Location: Aquatic Animal Health Research
2017 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 third year of a five year project that has two major objectives. Objective 1 Progress: Comparative bacterial genomics was performed on isolates belonging to the five different genetic types or genomovars. Phylogenetic analysis of the 16S ribosomal ribonucleic acid (rRNA), gyrase (gyrB), and nine concatenated housekeeping genes yielded four distinct clusters (genetic groups). There was an association between the three primary genetic groups and genomovar. Genetic groups 1, 2, and 3 were comprised of isolates assigned to genomovars I and I/II, genomovars II and II-B, and genomovar III, respectively. The fourth cluster (genetic group 4) was comprised of a single isolate in which genomovar assignment was not previously possible due to polymerase chain reaction (PCR) failure and may be unique. The results from this research establish the existence of four phylogenetically distinct genetic groups within the species Flavobacterium (F.) columnare.
There is a lack of knowledge of which genetic group(s) is most prevalent in the catfish industry and a better understanding of strain diversity and prevalence will aid in the development of better targeted control measures. Comparative bacterial genomics was used to identify regions in the genomes unique to each genetic group and these regions were exploited to develop a multiplex PCR assay. PCR primers were designed to amplify different sized amplicons specific for each genetic group. The multiplex PCR was tested on genomic deoxyribonucleic acid (gDNA) from a panel of F. columnare isolates of unknown genetic group and genomovar assignment was conducted in parallel. The results demonstrated 100% accordance between multiplex PCR results and genomovar assignment. Research demonstrated that DNA extracted from necrotic gill tissue and swabs taken from lesions of catfish exhibiting columnaris disease serve as suitable template for use in the multiplex PCR assay. The multiplex PCR reported here provides a useful tool for rapidly assigning an unknown isolate to genetic group and for determining the genetic group of F. columnare involved in individual diagnostic cases without the need for bacterial culture. Currently we are collaborating with stakeholder catfish farms and four diagnostic laboratories in the Southeastern USA, in which the labs are collecting swab samples from industry columnaris cases to determine which genetic groups of F. columnare are circulating and most predominant in the catfish industry.
Research is continuing to determine the capsular polysaccharide (CPS) and lipopolysaccharide (LPS) antigenic types of F. columnare isolates through western blotting. Several CPS antigenic types have been identified. The biosynthetic genes for the CPS of F. columnare were identified, and research is underway to perform comparative analyses of the CPS biosynthetic genes between isolates exhibiting different CPS antigenic types.
Two genetic group 2 isolates of F. columnare were grown in the presence of native (not heated) channel catfish and hybrid catfish mucus. The isolates selected differed in virulence with one isolate being highly virulent in channel catfish fry and fingerlings and the other isolate being moderately virulent. Both isolates were able to grow and survive in native catfish or hybrid catfish mucus as a nutrient source for more than 28 d. After demonstration of growth in catfish mucus, extracellular (secreted) proteolytic (protein degradation) activity was assayed using an in vitro test when the bacteria were grown in either laboratory culture media or catfish mucus. Extracellular proteolytic activity was significantly enhanced when the isolates were grown in the presence of catfish mucus versus media. Interestingly, the extracellular proteolytic activity of the highly virulent isolate grown in mucus/mucin was significantly higher (3-4 fold) when compared to the moderately virulent isolate grown under the same conditions. The identification of differential proteolytic activity may be related to virulence in that some F. columnare may have a greater ability to break down the protective mucus layer resulting in enhanced colonization, pathology and/or death. These studies demonstrated that F. columnare uses mucus and/or mucin as a nutrient source and provided new insight into the pathogenic mechanisms of F. columnare in catfish.
Objective 2 Progress: The CPS antigenic type of the parent F. columnare isolates used for mutagenesis was determined using antisera generated under Objective 1. These isolates were previously grown in the presence of a chemical known to modify the CPS of bacteria and the resultant mutagenized strains are ready for analysis. Research is ongoing to determine if the mutagenized isolates exhibit differences in the CPS. Once completed, isolates with modified CPS will be used to determine if these modifications resulted in reduced or loss of ability to cause columnaris disease in catfish.
