Characterization of an Attenuated Aeromonas hydrophila Vaccine and Molecular Mechanisms of Channel Catfish Immunity against Aeromonas hydrophila infection by Xingjiang Mu A dissertation submitted to the Graduate Faculty of Auburn University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Auburn, Alabama August 4, 2012 Keywords: vaccine, immunity, gene, expression, fish Copyright 2012 by Xingjiang Mu Approved by Yuping W. Pridgeon, Co-chair, Affiliate Associate Professor of Fisheries and Allied Aquacultures Zhanjiang Liu, Co-chair, Professor of Fisheries and Allied Aquacultures Phillip H. Klesius, Affiliate Professor of Fisheries and Allied Aquacultures Nannan Liu, Professor of Entomology and Plant Pathology Haishen Wen, Professor of College of Aquaculture, Ocean University of China ii Abstract An attenuated Aeromonas hydrophila AL09-71 N+R vaccine strain was compared to its virulent parent strain A. hydrophila AL09-71. The attenuated AL09-71 N+R strain was developed through selection of resistance to both novobiocin and rifampicin. The attenuated AL09-71 N+R strain had smaller colony size, slower growth and weaker chemotactic response compared to AL09-71. The motility and invasion ability of AL09-71 N+R were found to be abolished whereas that of AL09-71 was retained. The fatty acid methyl ester profiles of the attenuated strain AL09-71 N+R were detected to be different from that of AL09-71. However, at genomic DNA level, AL09-71 N+R appeared to be similar to that of AL09-71. To understand the molecular mechanisms of protection elicited by the attenuated AL09- 71 N+R vaccine strain in catfish, suppression subtractive hybridization (SSH) was used to identify genes up-regulated by the vaccine. A total of 22 unique genes were identified at 12 h post vaccination. Of the 22, six were confirmed to be significantly induced by vaccination. In addition, 88 channel catfish genes that were reported to be associated with host immunity were included in the expression analysis. Of the 88 genes, 14 were found to be significantly up- regulated by the vaccination. Expression profiles of the 20 genes at different time points showed that the pattern of gene up-regulation in vaccinated fish was similar to that in infected fish. To understand whether channel catfish response to secondary infection is similar to primary infection, SSH was used to identify genes up-regulated by secondary infection. Of the 28 unique genes identified by the library, eight were confirmed to be significantly induced by iii secondary infection compared to that by primary infection at 6 h post infection. In addition to the eight genes identified by SSH, 94 genes known to be associated with host immunity were also subjected to expression analysis. Of the 94 genes, 22 were identified to be induced and differential regulated at different time points. These results suggest that channel catfish host response play an important role in its immunity against A. hydrophila infection. iv Acknowledgments The author is very grateful to Dr. Yuping W. Pridgeon for her constant guidance, support and patience in the past years. The author has been very fortunate in having Dr. Yuping W. Pridgeon as my supervisor. Her scientific attitude and philosophy have been having a profound influence on me. The author is also indebted to his co-chair Dr. Zhanjiang Liu, committee members: Dr. Phillip H. Klesius, Dr. Nannan Liu, Dr. Haishen Wen, and dissertation outside reader, Dr. Xing Ping Hu, for their valuable advice and encouragement. The author is especially grateful to Dr. Phillip H. Klesius for his guidance and help in dissertation and for allowing access to the USDA-ARS Aquatic Animal Health Research Laboratory equipments and facilities. Sincere thanks also go to Beth Peterman and Dr. Yildirim-Aksoy for technical help. The author would like to acknowledge the contributions of other USDA-ARS Aquatic Animal Health Research Laboratory staff for their support. Sincere thanks also go to author?s parents for their endless love and support. v Table of Contents Abstract??????????????????????????????????. ii Acknowledgments?????????????????????????????.. ..iv List of Tables??????????????????????????????? .viii List of Figures .............................................................................................................................. ix List of Abbreviations ................................................................................................................... xi I. INTRODUCTION AND LITERATURE REVIEW?????????????? .??.1 Aeromonas hydrophila ? ????????????????????????..1 Motile aeromonad septicemia???????????????????? ..??. .2 Prevention and control?????????????????????????... 2 Important immune factors in channel catfish?????????????????.4 Reference?????????????????????????????...... 10 II. CHARACTERIZATION OF THE ATTENUATED AEROMONAS HYDROPHILA VACCINE STRAIN AL09-71 N+R COMPARED TO ITS PARENT STRAIN AL09-71?? ? ???????? ...????????????????????.. 21 Introduction ? ......................................................................................................??? 21 Materials and methods?????????????????????????.. 22 Results?????????? ??? ????????????????? ..?.29 Discussion????????????????????????? ?? .???31 Reference? ?????????????????????????????.. 35 vi III. TRANSCRITIONAL PROFILES OF MULTIPLE GENES IN THE ANTERIOR KIDNEY OF CHANNEL CATFISH VACCINATED WITH AN ATTENUATED AEROMONAS HYDROPHILA ??????????????????????????? ?? ..52 Introduction ???? ???????????????????????? .? 52 Materials and methods???????????????????????... .?.. 54 Results?? ???????????????????????????? .?.. 60 Discussion???????? ????????????????????? .? 64 Reference????????????????????????????? .?. 69 IV. USING SSH TO IDENTIFY GENES OVEREXPRESSED IN CHANNEL CATFISH AFTER SECONDARY EXPOSURE TO AEROMONAS HYDROPHILA COMPARED TO PRIMARY EXPOSURE? .????????????????..???????? .86 Introduction ?????????????????????????????.86 Materials and methods?????????????????????? ?...?... 87 Results??????????????? ???????????????.?..92 Discussion??????????????????????????????. 94 Reference? ?????? ???????????????????????.. 99 V. TRANSCRIPTIONAL PROFILES OF MULTIPLE KNOWN GENES IN CHANNEL CATFISH AFTER SECONDARY EXPOSURE TO AEROMONAS HYDROPHILA COMPARED TO PRIMARY EXPOSURE?????.. ?????????? .??.112 Introduction ??????????????????????????? ? ...112 Materials and methods???????????????????????... .? 114 Results????????????????????????? ?????? .119 Discussion?? ??????????????????????????? ...122 Reference??? ?????????????????????????? .? 131 VI. OVERALL RESULTS AND FUTURE DIRECTIONS?????????????... 148 Overall results and discussion??????????????????????. 148 vii Future directions???????????????????????????. 153 BIBLIOGRAPHY???????????????????????????? ..?154 viii List of Tables II. Table 1. Nomenclature of fatty acid methyl esters of mutant strain AL09-71 N+R and parent strain AL09-71?????????????????????????????. .42 II. Table 2. List of sequences isolated from mutant strain AL09-71 N+R versus parent strain AL09-71 bacterial genome subtractive library?????????????????. 