Location: Aquatic Animal Health Research
2018 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 fourth year of a five-year project that has two major objectives. Objective 1 Progress: Comparative bacterial genomics 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. Genomic deoxyribonucleic acid (gDNA) was extracted from representatives of each genetic group for complete genome sequencing to a single contiguous sequence. Once these sequences are finalized, comparative pan-genome analysis will be used to determine the genomic differences between the four genetic groups which may explain the host-pathogen associations previously documented. Additionally, the phenotypic and biochemical traits of isolates from each of the four genetic groups are being fully characterized to identify phenotypic differences among the four groups which will complement the genetic analyses.
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. A multiplex polymerase chain reaction (PCR) assay was developed for the ability to rapidly assign an unknown isolate of F. columnare to genetic group. 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. In collaboration with stakeholder catfish farms and four diagnostic laboratories in the southeastern USA over 100 samples have been collected from columnaris disease cases. The samples are being processed for DNA extraction and subsequently used in the multiplex PCR to determine which genetic groups of F. columnare are circulating and most predominant in the catfish and other aquaculture fish industries.
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 to determine if a PCR-based typing system is possible.
Objective 2 Progress: Research is being conducted to determine the attenuated phenotype and vaccine potential of the chemically passaged isolates of F. columnare with potential CPS modifications. Five isolates representing 3 of the 4 recently identified genetic groups known to be virulent in catfish and/or tilapia based on prior research studies are being tested. If one or more of the isolate(s) are attenuated and/or avirulent in channel catfish, reversion to virulence back-passage studies and efficacy trials will proceed.
Progress has been made towards developing DNA vaccines for Ichthyophthirius multifiliis (Ich). Two pilot studies were conducted to evaluate the expression of three DNA vaccines in channel catfish and to determine the optimum DNA vaccine dose for vaccination. A vaccine trial was conducted to determine whether immunization of catfish with these DNA vaccines induce immune responses and protection, and serum, kidney and spleen tissues were sampled from fish following vaccination to evaluate immune responses. Fish vaccinated with one of the DNA vaccines exhibited higher anti-Ich antibodies and survival than non-vaccinated control fish. Next, ARS scientists will determine if two doses of the DNA vaccine will provide catfish stronger protection against Ich.
Previous research under this objective has demonstrated that channel catfish develop immunity against infection with virulent Aeromonas (A.) hydrophila (vAh) after immunization with the pathogen’s extracellular proteins (ECP), and serum antibodies in these fish exhibited high agglutination titers against cells of vAh. Research was conducted to analyze the immune properties of this antiserum. The agglutinants of ECP and cells of vAh, elicited by antiserum, were analyzed and characterized. Five fish proteins were identified in ECP agglutinants, including two innate immunity associated proteins (serotransferrin and rhamnose-binding lectin), two immunoglobulin M (IgM) molecules (IgM heavy chain and light chain) and a constitutively-produced protein (warm temperature acclimation protein). More than 68 vAh proteins were agglutinated by the antiserum. Further, IgM molecules that agglutinated cells of vAh were isolated and characterized. The study revealed versatility of vAh antigens and catfish IgM, which will help develop strategic vaccines against vAh infection.
Aeromonas veronii is a Gram-negative bacterium commonly found in catfish ponds and has been frequently isolated not only from apparently healthy fish but also from fish with motile Aeromonas septicemia (MAS). It has been speculated that A. veronii is associated with MAS disease caused by vAh due to the observation that A. veronii was frequently isolated from vAh diseased fish. Research was conducted to determine the potential virulence of A. veroni in channel catfish and associations with vAh. Laboratory assays showed that some A. veronii isolates were highly hemolytic and toxic to cultured catfish cells. Seven phylogenetically different A. veronii isolates were subjected to genome sequencing and sequence analysis is underway to understand their potential pathogenicity. The aim of this study is to determine the potential involvement of A. veronii in MAS.
