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United States Department of Agriculture

Agricultural Research Service


Location: Cool and Cold Water Aquaculture Research

2010 Annual Report

1a. Objectives (from AD-416)
Endemic and emerging diseases limit U.S. aquatic farm-animal production. This project focuses on improving fish health by selectively breeding rainbow trout for increased disease resistance and utilizing divergent fish phenotypes to understand host resistance and pathogen virulence mechanisms under laboratory and farm conditions. 1: Conduct selective breeding and determine, under field and laboratory conditions, whether rainbow trout bred for bacterial cold water disease resistance exhibit superior performance traits. • 1.a. Evaluate and selectively breed NCCCWA rainbow trout for increased disease resistance and quantify improvement due to selection. • 1.b. Compare improved and reference lines of NCCCWA rainbow trout under farm conditions. 2: Characterize virulence determinants involved in the emergence of vaccine-resistant Yersinia ruckeri strains and develop novel therapeutics to treat or prevent disease. • 2.a. Elucidate the genetic basis of the biotype 2 phenotype. • 2.b. Evaluate the role of flagellar secretion in the function of the traditional ERM immersion vaccine and vaccine failure. • 2.c. Develop novel phage therapeutics for control of ERM and BCWD. 3: Identify genes and non-genetic factors in rainbow trout critical to innate and acquired immunity. • 3.a. Determine if heritable differences in spleen structure and function account for the superior BCWD survival and clearance traits of resistant fish. • 3.b. Determine whether orally-delivered immunostimulants modulate the gut immune system and whether variation exists between resistant and susceptible lines of rainbow trout. • 3.c. Initiate development of a rainbow trout immune-pathway database that is integrated with pathogen genomic information.

1b. Approach (from AD-416)
Our approach incorporates a comprehensive and multidisciplinary strategy that combines selective breeding, functional genomics of pathogenic bacteria, and immunological studies. In the first objective, we utilize conventional family-based selective breeding to increase bacterial cold water disease resistance and evaluate select and control fish performance in on-farm trials. In the second objective, we characterize indicators of virulence in emerging strains of vaccine-resistant Y. ruckeri using molecular and genomic approaches and develop novel therapeutics to treat or prevent bacterial disease. In the third objective, we identify genes and non-genetic factors in trout that are critical to expression of innate and acquired immune responses using molecular and immunological techniques. We will identify and measure mucosal immune system function and dietary immunomodulation of local immunity. The overall impact of this research is improved animal well-being, reduced antibiotic use and increased production efficiency.

3. Progress Report
This report documents progress for 1930-32000-005-00D which started 12/9/2009 and continues research from 1930-32000-002-00D and 1930-31000-007-00D. Previously rainbow trout were evaluated and selected for increased resistance to bacterial cold water disease (BCWD). In 2010, three year-old females from the resistant and reference lines of fish were mated and 38 viable crosses produced. Eyed eggs were shipped to three locations for field evaluation with cooperators Clear Springs Foods Inc (agreement #58-1930-0-038) and Utah State Fish and Wildlife Service (agreement # 58-1930-0-042N). Hatch rate was above 80% at each location. Fish survival and growth were measured, and microbiological analyses of water and fish samples conducted. Disease outbreaks occurred at two locations and mortality of resistant-line fish was less than 3%. Data analyses are continuing and additional trials are planned for 2011. Implementation of breeding programs is limited by an incomplete understanding of the immunogenetics of host resistance and correlated traits. Previously a family-based, phenotypic correlation between rainbow trout spleen somatic-index (SI) and family survival following F. psychrophilum challenge was observed. This year the genetic correlation between SI and BCWD were calculated from two independent trout lines evaluated over five year-classes. Spleen index was highly heritable and there was a positive genetic correlation with BCWD survival. In collaboration with Project # 1930-31000-009, the genetic architecture influencing spleen index was examined. Suggestive QTL for spleen weight and spleen index were identified on several chromosomes. Methods were developed and optimized for isolation of leukocytes from gill and gut tissues of rainbow trout. Flow cytometry analyses demonstrate few IgM positive cells in the gut tissue of rainbow trout under normal conditions. Several immune related genes were modulated in the gut following bacterial challenge. Immunostimulants and immunomodulators are emerging areas of interests in animal production to improve animal health and decrease antibiotic use. A standard rainbow trout diet was supplemented with chicken egg powder containing an anti-PLA2 antibody and observed improvement in weight gain compared to fish fed standard diet supplemented with control chicken egg powder. Initial studies indicate an 11% improvement in total weight gain over controls and approximately 21 fewer days to reach market weight. No significant differences in immune related mRNA expression were detected in the gut of treated rainbow trout. There was no difference in survival between anti-PLA2 and control fed fish following challenge with Yersinia ruckeri or Flavobacterium psychrophilium. Bacteriophages have potential as a means for pathogen biocontrol. A model system was developed for quantifying the fish-to-fish transmission of Yersinia ruckeri, a salmonid pathogen. The development of a model is a critical first step for future studies designed to assess and optimize the use of bacteriophage to minimize pathogen transmission in the aquaculture environment.

