Monoclonal antibodies against AsaP1, a major exotoxin of the fish pathogen Aeromonas salmonicida subsp. achromogenes, and their application in ELISA

Two monoclonal antibodies (Mabs) binding to a toxic extracellular metallo-proteinase of Aeromonas salmonicida subsp. achromogenes, AsaP1, were produced. Both reacted with common epitopes of the native enzyme and recognized this 20 kDa antigen on Western blots. One of these Mabs had an inhibitory effect on the caseinase activity of the exotoxin. A Mab-based ELISA was set up and evaluated for serological detection of AsaP1 in bacterial culture filtrates. The exotoxin was identified serologically in the extracellular products of 11 of 26 atypical Aer. salmonicida isolates, including the type strain for subsp. achromogenes NCIMB 1110. The ELISA was approximately 100-fold more sensitive in detecting AsaP1 compared with an azocasein assay. The established serological test enables AsaP1 to be quantified reliably with a lower detection limit of about 0.12 ng ml-1 and has a potential use for the phenotypic differentiation of atypical Aer. salmonicida isolates.     

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 .     

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.     

Genetic diversity of the fish pathogen Aeromonas salmonicida demonstrated by random amplified polymorphic DNA and pulsed-field gel electrophoresis analyses

The current taxonomy of Aeromonas salmonicida includes 4 subspecies. A. salmonicida subsp. salmonicida is associated with salmonid furunculosis, and A. salmonicida subsp. achromogenes, A. salmonicida subsp. masoucida, and A. salmonicida subsp. smithia are strains that show variation in some biochemical properties. This classification does not readily encompass isolates from a wide range of fish hosts currently described as atypical A. salmonicida. This study examined 17 typical strains, 39 atypical strains and 3 type A. salmonicida subspecies strains for genetic similarity using the random amplified polymophic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) techniques. On the basis of RAPD- and PFGE-derived profiles, similarity matrices and dendrograms were constructed. The results showed that species A. salmonicida constituted a genetically heterogeneous group of strains, encompassing within an homogeneous or clonal lineage comprised solely of typical strains and the A. salmonicida subsp. salmonicida type strain.     

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.     

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.     

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.     

Characterization of atypical Aeromonas salmonicida isolates by ribotyping and plasmid profiling

A total of 38 strains of atypical Aeromonas salmonicida , three oxidase-negative but otherwise typical Aer. salmonicida , three typical Aer. salmonicida , and two reference strains, isolated from several countries and fish species were examined with respect to rRNA gene restriction patterns (ribotypes) and plasmid profiles. Most epidemiologically unrelated strains had different ribotypes, whereas isolates from the same outbreak were identical. All strains, except one, carried one or more large plasmids (> 55 kbp) and all strains, except two, additionally carried one or more smaller plasmids. Many strains isolated from the same outbreak showed different plasmid profiles although some plasmids were identical. The results suggest the existence of several atypical Aer. salmonicida. It also seems that ribotypes are stable properties for these bacteria while the plasmids are more labile.     

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.     

Restriction endonuclease fingerprinting analysis of Canadian isolates of Aeromonas salmonicida

Restriction endonuclease fingerprinting (REF) analysis was used to examine total cellular DNA prepared from 56 independent field isolates of the fish pathogen, Aeromonas salmonicida. DNA was digested singly with the restriction enzymes EcoRI and HindIII, and the resulting fragments separated by polyacrylamide gel electrophoresis and visualized by silver staining. The REF patterns of typical isolates of A. salmonicida subsp. salmonicida were distinct from those of A. hydrophila, A. salmonicida subsp. achromogenes, A. salmonicida subsp. masoucida, and atypical isolates of A. salmonicida subsp. salmonicida. Differences between strains of typical A. salmonicida subsp. salmonicida could also be distinguished. Canadian isolates examined could be assigned to 1 of 12 different groups (REF groups), with the majority of the isolates belonging to REF groups 1 and 5. REF group 1 strains were isolated from British Columbia and New Brunswick while REF group 5 isolates were found in Ontario. None of the European strains examined had REF patterns identical to those of Canadian isolates. Based on REF analysis, there was little genetic heterogeneity detected among 23 isolates from two short-term studies of naturally occurring infections. Several different REF groups were seen among A. salmonicida collected over a 10-year period from coho salmon from the Credit River. Consistent with earlier biochemical and hybridization studies, the REF data suggest that A. salmonicida is a clonal pathogen. REF analysis can, however, permit the identification of subgroups, which may be useful in epidemiological studies.     

