Heme requirement for growth of fastidious atypical strains of Aeromonas salmonicida

The growth of fastidious atypical strains of the fish pathogen Aeromonas salmonicida on both solid and liquid media was dependent specifically on a source of heme which was apparently required for initiation of growth at low inoculum densities. Thus, hemin enhanced the plating efficiencies of such strains on solid medium and significantly reduced their inoculum-size-dependent lag times in broth. The heme requirement could also be satisfied by hematoporphyrin and, less effectively, by hemoglobin. Since the requirement was a stable property of all 17 strains tested, it may prove to be another taxonomic criterion by which the atypical strains can be differentiated from the typical strains of A. salmonicida.     

Heme requirement for growth of fastidious atypical strains of Aeromonas salmonicida.

The growth of fastidious atypical strains of the fish pathogen Aeromonas salmonicida on both solid and liquid media was dependent specifically on a source of heme which was apparently required for initiation of growth at low inoculum densities. Thus, hemin enhanced the plating efficiencies of such strains on solid medium and significantly reduced their inoculum-size-dependent lag times in broth. The heme requirement could also be satisfied by hematoporphyrin and, less effectively, by hemoglobin. Since the requirement was a stable property of all 17 strains tested, it may prove to be another taxonomic criterion by which the atypical strains can be differentiated from the typical strains of A. salmonicida.     

Genetic diversity among Lassa virus strains

The arenavirus Lassa virus causes Lassa fever, a viral hemorrhagic fever that is endemic in the countries of Nigeria, Sierra Leone, Liberia, and Guinea and perhaps elsewhere in West Africa. To determine the degree of genetic diversity among Lassa virus strains, partial nucleoprotein (NP) gene sequences were obtained from 54 strains and analyzed. Phylogenetic analyses showed that Lassa viruses comprise four lineages, three of which are found in Nigeria and the fourth in Guinea, Liberia, and Sierra Leone. Overall strain variation in the partial NP gene sequence was found to be as high as 27% at the nucleotide level and 15% at the amino acid level. Genetic distance among Lassa strains was found to correlate with geographic distance rather than time, and no evidence of a "molecular clock" was found. A method for amplifying and cloning full-length arenavirus S RNAs was developed and used to obtain the complete NP and glycoprotein gene (GP1 and GP2) sequences for two representative Nigerian strains of Lassa virus. Comparison of full-length gene sequences for four Lassa virus strains representing the four lineages showed that the NP gene (up to 23.8% nucleotide difference and 12.0% amino acid difference) is more variable than the glycoprotein genes. Although the evolutionary order of descent within Lassa virus strains was not completely resolved, the phylogenetic analyses of full-length NP, GP1, and GP2 gene sequences suggested that Nigerian strains of Lassa virus were ancestral to strains from Guinea, Liberia, and Sierra Leone. Compared to the New World arenaviruses, Lassa and the other Old World arenaviruses have either undergone a shorter period of diverisification or are evolving at a slower rate. This study represents the first large-scale examination of Lassa virus genetic variation.     

Assessment of genetic variability and relatedness among atypical Aeromonas salmonicida from marine fishes,using AFLP-fingerprinting

