Enhanced growth and decreased mortality in Atlantic salmon (Salmo salar) after probiotic bath.

AIMS: Disease in farmed Atlantic salmon occurs in all its life stages. Salmon are particularly vulnerable to infectious diseases at transition from the freshwater stage to the saltwater stage. Our aim in these studies reported was to investigate the possibility that waterborne delivery of a probiotic comprised of naturally occurring marine bacterial species would reduce the mortality and improve the health and growth of farmed Atlantic salmon. METHODS AND RESULTS: In three trials at two aquaculture production sites in Norway, isolates of Aliivibrio bacteria were added to the rearing water of Atlantic salmon. The fish were followed in 4-6 months after one single bath with observations and samplings. Growth, ulcers and survival were recorded. At the end of the studies growth was up to 31% larger in the probiotic enhanced groups and in trial 1 both mortality and prevalence of ulcer were significantly lower in the probiotic enhanced group compared to the control. Feed conversion rates were recorded in trial 1 and 2 and were from 9 to 28 % better for the probiotic enhanced groups compared to the control groups. CONCLUSION: Bathing of Atlantic salmon with probiotic Aliivibrio strains increased growth, reduced mortality and improved FCR in the postsmolt period. SIGNIFICANCE AND IMPACT OF THE STUDY: The study demonstrates the potential to enhance growth, prevent ulcers and decrease mortality in Atlantic salmon after adding probiotic strains of Aliivibrio spp. into the rearing water. The study can have impact on animal welfare, economy and sustainability in the aquaculture industry.

The future of therapeutic agents in aquaculture.

The implementation and monitoring of fish health regulations vary extensively in aquaculture throughout the world. In the main salmon-producing countries, there is strict regulatory oversight of the use of pharmaceutical drugs. Such controls have supported the sustainable growth of the aquaculture industry and, in Norway, aquaculture has been able to reduce its total consumption of antibiotics by more than 99% between 1995 and today, yet there has been a 20-fold rise in production volume. Other countries on other continents may have less control, with no mandatory prescription regulations and variable quality of the pharmaceutical products available. A good regulatory framework, with control and monitoring systems, should be established in all countries where aquaculture is practised and veterinary medicinal products should only be available under veterinary prescription and supervision. Many drug resistance genes have been identified, and molecular methods should be applied to control drug resistance in the microbial and parasitic populations of all major aquaculture-producing regions.

Les niveaux de mise en place et de suivi des réglementations applicables à la santé des poissons en aquaculture sont extrêmement variables d'une région à l'autre. Les pays où la salmoniculture prédomine pratiquent un contrôle réglementaire strict de l'utilisation de produits médicamenteux. Ces contrôles ont permis une croissance durable du secteur ; ainsi, en Norvège l'aquaculture a pu réduire sa consommation totale d'antibiotiques de plus de 99 % depuis 1995 tout en multipliant par vingt le volume de production. Le niveau des contrôles est parfois moindre dans d'autres pays d'autres continents, où aucune réglementation n'impose l'obligation d'une prescription vétérinaire et où les produits pharmaceutiques disponibles sont de qualité variable. Tous les pays ayant une production aquacole devraient mettre en place un cadre réglementaire solide doté de systèmes de contrôle et de suivi ; par ailleurs, les produits pharmaceutiques vétérinaires ne devraient pouvoir être obtenus que sous prescription et supervision vétérinaires. Plusieurs gènes responsables de résistances aux agents médicamenteux ont été identifiés ; il conviendrait que toutes les régions dédiées à l'aquaculture recourent à des méthodes moléculaires pour contrôler le phénomène de la résistance aux agents médicamenteux dans les populations de microbes et de parasites.

La aplicación de reglamentos de salud piscícola y las actividades de control reglamentario en la acuicultura varían sobremanera según el lugar del mundo de que se trate. En los principales países productores de salmón, el uso de productos farmacéuticos está sujeto a una estricta supervisión reglamentaria. Estos controles han favorecido el crecimiento sostenible del sector de la acuicultura, sector que en Noruega ha logrado reducir su consumo total de antibióticos en más de un 99% entre 1995 y la actualidad, pese a haber multiplicado por veinte su producción. En otros países de otros continentes hay a veces un menor control, debido a la ausencia de reglamentos de carácter prescriptivo y a la irregular calidad de los productos farmacéuticos disponibles. Sería menester que todos los países donde se practica la acuicultura contaran con un buen ordenamiento reglamentario, dotado de sistemas de control y seguimiento, y que en ellos solo se pudieran obtener productos de uso veterinario con receta y bajo supervisión veterinarias. Sabiendo que se han encontrado muchos genes que determinan resistencia a los medicamentos, convendría aplicar métodos moleculares para controlar la aparición de farmacorresistencias en las poblaciones microbianas y parasitarias de todas las regiones que albergan un importante sector acuícola.