Two disease phenotyping studies with Streptococcus (S.) iniae and S. agalactiae were completed using Nile tilapia families supplied by collaborators. The research demonstrated that the results from our previous studies were reproducible and confirmed additive genetic variation in resistance of Nile tilapia to S. iniae and S. agalactiae Ib. Assortative mating was also performed in which fish were produced based on high and low parental breeding values for both S. iniae and S. agalactiae Ib. As in our prior studies, resistance to S. iniae was heritable and this was confirmed for a third time. The results with S. agalactiae Ib demonstrated that resistance to S. agalactiae Ib is heritable and can be improved via selective breeding. Custom genetic material is produced by the collaborator and are being sold to commercial tilapia farmers to reduce disease caused by S. iniae and S. agalactiae.
Accomplishments
1. Identification of four distinct phylogenetic groups in Flavobacterium (F.) columnare with fish host associations. Columnaris disease, caused by the bacterium F. columnare, is one of the most prevalent fish diseases worldwide. An exceptionally high level of genetic diversity among isolates has long been recognized; however, little has been done to quantify or characterize this diversity in a systematic fashion. ARS researchers at Auburn, Alabama, and collaborators used high resolution methods to characterize the genetic diversity in F. columnare. The results established the existence of four phylogenetically distinct genetic groups within the species. Examination of isolate historical data indicated biological relevance to the identified genetic diversity, with some genetic groups isolated preferentially from specific fish species and/or geographic regions. This research highlights the importance of understanding the genetic diversity in F. columnare and has facilitated a standard nomenclature for these groups across the scientific community. The new knowledge gained from this research will aid in identifying which genetic type(s) of F. columnare are prevalent in different regions and/or aquaculture industries that will allow for the development of better control and treatment measures for columnaris disease.
2. Capsular typing of Streptococcus (S.) agalactiae from fish using multiplex PCR and serotyping. Streptococcus spp., including Streptococcus agalactiae (Lancefield group B streptococci) are considered emerging Gram-positive bacterial pathogens and are responsible for approximately $1 billion USD in annual losses to the global tilapia industry. The capsule is a sugar containing material on the surface of these bacteria that is important for virulence and antigenicity including serotype. There is an urgent need to identify different capsular types of S. agalactiae from farmed and wild fish to combat streptococcal disease. ARS researchers at Auburn, Alabama, used multiplex polymerase chain reaction (PCR) capsular typing assay and antiserum to assign capsular type to a total of forty S. agalactiae isolates. The multiplex PCR was modified to detect capsular types Ia, Ib, II and III, the common capsular types reported from fish and aquatic animals. Results demonstrated that most isolates from North, Central and South America were capsular type Ib. The modified multiplex PCR assay is used to determine the capsular type of S. agalactiae present on a farm and/or region and has assisted with disease management strategies including selective breeding and vaccines.
3. Virulent Flavobacterium (F.) columnare degrades catfish mucus. Flavobacterium columnare is an economically important bacteria that causes columnaris disease of farmed fish in the USA and abroad. Skin mucus is known to be an important factor in the early stages of columnaris disease; however, little is known about mucus composition in catfish and how columnaris bacteria respond to mucus. ARS researchers at Auburn, Alabama, in collaboration with Auburn University, determined the terminal glycosylation (sugar) pattern of catfish mucus and showed the sugars alpha-D-mannose/alpha-D-glucose were predominant in mucus and likely important for bacterial binding to the fish. They further demonstrated in multiple experiments that F. columnare can replicate and survive for up to 3 months in water containing catfish mucus. A highly virulent F. columnare isolate showed significantly elevated extracellular proteolytic activity (enzymes involved in disease) compared to a moderately virulent isolate following growth in catfish mucus. The identification of differential proteolytic ability is likely related to virulence in that some F. columnare isolates have a greater ability to break down the protective mucus layer resulting in enhanced colonization, pathology and/or death. The data provide new insights on the pathogenic mechanisms of F. columnare in columnaris disease.