Substantial progress has been made towards developing DNA vaccines for Ichthyophthirius multifiliis (Ich). First, our research team evaluated the expression of a suite of immune genes correlated with innate and adaptive immune responses in skin from channel catfish after immunization with live Ich theronts where immunized fish showed significantly higher survival (95%) than control fish. This study revealed that genes related to immunoglobulins, cytokines, inflammatory proteins, and cell receptors showed a rapid up-regulation soon after immunization. Following this examination of molecular immune mechanisms, our team set forth to determine the optimal conditions for transfecting catfish gill cells with two DNA vaccines encoding Ich antigens developed by unit scientists. Following a series of immunofluorescence staining approaches with immune fish sera, the transfected cells exhibited green florescence, which indicated the DNA vaccines could induce fish cells to produce Ich antigen protein. Building upon these in vitro studies, an experiment was conducted to determine whether immunization of catfish with the candidate DNA vaccines could induce detectable immune responses. Indeed, fish immunized with two of the DNA vaccines exhibited significantly higher anti-Ich antibodies than control fish immunized with the control DNA vaccine. These results demonstrated that immunization of catfish with the DNA vaccines induced production of Ich antigens in the fish and a specific immune response. Studies are currently being planned to determine if these promising DNA vaccine candidates will provide catfish protection against the Ich parasite.
A bacterium, Pseudomonas (P.) mosselii Gil3, was isolated from the gill tissue of a channel catfish that survived a lethal experimental infection of virulent Aeromonas hydrophila (vAh). When grown alongside vAh, P. mosselii Gil3 was shown to inhibit the growth of vAh. To explore the potential of P. mosselii Gil3 as a probiotic against vAh, we have sequenced the genome of this bacterium. Initial analyses showed that the genome of P. mosselii Gil3 features pathways related to the production of antimicrobial compounds. Trials are underway in the laboratory to determine whether this promising probiotic candidate can protect the catfish host against vAh.
The genome of a vAh strain, ML10-51K, was sequenced. Analyses of the genome sequence revealed that the catfish pathogen encodes a suite of proteins for chitin metabolism. Assays in vitro showed that vAh produced four chitinases, one chitobiase and one chitin binding protein that are able to degrade chitin and subsequently used for nutrients and growth. This study demonstrated that vAh is a highly chitinolytic bacterium and suggested that outbreaks of motile aeromonas septicemia disease caused by vAh in warm-water fish may be associated with high loads of organic (chitin) matter in fish ponds.
A study was performed to assess whether kaolin (a natural clay product) could be used to protect catfish against vAh infection. Using a waterborne challenge model developed earlier in this laboratory, initial experiments showed that water containing 0.1% kaolinite could reduce channel catfish mortality caused by vAh infection by nearly 30%. Additional varieties of commercially available kaolin products are being evaluated to determine the mechanism of disease protection and the potential for fish pond application.
Accomplishments
1. Nile tilapia resistance to Streptococcus (S.) iniae and S. agalactiae is heritable and can be markedly improved through selective breeding. Tilapia aquaculture worldwide is valued at about US $8 billion with a U.S. industry producing nearly 30 million pounds per year. Tilapia is an important source of protein in the US and abroad. Two gram positive bacteria, S. iniae and S. agalactiae, are responsible for around $1 billion in annual losses. ARS scientists in Auburn, Alabama, in collaboration with industry partners verified that resistance to S. iniae was heritable and selective breeding of superior individuals resulted in increased disease resistance of progeny. Research demonstrated that resistance to S. agalactiae was also heritable in Nile tilapia. Industry partners are now including selection for resistance to the two Streptococcus sp. in their breeding program along with increased harvest weight for a fast growing fish with reduced risks of disease. The improved tilapia are being sold throughout the Americas and abroad. Based on current production statistics and available models, representative gains from growing the improved tilapia on an average sized farm are U.S. $635,000 in additional revenue per farm assuming a conservative 5% increase in survival (which research trials indicate will be substantially higher). This research not only helps U.S. fish farmers but paves the way globally for reducing antibiotic use on farms leading to safer products entering the US. Tilapia can now be produced in a manner that reduces dependency on antibiotics resulting in a healthy, sustainable, and environmentally friendly product and in parallel, heightened food security in underdeveloped countries.