Zinc oxide enriched peat influence Escherichia coli infection related diarrhea, growth rates, serum and tissue zinc levels in Norwegian piglets around weaning: five case herd trials.

BACKGROUND: Zinc oxide (ZnO), commonly used to control post-weaning diarrhea in piglets, has been highlighted as of potential concern from an environmental perspective. The aim of this field trial was to examine effects of different sources and levels of ZnO added to peat on average daily weight gain (ADG), fecal score in pens and serum and tissue zinc (Zn) levels around time of weaning in order to reduce the environmental impact without loss of the beneficial effect of ZnO on intestinal health and growth. Five case herds with enterotoxic colibacillosis challenges were included. The piglets entered the study aged three or five weeks. All piglets received a commercial diet containing <150 mg Zn/ per kg of complete feed. Four treatment groups received commercial peat added A: uncoated ZnO, B: lipid microencapsulated ZnO, C: solely commercial peat or D: no peat (Farms 2 and 3). RESULTS: At Farms 1, 2 and 3, a significant effect of treatment was identified for fecal score (P < 0.05). Treatment A led to lower fecal scores compared to treatments C (P < 0.05) and D (P < 0.01). At Farms 2 and 3, there was a significant difference in individual average daily weight gain (iADG) between treatment A and D (P < 0.05). The iADG of piglets receiving treatment B did not differ significantly from treatment A. CONCLUSIONS: In 2016, The European Medicines Agency's Committee on Veterinary Medicinal Products concluded that the benefits of ZnO for the prevention of diarrhea in pigs do not outweigh the risks to the environment. Effective alternative measures to reduce the accumulation of Zn in the environment have not been identified. Our results imply that peat added low concentration of both coated and uncoated ZnO influences the gut health of weaned piglets reflected by enhanced weight gain and reduced occurrence of diarrhea. This preventive approach certainly represents a favourable alternative in the "One Health" perspective. It will also contribute to reduced antibiotic use in pig farming while diminishing the environmental consequences caused by ZnO.

The environmental and host-associated bacterial microbiota of Arctic seawater-farmed Atlantic salmon with ulcerative disorders.

The Norwegian aquaculture of Atlantic salmon (Salmo salar L.) is hampered by ulcerative disorders associated with bacterial infections. Chronic ulceration may provide microenvironments that disturb the normal microbial biodiversity of external surfaces. Studying the composition of microbial communities in skin ulcers will enhance our understanding of ulcer aetiology. To achieve this, we tested marine farmed Atlantic salmon and sampled the base and edge of ulcers at the end of winter (April) and end of summer (September), in addition to skin mucus of healthy individuals. In order to assess microbiota associated with the host and obtain insight into the environmental ecology, we also sampled sea water, the sediment layer underneath the farm facility and the distal intestine of Atlantic salmon. The skin microbiota of Atlantic salmon was different from that of the surrounding water. Residential Tenacibaculum and Arcobacter species persistently dominated the cutaneous skin and ulcer mucus surfaces of Atlantic salmon during both winter and summer periods. The intestinal microbiota was dominated by Mycoplasma with an increase in Aliivibrio and Alcaligenes abundance in the intestine of fish with ulcerative disorder at the end of winter. These findings suggest the presence of resilient microbes in the mucus surfaces of Atlantic salmon.

Cold-water vibriosis. The current status of knowledge.

