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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

2012 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:
1) On-going breeding efforts at the NCCCWA have produced three lines of rainbow trout that differ in resistance to bacterial cold water disease. Approximately 1,850 fish representing families from disease resistant (ARS-Fp-R), susceptible (ARS-Fp-S), and control lines (ARS-Fp-C) were evaluated for growth performance to 12.5 months of age (~1 kg). Disease resistance and pedigree data were used to identify families to breed in order to produce the 4th-generation of the resistant line. Pedigree information alone was used to develop breeding plans for propagating the susceptible and control lines. .
2)Completed field trials initiated in 2011 in Idaho and Utah measuring the performance of selectively-bred lines of rainbow trout. Outbreaks of bacterial cold water disease occurred at three field trial sites and infection severity and fish survival were measured. Over 300 bacterial isolates of F. psychophilum were collected, analyzed and archived.

Yersinia ruckeri is the causative agent of enteric red mouth disease and vaccine resistant strains of this bacterium have emerged within the U.S. Molecules that enable bacterial motility (flagella) or that make-up the carbohydrate outer membrane layer were purified from Y. ruckeri. These components will be used to vaccinate fish to investigate whether these components are important for inducing immunity. A mutant of Y. ruckeri that over-produces flagella proteins was identified, and this mutant will be used to determine whether flagella overproduction alters virulence and long-term immunity.

1) Fifty fish genes with similarity to human and mice immune genes were identified from sequence databases. For several genes, duplicates were identified in rainbow trout. These analyses will facilitate the development of a rainbow trout immune-gene database in order to improve understanding of disease resistance mechanisms..
2)An effective challenge procedure was established for infecting rainbow trout with Flavobacterium columnare. Optimized experimental parameters included bacterial growth conditions, fish exposure, challenge dose, and fish size. Challenge experiments routinely produce 30-70% mortality. The establishment of a standardized challenge protocol is a critical first step for future studies designed to measure differences in the innate immune response and also the response that occurs after vaccination.

Additional Progress:.
1)In collaboration with the NCCCWA genomics group, 15 select trout families were created and offspring challenged with F. psychrophilum to identify regions of chromosomes containing genes which affect disease resistance and spleen size..
2)In collaboration with the NCCCWA physiology group, 10 families of having a common genetic background but differing in their number of sets of chromosome (diploids, triploids) were examined for innate disease resistance following challenge with F. psychrophilum and Y. ruckeri. These studies further our understanding of the impact of chromosome set number on disease resistance.


4.Accomplishments
1. Release of germplasm from the ARS-Fp-R rainbow trout line to industry stakeholders. Bacterial cold water disease, caused by Flavobacterium psychrophilum, is a widely-distributed and economically-important disease that results in elevated mortality and deformity rates in rainbow trout aquaculture. Currently there are only limited chemotherapeutics, and no commercial vaccines, available for control or prevention of the disease. ARS researchers at the National Center for Cool and Cold Water Aquaculture at Leetown, West Virginia have developed the ARS-Fp-R rainbow trout line through multiple generations of genetic selection for improved disease resistance, and have demonstrated the line’s superior resistance to the bacterium in laboratory challenges and field trial evaluations. The release of 80,000 embryos from 52 pedigreed families to Clear Springs Foods Inc. (Buhl, ID) and Troutlodge Inc. (Sumner, WA), and approximately 50,000 embryos from 43 families to the Utah Division of Wildlife Resources, will allow industry and government stakeholders to propagate or introgress the line and produce future generations of fish with improved disease resistance.

2. Identification of Weissella sp. as the causative agent of a significant disease outbreak in farmed rainbow trout and development of an autogenous vaccine. Emerging pathogens are a significant threat to U.S. Aquaculture. An ARS researcher at Leetown, West Virginia isolated a new gram positive bacterial pathogen causing significant loss of rainbow trout in North Carolina. The pathogen is similar to Weissella sp. associated with recent disease outbreaks in farmed rainbow trout in both China and Brazil and this is the first report of this pathogen in the U.S. At the request of stakeholders, the NCCCWA developed and validated an effective autogenous vaccine which is now in commercial production and in use at North Carolina farms affected by this pathogen. Early pathogen detection and the rapid development and implementation of a vaccine will aid control efforts and reduce the likelihood of further pathogen dissemination in the U.S.

3. Quantified intestinal immune response to bacterial challenge. The intestinal immune response of trout following bacterial challenge is poorly understood. Two challenge routes, injection and immersion, were used by ARS researchers at Leetown, West Virgina to examine whether expression of immune genes changed in the lower intestine following bacterial exposure. Several immune genes representing both innate and acquired responses increased in abundance as high as 250-fold. These results demonstrate a robust immunological response in the lower intestine following challenge with bacterial pathogens Y. ruckeri or F. psychrophilum. These findings further our understanding of the pathogenesis of bacterial disease and will further the development of orally-delivered vaccines.


Review Publications
Evenhuis, J., Cleveland, B.M. 2012. Modulation of rainbow trout (Oncorhynchus mykiss) intestinal immune gene expression following bacterial challenge. Veterinary Immunology and Immunopathology. 146(1):8-17.

Lafrentz, B.R., Welch, T.J., Shoemaker, C.A., Drennan, J.D., Klesius, P.H. 2011. Modified live Edwardsiella ictaluri vaccine, AQUAVAC-ESC, lacks multidrug resistance plasmids. Journal of Aquatic Animal Health. 23(4):195-199.

Verner-Jeffreys, D.W., Haig, S.J., Welch, T.J., Pond, M.J., Stone, D., Davies, R., Gardner, R. 2011. Characterisation of a serotype O1 Yersinia ruckeri isolate from the Isle of Man: further evidence that O antigen serotype is not a reliable indicator of virulence. European Association of Fish Pathologists. 31(3):86-91.

Lafrentz, B.R., Lapatra, S.E., Call, D.R., Wiens, G.D., Cain, K.D. 2011. Identification of immunogenic proteins within distinct molecular mass fractions of Flavobacterium psychrophilum. Journal of Fish Diseases. 34:823-830.

Last Modified: 10/22/2014
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