4. Demonstrated molecular immune responses in mucosal and systemic immune tissues of channel catfish after vaccination with Ichthyophthirius multifiliis (Ich). Ich is a parasite of fish and causes severe losses aquaculture industries worldwide. Chemical treatment of Ich is costly and often not effective after the parasite penetrates the fish host skin and gill tissue. There is an urgent need for a better understanding of protective immune responses in fish to develop effective vaccines against the parasite. ARS researchers at Auburn, Alabama, investigated the expression of innate and adaptive immune-related genes in mucosal (skin, gill, intestine) and systemic (kidney, spleen, liver) tissues of channel catfish following vaccination with Ich. The research demonstrated significantly higher antibody levels and survival (95%) in vaccinated fish than non-vaccinated control fish. Results of this study revealed the molecular immune responses in vaccinated fish and offers new insights into the molecular responses that may govern protective immunity of catfish against Ich infection.
Review Publications
Zhao, H., Li, C., Hargrove, J.S., Bowen, B., Thongda, W., Zhang, D., Mohammed, H., Beck, B.H., Austin, J., Peatman, E. 2017. SNP marker panels for parentage assignment and traceability in the Florida bass (Micropterus floridanus). Aquaculture. 485:30-38.
Zhang, D., Beck, B.H., Mohammed, H., Zhao, H., Thongda, W., Ye, Z., Zeng, Q., Shoemaker, C.A., Fuller, S.A., Peatman, E. 2018. L-rhamnose-binding lectins (RBLs) in Nile tilapia, Oreochromis niloticus: characterization and expression profiling in mucosal tissues. Fish and Shellfish Immunology. 72:426-435.
Peatman, E., Mohammed, H., Kirby, A., Shoemaker, C.A., Aksoy, M., Beck, B.H. 2018. Mechanisms of pathogen virulence and host susceptibility in virulent Aeromonas hydrophila infections of channel catfish (Ictalurus punctatus). Aquaculture. 482:1-8.
Li, N., Zhu, Y., LaFrentz, B.R., Evenhuis, J., Hunnicutt, D.W., Conrad, R.A., Barbier, P., Gullstrand, C.W., Roet, J.E., Powers, J.L., Kulkami, S.S., Erbes, D.H., Garcia, J.C., Nie, P., McBride, M.J. 2017. The type IX secretion system is required for virulence of the fish pathogen Flavobacterium columnare. Applied and Environmental Microbiology. 83(23):e01769-17. https://doi.org/10.1128/AEM.01769-17.
Zhang, D., Thongda, W., Li, C., Zhao, H., Beck, B.H., Mohammed, H., Arias, C., Peatman, E. 2017. More than just antibodies: protective mechanisms of a muscosal vaccine against fish pathogen Flavobacterium columnare. Fish and Shellfish Immunology. 71:160-170.
Liu, Y., Zhang, Q., Xu, D., Fu, Y., Lin, D., Zhou, S. 2017. Antiparasitic efficacy of commercial curcumin against Ichthyophthirius multifiliis in grass carp (Ctenopharyngodon idellus). Aquaculture. 480:65-70.
Zhang, M., Qiao, G., Li, Q., Xu, D., Qi, Z., Wang, A., Xu, C., Huang, J. 2017. Transcriptome analysis and discovery of genes involved in immune pathways from coelomocytes of Onchidium struma after bacterial challenge. Fish and Shellfish Immunology. 72:528-543.
Zhang, M., Qiao, G., Xu, D., Li, Y., Qi, Z., Li, Q. 2018. Effect of different water biofloc contents on the growth and immune response of gibel carp cultured in zero water exchange and no feed addition system. Aquaculture Research. 49:1647-1656.
Xu, D., Moreira, G.S., Shoemaker, C.A., Zhang, D., Beck, B.H. 2017. Expression of immune genes in systemic and mucosal immune tissues of channel catfish vaccinated with live theronts of Ichthyophthirius multifiliis. Fish and Shellfish Immunology. 66:540-547.