The current review for the first time summarizes the findings of the 30 years of research on cold-water vibriosis (CWV). The diseased caused by Aliivibrio salmonicida (earlier known as Vibrio salmonicida) was for the first time described in 1986 and became one of the most important bacterial diseases in salmon aquaculture. The lack of appropriate vaccine hampered development of Atlantic salmon aquaculture until the late 1980s when a novel vaccine allowed dramatic increase in the Atlantic salmon farming. In December 2007, the genus Vibrio was split into two genera and several bacterial species including V. salmonicida were transferred to genus Aliivibrio. The change of the names create significant difficulties with the designation of the CWV disease agent since its abbreviation A. salmonicida became similar to another well-known salmon pathogen Aeromonas salmonicida (A. salmonicida). The disease was considered as controlled by vaccination, but reappeared at Atlantic salmon farms in 2011, this time affecting vaccinated Atlantic salmon. The current review summarizes the knowledge on pathogenesis, vaccination and treatment of CWV and proposes further directions for studying the disease.

vapA (A-layer) typing differentiates Aeromonas salmonicida subspecies and identifies a number of previously undescribed subtypes.

Sequence variation in a region of the virulence array protein gene (vapA; A-layer) was assessed in 333 ('typical' and 'atypical') isolates of the fish pathogenic bacterium Aeromonas salmonicida. Resulting similarity dendrograms revealed extensive heterogeneity, with nearly all isolates belonging to either of 14 distinct clusters or A-layer types. All acknowledged A. salmonicida subspecies (except ssp. pectinolytica, from which no vapA sequence could be obtained) were clearly separated, and notably, all isolates phenotypically identified as ssp. salmonicida formed a distinct and exclusive A-layer type. Additionally, an array of un-subspeciated atypical strains formed several equally prominent clusters, demonstrating that the concept of typical/atypical A. salmonicida is inappropriate for describing the high degree of diversity evidently occurring outside ssp. salmonicida. Most representatives assessed in this study were clinical isolates of spatiotemporally diverse origins, and were derived from a variety of hosts. We observed that from several fish species or families, isolates predominantly belonged to certain A-layer types, possibly indicating a need for host-/A-layer type-specific A. salmonicida vaccines. All in all, A-layer typing shows promise as an inexpensive and rapid means of unambiguously distinguishing clinically relevant A. salmonicida subspecies, as well as presently un-subspeciated atypical strains.

Genetic localization of a TetR-like transcriptional regulator gene in Pseudomonas fluorescens isolated from farmed fish.

Pseudomonas fluorescens isolates from Tanzanian tilapia ponds were found to possess a gene encoding a TetR-like transcriptional regulator protein. Phylogenetic analysis revealed close similarity to five previously reported GeneBank sequences which cluster separately from the other 70 members of this family. It is assumed that this TetR-like protein belongs to a new family of TetR-like proteins that has no direct link to the class 1 integron.

Multilocus variable number tandem repeat analysis of Edwardsiella piscicida isolates pathogenic to fish.

This study describes a novel multilocus variable number tandem repeat analysis (MLVA) based on six variable number of tandem repeat (VNTR) loci for genotyping of 37 Edwardsiella piscicida (previously Edwardsiella tarda) isolates from multiple sources. The number of alleles identified for each of the six VNTR loci ranged from 3 to 5 with VNTR loci 1 (DI = 0.632) and 3 (DI = 0.644), displaying the highest degrees of polymorphism. MLVA typing of the 37 E. piscicida isolates resulted in the identification of five major clusters consistent with their geographical origins, and were designated as MLVA types I, II, III, IV and V. Types III and V were resolved further into subtypes largely consistent with outbreak source. An MLVA profile comprising a string of integers representing the number of tandem repeats for each allele provided a unique identification for each MLVA type and/or strain. The MLVA protocol described in the current study is robust, relatively simple, has a higher power of resolution than multilocus sequence analysis (MLSA) and is capable of discriminating closely related isolates.

A brief multi-disciplinary review on antimicrobial resistance in medicine and its linkage to the global environmental microbiota.

The discovery and introduction of antimicrobial agents to clinical medicine was one of the greatest medical triumphs of the 20th century that revolutionized the treatment of bacterial infections. However, the gradual emergence of populations of antimicrobial-resistant pathogenic bacteria resulting from use, misuse, and abuse of antimicrobials has today become a major global health concern. Antimicrobial resistance (AMR) genes have been suggested to originate from environmental bacteria, as clinically relevant resistance genes have been detected on the chromosome of environmental bacteria. As only a few new antimicrobials have been developed in the last decade, the further evolution of resistance poses a serious threat to public health. Urgent measures are required not only to minimize the use of antimicrobials for prophylactic and therapeutic purposes but also to look for alternative strategies for the control of bacterial infections. This review examines the global picture of antimicrobial resistance, factors that favor its spread, strategies, and limitations for its control and the need for continuous training of all stake-holders i.e., medical, veterinary, public health, and other relevant professionals as well as human consumers, in the appropriate use of antimicrobial drugs.

