Dawn A. Austin

Recovery of a new biogroup of Yersinia ruckeri from diseased rainbow trout (Oncorhynchus mykiss, Walbaum)

Cultures of a new biogroup of Yersinia ruckeri, the causal agent of enteric redmouth (ERM), were recovered in England from diseased rainbow trout (Oncorhynchus mykiss, Walbaum), which had been previously vaccinated with a commercial ERM vaccine. The bacterial isolates were confirmed as Y. ruckeri by the results of sequencing the 16S rRNA, but differed from the characteristics of the taxon by positivity for the Voges Proskauer reaction and a general lack of motility, and could not be equated with any of the existing serovars. Cultures were pathogenic in laboratory-based infectivity experiments with 100% mortalities occurring in juvenile rainbow trout (average weight = 10 g) within 4-days of intraperitoneal or intramuscular injection with 10(5) cells/fish. Protection against disease was achieved using a formalin-inactivated whole vaccine prepared against a representative isolate.

Identification and typing of Vibrio anguillarum - a comparison of different methods.

Summary The majority (91%) of 260 isolates initially identified as Vibrio anguillarum , that were obtained from a wide range of hosts, habitats and geographical locations, were recovered in a single cluster based on the ribotype and were pathogenic to Atlantic salmon. A significant proportion of isolates (78% of the total) were allocated to 15 serogroups (O1–O10 and five previously undescribed groups referred to as VaNT1, VaNT2, VaNT4, NaNT5 and VaNT7). A minority of isolates (6%) reacted with more than one antiserum or were self-agglutinating, and the remainder did not react with any of the antisera tested. Good correlation was noted between serogroups and lipopolysaccharide profiles, particularly with respect to isolates belonging to serogroups O1, 02 and 04ߝ010. Plasmids were recognized in some serogroups. especially O1, which contained the 67 kb plasmid associated with virulence. However, the 19 profiles based on outer membrane protein patterns did not correspond to the results obtained with the other typing methods. Generally, the isolates were heterogeneous in their biochemical characteristics; 117 profiles were obtained with the API 20E system, and 9 and 32 clusters recognised from the results of BIOLOG fingerprinting and Biotype-100 biotyping methods, respectively. Three dominant dusters were defined from fatty acid methyl esters profiles.

Characterization of atypical Aeromonas salmonicida by different methods

Fifty two isolates of atypical Aeromonas salmonicida, recovered from a wide range of hosts and geographical locations, were heterogeneous in terms of molecular and phenotypic characteristics, and represented taxa which could not be accommodated by the current classification of four subspecies. Generally, there was incongruence between the molecular (PCR, RAPD and ribotyping) and phenotypic methods in terms of cluster membership. By PCR, 6 groups were described of which Group 1 encompassed 12 isolates including the type strain of A. salmonicida subsp. smithia. Group 2 accommodated 23 isolates including the reference cultures of subspecies achromogenes and masoucida. The named culture of Haemophilus piscium was recovered in Group 6. By ribotyping and RAPD, the reference cultures were recovered in separate groups. All methods pointed to the uniqueness of subspecies smithia. Most isolates contained 2-6 plasmids, of 2.3 to 150 kb in length. Nevertheless, all isolates possessed certain key characteristics, including Gram-negativity, and the absence of motility.

Taxonomic evidence that Vibrio carchariae Grimes et al. 1985 is a junior synonym of Vibrio harveyi (Johnson and Shunk 1936) Baumann et al, 1981.

A collection of 94 Vibrio isolates closely related to Vibrio harveyi, together with named reference and type strains, were investigated for phenotypic and genotypic properties. Using amplified fragment length polymorphism (AFLP), nine clusters were recognized. The largest cluster (n = 36), considered to be the bona fide V. harveyi group, contained the type strains of V. harveyi and Vibrio carchariae and most of the strains isolated from fish. The type strains of all other species, including Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio campbellii and Vibrio natriegens, clustered outside this group. By ribotyping, V. harveyi and V. carchariae patterns were very similar, insofar as they shared most bands. The V. campbellii type strain had several bands in common with the type strains of both V. harveyi and V. carchariae, whereas the other species were clearly distinct from these three species. DNA-DNA hybridization experiments showed 88% DNA binding between the type strains of V. harveyi and V. carchariae, whereas the DNA binding between V. harveyi and V. campbellii was 40%. Although the delineation of the species V. harveyi is still uncertain, the authors propose, on the basis of a number of tests, to delineate a core of V. harveyi strains which contained the type strains of both V. harveyi and V. carchariae. It is concluded that V. carchariae is the junior synonym of V. harveyi.

A comparison of methods for the typing of fish-pathogenic Vibrio spp.

