DNA probes specific for Aeromonas hydrophila (HG1)

Aeromonas hydrophila (HG1)-specific RAPD-PCR fragments were investigated for their potential as DNA probes. From 20 RAPD-PCR fragment bands, it was found that two were specific to all isolates of Aeromonas hydrophila (HG1) tested. Cloning and nucleotide sequence determination of one of these bands showed that co-migration of similar sized amplicons had occurred and that this band (designated '7e') contained at least four fragments of different sequences. Three of these individual amplicons had a sequence specific to Aer. hydrophila (HG1) isolates. The sequence of one of these amplicons ('7e5') was used to design primers for a specific polymerase chain reaction (PCR). The specificity of the PCR was achieved using a modified hot-start procedure. The identity of the PCR amplicons was confirmed by high stringency hybridization with a digoxygenin-labelled 7e5 probe.     

The use of PCR to aid in the rapid identification of Vibrio harveyi isolates

AIMS: Vibrio harveyi is an important pathogen, causing potential devastation to marine aquaculture. This organism, however, is extremely difficult to identify because it is phenotypically diverse. Biochemical identification can involve many tests and take weeks to perform. The aim of this work is to develop a PCR that can reduce the number of biochemical tests, and the time taken, to get a definitive identification of this organism. METHODS AND RESULTS: The PCR was developed using 16S rDNA sequences from a number of V. harveyi strains, and other vibrios. The described test gave positive results for all strains of V. harveyi tested. However, some strains of V. alginolyticus also gave positive results and a small number of biochemical tests were required to differentiate between these two species. This indicated that preisolation of the bacteria was needed and therefore the test was not applicable to the testing of mixed populations directly. CONCLUSION, SIGNIFICANCE AND IMPACT OF THE STUDY: The duration of identification of this species was significantly reduced from a number of weeks to a few days. Hence, diagnosis of affected animals will be faster and earlier treatment can be administered which may increase the survival rate from vibriosis.     

A new bacteriophage, VHML, isolated from a toxin-producing strain of Vibrio harveyi in tropical Australia

Some strains of Vibrio harveyi are known to be pathogenic for fish and many invertebrates including crustaceans. Despite their importance, their modes of virulence have yet to be fully elucidated. Here, we present a previously unreported bacteriophage extracted from a toxin-producing strain of V. harveyi isolated from moribund prawn larvae in tropical Australia. Classification into the family Myoviridae was based upon morphological characteristics (an icosahedral head, a neck/collar region and a sheathed rigid tail) and nucleic acid characteristics (double-stranded linear DNA). We have termed the bacteriophage VHML (Vibrio Harveyi Myovirus Like). VHML is a temperate bacteriophage that has a narrow host range and shows an apparent preference for V. harveyi above other vibrios (63 Vibrio isolates tested) and other genera (10 other genera were tested). The conventional methods for phage concentration and extraction of nucleic acids from phage particles were not efficient and the alternative methods that were used are discussed.     

Detection of Chlamydiaceae DNA in veterinary specimens using a family-specific PCR

AIMS: The aim of this work was to develop a rapid molecular test for the detection of the Chlamydiaceae family, irrespective of the species or animal host. METHODS AND RESULTS: The method described herein is a polymerase chain reaction targeting the 16S rRNA gene of the Chlamydiaceae family, and the results demonstrate that the test reacts with five reference Chlamydiaceae but none of the 19 other bacterial species or five uninfected animal tissues tested. The results also indicate the enhanced sensitivity of this test when compared with conventional culture or serology techniques. This is demonstrated through parallel testing of six real clinical veterinary cases and confirmatory DNA sequence analysis. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: This test can be used by veterinary diagnostic laboratories for rapid detection of Chlamydiaceae in veterinary specimens, with no restriction of chlamydial species or animal host. The test does not differentiate chlamydial species, and if required, speciation must be carried out retrospectively using alternate methods. However, for the purpose of prescribing therapy for chlamydiosis, this test would be an invaluable laboratory tool.     

Joint modeling of additive and non-additive (genetic line) effects in multi-environment trials

A statistical approach for the analysis of multi-environment trials (METs) is presented, in which selection of best performing lines, best parents, and best combination of parents can be determined. The genetic effect of a line is partitioned into additive, dominance and residual non-additive effects. The dominance effects are estimated through the incorporation of the dominance relationship matrix, which is presented under varying levels of inbreeding. A computationally efficient way of fitting dominance effects is presented which partitions dominance effects into between family dominance and within family dominance line effects. The overall approach is applicable to inbred lines, hybrid lines and other general population structures where pedigree information is available.