Background: Aeromonas salmonicida subsp. salmonicida is a Gram-negative bacterium that is the causative agent of furunculosis, a bacterial septicaemia of salmonid fish. While other species of Aeromonas are opportunistic pathogens or are found in commensal or symbiotic relationships with animal hosts, A. salmonicida subsp. salmonicida causes disease in healthy fish. The genome sequence of A. salmonicida was determined to provide a better understanding of the virulence factors used by this pathogen to infect fish.

The recently described type III secretion system (TTSS) of Aeromonas salmonicida subsp. salmonicida has been linked to virulence in salmonids. In this study, three TTSS effector genes, aexT, aopH or aopO, were inactivated by deletion, as was ascC, the gene encoding the outer-membrane pore of the secretion apparatus. Effects on virulence were assayed by live challenge of Atlantic salmon (Salmo salar). The DascC mutant strain was avirulent by both intraperitoneal (i.p.) injection and immersion, did not appear to establish a clinically inapparent infection and did not confer protection from subsequent rechallenge with the parental strain.

Renibacterium salmoninarum, the causative agent of bacterial kidney disease (BKD) in salrnonids, can be transmitted vertically, i.e. from parent to progeny via the egg. The usual procedure to determine the likelihood of egg infection has been to examine non-egg tissues from mature female fish for evidence of R. sahoninarum cells or antigens. In the present study we examined the feasibility of detecting the pathogen directly within individual salmonid eggs by use of the polymerase chain reaction (PCR) to amplify segments of its DNA. Total DNA was extracted from the contents of eggs taken from several coho and chinook salmon (Oncorhynchus lusutch and 0 . tshawytscha), including coho eggs that had been injected with numbers of R. salmoninarum cells ranging from 2 to 2000 cells per egg. We also isolated DNA from kidney samples taken from these fish. Utilizing the PCR, two 24-base oligonucleotide primers were used to amplify a 501 base-pair region of the gene encoding a 57 kDa soluble protein ('p577 produced by R. salmoninarum. A segment of DNA of the appropriate size (501 base-pairs) from R. salmoninarum was detected by the PCR in all of the injected eggs. R. salrnoninarum DNA was also detected in some of the eggs taken from the coho and chinook samples that had previously been diagnosed as 'high-BKD' or 'negative-BKD' fish. Direct sequencing of the heatdenatured, double-stranded amplification product was carried out by dideoxy termination using T7 DNA polymerase and 35S labelling. There was 100 % homology between the partial sequence obtained from the amplified DNA fragment and the sequence of the p57 gene. The DNA extracted from other bacterial fish pathogens did not yield a positive result. These results indicate that the PCR is able to detect as few as 2 R. salmoninarum cells per egg and that it may be feasible as a broodstock screening technique specific for R. s a l m o n i n a m .

Aeromonas salmonicida subsp. salmonicida, a bacterial pathogen of Atlantic salmon, has no visible pili, yet its genome contains genes for three type IV pilus systems. One system, Tap, is similar to the Pseudomonas aeruginosa Pil system, and a second, Flp, resembles the Actinobacillus actinomycetemcomitans Flp pilus, while the third has homology to the mannose-sensitive hemagglutinin pilus of Vibrio cholerae. The latter system is likely nonfunctional since eight genes, including the gene encoding the main pilin subunit, are deleted compared with the orthologous V. cholerae locus. The first two systems were characterized to investigate their expression and role in pathogenesis. The pili of A. salmonicida subsp. salmonicida were imaged using atomic force microscopy and Tap-and Flp-overexpressing strains. The Tap pili appeared to be polar, while the Flp pili appeared to be peritrichous. Strains deficient in tap and/or flp were used in live bacterial challenges of Atlantic salmon, which showed that the Tap pilus made a moderate contribution to virulence, while the Flp pilus made little or no contribution. Delivery of the tap mutant by immersion resulted in reduced cumulative morbidity compared with the cumulative morbidity observed with the wild-type strain; however, delivery by intraperitoneal injection resulted in cumulative morbidity similar to that of the wild type. Unlike the pili of other piliated bacterial pathogens, A. salmonicida subsp. salmonicida type IV pili are not absolutely required for virulence in Atlantic salmon. Significant differences in the behavior of the two mutant strains indicated that the two pilus systems are not redundant.Aeromonas salmonicida subsp. salmonicida is a gram-negative, nonmotile, rod-shaped bacterium that is the etiologic agent of an infectious bacteremia-septicemia of salmonids known as furunculosis. Furunculosis is a complex disease that exists in different forms depending on the health, age, and species of fish. Many cell-associated and secreted factors have been implicated as virulence determinants in this bacterium (for reviews, see references 5 and 7). Despite this, much of the pathogenesis of A. salmonicida subsp. salmonicida remains poorly understood, and no single characteristic or phenotype was found only in virulent strains (16,17,30,42) until the recent description of an A. salmonicida subsp. salmonicida type III secretion system (6, 11).In order to better understand the virulence strategies employed by A. salmonicida subsp. salmonicida, we have focused on the initial stages of infection: adherence and invasion. Bacteria use complex intercellular mechanisms and specific and nonspecific adhesins to achieve these aims (34). The most well-studied A. salmonicida subsp. salmonicida adhesin is the surface layer or S-layer, sometimes referred to as the A-layer (additional layer) (33). This layer is a nonspecific but important factor for adherence due to its hydrophobic nature.Pili allow bacteria to attach to solid surfaces, including host tissues, and are considered impor...

O-acetylation is one of the major modifications of sialic acids that significantly alters biological properties of the parent molecule. These O-acetylated forms are components of the cellular membrane and can affect physiological and pathological responses. Understanding the role of N-glycans in physiology is of increasing relevance to cellular biologists in various disciplines who study glycoproteomics yet lack information regarding the function of the attached glycans. It is well known that stress may decrease immune function in fish; however, there are only few suitable biomarkers available to monitor the physiological responses under the stress conditions. This study is the first report on the effect of stress on the profile of O-acetylation of sialic acids in fish serum. In order to preserve the relevant structural characteristics as much as possible, native N-glycans were directly analyzed using CE-MS. We have characterized the N-glycans in serum of salmon (Salmo salar) exposed to long-term handling stress (15 s out of the water, daily for 4 wk) and compared with the results obtained from sera of control fish. The results indicated that major N-glycans in salmon serum contained mono-acetylated sialic acids (83%), and that the O-acetylation pattern of sialic acids could be altered by long-term stress.