RAPD analysis of Aeromonas salmonicida and Aeromonas hydrophila

The randomly amplified polymorphic DNA (RAPD) technique was used to analyse the genetic differentiation of 13 strains of Aeromonas salmonicida subsp. salmonicida , and seven strains of Aer. hydrophila. Reproducible profiles of genomic DNA fingerprints were generated by polymerase chain reaction (PCR) using a single randomly designed primer. The RAPD profiles of all the non-motile aeromonads, Aer. salmonicida subsp. salmonicida were identical. However, profiles of the motile aeromonads, Aer. hydrophila differed between isolates. These findings reveal genomic homogeneity in Aer. salmonicida subsp. salmonicida and genetic variety in Aer. hydrophila strains.     

Virulence studies based on plasmid profiles of the fish pathogen Vibrio salmonicida

Strains of Vibrio salmonicida isolated from Atlantic salmon (Salmo salar) and rainbow trout (Salmo gairdneri) suffering from cold-water vibriosis could be divided on the basis of plasmid profiles into four different categories. Of 32 strains, 19% harbored three plasmids of 24, 3.4, and 26 megadaltons (MDa), 69% harbored the 24- and 3.4-MDa plasmids but not the 2.6-MDA plasmid, and 9% harbored only the 24-MDA plasmid. The fourth category, which consisted of only one strain, harbored a plasmid of 10 MDa. In spite of different plasmid patterns, the strains of V. salmonicida were very similar with respect to biochemical reactions. The one-third of the V. salmonicida strains which were serotyped were of the same type. The 50% lethal doses, which were determined by intraperitoneal injection, ranged from 4 x 106 to 1 x 108 CFU per fish.     

Virulence studies based on plasmid profiles of the fish pathogen Vibrio salmonicida.

Strains of Vibrio salmonicida isolated from Atlantic salmon (Salmo salar) and rainbow trout (Salmo gairdneri) suffering from cold-water vibriosis could be divided on the basis of plasmid profiles into four different categories. Of 32 strains, 19% harbored three plasmids of 24, 3.4, and 26 megadaltons (MDa), 69% harbored the 24- and 3.4-MDa plasmids but not the 2.6-MDA plasmid, and 9% harbored only the 24-MDA plasmid. The fourth category, which consisted of only one strain, harbored a plasmid of 10 MDa. In spite of different plasmid patterns, the strains of V. salmonicida were very similar with respect to biochemical reactions. The one-third of the V. salmonicida strains which were serotyped were of the same type. The 50% lethal doses, which were determined by intraperitoneal injection, ranged from 4 x 106 to 1 x 108 CFU per fish.     

Characteristics of 'atypical', cytochrome oxidase-negative Aeromonas salmonicida isolated from ulcerated flounders (Platichthys flesus (L.))

'Atypical', cytochrome oxidase-negative variants of the fish pathogen Aeromonas salmonicida , isolated from ulcerated flounder ( Platichthys flesus ), were studied using different methods. Two of the strains possessed a protein that corresponded to the A-layer protein of Aer. salmonicida . The strains reacted with antibodies against the A-layer and monoclonal antibodies against the O-antigen of typical Aer. salmonicida . These tests confirm that the isolates from flounder should be classified as Aer. salmonicida . Analysis of the fatty acids showed that the isolates were rather homogenous but the values of the guanine plus cytosine content of the DNA of the bacteria varied too much for any conclusion to be drawn on their taxonomic location. The strains examined exhibited several biochemical characters that differed from those of the type strains of Aer. salmonicida subsp. salmonicida and Aer. salmonicida , subsp. achromogenes . The results suggest that these 'atypical', cytochrome oxidase-negative variants may form a new subspecies of Aer. salmonicida .     

Evidence for the existence of distinct populations of Vibrio anguillarum serogroup O1 based on plasmid contents and ribotypes.

A total of 103 Vibrio anguillarum serogroup O1 strains displaying 15 different plasmid profiles were characterized with respect to biochemical properties and ribotypes. The results confirmed that V. anguillarum O1 is a biochemically homogeneous group. The 103 strains could be allocated to three main clusters with high similarity coefficients. None of the biochemical properties were connected with the presence of plasmids. In total, 12 different ribotypes were demonstrated, with HindIII being used as the restriction enzyme. Forty of the strains were isolated from the same Danish fish farm, some from the kidneys of diseased fish and some from the environment, and some strains were isolated from the mucus, gills, and feces of healthy fish. Nineteen of these isolates possessed the 67-kb virulence plasmid alone or in combination with other plasmids, while 21 had no plasmids. All strains isolated from the kidneys of diseased fish on this farm had plasmids. Irrespective of their origin (kidneys, gills, or mucus), all 19 strains carrying the 67-kb virulence plasmid had the same ribotype, profile 1, while isolates without plasmids belonged to five different profiles, all different from profile 1. These results suggest that pathogenic V. anguillarum O1 strains possessing a virulence plasmid and nonpathogenic strains without plasmids from a small geographical area and even from the same fish may constitute two essentially distinct populations. Thus, it may be suggested that an exchange of virulence plasmids among strains is unlikely to occur in vivo.     

