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Yersinia Ruckeri Isolates Recovered from Diseased Atlantic Salmon

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Yersinia Ruckeri Isolates Recovered from Diseased Atlantic Salmon View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Enlighten crossmark Yersinia ruckeri Isolates Recovered from Diseased Atlantic Salmon (Salmo salar) in Scotland Are More Diverse than Those from Rainbow Trout (Oncorhynchus mykiss) and Represent Distinct Subpopulations Michael J. Ormsby,a Thomas Caws,a Richard Burchmore,b Tim Wallis,c David W. Verner-Jeffreys,d Robert L. Daviesa Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdoma; Glasgow Downloaded from Polyomics, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdomb; Ridgeway Biologicals Ltd., Compton, Berkshire, United Kingdomc; Cefas Weymouth Laboratory, The Nothe, Weymouth, Dorset, United Kingdomd ABSTRACT Yersinia ruckeri is the etiological agent of enteric redmouth (ERM) disease of farmed salmonids. Enteric redmouth disease is traditionally associated with rainbow trout (Oncorhynchus mykiss, Walbaum), but its incidence in Atlantic salmon (Salmo salar) is increasing. Yersinia ruckeri isolates recovered from diseased Atlantic salmon have been poorly characterized, and very little is known about the relationship of the isolates associated with these two species. Phenotypic approaches were used to char- http://aem.asm.org/ acterize 109 Y. ruckeri isolates recovered over a 14-year period from infected Atlantic salmon in Scotland; 26 isolates from in- fected rainbow trout were also characterized. Biotyping, serotyping, and comparison of outer membrane protein profiles identi- fied 19 Y. ruckeri clones associated with Atlantic salmon but only five associated with rainbow trout; none of the Atlantic salmon clones occurred in rainbow trout and vice versa. These findings suggest that distinct subpopulations of Y. ruckeri are associated with each species. A new O serotype (designated O8) was identified in 56 biotype 1 Atlantic salmon isolates and was the most common serotype identified from 2006 to 2011 and in 2014, suggesting an increased prevalence during the time period sampled. Rainbow trout isolates were represented almost exclusively by the same biotype 2, serotype O1 clone that has been responsible for the majority of ERM outbreaks in this species within the United Kingdom since the 1980s. However, the identification of two on November 7, 2016 by Glasgow University Library biotype 2, serotype O8 isolates in rainbow trout suggests that vaccines containing serotypes O1 and O8 should be evaluated in both rainbow trout and Atlantic salmon for application in Scotland. IMPORTANCE Vaccination plays an important role in protecting Atlantic salmon against the bacterial pathogen Yersinia ruckeri, but, in recent years, there has been an increasing incidence of vaccine breakdown in salmon. This is largely because current vaccines are aimed at rainbow trout and are based on serotypes specific for this species. A wider range of serotypes is responsible for infection in Atlantic salmon, but very little is known about the diversity of these strains and their relationships to those recovered from rain- bow trout. In the present study, we demonstrate that Y. ruckeri isolates recovered from diseased Atlantic salmon in Scotland are more diverse than those from rainbow trout; furthermore, isolates from the two species represent distinct subpopulations. In addition, a new O serotype was identified that is responsible for a significant proportion of the disease in Atlantic salmon. Our findings are likely to have important implications for the development of improved vaccines against Y. ruckeri. he Gram-negative bacterium Yersinia ruckeri is the etiological (11, 12), and Scotland, where salmon production is of significant Tagent of enteric redmouth (ERM) disease of cultured sal- economic importance. Indeed, a less severe form of the disease, monids and causes significant economic losses to the fish-farming known as yersiniosis or salmon blood spot disease, has been rec- industry. Yersinia ruckeri was first isolated in 1956 from diseased ognized in Atlantic salmon in Australia since 1980 (7, 8, 13). Ye- rainbow trout (Oncorhynchus mykiss, Walbaum) in the Hagerman rsiniosis is characterized by a marked unilateral or bilateral exoph- Valley in Idaho (1, 2) but has since become widely disseminated and is present in fish populations in large areas of North and South America, Europe, Australia, and South Africa (3). Enteric red- Received 10 May 2016 Accepted 27 June 2016 mouth disease is an acute infection primarily of rainbow trout and Accepted manuscript posted online 22 July 2016 is typically characterized by a hemorrhagic septicemia, which Citation Ormsby MJ, Caws T, Burchmore R, Wallis T, Verner-Jeffreys DW, Davies RL. manifests as subcutaneous hemorrhages in and around the oral 2016. Yersinia ruckeri isolates recovered from diseased Atlantic salmon (Salmo cavity, the latter giving rise to the name “redmouth” disease (1, salar) in Scotland are more diverse than those from rainbow trout (Oncorhynchus 4–6). Internally, petechial hemorrhages on the surfaces of the mykiss) and represent distinct subpopulations. Appl Environ Microbiol 82:5785–5794. doi:10.1128/AEM.01173-16. liver, pancreas, pyloric ceca, swim bladder, and lateral muscula- Editor: H. L. Drake, University of Bayreuth ture may occur, and the spleen and lower intestine are often in- Address correspondence to Robert L. Davies, [email protected]. flamed, with the lower intestine filled with an opaque yellowish Supplemental material for this article may be found at http://dx.doi.org/10.1128 fluid (4, 6). However, Y. ruckeri is also becoming increasingly re- /AEM.01173-16. sponsible for infections in Atlantic salmon (Salmo salar), particu- Copyright © 2016, American Society for Microbiology. All Rights Reserved. larly in countries, such as Australia (7, 8), Chile (9, 10), Norway October 2016 Volume 82 Number 19 Applied and Environmental Microbiology aem.asm.org 5785 Ormsby et al. thalmos, often with patches (the blood spots) of hemorrhagic salmon and 26 from rainbow trout) recovered in the United Kingdom congestion on the iris of the eye (7). Histopathological features are between 2001 and 2014 are presented in Tables S1 and S2 in the supple- those of a typical septicemia; bacteria are readily detected in the mental material, respectively. All isolates were recovered from individual blood and in circulating macrophages and may also localize at moribund or dead fish that were showing clinical signs of ERM/yersini- sites of tissue hemorrhage (7). osis; in some cases, isolates were recovered from two or more fish at a Serotyping is particularly important for strain differentiation particular site. The geographic origins of the field isolates are shown in Fig. S1 in the supplemental material. Bacteria were stored at Ϫ80°C in 50% of Y. ruckeri, and the O-serotyping scheme of Davies (14)isthe glycerol (vol/vol) in tryptone soya broth (TSB; Oxoid) and were routinely most commonly used (12, 15–17). In this scheme, five O sero- subcultured on tryptone soya agar (TSA; Oxoid) at 22°C for 48 h. Liquid types, O1, O2, O5, O6, and O7, are recognized by rapid slide cultures were prepared by inoculating three or four colonies into 10-ml agglutination assay using defined O antigens (14); the individual volumes of TSB and incubating overnight at 22°C with shaking at 120 Downloaded from O serotypes correspond to discrete lipopolysaccharide (LPS) types rpm. For the production of OMPs, 400-␮l overnight cultures were inoc- as identified by SDS-PAGE and Western blotting (18). It is well ulated into 400-ml volumes of TSB in 2-liter Erlenmeyer flasks. Cultures established that serotype O1 strains of Y. ruckeri are responsible were grown aerobically at 22°C for ϳ16 h with shaking at 120 rpm or until for the majority of disease outbreaks in rainbow trout worldwide a minimum optical density at 600 nm (OD600) of 1.2 (mid-log phase) was (3, 14, 19–21). However, yersiniosis of Atlantic salmon in Austra- achieved. Anaerobic conditions were created by growing bacteria stati- lia (7, 8) and Chile (9) is associated with a serotype O1 subtype cally in 1,000-ml volumes of TSB in 1-liter Erlenmeyer flasks overlaid with (O1b). Historically, serotype O1 isolates associated with ERM dis- sterile mineral oil. For serotyping, isolates were grown as bacterial lawns on TSA at 22°C for 48 h. ease in North America and Europe were motile and classified as http://aem.asm.org/ Biotyping. Isolates grown overnight as liquid cultures were viewed for the Hagerman strain. However, in 1990, Davies and Frerichs (22) motility by the hanging drop method using phase-contrast microscopy. described, for the first time, a nonmotile serotype O1 variant that The presence or absence of flagella was confirmed in selected isolates by also lacked the ability to hydrolyze Tween 20 and 80; conversely, transmission electron microscopy (TEM) as previously described (22). To motile serotype O1 isolates were able to hydrolyze both substrates. detect lipase activity, isolates were grown on TSA supplemented with The motile lipase-positive and nonmotile lipase-negative isolates 0.1% (vol/vol) Tween 20 and 80 at 22°C for 48 h as previously described were designated biotypes 1 and 2, respectively (22). Since the first (22, 35). A positive result was recorded when clear zones were observed identification of biotype 2 isolates in the United Kingdom, non- around the colonies. motile variants have been described elsewhere in Europe (16, 17, O serotyping. O serotyping of isolates was conducted by slide agglu- 23–25), the United States (26), and Australia (7). Indeed, biotype tination as previously described (14). Cross-absorbed anti-O1 and an- on November 7, 2016 by Glasgow University Library 2 Y. ruckeri strains are causing increasing concern because they are ti-O5 antisera were prepared against the cross-agglutinating isolate responsible for disease in fish previously vaccinated against bio- RD426 as previously described (14). type 1 strains (17, 20, 23, 26, 27). A typing scheme based on the Isolation of outer membrane proteins.
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