海洋弧菌中分布的研究

哈维氏弧菌(V.harveyi)的VHH溶血素是对海水养殖鱼类的潜在致病因子。哈维氏弧菌的VHH溶血素基因与副溶血弧菌(V.parahaemolyticus)的TLH热不稳定性溶血素基因具有高度相似性,其氨基酸序列的相似性达到85.6 %。根据哈维氏弧菌vhhA溶血素基因序列,合成一个地高辛标记的VHH基因探针,利用其进行Southern Blot ,检测VHH溶血素基因在57株弧菌(包括26株国际标准菌株,20株哈维氏弧菌,11株副溶血弧菌)中的分布情况。结果显示,VHH基因探针与13株弧菌标准菌株有强杂交信号,包括2株溶藻胶弧菌(V.alginolyticus) ,2株哈维氏弧菌以及1株霍氏格里蒙菌(Grimontia hollisae) ,坎贝氏弧菌(V.campbellii) ,辛辛那提弧菌(V.cincinatiensis) ,费氏弧菌(V.fischeri) ,拟态弧菌(V.mimicus) ,飘浮弧菌(V.natriegens) ,副溶血弧菌,解蛋白弧菌(V.proteolyticus)和火神弧菌(V.logei)。与6株弧菌标准菌株有弱杂交信号,包括鳗弧菌(V.anguillarum) ,河口弧菌(V.aestuarianus) ,美人鱼发光杆菌(Photobacterium damselae subsp.damselae) ,河弧菌(V.fluvialis) ,弗尼斯弧菌(V.furnissii)和创伤弧菌(V.vulnificus) ,而另外7株弧菌标准菌株中无杂交信号。所有的哈维氏弧菌菌株至少含有一条杂交带,其中菌株VIB645 , VIB 648和SF-1分别含有2条杂交带。11株副溶血弧菌中均含有一条杂交带。上述数据表明,vhh/tlh溶血素基因广泛分布于弧菌中,尤其是哈维氏弧菌相关菌株和费氏弧菌相关菌株中。另外对鳗弧菌VIB 72 ,坎贝氏弧菌VIB 285 ,飘浮弧菌VIB 299和哈维氏弧菌VIB 647的vhh/tlh溶血素基因进行克隆并测序,其氨基酸序列与VHH溶血素和TLH溶血素氨基酸序列的同源性分别为67 %~99 %和69 %~91 %。对vhh/tlh溶血素基因在弧菌中的分布研究,将有助于进一步确定这类溶血素基因在病原弧菌致病性中的作用。     

半滑舌鳎病原菌(发光杆菌杀鱼亚种)的分离与鉴定

2006年夏,山东青岛某渔场养殖半滑舌鳎(Cynoglossus semilaevis Gunther)大量死亡。症状主要表现为体表溃烂,鳍基部出血等,解剖可见胆囊发黑,肾脏发黄。从患病半滑舌鳎胆囊分离出优势菌并命名为WY06。人工感染试验证实WY06对半滑舌鳎及模式动物斑马鱼都具有很强的致病性,其半数致死量分别为5.5×103cfu/克鱼(5.2×105cfu/条鱼)和1.9×103cfu/克鱼(8.9×102cfu/条鱼)。该病原菌革兰氏染色阴性,菌体呈杆状。综合该菌在形态、生理生化特征及16S rDNA同源性等方面的结果,确认WY06为美人鱼发光杆菌杀鱼亚种(Photobacterium damselae subsp.piscicida)。该菌对头孢呋肟、菌必治等抗生素敏感。美人鱼发光杆菌杀鱼亚种在美国、日本、欧洲的海水养殖中为常见的病原菌,但作为鱼类病原菌在中国属首次报道。     

Purification and characterization of an antibacterial protein in the skin secretion of rockfish Sebastes schlegeli

An antibacterial protein in the skin secretion of rockfish (Sebastes schlegeli) was purified by lectin affinity chromatography on Con A-Sepharose and gel filtration on TSKgel G3000SW. The antibacterial protein featured the high molecular mass and selective action against Gram-negative bacteria. The molecular mass of the protein was estimated to be approximately 150 kDa in gel filtration and approximately 75 kDa by SDS-PAGE, suggesting that it is dimeric. The antibacterial principle was an acidic glycoprotein with pI 4.5, 3.4% reducing sugar and 2.8% amino sugar. Its sugar chains had N-type (high mannose-type) oligosaccharide and sialic acid components. It inhibited strongly the growth of Aeromonas salmonicida, Photobacterium damselae and Shewanella putrefaciens with a minimum inhibitory concentration (MIC) of approximately 3 microg/ml, and moderately the growth of Vibrio parahaemolyticus and A. hydrophila with a MIC of 12.5 microg/ml and 25 microg/ml, respectively. The values of the minimum bactericidal concentration were almost equivalent to those of MIC. The potent sensitivity against virulent pathogens such as A. hydrophila, A. salmonicida and P. damselae may contribute considerably to the innate host defense mechanism to combat microbes on the mucosal surfaces of the rockfish.