Brian Austin

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.

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.

Flavobacterium scophthalmum sp. nov., a Pathogen of Turbot (Scophthalmus maximus L.)

Fifty orange-pigmented, gram-negative, rod-shaped isolates were recovered from healthy and diseased turbot and from coastal waters (collected in Scotland). On the basis of the results of an examination of 125 phenotypic characteristics and the results of DNA-DNA and DNA-rRNA hybridization experiments, we concluded that these isolates are members of a new species in the genus Flavobacterium, for which the name Flavobacterium scophthalmum is proposed. The type strain is CCM 4109 (= LMG 13028).

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.

Diversity of Vibrio anguillarum Isolates from Different Geographical and Biological Habitats, Determined by the Use of a Combination of eight Different Typing Methods

Summary In the present investigation we have studied 260 presumed Vibrio anguillarum isolates from a wide range of habitats, ,using a combination of eight different typing methods. The aims of the study were to investigate the diversity of V. anguillarum, as indicated by the use of combined typing, and to determine if strains with identical or similar characteristics were present in certain geographical locations, or in certain fish species. We also present a simple numerical method to analyse data obtained from combined typing. Two hundred and sixty isolates named as V. anguillarum from various fish species, rotifers, Artemia, water and sediment were subjected to the following eight assays: Species identification using ribotyping and the BIOLOG GN plates, subtyping using determination of outer membrane profiles, plasmid typing, serotyping, determination of lipopolysaccharide profiles and biotyping with API 20E, and biochemical fingerprinting with the PhenePlate system. The diversity among all isolates, calculated as Simpsons diversity index (Di), varied between 0.19 (ribotyping), i.e. most isolates belonged to the same type, and 0.98 (PhP), i.e. most isolates different. Upon combination of all methods, where a difference between two strains in at least two methods was regarded as significant, a diversity of 0.92 was obtained. Isolates collected from fish showed lower diversity (Di = 0.89) than those collected from other sources (environment, rotifers, Artemia) (Di = 0.98). The lowest diversities were found among isolates collected from rainbow trout (Oncorhynchus mykiss), salmon (Salmo salar) and turbot (Scophthalmus maximus). Isolates from close geographical locations were also less diverse than isolates obtained from more distant locations. We conclude that the diversity of V. anguillarum is high, as shown by the combined typing methods. However, it seems that strains with specific characteristics are associated with certain geographic areas, and also with certain fish species. This could only be detected by applying a combination of several typing methods. Our results emphasise the need to use several different typing methods for studies of bacterial diversity.

New Methods for the Analysis of Binarized BIOLOG GN Data of Vibrio species: Minimization of Stochastic Complexity and Cumulative Classification

We apply minimization of stochastic complexity and the closely related method of cumulative classification to analyse the extensively studied BIOLOG GN data of Vibrio spp. Minimization of stochastic complexity provides an objective tool of bacterial taxonomy as it produces classifications that are optimal from the point of view of information theory. We compare the outcome of our results with previously published classifications of the same data set. Our results both confirm earlier detected relationships between species and discover new ones.

Typing of the Fish Pathogen Listonella (Vibrio) anguillara by Pyrolysis Mass Spectrometry

Twenty-eight representatives of Listonella (Vibrio) anguillara serovars O1, O2 and O3 were compared by Curie-point pyrolysis mass spectrometry (PyMS). The representatives of serovars O1 and O3 formed discrete, homogeneous groups in ordination plots of the PyMS data. Strains from serovar O2 were recovered in two groups, one of which encompassed six strains including the type strain of the species and the reference strain for serovar O2, and the other included two strains which showed cross-reactions between serovars O2 and O5. The almost complete agreement found between the PyMS and the serological data suggests that pyrolysis mass spectrometry will prove to be an effective method for interstrain comparison within the species Listonella anguillara.