Plasmid profiles and randomly amplified polymorphic DNA analysis of Salmonella enterica serotype Enteritidis strains from outbreaks and sporadic cases in Turkey

The aim of the study was to investigate the characteristics of Salmonella serotype Enteritidis strains isolated from outbreaks and sporadic cases in Turkey by plasmid profiles and randomly amplified polymorphic DNA (RAPD) patterns. A total of 64 S. Enteritidis clinical strains were selected from the culture collection of the Enterobacteria Laboratory of Ankara University Medical School Department of Microbiology and Clinical Microbiology for molecular analysis using the plasmid profiles and RAPD method. Fifty-six isolates (88%) harbored one to four plasmids ranging in size from 2.5 to 100 kbp. 57 kbp plasmids were the most common plasmids, and forty-four strains (69%) carried 57 kbp plasmids alone or together with other plasmids. The outbreak strains carried the same plasmid profile: three plasmids sized 57, 40, 3.0 kbp. None of the strains analyzed displayed any RAPD bands with the primer OPB-17. By using primer p-1254, 42 strains (66%) were divided into fourteen RAPD patterns. Ten of the outbreak strains (77%) showed >80% similarity by cluster analysis program. Analysis of RAPD-PCR with primer p-1254 proved an easy, rapid and discriminative method complementing antibiogram and plasmid profiles in routine laboratories, and may contribute to the investigations of S. Enteritidis which still cause outbreaks in Turkey. This study presents the first report on S. Enteritidis isolates in Turkey investigated by plasmid profiles and RAPD methods.     

Comparison of extracellular proteases produced by Aeromonas salmonicida strains, isolated from various fish species

Extracellular products (ECPs) of five typical and 25 atypical Aeromonas salmonicida isolates from various fish species and geographical locations were analysed by substrate specificity, inhibition of proteolytic activity and substrate SDS-PAGE. The type strains of Aer. salmonicida subsp. salmonicida and Aer. salmonicida subsp. achromogenes were included for comparison. The results indicated that the strains formed six protease groups. The proteases produced by the two type strains were of a different nature. All the typical strains belonged to one group and showed proteolytic activities comparable to P1 and P2 proteases. Three atypical (oxidase-negative) strains secreted a protease comparable to P1. With the exception of these three, all strains produced metallo-gelatinases. A metallo-caseinase (AsaP1) was detected in the ECP of subsp. achromogenes type strain and 10 of the atypical strains. A number of proteolytic components with different apparent molecular weights (AMWs) were identified. These include caseinases with AMWs of > 100, 80, 60 and 30 kDa and gelatinolytic components with different AMWs, including some with AMW higher than P1 and lower than P2. The protease production of the isolates was not found to be host specific.     

Atypical Aeromonas salmonicida Isolated from Diseased Turbot (Scophtalmus maximus L.)

Atypical Aeromonas salmonicida were isolated from 3 outbreaks of disease among farmed turbot (Scophthalmus maximus L.) in 3 different farms, 1 from Norway (Nl) and 2 from Denmark (DK1 and DK2). In all 3 cases, the incidence of disease and mortality was high and the main characteristic pathological finding was skin ulcers and septicaemia. The isolated bacteria were subjected to a thorough phenotypic and genotypic examination and comparison in the laboratory. All 3 isolates belonged to A. salmonicida but dis-played some very different biochemical properties. However, the 2 Danish strains, DK1 and DK2 had identical ribotypes but different from that of Nl, whereas the plasmid pro-files of DK1 and Nl were identical but different from that of DK2. These observations emphasize the need for an improvement of our understanding of the taxonomy and epi-demiology of atypical A. salmonicida.     

