J Carson

Bacterial decontamination of on-grown Artemia

The bacterial load of on-grown Artemia was manipulated using a variety of commercially available enrichment DHA boosters, selected algal species (Skeletonema costatum; Nannochloropsis oculata; Tetraselmis suecica; Chaetoceros muelleri), and ozone to decontaminate enteric and external surfaces, respectively. Enrichment in C. muelleri over a 6-h period, with an additional algal exchange mid-enrichment, provided the most efficient method for enteric decontamination as measured by total viable counts. Direct exposure to ozone at 4 ppm for 5 min provided further bacterial reduction, resulting in a combined bacterial load reduction of 99.5% without compromising Artemia viability. The commercial enrichment A1 DHA Selcok (TM), containing antibacterial compounds, provides an alternative to algal-based enrichments, however, its decontaminating properties were inferior. Fluorescence in situ hybridisation (FISH) was used on occasions to verify total bacterial abundance estimates obtained by standard plating procedures. In all except one case, Johnsons Marine Agar (JMA) provided results comparable to direct counts by FISH. This indicates that the bacterial community present in on-grown Artemia cultures is dominated by several fast-growing r-strategists amenable to culture on conventional plates. Underestimation of bacterial abundance using marine agar was therefore unlikely.

Rapid, high-throughput extraction of bacterial genomic DNA from selective-enrichment culture media.

Aims: To create a fast, sensitive and inexpensive high‐throughput method for the extraction of bacterial genomic DNA from selective‐enrichment culture media.

The histopathological effects of a levamisole-adjuvanted Vibrio anguillarum vaccine on Atlantic salmon (Salmo salar L.)

Abstract Atlantic salmon (Salmo salar L.) were administered a levamisole-adjuvanted or non-adjuvanted Vibrio anguillarum vaccine by either bath or intraperitoneal (IP) injection. The potential side effects of vaccination were investigated at a histological level, with the skin, gills, anterior kidney and spleen all examined. At a gross level, no pathological changes were evident in any organs. Observations of histological structures found that the skin, anterior kidney and spleen were not affected by vaccination. However pathological changes were observed in the gills of fish treated with the adjuvanted vaccine by both routes of administration. Pathology of the gills was multifocal and included hyperplasia of mucous cells, lamellar oedema, proliferation of chloride cells, inflammation and necrosis. Hyperplasia and hypertrophy of epithelial cells resulted in lamellar fusion. The abundance of lamellar associated mucous cells significantly increased in the gills of fish from groups administered the levamisole-adjuvanted vaccine (IP and bath) (P 0.05).

Miniaturized tests for computer‐assisted identification of motile Aeromonas species with an improved probability matrix

J. CARSON, T. WAGNER, T. WILSON AND L. DONACHIE. 2001.

Vibrio jasicida sp. nov., a member of the Harveyi clade, isolated from marine animals (packhorse lobster, abalone and Atlantic salmon)

Six isolates of a facultatively anaerobic bacterium were recovered in culture from marine invertebrates and vertebrates, including packhorse lobster (Jasus verreauxi), abalone (Haliotis sp.) and Atlantic salmon (Salmo salar), between 1994 and 2002. The bacteria were Gram-negative, rod-shaped and motile by means of more than one polar flagellum, oxidase-positive, catalase-positive and able to grow in the presence of 0.5-8.0% NaCl (optimum 3.0-6.0%) and at 10-37 °C (optimum 25-30 °C). On the basis of 16S rRNA gene sequence analysis and multilocus sequence analysis (MLSA) using five loci (2443 bp; gyrB, pyrH, ftsZ, mreB and gapA), the closest phylogenetic neighbours of strain TCFB 0772(T) were the type strains of Vibrio communis (99.8 and 94.6 % similarity, respectively), Vibrio owensii (99.8 and 94.1%), Vibrio natriegens (99.4 and 88.8%), Vibrio parahaemolyticus (99.4 and 90.3%), Vibrio rotiferianus (99.2 and 94.4%), Vibrio alginolyticus (99.1 and 89.3%) and Vibrio campbellii (99.1 and 92.3%). DNA-DNA hybridization confirmed that the six isolates constitute a unique taxon that is distinct from other known species of Vibrio. In addition, this taxon can be readily differentiated phenotypically from other Vibrio species. The six isolates therefore represent a novel species, for which the name Vibrio jasicida sp. nov. is proposed; the novel species is represented by the type strain TCFB 0772(T) ( = JCM 16453(T)  = LMG 25398(T)) (DNA G+C content 45.9 mol%) and reference strains TCFB 1977 ( = JCM 16454) and TCFB 1000 ( = JCM 16455).