Pathogenicity of atypical Aeromonas salmonicida in Atlantic salmon compared with protease production

The pathogenicity of extracellular products (ECPs) from 24 atypical Aeromonas salmonicida strains was studied with respect to : lethality in Atlantic salmon, pathogenic effect in muscle, haemolytic activity, cytotoxicity in two fish cell lines and proteolytic activities. Furthermore, the relationship between lethality of ECPs and mortality caused by bacterial challenge was examined. Correlation was demonstrated between the pathogenic properties and proteolytic activities of the ECPs. Cytolytic (GCAT) activity comparable with that of the typical reference strain used (NCMB 1102) was not detected in ECPs of any of the atypical strain tested. An extracellular metallo-caseinase, AsaP1, was linked with lethal toxicity and a strong pathogenic effect. Furuncular-like lesions were produced by ECPs containing AsaP1 activity. One strain produced a lethal toxin which was neither caseinolytic nor with GCAT comparable activity. The examined atypical strains form at least three distinct groups based on different virulence mechanisms and extracellular proteases.     

Protease activation of the entomocidal protoxin of Bacillus thuringiensis subsp. kurstaki

Two isolates of Bacillus thuringiensis subsp. kurstaki were examined which produced different levels of intracellular proteases. Although the crystals from both strains had comparable toxicity, one of the strains, LB1, had a strong polypeptide band at 68,000 molecular weight in the protein from the crystal; in the other, HD251, no such band was evident. When the intracellular proteases in both strains were measured, strain HD251 produced less than 10% of the proteolytic activity found in LB1. These proteases were primarily neutral metalloproteases, although low levels of other proteases were detected. In LB1, the synthesis of protease increased as the cells began to sporulate; however, in HD251, protease activity appeared much later in the sporulation cycle. The protease activity in strain LB1 was very high when the cells were making crystal toxin, whereas in HD251 reduced proteolytic activity was present during crystal toxin synthesis. The insecticidal toxin (molecular weight, 68,000) from both strains could be prepared by cleaving the protoxin (molecular weight, 135,000) with trypsin, followed by ion-exchange chromatography. The procedure described gave quantitative recovery of toxic activity, and approximately half of the total protein was recovered. Calculations show that these results correspond to stoichiometric conversion of protoxin to insecticidal toxin. The toxicities of whole crystals, soluble crystal protein, and purified toxin from both strains were comparable.     

Polymerase chain reaction (PCR)-based typing analysis of atypical isolates of the fish pathogen Aeromonas salmonicida.

Two hundred and five isolates of atypical Aeromonas salmonicida, recovered from a wide range of hosts and countries were characterized by polymerase chain reaction (PCR) targeting four genes. The chosen genes were those encoding the extracellular A-layer protein (AP), the serine protease (Sprot), the glycerophospholipid:cholestrol acetyltransferase protein (GCAT), and the 16S rRNA (16S rDNA). All the atypical A. salmonicida isolates could be assigned to 4 PCR groups. Group 1 comprised 45 strains which tested positive for PCR amplification, using the 16S rDNA, GCAT2, Sprot2, and AP primer-sets. Group 2 comprised 88 strains with produced PCR products using the 16S rDNA, GCAT2 and AP primer-sets. Group 3 comprised 21 strains which produced PCR products using 16S rDNA, GCAT2 and Sprot2 primer-sets, and group 4 comprised 51 strains which produced PCR products using the 16S rDNA and GCAT2 primer-sets only. A. salmonicida subsp. salmonicida isolates tested, belonged to group 1. The PCR primer-sets separated A. salmonicida from other reference strains of Aeromonas species and related bacteria with the exception of Aeromonas hydrophila. The results indicated that PCR typing is a useful framework for characterization of the increasing number of isolations of atypical A. salmonicida.     

Protease activation of the entomocidal protoxin of Bacillus thuringiensis subsp. kurstaki.

Two isolates of Bacillus thuringiensis subsp. kurstaki were examined which produced different levels of intracellular proteases. Although the crystals from both strains had comparable toxicity, one of the strains, LB1, had a strong polypeptide band at 68,000 molecular weight in the protein from the crystal; in the other, HD251, no such band was evident. When the intracellular proteases in both strains were measured, strain HD251 produced less than 10% of the proteolytic activity found in LB1. These proteases were primarily neutral metalloproteases, although low levels of other proteases were detected. In LB1, the synthesis of protease increased as the cells began to sporulate; however, in HD251, protease activity appeared much later in the sporulation cycle. The protease activity in strain LB1 was very high when the cells were making crystal toxin, whereas in HD251 reduced proteolytic activity was present during crystal toxin synthesis. The insecticidal toxin (molecular weight, 68,000) from both strains could be prepared by cleaving the protoxin (molecular weight, 135,000) with trypsin, followed by ion-exchange chromatography. The procedure described gave quantitative recovery of toxic activity, and approximately half of the total protein was recovered. Calculations show that these results correspond to stoichiometric conversion of protoxin to insecticidal toxin. The toxicities of whole crystals, soluble crystal protein, and purified toxin from both strains were comparable.     

