Andrew J. Evenden

Qualitative assessment of genotoxicity using random amplified polymorphic DNA: Comparison of genomic template stability with key fitness parameters in Daphnia magna exposed to benzo[a]pyrene

A method of DNA profiling using the random amplified polymorphic DNA (RAPD) was used to assess toxicant-induced DNA effects in laboratory populations of Daphnia magna exposed to varying concentrations of the genotoxic hydrocarbon benzo[a]pyrene. These effects, represented by changes in the RAPD profiles, were compared with a number of key ecological fitness parameters (age-specific survival, age-specific fecundity, net reproductive rate, and intrinsic rate of population increase). Not only was the RAPD profiling method shown to be a rapid and reproducible assay of toxicant-induced DNA effects, but the qualitative measure of genomic template stability compared favorably with the traditional indices of fitness. The RAPD profiles, however, exhibited higher sensitivity in detecting toxic effects. The significance of these findings for future ecotoxicological studies is discussed.

Comparison of ultraviolet-induced genotoxicity detected by random amplified polymorphic DNA with chlorophyll fluorescence and growth in a marine macroalgae, Palmaria palmata.

The random amplified polymorphic DNA (RAPD) technique was used to detect DNA damage in the sublittoral macroalgae Palmaria palmata (Rhodophyta) exposed to both ambient and elevated irradiances of UV-B (280-315 nm). To investigate the potential of this method in ecotoxicological assessments, the qualitative and quantitative modifications in RAPD profiles were compared with changes in a number of physiological and fitness parameters. RAPD detectable modifications in DNA profiles were observed in all UV exposed individuals compared with controls. Changes in chlorophyll fluorescence (F(v)/F(m) ratio), in vivo pigment absorptance, thallus growth and RAPD profiles, examined simultaneously, provided a sensitive measure of UV-induced toxicity. In conclusion, the application of the RAPD method in conjunction with other suitable physiological and fitness measurements, may prove to be a valuable tool for investigating the specific effects of genotoxic agents upon marine algal populations. Ultimately, this methodology may allow the ecotoxicological examination of the link between molecular alterations and measurable adverse effects at higher levels of biological organisation.

Renibacterium salmoninarum and bacterial kidney disease — the unfinished jigsaw

Abstract Bacterial kidney disease (BKD) is one of the most important diseases of wild and cultured salmonid fish, and has been reported from many different countries. Despite considerable effort, many pieces are still missing from the “jigsaw puzzle” which represents our knowledge of the disease, and its etiological agent Renibacterium salmoninarum . The purpose of this review is to consider current knowledge about this bacterial pathogen and the pathogenesis of BKD. It is our intention to construct a picture of the possible ways in which pathogen and host interact, in particular by high-lighting the areas where our understanding is poor and indicating how recent advances in methodology offer the prospect of improvement. The exact status of R. salmoninarum as an obligate fish pathogen, and the implications for transmission, entry, colonization, and disease progression are considered. We review the considerable progress that has been made recently through work on the molecular determinants of pathogenicity, particularly attachment, cellular invasion mechanisms, intracellular survival of the bacterium, and its interactions with the host defense mechanisms and immune system. Finally, we consider ways in which the future control of BKD through improved diagnosis, chemotherapy, and vaccination may be realized through an integrated approach to the study of BKD.

Host responses to Renibacterium salmoninarum and specific components of the pathogen reveal the mechanisms of immune suppression and activation

During infection, Renibacterium salmoninarum survives within the pronephric macrophages of salmonid fish. Therefore, to study the initial phases of the interaction we infected macrophages with live bacteria and analysed the responses of host and pathogen. It was found that the expression of msa encoding the p57 antigen of R. salmoninarum, was constitutive, while the expression of hly and rsh, encoding haemolysins, and lysB and grp was reduced after infection. Macrophages showed a rapid inflammatory response in which the expression of interleukin‐1β (IL‐1β), major histocompatibility complex class II (MHC II), inducible cyclo‐oxygenase (Cox‐2), and inducible nitric oxide synthase (iNOS) was enhanced, but tumour necrosis factor‐α (TNF‐α) expression was greatly reduced initially and then increased. After 5 days, except for TNF‐α and MHC II, expression returned to levels approaching those of uninfected macrophages. We propose that R. salmoninarum survives initial contact with macrophages by avoiding and/or interfering with TNF‐α‐dependent killing pathways. The effects of specific R. salmoninarum components were studied in vivo by injecting fish with DNA vaccine constructs expressing msa, hly, rsh, lysB, or grp. We found that msa reduced the expression of IL‐1β, Cox‐2, and MHC II but stimulated TNF‐α while hly, rsh and grp stimulated MHC II but down‐regulated TNF‐α. Constructs expressing hly or lysB stimulated iNOS expression and additionally, lysB stimulated TNF‐α. The results show how p57 suppresses the host immune system and suggest that the immune mechanisms for the containment of R. salmoninarum infections rely on MHC II‐ and TNF‐α‐dependent pathways. Moreover, prolonged stimulation of TNF‐α may contribute to the chronic inflammatory pathology of bacterial kidney disease.

