2012 Annual Report
1a.Objectives (from AD-416):
Objective 1 - Determine population and strain responses to vaccines and infectious pathogens (e.g., Edwardsiella ictaluri, Flavobacterium columnare), using genetically characterized fish.
Objective 2 - Determine characteristics of coinfections and their role in disease processes in aquaculture and aquatic environments.
Objective 3 - Identify microbial pathogen genes and pathways critical for host pathogenesis and immunity.
Objective 4 - Develop and validate new and novel pathogen detection tests for Edwardsiella ictaluri, Flavobacterium columnare, Streptococcus iniae and S. agalactiae using genomic, proteomic, microbiological and immunological approaches.
1b.Approach (from AD-416):
Fish farmers continue to identify disease as a significant negative impact on profitability. Therefore, the goal of this project is to improve fish health and reduce this negative impact. Using a multi-disciplinary approach, we will accomplish four objectives that address important questions about bacterial diseases that affect the catfish (e.g., Edwardsiella ictaluri, Flavobacterium columnare) and tilapia (e.g., Streptococcus iniae, S. agalactiae) industries. Studies will be conducted at the gene, protein, individual, and/or population levels. Questions remain about some barriers to optimum vaccine efficacy in the field and about the responses of current and future strains of fish to pathogens and vaccines. Therefore, Objective 1 will determine population and strain responses to vaccines and infectious pathogens (e.g., E. ictaluri, F. columnare), using genetically characterized fish. In most intensive aquaculture production systems, multiple pathogens are present and result in mortality. Objective 2 will determine characteristics of coinfections and their role in disease processes in aquaculture and aquatic environments. Objective 3 will identify microbial pathogen genes and pathways critical for host pathogenesis and immunity that will provide important information for future vaccine development. Objective 4 will develop and validate new and novel pathogen detection tests for E. ictaluri, F. columnare, S. iniae and S. agalactiae so that these can be used in fish health management. The results from this work will contribute to present and future vaccine development, provide useful management information about farm use of vaccines and coinfections, and leverage development of future catfish strains being developed for the industry.
Progress was made on all five objectives and their subobjectives, all of which fall under National Program 106. Under Objective 1A, significant progress was made to evaluate the acquired immune response of channel catfish using microarray and genomic technologies following immunization with modified live Edwardsiella (E.) ictaluri vaccines. Microarray and genomic data have been obtained and analyzed. Two manuscripts were published under this objective. Under Objective 1B, significant progress was made to evaluate channel catfish families which have been selectively bred for resistance against E. ictaluri for susceptibility to Flavobacterium (F.) columnare. All experiments have been completed and no significant disease resistance differences were found between families. Under Objective 2A, significant progress was made to determine the effect of parasitism on the susceptibility of channel catfish to F. columnare infection and it has been found that dual infection of parasite and F. columnare significantly increased fish mortality compared to fish only infected by F. columnare or only infected by parasites. Under Objective 2B, significant progress was made to determine whether parasites can be used to vector E. ictaluri in channel catfish. Polymerase chain reaction (PCR) assays have been successfully modified and developed to detect E. ictaluri in fish tissues. One manuscript was published. Under Objective 3A, significant progress was made to identify proteins of Flavobacterium columnare that are differentially regulated following growth in vivo or in media mimicking in vivo conditions and determine their role in pathogenesis. Identification of F. columnare proteins differentially regulated during in vivo growth are current underway. Under Objective 3B, significant progress was made to identify immune genes critical for tilapia immunity against Streptococcus iniae vaccine and S. iniae infection. Two manuscripts were published. Under Objective 4B, significant progress was made to obtain distinct extracellular protein profiles among virulent and avirulent strains of Aeromonas hydrophila. Under Objective 5A, significant progress was made to develop and evaluate attenuated vaccines to protect catfish and tilapia against S. agalactiae. Attenuated S. agalactiae vaccines have been developed and invention disclosures have been filed. Under Objective 5B, significant progress was made to develop and evaluate recombinant Deoxyribonucleic acid (DNA) vaccine against the protozoan parasite Ichthyophthirius (Ich). The efficacy studies on these recombinant DNA vaccines have been completed.
Vaccines developed to protect tilapia against Streptococcosis. Streptococcosis is a hyperacute systematic disease that affects both cultured and wild fish species in various aquatic environments (freshwater, estuarine, and marine). ARS researchers at Auburn, Alabama, developed attenuated vaccines to protect tilapia against both Streptococcosis agalactiae and Streptococcus (S.) iniae. The attenuated S. iniae vaccine offered tilapia 100% protection at 14, 28, and 60 days post vaccination. In addition, the attenuated S. iniae vaccine offered 78% to 100% protection against multiple isolates of S. iniae. The use of these vaccines will protect tilapia against streptococcosis, which will decrease the economic losses caused by streptococcosis affecting the aquaculture industry.
