Location: Aquatic Animal Health Research
2016 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 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 second year of a five year project that has two major objectives. Objective 1 Progress: Draft genome sequencing of thirteen isolates of Flavobacterium (F.) columnare has been completed. The sequence reads for each isolate have been assembled to contiguous sequences and annotated. Comparative bacterial genomics is being performed on isolates belonging to the different genetic types or genomovars.
Antisera was generated against nine representative isolates of F. columnare and research is underway to determine the capsular polysaccharides (CPS) and lipopolysaccharides (LPS) antigenic types through western blotting. Approximately 50 isolates of F. columnare were collected from columnaris disease cases at stakeholder catfish farms in Mississippi and Alabama. These isolates will be included into this research.
Initial studies demonstrated that F. columnare isolates of different genomovars could replicate and survive in formulated water (water with added salts) supplemented with autoclaved tilapia mucus and porcine gastric mucin. Recent work demonstrated that a genomovar II isolate of F. columnare replicated and survived more than 125 days in sterile filtered native catfish mucus that was added to formulated water. These studies demonstrated that F. columnare uses mucus and/or mucin as a nutrient source and can survive for extended periods in the aquatic environment.
Objective 2 Progress: Six selected F. columnare isolates of various genomovars were successfully passaged on medium amended with a chemical suggested to change the LPS or CPS. Studies are underway to characterize potential changes to the LPS and/or CPS of the isolates passed on the media. Bacterial cells from archived parent and isolates passed on the chemical 48 times were collected for carbohydrate extractions and subsequent analysis.
Two DNA vaccine plasmids, each containing a gene that encodes an immobilization antigen (i-antigen) of Ichthyophthirius multifiliis (Ich), have been extracted and purified. Research is being conducted to determine optimal conditions for transfecting a catfish gill cell line and whether the DNA vaccine plasmids are expressed in vitro following transfection. Different concentrations of Lipofectamine, plasmid DNA concentrations, and combinations thereof are being evaluated to optimize cell transfection. The two Ich i-antigen genes have been cloned into a plasmid vector and transformed in expression host strains of Escherichia (E.) coli for the production of recombinant i-antigen protein.
ARS scientists reported on the development of a reproducible waterborne disease model for virulent Aeromonas (A.) hydrophila (vAh) and demonstrated that portals of entry for the bacterium are a key factor that predispose catfish to vAh disease in the laboratory. The disease model was used for a comparative virulence study using selected isolates of vAh (Alabama genotype; Mississippi genotype and Chinese grass carp genotype). Analysis of data obtained following waterborne challenge suggested that the genotypes of vAh differ in virulence, with the Chinese grass carp genotype resulting in significantly less mortality in channel catfish than the Alabama and Mississippi genotypes. The disease model was also used to study the pathogenesis of vAh in channel catfish and ARS scientists reported on the distribution of the bacterium following waterborne exposure. The research suggested that vAh was able to rapidly spread through the fish blood circulation system and proliferate causing mortality within eight to twenty-four hours.
Collaborative research with a tilapia producer in the United States and geneticists in Norway has determined the feasibility of selectively breeding Nile tilapia for resistance to Streptococcus (S.) iniae, a gram-positive bacterium that causes losses in tilapia aquaculture. Substantial additive genetic variation in resistance to S. iniae was observed when fish were infected, and this suggests promise for genetic improvement of tilapia for resistance to S. iniae through selective breeding. Additional research included two disease phenotyping studies with S. iniae and S. agalactiae 1b using Nile tilapia families supplied by the collaborator. The research confirmed the previous S. iniae results and also demonstrated considerable additive genetic variation in resistance of the Nile tilapia families to Streptococcus agalactiae 1b. The potential impact is custom genetic material produced by our collaborator for use on commercial tilapia farms to reduce disease caused by S. iniae and S. agalactiae.
