Performance of serum-free broth media for growth of  Renibacterium salmoninarum

Starliper, Clifford E.; Schill, William B.; Mathias, Jay

doi:10.3354/dao034021

Cited by 5 publications

(4 citation statements)

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“…These modifications combined the observations that each of these supplements enhance and/or select R. salmoninarum growth [14][15][16]20]. The modified medium will be referred to as the modified KDM2 (MKDM).…”

Section: Methodsmentioning

confidence: 99%

“…The improved growth was attributed to metabolites secreted by the initial culture. Using the same concept, Teska [15] and Starliper et al [16] incorporated R. salmoninarum spent medium (1% v/v) into KDM2 for related BKD studies. While these modifications have supported the subculturing of previously isolated R. salmoninarum strains, direct isolation from infected tissues, particularly from carrier fish, yielded inconsistent results.…”

Section: Introductionmentioning

confidence: 99%

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Recovery of Renibacterium salmoninarum from naturally infected salmonine stocks in Michigan using a modified culture protocol

Faisal

Eissa

Starliper

2010

Journal of Advanced Research

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Renibacterium salmoninarum, the causative agent of bacterial kidney disease (BKD), is a fastidious and slow-growing bacterium that is extremely difficult to grow in vitro. Herein, we describe a modified primary culture protocol that encompasses a modified bacteriological culture medium and a tissue processing procedure. In order to facilitate the release of R. salmoninarum from granulomatous tissues, kidneys of infected fish were homogenized in a high speed stomacher. The kidney disease medium (KDM2), routinely used for primary culture of R. salmoninarum was modified by the addition of antibiotics and metabolites. When a relatively large inoculum of diluted kidney homogenate was streak-plate inoculated onto the modified KDM2, colonial growth of R. salmoninarum was achieved within 5-7 days, compared to the standard of two weeks or more. The modified procedure was then used to determine the prevalence of R. salmoninarum among representative captive and feral salmonid stocks in Michigan. Prevalence and clinical manifestations varied among species, strains of fish, and locations; however, R. salmoninarum isolates were biochemically homogenous. The improved primary culture procedure described in this study enabled selective and quick isolation of R. salmoninarum. Also, the isolates retrieved in this study constitute a unique biological resource for future studies of R. salmoninarum in the Laurentian Great Lakes.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Recovery of Renibacterium salmoninarum from naturally infected salmonine stocks in Michigan using a modified culture protocol

Faisal

Eissa

Starliper

2010

Journal of Advanced Research

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“…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).…”

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confidence: 99%

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

Coady

Murray

Elliott

et al. 2006

Appl Environ Microbiol

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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...

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“…In Germany, BKD was first recorded in farmed salmon and trout by Hoffmann et al (1984) and has since been reported elsewhere in Europe, Japan, South America and many states of the USA. R. salmoninarum is a small, Gram-positive, nonmotile diplobacillus that has fastidious nutritional requirements (Austin et al 1983, Daly & Stevenson 1993, Teska 1994, Starliper et al 1998). Acute BKD is characterized by dark colouration of the fish, bloody ascites, exophathalmia, and granulomatous lesions of internal organs such as the kidney, whereas asymptomatic carriers can complete an entire life cycle and successfully spawn (Fryer & Lannan 1993).…”

Section: Introductionmentioning

confidence: 99%

Loop-mediated isothermal amplification (LAMP) for rapid detection of Renibacterium salmoninarum, the causative agent of bacterial kidney disease

Saleh

Soliman²,

El-Matbouli³

2008

Dis. Aquat. Org.

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A loop-mediated isothermal amplification (LAMP) assay was developed for rapid, specific and sensitive detection of Renibacterium salmoninarum in 1 h without thermal cycling. A fragment of R. salmoninarum p57 gene was amplified at 63°C in the presence of Bst polymerase and a specially designed primer mixture. The specificity of the BKD-LAMP assay was demonstrated by the absence of any cross reaction with other bacterial strains, followed by restriction digestion of the amplified products. Detections of BKD-LAMP amplicons by visual inspection, agrose gel electrophoresis, and real-time monitoring using a turbidimeter were equivalently sensitive. The BKD-LAMP assay has the sensitivity of the nested PCR method, and 10 times the sensitivity of one-round PCR assay. The lower detection limit of BKD-LAMP and nested PCR is 1 pg genomic R. salmoninarum DNA, compared to 10 pg genomic R. salmoninarum DNA for one-round PCR assay. In comparison to other available diagnostic methods, the BKD-LAMP assay is rapid, simple, sensitive, specific, and cost effective with a high potential for field application. KEY WORDS: Renibacterium salmoninarum · BKD · LAMP · Diagnosis Resale or republication not permitted without written consent of the publisherDis Aquat Org 81: [143][144][145][146][147][148][149][150][151] 2008 bacterium salmoninarum, culture is often impractical for routine diagnosis (Benediktsdottir et al. 1991). Alternate techniques have been developed for detection of the bacterium including direct and indirect fluorescent antibody assay (Bullock & Stuckey 1975, Austin & Austin 1993, and the enzyme-linked immunosorbent assay (ELISA) (Pascho et al. 1987, Jansson et al. 1996. These techniques also have some drawbacks as they are not sensitive enough to detect low levels of the pathogen in asymptomatic fish and, in the case of the fluorescent antibody assay, may give false positive reactions (Austin et al. 1985, Armstrong et al. 1989. Conversely, inconsistent results may arise with ELISA due to the variable quality of antibody lots and cross reactivity with other bacterial species (Scott & Johnson 2001, Powell et al. 2005. To overcome the drawbacks of these methods, several polymerase chain reaction (PCR) assays have been developed for sensitive and rapid detection of BKD in infected fish tissues and eggs (Brown et al. 1994, Leon et al. 1994, Magnusson et al. 1994, Chase & Pascho 1998, Powell et al. 2005, Chase et al. 2006, Rhodes et al 2006, Suzuki & Sakai 2007.Although PCR assays are powerful and sensitive tools for diagnosis of BKD, they require expensive equipment, precision thermocycling and laboratory training, which limits their use as routine diagnostic tools in the field.Loop-mediated isothermal amplification (LAMP) is a technique developed recently to amplify nucleic acid under isothermal conditions. It offers a rapid, inexpensive and accurate tool for all life sciences, including diagnosis of pathogens and detection of genetic disorders (Notomi et al. 2000). Unlike PCR, LAMP does not require a denatured ...

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Performance of serum-free broth media for growth of Renibacterium salmoninarum

Cited by 5 publications

References 9 publications

Recovery of Renibacterium salmoninarum from naturally infected salmonine stocks in Michigan using a modified culture protocol

Recovery of Renibacterium salmoninarum from naturally infected salmonine stocks in Michigan using a modified culture protocol

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

Loop-mediated isothermal amplification (LAMP) for rapid detection of Renibacterium salmoninarum, the causative agent of bacterial kidney disease

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