Fu, Y., Wang, B., Zhang, Q., Xu, D., Lin, D., Yang, X., Zhu, S., Pan, J., Deng, Q., Liu, Y., Zhou, S. 2017. Combined effects of Chinese medicine feed and ginger extract bath on co-infection of Ichthyophthirius multifiliis and Dactylogyrus ctenopharyngodonid in grass carp. Parasitology Research. 116(7):2017-2025.
Li, J., Fu, Y., Zhang, Q., Xu, D., Liu, Y., Zhou, S., Lin, D. 2018. Grass carp which survive Dactylogyrus ctenopharyngodonid infection also gain partial immunity against Ichthyophthirius multifiliis. Diseases of Aquatic Organisms. 129:63-70.
Fuller, S.A., Beck, B.H., McEntire, M.E., Peatman, E., Abernathy, J.W. 2018. Heritability of growth traits and correlation with hepatic gene expression among hybrid striped bass exhibiting extremes in performance. Cogent Biology. 4:1453319.
Lafrentz, B.R., Garcia, J.C., Waldbieser, G.C., Evenhuis, J., Loch, T.P., Liles, M.R., Wong, F.S., Chang, S.F. 2018. Identification of four distinct phylogenetic groups in Flavobacterium columnare with fish host associations. Frontiers in Microbiology. 9:452. https://doi.org/10.3389/fmicb.2018.00452.
Garcia, J.C., LaFrentz, B.R., Waldbieser, G.C., Wong, F., Chang, S. 2018. Characterization of atypical Flavobacterium columnare and identification of a new genomovar. Journal of Fish Diseases. 41:1159-1164.
Shoemaker, C.A., Xu, D., Garcia, J.C., LaFrentz, B.R. 2017. Capsular typing of Streptococcus agalactiae (Lancefield group B streptococci) from fish using multiplex PCR and serotyping. Bulletin of the European Association of Fish Pathologists. 37(5):190-197.
Shoemaker, C.A., LaFrentz, B.R., Peatman, E., Beck, B.H. 2018. Influence of native catfish mucus on Flavobacterium columnare growth and proteolytic activity. Journal of Fish Diseases. https://doi.org/10.111/jfd.12833.
Mohammed, H.H., Brown, T.L., Beck, B.H., Aksoy, M., Eljack, R.M., Peatman, E. 2018. The effects of dietary inclusion of a Saccharomyces cerevisiae fermentation product in a commercial catfish ration on growth, immune readiness, and columnaris disease susceptibility. Journal of Applied Aquaculture. 31(3):193-209. https://doi.org/10.1080/10454438.2018.1499576.
Reichley, S.R., Ware, C., Steadman, J., Gaunt, P.S., Garcia, J.C., LaFrentz, B.R., Thachil, A., Stine, C., Waldbieser, G.C., Arias, C.R., Lock, T., Welch, T.J., Cipriano, R.C., Greenway, T.E., Khoo, L.H., Wise, D.J., Lawrence, M.L., Griffin, M.J. 2017. Comparative phenotypic and genotypic analysis of Edwardsiella spp. isolates from different hosts and geographic origins with an emphasis on isolates formerly clasified as E. tarda and an evaluation of diagnostic methods. Journal of Clinical Microbiology. 55:3466-3491.
Soto, E., Griffin, M.J., Morales, J.A., Calvo, E.B., Sebastiao, F.A., Porras, A.L., Viquez-Rodriguez, X., Reichley, S.R., Rosser, T.G., Ware, C., Byrne, B.A., Lafrentz, B.R., Garcia, J.C., Camus, A.C. 2018. Francisella marina sp. nov., etiologic agent of systemic disease in cultured spotted rose snapper (Lutjanus guttatus) in Central America. Applied and Environmental Microbiology. 84(16):e00144-18. https://doi.org/10.1128/AEM.00144-18.