Edwardsiella piscicida sp. nov., a novel species pathogenic to fish.

AIMS: This study describes a novel species within the genus Edwardsiella based on phenotypic and genetic characterization of fish pathogenic Edwardsiella isolates previously identified as E. tarda. METHODS AND RESULTS: Phenotypic characterization, DNA-DNA hybridization and phylogenetic analysis of representative Edwardsiella isolates from fish previously identified as E. tarda were conducted and compared with E. tarda type strain (ATCC 15947(T)). Phenotypically, strains from fish grow with pin-point colonies producing slight ß-haemolysis under the colony. In contrast to the E. tarda type strain, fish strains did not [corrected] degrade ß-methyl-D-glucoside [corrected] (with the exception of NCIMB 2034), citric acid and L-proline. [corrected]. With the exception of strain ETK01, all fish strains were highly pathogenic to zebra fish, while ATCC 15947(T) and NCIMB 2034 were nonpathogenic. DNA-DNA hybridization (DDH) levels between representative fish isolates and the E. tarda type strain ranged from 15 to 43·6%, while NCIMB 2034 hybridised with the type strain at the level of 63·2%. DDH values between the various fish isolates ranged from 68·2 to 93·9% defining a new and separate DNA hybridization group differing from the E. tarda type strain consistent with the findings of phylogenetic analysis, in which the fish isolates comprised a separate clade. CONCLUSIONS: Phenotypical and genetic characterizations demonstrated that Edwardsiella isolates from fish described in this study do not belong to the species E. tarda or any of the previously established taxa within the genus Edwardsiella. The fish related strains studied here (excluding NCIMB 2034) represent, therefore, a novel species within the genus Edwardsiella for which we propose the name Edwardsiella piscicida sp. nov, with strain ET883(T) (NCIMB 14824(T) = CCUG 62929) as the type strain. SIGNIFICANCE AND IMPACT OF THE STUDY: The current finding will improve the diagnosis, understanding of the epidemiology and in establishment of effective control measures against this serious fish pathogen.

A novel gold nanoparticles-based assay for rapid detection of Melissococcus plutonius, the causative agent of European foulbrood.

European foulbrood (EFB) is a severe bacterial brood disease of honey bees (Apis mellifera) caused by Melissococcus plutonius. Diagnosis of EFB in the field is based on visual inspection of brood-combs and detection of diseased larvae. However, symptoms of EFB may be easily obscured by other diseases or abnormalities in the brood, making definitive diagnosis difficult. Hence, confirmatory laboratory assays, such as PCR and real-time PCR, are used to verify the presence of M plutonius in suspected colonies. While these methods are accurate and specific, they are time consuming and labour intensive. Herein, we report development of a label-free colorimetric nanodiagnostic method for direct detection of unamplified M plutonius DNA using unmodified gold nanoparticles. Under appropriate conditions, the DNA probes hybridised with their complementary target sequences in the sample DNA, which resulted in aggregation of the gold nanoparticles and a concomitant red to blue colour change, which was observed visually. The assay could detect as few as 25 copies of the M plutonius cell wall-associated protease gene within 20 minutes. The assay results were in 100 per cent concordance with real-time PCR-positive and PCR-negative samples. Our study demonstrated that the gold nanoparticles-based assay is a specific and sensitive tool for rapid detection of M plutonius.

Feeding Jerusalem artichoke reduced skatole level and changed intestinal microbiota in the gut of entire male pigs.

