Coordinated Bacterial Virulence: Relevance in Winter Ulcer

Controlling infectious diseases is one of the key factors in aquaculture production. Ulcerative disorders and skin health have received increased focus in recent years as challenges are faced from sea transfer of smolt to harvest of Atlantic salmon. Winter-ulcers are typically observed during low seawater temperatures and are associated with the bacterium Moritella viscosa and Tenacibaculum. Functional studies in combination with genome analysis have been performed to investigate how M. viscosa can be an ulcerative pathogen in this project. Previously, two major (typical and variant) genetic clades have been demonstrated within M. viscosa. In this project we have demonstrated that "typical" M. viscosa isolated from Norwegian Atlantic salmon is highly virulent in this fish species but resulted in lower levels of mortality in rainbow trout. "Variant" M. viscosa isolated from rainbow trout resulted in modest mortality levels in both Atlantic salmon and rainbow trout. To investigate the possible genetic background for inter-strain virulence differences, 38 M. viscosa isolates of diverse geographical origin and host species were investigated for the presence/absence of 11 putative virulence related homologs and membrane structures. Microscopy investigation revealed pili and flagella surface structures on both typical and variant M. viscosa. A putative insecticidal toxin complex was detected exclusively in "typical" Atlantic salmon isolates, and provide support for the existence of host-specificity/high virulence in "typical" M. viscosa related to Atlantic salmon. From comparative genomic analysis of twelve M. viscosa isolates it is further suggested the delineation of M. viscosa into several phylogenetic lineages. The majority of the genomic variations is associated to patterns of mobile genetic elements and the defense systems against such invading genetic elements, which is likely important in the genome evolution of M. viscosa. Also Aliivibrio wodanis is repeatedly isolated from farmed fish with winter-ulcer disease. A cell culture infection model showed that A. wodanis adhered to, but did not invade the fish cells. The culture supernatant of A. wodanis is also cytopathogenic to fish cells, and rearrange the actin filaments of the cytoskeleton. These observations suggest that the bacterium secretes toxins into the extracellular environment. Both M. viscosa and A. wodanis were re-isolated from external surfaces and internal organs from live and deceased co-infected fish, reproducing field observations. It is further hypothesized that A. wodanis colonization might influence the progression of a M. viscosa infection. Little is known about the interaction between A. wodanis and M. viscosa and how the presence of one bacterial species affects the behavior of the other. We have sequenced the complete genomes of these two Gram-negative species, and shown by in vitro growth studies that A. wodanis inhibits M. viscosa, Using bacterial implants in the fish abdomen, we demonstrate further from expression profiling of the transcriptomes, that the presence of A. wodanis is altering the global gene expression of M. viscosa. The project has successfully developed fully defined media for both M. viscosa and A. wodanis suited for reproducible growth in chemostat cultivations. One of the goals in the project has been to perform gene expression analyses of A. wodanis and M. viscosa. The defined media have been used to characterize growth characteristics, tolerance to metal elements, temperature and to study the influence of one bacterium on the gene expression of the other. The project has also initiated a microbiota study focusing on winter ulcers, the water column, and sediments around net pens. Because mucus of fish skin is in constant contact with the surrounding water of temporal conditions, the prevailing view is that transient populations structure the microbial community. The microbiota of skin and ulcers were different from that of the surrounding water that could suggest a potential regulated microbial-host relationship in the mucosal surfaces of Atlantic salmon.

The bacterium Moritella viscosa is considered the main agent of winter ulcer a disease that affects cultured fish in marine waters. In winter ulcer outbreaks, co-infection with M. viscosa and Vibrio wodanis is common. Little is known about the interplay b etween the two bacteria. We plan to identify key components that contribute to the communication between them that are relevant for pathogenicity. The genomes of one isolate of M. viscosa and a co-isolate of V. wodanis from the same salmon are sequenced. From the genome of M. viscosa several putative virulence factors were identified, however, little is known about the expression of these factors. A transcriptome analysis for their identification under condition relevant to M. viscosa pathogenesis will be performed and compared to gene expression when co-cultured with V. wodanis. A glimpse into the V. wodanis genome also showed genes encoding potential virulence factors in addition to quorum sensing systems (QSs). No QSs system has been detected in the ge nome of M. viscosa. In an attempt to understand and identify factors important to the relations between M. viscosa and V. wodanis during infections we plan the construction of transposon mutant libraries of both organisms. These libraries will be used for screening and identification of genetic elements relevant in disease development. For such purposes various bioassays will be performed made possible by high throughput screening technology. We will then identify and complement the various mutated genes before we finally test a selection of strains in cell culture assays.