Location: Aquatic Animal Health Research
2019 Annual Report
Objectives
The additional funds will enhance research on current Objective 2 and two new objectives that are presently going through OSQR review at this time which are as follows:
Current Objective 2: Develop vaccines and probiotics that provide protection against bacterial and parasitic pathogens and identify mechanisms of immunity by identifying and characterizing host mechanisms responding to infection and host-pathogen interactions that can be used to develop approaches that reduce losses to disease.
New Objective 1: Identify virulence factors critical for pathogenesis of major catfish pathogens to guide the development of novel and cost-effective disease interventions.
New Objective 2: Improve prevention and control strategies for bacterial and parasitic diseases of catfish and shrimp.
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 by identifying and characterizing host mechanisms responding to infection and host-pathogen interactions that can be used to develop approaches that reduce losses to disease.
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
This is the fourth year of a five-year project that has two major objectives. Objective 1 Progress: Through multilocus phylogenetic analyses, we previously established the existence of four phylogenetically distinct genetic groups within the species Flavobacterium (F.) columnare. Research was conducted to further understand the genetic and phenotypic differences between the four groups. We sequenced the whole genomes of one genetic group 3 isolate and two genetic group 4 isolates to single contiguous sequences. Comparative pan-genome analyses are being conducted to determine the genetic differences between the four groups. Through these sequencing efforts we have identified the first plasmid reported in F. columnare. Isolates from each of the four genetic groups were analyzed by MALDI-TOF. Methods for sample preparation were optimized and found to be highly reproducible. The spectra generated are being analyzed to determine if this technique may be useful for identification of isolates belonging to the four genetic groups. Findings from this research may explain the host-pathogen associations previously documented for the genetic groups.
A better understanding of the prevalence of the four genetic groups of F. columnare in the catfish industry will aid in the development of better targeted control measures such as vaccines. We previously developed a multiplex polymerase chain reaction assay (PCR) to assign an unknown isolate of F. columnare to genetic group. The multiplex PCR was optimized to be able to identify all four groups in a single reaction and the assay was validated. The PCR assay is currently being used to determine the genetic group of F. columnare involved in individual diagnostic cases from the catfish as well as other aquaculture industries. To facilitate this, the ability to use swabs for collecting deoxyribonucleic acid (DNA) samples from infected catfish was found to be a feasible means for high throughput sampling. Methods to extract DNA from the swabs were optimized. In collaboration with stakeholder catfish farms and four diagnostic laboratories in the Southeastern USA over 100 samples have been collected from columnaris disease cases. Preliminary data indicates that isolates belonging to genetic groups 1, 2, and 3 are involved in catfish columnaris disease cases in the southeast U.S. and in some instances co-infections of the three groups occur. Genetic group 2 isolates appear to be most predominant in the catfish industry.
Knowledge gaps remain with respect to how virulent Aeromonas (A.) hydrophila (vAh) establishes infection and results in severe losses seen in catfish ponds. Large-scale losses of catfish to vAh are common in commercial aquaculture; however, this pathogen is challenging to work with in the laboratory because the experimental exposure of healthy catfish to vAh results in little to no disease unless a portal of entry is provided. Recently, we found that vAh can use the naturally occurring biopolymer chitin and chitin derivatives as sole carbon and nitrogen sources for vigorous growth. This suggests that the persistently high vAh populations that support outbreaks of MAS could be related to the organic load in catfish ponds. Research was conducted to determine the effect of chitin and other organic matter on the pathogenicity of vAh. Using an in-tank bacterial culture method, in which vAh was inoculated in tank water supplemented with organic nutrients, it was found that the pathogen could reach very high levels within 24 hours that caused more than 70% mortality of fish in which a portal of entry was not provided. The research may indicate that vAh is more virulent in the presence of organic matter and may explain the high virulence seen in catfish aquaculture.
In collaboration with a stakeholder farm, several epizootics involving largemouth bass were investigated. Samples from various tissues were streaked onto various media and resultant colonies were characterized via a combination of biochemical and molecular methods. Aeromonas veronii was the predominant bacterium recovered from fish; however, F. columnare was also isolated from some fish. Our research suggests the possibility of A. veronii as a primary pathogen in largemouth bass aquaculture and research is underway to explore treatment and control options.
Objective 2 Progress: Progress has been made towards developing DNA vaccines for Ichthyophthirius multifiliis (Ich). We completed a vaccine trial to evaluate the protective effect of three DNA vaccines encoding Ich immobilization antigens. The research identified one DNA vaccine that provided protection to catfish following pathogen challenge. Research was conducted to evaluate whether higher doses or multiple doses of the DNA vaccine would provide better protection for catfish against Ich. Research demonstrated fish vaccinated with one high dose or two low doses of the DNA vaccine exhibited elevated levels of anti-Ich antibodies and protection following Ich challenge. Vaccination with two low doses conferred a higher level of protection. Results of this study revealed that the DNA vaccine could enhance fish immune responses and increase catfish survival against Ich.