Small subunit rRNA gene sequences of Aeromonas salmonicida subsp. smithia and Haemophilus piscium reveal pronounced similarities with A. salmonicida subsp. salmonicida

The small subunit ribosomal RNA (SSU rRNA) encoding genes from reference strains of Aeromonas salmonicida subsp. smithia and Haemophilus piscium were amplified by polymerase chain reaction and cloned into Escherichia coli cells. Almost the entire SSU rRNA gene sequence (1505 nucleotides) from both organisms was determined. These DNA sequences were compared with those previously described from A. salmonicida subsp. salmonicida, subsp. achromogenes and subsp. masoucida. This genetic analysis revealed that A. salmonicida subsp. smithia and H. piscium showed 99.4 and 99.6% SSU rRNA gene sequence identity, respectively, with A. salmonicida subsp. salmonicida.     

Carbohydrate analysis and serological classification of typical and atypical isolates of Aeromonas salmonicida: a rationale for the lipopolysaccharide-based classification of A. salmonicida

The cell envelope of Aeromonas salmonicida contains a lipopolysaccharide (LPS) essential for the physical integrity and functioning of bacterial cell membrane. Using a recently developed in-source fragmentation technique, we screened 39 typical and atypical isolates of A. salmonicida and established their O-chain polysaccharide structure by capillary electrophoresis-mass spectrometry (CE-MS), compositional and linkage analyses and comparison to the previously determined O-chain polysaccharide structure of A. salmonicida strain A449. These studies have demonstrated that A. salmonicida isolates fall into three distinct structural types, types A-C, based on chemical structures of their respective O-chain polysaccharide components. Subsequent immunoblotting and serological studies with salmon polyclonal antisera produced to formalin-fixed cells of A. salmonicida strains A449, N4705 and 33659 representing three structural types A-C revealed that variations in the O-chain polysaccharide structure have led to significant serological differences between strains belonging to type A and non-type A, where non-type A species include chemically separated structural types B and C. Due to the presence of common antigenic determinants shared by their respective O-chain polysaccharide components, serological cross-reactions were observed between A. salmonicida strains belonging to structural types B and C. These findings suggest the possibility of developing LPS-based classification system of A. salmonicida sub-species consisting of two serologically distinct types, type A and non-type A.     

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.     

Pulsed-field gel electrophoresis in the study of mesophilic and psychrophilic Aeromonas spp.

Pulsed-field gel electrophoresis (PFGE) was used to study the genetic diversity of mesophilic Aeromonas hybridization group (HG) 1, HG 2, HG 3, HG 4, HG 5, HG 6, HG 7, HG 8/10and HG 11, psychrophilic Aeromonas salmonicida subsp. salmonicida and atypical Aerom. salmonicida strains. Xba I was chosen for restriction because it producedfragments whose numbers and size were appropriate for PFGE analysis of all studied HGs. Allmesophilic Aeromonas strains within an HG had different banding patterns. No sharedbands which could be used for identification of an HG were found. Pulsed-field gelelectrophoresis analysis further confirmed the known genetic homogeneity of Aerom.salmonicida subsp. salmonicida . Pulsed-field gel electrophoresis pattern analysissuggested that the genomic size of Aerom. salmonicida subsp. salmonicida issmaller than that of mesophilic Aeromonas spp. or atypical Aerom. salmonicida . Aeromonas salmonicida subsp. salmonicida had only one large restriction fragment (310kb) and lacked other large fragments (>160 kb). Although the PFGE patterns of atypical Aerom. salmonicida resembled the banding patterns of mesophilic Aeromonas spp.they had several small fragments (15–50 kb) shared with Aerom. salmonicida subsp. salmonicida suggesting genetic relatedness.     

Quorum sensing signals are produced by Aeromonas salmonicida and quorum sensing inhibitors can reduce production of a potential virulence factor

Many pathogens control production of virulence factors by self-produced signals in a process called quorum sensing (QS). We demonstrate that acyl homoserine lactone (AHL) signals, which enable bacteria to express certain phenotypes in relation to cell density, are produced by a wide spectrum of Aeromonas salmonicida strains. All 31 typical strains were AHL producers as were 21 of 26 atypical strains, but on a strain population basis, production of virulence factors such as protease, lipase, A-layer or pigment did not correlate with the production and accumulation of AHLs in the growth medium. Pigment production was only observed in broth under highly aerated conditions. Quorum sensing inhibitors (QSIs) are compounds that specifically block QS systems without affecting bacterial growth and 2 such compounds, sulphur-containing AHL-analogues, reduced production of protease in a typical strain of Aeromonas salmonicida. The most efficient compound N-(heptylsulfanylacetyl)-L-homoserine lactone (HepS-AHL), reduced protease production by a factor of 10. Five extracellular proteases were detected on gelatin-containing sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gels and 3 of these were completely down regulated by HepS-AHL. Hence, QSIs can curb virulence in some strains and could potentially be pursued as bacterial disease control measures in aquaculture.