Accomplishments
1. Improvement of disease resistance in Nile tilapia is possible through selective breeding. Intensification of tilapia production has resulted in disease outbreaks that negatively affect commercial fish farmers and one common bacterial pathogen is Streptococcus (S.) iniae. Control and prevention of S. iniae can be difficult and requires management practices, use of antibiotics, and vaccination. Selective breeding for resistance to disease is a complimentary strategy, but the potential for this was unknown. ARS scientists in Auburn, Alabama, in collaboration with industry stakeholders demonstrated that resistance to S. iniae is moderately heritable, indicating that is should be possible to develop an improved line of tilapia that are more resistant to disease. The long term goal of this research is to provide fish farmers with a more resistant stock of tilapia as an additional management tool for reducing production losses due to Streptococcus spp.
2. Phytase addition to a commercial catfish diet improved mineral uptake and blood parameters in catfish. Catfish feeds are plant-based and can feature high levels of phytate, which is a molecule known to bind iron and other minerals making them unavailable to the fish. ARS scientists in Auburn, Alabama, and Stuttgart, Arkansas, collaborated with Auburn University and demonstrated that coating feed with the enzyme phytase, which destroys phytate, could boost the uptake of iron and other key minerals. Scientists also showed that fish fed the phytase-treated diet had greater numbers of red blood cells, hemoglobin (the oxygen carrying molecule within red cells) better growth rates, and feed conversion. Phytase is currently used in poultry and swine diets to destroy phytate. This research suggests that phytase amended diets could improve the health and production efficiency of farmed catfish.
3. Reproducible waterborne disease model for virulent Aeromonas (A.) hydrophila (vAh). The catfish industry in the Southern United States has been greatly impacted by motile Aeromonas septicemia (MAS) caused by virulent vAh. This disease is responsible for the loss of approximately three million pounds of market-size fish annually since 2009. ARS scientists in Auburn, Alabama, demonstrated that portals for bacterial entry is a prerequisite for vAh infection via waterborne exposure in the laboratory and developed a reproducible disease model. The waterborne disease model will facilitate urgently-needed studies of vAh prevention and treatment.
4. Developed in vitro methods to screen parasiticides against Ich. Ichthyophthirius multifiliis, commonly called Ich, is a severe ciliate parasite that infects most fresh water fish worldwide resulting in heavy economic loss for aquaculture. Currently available parasiticides to control this parasite are limited. Ich is an obligated parasite, requires a fish host to survive and cannot be cultured in vitro. Tetrahymena (T.) thermophila is a free living protozoa in water similar to Ich and can be easily cultured in large quantities within a short period of time. ARS scientists in Auburn, Alabama, evaluated whether T. thermophila could be used to screen potentially effective parasiticides against Ich. The results demonstrated that the parasiticides that killed T. thermophila would kill Ich, thus the in vitro method using T. thermophila can be used to screen novel parasiticides effective against Ich.
5. Molecular immune response of channel catfish following immunization with live Ichthyophthirius. The parasite Ichthyophthirius multifiliis (Ich) has resulted in severe losses to both food and aquarium fish production worldwide. Fish surviving natural infections or immunized with live theronts develop strong immune responses. Little is known about how immune genes are induced or how they interact and lead to specific immunity against Ich in channel catfish. ARS scientists in Auburn, Alabama, in collaboration with scientists from Brazil and China, evaluated the differential expression of immune genes in catfish after immunization with Ich and demonstrated that the immunized fish showed significantly higher anti-Ich antibody and survival (95%) than non-immunized fish. Results showed that immune genes involved in the specific or non-specific immune response were up-regulated post immunization. This study has led to a better understanding of the molecular immune response following immunization of catfish against Ich.
None.
Review Publications
Beck, B.H., Fuller, S.A., Li, C., Green, B.W., Rawles, S.D., Webster, C.D., Peatman, E. 2016. Hepatic transcriptomic and metabolic responses of hybrid striped bass (Morone saxatilis × Morone chrysops) to acute and chronic hypoxic insult. Comparative Biochemistry and Physiology. 18(Part D):1-9.