43 II. Table 3. Gene-specific primers used in PCR??????????????????.. 44 III. Table 1. List of the 22 genes isolated from the Aeromonas hydrophila AL09-71 N+R vaccinated versus non-vaccinated channel catfish anterior kidney subtractive cDNA library????????????????????????????????... 77 III. Table 2. Gene-specific primers used in qPCR ?????????????????..78 III. Table 3. List of the 20 genes identified to be significantly upregulated at 12h post vaccination in the anterior kidney of channel catfish???????????????.????. 79 IV. Table 1. List of genes isolated from the secondary infected versus primary infected catfish subtractive cDNA library???????????????????????? ....106 IV. Table 1. Continue??? ???????????????????????? ? .107 IV. Table 2. Gene-specific primers used in qPCR???????????????? .?108 V. Table 1. Gene-specific primers used in qPCR?????????????????..141 ix List of Figures II. Figure 1. Surface plating of Aeromonas hydrophila parent AL09-71 and mutant AL09-71 N+R. Quadruplicate plates for per strain were incubated overnight at 28?C??.................46 II. Figure 2. Growth curve of Areomonas hydrophila parent AL09-71 and mutant AL09-71 strains for 25h. Four dilutions as follows: 1:40 (A), 1:80 (B), 1:160 (C), and 1:320 (D) with triplicate. ??????????? ?????? ????????????? ...? .47 II. Figure 3. Motility assay of Aeromonas hydrophila parent AL09-71 showed diffuse growth throughout the entire medium and mutant AL09-71 N+R grown only along the line of inoculation. Quadruplicate tubes were inoculated for per strain???????????48 II. Figure 4. Fatty acid composition (%) of total lipid from Aeromonas hydrophila parent AL09- 71 and mutant AL09-71 N+R. Values are means of four replicates per strain. Significant deference between parent and mutant strain for the same fatty acid are labeled with a, b? 48 II. Figure 5. The alignment results of part sequences from parent AL09-71 (P) and mutant AL09- 71 N+R (N+R). * Clone no??????????????????????? ? ....49 II. Figure 6. Chemotactic response of A. hydrophila AL09-71 and AL09-71 N+R to channel catfish mucus. Data were presented as mean ? standard deviation (S.D.) from four replicates. Significant difference (P<0.05) was marked by asterisk? ...???????.. .50 II. Figure 7. In vitro invasion of A. hydrophila AL09-71 and AL09-71 N+R to G1B catfish gill cells. Data were presented as mean ? standard deviation (S.D.) from four replicates. Significant difference (P<0.05) was marked by asterisk??? .??????????..51 III. Figure 1. Effect of Aeromonas hydrophila treatment on the transcriptional kinetics of the six genes identified by suppression subtractive hybridization.?? ?????????... ? 82 III. Figure 2. Effect of Aeromonas hydrophila treatment on the transcriptional kinetics of the seven genes induced by attenuated Edwardsiella ictaluri.????????????? 83 III. Figure 3. Effect of Aeromonas hydrophila treatment on the transcriptional kinetics of the other seven genes selected from literature.??????????????????.. ..84 III. Figure 4. Daily mean percent cumulative mortality of channel catfish intraperitoneally vaccinated with or without the Aeromonas hydrophila AL09-71 N+R and challenged with x their respective virulent parent isolates of A. hydrophila through intraperitoneal injection at 14 days post vaccination. Data are presented as mean ? S.D. from three trials????? .85 III. Figure 5. Relative transcriptional levels of the 20 genes in the anterior kidney of channel catfish at 14 days post vaccination. Data are presented as means ? S.D. from three replicates. Differences were considered statistically significant between vaccinated or infected and control fish at P value < 0.05. Significant difference is marked by an asterisk. TSB: tryptic soy broth; N+R: A. hydrophila AL09-71 N+R; parent: A. hydrophila AL09-71? ???.. .85 IV. Figure 1. Effect of Aeromonas hydrophila treatment on the transcriptional levels of the 8 ESTs at 6 h post-infection including primary, secondary infections and TSB control. Data are presented as means ?S.D. from three replicates????????..? ?????.. .110 IV. Figure 2. Effect of Aeromonas hydrophila treatment on expression kinetics of the 8 genes at different time points post infection including primary, secondary infections and TSB control. Data are presented as means ?S.D. from three replicates??????? ?????? .111 V. Figure 1. Transcription profiles of 5 TLRs in anterior kidney of channel catfish at different time points including primary, secondary infections and TSB control. Data are presented as means ?S.D. from three replicates???????????? ?????. ?...??? 143 V. Figure 2. Transcription profiles of 8 AMPs in anterior kidney of channel catfish at different time points including primary, secondary infections and TSB control. Data are presented as means ?S.D. from three replicates??????? ?????. ?????????...144 V. Figure 3. Transcription profiles of 4 cytokines in anterior kidney of channel catfish at different time points including primary, secondary infections and TSB control. Data are presented as means ?S.D. from three replicates?????????? ?.. ?????.145 V. Figure 4. Transcription profiles of 5 reported genes in anterior kidney of channel catfish at different time points including primary, secondary infections and TSB control. Data are presented as means ?S.D. from three replicates?????? ?.. ????????.....146 V. Figure 5. Plasma lysozyme activity in primary, secondary infected and TSB control fish. Data are presented as means ?S.D. from three replicates????????? ?? ??. ?....147 xi List of Abbreviations AMPs Antimicrobial peptides bp Base pair BPI Bactericidal permeability-increasing protein cDNA Complementray DNA CELSR1 Cadherin EGF LAG seven-pass G-type receptor 1 CFU Colony forming unit Ct Cycle threshold DNA Deoxyribose nucleic acid dpi Day post infection dpv Day post vaccination EST Expressed sequence tags FAMEs Fatty acid methyl esters hpi Hour post infection hpv Hour post vaccination Ig Immunoglobulin IL Interleukin IP Intraperitoneally LB Luria?Bertani LD50 Lethal dosage necessary to kill 50% of a test population xii LEAP2 Liver-expressed AMP 2 MAS Motile aeromonad septicemia NKL NK-lysin PADII Protein-arginine deiminasae type II-like PCR Polymerase chain reaction QPCR Quantitative PCR RPS Relative percent of survival SSH Suppression subtractive hybridization TLRs Toll-like receptors TNF Tumor necrosis factor TNFAIP2 Tumor necrosis factor alpha, alpha-induced protein 2 TOPK Lymphokine-activated killer T-cell-originated protein kinase-like TSA Tryptic soy agar TSB Tryptic soy broth VLIG Very large inducible GTPase 1 1 I. INTRODUCTION AND LITERATURE REVIEW Aeromonas hydrophila Aeromonas hydrophila is a gram-negative bacterium classified as a member of the family Vibrionaceae whose normal habitat is soil and water (Popoff and Veron, 1976). Historically, the genus Aeromonas has been divided into two groups: nonmotile group (psychrophilic species) and motile group (mesophilic species). The nonmotile group can be best represented by Aeromonas salmonicida, which are generally only associated with fish disease. However, the motile group is associated with human disease (Seshadri et al., 2006). Aeromonas hydrophila is in the motile group category, which has the unusual potential to infect both cold- and warm-blooded organisms. Thus, it causes devastating epidemics in fish, reptile, and amphibian populations (Schubert, 1967; Shotts et al., 1975) as well as sporadic water-associated human disease (Schubert, 1967). Channel catfish (Ictalurus punctatus) is the most important cultured fish species in the United States. In West Alabama, a motile aeromonad septicemia (MAS) disease outbreak caused by A. hydrophila in 2009 alone led to an estimated loss of more than 3 million pounds of food size channel catfish (Pridgeon and Klesius, 2011a). Virulence studies have revealed that AL09-71, a 2009 West Alabama isolate of A. hydrophila, is highly virulent to channel catfish, killing fish within 24 h post exposure (Hemstreet, 2010; Pridgeon and Klesius, 2011b). In 2010, A. hydrophila continued to cause disease outbreaks in channel catfish grown in Alabama (Hemstreet, 2010). 