Atypical strains of Aeromonas salmonicida are the causal agent of atypical furunculosis or ulcer disease in various fish species, including spotted wolffish Anarhichas minor, which is a promising species in the Norwegian fish-farming industry. Isolates of atypical A. salmonicida comprise a very heterogenous group showing large variety in biochemical, molecular and virulence characteristics. The genetic variability among atypical isolates from wolffish was characterised using amplified fragment length polymorphism analysis: AFLP-fingerprinting. Additional isolates from halibut Hippoglossus hippoglossus, turbot Scophthalmus maximus, cod Gadus morhua and several salmonid fishes were included for assessment of variability and relatedness among a total of 56 atypical isolates of A. salmonicida. They were compared to reference strains of A. salmonicida subspecies and to other Aeromonas species pathogenic in fishes. AFLP-fingerprints subjected to similarity analysis yielded a grouping of the isolates into several clusters, revealing genetic heterogeneity among the isolates. There seems to be a correlation between genetic similarity among isolates and the fish host. The Icelandic isolates, mainly from cod, formed a very homogeneous subcluster, which was closely related to the wolffish isolates. All atypical isolates from spotted and common wolffish grouped together in a large cluster and appear to be very homogeneous, even though they had been isolated over a period of 8 yr at different locations in Norway. On the other hand, most of the isolates from turbot and halibut grouped together into 2 different clusters, while the 9 atypical isolates from salmonids appeared in 4 different clusters. Thus, the atypical isolates of A. salmonicida from halibut, turbot and salmonid fishes seem to be more genetically diverse than those from wolffish and cod.     

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.     

Genetic diversity among Botulinum Neurotoxin-producing clostridial strains

Clostridium botulinum is a taxonomic designation for many diverse anaerobic spore-forming rod-shaped bacteria that have the common property of producing botulinum neurotoxins (BoNTs). The BoNTs are exoneurotoxins that can cause severe paralysis and death in humans and other animal species. A collection of 174 C. botulinum strains was examined by amplified fragment length polymorphism (AFLP) analysis and by sequencing of the 16S rRNA gene and BoNT genes to examine the genetic diversity within this species. This collection contained representatives of each of the seven different serotypes of botulinum neurotoxins (BoNT/A to BoNT/G). Analysis of the16S rRNA gene sequences confirmed previous identifications of at least four distinct genomic backgrounds (groups I to IV), each of which has independently acquired one or more BoNT genes through horizontal gene transfer. AFLP analysis provided higher resolution and could be used to further subdivide the four groups into subgroups. Sequencing of the BoNT genes from multiple strains of serotypes A, B, and E confirmed significant sequence variation within each serotype. Four distinct lineages within each of the BoNT A and B serotypes and five distinct lineages of serotype E strains were identified. The nucleotide sequences of the seven toxin genes of the serotypes were compared and showed various degrees of interrelatedness and recombination, as was previously noted for the nontoxic nonhemagglutinin gene, which is linked to the BoNT gene. These analyses contribute to the understanding of the evolution and phylogeny within this species and assist in the development of improved diagnostics and therapeutics for the treatment of botulism.     

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.     

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.     

Colonization of turbot tissues by virulent and avirulent Aeromonas salmonicida subsp. salmonicida strains during infection

Preventing disease outbreaks in cultured turbot Psetta maxima L. caused by Aeromonas salmonicida subsp. salmonicida (ASS) requires a better understanding of how this pathogen colonizes its host. Distribution of 1 virulent and 2 avirulent ASS strains in turbot tissues was investigated during early and late stages of infection following an immersion challenge. To track bacteria within the turbot, the ASS strains were tagged with green fluorescent protein (GFP). Both virulent and avirulent strains colonized the epidermal mucus, gills, and intestine within the first 12 h post challenge, suggesting that these sites may serve as points of entry into turbot. Although the avirulent strains colonized these initial sites in the turbot tissues, they were rarely found in the internal organs and were cleared from the host 4 d post challenge. In contrast, the virulent ASS strain was found in the liver and kidney as early as 12 h post challenge and was found in the muscle tissue at very late stages of infection. The virulent strain persisted in all tested host tissues until death occurred 7 d post challenge, suggesting that ASS must colonize and survive within the turbot tissues for an infection to result in death of the fish. Comparisons of the distribution profiles of both virulent and avirulent strains during early and late stages of an infection in turbot has provided important information on the route and persistence of an ASS infection in this host.     

