Immunochemical analyses of Vibrio anguillarum strains isolated from cod, Gadus morhua L., suffering from vibriosis

Abstract. Vibrio anguillarum isolates were sampled from cod fry showing clinical signs of vibriosis at different cod farms along the western coast of Norway. Except for one isolate, all were shown to cause mortality in laboratory challenge experiments. Using biochemical analysis and specific rabbit antisera (O1–O5), all the pathogenic isolates were classified as typical V. anguillarum serotype O2. SDS-PAGE and Western blot analysis of both whole bacterial cells and purified lipopolysaccharides (LPS) showed that the LPS structures were heterogeneous. ELISA analyses using adsorbed antisera identified the presence of two different subgroups within serogroup O2, which correspond with O2a/O2α and O2b/O2β described previously.     

发病鲆鲽类分离菌株的16S rRNA基因测序分析

细菌性疾病是中国海水养殖鲆鲽类的主要病害,为全面了解病原菌种类,本研究对1999~2012年从山东、江苏、河北、天津等沿海地区养殖场发病鲆鲽鱼类中分离得到的124株优势菌株进行了16S rRNA基因测序和系统发育学分析。将基因序列与GenBank核酸序列数据库进行相似度比对分析,结果显示,有83株与弧菌属(Vibriosp.)细菌相似度最高,11株与气单胞菌属(Aeromonas sp.)细菌相似度最高,4株与爱德华氏菌属(Edwardsiella sp.)细菌相似度最高,26株为其他15种属的细菌。根据系统发育学分析结果,进一步将66株菌鉴定为16个种,优势种为溶藻弧菌(V.alginolyticus)、哈氏弧菌(V.harveyi)、鳗弧菌(V.anguillarum)、杀鲑气单胞菌(A.salmonicida)和迟缓爱德华氏菌(E.tarda)。选择其中的9株鳗弧菌和4株迟缓爱德华氏菌进行人工感染实验,结果显示,其中7株鳗弧菌和3株迟缓爱德华氏菌对大菱鲆(Scophthalmus maximus)有较强的致病性。研究结果可为阐明中国海水养殖鲆鲽类的流行病发生历史、病原种类、病原监测及疾病控制提供重要参考。     

Evaluation of the AQUARAPID-Va, AQUAEIA-Va and dot-blot assays for the detection of Vibrio anguillarum in fish tissues

The comparative accuracy of the serological assays AQUARAPID-Va, AQUAEIA-Va (BIONOR AS), and dot-blot for a rapid diagnosis of vibriosis in fish was evaluated. Twenty-one Vibrio anguillarum strains, representative of pathogenic and environmental serotypes, and 13 strains of other fish pathogenic bacteria were used to assess the sensitivity and specificity of the detection methods. The serological assays tested detected all the strains of V. anguillarum serotypes O1 and O2. The dot-blot assay was the most specific and sensitive method, detecting almost all isolates from serotypes O1, O2 and O3, with an average sensitivity of 1 x 10(6) bacteria g(-1) of fish tissue. The AQUARAPID-Va and the AQUAEIA-Va systems were able to detect 5 x 10(6) and 5 x 10(7) bacteria g(-1) of fish tissue, respectively. The simplicity, effectiveness and speed of the AQUARAPID-Va system confirmed this method as the most suitable serological test for the detection of V. anguillarum in field analysis and small-scale laboratory studies.     

Serotyping of Vibrio anguillarum isolated from diseased freshwater fish in Japan

Abstract. During the period from 1965 to 1980, 263 Vibrio anguillarum strains from ayu, Plecoglossiis altivelis (Temminck & Schlegel), two from rainbow trout. Salmo gairdneri Richardson, and two from eel, Anguilla japonica (Temminck & Schlegel), were collected from fish suffering from vibriosis in various parts of Japan. On the basis of cross-agglutination and cross-absorption tests with thermostable (O) antigens, six distinct serotypes (A, B, C, D, E and F) were established among 12 selected strains of V. anguillarum . 241 strains isolated from ayu and two strains from rainbow trout belonged to serotype A, six strains from ayu and one strain from eel to serotype B, 12 strains from ayu to serotype C, three strains from ayu to serotype D, one strain from ayu to serotype E, and one strain from eel to serotype F. V. anguillarum strains belonging to serotypes D, E and F have not been detected from ayu, rainbow trout and eel since 1973; these serotypes appear to be minor types. V. anguillarum strain NCMB 6 and 1669 belong to our serotype A and V. anguillarum 813 to our serotype C.     