2 Motile aeromonad septicemia Aeromonas hydrophila is the causative agent of MAS. This disease occurs mostly from February to July, with some outbreaks occurring in September and November (Woo and Bruno, 2002). It was the most frequently diagnosed bacterial fish disease and the most severe disease problem among cage cultured channel catfish in the USA between 1972 and 1980 (Plumb, 1994). Subsequently, it became one of the most common bacterial infections (1987-1991) among cage- cultured channel catfish in the USA, accounting for 13-22% of disease outbreaks (Duarte et al., 1993). MAS causes diverse pathological conditions including acute and chronic infections. The disease is usually acute in very young fish and chronic in adult fish (Plumb, 1994). Infected fish lose their appetite, become lethargic and swim near the surface. The clinical signs of A. hydrophila infections include swelling of tissues, dropsy, red sores, necrosis, ulceration, and hemorrhagic septicemia (Karunasagar et al., 1989, Azad et al, 2001). However, in the acute form of MAS, the fish can die so rapidly before they have time to develop clinical signs but a few gross signs of disease. The infection of A. hydrophila on channel catfish can be divided into three categories: (i) motile aeromonad septicemia with external signs; (ii) cutaneous, manifesting lesions that are limited to the skin and underlying muscle; and (iii) latent septicemia with no external signs (Grizzle and Kiryu, 1993). Internal clinical signs include oedema, haemorrhage, and necrosis (Woo and Bruno, 2002). Prevention and control MAS outbreaks are generally related to environmental stress such as elevated water temperature, a decrease in dissolved oxygen concentration, low pH, and increased levels of 3 ammonia and carbon dioxide (Walters and Plumb, 1980; Lio-Po et al., 1986). Environmental variables are monitored for stressful situations and possible avoidance of outbreaks. Oxytetracycline (Terramycin) has been the drug of choice for treating MAS in fishes, which is approved for use with pond fishes, channel catfish, and salmonids (Cipriano and Austin, 2011). Medicated feed with 2?4 g oxytetracycline kg-1feed (50?100 mg/kg fish) for 14 days is recommended (Plumb, 1994). However, drug-resistant strains of A. hydrophila may evolve. The antibiotic and chemical resistance development is a persistent problem in A. hydrophila management in aquaculture. Vaccination is regarded as a good method to prevent MAS disease caused by A. hydrophila (Evelyn, 1997). Formalin or heat-killed bacteria of pathogenic A. hydrophila strains show limited success in protection (Areechon et al., 1992; Chandran et al., 2002; John et al., 2002). Vaccination with crude lipopolysaccharide (LPS) induced better protection against A. hydrophila infection in the common carp, C. carpio, than the formalin killed vaccine (Baba et al., 1988). Additionally, live attenuated vaccines have been studied to be effective in protection against homologous A. hydrophila challenge such as aroA mutant and transposon Tn916- generated mutant (Hernanz Moral et al., 1998; Liu et al., 2007). Furthermore, recombinant protein vaccines, developed by using protein OmpTS or S-layer protein have been reported to confer protection against A. hydrophila challenges (Khushiramani et al., 2007; Poobalane et al., 2010). However, it is well known that A. hydrophila is very heterogeneous biochemically and serologically, which is the biggest obstacle in developing effective commercial vaccine against A. hydrophila (Poobalane et al., 2010). Recently, an attenuated vaccine AL09-71 N+R specifically targeting A. hydrophila AL09-71 was developed to prevent future disease outbreaks 4 caused by the highly virulent West Alabama 2009 isolates of A. hydrophila (Pridgeon and Klesius, 2011c). Important immune factors in channel catfish Immune system has been divided into two parts: innate immunity and adaptive immunity. In teleosts, both innate and adaptive immune responses are initiated in against bacterial infection. The innate immune system is of prime importance in the immune defense of fish, which provides the first line of immune defense, whereas the adaptive immunity relies on the generation of random and highly diverse repertoires of T and B-lymphocyte receptors contributes to a more specific and efficient response against infections (McGuinness et al., 2003; Medzhitov, 2007). However, this dichotomy between innate and adaptive systems has been challenged by increasing evidences of the integration of different immune mechanisms into a multilevel network (Flajnik and Du Pasquier, 2004). The immune system not only protects an organism against diseases by identifying and eliminating the pathogen, but also play an important role in processes that maintain stable conditions (homeostasis) following inflammatory reaction or tissue damage (Magnad?ttir, 2010). Numerous immune-relevant genes involved in innate and/or adaptive immunity have been characterized from channel catfish, include the following: 1) Toll-like receptors: There is a growing interest in Toll-like receptors (TLRs), which is demonstrated by the 2011 Nobel Prize in medicine awarded to BA Beutler and JA Hoffmann for their studies on TLR role in physiology and pathology. Innate immune initiation relies on the recognition of pathogen- associated molecular patterns (PAMPs) by pathogen recognizing receptors (PRRs) that induces subsequent host immunity through multiple signaling pathways that contribute to the eradication of the pathogen (Janeway and Medzhitov, 2002). There are several functionally distinct classes 5 of PRRs, but the best characterized are Toll-like receptors (TLRs). All TLRs are type I transmembrane proteins which were composed by three parts: an N-terminal ectodomain containing leucine-rich repeats (LRR) that mediate the recognition of PAMPs, a transmembrane region with one a-helix, and a C-terminal intracellular Toll-IL-1 receptor (TIR) domain that activates downstream signaling pathways (Kawai and Akira, 2011). Following recognition of ligands, TLRs initiate both cell?cell interaction and signaling events that