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United States Department of Agriculture

Agricultural Research Service

2009 Annual Report

1a.Objectives (from AD-416)
1. Characterize virulence determinants produced by the rainbow trout pathogens Yersinia ruckeri and Flavobacterium psychrophilum. 2. Identify rainbow trout genes and factors that are critical to expression of innate and acquired immunity against Y. ruckeri and F. psychrophilum. 2a. Molecular identification and sequencing of antibiotic resistance and virulence genes in bacterial pathogens of aquaculture species. 3. Identify water quality factors that influence host susceptibility and pathogen refuge within a recirculating system, and evaluate the use of bacteriophage as a targeted antibacterial strategy in recirculating and serial reuse aquaculture systems.

1b.Approach (from AD-416)
Virulence determinants and antibiotic resistance plasmids of Yersinia ruckeri and Flavobacterium psychrophilum will be identified using molecular genetic approaches. Y. ruckeri virulence factors will be established by screening transposon-induced mutants for attenuation of virulence. For F. psychrophilum, we will completely sequence the genome and target potential virulence genes for mutation. These studies will generate candidates for vaccines and rapid diagnostic assays. Host factors involved in innate and acquired immunity will be identified by bioinformatics and gene expression analyses. The expression of trout immune genes will be measured in vaccinated and challenged fish by RT-PCR and immune gene microarrays. These studies will identify targets for diagnostic assays measuring protective immunity. The impact of organic constituents on host disease susceptibility and microbial growth will be assessed in recirculating aquaculture systems. Pathogen refuges within culture systems will be identified to aid targeted disinfection strategies. These multidisciplinary efforts will define the important parameters governing infectious disease in order to develop effective vaccines, novel biotherapeutics and strategies for maintaining healthier fish rearing conditions for the aquaculture industry.

3.Progress Report
Enteric red mouth disease in salmonid aquaculture has been successfully controlled by immersion vaccination for over two decades. However, an emergent biotype of Yersinia ruckeri (biotype.
2)has caused recent outbreaks in vaccinated fish in both the United States and Europe. Complementation, sequence and mutation analyses were used to identify the primary genetic differences that define the US lineage of Y. ruckeri biotype 2. Research elucidating the mechanism(s) causing this apparent vaccine failure is in progress.

The fish immune response to Yersinia ruckeri biotype 2 is poorly understood. Rainbow trout were challenged with Y. ruckeri biotype 2 and bacterial numbers and fish gene expression were measured. Temporal and dose-dependent changes in gene expression were identified over a 7 day challenge period. Correlation of gene expression patterns and infection severity was determined. We indentified increased abundance of four cytokine gene transcripts and no-change or down-regulation of three other immune-relevant genes. These findings increase our understanding of the immune response to Y. ruckeri biotype 2 infection.

Bacterial coldwater disease is a long-standing problem in trout aquaculture and there are limited methods available for control. Since 2005, the NCCCWA has been selectively breeding rainbow trout for improved disease resistance. This year, we evaluated 96 select-line and 18 susceptible-line families, 3 pure strains, 2 unique mixed-family control lots and one sham injected group by experimental challenge. After two generations of selection, survival has increased approximately 45 % compared to unselected control fish. In addition, vaccine prototypes were evaluated in resistant and susceptible crosses of fish. Research combining vaccination with selective breeding is underway with the goal of enhancing both the innate and acquired immune response.

Bacteriophage have potential as safe and effective antimicrobials that offer the aquaculture industry an alternative to traditional antibiotics. Using a bacteriophage specific to the fish pathogen Y. ruckeri we have identified a novel bacteriophage-produced enzyme activity that neutralizes the virulence of this pathogen by degrading bacterial components that are essential for survival during infection.

Immunostimulants and probiotics have potential for improving animal health and decreasing antibiotic use. This year, we investigated supplementation of standard rainbow trout feed with chicken egg powder containing an anti-PLA2 antibody. Modest improvement in weight gain and modulation of immune-related gene expression were observed. Further studies on leukocyte population changes in mucosal tissue are currently in progress in collaboration with an industry partner.

1. Antibiotic Resistance Plasmid Identified: An improved understanding of mobile antimicrobial resistance plasmids is essential for both predicting the utility of antimicrobial drugs and understanding the risks associated with their use. This year, we reported the identification and characterization of the first florfenicol-resistant bacterial fish pathogen in the United States. The resistant isolate carried a self-transmissible IncA/C multi-drug resistance plasmid that conferred resistance to all three drugs currently approved for use in U.S. aquaculture. The further dissemination of this plasmid could have an impact on the performance of therapeutic antimicrobial drugs in U.S. aquaculture.

2. Potential Basis for the Emergence of biotype 2 Yersinia Ruckeri Identified: Disease outbreaks of Yersinia ruckeri Biotype 2 are an increasing problem in finfish aquaculture and the mechanism behind this emergence is unclear. We have found that mutation of a biotype 1 strain can cause the same phenotypic changes indicative of biotype 2 strains. Our results also show that this mutation has no effect on virulence in rainbow trout. These data demonstrate that a single mutational change is all that is required for the switch from biotype 1 to 2 and therefore reveal a potential molecular basis for the natural emergence of biotype 2 Y. ruckeri. The strains developed in this study will be useful for understanding vaccine failure and for developing a vaccine that is effective against biotype II strains.

6.Technology Transfer

Number of the New/Active MTAs (providing only)1

Review Publications
Fricle, F., Welch, T.J., Mcdermott, P., Mammel, M., Leclerc, E., White, D., Cebula, T., Ravel, J. 2009. Comparative genomics of the IncA/C multidrug resistance plasmid family. Journal of Bacteriology. 191(15):4750-4757.

Wheeler, R.W., Davies, R.L., Dalsgaard, I., Garcia, J., Welch, T.J., Waqley, S., Bateman, K.S., Verner-Jeffreys, D.W. 2009. Characterization of geographically and temporally diverse Yersinia ruckeri isolates: evidence that UK and mainland European biotype 2 isolates represent different clonal groups. Diseases of Aquatic Organisms. 84:25-33.

Welch, T.J., Evenhuis, J., White, D.G., Mcdermott, P.F., Harbottle, H., Miller, R., Griffin, M., Wise, D. 2008. IncA/C Plasmid-Mediated Florfenicol Resistance in the Catfish Pathogen Edwardsiella ictaluri. Antimicrobial Agents and Chemotherapy. 53(2):845-846.

Shah, D.H., Cain, K.D., Wiens, G.D., Call, D.R. 2008. Challenges associated with heterologous expression of Flavobacterium psychrophilum proteins in Escherichia coli. Marine Biotechnology. 10(6):719-730. Epub 2008 Jun 13.

Wiens, G.D., Dale, O. 2009. Renibacterium salmoninarum p57 antigenic variation is restricted in geographic distribution and correlated with genomic markers. Diseases of Aquatic Organisms. 83:123-131.

Wiens, G.D., Rockey, D.D., Wu, Z., Crane, S., Chen, D.S., Capri, G.R., Burnett, J.R., Ponnerassary, S., Schimpma, M.J., Burd, H., Bhattacharyya, A., Rhodes, L.D., Strom, M.S. 2008. The genome sequence of Renibacterium salmoninarum suggests reductive evolution away from an environmental Arthrobacter ancestor. Journal of Bacteriology. 190(21):6970-6982.

Last Modified: 11/27/2014
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