Antibody specificities in Atlantic salmon, Salmo salar L., against the fish pathogens Vibrio salmonicida and Vibrio anguillarum

Abstract. Specificities of polyclonal salmon antisera made against the fish pathogens Vibrio salmonicida and Vibrio anguillarum were studied. Using ELISA and Western blot techniques, antisera made against V. salmonicida or V. anguillarum serovar 1 demonstrated high responses against the homologous bacterium or its isolated LPS. In contrast, antisera obtained after immunization with V. anguillarum serovar 2 displayed low antibody titres against homologous antigens. Elcctrophoretic transfer of SDS-PAGE separated V. salmonicida LPS antigen to nitrocellulose strips and subsequent immunostaining with salmon antisera revealed a strong reaction exclusively in the low molecular weight region (<14kD). On the other hand, immunoblots of V. anguillarum LPS preparations using salmon immunesera raised against this species showed a heterogenous staining pattern ranging from high to medium LPS-size. In addition, most of the salmon antisera made against V. anguillarum serovar 2 also reacted with a low molecular weight LPS antigen band.     

Novel non-motile phenotypes of Yersinia ruckeri suggest expansion of the current clonal complex theory

The biochemical and cell surface characteristics of 63 non‐motile isolates of Yersinia ruckeri from various sources were compared using the API 20E rapid identification system and conventional phenotypic methods. Eight individual phenotypic groups from a variety of fish species were observed from the data set. Non‐motile isolates were not exclusively observed from serogroup O1; membership of biotype 2 was recorded for representatives from serogroups O2–O7. Variations in phenotypes highlights that new clonal groups are arising and that the current typing scheme requires expansion. Previously, it was hypothesized that disease was caused by a few virulent clones; data in this paper suggests that this assumption is not the case. The lipopolysaccharide (O antigen) type in the non‐motile biotype was different from other isolates of Y. ruckeri.     

The use of API 20E for the identification of Vibrio anguillarum and V. ordalii

Abstract. Sixty-eight strains of Vibrio anguillarum , five of V. ordalii and the type strains of V. alginolyticus, V. carchariae, V. damsela and V. parahaemolyticus were compared using the API 20E gallery. Within the V. anguillarum strains, distinct groups could be separated mainly on the basis of their reaction on indole production and the fermentation of amygdalin and arabinose. Vibrio ordalii , the former V. anguillarum biotype 2, could easily be separated from V. anguillarum and from the other fish pathogenic Vibrio spp.     

Vaccination experiments and studies of the humoral immune responses in cod, Gadus morhua L., to four strains of monoclonal-defined Vibrio anguillarum

Abstract. Vibrio anguillarum isolated from diseased cod, Gadus morhua L., were serotyped by use of a panel of monoclonal antibodies. Four serotypes could be distinguished, having different lipopolysaccharide determinants. These phenotypic differences were also reflected in the genetic map, as revealed by fingerprinting of bacterial DNA. Antisera were raised in cod after immunization with the V. anguillarum serotypes, and Western blot techniques demonstrated production of specific antibodies mainly to LPS-antigens. The immune system in cod discriminates to a eertain degree between the four serotypes as shown by crossreactions of the immune sera in elisa . Moreover, it was also shown that natural antibodies to bacterial antigens are present in non-immune sera, but these specificities are non-LPS in nature. As a consequence of the heterogeneity of the V. anguillarum strains, vaccination experiments were performed under laboratory conditions to compare the effectiveness of bacterins based on either single vaccines or polyvaccines. The results from these experiments were promising since challenge with one strain demonstrated 100% protection both in fish vaccinated with the homologous serotype as well as a mixture of all the four serotypes.     

