Both msa Genes in Renibacterium salmoninarum Are Needed for Full Virulence in Bacterial Kidney Disease

Abstract

Renibacterium salmoninarum, a gram-positive diplococcobacillus that causes bacterial kidney disease among salmon and trout, has two chromosomal loci encoding the major soluble antigen (msa) gene. Because the MSA protein is widely suspected to be an important virulence factor, we used insertion-duplication mutagenesis to generate disruptions of either the msa1 or msa2 gene. Surprisingly, expression of MSA protein in broth cultures appeared unaffected. However, the virulence of either mutant in juvenile chinook salmon (Oncorhynchus tshawytscha) by intraperitoneal challenge was severely attenuated, suggesting that disruption of the msa1 or msa2 gene affected in vivo expression.Bacterial kidney disease (BKD) has been reported in most areas of the world where salmonid species are found (4,11,13,20,27,28,35,37,42,46), and it is a principal source of morbidity and mortality among cultured salmon and trout. Acute BKD is characterized by ascites, swollen kidneys, exophthalmia, melanosis, and granulomatous lesions of internal organs such as the kidney, whereas asymptomatic carriers can complete an entire life cycle and successfully spawn (16). The infectious agent of BKD, Renibacterium salmoninarum, can be transmitted both horizontally among cohorts and vertically by intraovum inclusion (2,14). Current disease controls include broodstock segregation or culling, antibiotic treatment, and vaccination, but these methods have only moderate efficacy in preventing or treating the disease (1,30,32).R. salmoninarum is a gram-positive diplococcobacillus that grows very slowly and has fastidious nutritional requirements (8,12,39,40). R. salmoninarum produces abundant quantities of an extracellular, 57-kDa protein called p57 or major soluble antigen (MSA) (7,17,41). MSA is associated with the bacterial surface and is released into surrounding tissues, and it has been associated with leukocyte agglutination (7, 43, 44) and immunomodulation (3, 15, 41, 45). A variant strain of R. salmoninarum displays both reduced MSA expression and lowered mortality upon infection (5, 24, 38), whereas R. salmoninarum isolates possessing an additional copy of the gene encoding MSA are more virulent than isolates lacking the additional copy (32). MSA is encoded by duplicate chromosomal loci (25), and both loci are expressed (33). These observations suggest that MSA is a significant virulence determinant, and we hypothesized that disruption of either chromosomal locus would result in a less virulent strain of R. salmoninarum. The goals of the present study were to generate and characterize R. salmoninarum clones bearing disruptions of either msa1 or msa2, to compare the production of MSA protein in the mutants and the parental R. salmoninarum strain, and to evaluate the virulence of the mutant and parental R. salmoninarum strains for chinook salmon, Oncorhynchus tshawytscha. MATERIALS AND METHODSBacterial strains, media, and culture conditions. The parental R. salmoninarum strain used for transformation was the ATCC type strain 33209 (American Type Cult...

Experimental challenges with Renibacterium salmoninarum in Arctic charr Salvelinus alpinus

“…In contrast, the ELISA results for kidney samples showed an abundance of bacterial antigens at all times, indicating accumulation as well as persistence of antigens. Similar results were ob tained in a study run for 115 d where live bacteria were intraperitoneally injec ted (Coady et al 2006) and in a study run for 110 d where killed bacteria were used (Pascho et al 1997). All kidney samples, except 1, were positive in snPCR and/or qPCR at Weeks 2 and 7, while there were few and weakly positive samples at Weeks 25 and 34, suggesting considerable clearance of the bacterium.…”

“…injection trial, infection was already established in every fish tested at the first sampling 2 wk post-injection. Intraperitoneal injection is a widely used, standardized and fast infection method (Jones & Moffitt 2004, Rhodes et al 2004, Coady et al 2006, Jones et al 2007, Jansson et al 2008 many factors, such as strain of bacteria, method of cultivation, conditions in aquaria, different fish species and sizes, may influence the results obtained. R. salmoninarum was isolated from all samples at Week 2 and 75% of samples at Week 7, but there were no bacterial isolations at Weeks 25 and 34.…”

Genome Sequence of the Fish Pathogen Renibacterium salmoninarum Suggests Reductive Evolution away from an Environmental Arthrobacter Ancestor

“…MSA is found in culture supernatants, in fish tissues, and on the cell surface as an intact 57-kDa protein, and as defined fragments of the full-length molecule (69). This protein has immunosuppressive properties and is considered a major virulence factor (9). Previous work demonstrated that the MSA gene is duplicated in the genomes of many R. salmoninarum strains, including ATCC 33209 (44,70), and this was confirmed by genome sequencing.…”

Comparison and evaluation of Renibacterium salmoninarum quantitative PCR diagnostic assays using field samples of Chinook and coho salmon

“…When only the CRH samples were included, the msa/NFQ assay had a greatly increased geometric mean quantity (1.9 × 10 7 ) and the C q ratio declined to 0.82, suggesting that it performed better among these samples, almost all of which had ELISA ODs > 1.2 (Table 5). The cause of this result is uncertain; the CRH samples were from heavily infected fish that were likely to contain Renibacterium salmoninarum strains with multiple msa gene copies, which could account for their virulence (Coady et al 2006) and may explain the increase in amplification. However, log scale differences in gene quantity which may be attributable to multiple copies of the msa gene would still only result in minor changes in the resulting C q value.…”