Effects of copper-based antifouling treatment on the presence of Neoparamoeba pemaquidensis Page, 1987 on nets and gills of reared Atlantic salmon (Salmo salar)

Abstract Amoebic gill disease (AGD) is the main disease affecting the salmon industry in Australia. Little information is available on the epidemiology of AGD and the biology of Neoparamoeba pemaquidensis [Page, 1987], the disease-causing organism of AGD. In previous studies, N. pemaquidensis was found on biofouled netting of sea cages, and a reduction in AGD prevalence was achieved with increasing number of net changes. Presently, it is not known if N. pemaquidensis on netting is able to induce AGD. To reduce biofouling on nets, antifouling paints are commonly used on Tasmanian salmon farms. This study investigated the effects of a copper-based antifouling paint on the N. pemaquidensis densities on nets and the AGD prevalence of Atlantic salmon reared in these nets. Four sea cages stocked with 5–9 kg/m 3 Atlantic salmon were used in this study. Two nets were coated with a copper-based antifouling paint and two nets were not treated and used as a control. Fish were sampled every 2 weeks for 10 weeks. A gross gill score was determined and gill mucus samples were taken for dot blot analysis to determine the presence of N. pemaquidensis for each fish. Biofouling samples from netting were inoculated onto 75% malt yeast agar culture plates, and presence of N. pemaquidensis assessed using conventional culture techniques, followed by indirect immunofluorescent antibody test (IFAT). The presence of N. pemaquidensis was confirmed from culture-enriched biofouling samples from weeks 2 and 8 were tested using nested PCR. Results suggest that copper paint treated cages had significantly higher paramoebae ( P =0.002) and AGD ( P =0.014) prevalence compared to the control cages. No treatment effect was found on the intensity of infection, determined by gross gill scores ( P =0.243). At the end of the study, the paramoebae prevalence of net samples was 58.5% (S.E. 1.5) and AGD prevalence was 42.5% (S.E. 2.5) for copper treated nets, while no paramoebae were found on control nets and AGD prevalence was 35.0% (S.E. 5.0). Nets could be the source of N. pemaquidensis infection of fish with AGD, and therefore copper paint treated nets could be a risk factor for AGD.

Effect of extracellular products of Tenacibaculum maritimum in Atlantic salmon, Salmo salar L.

Keywords: Atlantic salmon, extracellular products, LD50, Tenacibaculum maritimum, toxins. Tenacibaculum maritimum (formerly Flexibacter maritimus) is a well-known pathogen in a number of cultured fish species worldwide (Wakabayashi, Hikida & Masumura 1986; Alsina & Blanch 1993; Chen, Henry-Ford & Groff 1995; Handlinger, Soltani & Percival 1997; Ostland, LaTrace, Morrison & Ferguson 1999). It is a marine bacterium that causes necrotic lesions on the body, head, fins and gills, with erosive lesions on the external surface as the prominent clinical sign (Carson, McCosh & Schmidtke 1992). In Australia, the main species affected are Atlantic salmon, Salmo salar L., and rainbow trout, Oncorhynchus mykiss (Walbaum), in sea-cage culture in Tasmania (Handlinger et al. 1997). Experimental investigation into the pathogenesis of T. maritimum showed that challenge at higher doses (c. 1 · 108 cells mL)1) had an acute lethal effect on Atlantic salmon (van Gelderen 2007). Mortalities occurred within days and the clinical sign was the disintegration of the epithelium. In addition, a lack of an inflammatory response is characteristic of early flexibacteriosis lesions. Handlinger et al. (1997) suggested that this was the result of powerful exotoxins that prevent a host response. These findings pointed to a possible role of toxins in the pathogenicity of T. maritimum in Atlantic salmon. Effects of T. maritimum toxins have been explored in red and black sea bream, Pagrus major (Temminck and Schlegel), and Acanthopagrus schlegeli (Bleeker) (Baxa, Kawai & Kusuda 1988). In both fish species, extracellular products (ECP) showed insignificant in vitro activity; however, this did not correspond with the toxic effects observed in vivo with ECP recording the lowest LD50. Baxa et al. (1988) did indicate that the pathogenicity of T. maritimum in black and red sea bream may be ascribed in part to ECP. The current study investigated ECP toxicity in vivo to observe direct effects rather than in vitro activity of different toxins. Further, this study provides the first observations of T. maritimum ECP toxicity in Atlantic salmon.