Use of the random amplified polymorphic DNA (RAPD) assay for the detection of DNA damage and mutations: possible implications of confounding factors.

The aim of this study was to evaluate the potential of the random amplified polymorphic DNA (RAPD) assay to qualitatively detect the kinetics of benzo[a]pyrene (B[Ma]P)-induced DNA effects in the water flea Daphnia magna exposed to 25 and 50 μg l-1 B[a]P for 7 and 6 days, respectively. Mortality was recorded on a daily basis in both experiments, and RAPD analysis was performed on samples collected every day following isolation of genomic DNA. The main changes occurring in RAPD profiles produced by the population of Daphnia magna exposed to 25 and 50μg l-1 B[a]P was a decrease and increase in band intensity, respectively. Most of the changes occurring in the RAPD patterns were likely to be the result of B[a]P-induced DNA damage (B[a]P DNA adducts, oxidized bases, DNA breakages) and/or mutations (point mutations and large rearrangements). In addition, reproducible changes also occurred in the profiles generated by control Daphnia magna. The results lead us to suggest that, in addition to B[a]P-induced DNA damage and mutations, factors such as variation in gene expression, steady levels of genetic alterations and changes in metabolic processes could induce some changes in RAPD patterns. Nevertheless, our data suggest that DNA damage and mutations appear to be the main factors influencing RAPD patterns. This study also emphasizes that unexpected variation in control profiles is not always associated with artefacts.

Application of the arbitrarily primed polymerase chain reaction for the detection of DNA damage

Abstract The technique of arbitrarily primed polymerase chain reaction (AP-PCR) shows potential as a selective and sensitive assay for the detection of xenobiotic-induced DNA damage. Problems, however, may occur in AP-PCR, diminishing its discriminative abilities. These problems include the presence of spurious amplification products in non-template-containing negative control reactions, and a lack of reproducibility amongst amplification patterns. Experiments designed to remove contaminated nucleic acids by ultraviolet (UV) treatment indicated that spurious bands are the result of aberrant primer-induced polymerisation, an event shown to be influenced by the concentration of deoxynucleotide triphosphates (dNTP) present in the reaction mixtures. Optimisation of dNTP concentration from 0.22 to 0.33 m m resulted in clear negative controls and highly reproducible amplification patterns with all DNA templates. As an example of the application of the method, in the present study, the macroalga Palmaria palmata (Rhodophyta) was exposed to UV A and B radiations. The study shows that the AP-PCR method can detect DNA damage and may be useful in detecting such damage following exposure of cells to xenobiotics.

A gene from Renibacterium salmoninarum encoding a product which shows homology to bacterial zinc-metalloproteases

A genomic library constructed from Renibacterium salmoninarum isolate MT444 DNA in the plasmid vector pBR328 was screened using Escherichia coli host strain DH1 for the expression of genes encoding putative virulence factors. A single haemolytic clone was isolated at 22 degrees C and found to contain a 3.1 kb HindIII fragment of inserted DNA. This fragment was present in seven isolates of R. salmoninarum which were examined. Western blots of extracts from clones exhibiting haemolytic activity were performed with antisera raised against either cellular or extracellular components of R. salmoninarum and failed to identify any additional proteins compared to control E. coli containing pBR328. However, minicell analysis revealed that a polypeptide with an apparent molecular mass of 65 kDa was associated with a haemolytic activity distinct from that previously described for R. salmoninarum. The nucleotide sequence of the gene encoding this product was determined and the amino acid sequence deduced. The product was 548 amino acids with a predicted molecular mass of 66757 Da and a pl of 5.57. The deduced amino acid sequence of the gene possessed strong similarities to those of a range of secreted bacterial zinc-metalloproteases and was tentatively designed hly. Neither protease nor lecithinase activities were detectable in E. coli recombinants expressing gene hly. Haemolytic activity was observed from 6 degrees C to 37 degrees C for erythrocytes from a number of mammalian species and also from fish. Gene hly was expressed in E. coli as a fusion protein consisting of maltose-binding protein at the N-terminus linked to all but the first 24 amino acids, largely constituting the putative signal peptide, of the N-terminus of Hly. The soluble fusion protein was produced and purified by affinity chromatography. Antiserum raised against the purified fusion protein was used to probe Western blots of cell lysates and extracellular products from seven isolates of R. salmoninarum cultured under conditions of iron-sufficiency or iron-restriction. The results indicate that the availability of iron modulates the expression of the hly gene.