Virulence factors of Aeromonas salmonicida subsp. salmonicida strains associated with infections in turbot Psetta maxima

Virulence factors for Aeromonas salmonicida subsp. salmonicida (ASS) strains isolated from cultured turbot Psetta maxima L. are unknown with regard to this host. The presence of virulence genes associated with different stages of ASS infection in salmonids (vapA, tapA, fla, ascV, ascC, aexT, satA and aspA) was analysed using a polymerase chain reaction (PCR) technique in ASS strains isolated from turbot. Other ASS strains isolated from salmonids and environmental A. salmonicida (AS) strains were included for comparison. The presence of the genes was evaluated with respect to ASS virulence in turbot based on intraperitoneal and bath challenges. The genetic profile, including all of the genes studied, that was linked to virulent behaviour after intraperitoneal challenge was significantly more frequent in strains isolated from turbot than in those from salmonids or the environment. The data prove that it is not possible to predict the virulence of ASS in turbot based only on the presence of all genes tested. Moreover, the combined PCR results of vapA, aexT, ascV and ascC were useful for separating most of the ASS from environmental A. salmonicida strains. An association between virulence or genetic profile and the geographical or facility origin of the strains was not found.     

Characterization of pRAS1-like plasmids from atypical North American psychrophilic Aeromonas salmonicida

Atypical psychrophilic Aeromonas salmonicida isolates were obtained from farmed and wild fish in Northeastern North America. These bacteria were isolated between 1992 and 2001 and carried tetracycline resistance (Tc(r)) plasmids of approximately 58 kb. The nine isolates had plasmids which could be divided into four groups based on the specific tetracycline resistance (tet) gene carried [tet(A) or tet(B)], incompatibility of the plasmid [IncU or other], whether the plasmid carried the IS6100 sequences, the sul1 gene, coding for sulfonamide resistance, the dfrA16 gene, coding for trimethoprim resistance, and/or carried a complete Tn1721, and their ability to transfer their Tc(r) plasmids to an Escherichia coli recipient at 15 degrees C. Five of the isolates, with genetically related Tc(r) plasmids, were able to transfer their plasmids to an E. coli recipient at frequencies ranging from 5.7x10(-4) to 2.8x10(-6) per recipient. The 1992 isolate carried a genetically distinct plasmid, which transferred at a slightly higher rate. The three remaining isolates carried one of two genetically different plasmids, which were unable to transfer to an E. coli recipient. Conjugal transfer at 15 degrees C is the lowest temperature that has been documented in bacteria.     

Characterization of Aeromonas salmonicida subsp. salmonicida: a comparative study of strains of different geographic origin

A total of 130 strains of the fish pathogen Aeromonas salmonicida isolated in Denmark, Norway, Scotland, Canada and the USA were examined. The strains originated from farmed salmonid fish. The biochemical, physiological and serological characteristics, antibiotic resistance patterns and cell surface-related properties were compared. Aeromonas salmonicida was found to be remarkably consistent in general cultural and biochemical characteristics. It is noteworthy that the strains were positive in the fermentation of L-arabinose and were negative in the fermentation of D-arabinose. All the strains were highly proteolytic. It was observed, however, that 5% of the strains did not digest calf and trout serum and the production of haemolysin and degradation of casein by the same strains were delayed compared with the other strains. Common to all of the rough strains were auto-aggregation and ability to bind the dyes Coomassie brilliant blue and Congo red and the majority of these strains were highly hydrophobic. The strains were tested for their susceptibility to 22 antibacterial agents. Antibiotic resistance profiles of Aer. salmonicida indicated that resistance to the quinolones and oxytetracycline was increasing and that multi-resistant strains were found in several countries. The variation found in antibiograms could have potential as epidemiological markers in certain geographic areas.     

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.     

Electrostatic mechanism of survival of virulent Aeromonas salmonicida strains in river water.