Different levels of dried Jerusalem artichoke were fed to entire male pigs 1 week before slaughter. The objective was to investigate the effect on skatole level in the hindgut and in adipose tissue, as well as the effect on microflora and short-chain fatty acids (SCFA) in the hindgut. Five experimental groups (n = 11) were given different dietary treatments 7 days before slaughtering: negative control (basal diet), positive control (basal diet + 9% chicory-inulin), basal diet + 4.1% Jerusalem artichoke, basal diet + 8.1% Jerusalem artichoke and basal diet + 12.2% Jerusalem artichoke. Samples from colon, rectum, faeces and adipose tissue were collected. Effect of dietary treatment on skatole, indole and androstenone levels in adipose tissue and on skatole, indole, pH, dry matter (DM), microbiota and SCFA in the hindgut was evaluated. Feeding increasing levels of Jerusalem artichoke to entire male pigs reduced skatole in digesta from colon and in faeces (linear, P < 0.01). There was also a tendency towards a decreased level of skatole in adipose tissue (linear, P = 0.06). Feeding Jerusalem artichoke decreased DM content in colon and faeces and pH in colon (linear, P < 0.01). Increasing levels of Jerusalem artichoke resulted in a reduced level of Clostridium perfringens in both colon and rectum (linear, P < 0.05) and a tendency towards decreased levels of enterobacteria in colon (linear, P = 0.05). Further, there was an increase in total amount of SCFA (linear, P < 0.05), acetic acid (linear, P < 0.05) and valerianic acid (linear, P < 0.01) in faeces. In conclusion, adding dried Jerusalem artichoke to diets for entire male pigs 1 week before slaughter resulted in a dose-dependent decrease in skatole levels in the hindgut and adipose tissue. The reduced skatole levels might be related to the decrease in C. perfringens and the increase in SCFA with subsequent reduction in pH.

Multi-locus Sequence Analysis (MLSA) of Edwardsiella tarda isolates from fish.

Edwardsiella tarda is an enteric fish pathogen that has caused significant economic losses in a range of fish species residing in diverse ecological conditions. Several molecular methods relying on DNA fingerprinting (RAPD, RFLP and ERIC-PCR) and the gyrB gene marker have been used to characterize E. tarda isolates. However, all had drawbacks in resolving power and reproducibility. The present study was aimed at developing a novel Multi-locus Sequence Analysis (MLSA) scheme for genetic characterization of E. tarda isolates originating from multiple sources. MLSA has been described as an effective molecular tool with superior discriminatory power and reproducibility for exploring intra-species genetic diversity of several bacterial species. Nucleotide sequence fragments of eight protein coding housekeeping genes (gyrB, mdh, adk, dnaK, phoR, metG, pyrG and aroE2) were obtained from 23 fish pathogenic E. tarda isolates of different geographical origins, one human isolate and 3 reference strains. The phylogenetic relationships between isolates in individual gene analyses were not consistent, although some common patterns were apparent. Phylogenetic analysis based on concatenated sequences of seven gene loci, however, buffered the conflicting phylogenetic signals and resolved isolates according to their geographical origin and/or fish host. The MLSA revealed two major genetically diverging clusters in E. tarda isolates examined, one cluster representing isolates from fish and the other representing (in the main) human isolates, with E. ictaluri cluster situated in between. The results suggest, therefore, that the fish pathogenic E. tarda isolates may have been previously misclassified and probably represent one or more as yet unrecognized taxa within the genus Edwardsiella. The MLSA described here was robust enough in discriminating E. tarda isolates not only with respect to their geographical origins but also within different hosts from the same geographical location, high-lighting its potential application in tracing the source of infection and understand the epidemiological relationships among isolates of environmental, fish, other domestic animals or human origins.

Tenacibaculum sp. associated with winter ulcers in sea-reared Atlantic salmon Salmo salar.

Coldwater-associated ulcers, i.e. winter ulcers, in seawater-reared Atlantic salmon Salmo salar L. have been reported in Norway since the late 1980s, and Moritella viscosa has been established as an important factor in the pathogenesis of this condition. As routine histopathological examination of winter ulcer cases in our laboratory revealed frequent presence in ulcers of long, slender rods clearly different from M. viscosa, a closer study focusing on these bacteria was conducted. Field cases of winter ulcers during 2 sampling periods, 1996 and 2004-2005, were investigated and long, slender rods were observed by histopathological examination in 70 and 62.5% of the ulcers examined, respectively, whereas cultivation on marine agar resulted in the isolation of yellow-pigmented colonies with long rods from 3 and 13% of the ulcers only. The isolates could be separated into 2 groups, both identified as belonging to the genus Tenacibaculum based on phenotypic characterization and 16S rRNA sequencing. Bath challenge for 7 h confirmed the ability of Group 1 bacterium to produce skin and cornea ulcers. In fish already suffering from M. viscosa-induced ulcers, co-infection with the Group 1 bacterium was established within 1 h. Ulcers from field cases of winter ulcers and from the transmission experiments tested positive by immunohistochemistry with polyclonal antiserum against the Group 1 bacterium but not the Group 2 bacterium. Our results strongly indicate the importance of the Group 1 bacterium in the pathogenesis of winter ulcers in Norway. The bacterium is difficult to isolate and is therefore likely to be underdiagnosed based on cultivation only.