Ribotyping and plasmid profiling of Vibrio anguillarum serovar O2 and Vibrio ordalii

One hundred and twenty-nine strains of Vibrio anguillarum serovar O2 and 14 strains of Vibrio ordalii were ribotyped and examined for plasmid contents. A total of 35 different ribotypes were detected. The V. anguillarum serovar O2 strains were divided into 32 different ribotypes. The V. ordalii strains showed three different ribotypes, clearly distinct from those of the V. anguillarum strains.
Ribotypes were separated into seven clusters, of which one comprised the V. ordalii strains. Clustering of the strains indicated a genetic difference between North European and South European V. anguillarum O2 strains. Sero-subgroups O2a and O2b shared ribotypes; however, three of the clusters did not include O2a strains.
All V. ordalii strains had a plasmid of 32 kb. This plasmid was not detected in any of the V. anguillarum strains. Seventeen different plasmid profiles with 17 different sized plasmids were detected among the V. anguillarum strains. Most of the plasmids were small (< 6 kb) and found in several strains. Except for one South European strain, plasmids were detected only in the North European strains of V. anguillarum O2.     

A global non-conjugative Tet C plasmid,pRAS3, from Aeromonas salmonicida

Two 11.8 kb non-conjugative, but mobilizable R plasmids designated pRAS3.1 and pRAS3.2 were isolated from Aeromonas salmonicida subspecies salmonicida and atypical A. salmonicida, respectively. Differences between the plasmids were of minor extent and they are considered as being variants of the same plasmid, pRAS3. The genes repA, repB, mobA, mobC, mobD, and mobE were organized similar to corresponding genes in the small, mobilizable plasmid pTF-FC2 isolated from Acidithiobacillus ferrooxidans (previously Thiobacillus ferrooxidans). The nucleotide identity between these genes from pRAS3.1 and pTF-FC2 ranged from 89.5 to 98.2%. The tetA(C), tetR(C), and approximately 960 base pairs adjacent to tetR(C) were highly similar to the nucleotide sequence in pSC101. Plasmid pRAS3 was also found in a Scottish A. salmonicida strain, and appears to be identical to the R plasmid pJA8102-2 isolated from A. salmonicida in Japan.     

Grouping by plasmid profiles of atypical Aeromonas salmonicida isolated from fish, with special reference to salmonid fish

Plasmid profile analyses were performed for 113 strains of atypical Aeromonas salmonicida and the reference strain A. salmonicida subsp. salmonicida ATCC 14174. The atypical A. salmonicida strains comprised 98 strains obtained from fish originating from 54 farms and 2 lakes in Norway, 10 strains from Canada (2), Denmark (2), Finland (1), Iceland (1) and Sweden (4), the reference strains NCMB 1109 and ATCC 15711 (Haemophilus piscium) of A. salmonicida subsp. achromogenes, and the type cultures A. salmonicida subsp. achromogenes NCMB 1110, A. salmonicida subsp. masoucida ATCC 27013 and A. salmonicida subsp. smithia CCM 4103. A total of 95 strains of atypical A. salmonicida were separated into 7 groups (I to VII) based on the plasmid profiles. Eighteen strains of atypical A. salmonicida had no common plasmid profile. The type strain NCMB 1110 and the reference strain NCMB 1109 were included in group IV, and the type strain ATCC 27013 in group V, but the other reference and type strains had plasmid profiles different from all the other strains. An epidemiological link was documented between strains collected from different farms/localities in each of groups I, III, V and VII. Physiological and biochemical characterizations were performed for 93 of the strains to investigate phenotypic differences between the plasmid groups. Group VII strains and 3 strains with no common plasmid profile differed from the other groups in being catalase-negative. Differences in phenotypic characteristics were shown between the plasmid groups. However, significant variations in reactions for several phenotypic characteristics also occurred within each of the groups I to VII. The present study indicates that plasmid profiling may give useful epidemiological information during outbreaks of atypical A. salmonicida infections in fish. Additional comprehensive phenotypic characterisation is of limited value since the phenotypic characteristics in each plasmid group are not uniform.     

Use of plasmid profiling as a typing method for epidemiologically related Clostridium perfringens isolates from food poisoning cases and outbreaks

Plasmid profiling was used for the characterization of Clostridium perfringens isolates involved in disease outbreaks. The usefulness of this technique was demonstrated by the retrospective examination of food and patient isolates from 10 cases and outbreaks from 1984 to 1991. The origin of three outbreaks could be clearly confirmed due to identical plasmid profiles in all isolates. In one outbreak identical plasmid patterns were found between one food and one patient isolates, while one plasmid was missing in the second patient isolate. In an additional two cases a relationship between food and patient isolates is likely, if the possibility of the loss of one plasmid in one of the isolated strains is considered. In one outbreak two faecal isolates could be related to an isolate from one of the two foods implicated as outbreak source; isolates from the other food and a third faecal sample could not be linked to any other isolate. The results from three outbreaks were largely inconclusive because plasmids were not present either in all or in some of the isolates.     