2. Evaluation of virulent Aeromonas (A.) hydrophila demonstrated genomic diversity but little differences in virulence among U.S. isolates. Aquaculture industries across the world have been impacted by virulent Aeromonas hydrophila (vAh), a Gram negative bacterium, and economic impacts of the disease to the U.S. catfish industry are around $10 million yearly. ARS scientists in Auburn, Alabama, in collaboration with scientists from Auburn University and Mississippi State University evaluated the genetic diversity of vAh isolates from Alabama, Mississippi, and China and found that isolates from Alabama were very similar (i.e., clonal) but isolates from Mississippi exhibited genetic diversity and were more like the isolate from China that originated from a carp. To evaluate the biological significance of the identified genetic diversity, comparative virulence studies were conducted with representatives of different vAh genotypes. These studies revealed that isolate ZC1 (Asian grass carp isolate) yielded significantly lower mortality (~27%) in channel catfish, relative to Alabama and Mississippi vAh isolates (greater than or equal to 60% mortality). Results based on genomic data also suggest that virulence components vary among the isolates which will be important for future work in designing effective control strategies including vaccines.
3. Naturally occurring non-antibiotic compounds kill bacterial fish pathogens. Fish producers are eager for new non-antibiotic strategies to prevent and treat costly diseases on farms. Three of the most problematic pathogens of the United States farmed catfish industry are Aeromonas hydrophila, Edwardsiella ictaluri and Flavobacterium columnare. ARS scientists in Auburn, Alabama, evaluated the antibacterial activity of two naturally occurring non-antibiotic compounds called chitosan and chitosan oligosaccharide lactate, which are derived from the shells of invertebrates such as shrimp. The ARS investigators found that both of these compounds were effective at halting bacterial growth and directly killing bacteria in laboratory tests. It was determined that these compounds act by coating bacteria and disrupting the protective barrier function of their outer membranes. These findings are a crucial first step in developing strategies to exploit the antibacterial activity of chitosan-based approaches for combating disease outbreaks in aquaculture.
4. Catfish feeding practices may impact susceptibility to virulent Aeromonas (A.) hydrophila (vAh). An emerging bacterial pathogen called vAh has been responsible for widespread farm losses in the U.S. catfish industry over the last eight years. While the genetic and biochemical understanding of vAh has been greatly enhanced during this period, the environmental or host-derived factors leading to disease outbreaks have remained elusive. Taking cues from observed farm conditions associated with outbreaks, ARS scientists in Auburn, Alabama, in collaboration with Auburn University, evaluated nutritional factors tentatively associated with the vAh disease process. Investigators determined that the time between the last feeding and an experimental infection was a critical driver of catfish susceptibility to vAh. Indeed, fish recently fed and with a full gastrointestinal tract had dramatically lower survival than those in a fasted state where food was withheld for 24 hours prior to the challenge. Taken together, these results not only provide a more robust challenge model, but offer actionable insights into pond level host-pathogen interactions underlying vAh disease development.
5. Sensing of iron by virulent Aeromonas (A.) hydrophila (vAh) may trigger increased ability to kill catfish. Iron is a vital nutrient for bacteria, so it is no surprise that virulent bacterial pathogens are characterized by a dizzying array of mechanisms by which they can acquire iron from host animals such as catfish, but also at the expense of other bacteria in their aquatic environment. Among these mechanisms are the production and secretion of siderophores, small molecules with a high binding affinity for iron that are also regarded as key virulence factors for a number of bacteria. ARS scientists in Auburn, Alabama, in collaboration with Auburn University, determined that vAh possesses a defense mechanism whereby the presence of siderophores from other microbes, such as those in production ponds, constitute a danger signal, alerting the pathogen to the presence of competing bacteria or algae and oncoming iron limited conditions. In response to the presence of competing microbial siderophores, the investigators found that vAh virulence dramatically increased which doubled mortality of catfish in laboratory challenges.
Review Publications
Moreira, G.S., Shoemaker, C.A., Zhang, D., Xu, D. 2017. Expression of immune genes in skin of channel catfish immunized with live theronts of Ichthyophthirius multifiliis. Parasite Immunology. 39:e12397.