Atlantic salmon bath challenged with Moritella viscosa--pathogen invasion and host response.

The Gram-negative bacterium Moritella viscosa is considered to be the main causative agent of winter ulcer, a disease that primarily affects salmonid fish in sea water during cold periods. The disease is initially characterised by localised swelling of the skin followed by development of lesions. To gain more knowledge of the role of M. viscosa in the pathogenesis of winter ulcer, 159 Atlantic salmon (80-110 g) were exposed to a bath challenge dose of 7 x 10(5) cfu ml(-1) for 1 h at 8.9 degrees C. The first mortalities were registered two days post-challenge and the mortality rate increased rapidly. Multi-organ samples were taken throughout the challenge for culture, immunohistochemistry and PCR analysis. Using real-time PCR, M. viscosa DNA was first detected in the gills of all fish examined 2, 6 and 12 h after challenge. From day 2, the bacterium was detected in the muscle/skin, head kidney, spleen and liver. This was in correlation with positive cultured samples and confirmed systemic infection. The early and consistent detection of M. viscosa DNA in gill samples, and less or not in muscle/skin or intestine, could suggest gills as a port of entry for the bacterium. Immunohistochemical analysis using a polyclonal antiserum against M. viscosa demonstrated generalised staining in the lumen of blood vessels and some positive mononuclear cells. The antigens recognised by the antiserum may have originated from extracellular bacterial products and be part of a bacterial invasion strategy. To better understand the immune response in salmon to M. viscosa infection, the expression profiles of the immune genes IL1 beta, C3, ISG15 and CD83 were studied. Increased expression of IL1 beta and C3 was not induced until day 7, which may suggest that M. viscosa might utilize escape mechanisms to evade the host's immune system by suppressing relevant immune responses.

Mycoplasma canis and urogenital disease in dogs in Norway.

Mycoplasmas identified as Mycoplasma canis were isolated from nine dogs with clinical signs of urogenital disease in Norway over a period of 20 months. Some of the dogs had been treated unsuccessfully with antibiotics, and three were euthanased as a result of severe persistent disease. Seven of the dogs had a urinary tract infection, one had chronic purulent epididymitis and one had chronic prostatitis. Overt haematuria was frequently observed among the dogs with cystitis. M canis was isolated in pure culture from seven of the dogs and in mixed culture from the other two. In three cases the mycoplasma was cultivated only from urinary sediment, and it was typically obtained in smaller numbers than would be considered indicative of a urinary tract infection. In contrast with most mycoplasmas, the M canis isolated from all the dogs grew on ordinary blood agar plates used for routine bacteriological cultivation. Specific mycoplasma media were not used and the presence of other Mycoplasma or Ureaplasma species cannot be excluded.

Organization of the antiseptic resistance gene qacA and Tn552-related beta-lactamase genes in multidrug- resistant Staphylococcus haemolyticus strains of animal and human origins.

A part (12 kb) of a plasmid containing the beta-lactamase genes of Tn552, the disinfectant resistance gene qacA, and flanking DNA has been cloned from a Staphylococcus haemolyticus isolate and sequenced. This region was used to map the corresponding regions in six other multiresistant S. haemolyticus isolates of human and animal origin. The organizations of the genetic structures were almost identical in all isolates studied. The beta-lactamase and qacA genes from S. haemolyticus have >99.9% identities at the nucleotide level with the same genes from S. aureus, demonstrating that various staphylococcal species able to colonize animal and human hosts can exchange the genetic elements involved in resistance to antibiotics and disinfectants. The use of antibiotics and disinfectants in veterinary practice and animal husbandry may also contribute to the selection and maintenance of resistance factors among the staphylococcal species. Different parts of the 12-kb section analyzed had high degrees of nucleotide identity with regions from several other different Staphylococcus aureus plasmids. This suggests the contribution of interplasmid recombination in the evolutionary makeup of this 12-kb section involving plasmids that can intermingle between various staphylococcal species. The lateral spread of resistance genes between various staphylococcal species is probably facilitated by the generation of large multiresistance plasmids and the subsequent interspecies exchange of them.