result in acute innate responses. TLRs are also responsible for initiation of adaptive immune responses against pathogen-derived antigens primarily through triggering dendritic cell activation (Pasare et al., 2004). At present, at least 17 TLRs (TLR1, 2, 3, 4, 5, 5S, 7, 8, 9, 13, 14, 18, 19, 20, 21, 22, 23) were identified in teleost species, among which TLR14, TLR19, TLR20, TLR21, TLR22, and TLR23 are non-mammalian TLRs and TLR5S is a soluble isoform of TLR5 that appears to be unique in fish (Palti, 2011). The best characterized ligand that TLRs recognize include: (1) lipoteichoic acid and lipoproteins by TLR2; (2) dsRNA by TLR3; (3) lipopolysaccharide (LPS) by TLR4; (4) bacterial flagellin by TLR5, (5) single stranded RNA (ssRNA) by TLR7, and (6) dsDNA by TLR9 (Baoprasertkul et al., 2007b; Iwasaki and Medzhitov, 2010). Additionally, TLRs were also reported to have multi-functions and act together in pathogen recognition and signaling (Ishii et al., 2005; Baoprasertkul et al., 2007a). Five TLRs (TLR2, TLR3, TLR5, TLR20 and TLR21) have been reported in channel catfish (Bilodeau and Waldbieser, 2005; Baoprasertkul et al., 2007a; Baoprasertkul et al., 2007b). TLR2 belong to TLR1 family which was found to recognize lipopeptides (Rebl et al., 2010). Specific lipopeptide derivates are recognized by combinations of different members of the TLR1 family in mammals (Palsson-McDermott and O?Neill, 2007). Regarding teleosts, Chang and Nie (2008) reported a cooperation of TLR2 and the peptidoglycan recognition proteins 6 (PGRP). In adult zebrafish, the expressions of both TLR2 and TLR1 were induced by Gram- positive Mycobacterium marinum at eight weeks post infection (Meijer et al., 2004). In channel catfish and blue catfish (Ictalurus furcatus), TLR2-encoding mRNA concentration was found to increase one day post-infection by Gram-negative Edwardsiella ictulari (Baoprasertkul et al., 2007a). Recently, Pridgeon et al. (2010) reported that the expression of TLR2 in the head kidney was significantly induced by the infection of E. ictulari at 6 hour post infection (hpi). In addition, TLR2 expression was detected to be significantly induced at 12 hpi in the kidney of Indian major carp, Cirrhinus mrigala, infected by A. hydrophila (Basu et al., 2012a). Since the expression of TLR2 was shown to be up-regulated by infection of both Gram-positive and Gram-negative pathogens, TLR2 may function more widely in teleosts than previously assumed (Rebl et al., 2010). Matsuo et al. (2008) showed that TLR3 can recognize relatively short dsRNA in the pufferfish. Several studies reported upregulation of fish TLR3s mRNA in response to infections with dsRNA viruses (Phelan et al., 2005; Rodriguez et al., 2005; Su et al., 2008). Interestingly, the increasing expression of TLR3 induced by Gram-negative bacteria was also been detected in zebrafish and channel catfish after infection with the Gram-negative E. tarda and E. ictulari, respectively (Phelan et al., 2005; Bilodeau and Waldbieser, 2005; Pridgeon et al., 2010). TLR5 has been identified in bony fish and plays an important role in recognizing the flagellin of bacterial pathogens. In fish, TLR5 was identified to have two forms, membrane- bound TLR5 and soluble TLR5S. Although soluble forms of TLR4S and TLR2S have already been identified in mammals (Iwami et al., 2000; LeBouder et al., 2003), so far no soluble TLR5S was found in mammalian genomes (Rebl et al., 2010). In channel catfish, both, the membrane- bound TLR5 and the soluble TLR5S were characterized and found up-regulated in different 7 tissues by infection of E. ictulari (Bilodeau and Waldbieser, 2005; Baoprasertkul et al., 2007b; Pridgeon et al., 2010). TLR20 and TLR21 belong to so-called ?fish-specific? TLR family, which were not identified in mammalian (Rebl et al., 2010). The channel catfish TLR20 and TLR21 were characterized by Baoprasertkul et al. (2007b). Although they appear to branch with the murine TLR11, 12 and 13 in phylogenetic analyses, they form distinct branches (Baoprasertkul et al., 2007b; Palti, 2011). No direct evidence of ligand specificity has been identified for TLR20 and TLR21 (Palti, 2011). The roles of TLR20 and TLR21 are also unknown in channel catfish. However, TLR20 and TLR21 in the head kidney were significantly upregulated in channel catfish infected by Edwardsiella ictaluri (Pridgeon et al., 2010), indicating that they may play an important role in immune response against Gram-negative bacteria pathogens. 2) Antimicrobial peptides (AMPs) It is well known that fish is able to secrete a lot of different kinds of antimicrobial peptides (AMPs) that are positively charged short amino-acid-chain molecules. AMPs, also known as host defense peptides, play major roles in the innate immune system, and protect against a wide variety of bacterial, fungal, viral, and other pathogenic infections. To answer how AMPs works in the immune system, two different viewpoints about the anti-bacterial mechanisms of AMPs have been proposed: a) the amphiphilic structure of AMPs can selectively bind to the bacterial membrane and form transmembrane channels, which destruct of their membrane integrity and kill incursive bacteria; and b) AMPs can directly enter the bacterial cell to interact with specific intracellular targets to interfere with bacterial growth and metabolism, thus playing a role in bacterial death (Wimley, 2010; Zhu et al., 2012). Except for the central role in infection and inflammation, AMPs also have other important functions since they are 8 multifunctional molecules (Lai et al., 2007). For example, some AMPs influence diverse cellular processes including cytokine release, chemotaxis, antigen presentation, angiogenesis and wound healing through interacting with its receptors on the membrane or altering the properties of the mammalian membrane (Lai et al., 2007). These functions can indirectly support the immune system to eliminate bacteria pathogens and help the host maintain stable conditions (homeostasis) and repair damaged tissues. Now, an increasing number of AMPs have been isolated from fish and with their abundance in many tissues, they may represent the most important innate defense in fish (Noga et al., 2011). Hepcidin, a cysteine-rich amphipathic peptide, is the most widely studied AMP in fish. Hepcidin has been found to play an important role in regulation of iron metabolism and indirect host defense by binding to ferroportin (a key iron exporter on macrophages) and inducing ferroportin-mediated endocytosis and proteolysis (Zhu et al., 2012). Recently, hepticdin were reported to be strikingly induced after challenged by E. ictaluri (Pridgeon et al., 2012). Transferrin also has a critical role in iron metabolism, maintaining low levels of extracellular free iron and transporting iron to tissues as required, which also participates in a wide variety of metabolic processes, including immune regulation, antimicrobial and antioxidant activity, DNA synthesis, cytoprotection, and electron transport (Stafford et al., 2003; Ong et al., 2006). In channel catfish, transferrin was identified, sequenced, and characterized by Liu et al. (2010). Six other AMPs [(NK-lysin type 1, NK-lysin type 2, NK-lysin type 3, bactericidal permeability- increasing protein (BPI), cathepsin D and liver-expressed AMP 2 (LEAP2)] have also been reported in channel catfish. The trancriptional profiles of the three types of NK-lysin gene and LEAP2 have been demonstrated to be different in various tissues of normal channel catfish (Bao et al., 2006; Wang et al., 2006ab). The expression profiles of cathepsin D and BPI in response to 9 pathogen infections at 24 h or longer post-infection have also been reported (Cho et al., 2002; Xu et al., 2005). Recently, the transcriptional files of these six AMPs were analyzed in the anterior kidney of channel catfish infected by E. ictaluri (Pridgeon et al., 2012). 