Location: Aquatic Animal Health Research
2015 Annual Report
Objectives
Objective 1: Identify and characterize genetic diversity, mechanisms of pathogenesis and virulence factors in Flavobacterium (F.) columnare.
Subobjective 1.A.: Comparative bacterial genome analysis of Flavobacterium columnare isolates of different genetic types and virulence.
Subobjective 1.B.: Molecular basis of lipopolysaccharide (LPS) and capsular polysaccharide (CPS) antigenic diversity in Flavobacterium columnare.
Subobjective 1.C.: Characterize the growth and survival of F. columnare in fish mucus.
Objective 2: Develop vaccines and probiotics that provide protection against bacterial and parasitic pathogens and identify mechanisms of immunity.
Subobjective 2.A.: Chemical mutagenesis of Flavobacterium columnare to modify the capsular polysaccharide (CPS) to develop attenuated vaccines.
Subobjective 2.B.: Evaluate the protective effect of a DNA vaccine encoding Ich immobilization antigens to protect catfish against Ich.
Subobjective 2.C.: Develop a waterborne challenge model and recombinant protein vaccine to protect channel catfish against virulent Aeromonas hydrophila.
Approach
Catfish and tilapia farmers continue to identify disease as a major problem in their industries. For instance, columnaris disease, caused by Flavobacterium (F.) columnare, is one of the top two diseases diagnosed in the industry. Since 2009, a virulent strain of Aeromonas (A.) hydrophila has greatly impacted the catfish industry and resulted in losses of greater than 12 million U.S. dollars. Ichthyophthirius (I.) multifiliis, the parasite that causes Ich, is responsible for annual losses of 1.2 million U.S. dollars to the catfish industry alone. An increased understanding of the pathogen, host responses to the pathogen, and host-pathogen interactions is necessary for disease prevention and control. This in-house project will expand our knowledge of these and will use new and existing knowledge to develop approaches to reduce disease losses in catfish and tilapia aquaculture. Development of disease prevention strategies will increase the profitability and sustainability of these important aquaculture industries.
Objective 1 recognizes that although columnaris disease has been intensely studied in the past, important questions concerning genetic diversity of isolates impacting aquaculture and mechanisms of pathogenesis have newly emerged. A greater understanding of these factors will enhance our ability to improve existing and develop new prevention strategies practical for use in the catfish and tilapia industries. Research conducted in this objective will utilize comparative genome analyses to identify genetic similarities and differences among F. columnare isolates of different genomovars (genetic types) and will correlate the genetic differences with variation in virulence. The genome sequences will be exploited to determine the molecular basis of lipopolysaccharide and capsular polysaccharide antigenic diversity in F. columnare. The growth of F. columnare in catfish mucus will be used as a model to determine the proteomic changes that occur in F. columnare during the colonization of catfish and how these changes are involved in virulence.
Objective 2 acknowledges that even though there is a commercially available vaccine for F. columnare and experimental vaccines exist for other bacterial and parasite pathogens, there is a need to develop improved disease prevention methods and identify the mechanisms responsible for protective immunity. Research conducted will utilize chemical mutagenesis to modify the capsular polysaccharide of F. columnare to develop more effective attenuated vaccines. A DNA vaccine for I. multifiliis will be developed based on proteins of the parasite that have been previously demonstrated to be protective. A reproducible waterborne challenge model for virulent A. hydrophila will be developed and will allow for more effective testing of treatment or prevention strategies. A recombinant protein vaccine for A. hydrophila will be developed based on secreted proteins of the bacterium that are identified as protective. This research will increase our understanding of the host immune responses against pathogens and will develop improved and new vaccines for prevention of disease in catfish and tilapia aquaculture.
Progress Report
Considerable progress has been made under Objective 1 to identify and characterize genetic diversity, mechanisms of pathogenesis and virulence factors in Flavobacterium (F.) columnare. Thirteen isolates of F. columnare, encompassing different genomovars (genetic type) and virulence, were selected for whole genome sequencing. The genomic DNA was extracted from each and were submitted for sequencing. The sequence reads obtained are being assembled to contiguous sequences and annotated. Once completed, comparative genome analyses will be performed.
To investigate the antigenic diversity of the capsular polysaccharides (CPS) and lipopolysaccharides (LPS) of F. columnare, carbohydrates were extracted from representative isolates and antisera is in the process of being generated. Once the antisera has been generated, the CPS and LPS antigenic types of each isolate will be determined.