The ecological mechanism of survival of Aeromonas salmonicida, the bacterial pathogen of fish furunculosis, in river water was investigated by laboratory-based experiments with two virulent strains (which were autoagglutinating) and two virulent strains (which were nonagglutinating). A difference in net electrical charge of A. salmonicida cells was detected by electrophoresis; cells of the virulent strains were negative, whereas cells of the avirulent strains were positive. Despite the loss of viable cells within a week in distilled water and physiological saline (0.85% sodium chloride), the cells of the virulent strains survived for more than 15 weeks in the presence of diluted humic acid (10 micrograms/ml), tryptone (10 micrograms/ml), and cleaned river sand (100 g/100 ml of medium), but loss of viable cells occurred within 5 weeks in the absence of sand. The cells of the avirulent strains lost viability within 2 weeks with no relation to the presence of sand. Using ion-exchange columns, humic acid and the amino acids of tryptone were found to be anionic and cationic in water (pH 7.0), respectively. Sand particles had a high capacity to adsorb humic acid alone and amino acid-humic acid complexes. Thirty to fifty times the environmental concentration of amino acids (10 micrograms/ml) were accumulated on the surface of sand particles, thereby permitting only bacterial cells carrying net negative electrical charges (virulent cells) to survive for a long period on the surface of the sand particles. These electrostatic interrelationships among river sand, humic acid, and bacterial cells are closely implicated in the mechanism of long-term survival of virulent A. salmonicida in river sediments.     

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.     

Molecular characterization and quantitative analysis of superoxide dismutases in virulent and avirulent strains of Aeromonas salmonicida subsp. salmonicida

Aeromonas salmonicida subsp. salmonicida is a facultatively intracellular gram-negative bacterium that is the etiological agent of furunculosis, a bacterial septicemia of salmonids that causes significant economic loss to the salmon farming industry. The mechanisms by which A. salmonicida evades intracellular killing may be relevant in understanding virulence and the eventual design of appropriate treatment strategies for furunculosis. We have identified two open reading frames (ORFs) and related upstream sequences that code for two putative superoxide dismutases (SODs), sodA and sodB. The sodA gene encoded a protein of 204 amino acids with a molecular mass of approximately 23.0 kDa (SodA) that had high similarity to other prokaryotic Mn-SODs. The sodB gene encoded a protein of 194 amino acids with a molecular mass of approximately 22.3 kDa that had high similarity to other prokaryotic Fe-SODs. Two enzymes with activities consistent with both these ORFs were identified by inhibition of O(2)(-)-catalyzed tetrazolium salt reduction in both gels and microtiter plate assays. The two enzymes differed in their expression patterns in in vivo- and in vitro-cultured bacteria. The regulatory sequences upstream of putative sodA were consistent with these differences. We could not identify other SOD isozymes such as sodC either functionally or through data mining. Levels of SOD were significantly higher in virulent than in avirulent strains of A. salmonicida subsp. salmonicida strain A449 when cultured in vitro and in vivo.     

Changes of iron content in eels (Anguilla japonica) infected with atypical Aeromonas salmonicida

1. Hb, MCHC, Na, Ca, Mg and Cu in plasma decreased with atypical Aeromonas salmonicida infection. Whilst plasma K and NPN increased.2. Plasma Fe decreased markedly with infection. No statistically significant change in UIBC was found, though moribund eels showed slightly lower values compared to that of the control. The rate of decrease in TIBC is similar to that in most elements in plasma.3. Fe content in most tissues decreased with infection. It increased in the gills and head muscle (infection site). No significant change was found in the intestine.4. Water content in various tissues increased with infection.     

Genetic diversity among Rhizobium strains from Cicer arietinum L.

Following bacteriological cloning and determination of their symbiotic performance, 15 representative, diverse strains of chickpea rhizobia were genetically analysed for restriction fragment length polymorphisms. Analyses of genomic DNAs showed several different groups. Almost half (7) of the strains examined were very similar and clearly represented a single species. There was a related group of four strains which could be a subspecies. There was also one distinct group of four strains which were apparently unrelated to the reference strain 3377. This latter group may constitute a separate species. Phenotypic differences should be investigated further.