Identification of atypical Aeromonas salmonicida: inter-laboratory evaluation and harmonization of methods

The atypical isolates of Aeromonas salmonicida are becoming increasingly important as the frequency of isolation of bacteria belonging to this group continues to rise. The primary object of this study was to compare and evaluate the results obtained in various laboratories concerning the biochemical identification of atypical Aer. salmonicida before and after standardization of media and methods. Five laboratories examined 25 isolates of Aer. salmonicida from diverse fish species and geographical locations including the reference strains of Aer. salmonicida subsp. salmonicida (NCMB 1102) and Aer. salmonicida subsp. achromogenes (NCMB 1110). Without standardization of the methods, 100% agreement was obtained only for two tests: motility and ornithine decarboxylase. The main reason for the discrepancies found was the variation of the incubation time prior to reading the biochemical reactions. After standardization, improvement was obtained with the identification; however, disagreement was still observed between the different laboratories. These findings demonstrate the difficulties involved in a proper identification of atypical Aer. salmonicida and also that data presented in the literature on various strains of Aer. salmonicida are not readily comparable. This paper seems to be the first on standardization of microbiological tests for identification of fish pathogens and the results obtained show the need for standardization of methods both within and between laboratories.     

Plasmid content and homology of 16 strains of filamentous,nonheterocystous cyanobacteria

We have developed several strain-specific, rapid, small-scale plasmid isolation procedures in order to characterize the plasmid profiles of 16 filamentous, nonheterocstous cyanobacteria. At least one distinct plasmid was found in eight strains, with seven of these containing two or more different plasmids. Eight strains were found to be without plasmid DNA. Both the large, 12.9 kb, and the small, 1.6 kb, plasmids fromPlectonema boryanum 581 were isolated, purified, and cloned. Southern blots of plasmid DNAs from the eight strains were probed with these cloned DNAs and also with ultra-pure plasmid DNA fromPhormidium liridum 426. Four strains ofP. boryanum (485, 581, 594, 1542) andP. luridum 426 have identical plasmid profiles, and plasmid homology is extensive.     

Plasmid profiling of Vibrio salmonicida for epidemiological studies of cold-water vibriosis in Atlantic salmon (Salmo salar) and cod (Gadus morhua).

In 1988, a new plasmid profile was observed for Vibrio salmonicida isolated from cod (Gadus morhua) and Atlantic salmon (Salmo salar) in fish farms in northern Norway. This new plasmid profile, which consisted of plasmids of 61, 21, 3.4, and 2.8 megadaltons, is 1 of 11 plasmid profiles which have so far been observed for V. salmonicida. Plasmid profiling and plasmid DNA hybridization were used in epidemiological studies of cold-water vibriosis. Our results indicate that V. salmonicida was transmitted from Atlantic salmon to cod and vice versa. The 61-megadalton plasmid was found exclusively in V. salmonicida strains originating from northern Norway, which is the only area in which this plasmid has ever been observed. Plasmid DNA hybridization and restriction endonuclease analysis show that the plasmid DNA of V. salmonicida remained stable throughout a 7-year survey.     

Distinct plasmid profiles of Pasteurella haemolytica serotypes and the characterization and amplification in Escherichia coli of ampicillin-resistance plasmids encoding ROB-1 beta-lactamase.

Thirty-five isolates of Pasteurella haemolytica from cattle or sheep were screened for the presence of plasmids and for resistance to a range of antibiotics. Eight strains (four of serotype A1, three of serotype A2 and one untypable) contained plasmid DNA and isolates of the same serotype had similar plasmid profiles, which were different from those of the other serotypes. All but one of the plasmid-bearing strains were isolated from pneumonic animals or from animals in contact with pneumonic cattle or sheep. In A2 and untypable strains, there was no obvious correlation between antibiotic resistance and the presence of a specific plasmid. In contrast, all plasmid-bearing A1 strains exhibited ampicillin resistance (ApR), which was shown by transfer studies to be plasmid-mediated. Plasmid DNA prepared from E. coli transformants was not routinely detected on ethidium-bromide-stained agarose gels, but could be amplified to detectable levels by treatment of cultures with chloramphenicol (Cm) or by modifying the growth conditions. The ApR plasmids from P. haemolytica were identical by restriction enzyme analysis. Restriction analysis and hybridization data indicated that these plasmids were closely related to the prototype ROB-1 beta-lactamase-encoding plasmid, originally isolated from Haemophilus influenzae. From substrate profiles and isoelectric focusing data, the beta-lactamases encoded by the P. haemolytica plasmids were indistinguishable from the ROB-1 beta-lactamase.     

