Experimental induction of gill disease in Atlantic salmon Salmo salar smolts with Tenacibaculum maritimum

Abstract

An experimentally induced bacterial infection of marine Atlantic salmon Salmo salar smolt gills was developed using strains of Tenacibaculum maritimum originally isolated from disease outbreaks in Tasmania. The gills of salmon were inoculated with a high concentration of bacteria (4 × 10 11 cells per fish) of either strain 00/3280 or 89/4747 T. maritimum. Gentle abrasion of the gills was used to enhance the progression of gill disease. One strain (00/3280) was highly pathogenic, causing morbidity and mortality within 24 h post-inoculation, and produced acute focal branchial necrosis associated with significant increases in plasma osmolality and lactate concentration compared with controls (non-inoculated) or strain 89/4747-inoculated fish. There were no differences in the whole body net ammonium flux between control (non-inoculated) and strain 00/3820-inoculated fish. Gill abrasion resulted in acute telangiectasis and focal lamellar hyperplasia in all fish regardless of bacterial inoculation. This work provides the basis of a challenge model suitable for investigating the pathophysiological processes associated with acute branchial necrosis in marine fish, suggesting that osmoregulatory and possibly respiratory dysfunction are the primary consequences of infection. KEY WORDS: Atlantic salmon · Tenacibaculum maritimum · Pathophysiology · Gill disease · Osmoregulation · Respiration Resale or republication not permitted without written consent of the publisherDis Aquat Org 61: [179][180][181][182][183][184][185] 2004 MATERIALS AND METHODS Preparation of bacterial cultures. Cultures ofTenacibaculum maritimum were isolated by the Department of Primary Industry, Water and Environment from the skin of farmed salmon from Tasmania, Australia, with clinical cases of cutaneous erosion disease. The cultures were designated 89/4747 (Atlantic salmon) and 00/3280 (rainbow trout) and were isolated in 1989 and 2000 respectively. The bacteria were isolated on the medium of Anacker & Ordal (1959), formulated with seawater. Isolates were identified using a 16S ribosomal RNA (rRNA) PCR primer set specific for T. maritimum (Carson 1998). Cultures were stored frozen at -80°C on MicroBank beads (Pro-Lab Diagnostics) until required.Cultures for infection trials were prepared by inoculating 200 ml of Shieh's medium (Song et al. 1988) formulated with seawater (mineral salts buffer, MSB) in a 1 l conical flask and incubated with gentle agitation (30 cycles min -1 ) at 20 to 22°C for 48 h. The cell suspension was harvested by centrifugation at 2500 µg RCF (relative centrifugal force) for 20 min and the pellet washed twice with sterile seawater. Harvested cells were resuspended in 15 ml of sterile seawater.Experimental series 1: Infection study. Atlantic salmon smolts of mean mass (± SE) 77.0 ± 2.9 g were acclimated to full strength seawater (35 ppt) over a period of 10 d, then allocated to triplicate tanks (4 fish per tank, n = 12 per treatment) and allowed to habituate for 24 h prior to anaesthetization with AQUI-S (0.04 ml l...

Experimentally induced marine flexibacteriosis in Atlantic salmon smolts Salmo salar. I. Pathogenicity

“…Strain 89/4747 was non-pathogenic, causing no infection or mortalities throughout the experimental period, which is consistent with the findings of Powell et al (2004). Interestingly, Carson et al (1992) described this strain as highly virulent, with all fish becoming infected and developing lesions, including controls.…”

“…Anecdotal evidence from the present study suggests that attenuation occurs quickly in Tenacibaculum maritimum isolates (R. van Gelderen unpubl.). The cause of the non-pathogenic results of strain 89/4747 in the present study may be attenuation, but this requires further investigation as there are many other factors to consider, such as preservation techniques, age of isolate, culture media and storage over time (Michel & Garcia 2003, O'Keefe et al 2006.In contrast, strains 00/3280 and 89/4762 were highly virulent, resulting in 100% mortalities in 3 d. Both strains have previously been demonstrated as highly pathogenic (Carson et al 1992, Handlinger et al 1997, Powell et al 2004. Experimental application of Tenacibaculum maritimum to the gills of Atlantic salmon using strain 00/3280 caused high levels of mortalities within 48 h (Powell et al 2004).…”

“…In contrast, strains 00/3280 and 89/4762 were highly virulent, resulting in 100% mortalities in 3 d. Both strains have previously been demonstrated as highly pathogenic (Carson et al 1992, Handlinger et al 1997, Powell et al 2004. Experimental application of Tenacibaculum maritimum to the gills of Atlantic salmon using strain 00/3280 caused high levels of mortalities within 48 h (Powell et al 2004).…”

“…Experimental application of Tenacibaculum maritimum to the gills of Atlantic salmon using strain 00/3280 caused high levels of mortalities within 48 h (Powell et al 2004). Strain 89/4762 was highly pathogenic in rainbow trout and Atlantic salmon when challenged by immersion (Carson et al 1992, Soltani 1995, Handlinger et al 1997.…”

“…Salmonids are competitive and will bite and harass each other when held in captivity (Mork et al 1999, MacLean et al 2000. The observed size difference of fish in each experiment would have contributed to this behaviour, which is likely to facilitate bacterial invasiveness, as damage to the epithelium would create a portal of entry for the bacteria to colonise and proliferate (Handlinger et al 1997).Other successful methods of inducing marine flexibacteriosis are usually associated with direct application to various areas of the body and scarification by different means (Masumura & Wakabayashi 1977, Wakabayashi et al 1984, Baxa et al 1987, Carson et al 1992, Bernardet et al 1994, Powell et al 2004. While intraperitoneal and intramuscular injections are ineffective (Campbell & Buswell 1982, Baxa et al 1987, Carson et al 1992, subcutaneous injection has worked on occasion, which is probably a result of injecting into the dermal layer, where bacteria are found in natural outbreaks (Masumura & Wakabayashi 1977, Wakabayashi et al 1984, Alsina & Blanch 1993, Bernardet et al 1994, Soltani 1995, Avendaño-Herrera et al 2006.…”