The halophilic bacterium Vibrio parahaemolyticus is widely distributed as a natural inhabitant of marine and estuarine environments throughout the world. Many strains are avirulent in humans, but virulent strains are the causative agent of gastroenteritis acquired through consumption of contaminated raw or undercooked seafood. Therefore, it is likely that most V. parahaemolyticus isolates from the marine environment are harmless to humans. Virulent isolates are difficult to differentiate from the majority of avirulent environmental isolates. A total of 55 V. parahaemolyticus isolates (22 clinical, 31 environmental and 2 reference strains) were examined for the carriage of plasmids, whole cell protein profiles; their haemolytic, lipase, phospholipase, protease and urease activities; and the cytotoxicity of their extracellular products (ECPs). Analysis of variance for V. parahaemolyticus isolates showed that Kanagawa haemolysis, phospholipase, protease and urease activities were significantly related to cytotoxicity as assayed by measuring relative extracellular lactate dehydrogenase as an indicator of damage of CHO-K1 cells. The difference between the mean values of cytotoxicity of ECPs of clinical and environmental isolates was significant at a p value of < 0.05.

Vibrio parahaemolyticus is a marine bacterium and some strains cause gastroenteritis in humans. Clinical isolates are thought to possess virulence factors that are absent from the majority of environmental isolates. Use of randomly amplified polymorphic DNA (RAPD)-PCR produced a unique 600 bp amplicon (band Y) in the majority of clinical isolates and rarely in environmental isolates tested. The DNA from band Y was cloned and sequenced and found to code for an outer membrane protein (OMP). Two polymerase chain reaction (PCR) primers were designed to specifically amplify a 200 bp unique sequence from presumptive virulent strains (PCR-OMP). The virulence of 23 clinical and 32 environmental isolates was assessed in cytotoxicity tests by treatment of Caco-2 cells with extracellular products (ECPs). All but two of the clinical isolates (91%) were positive for the 200 bp PCR-OMP and their ECPs produced a significantly higher ( p < 0.05) lactate dehydrogenase (LDH) release (mean 72.88%) than the ECPs of environmental isolates (mean 15.3%) with the exception of one environmental isolate that produced the 200 bp amplicon. A positive 200 bp PCR-OMP is strongly correlated with virulence, as determined by the cytotoxicity assay, and identified virulent forms better than current PCR tests for tdh, trh or T3SS2.

During infection, Renibacterium salmoninarum survives within the pronephric macrophages of salmonid fish. Therefore, to study the initial phases of the interaction we infected macrophages with live bacteria and analysed the responses of host and pathogen. It was found that the expression of msa encoding the p57 antigen of R. salmoninarum , was constitutive, while the expression of hly and rsh , encoding haemolysins, and lysB and grp was reduced after infection. Macrophages showed a rapid inflammatory response in which the expression of interleukin-1beta (IL-1beta), major histocompatibility complex class II (MHC II), inducible cyclo-oxygenase (Cox-2), and inducible nitric oxide synthase (iNOS) was enhanced, but tumour necrosis factor-alpha (TNF-alpha) expression was greatly reduced initially and then increased. After 5 days, except for TNF-alpha and MHC II, expression returned to levels approaching those of uninfected macrophages. We propose that R. salmoninarum survives initial contact with macrophages by avoiding and/or interfering with TNF-alpha-dependent killing pathways. The effects of specific R. salmoninarum components were studied in vivo by injecting fish with DNA vaccine constructs expressing msa , hly , rsh , lysB , or grp . We found that msa reduced the expression of IL-1beta, Cox-2, and MHC II but stimulated TNF-alpha while hly , rsh and grp stimulated MHC II but down-regulated TNF-alpha. Constructs expressing hly or lysB stimulated iNOS expression and additionally, lysB stimulated TNF-alpha. The results show how p57 suppresses the host immune system and suggest that the immune mechanisms for the containment of R. salmoninarum infections rely on MHC II- and TNF-alpha-dependent pathways. Moreover, prolonged stimulation of TNF-alpha may contribute to the chronic inflammatory pathology of bacterial kidney disease.