Lafrentz, B.R., Lozano, C.A., Shoemaker, C.A., Garcia, J.C., Xu, D., Lovoll, M., Rye, M. 2016. Controlled challenge experiment demonstrates substantial additive genetic variation in resistance of Nile tilapia (Oreochromis niloticus) to Streptococcus iniae. Aquaculture. 458:134-139.
Xu, D., Zhang, Q., Shoemaker, C.A., Zhang, D., Moreira, G.S. 2016. Molecular immune response of channel catfish immunized with live theronts of Ichthyophthirius multifiliis. Fish and Shellfish Immunology. 54:86-92.
Bartelme, R.P., Newton, R.J., Zhu, Y., Li, N., Lafrentz, B.R., Mcbride, M.J. 2016. Complete genome sequence of the fish pathogen Flavobacterium columnare strain C#2. Genome Announcements. 4(3):e00624-16. doi:10.1128/genomeA.00624-16.
Xu, D., Shoemaker, C.A., Zhang, D. 2015. Treatment of Trichodina sp. reduced load of Flavobacterium colummnare and improved survival of hybrid tilapia. Aquaculture Report. 2:126-131.
Fu, Y., Zhang, Q., Xu, D., Wang, B., Liang, J., Lin, D. 2015. Cynatratoside-C efficacy against Ichthyophthirius multifiliis, and toxicity tests on grass carp and mammal blood cells. Diseases of Aquatic Organisms. 117:13-20.
Zhang, D., Bland, J.M., Xu, D., Chung, S. 2015. Degradation of chitin and chitosan by a recombinant chitinase derived from a virulent Aeromonas hydrophila isolated from diseased channel catfish. Advances in Microbiology. 5:611-619.
Lin, D., Zhang, Q., Xu, D., Fu, Y., Liu, Y., Zhou, S. 2016. Evaluation of medicated feeds with antiparasitical and immune-enhanced Chinese herbal medicines against Ichthyophthirius multifiliis in grass carp (Ctenopharyngodon idellus). Parasitology Research. 115:2473-2483.
Dong, T.H., Lafrentz, B.R., Pirarat, N., Rodkhum, C. 2015. Phenotypic characterization and genetic diversity of Flavobacterium columnare isolated from red tilapia, Oreochromis sp. in Thailand. Journal of Fish Diseases. 38:901-913.
Dong, H.T., Senapin, S., Lafrentz, B.R., Rodkhum, C. 2016. Virulence assay of rhizoid and non-rhizoid morphotypes of Flavobacterium columnare in red tilapia, Oreochromis sp., fry. Journal of Fish Diseases. 39:649-655.
Xu, D., Zhang, Q., Zhang, D. 2015. Two in vitro methods for screening potential parasiticides against Ichthyophthirius multifiliis using Tetrahymena thermophila. Journal of Fish Diseases. 39:285-294.
Zhang, D., Moreira, G.S., Shoemaker, C.A., Newton, J.C., Xu, D. 2016. Detection and quantification of virulent Aeromonas hydrophila in channel catfish tissues following waterborne challenge. FEMS Microbiology Letters [online]. 363(9). Available: http://femsle.oxfordjournals.org/content/363/9/fnw080. doi:10.1093/femsle/fnw080.
Zhang, D., Xu, D., Shoemaker, C.A. 2015. Immunization with recombinant aerolysin and hemolysin protected channel catfish against virulent Aeromonas hydrophila. Aquaculture Research. doi: 10.111/are.12931.
Zhang, D., Xu, D., Shoemaker, C.A. 2016. Experimental induction of motile Aeromonas septicemia in channel catfish by waterborne challenge with virulent Aeromonas hydrophila. Aquaculture Reports. 3:18-23.