Plasmid profiling of Vibrio salmonicida for epidemiological studies of cold-water vibriosis in Atlantic salmon (Salmo salar) and cod (Gadus morhua)

In 1988, a new plasmid profile was observed for Vibrio salmonicida isolated from cod (Gadus morhua) and Atlantic salmon (Salmo salar) in fish farms in northern Norway. This new plasmid profile, which consisted of plasmids of 61, 21, 3.4, and 2.8 megadaltons, is 1 of 11 plasmid profiles which have so far been observed for V. salmonicida. Plasmid profiling and plasmid DNA hybridization were used in epidemiological studies of cold-water vibriosis. Our results indicate that V. salmonicida was transmitted from Atlantic salmon to cod and vice versa. The 61-megadalton plasmid was found exclusively in V. salmonicida strains originating from northern Norway, which is the only area in which this plasmid has ever been observed. Plasmid DNA hybridization and restriction endonuclease analysis show that the plasmid DNA of V. salmonicida remained stable throughout a 7-year survey.     

Genetic diversity among A-proteins of atypical strains of Aeromonas salmonicida

The virulence array protein gene A (vapA) encoding the A-protein subunit of the surface layer of 23 typical and atypical strains of Aeromonas salmonicida from salmonids and marine fish species were sequenced, and the deduced A-protein sequences compared. The A-proteins of the typical A. salmonicida ssp. salmonicida strains were shown to be identical, while amino acid variability was revealed among A-proteins of atypical strains. The highest amino acid variability appears to be in a predicted surface exposed region and is believed to result in antigenic differences among the atypical strains of A. salmonicida.     

The development of random DNA probes specific for Aeromonas salmonicida

RAPD-PCR has been used to produce DNA probes for Aeromonas salmonicida . DNA hybridization studies showed that RAPD-PCR fragments of the same size did not necessarily hybridize to each other and therefore these sequences were not always homologous. However, a single RAPD-PCR fragment (designated 15e) was identified as being common to Aer. salmonicida . Subsequently, 15e was found to comprise five DNA fragments of similar size which differed in their nucleotide sequences. All five fragments were evaluated as DNA probes for the specific detection of Aer. salmonicida DNA: two hybridized specifically to DNA of all Aer. salmonicida isolates tested, including the four current subspecies and atypical isolates; one hybridized to subspecies salmonicida , achromogenes and masoucida , but not subspecies smithia ; one hybridized to subspecies salmonicida and achromogenes , but not subspecies masoucida or smithia ; and one hybridized to subspecies salmonicida , achromogenes and smithia , but not subspecies masoucida . It is believed that these fragments could be useful as non-radioactive probes for the safe and rapid diagnosis of these fish pathogens.     

Atypical strains of Aeromonas salmonicida contain multiple copies of insertion element ISAsa4 useful as a genetic marker and a target for PCR assay

The species Aeromonas salmonicida includes a quite complex group of pathogens that cause a variety of diseases in fishes. Best studied strains of this species are those of the subspecies salmonicida also referred to as 'typical' A. salmonicida, which cause furunculosis in salmonids. Less completely understood are bacteria assigned to other subspecies, e.g. achromogenes and masoucida, or those that cannot be assigned to a recognized subspecies. These strains are referred to collectively as 'atypical' A. salmonicida and cause diseases distinct from furunculosis, primarily affecting non-salmonids. In the course of a study to investigate the suitability of the gene product of tapA as a subunit vaccine, we discovered several atypical strains of A. salmonicida in which the tapA gene was interrupted by an insertion sequence (IS). Subsequent Southern blot analyses indicated that nearly all atypical strains (27 of 29) examined carry many copies of this IS, which we named ISAsa4. Genetic characterization of this IS element revealed it to be a member of the IS5 family, subgroup IS903. Aside from the presence of ISAsa4 in several atypical strains, the nucleotide sequence of tapA was virtually identical to that found in typical strains. This finding suggests that ISAsa4 might be a major source of genetic diversity among atypical strains which, unlike typical strains, are genetically heterogeneous. The presence of ISAsa4 in atypical strains may also help explain the host tropism of atypical strains of this bacterium. Using information on the nucleotide sequences of ISAsa4 from atypical strains of A. salmonicida, primers were designed to selectively amplify genomic DNA from most atypical strains.