Streptococcus phocae subsp. salmonis is a Gram-positive bacterium that causes mortality only in Atlantic salmon (Salmo salar) farmed in Chile, even when this species is co-cultured with rainbow trout (Oncorhynchus mykiss). This susceptibility could be determined by innate immune response components and their responses to bacterial infection. This fish pathogen shares subspecies status with Streptococcus phocae subsp. phocae isolated from seals. The present study compared innate immune system mechanisms in Atlantic salmon and rainbow trout when challenged with different S. phocae, including two isolates from Atlantic salmon (LM-08-Sp and LM-13-Sp) and two from seal (ATCC 51973T and P23). Streptococcus phocae growth was evaluated in the mucus and serum of both species, with rainbow trout samples evidencing inhibitory effects. Lysozyme activity supported this observation, with significantly higher (p < 0.01) expression in rainbow trout serum and mucus as compared to Atlantic salmon. No differences were found in phagocytic capacity between fish species when stimulated with ATCC 51973T and P23. Against all S. phocae strains, rainbow trout and Atlantic salmon showed up to two-fold increased bactericidal activity, and rainbow trout demonstrated up to three-fold greater reactive oxygen species production in macrophages. In conclusion, the non-specific humoral and cellular barriers of Atlantic salmon were immunologically insufficient against S. phocae subsp. salmonis, thereby facilitating streptococcosis. Moreover, the more robust response of rainbow trout to S. phocae could not be attributed to any specific component of the innate immune system, but was rather the consequence of a combined response by the evaluated components.

ABSTRACT A means for distinguishing between clinical isolates of Renibacterium salmoninarum that is based on the PCR amplification of length polymorphisms in the tRNA intergenic spacer regions (tDNA-ILPs) was investigated. The method used primers specific to nucleotide sequences of R. salmoninarum tRNA genes and tRNA intergenic spacer regions that had been generated by using consensus tRNA gene primers. Twenty-one PCR products were sequenced from five isolates of R. salmoninarum from the United States, England, and Scotland, and four complete tRNA genes and spacer regions were identified. Sixteen specific PCR primers were designed and tested singly and in all possible pairwise combinations for their potential to discriminate between isolates from recent clinical outbreaks of bacterial kidney disease (BKD) in the United Kingdom. Fourteen of the isolates were cultured from kidney samples taken from fish displaying clinical signs of BKD on five farms, and some of the isolates came from the same farm and at the same time. The tDNA-ILP profiles separated 22 clinical isolates into nine groups and highlighted that some farms may have had more than one source of infection. The grouping of isolates improved on the discriminatory power of previously reported typing methods based on randomly amplified polymorphic DNA analysis and restriction fragment length profiles developed using insertion sequence IS 994 . Our method enabled us to make divisions between closely related clinical isolates of R. salmoninarum that have identical exact tandem repeat (ETR-A) loci, rRNA intergenic spacer sequences, and IS 994 profiles.

The molecular diversity among 60 isolates of Renibacterium salmoninarum which differ in place and date of isolation was investigated by using randomly amplified polymorphic DNA (RAPD) analysis. Isolates were grouped into 21 banding patterns which did not reflect the biological source. Four 16S-23S rRNA intergenic spacer (ITS1) sequence variations and two alleles of an exact tandem repeat locus, ETR-A, were the bases for formation of distinct groups within the RAPD clusters. This study provides evidence that the most common ITS1 sequence variant, SV1, possesses two copies of a 51-bp repeat unit at ETR-A and has been widely dispersed among countries which are associated with mainstream intensive salmonid culture.

ABSTRACT Renibacterium salmoninarum is a genospecies that is an obligate pathogen of salmonid fish and is capable of intracellular survival. Conventional typing systems have failed to differentiate isolates of R. salmoninarum . We used two methods to assess the extent of molecular variation which was present in isolates from different geographic locations. In one analysis we investigated possible polymorphisms in a specific region of the genome, the intergenic spacer (ITS) region between the 16S and 23S rRNA genes. In the other analysis we analyzed differences throughout the genome by using randomly amplified polymorphic DNA (RAPD). We amplified the spacer region of 74 isolates by using PCR and performed a DNA sequence analysis with 14 geographically distinct samples. The results showed that the 16S-23S ribosomal DNA spacer region of R. salmoninarum is highly conserved and suggested that only a single copy of the rRNA operon is present in this slowly growing pathogen. DNA sequencing of the spacer region showed that it was the same length in all 14 isolates examined, and the same nucleotide sequence, sequevar 1, was obtained for 11 of these isolates. Two other sequevars were found. No tRNA genes were found. We found that RAPD analysis allows reproducible differentiation between isolates of R. salmoninarum obtained from different hosts and different geographic regions. By using RAPD analysis it was possible to differentiate between isolates with identical ITS sequences.