3) Cytokines Cytokines are a family of low molecular weight proteins, which are secreted by activated immune-related cells and related to both innate and adaptive responses (Salazar-Mather and Hokeness, 2006). They can be divided into five types: interferons (IFNs), interleukins (ILs), tumor necrosis factors (TNFs), colony stimulating factors, and chemokines (Savan and Sakai, 2006). Cytokines can modulate immune responses through an autocrine or paracrine manner upon binding to their corresponding receptors (Zhu et al., 2012). Interleukin-1? (IL-1?) gene is a well studied gene in teleost, which belongs to interleukin-1 (IL-1) family. IL-1 is an important early response pro-inflammatory cytokine that regulate both innate and adaptive immune response. IL-1 could be secreted by monocytes, activated macrophages, granulocytes, endothelial cells, activated Tlymphocytes, and many other cell types (Zhu et al., 2012). So far, IL-1? genes have been identified in various teleost fish species, including rainbow trout (Oncorhynchus mykiss), carp (Cyprinus carpio), seabass (Dicentrarchus labrax), channel catfish (I. punctatus) and yellowfin sea bream (Acanthopagrus latus) (Zou et al., 1999; Fujiki et al., 2000; Scapigliati et al., 2001; Wang et al., 2006c; Jiang et al., 2008). In general, only one IL-1b gene seems to exist in fish. However, two IL-1-b-like genes encoding 280-amino acid peptides with high identity (94.3%) with each other have been cloned from channel catfish (Wang et al., 2006c). 10 Reference: Areechon, N., Kitancharoen, N., Tonguthai, K., 1992. 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Advances in research of fish immune- relevant genes: a comparative overview of innate and adaptive immunity in teleosts. Dev. Comp. Immunol. (http://dx.doi.org/10.1016/j.dci.2012.04.001) Zou, J., Grabowski, P.S., Cunningham, C., Secombes, C.J., 1999. Molecular cloning of interleukin 1beta from rainbow trout Oncorhynchus mykiss reveals no evidence of an ice cut site. Cytokine. 11, 552?560. 21 II. CHARACTERIZATION OF THE ATTENUATED AEROMONAS HYDROPHILA VACCINE STRAIN AL09-71 N+R COMPARED TO ITS PARENT STRAIN AL09-71 Introduction Aeromonas hydrophila is the causative agent of motile aeromonad septicemia (MAS) (Harikrishnan et al., 2003) in fish which is also known as epizootic ulcerative syndrome (EUS) (Mastan and Qureshi, 2001). Although usually considered as a secondary pathogen, A. hydrophila can be a primary pathogen, which may cause outbreaks in fish farms with high mortality rates, resulting in severe economic losses to the aquaculture industry worldwide (Thore and Roberts, 1972; Nielsen et al., 2001; Fang et al., 2004). In Alabama, MAS disease outbreaks caused by A. hydrophila in 2009 led to an estimated loss of more than 3 million pounds of food size channel catfish (Pridgeon and Klesius, 2011a). Virulence studies have revealed that the AL09-71 strain, a 2009 Alabama isolate of A. hydrophila, is highly virulent to channel catfish, killing fish within 24 h post exposure (Hemstreet, 2010; Pridgeon and Klesius, 2011b). In 2010, virulent strains of A. hydrophila were also associated with major disease outbreaks in Alabama (Hemstreet, 2010). Due to the multiple medications resistance of A. hydrophila, vaccination may be a better choice for the farmer to protect fish from the infection with A. hydrophila. An attenuated vaccine specifically against A. hydrophila AL09-71 was developed through selection for resistance to both novobiocin and rifampicin (Pridgeon and Klesius, 2011c). 22 The attenuated AL09-71 N+R vaccine provided 80?100% protection against challenges with virulent parent A. hydrophila AL09-71 (Pridgeon and Klesius, 2011c). However, the detailed morphological and growth profiles of this attenuated A. hydrophila strain has not been well characterized. In addition, whether there is a difference at genomic DNA level between the attenuated AL09-71 N+ R strain and its parent AL09-71 strain is currently unknown. Therefore, the objectives of this study were: 1) To characterize the morphological and biological characters of the attenuated AL09-71 N+R vaccine strain compared to its virulent parent AL09-71 strain; and 2) To understand whether the vaccine strain AL09-71 N+R is different from that of the parent strain AL09-71 at genomic DNA level. Materials and methods Bacteria source and growth conditions The A. hydrophila AL09-71 was collected from diseased food-size channel catfish from West Alabama in August 2009. The isolate was cultured on tryptic soy agar (TSA) plates following the procedures described by Panangala et al. (2007). The isolate AL09-71 was then confirmed as A. hydrophila through biochemical analysis with standard biochemical tests as well as API-20E (Biom?rieux, Durham, NC, USA) and molecular identification using gene-specific primers for four A. hydrophila genes: 16S?23S rDNA intergenic spacer region, 60 kDa chaperonin, DNA gyrase B subunit and RNA polymerase sigma factor RpoD (Pridgeon and Klesius, 2011a). The attenuated A. hydrophila AL09-71 N+R mutant was produced from the virulent parent strain of A. hydrophila AL09-71 through selection for resistance to both novobiocin and rifampicin (Pridgeon and Klesius, 2011c). The isolates were maintained on TSA plates or in tryptic soy broth (TSB) (Difco, Sparks, MD, USA) for 23 18?24 h at 28 ?C. Bacteria were stored as frozen cultures at -80 ?C in TSB containing 25% (v/v) glycerol. Colony forming unit and growth rate Absorbance readings of overnight sample cultures were adjusted to 1.0 at 540 nm using Thermospectronic spectophotomer (Fisher Scientific, Pittsburgh, PA). Serial dilutions (1:10) of each samples were prepared in TSB and immediately 0.1 ml of dilutions in quadruplicate plated onto TSB plates. After incubating the plates overnight at 28 ?C, number of colonies was counted and the average number of colony forming unit (FU) ml-1 were calculated for both samples. For the determination of growth rate, same bacterial cultures above were used. Serial dilutions of 1:2 (starting from 1:10 dilution) up to 1:640 were made in triplicate using sterile 96-microtiter plates for assay. Plates were incubated at 28?C with constant shaking and absorbance measured at time intervals at 540 nm for growth rate using ELISA spectrophotometer. Optical reading were blanked with 0 h readings and