Host preference of adult Caligus elongatus Nordmann in the laboratory and its implications for Atlantic cod aquaculture

The sea louse Caligus elongatus utilizes a range of marine fish species as hosts, and it has been suggested that fish farms are infected with adult C. elongatus from wild fish hosts. We assessed experimentally whether dislodged lice return to their original host species. Adult lice from wild lumpfish and saithe were presented to lumpfish, sea trout, Atlantic cod, saithe and European plaice. All fish species became infected. Lumpfish and cod were strongly preferred, followed by saithe, trout and plaice. Lice from wild saithe showed a preference for saithe and lumpfish, followed by cod, sea trout and plaice. The mtCO1 gene was used to determine the genotypes of the lice. Lice from lumpfish were genotype 1. Lice from saithe were mainly genotype 2. The two genotypes of C. elongatus differed slightly in their host preferences: lice from saithe were less discriminating. The data support the hypothesis that adult C. elongatus may transfer between different host species. Cod is an attractive host for lice from both lumpfish and saithe, which suggests that C. elongatus may become a serious pest of farmed cod.     

Application of the rpoS gene for the detection of Vibrio anguillarum in flounder and prawn by polymerase chain reaction

Vibrio anguillarum , an opportunistic fish pathogen, is the main species responsible for vibriosis, a disease that affects feral and farmed fish and shellfish, and causes considerable economic losses in marine aquaculture. In this study, we used polymerase chain reaction (PCR) to detect V. anguillarum . PCR specificity was evaluated by amplifying the rpoS gene, a general stress regulator, in six strains of V. anguillarum and 36 other bacterial species. PCR amplified a species-specific fragment (689 bp) from V. anguillarum . Furthermore, the PCR assay was sensitive enough to detect rpoS expression from 3 pg of genomic DNA , or from six colony-forming units (CFU) mL⁻¹ of cultured V. anguillarum . However, the assay was less sensitive when genomic DNA from the infected flounder and prawn was used (limit of detection, 50 ng and 10 ng g⁻¹ tissue, respectively). These data demonstrate that PCR amplification of the rpoS gene is a sensitive and species-specific method to detect V. anguillarum in practical situations.     

Yersiniosis in Atlantic cod,Gadus morhua (L.), characterization of the infective strain and host reactions

A disease outbreak in farmed Atlantic cod caused by Yersinia ruckeri is reported. Mortality started following vaccination of cod reared in two tanks (A and B). The accumulated mortality reached 1.9% in A and 4.8% in B in the following 30 days when treatment with oxytetracycline was applied. Biochemical and molecular analysis of Y. ruckeri isolates from the cod and other fish species from fresh and marine waters in Iceland revealed a high salinity‐tolerant subgroup of Y. ruckeri serotype O1. Infected fish showed clinical signs comparable with those of Y. ruckeri ‐infected salmonids, with the exception of granuloma formations in infected cod tissues, which is a known response of cod to bacterial infections. Immunohistological examination showed Y. ruckeri antigens in the core of granulomas and the involvement of immune parameters that indicates a strong association between complement and lysozyme killing of bacteria. Experimental infection of cod with a cod isolate induced disease, and the calculated LD₅₀ was 1.7 × 10⁴ CFU per fish. The results suggest that yersiniosis can be spread between populations of freshwater and marine fish. Treatment of infected cod with antibiotic did not eliminate the infection, which can be explained by the immune response of cod producing prolonged granulomatous infection.     

