In an effort to develop a rapid diagnostic test for the fish pathogen Aeromonas salmonicida, the performance of 2 polymerase chain reaction (PCR) primer sets (AP and PAAS) targeting the fish pathogen A. salmonicida and 1 PCR primer set (MIY) targeting A. salmonicida subsp. salmonicida were evaluated. Initially, the PCR assays were used to screen purified DNA extracted from 308 A. salmonicida isolates. The AP and PAAS PCR tests were demonstrated to be 100% specific for the species A. salmonicida and did not cross-react with any of the non-target organisms (bacterial species other than A. salmonicida) used in this study. The combined sensitivity of the AP and PAAS tests was 99.4% and offered the best coverage in terms of identifying the target organism. The MIY PCR appeared to be 100% sensitive and specific for A. salmonicida subsp. salmonicida. Studies with tissues, spiked with known quantities of bacteria, were conducted to determine the lower detection limit of the PCR tests, and then the ability of these PCR tests to detect A. salmonicida in experimentally infected salmonids was assessed.

Culture‐independent modes of analysis were chosen to investigate a microbial mat associated with the thermal waters of the Great Artesian Basin (GAB). 16S rDNA was amplified from total genomic mat DNA, and used to construct a clone library. Use of plasmid‐specific primers to amplify the inserts from 92 selected recombinants proved to be an effective approach, and demonstrated that there were four size categories of insert: 1500 bp (62% of clones examined), 1400 bp (25% of clones examined), 500 bp (5% of clones examined) and 240 bp (8% of clones examined). Restriction enzyme analysis was evaluated for its ability to group the 1500 bp and 1400 bp size inserts into operational taxonomic units. Clone inserts were presumptively identified by analysis of partial sequence data, and each operational taxonomic unit was found to be phylogenetically cohesive. Phylogenetic analyses of the sequence data indicated the presence of a broad range of bacteria related to the cyanobacteria, Thermus species, thiobacilli, planctomycetes, thermophilic hydrogen oxidisers, thermotogales, clostridia, actinomycetes, and β and δ subclasses of the proteobacteria. Use of the restriction enzyme analysis protocol also enabled the extent of insert repetition within the library to be monitored, and would thus have eliminated 70% of sequencing expenses. This is the first time a community analysis has been performed on this extreme environment.

Culture-independent modes of analysis were chosen to investigate a microbial mat associated with the thermal waters of the Great Artesian Basin (GAB). 16S rDNA was amplified from total genomic mat DNA, and used to construct a clone library. Use of plasmid-specific primers to amplify the inserts from 92 selected recombinants proved to be an effective approach, and demonstrated that there were four size categories of insert: 1500 bp (62% of clones examined), 1400 bp (25% of clones examined), 500 bp (5% of clones examined) and 240 bp (8% of clones examined). Restriction enzyme analysis was evaluated for its ability to group the 1500 bp and 1400 bp size inserts into operational taxonomic units. Clone inserts were presumptively identified by analysis of partial sequence data, and each operational taxonomic unit was found to be phylogenetically cohesive. Phylogenetic analyses of the sequence data indicated the presence of a broad range of bacteria related to the cyanobacteria, Thermus species, thiobacilli, planctomycetes, thermophilic hydrogen oxidisers, thermotogales, clostridia, actinomycetes, and L and N subclasses of the proteobacteria. Use of the restriction enzyme analysis protocol also enabled the extent of insert repetition within the library to be monitored, and would thus have eliminated 70% of sequencing expenses. This is the first time a community analysis has been performed on this extreme environment. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V.

Two Aeromonas salmonicida-specific polymerase chain reaction (PCR) tests and 1 A. salmonicida subsp. salmonicida-specific PCR test were used to screen salmonid populations that were either overtly or covertly infected with A. salmonicida subsp. salmonicida. It was demonstrated that these PCR assays could be used to replace the biochemical testing currently employed to confirm the identity of A. salmonicida isolates cultured from infected fish. The AP and PAAS PCR assays were also capable of direct detection of A. salmonicida in overtly infected fish, with mucus, gill and kidney samples most likely to yield a positive result. Culture was a more reliable method for the direct detection of A. salmonicida in covertly infected salmonids than was the direct PCR testing of tissue samples, with the AP and PAAS PCRs having a lower detection limit (LDL) of approximately 4 × 10 5 colony-forming units (CFU) g -1 sample.