log plotted versus incubation time. In vitro motility assay Agar was used at a low concentration (2.5 g L-1) as a solidifying agent for the differentiation of parent and mutant on the basis of motility. Tubes containing semi-solid agar were inoculated by stabbing through center of the medium with inoculating needle. Quadruplicate samples of test tubes were incubated at 28 ?C for 24 h. Motility was observed visually by diffuse growth spreading from the line of inoculation. In vitro chemotactic response of AL09-71 N+R and AL09-71 to catfish mucus 24 In vitro chemotaxis assays were performed according to procedures (Klesius et al., 2010) with slight modifications. Briefly, healthy channel catfish were anesthetized with 100 mg L-1 tricane methanesulfonate (Agent Chemicals, Redmond, CA). The anesthetized fish were held vertically and mucus was collected from the skin into a petri dish by pressing the edge of petri dish gently against the skin. Mucus samples were then transferred form petri dish into a 1.5 ml tube. The mucus samples were centrifuged at 6000 g for 15 min and the pellets (epithelium cells and cellular debris) were discarded. The mucus protein concentration was then adjusted to 0.2 mg ml-1 with PBS. The pooled mucus samples were stored at -20?C until use. Chemotaxis assay was performed using blind-well chemotaxis chambers (Corning CoStar, Cambridge, MA). Briefly, the bottom chambers were filled with 200 ?l of either A. hydrophila (1x107 CFU ml-1) parent or mutant strain. The bottom chamber was separated from the upper chamber by an 8-?m pore diameter polycarbonate membrane filter (Nucleopore, Pleasontan, CA) and the chambers were assembled. Triplicate mucus (0.2 mg protein ml-1) samples were added to the upper chamber of each parent and mutant. As negative controls, lower chamber with tryptic soy broth (TSB) and upper chamber with mucus samples was also included in the assay. The chambers were incubated for 3 h at 28?C. Following incubation, 100 ?l mucus from the upper chambers were transferred to a flat- bottom 96-well microtiter plate (Thermo Scientific, Milford, MA). The number of viable bacterial cells in each well was then determined by CellTiter 96 ? AQueous Non-Radioactive Cell Proliferation Assay (MTS) (Promega, Madison, WI). Soon after 20 ?l of MTS was added to each well, the OD at 490 nm was recorded. The plate was then incubated at 28?C for 25 20 min. After the incubation, the OD was measured again at 490 nm. Relative increased OD value was calculated using the following formula: ? OD 490 nm value (sample) = OD 490 nm value (after incubation) - OD 490 nm value (0 h of the incubation). The relative chemotactic index of AL09-71 or AL09-71 N+R to fish mucus was calculated using the following formula: ?? chemotactic index = ? OD 490 nm value of sample (AL09-71 or AL09-71 N+R) - ? OD 490 nm value of sample alone (without bacteria in the lower chamber, negative control). The experiments were repeated four times. In vitro invasion of A. hydrophila to G1B catfish gill cells Invasion assays were performed as described by Thiagarajan et al. (1996) with slight modifications made by Pridgeon et al. (2011). Briefly, G1B gill cells in a total volume of 1 ml were split into 96 ?well tissue culture plates with final concentration of 5x104 cells per well and grown at 25?C for 24 h. Overnight culture of A. hydrophila parent and mutant strains were adjusted to same OD reading at 540nm (0.80) and, then, were diluted to 1:10 and mixed with cells at ratio 1:5. Gill cells in the absence of any bacteria were used as negative control. Plates were incubated at 25?C for 1h. For the invasion assay, a total volume of 0.2ml of culture media containing 5mg ml-1 gentamicin (Sigma-Aldrich, St. Louis, MO, USA) was added to each well to kill any extracellular bacteria. Plates were incubated at 25?C for 1h. The culture medium containing gentamicin and any extracellular bacteria were gently removed. The 0.2 new fresh medium were added into each well. The number of viable bacterial cells in each well was then determined by CellTiter 96 ? AQueous Non-Radioactive Cell Proliferation Assay (MTS) (Promega, Madison, WI). Soon after 20 ?l of MTS was added to each well, the OD at 490 nm 26 was recorded. The plate was then incubated at 28?C for 30 min. After the incubation, the OD was measured again at 490 nm. Relative increased OD value was calculated using the following formula: ? OD 490 nm value (sample) = OD 490 nm value (after incubation) - OD 490 nm value (0 h of the incubation). The relative invasion index of AL09-71 or AL09-71 N+R to G1B gill cells was calculated using the following formula: ?? invasion index = ? OD 490 nm value of sample (AL09-71 or AL09-71 N+R) - ? OD 490 nm value of cell alone (negative control). The experiments were repeated four times. Whole cellular fatty acid profile Preparation of fatty acid methyl esters (FAMEs) from bacteria grown at 28 ?C on sheep blood agar plates was done according to the Microbial Identifications Systems (MIS) (MIDI, Newark, DE, USA) version 4.5 (Shoemaker et al. 2005). Briefly, overnight culture of 25-30 mg of bacteria were harvested and placed in 13 mm x 100 mm glass tubes. Bacterial cells were saponifed in 1 ml of a sodium hydroxide and methanol solution while boiling in a water bath for 30 min. After cooling, the fatty acids were methylated in 2 ml of hydrochloric acid and methanol reagent for 10min in an 80?C water bath. Then, the FAMEs were extracted in hexane and methyl tert-butyl ether (1.25 ml). Any residual fatty acids and reagents were removed from the organic extract by washing with 3.0 ml of a 0.3 M sodium hydroxide solution for 5 min. Final, top 2/3 phase was removed and transferred to GC vial and FAMEs were injected into an Agilent Technology 6850 gas chromatograph for analysis following the MIS rapid protocol (RCLN50). FAMEs were identified by comparison of their retention times with those of authentic standards obtained from Microbial ID, Inc. (Newark, DE, USA). FAMEs in hexane were used as calibration standards. 27 Suppression subtractive hybridization (SSH) Bacterial genomic DNA was extracted according to the manual of DNeasy kit (Qiagen, Valencia, CA, USA). All DNAs were eluted with distilled water and quantified on a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, Rockland, DE). SSH was carried out using PCR-Select Bacterial Genome Subtraction Kit (Clontech, Palo Alto, CA, USA). Genomic DNA of the most virulent 2009 isolate AL09-71was used as tester and that of attenuated AL09-71 N+R was used as driver. According to the manufacturer?s instructions, equal amounts (2 mg) of genomic DNA from both tester and driver were digested by RsaI at 37 ?C overnight. The digested tester DNAs were purified, subdivided equally and ligated with two different adaptors (adaptor 1 and adaptor 2R supplied by the kit) respectively. Two hybridizations were performed. In the first, an excess of driver was added to each adaptor-ligated tester sample followed by 98 ?C for 1.5 min, 63 ?C for 6 h. In the second hybridization, the denatured driver was added into two primary hybridization samples which were not denatured followed by 63 ?C overnight. Finally, the ratio of driver DNA: tester DNA sample was 50:1. After filling in the adapter ends with DNA polymerase, the entire population of molecules was then subjected to PCR to amplify the tester-specific sequences as described in the manual. The secondary PCR amplification product was cloned into pGEM-T Easy vector (Promega, Madison, WI, USA) following manufacturer?s instructions and transformed into one Shot? TOP10 chemically competent E. coli (Invitrogen, Carlsbad, CA) according to the manual. Transformed cells were then plated on Luria?Bertani (LB) plates containing ampicillin (100 ?g/ml) and X-Gal (5-bromo-4-chloro-3-indolyl-beta- D-galactopyranoside) (40 ?g/ml). 