The top challenge for shrimp producers is maintaining profitability in the face of massive disease-related losses. Large scale losses are not uncommon, and the causative agents are numerous and diverse. At or near the top of the list is an emerging bacterial disease called acute hepatopancreatic necrosis disease (AHPND) most commonly caused by Vibrio (V.) parahaemolyticus, which harbors a plasmid expressing a Pir A/B toxin. These toxins have been reported to be responsible for mortalities associated with AHPND. Research was conducted to evaluate the toxicity of the protoxins PirA and PirB derived from V. parahaemolyticus. Recombinant PirA and PirB (rPirA and rPirB) proteins were produced in Escherichia coli expression systems and then shrimp were exposed to the recombinant proteins. The results demonstrated that shrimp exposed to both toxins simultaneously exhibited high mortality, confirming their role in the pathogenesis of AHPND. Mineral-based approaches are also being evaluated in both the water and feed to determine the extent to which various minerals can bind toxin and minimize mortality.
Francisella (F.) noatunensis subsp. orientalis (FNO) causes francisellosis in tilapia aquaculture and is responsible for large economic losses. Research was conducted to develop an experimental challenge model for FNO for use in laboratory disease studies. Two disease phenotyping studies with FNO and Streptococcus (S.) agalactiae were completed using Nile tilapia families supplied by collaborators. Substantial additive genetic variation in resistance to FNO was observed, suggesting that genetic improvement of tilapia for resistance to FNO should be possible through selective breeding. Research results confirmed additive genetic variation found previously for survival to S. agalactiae and survival results for assortative mating groups supported this. Collaborator has incorporated selective breeding for these important tilapia pathogens into their selective breeding program.
Accomplishments
1. A killed Aeromonas hydrophila vaccine delivered via immersion protects catfish against motile Aeromonas septicemia. Outbreaks of motile Aeromonas septicemia (MAS) in West Alabama and East Mississippi have cost U.S. catfish aquaculture an estimated $60-70 million due to death, lost feeding days and costly chemical and antibiotic treatments. Control of virulent Aeromonas (A.) hydrophila (vAh) is problematic because fish kills on farms are often rapid and the mortality is typically seen in larger and highly valuable market-sized fish. Little time is available to initiate antibiotic therapy and the withdrawal period after antibiotic feeding requires additional time and economic input prior to harvest. Alternative control strategies such as vaccination are desperately needed at the farm level. Due to the ease of manufacture and, ultimately, the licensing of a killed vaccine for farm use, ARS scientists in Auburn, Alabama designed and evaluated the effectiveness of a simple vAh bacterin (killed vaccine) delivered via immersion (waterborne route) to hybrid catfish. Results demonstrated strong protection of hybrid catfish for at least 7 weeks following vaccination with this simple preparation against two vAh strains.
2. Waterborne exposure to select clay minerals protects catfish against virulent Aeromonas hydrophila infections. Aeromonas hydrophila is one of the most widespread bacterial pathogens affecting freshwater fish and a new strain of A. hydrophila has severely impacted the catfish industry over the last decade. ARS scientists in Auburn, Alabama, evaluated the effect of treatment with kaolin, an inert clay, for controlling A. hydrophila outbreaks. Tests revealed that kaolin clay significantly blocked the movement and binding ability of A. hydrophila to catfish mucus. Kaolin treatment at a level of 0.1% led to a significant improvement in survival (66.7%) of experimentally infected catfish as compared to untreated fish (28.9%). Kaolin treatment did not alter the growth of A. hydrophila, but bacterial levels in test suspensions were significantly reduced by kaolin treatment within 15 minutes, indicating the rapid formation of settleable complexes between kaolin and bacteria. These findings suggest that the integration of kaolin-based approaches into some production settings may be beneficial, particularly in scenarios where the large-scale use of antibiotics is not appropriate or advisable, or when it is likely that an Aeromonas outbreak is going to occur following stressors such as grading, stocking, or transport of fish.