A cell culture system, employing the fish cell line Epithelioma papillosum cyprini (EPC), was developed to study the synthesis of intracellular antigen and the expression of putative virulence factors by Renibacterium salmoninarum. EPC cultures infected with R. salmoninarum could be maintained for 7 weeks, during which the pathogen multiplied intracellularly. Immunohistochemical examination of infected cultures revealed the production of the p57 antigen, haemolysin and cytolysin. The intracellular nature of the infection was confirmed by transmission electron microscopic examination of EPC monolayers. A comparison of the relative virulence of bacterial cells cultured in EPC cells and on agar plates revealed that the former were markedly more virulent in challenge experiments with juvenile rainbow trout ( Oncorhynchus mykiss Walbaum). The EPC cell culture model provided a system for the study of R. salmoninarum under more natural conditions than those achieved with plate culture techniques.

A DNA fragment containing all but the first 90 base pairs of gene msa encoding p57, the major soluble antigen of Renibacterium salmoninarum, was cloned in the plasmid vector pUC18 and subsequently a soluble fusion protein was produced using the pMAL expression vector system. The fusion protein retained major epitopes shared with the native p57 molecule and provided a source of protein suitable for further immunological analysis which was independent of in vitro cultures of this slow growing organism. Antiserum raised against the purified fusion protein was used to probe Western blots of cell extracts and extracellular products derived from R. salmoninarum cultured in vitro. The results show that under conditions of iron-restriction, both the production and processing of p57 are reduced.

Injection of extracts derived from adult caligid copepods induced a partial immunity to Lepeophtheirus salmonis in Atlantic salmon. Antigens were derived from the supernatant of adult lice extracts and were partially purified by Con A affinity chromatography. The antigens were also present in the pellet derived from adult lice extracts, Immunohistochemistry showed that antibodies present in the serum of rats immunized with the Con A purified extract bound predominantly to the gut of L. salmonis. Components of apparent sizes > 205 000, 165 000, 133 000, 130 000, 125 000, 114 000, 110 000, 96 000, 82 000, 78 000, 65 000, 46 000, 35 000, 31 000 and < 29 000 were present in the extract. Although the numbers of attached lice were initially higher on those fish in the immunized group, throughout the course of the challenge experiment the total numbers of lice did not vary greatly between the immunized or control groups of fish. Initially, an overall average of about 17 attached stages were recorded from each fish sampled and by the end of the experiment this figure had fallen to about five adult lice per fish. However, compared with control fish fewer gravid female lice were present on immunized fish and furthermore these lice possessed fewer eggs (P<0.01). No major differences in egg hatching success were recorded. Further exploitation of this would require the isolation and purification of the antigens responsible for the observed effects. The possibilities for the development of a salmon louse vaccine are promising.

Gene rsh encoding a novel hemolysin of Renibacterium salmoninarum was fused to the lacZ gene of the pAX5+ plasmid vector to facilitate production of a 160 kDa beta-galactosidase fusion protein. The soluble fusion protein was produced cytoplasmically in Escherichia coli XL1-Blue and purified using affinity chromatography. The fusion protein retained epitopes which were identified in components present in in vitro cultures of R. salmoninarum. Western blots of R. salmoninarum cell extracts which were probed with an antiserum raised against the affinity purified fusion protein predominantly recognized bands of apparent molecular weights 82 and 78 kDa as well as a number of other bands which may represent breakdown products of the native protein. No components were detected in extracellular products and iron-restricted culture conditions did not obviously affect the production of these components.

Renibacterium salmoninarum is the etiologic agent of bacterial kidney disease in salmonid fish. The virulence of R. salmoninarum has been associated with hydrophobicity although little is known of the means by which this organism acquires iron. In this study, 7 strains of R. salmoninarum were grown in iron-restricted media. Siderophore production was not detected by chemical assays. The growth of all strains, but particularly non-hydrophobic strains, was considerably reduced in the presence of the high-affinity iron chelators ethylenediamine di(o-hydroxyphenylacetic acid) or alpha,alpha'-dipyridyl or when cultured in Chelex-treated medium. Culture supernatant from both hydrophobic and nonhydrophobic strains was found to inhibit the binding of iron by bovine transferrin and this inhibition was most pronounced in supernatants derived from iron-sufficient cultures. A strong iron reductase activity was detected in R. salmoninarum cells. Maximum reductase activity occurred under iron-restricted culture conditions with reduced beta nicotinamide adenine dinucleotide as reductant. The results suggest that iron reductase is an important component of the iron acquisition mechanism of R. salmoninarum.