Identification of bacteria from the normal flora of perch, Perca fluviatilis L., and evaluation of their inhibitory potential towards Aeromonas species

Pathogenic Aeromonas sobria has been identified as a causative agent of ulcerative disease in farmed European perch, Perca fluviatilis L. To study the effect of the normal intestinal bacterial flora of perch against A. sobria, we sampled 193 bacterial isolates from the perch digestive tract. The isolates were identified by sequence analysis of the 16S rRNA gene and their inhibitory potential against A. sobria was evaluated in vitro. Nineteen of the strains isolated showed inhibition and were also tested against other aeromonad and non-aeromonad fish pathogens including Yersinia ruckeri and Vibrio anguillarum. Isolates showing inhibition were primarily Pseudomonas spp.; however, inhibitory Shewanella spp., and Delftia sp. were also identified. A Pseudomonas chlororaphis isolate showed inhibition against all fish pathogens tested.     

Identification of pathogenic bacteria associated with the occurrence of 'red spot' in sea mullet, Mugil cephalus L., in south-eastern Queensland

Abstract. An investigation was undertaken to determine the bacterial species associated with the fish disease known as 'red spot' which affected wild fish stocks in south-eastern Queensland. Outbreaks of the disease in the Noosa River were examined with particular reference to the sea mullet, Mugil cephalus. The bacterial flora of the skin of healthy fish as well as those exhibiting early and advanced lesions was determined. In the lower Noosa River estuary, Vibrio anguillarum was the sole organism associated with very early lesions. It was later shown that V. alginolyticus was also associated with V. anguillarum in early lesions, but was considered to be a secondary invader. Aeromonas hydrophila was isolated from advanced lesions on fish taken in freshwater reaches of the river. This organism was also regarded as a secondary invader.     

Surveillance of health status on eight marine rainbow trout, Oncorhynchus mykiss (Walbaum), farms in Denmark in 2006

The health status of eight marine rainbow trout farms was followed from mid-June to mid-September 2006 by sampling both dead and healthy fish approximately every 2 weeks for bacteriological and virological investigation. No fish pathogenic viruses were detected, but all farms experienced disease and mortality as a result of various bacterial infections. Yersinia ruckeri was found on four and Renibacterium salmoninarum on five of the farms, but only during the first part of the surveillance period. This indicates that the fish carried the infection from fresh water, and cleared the infection in salt water. Aeromonas salmonicida subsp. salmonicida caused mortality on five farms, but persisted throughout the sampling period. Although A. salmonicida was probably carried from fresh water, the fish were not able to clear the infection in the sea. Vibrio anguillarum caused mortality on six of the farms throughout the sampling period, O1 being the dominant serovar, and Photobacterium damselae subsp. damselae was found on seven farms as a cause of disease. During the period of highest water temperatures Vibrio parahaemolyticus and Vibrio vulnificus were detected in dead fish in five and two farms, respectively, although their significance as causative pathogens is questionable. Vibrio vulnificus has not previously been found in rainbow trout in Denmark. Both mortality and number of antimicrobial treatments during the period were considerably higher in unvaccinated compared with vaccinated fish. Resistance to commonly used antimicrobials was low or absent.     

舟山崎头洋海域春秋季鱼类种类组成及数量分布

本研究利用2012年5月（春季）、10月（秋季）2个季度月的单船底拖网渔获的鱼类样品,对舟山渔场崎头洋海域鱼类种类组成、区系特点及数量分布等进行了研究。结果如下：（1）共获得鱼类50种,隶属于11目27科,以石首鱼科和缎虎鱼科的种类最多,各有7种,分别占总种数的14．0％;（2）根据适温性可将其划分为暖水性和暖温性2种适温类型,以暖温性为主,有36种,占鱼类总种数的72．0％;（3）春季主要集中在舟山本岛一小干岛海域,而秋季则集中在舟山本岛一小干岛海域以及穿山半岛一六横岛西南海域;（4）春季优势种有六丝矛尾锻虎鱼Chaeturichthys hexanema、带鱼Trichiurus haumela、鲈鱼Lateolabrax japonicus、棘头梅童鱼Collichthys lucidus、龙头鱼Harpodon nehereus等5种,而秋季优势种仅龙头鱼1种;（5）通过分析优势种的种群组成和底层温盐数据,发现该海域在春季可能为带鱼、棘头梅童鱼和鲈鱼等幼鱼的索饵场。     