Vaccine developed to protect tilapia against Vibrio vulnificus. Vibrio (V.) vulnificus, a Gram-negative bacterium, has emerged as a pathogen to tilapia. ARS researchers at Auburn, Alabama, developed a formalin-inactivated vaccine to protect tilapia against V. vulnificus. The vaccine offered tilapia 60% to 73% protection against the homologous isolate challenge and up to 88% protection against a heterologous isolate. The use of this vaccine will protect tilapia against V. vulnificus infection, which will benefit the aquaculture industry.
Global gene expression elucidated in channel catfish after immersion vaccination with AquaVac ESC vaccine. Enteric septicemia of catfish (ESC), the most prevalent disease affecting farm-raised channel catfish, is caused by Edwardsiella ictaluri, a Gram negative bacterium. To control ESC, a live attenuated AquaVac-ESC vaccine has been developed by ARS researchers at Auburn, Alabama. The vaccine offers protection to channel catfish against ESC with a single bath immersion. To understand the global gene expression in channel catfish after immersion vaccination with AquaVac ESC vaccine, ARS researchers at Auburn, Alabama, performed microarray analysis and found that 52 genes were up-regulated by the vaccine at 48h post vaccination whereas 129 genes were down-regulated. These research results will lead to a better understanding on how AquaVac-ESC vaccine protects channel catfish at molecular level.
Parasite vector for a bacterial pathogen in channel catfish. There is limited information on whether parasites act as vectors to transmit pathogenic bacteria in fish. Scientists at the Aquatic Animal Health Research Laboratory at Auburn, Alabama, conducted studies with Ichthyophthirius multifiliis (Ich, parasite) and Edwardsiella (E.) ictaluri (bacterium) to determine the interactions between the parasite, the bacteria and the catfish. This study provided evidence that Ich can vector E. icaluri into channel catfish. Tomonts (the reproductive parasite stage) exposed to E. ictaluri could pass E. ictaluri to infective theronts released from the tomonts. Subsequently, the theronts transmitted the bacterium to channel catfish. The vectoring ability of parasites is particularly important at fish farms because the introduction of parasites either from wild fish or from other farms could concomitantly involve the introduction and/or transmission of bacterial diseases.
Vaccines developed to protect catfish and tilapia against Motile Aeromonad Septicemia. Aeromonas (A.) hydrophila, a Gram-negative bacterium widely distributed in aquatic environments, is a causative agent of motile aeromonad septicemia (MAS). In West Alabama, disease outbreaks caused by A. hydrophila in 2009 and 2010 has led to economic loss estimated at more than $3 million annually. ARS researchers at Auburn, Alabama, have developed attenuated vaccines to protect catfish and tilapia against MAS. Vaccination of channel catfish with these attenuated vaccines offered 86% to 100% protection at 14 days post vaccination. Vaccination of Nile tilapia with these attenuated vaccines offered 100% protection at 14, 28, and 56 days post vaccination. The use of these vaccines will prevent future disease outbreaks caused by the highly virulent West Alabama isolates of A. hydrophila.
Lafrentz, B.R., Welch, T.J., Shoemaker, C.A., Drennan, J.D., Klesius, P.H. 2011. Modified live Edwardsiella ictaluri vaccine, AQUAVAC-ESC, lacks multidrug resistance plasmids. Journal of Aquatic Animal Health. 23(4):195-199.
Bebak, J.A., Evans, J.J., Weber, E., Wolf, J. 2012. Pathology in practice: Peripheral nerve sheath tumor in a Shubunkin goldfish. Journal of the American Veterinary Medical Association. 240(7):827-829.
Bebak, J.A., Wagner, B. 2012. Use of vaccination against enteric septicemia of catfish and columnaris disease by the US catfish industry. Journal of Aquatic Animal Health. 24:30-36.
Xu, D., Wei Pridgeon, Y., Klesius, P.H., Shoemaker, C.A. 2011. Parasitism by protozoan Ichthyophthirius multifiliis enhanced invasion of Aeromonas hydrophila in tissues of channel catfish. Veterinary Parasitology. 184:101-107.