3. Pacific white shrimp (Litopenaeus vannamei) cultured in onshore tanks in low salinity waters showed that overall performance was acceptable regardless of hatchery source. Inland, low salinity shrimp farmers in west Alabama, that produce the Pacific white shrimp, Litopenaeus vannamei, have recently reported abnormally low survivals at harvest. Reduced survival has also been reported by farms in Florida and Texas. Multiple theories exist as to the cause of increased mortality including disease, toxic algae blooms, water quality, shrimp source, and reduced robustness of shrimp in later stages of production. To compare performance of shrimp from different sources, shrimp were obtained from three different hatcheries and stocked on the same day in three different flow-through systems. One tank system (TS) was installed on one farm (Farm 1-TS) and two systems were installed on two different pond banks of another farm (Farm 2-TS1; Farm 2-TS2). Following 107 days of culture on Farm 2-TS1 and Farm 2-TS2 there were no differences in survival (72.8 – 91.2 %) or final weight (19.8 – 24.6 g) among shrimp sourced from three different hatcheries. At Farm 1-TS, following 111 days of culture there were differences in survival from shrimp sourced from one hatchery (40.5%) compared to the other two hatcheries (61.0 – 69.8%). A large percentage of the mortality in the trial occurred in the first thirty days for two of the tank systems (Farm 2-TS1 and Farm 1-TS). Results of this trial by ARS scientists in Auburn, Alabama, demonstrate that while hatchery source did influence survival on one farm, overall performance was acceptable from all three sources compared to performance of shrimp reared in the production ponds in which the tank systems were housed and drawing water.
Review Publications
Fu, Y., Wang, B., Zhang, Q., Xu, D., Liu, Y., Hou, T., Guo, S. 2019. Efficacy and antiparasitic mechanism of 10-gingerol isolated from ginger Zingiber officinale against Ichthyophthirius multifiliis in grass carp. Veterinary Parasitology. 265:74-84.
Roy, L., Teichert-Coddington, D., Laramore, S., Dahl, S., James, J., Whitis, G.N., Beck, B.H., Shoemaker, C.A. 2018. Commercial demonstration of a probiotic to enhance pacific white shrimp production in inland ponds of Alabama and Florida. Journal of the World Aquaculture Society. 49(4):42-49.
Aksoy, M., Mohammed, H., Peatman, E., Fuller, S.A., Beck, B.H. 2018. Influence of Kaolin Clay on Aeromonas hydrophila Growth, Chemotaxis, and Virulence to Channel Catfish. North American Journal of Aquaculture. 80(4):427-435. https://doi.org/10.1002/naaq.10059.
Shoemaker, C.A., Mohammed, H., Bader, T.J., Peatman, E., Beck, B.H. 2018. Immersion vaccination with an inactivated virulent Aeromonas hydrophila bacterin protects hybrid catfish (Ictalurus punctatus X Ictalurus furcatus) from motile Aeromonas septicemia. Fish and Shellfish Immunology. 82:239-242. https://doi.org/10.1016/j.fsi.2018.08.040.
He, Z., Zhang, D., Cao, H. 2018. Protein profiling of water and alkali soluble cottonseed protein isolates. Scientific Reports. 8:9036. https://doi.org/10.1038/s41598-018-27671-z.
Roy, L.A., Teichert-Coddington, D., Dahl, S., Beck, B.H., Shoemaker, C.A., Whitis, G.N., James, J. 2019. On-farm evaluation of three different hatchery sources of Pacific white shrimp (Litopenaeus vannamei) cultured in on-levee tanks in low salinity waters of west Alabama. Journal of Applied Aquaculture. https://doi.org/10.1080/10454438.2019.1614510.
Declercq, A., Cai, C., Naranjo, W., Thongda, W., Eeckhaut, V., Bauwens, E., Arias, C., De La Fuente, L., Beck, B.H., Lange, M.D., Peatman, E., Aerts, J., Haesebrouck, F., Decostere, A. 2019. Evidence that the stress hormone cortisol regulates biofilm formation differently among Flavobacterium columnare isolates. Veterinary Research. 50(1):24. https://doi.org/10.1186/s13567-019-0641-3.
Beck, B.H., Aksoy, M., Shoemaker, C.A., Fuller, S.A., Peatman, E. 2019. Antimicrobial activity of the biopolymer chitosan against Streptococcus iniae. Journal of Fish Diseases. 42:371-377.
Thurlow, C.M., Hossain, M.J., Sun, D., Barger, P., Foshee, L., Beck, B.H., Newton, J.C., Terhune, J.S., Saper, M.A., Liles, M.R. 2019. The gfc operon is involved in the formation of the O antigen capsule in Aeromonas hydrophila and contributes to virulence in channel catfish. Aquaculture. https://doi.org/10.1016/j.aquaculture.2019.734334.
Sebastiao, F., Lafrentz, B.R., Shelley, J.P., Stevens, B., Marancik, D., Dunker, F., Reavil, D., Soto, E. 2019. Flavobacterium inkyongense isolated from ornamental cichlids. Journal of Fish Diseases. 42:1309-1313. https://doi.org/10.1111/jfd.13043.