4. Accomplishments
1. Discovered genetic basis for Biotype 2 (BT2) disease caused by Yersinia ruckeri: Biotype 2 variants of the bacterium Yersinia ruckeri are an emerging disease problem in US and European finfish aquaculture. Scientists at the National Center for Cool and Cold Water Aquaculture identified four mutations, each in a different strain, that cause the loss of motility and enzyme (lipase) secretion that define Biotype 2 Yersinia ruckeri strains. All four mutations were in genes predicted to encode essential components of the flagellar secretion apparatus which allows the bacterium to swim and secrete certain enzymes. Our results demonstrate that the Biotype 2 phenotype has emerged separately at least four times. This differentiation of pathogen lineages is being used to assess the risk of BT2 disease and for development of specific vaccines and treatment strategies.

Review Publications
Evenhuis, J., Lapatra, S.E., Verner-Jefferys, D.W., Dalsgaard, I., Welch, T.J. 2009. Identification of flagellar motility genes in Yersinia ruckeri by transposon mutagenesis. Applied and Environmental Microbiology. 75(20):6630-6633.

Lindsey, R.L., Frye, J.G., Cray, P.J., Welch, T.J., Meinersmann, R.J. 2010. An oligonucleotide microarray to characterize multidrug resistant plasmids. Journal of Microbiological Methods. 81(2):96-100.

Moulana, M., Evenhuis, J., Kountikov, E.I., Wilson, M., Bengten, E., Miller, N.W., Mcconnell, T. 2008. Characterization of anti-channel catfish MHC class II monoclonal antibodies. Veterinary Immunology and Immunopathology. 126:120-130.

Wiens, G.D. 2009. Microbial Genomics of Aquaculture Pathogens. In: Overturf, K., editor. Current Status of Molecular Techniques in Aquaculture. Hobolen, NJ: Wiley, John & Sons, Inc. p. 315-335.

Lafrentz, B.R., Lapatra, S.E., Call, D.R., Wiens, G.D., Cain, K.D. 2009. Proteomic analysis of Flavobacterium psychrophilum cultured in vivo and in iron-limited media. Diseases of Aquatic Organisms. 87:171-182.

Wiens, G.D., Vallejo, R.L. 2010. Temporal and pathogen-load dependent changes in rainbow trout (Oncorhynchus mykiss) immune response traits following challenge with biotype 2 Yersinia ruckeri. Fish and Shellfish Immunology. doi:10.1016/j.fsi.2010.06.010 29:639-647.

Verner-Jefferies, D., Welch, T.J., Schwarz, T., Pond, M.J., Woodward, M.J., Haig, S.J., Rimmer, G.S., Roberts, E., Morrison, V., Baker-Austin, C. 2009. High prevalence of multidrug-tolerant bacteria and associated antimicrobial resistance genes isolated from ornamental fish and their carriage water. PLoS One. 4(12):1-9.

Haig, S., Davies, R.L., Welch, T.J., Reese, A.R., Verner-Jefferies, D. 2010. Comparative susceptibility of Atlantic salmon and rainbow trout to Yersinia ruckeri: relationship to O antigen serotype and resistance to serum killing. Veterinary Microbiology. doi:10.1016/j.vetmic.2010.06.022

Last Modified: 2/23/2016
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