Progress has been made under Objective 2 to develop vaccines and probiotics that provide protection against bacterial and parasitic pathogens and identify mechanisms of immunity. Five isolates of F. columnare of different genomovars were selected for chemical mutagenesis and passaged 57–69 times on medium amended with the chemical. The parent and passaged bacterial isolates were archived for future testing to determine whether the capsular polysaccharides have been modified and if the passaged isolates are attenuated.
Progress has been made towards developing a deoxyribonucleic acid (DNA) vaccine for Ichthyophthirius multifiliis (Ich). Two genes encoding immobilization antigens (i-antigens) of Ich have been optimized for protein expression in channel catfish and modified for expression in E. coli. The genes were synthesized and cloned into a vector commonly used in DNA vaccine studies in fish. The recombinant vectors were transformed into E. coli and positive transformants containing the Ich immobilization genes have been selected for DNA vaccine plasmids. These vaccine plasmids have been sent to USDA-ARS Southeast Area Genomic Laboratory in Stoneville, MS, for sequencing to confirm the proper insertion and correct sequence of synthesized genes. Next, ARS scientists will evaluate the ability of the vaccine plasmids to express in fish cell lines and in channel catfish to determine if they provide protection against Ich.
Substantial progress has been made towards developing a waterborne disease method for virulent Aeromonas (A.) hydrophila (vAh). ARS scientists demonstrated that portals of entry for the bacterium are one of the key factors that predispose catfish to vAh infection via laboratory infection models, and a reproducible waterborne challenge model was developed. It was also demonstrated that mortality caused by vAh is significantly associated with bacterial concentration, exposure time and water temperature. In collaboration with Auburn University and Mississippi State University, a comparative virulence study was completed using different genotypes of vAh (Alabama genotype, Mississippi genotype and Chinese grass carp genotype). The median lethal dose for these isolates was determined with the waterborne challenge model. Preliminary data demonstrated the reproducibility of the developed challenge model in inducing vAh disease in channel catfish and suggested that the genotypes differ in virulence, with the Chinese grass carp genotype being much less virulent than the Alabama and/or Mississippi genotypes.
Progress has been made towards developing a recombinant protein vaccine to protect channel catfish against vAh. Two virulence-associated proteins (aerolysin and hemolysin) in A. hydrophila were demonstrated to be promising immunogens for vaccine development. Vaccination of catfish with the recombinant proteins provided protective immunity (relative percent survival of approximately 70-75%) against A. hydrophila infection. Additionally, genes encoding aerolysin and hemolysin were cloned into eukaryotic-expression vectors to determine their potential as DNA vaccines.
A collaboration has been established with a tilapia producer in the United States and geneticists in Norway. Research was initiated to determine the feasibility of selectively breeding Nile tilapia for resistance to Streptococcus (S.) iniae, a gram-positive bacterium that causes multi-million dollar losses in tilapia aquaculture.
Accomplishments
1. Flavobacterium columnare utilizes fish mucus as a nutrient source. Flavobacterium (F.) columnare is an economically important gram-negative bacterium that infects most freshwater farmed fish worldwide. The bacterium colonizes the skin and gills of fish in the initial steps of infection. The fish’s surface is coated with mucus made up of high molecular weight glycoproteins. ARS scientists in Auburn, Alabama, demonstrated the ability of different F. columnare isolates to grow and survive for long periods of time in water containing tilapia mucus or porcine gastric mucin (mucus purified from pig intestine) as a nutrient source. Presence of mucus and/or mucin proteins likely regulate the gene expression and virulence of F. columnare.
Review Publications
Shoemaker, C.A., Lafrentz, B.R. 2015. Growth and survival of the fish pathogenic bacterium, Flavobacterium columnare, in tilapia mucus and porcine gastric mucin. FEMS Microbiology Letters. 362(4):1-5.
Shoemaker, C.A., Xu, D., Lafrentz, B.R., Lapatra, S. 2015. Overview of fish immune system and infectious diseases. In: Lee, C.S., Lim, C., Gatlin, D.M., Webster, C.D. Dietary Nutrients, Additive, and Fish Health. Hoboken, New Jersey:John Wiley & Sons, Inc. 1-24.
