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United States Department of Agriculture

Agricultural Research Service

Research Project: Integrated Approaches for Improving Aquatic Animal Health in Cool and Cold Water Aquaculture

Location: Cool and Cold Water Aquaculture Research

2013 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:
One hundred resistant- (ARS-Fp-R), 28 control- (ARS-Fp-C), and 21 susceptible-line (ARS-Fp-S) families were produced and evaluated at approximately 2.5 grams for bacterial cold water disease resistance (BCWD) in a standardized 21-day laboratory challenge. Mean family survival rates were 77.0% (ARS-Fp-R), 34.2% (ARS-Fp-C), and 14.5% (ARS-Fp-S). Through four generations, selection response in the ARS-Fp-R line has averaged approximately +14 percentage points (survival rate) per generation when estimated using genetic trend or comparison with randomly-mated control lines. Approximately 417,500 embryos from the ARS-Fp-R line were distributed to four stakeholders/collaborators in West Virginia, Montana, Idaho, and Utah for the purposes of field trials, breeding, and research. In two field trials, survival rate from the embryo stage to approximately 60 days post hatch ranged between 65 and 75%, and no epizootic has been reported to date. Draft genome sequencing was used to identify a large cluster of O-antigen biosynthetic genes specific to serotype O1 Yersinia ruckeri strains. This cluster primarily consisted of genes encoding proteins predicted to function in O-antigen and LPS biosynthesis and included proteins predicted to function in the biosynthesis of Legionamic acid, a nonulosonic acid know to be part of the O-polysaccharide repeat of O1 Y. ruckeri. Mutation of one of the identified nonulosonic acid biosynthesis genes (nab2) resulted in loss of both LPS synthesis and cross reactivity with a commercially available anti-O1 serotyping antibody. This loss of LPS biosynthesis was also shown to cause a dramatic reduction in serum resistance and a complete loss of virulence in a rainbow trout challenge model. Vaccination and challenge experiments using bacterin vaccines derived from the nab2 mutant and its wild type parent strain demonstrated that the presence of LPS is required to mount a protective response against Y. ruckeri challenge. Rainbow trout vaccinated with a flagellated biotype 1 Y. ruckeri vaccine mounted a robust IgM serum response. Measurable amounts of Y. ruckeri specific antibody were completely absorbed with a non-flagella expressing biotype 2 Y. ruckeri strain. This result suggests that lipopolysaccharide or another common surface structure, and not the flagella, is the major antigen recognized by the trout immune system. We previously observed that larger spleen size correlated with increased disease resistance. Surgical splenectomy or sham operation were randomly performed on resistant/susceptible line fish, and following recovery, fish were challenged with F. psychrophilum. Spleen removal did not alter the survival phenotypes, however our results indicate that spleen size is an indirect indicator of disease resistance and immunological status.


4.Accomplishments
1. New diagnostic test detects a serious bacterial disease of rainbow trout. Bacterial cold water disease, caused by Flavobacterium psychrophilum, is a widely-distributed and economically-important disease that results in elevated mortality in rainbow trout aquaculture. ARS researchers at Leetown, West Virginia developed a highly sensitive polymerase chain reaction (PCR) assay to accurately measure small amounts of this pathogen in fish tissue. The test recognizes a unique gene sequence that is only found in F. psychrophilum. The assay successfully identified more than 200 different isolates collected from farms where fish suffered from the disease. The assay was used to quantify bacterial numbers in experimentally and naturally infected rainbow trout tissues. Assay protocol and DNA standard have been distributed to government and university fish health researchers to enhance efforts to detect and control bacterial cold water disease.

2. High survival of bacterial cold water disease resistant rainbow trout line in farm trials. Bacterial cold water disease (BCWD) is a frequent cause of fresh water farmed trout loss. ARS researchers at Leetown, West Virginia have developed a BCWD resistant rainbow trout line, designated ARS-Fp-R, through multiple generations of genetic selection for improved disease resistance. Three consecutive years of performance testing of these fish were carried out under farm conditions. In five completed trials to date, in which age-matched control fish were diagnosed with BCWD, survival of the ARS-Fp-R line has been 95% from initial feeding through the early rearing phase. This was significantly greater than reference control and hatchery populations. In addition to greater survival, the ARS-Fp-R line had a smaller percentage of fish that tested positive for the pathogen that causes BCWD. These findings support the release of germplasm to stakeholders and the continued evaluation of the ARS-Fp-R genetic line in large-scale production trials.


Review Publications
Wiens, G.D., Lapatra, S.E., Welch, T.J., Evenhuis, J., Rexroad III, C.E., Leeds, T.D. 2013. On-farm performance of rainbow trout (Oncorhynchus mykiss) selectively bred for resistance to bacterial cold water disease: effect of rearing environment on survival phenotype. Aquaculture. 388-391:128-136.

Marancik, D.P., Wiens, G.D. 2012. A real-time polymerase chain reaction assay for identification and quantification of Flavobacterium psychrophilum and application to disease resistance studies in selectively bred rainbow trout. FEMS Microbiology Letters. doi: 10.1111/1574-6968.12061.

Ladner, J.T., Welch, T.J., Whitehouse, C.A., Palacios, G.F. 2013. Genome sequence of Weissella ceti NC36, an emerging pathogen of farmed rainbow trout in the United States. Genome Announcements. 1(1) e00187-12: 1-2.

Evenhuis, J., Welch, T.J., Booth, N.J. 2013. Transferable green fluorescence-tagged pEI2 in Edwardsiella ictaluri. Diseases of Aquatic Organisms. 105:75–79.

Last Modified: 9/10/2014
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