Production of proteinase during experimental infection of Ostrea edulis L. larvae with Vibrio alginolyticus NCMB 1339 and the antigenic relationship between proteinases produced by marine vibrios pathogenic for fish and shellfish

Abstract. Seven marine Vibrio strains pathogenic for fish or shellfish (including strains of Vibrio alginolyticus, V. anguillarum, V. tubiashi and V. ordalii ) were categorized into three groups on the basis of their extracellular proteinases. Antiserum raised against purified Vibrio alginolyticus NCMB 1339 proteinase neutralized the enzymes produced by all seven Vibrio strains and, on immunodiffusion, the V. alginolyticus proteinase gave a reaction of partial antigenic identity with the other Vibrio strains. In experimental infections of Ostrea edulis larvae with V. alginolyticus , the production of proteinase was demonstrated by immunofluorescence with fluorescein-labelled anti-serum. The toxicity of purified proteinase to larval O. edulis was significantly reduced by preincubation with antiserum but, when larvae were challenged with V. alginolyticus culture supernate containing a similar proteinase concentration, preincubation with antiserum had no protective effect.     

A spatial linkage between dams and non-native fish species in Hokkaido, Japan

Abstract –  The native and non-native freshwater fish species richness as well as the proportion of native fish species to overall fish species were examined to test a hypothesis of a spatial linkage between the distributions of dams and non-native fishes based on survey data collected in rivers and streams in Hokkaido, Japan. Non-native fish species have steadily increased in Hokkaido since the mid 1970s, as indicated by the decreasing proportion of native fish species per fish survey. Non-native species richness was significantly higher in areas above dams (i.e., reservoirs and their inlet streams) compared to areas without dams. As a result, the proportion of native fish species was lower above dams. This proportion was lower in developed and agricultural areas compared with forest areas, although both native and non-native species richness were higher in areas with development and agriculture. Non-native cyprinids, catfish and snakehead occurred primarily in a particular river basin with the largest drainage area, the longest history of reclamation and highest human population in the island. Non-native salmonids on the other hand, occurred in forest areas at higher elevations and their occurrence was associated with the existence of dams. There was a spatial linkage between the distribution of dams and non-native species, although it was applicable only to salmonids. The management of dams and reservoirs needs a strategy to prevent further expansion of exotic salmonids such as rainbow and brown trout. These species can be a significant threat to native Japanese salmonids as a result of various biological mechanisms.     

Growth inhibition of the salmon pathogen Vibrio ordalii by a siderophore produced by Vibrio anguillarum strain VL4355

Abstract. Thirty strains of V. anguillarum were tested for the production of inhibitory substances against closely-related bacteria using the deferred antagonism test. Only one strain, Vibrio anguillarum VL4355, inhibited strains of V. ordalii and this effect was blocked by the addition of iron salts to the culture medium. Siderophore production was investigated for this strain. Results from bioassays suggested that strain VL4355 produced a siderophore related to anguibactin, the plasmid-encoded phenolate siderophore produced by V. anguillarum strain 775. However, when plasmid DNA was compared for strains 775 and VL4355 the Bam HI-generated restriction profiles were different, although hybridization experiments indicated some homology. Using the chrome-azurol sulphate assay to measure siderophore production, strain VL4355 yielded significantly higher values than other V. anguillarum strains. Amberlite XAD-2 was used to produce concentrated siderophore preparations from strains VL4355 and 775. Both preparations were inhibitory to the growth of strains of V. ordalii , but not V. anguillarum , as were solutions of the iron chelator ethylenediamine-di(o-hydroxyphenylacetic acid). The difference in sensitivity to iron-limiting conditions for V. ordalii and V. anguillarum , coupled with the inability of V. ordalii to utilize ferric-anguibactin, could reflect different mechanisms of iron uptake for these two organisms.