Effect of vaccination against yersiniosis on the relative percent survival, bactericidal and lysozyme response of Atlantic salmon, Salmo salar

Abstract

The bacterium Yersinia ruckeri serovar O1b causes yersiniosis in Atlantic salmon, Salmo salar, in the southern hemisphere. Despite vaccination this disease has resulted in significant hatchery losses in the Tasmanian Atlantic salmon aquaculture industry. A poor response to vaccination in juveniles, 1–5 g, has lead to the investigation of the suitability of the current formalin killed whole-cell vaccine Yersinivac-B. In this study trypsin was added to the Yersinivac-B to expose the bacteria's protective O-antigen to make the vaccine more immunogenic. At six weeks post vaccination, the effect of Yersinivac-B and the novel trypsinated Yersinivac-B vaccine on body mucus lysozyme and mucus and serum bactericidal activity of fish was determined over a 48 h period following challenge with Y. ruckeri. Body and gill mucus lysozyme and mucus and serum bactericidal activity was also determined in surviving fish at 10 weeks post Y. ruckeri challenge. Following the challenge period of 14 days the trypsinated Yersinivac-B fish demonstrated a significantly higher percent survival compared to the Yersinivac-B and control unvaccinated fish. Body mucus lysozyme concentration was also significantly elevated at 8 h post challenge in the trypsinated Yersinivac-B fish compared to controls. This variable however appears unlikely to play a significant role in protection as positive bactericidal activity was not found in the mucus of any fish following challenge. Bactericidal activity was not observed in the serum or mucus of any challenge survivors. At 8 h post challenge the trypsinated Yersinivac-B fish demonstrated the highest serum bactericidal activity. However, the unvaccinated control fish also displayed positive serum bactericidal activity despite being unlikely to have been previously exposed to Y. ruckeri. A significantly higher gill mucus lysozyme concentration in control survivors compared to vaccinated fish suggests that this response may be important in the protection of unvaccinated fish against yersiniosis. This research has highlighted the potential use of trypsin to increase the efficacy of Yersinivac-B. It has also contributed to better understanding of the role of humoral immune responses during a Y. ruckeri challenge.

Introduction

The Gram-negative bacterium Y. ruckeri is the causative agent of enteric redmouth (ERM) disease predominantly in rainbow trout, Oncorhynchus mykiss, in the northern hemisphere and is the cause of yersiniosis predominately in Atlantic salmon, Salmo salar, in southern hemisphere (Carson and Wilson, 2008). Yersiniosis caused the mortality of half a million juvenile fish within six months during 2007 in a single Tasmanian hatchery despite vaccination. The poor level of protection has lead to investigations to assess vaccination strategies and host response in juvenile fish vaccinated with a conventional formalin inactivated whole-cell bacterin.

In fish, first contact with pathogens is via the epithelial surfaces of the skin, gills and alimentary tract. A layer of mucus secreted by the mucous cells plays an integral role in the trapping and sloughing of pathogens and contains humoral immune parameters such as lysozyme, complement and antibodies (Narvaez et al., 2010). Previous studies have shown that the gills may be an important portal of entry for Y. ruckeri (Zapata et al., 1987, Torroba et al., 1993, McIntosh et al., 2000, Tobback et al., 2009). Other sites of entry however including the skin and gut however may also be important for this bacterium (Busch and Lingg, 1975, Valtonen et al., 1992). Lysozyme is an important innate immune response in fish (Magnadóttir, 2006). Lysozyme can be found in body surface secretions including skin and gill mucus, as well as in the intestinal tract and blood (Saurabh and Sahoo, 2008). Lysozyme is particularly effective against gram positive bacteria where is splits the β (1–4) linkages between N-acetylmuramic acid and N-acetylglucosamine in the cell walls (Saurabh and Sahoo, 2008). Lysozyme can also be effective against gram negative bacteria once the inner petidoglycan layer has been exposed via the action of complement and/or other enzymes (Saurabh and Sahoo, 2008). In fish, spontaneous bactericidal activity is also considered one of the major defence mechanisms in the early stages of microbial infections (Hollebecq et al., 1995). Furthermore, bactericidal activity has been found to be a reliable biological marker of resistance against furunculosis (Hollebecq et al., 1995).

Most Tasmanian Atlantic salmon are currently vaccinated with Yersinivac-B, a formalin killed whole-cell vaccine by bath immersion at 1 g, followed by a booster at 5 g. However, the industry goal is a single vaccination that affords protection over 18 months. The gills are an efficient uptake system during immersion vaccination and this type of vaccination best imitates the natural route of infection (Raida and Buchmann, 2008). Understanding how the immune system functions after vaccination against yersiniosis is important for improved protection, husbandry and health management of the species (Whyte, 2007). Experimental fish vaccines can be improved through the addition of the protease trypsin, which can help expose the bacteria's protective O-antigen in order to make the vaccine more efficient (Egidius and Andersen, 1979). Therefore, with a view to improving the current vaccine, as well as the understanding of yersiniosis in Atlantic salmon, the main aims of this study were to assess a novel trypsinated vaccine and determine the role of mucosal lysozyme in both vaccinated and non-vaccinated fish. Identification of a possible biomarker of effective vaccination might lead to an improved vaccine and increased industry sustainability and fish welfare, as fewer challenge trials that sacrifice large amounts of fish would need to be conducted in the future (Marsden et al., 1996). Therefore bactericidal activity in both serum and mucus was also assessed as a possible indicator of effective vaccination.