Summary The validity and distinctiveness of Vibrio anguillarum (= Listonella anguillara), V. (= Photobacterium) damsela, V. ordalii and V. salmonicida was confirmed. However, strains received as V. cholerae and V. splendidus were heterogeneous. Ribotyping, phenotypic (BIOLOG-GN fingerprints and API 20E profiles), chemotaxonomic (lipopolysaccharide [LPS] and outer membrane proteins [OMP]), serogrouping and plasmid profiling data were not always congruent. V. anguillarum isolates were recovered in a single ribotype cluster, but many serogroups. There was little variation in OMP profiles, but not so for LPS profiles and plasmid composition. Heterogeneity was recorded in the phenotypic characters, particularly with the API 20E rapid identification system. V. damsela displayed heterogeneity by ribotyping, but homogeneity by BIOLOG-GN fingerprints and API 20E profiles. Four serogroups were defined, but only one LPS profile was recognised. V. ordalii was homogeneous by ribotyping, serogrouping and plasmid profiling, was accommodated in two LPS groups, but was more heterogeneous by BIOLOG-GN and API 20E. Despite its more exacting cultural requirements, V. salmonicida autoagglutinated and could not be serogrouped, but was accommodated in a single LPS group showing a profile associated with rough strains, and contained plasmids of 4.7 and 42 kb. Heterogeneity was recorded with the API 20E rapid identification system.

Identification and pathogenicity to rainbow trout, Oncorhynchus mykiss (Walbaum), of some aeromonads.

Twelve strains of fish pathogenic aeromonads were identified by 16S rRNA sequencing as Aeromonas bestiarum, A. hydrophila, A. hydrophila subsp. dhakensis, A. salmonicida subsp. salmonicida, A. sobria biovar sobria and A. veronii biovar sobria. Following intramuscular injection, A. hydrophila subsp. dhakensis caused dark liquefying, raised furuncle-like lesions in rainbow trout within 48 h. Extracellular products of all cultures contained gelatinase and lecithinase, and most revealed lipase. Congo red absorption and siderophore production was recorded, but not so the suicide phenomenon or slime production. Sodium dodecyl sulphate polyacrylamide gel electrophoresis profile of the outer membrane proteins (OMP) revealed 10-25 bands, of which major bands were seen in the region of 32.5-47.5 and 62-83 kDa. Marked heterogenicity of the OMP and whole cell protein (WCP) profiles within and among the species was observed. Polypeptides of 83-173 kDa were detected in the WCP profile of the cultures, but they were not expressed in OMP fractions.

Pathogenicity of Vibrio anguillarum serogroup O1 strains compared to plasmids, outer membrane protein profiles and siderophore production.

The virulence of 18 strains of Vibrio anguillarum serogroup O1 was compared to plasmid content, expression of siderophores and outer membrane proteins. All strains, irrespective of plasmid content, produced siderophores and inducible outer membrane proteins under iron‐limited conditions. Only strains that carried the 67 kbp virulence plasmid or derivatives of it produced the outer membrane protein, OM2. All virulent strains harboured the 67 kbp plasmid or derivatives of it, indicating its importance for virulence. However, some strains carrying the virulence plasmid or a derivative of it, produced siderophores as well as OM2 but were non‐pathogenic to fish. Likewise, among the virulent strains, considerable variation in LD50 values was recorded. Plasmid profiling and restriction analysis showed that the virulence plasmid existed in various molecular weights from 26 to 80 kbp, with 65–67 kbp being the most common, and that this plasmid displayed various restriction profiles. The presence of other plasmids did not seem to affect the pathogenic properties.

An improved growth medium for Flavobacterium psychrophilum

Supplementing cytophaga agar and broth with 0·5 g l−1 each of d(+) galactose, d(+) glucose, l‐rhamnose and skimmed milk gave a dramatic improvement in the isolation of the fish pathogen Flavobacterium psychrophilum. By means of spread‐plating, approximately double the number of colonies of larger size were obtained on the improved medium compared to cytophaga agar alone. In supplemented cytophaga broth, growth of Fl. psychrophilum was more rapid and generated greater biomass.

Aeromonadaceae Representative (Aeromonas salmonicida)

Aeromonas salmonicida is a significant pathogen of salmonids, and in its atypical form has spread into cyprinids and marine flatfish. Although Aeromonas salmonicida subsp. salmonicida is homogeneous, atypical isolates are more heterogeneous and do not fit into the current subspecies classification. Questions about the ecology of the organism remain but the consensus is that despite earlier work, cells exist in the aquatic environment although largely in a nonculturable form. Diagnostics have moved towards the use of sensitive and specific molecular methods. Disease control has focused on prophylaxis principally by vaccination, probiotics and immunostimulants.

Non-adjuvanted flagellin elicits a non-specific protective immune response in rainbow trout (Oncorhynchus mykiss, Walbaum) towards bacterial infections

Enteric redmouth disease, caused by Yersinia ruckeri, may result in high mortalities in farmed salmonids. Prophylaxis has been achieved with an immersion vaccine comprised of inactivated serovar 1 biotype 1 (motile) Y. ruckeri cultures. However, there has been a growing number of enteric redmouth outbreaks in vaccinated livestock associated with serovar 1 biotype 2 (non-motile) Y. ruckeri strains which do not produce flagellin. It was the aim of this study to evaluate the protective role of flagellin in enteric redmouth vaccines. Results showed that flagellin in the inactivated whole-cell vaccine were not the main immunoprotective molecule in eliciting a protective immune response towards infection. However, use of non-adjuvanted flagellin as a sub-unit vaccine, both in the native and recombinant form, resulted in a potent non-specific protective function towards challenge with biotype 1 (flagellin-producing) and biotype 2 (flagellin-devoid) Y. ruckeri. This vaccine can also protect rainbow trout against other microbial fish pathogens, for example Aeromonas salmonicida. Thus non-adjuvanted flagellin may have potential as a non-specific vaccine for fish towards bacterial pathogens.