Optimisation of one-tube PCR-ELISA to detect femtogram amounts of genomic DNA

A simple, high-throughput, low-cost polymerase chain reaction-enzyme linked immunosorbent assay (PCR-ELISA) protocol that detects the presence of 4 fg of DNA from four bacterial fish pathogens Yersinia ruckeri, Tenacibaculum maritimum (formerly Flexibacter maritimus), Lactococcus garvieae and Aeromonas salmonicida was developed. DNA amplification was undertaken in a biphasic system with free and bound PCR that are achieved in the one NucleoLink tube. Solid-phase amplicons were detected using biotin labelled hybridization probes and visualised colourimetrically with streptavidin-alkaline phosphatase and p-nitrophenylphosphate as substrate. PCR and hybridization took less than 8 h to perform with maximum signal output for femtogram amounts of template DNA achieved within 24 h. Implementation and optimization of the protocol is discussed.

Culturable microbiota of ranched southern bluefin tuna (Thunnus maccoyii Castelnau)

The Australian tuna industry is based on the ranching of wild southern bluefin tuna (SBT, Thunnus maccoyii). Within this industry, only opportunistic pathogens have been reported infecting external wounds of fish. This study aimed to identify different culturable bacteria present in three cohorts of SBT and to determine normal bacteria and potential pathogens in isolates from harvest fish and moribund/dead fish. Post‐mortem changes in the microbiota were also studied.

Quality control ranges for testing broth microdilution susceptibility of Flavobacterium columnare and F. psychrophilum to nine antimicrobials

A multi-laboratory broth microdilution method trial was performed to standardize the specialized test conditions required for the fish pathogens Flavobacterium columnare and F. psychrophilum. Nine laboratories tested the quality control (QC) strains Escherichia coli ATCC 25922 and Aeromonas salmonicida subsp. salmonicida ATCC 33658 against 10 antimicrobials (ampicillin, enrofloxacin, erythromycin, florfenicol, flumequine, gentamicin, ormetoprim/sulfadimethoxine, oxolinic acid, oxytetracycline, and trimethoprim/sulfamethoxazole) in diluted (4 g l-1) cation-adjusted Mueller-Hinton broth incubated at 28 and 18°C for 44-48 and 92-96 h, respectively. QC ranges were set for 9 of the 10 antimicrobials. Most of the minimal inhibitory concentration (MIC) distributions (16 of 18, 9 drugs at both temperatures) for A. salmonicida ATCC 33658 were centered on a single median MIC ± 1 two-fold drug dilution resulting in a QC range that spanned 3 dilutions. More of the E. coli ATCC 25922 MIC distributions (7 of 16) were centered between 2 MIC dilutions requiring a QC range that spanned 4 dilutions. A QC range could not be determined for E. coli ATCC 25922 against 2 antimicrobials at the low temperature. These data and their associated QC ranges have been approved by the Clinical and Laboratory Standards Institute (CLSI), and will be included in the next edition of the CLSI M49-A Guideline. This method represents the first standardized reference method for testing fish pathogenic Flavobacterium spp.