28 Plasmid DNA isolation and sequencing From the library, a total of 96 white colonies were subsequently picked and cultured overnight in LB broth in the presence of ampicillin (100 ?g/ml) in the InnovaTM 4000 Incubator Shaker (New Brunswick Scientific, Edison, NJ) at 37 ?C and 235 rpm settings, respectively. Overnight cultures were then sent to USDA-ARS Mid South Genomics Laboratory in Stoneville, MS for plasmid DNA extraction and DNA sequencing was carried out with an ABI 3730 Genetic Analyzer (Applied Biosystems, Foster City, CA). Sequences were trimmed and analyzed using the National Center for Biotechnology Information (NCBI) BlastX program to search for sequence homologies. Primer design and polymerase chain reaction Sequencing results of different clones were used to design gene-specific primers by using Primer3 program (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). PCR was performed in a 10 ?l mixture consisting of 5 ?l of Taq PCR Master Mix (Qiagen, Valencia, CA), 3 ?l of nuclease-free H2O, 1 ?l of A. hydrophila genomic DNA (10 ng/?l), 0.5 ?l of forward primer (5 ?M), and 0.5 ?l of reverse primer (5 ?M). All PCRs were carried out in a Biometra T Gradient thermocycler (Biometra, Goettingen, Germany). PCR products were analyzed on 1% agarose gel by electrophoresis. Gel purification and sequencing analysis PCR products were analyzed on 1% agarose gel by electrophoresis and purified with QIAquick Gel Extraction Kit (Qiagen, Valencia, CA). The purified DNA products and correlated primers were then sent to USDA-ARS Mid South Genomics Laboratory in Stoneville for DNA with an ABI 3730 Genetic Analyzer (Applied Biosystems, Foster City, 29 CA). Sequences were analyzed by using the National Center for Biotechnology Information (NCBI) BlastT, BlastX program to search for sequence homologies and CLUSTALW (Thompson et al., 1994) to do the alignment. Statistical analysis Growth rate data and cellular fatty acid profile of parent and mutant strains were analyzed by ANOVOA using Student?s t test with a significance level of P<0.05 (SAS, version 9.2, Cary, NC). Results Colony, growth rate and motility The colony morphology of parent strain (AL09-71) and attenuated mutant strain (AL09-71 N+R) are shown (Fig. 1). AL09-71 N+R had smaller colony size than its parent, however, no other obvious difference was observed. A comparison on the growth of the attenuated mutant and its parent was determined by turbidity at 540nm. The results showed that the parent strain grew faster than that of AL09-71 N+R at all dilutions tested (1:10, 1:20, 1:40, 1:80, 1:160, 1:320, and 1:640) (Fig. 2). The vitro motility test revealed that the parent strain was motile whereas the vaccine strain was not motile (Fig. 3). In vitro chemotactic response of AL09-71 N+R and AL09-71 to catfish mucus The results of the in vitro chemotactic response of AL09-71 N+R and AL09-71 to catfish mucus are summarized in Figure 7. The average chemotactic index of AL09-71 to catfish mucus was 1.17?0.14, which was significantly (P<0.05) higher than that of AL09-71 N+R. In vitro invasion of A. hydrophila to G1B catfish gill cells 30 The results of the in vitro invasion of AL09-71 N+R and AL09-71 to G1B catfish gill cells are summarized in Figure 7. The average invasion index of AL09-71 to G1B catfish gill cells was 0.31?0.056, which was significantly (P<0.05) higher than that of AL09-71 N+R. The average invasion index of AL 09-71 N+R was only 0.016?0.009, which was almost zero. Whole cellular fatty acid profile The major cellular fatty acids associated with the parent (AL09-71) and attenuated mutant (AL09-71 N+R) strain are shown in Table1. The most abundant fatty acids present in both were 16:0, 18:1 w7c, summed feature 2 and summed feature 3, which collectively accounted for over 80% of total peak area (Fig 4.). The percentage of 16:00 and18:1 w7c fatty acid was significantly higher whereas the percentage of summed feature 3 was significantly lower in total cellular lipid of attenuated mutant compared to patent strain (Fig 4.). No significant differences were detected in the saturated cellular fatty acids between parent and attenuated AL09-71 N+R on dodecanoic acid and tetradecanoic acid. Characteristics of the subtractive genomic DNA library A total of 96 clones were obtained from the subtractive genomic DNA library using the highly virulent 2009 isolate of A. hydrophila AL09-71 as tester and attenuated vaccine AL09-71 N+R as driver. All 96 clones were subjected to sequencing. Of the 96 clones, 94 contained inserts. After amputation of the vector sequences, BlastX sequence homology analyses were performed by using the BlastX network service of the National Center for Biotechnology Information (NCBI). Of these 94 sequences, 62 sequences were discarded due to redundancy. A total of 32 unique DNA sequences (34%) were obtained from the 94 clones (Table 2). The insert sizes of the 32 unique sequences ranged in size from 147 bp to 31 840 bp. The average insert size was 406 bp (Table 2). According to the BlastX result unique sequences (94%) in 32 unique DNA were found had an e-value lower than 10-20(Table 2). Twenty-four of the 32 sequences shared high homologies with the genome of A. hydrophila ATCC7966 strain deposited at GenBank. PCR analysis, gel purification and sequence analysis of the 32 genome sequences obtained from the subtractive library In order to determine the specificity of 32 unique DNA sequences to the highly virulent 2009 isolate of A. hydrophila AL09-71, one set of primers (forward primer and reverse primers) was designed for each DNA sequence. Primers used in PCR are listed in Table 3. Genomic DNA from AL09-71 and AL09-71 N+R was used as template in PCR. However, all PCR products using AL09-71 as template yielded similar size to that using AL09-71 N+R as template. The PCR products using the genomic DNA of AL09-71 or AL09- 71 N+R as templates were then subjected to sequencing. Sequence analysis of the 64 PCR products revealed that there was no difference between AL09-71 and AL09-71 N+R (Fig 5.). Discussion In this study, the attenuated vaccine strain had smaller colony size and significantly (P<0.05) lower growth rate compared to the parent strain. The reduced bacterial growth rate is typically regarded as a fitness cost of most antibiotic resistance mechanisms (Andersson and Hughes, 2010). Rifampicin resistance