Wei Pridgeon, Y., Mu, X., Klesius, P.H. 2012. Expression profiles of seven channel catfish antimicrobial peptides in response to Edwardsiella ictaluri infection. Journal of Fish Diseases. 35:227-237.
Martins, M.L., Xu, D., Shoemaker, C.A., Klesius, P.H. 2011. Temperature effects on immune response and hematological parameters of channel catfish Ictalurus punctatus vaccinated with live theronts of Ichthyophthirius multifiliis. Fish and Shellfish Immunology. 31:774-780.
Lafrentz, B.R., Lapatra, S.E., Call, D.R., Wiens, G.D., Cain, K.D. 2011. Identification of immunogenic proteins within distinct molecular mass fractions of Flavobacterium psychrophilum. Journal of Fish Diseases. 34:823-830.
Yeh, H., Klesius, P.H. 2011. Over-expression, purification and immune responses to Aeromonas hydrophila AL09-73 flagellar proteins. Fish and Shellfish Immunology. 31:1278-1283.
Wei Pridgeon, Y., Klesius, P.H. 2011. Development and efficacy of novobiocin and rifampicin-resistant Aeromonas hydrophila as novel vaccines in channel catfish and Nile tilapia. Vaccine. 29:7896-7904.
Wei Pridgeon, Y., Klesius, P.H., Mu, X. 2012. Evaluation of an in vitro cell assay to select attenuated bacterial mutants of Aeromonas hydrophila and Edwardsiella tarda to channel catfish. Journal of Applied Microbiology. 111:1310-1318.
Mu, X., Wei Pridgeon, Y., Klesius, P.H. 2011. Transcriptional profiles of multiple genes in the anterior kidney of channel catfish vaccinated with an attenuated Aeromonas hydrophila. Fish and Shellfish Immunology. 31:1162-1172.
Wei Pridgeon, Y., Klesius, P.H., Mu, X., Yancey, R.J., Kievit, M.S., Dominowski, P.J. 2011. Efficacy of QCDCR formulated CpG ODN 2007 in Nile tilapia against Streptococcus iniae and identification of upregulated genes. Veterinary Immunology and Immunopathology. 145:179-190.
Xu, D., Shoemaker, C.A., Martins, M., Wei Pridgeon, Y., Klesius, P.H. 2012. Enhanced susceptibility of channel catfish to the bacterium Edwardsiella ictaluri after parasitism by Ichthyophthirius multifiliis. Veterinary Microbiology. 158:216-219.
Wei Pridgeon, Y., Yeh, H., Shoemaker, C.A., Mu, X., Klesius, P.H. 2012. Global gene expression in channel catfish after vaccination with an attenuated Edwardsiella ictaluri. Fish and Shellfish Immunology. 32:524-533.
Xu, D., Shoemaker, C.A., Klesius, P.H. 2012. Ichthyophthirius multifiliis as a potential vector of Edwardsiella ictaluri in channel catfish. FEMS Microbiology Letters. 329:160-167.
Wei Pridgeon, Y., Yeh, H., Shoemaker, C.A., Klesius, P.H. 2012. Global transcription analysis of vaccinated channel catfish following challenge with virulent Edwardsiella ictaluri. Veterinary Immunology and Immunopathology. 146:53-61.
Pasnik, D.J., Evans, J.J., Klesius, P.H. 2011. Specific serum antibody responses in channel catfish (Ictalurus punctatus) provide limited protection against Streptococcus ictaluri challenge. Veterinary Immunology and Immunopathology. 144:144-146.
Wei Pridgeon, Y., Klesius, P.H., Mu, X., Carter, D., Fleming, K., Xu, D. 2011. Identification of unique DNA sequences present in highly virulent 2009 Alabama isolates of Aeromonas hydrophila. Veterinary Microbiology. 152:117-125.
Qiao, G., Li, H., Xu, D., Parks, S. 2012. Modified a colony forming unit microbial adherence to hydrocarbons assay and evaluated cell surface hydrophobicity and biofilm production of Vibrio scophthalmi. J Bacteriol Parasitol. 3:130. doi:10.4172/2155-9597.1000130.
Wei Pridgeon, Y., Klesius, P.H. 2011. Identification and expression profile of multiple genes in Nile tilapia in response to formalin killed Streptococcus iniae vaccination. Veterinary Immunology and Immunopathology. 142:201-206.
Wei Pridgeon, Y., Klesius, P.H. 2011. Molecular identfication and virulence of three Aeromonas hydrophila isolates cultured from infected channel catfish during a disease outbreak in West Alabama (USA) in 2009. Diseases of Aquatic Organisms. 94:249-253.