A genomic library constructed from Renibacterium salmoninarum isolate MT444 DNA in the plasmid vector pBR328 was screened using Escherichia coli host strain DH1 for the expression of genes encoding putative virulence factors. A single haemolytic clone was isolated at 22 C and found to contain a 31 kb Hindlll fragment of inserted DNA. This fragment was present in seven isolates of R. salmoninarum which were examined. Western blots of extracts from clones exhibiting haemolytic activity were performed with antisera raised against either cellular or extracellular components of R. salmoninarum and failed to identify any additional proteins compared to control E. coli containing pBR328. However, minicell analysis revealed that a polypeptide with an apparent molecular mass of 65 kDa was associated with a haemolytic activity distinct from that previously described for R. salmoninarum. The nucleotide sequence of the gene encoding this product was determined and the amino acid sequence deduced. The product was 548 amino acids with a predicted molecular mass of 66757 Da and a pl of 557. The deduced amino acid sequence of the gene possessed strong similarities to those of a range of secreted bacterial zinc-metalloproteases and was tentatively designated hly. Neither protease nor lecithinase activities were detectable in E. coli recombinants expressing gene hly. Haemolytic activity was observed from 6 C to 37 C for erythrocytes from a number of mammalian species and also from fish. Gene hly was expressed in E. coli as a fusion protein consisting of maltose-binding protein at the N-terminus linked to all but the first 24 amino acids, largely constituting the putative signal peptide, of the N-terminus of Hly. The soluble fusion protein was produced and purified by affinity chromatography. Antiserum raised against the purified fusion protein was used to probe Western blots of cell lysates and extracellular products from seven isolates of R. salmoninarum cultured under conditions of iron-sufficiency or iron-restriction. The results indicate that the availability of iron modulates the expression of the hly gene.

Bacterial kidney disease (BKD) is one of the most important diseases of wild and cultured salmonid fish, and has been reported from many different countries. Despite considerable effort, many pieces are still missing from the “jigsaw puzzle” which represents our knowledge of the disease, and its etiological agent Renibacterium salmoninarum. The purpose of this review is to consider current knowledge about this bacterial pathogen and the pathogenesis of BKD. It is our intention to construct a picture of the possible ways in which pathogen and host interact, in particular by high-lighting the areas where our understanding is poor and indicating how recent advances in methodology offer the prospect of improvement. The exact status of R. salmoninarum as an obligate fish pathogen, and the implications for transmission, entry, colonization, and disease progression are considered. We review the considerable progress that has been made recently through work on the molecular determinants of pathogenicity, particularly attachment, cellular invasion mechanisms, intracellular survival of the bacterium, and its interactions with the host defense mechanisms and immune system. Finally, we consider ways in which the future control of BKD through improved diagnosis, chemotherapy, and vaccination may be realized through an integrated approach to the study of BKD.

Strains of the bacterial fish pathogen Yersinia ruckeri were identified with the API 20E system and distinguished on the basis of whole cell agglutination with antisera, sorbitol fermentation and polymyxin B sensitivity. Strains which were shown to possess the virulence-associated heat-sensitive factor (HSF) were shown to grow preferentially on culture media containing sodium dodecyl sulphate (SDS) and to produce a creamy deposit around the colonies. By contrast, strains lacking this factor (HSF-) grew poorly and without forming a deposit. Enhancement of the differentiation between the two types was shown by the incorporation of Coomassie brilliant blue dye into agar containing 1 % SDS, and the uptake of Coomassie blue and Congo red was shown to be temperature-dependent. Most strains tested were shown to belong to serotype I, and were sensitive to polymyxin and did not ferment sorbitol. With the medium developed most serotype I strains but not those of other serotypes were shown to possess HSF. It is suggested that the medium is used in epidemiological studies of Y. ruckeri.

A gene encoding haemolytic activity from Renibacterium salmoninarum (strain PPD) was cloned into Escherichia coli using the cosmid vector pHC79, and subsequently subcloned on a 1.6 kbp SAlI fragment into pBR328. Southern blot hybridisation revealed that a homologous sequence is found in other strains of R. salmoninarum.