Cloning, characterisation and phylogenetic analysis of the fur gene in Vibrio salmonicida and Vibrio logei.

The gene encoding the ferric uptake regulator protein (fur gene) of Vibrio salmonicida 87/09/1193 was located following hybridisation of an EcoRI digest of chromosomal V. salmonicida DNA with a 316 base pairs (bp) probe internal to the fur gene of Vibrio anguillarum. A 2088 bp fragment including an open reading frame of 441 bp, encoding a protein of 147 amino acids, and homologous with fur, was identified, cloned and sequenced. A plasmid bound V. salmonicida fur gene was found capable of complementing the fur mutation of Escherichia coli H1681. Although no 'iron-box' was identified upstream of the start-codon, beta-galactosidase activity in E. coli H1681 was regulated by iron availability in the media, indicating that in V. salmonicida fur, as in other fur genes, iron functions as a co-repressor. Southern blot hybridizations demonstrated that fur is conserved amongst V. salmonicida strains and several other closely related Vibrio strains in which fur remains as yet, uncharacterized. The fur gene of Vibrio logei NCIMB 2252 was subsequently amplified using polymerase chain reaction primers external to the V. salmonicida fur gene. Comparison of phylogenetic analyses using fur and 16S DNA coding for rRNA sequences, confirmed the usefulness of fur as an evolutionary marker within the genus Vibrio.

Relevance of incubation temperature for Vibrio salmonicida vaccine production.

AIMS: To investigate the relationships between water temperature, bacterial growth, virulence and antigen expression in Vibrio salmonicida, the causal agent of cold water vibriosis in Atlantic salmon (Salmo salar L.). METHODS AND RESULTS: The significance of sea temperature was investigated using historical clinical and oceanographic data. An upper threshold for disease of approx. 10 degrees C was established. The effects of culture temperature and media type on bacterial growth were studied on solid and in liquid media. The highest rates of cell division were identified at 15 degrees C on solid media and 10 degrees C in liquid media. Outer membrane protein (OMP) expression and serological response in Atlantic salmon were studied using sodium dodecyl sulphate-polyacrylamide gel electrophoresis, Western blotting and enzyme-linked immunosorbent assay. A novel 76-kDa OMP produced in unshaken cultures at 10 degrees C was not found to stimulate a specific humoral response. CONCLUSIONS: Diagnostic agar plate-based incubation of suspected V. salmonicida should be carried out at 15 degrees C. High yield broth cultures for vaccine production should be incubated at 10 degrees C or lower. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is, to the best of our knowledge, the first to identify different optimal temperatures in a bacterial species cultured on physically different types of media. The evidence presented suggests that V. salmonicida and possibly other bacteria destined for vaccine use in poikilothermic organisms should be cultured at temperatures consistent with that at which disease occurs.

Temperature dependent siderophore production in Vibrio salmonicida.

Vibrio salmonicida is the causative agent of cold water vibriosis, a haemorrhagic septicaemia of Atlantic salmon (Salmo salar). The disease is observed only at low water temperatures and not normally above 10 degrees C. Siderophore production and iron regulated outer membrane protein expression was studied at various temperatures. Although in iron-limited media optimal cell growth was identified at 12 degrees C, significant quantities of a single hydroxamate siderophore were produced only at 10 degrees C or below. Dependent on inoculant size, good growth without significant siderophore production was also observed in iron-limited media at temperatures above and below 10 degrees C. It is therefore likely that V. salmonicida also possesses one or more non-siderophore based iron assimilation systems. Expression of high molecular weight iron-regulated outer membrane proteins appeared to be suppressed at 15 degrees C compared to expression at 6 and 10 degrees C. It is proposed that temperature sensitive iron sequestration may constitute a significant virulence factor in V. salmonicida and may have implications for vaccine manufacture.