Liu, Y., Zhang, Q., Xu, D., Fu, Y., Lin, D., Zhou, S., Li, J. 2017. Antiparasitic efficacy of curcumin from Curcuma longa against Ichthyophthirius multifiliis in grass carp. Veterinary Parasitology. 236:128-136.
Faisal, M., Diamanka, A., Loch, T., Lafrentz, B.R., Winters, A., Garcia, J.C., Togubaye, B. 2017. Isolation and characterization of Flavobacterium columnare strains infecting fishes inhabiting the Laurentian Great Lakes basin. Journal of Fish Diseases. 40:637-648.
Shoemaker, C.A., Xu, D., Soto, E. 2017. Streptococcus iniae and Streptococcus agalactiae. In: Woo, P.T.K., Cipriano, R.C.,editors. Fish Virus and Bacteria: Pathobiology and Protection. Boston, MA: CAB International. p. 298-313.
Shoemaker, C.A., Lozano, C.A., Lafrentz, B.R., Garcia, J.C., Soto, E., Xu, D., Beck, B.H., Rye, M. 2017. Additive genetic variation in resistance of Nile tilapia (Oreochromis niloticus) to Streptococcus iniae and S. agalactiae capsular type Ib: is genetic resistance correlated? Aquaculture. 468:193-198.
Peatman, E., Beck, B.H. 2016. From floor sweepings to fish flesh: Phytase superdosing in the US catfish industry. In: Walk, C.L., Kuhn, I., Stein, H.H., Kidd, M.T., Rodehutscord, M., editors. Phytate destruction consequences for precision animal nutrition. The Netherlands. Wageningen Academic Publishers. p. 237-250.
Zhang, D., Beck, B.H., Lange, M.D., Zhao, H., Thongda, W., Ye, Z., Li, C., Peatman, E. 2016. Impact of oral and waterborne administration of rhamnolipids on the susceptibility of channel catfish (Ictalurus punctatus) to Flavobacterium columnare infection. Fish and Shellfish Immunology. 60:44-49.
Zhang, D., Xu, D., Qiu, J., Rasmussen-Ivey, C.R., Liles, M.R., Beck, B.H. 2016. Chitin degradation and utilization by virulent Aeromonas hydrophila strain ML10-51K. Archives Of Microbiology. 199(4):573-579. doi:10.1007/s00203-016-1326-1.
Zhang, D., Xu, D., Qiu, J., Rasmussen-Ivey, C.R., Liles, M.R., Beck, B.H. 2017. Draft genome sequence of Pseudomonas mosselii Gil3, isolated from catfish and antagonistic against hypervirulent Aeromonas hydrophila. Genome Announcements. 4(6):e0135-16.
Rasmussen-Ivey, C.R., Hossain, M.J., Odom, S.E., Terhune, J.S., Hemstreet, W.G., Shoemaker, C.A., Zhang, D., Xu, D., Griffin, M.J., Liu, Y. 2016. Classification of a hypervirulent Aeromonas hydrophila pathotype responsible for epidemic outbreaks in warm-water fishes. Frontiers in Microbiology. 7:1615.
Straus, D.L., Farmer, B.D., Ledbetter, C.K., Beck, B.H., Williams, R.S., Clark, M.L., Freeze, M.T. 2016. Use of copper sulfate to control Saprolegniasis at a commercial sunshine bass hatchery. North American Journal of Aquaculture. 78:243-250.
Evenhuis, J., Lafrentz, B.R. 2016. Virulence of Flavobacterium columnare genomovars in rainbow trout Oncorhynchus mykiss. Diseases of Aquatic Organisms. 120(3):217-224.
Aksoy, M., Beck, B.H. 2017. Antimicrobial activity of chitosan and a chitosan oligomer against bacterial pathogens of warmwater fish. Journal of Applied Microbiology. 122:1570-1578.
Lafrentz, B.R., Garcia, J.C., Dong, H., Waldbieser, G.C., Rodkhum, C., Wong, F., Chang, S. 2017. Optimized reverse primer for 16S-RFLP analysis and genomovar assignment of Flavobacterium columnare. Journal of Fish Diseases. 40:1103-1108.