is caused by mutations in DNA-directed RNA polymerase subunit-?. It has been reported that the tested rifampicin-resistant mutants have a reduced fitness on growth rate (Reynolds, 2000; Enne et al., 2004). The slower growth has also been reported as a fitness cost in novobiocin-resistant Streptococcus iniae (Pridgeon and 32 Klesius, 2011d). In addition, resistant mutants usually show decreased fitness as well as decreased virulence (Cohen et al., 2003; Andersson and Hughes, 2010). For example, mutant strains of Mycobacterium bovis and M. tuberculosis that are resistant to isoniazid was shown to have significantly reduced virulence as measured by host killing and by histopathology (Wilson et al., 1995; Li et al., 1998). Furthermore, Andersson and Levin (1999) reported that mutations that confer antibiotic resistance resulted in reduce fitness, such as decreased virulence and slowed growth rate. Recently, the novobiocin-resistant Streptococcus iniae strain was reported to be attenuated with smaller colony size and slower growth rate compared to its parent (Pridgeon and Klesius, 2011d). Taken together, our results suggest that the smaller colony size and slower growth rate associated with the attenuation of virulence of AL09-71 N+R may be regarded as fitness costs related to its resistance to novobiocin and rifampicin. The ability of a pathogen to attach to, invade and subsequently infect a susceptible host is directly related to its virulence. Motility and the presence of flagella have been related to different early aspects of bacterial pathogenesis, predominantly adherence to and invasion to eukaryotic cells (Yao et al., 1994). Motility is considered to be an important virulence factor in the pathogenesis of Aeromonas-associated infections, as it facilitates pathogens to adhere and invade the host cells (Kirov et al., 2002; Khajanchi et al., 2012). Studies have demonstrated the requirement of motility for the virulence of Helicobacter felis (Josenhans et al., 1999) and Pseudomonas aeruginosa (Feldman et al., 1998). In addition, Merino et al. (1997) reported that motility in A. hydrophila of serogroup O:34 strains was important for its adherence to host. Furthermore, a mutant A. hydrophila was found to be diminished motility 33 and attenuated (Khajanchi et al., 2012). In the present study, our result revealed that the motility of AL09-71 N+R strain was impaired, which might, at least, partially explain why AL09-71 N+R strain does not show virulence to channel catfish. In order to act as a pathogen, bacteria need to have contact with the host. This can be coincidental or the result of directed chemotactic movement. Hazen et al (1982) reported that skin mucus was a chemoattractant for A. hydrophila. Based on the difference of chemotactic behaviour of the isolates, it was suggested that there could be a difference in pathogenicity between A. hydrophila strains (Hazen et al.,1982). In addition, Ascencio et al. (1998) stated that mucus can serve as a carbon and nitrogen source for A. hydrophila. In this study, the in vitro chemotaxis assays revealed that AL09-71 had significantly higher chemotactic response to catfish mucus than AL09-71 N+R. Furthermore, our in vitro invasion studies revealed that the invasion rate of AL09-71 to G1B gill cells was significantly higher than that of AL09-71 N+R. The attenuated vaccine strain almost lost their invasion ability with much lower invasion index which is near zero. Although cell motility and invasion ability are well known virulence factors (Josenhans and Suerbaum 2002; Zakikhany et al. 2008), the importance of chemotaxis affecting the virulence of pathogens are not extensively studied. It has been suggested that chemotaxis is not necessary for A. hydrophila to become pathogenic to common carp, but may be a necessary parameter for A. hydrophila to become an obligate pathogen (Van der Marel et al. 2008). The fatty acid profiles of Aeromonas species were described (Canonica and Pisano, 1988; Hansen et al., 1991; Huys et al., 1994). In the present study, the most abundant fatty acids in both strains were hexadecanoic acid (16:0), cis-7-Octadecenoic acid (18:1 w7c), and 34 summed feature 3(2-Hydroxypentadecanoic acid or cis-7-Hexadecenoic acid) (SF3). These three major components accounted for approximately 80% of the total cellular fatty acids, which confirms with the fatty acid profiles described before. Lambert et al. (1983) reported that A. hydrophila and A. salmonicida were differentiated from other members of the Vibrionaceae because they did not contain 13:l iso and contained only trace amounts of 12:0 3OH. In our study, the fatty acid profiles of both strains do not inculde 13:l iso, but, only AL09-71 N+R contains up to 0.18% 12:0 3OH of total fatty acids. In general, as long as the growth rate was relatively normal, the fatty acid composition is highly conserved (Campbell and Cronan, 2001; Heath et al., 2002). Slight but significantly (P<0.05) higher level of hexadecanoic acid (16:0), cis-7-Octadecenoic acid (18:1 w7c) and lower level of SF 3 in the attenuated AL09-71 N+R were found, suggesting that some kinds of mutants may exist on genomic or transcriptional level in AL09-71 N+R , which can affect the fatty acid biosynthesis. Suppression subtractive hybridization (SSH) has been widely used to identify sequences that are unique to one genome but absent in another (Straus and Ausubel, 1990; Mahairas et al., 1996; Zhang et al., 2000; Olivares-Fuster and Arias, 2008; Dai et al., 2010). Pridgeon et al. (2011) identified 2 sequences only present in highly virulent A. hydrophila strains, but absent in avirulent strains by using SSH. However, in this study, no different sequence is detected out between the attenuated mutant and parent strain by SSH. The fact that no difference is identified and confirmed could be due to the following reasons. Firstly, when the samples under comparison have relatively small number of differential sequences and the background is high, it will be easier to get the false positive results (Buzdin and 35 Lukyanov, 2007). Secondly, the mutant on the genome level may be slight such as point mutation, which is not easier to detect with SSH. Finally, the difference between the AL09- 71 and AL09-71 N+R may happen on the transcriptional level. In conclusion, our results indicate that the attenuated vaccine strain A. hydrophila AL09-71 N+R has smaller colony size and slower growth rate compared to its parent AL09- 71. In vitro motility assay revealed that AL09-71 N+R was immotile whereas AL09-71was motile. The chemotactic response of AL09-71 N+R to channel catfish mucus was significantly lower than that of AL09-71. The ability of AL09-71 N+R to invade catfish gill cells was significantly lower than that of AL09-71. 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Molecular analysis of genetic differences between virulent and avirulent strains of Aeromonas hydrophila isolated from diseased fish. Microbiology. 146, 999?1009. 42 Table 1. Nomenclature of fatty acid methyl esters of mutant strain AL09-71 N+R and parent strain AL09-71. *Summed feature (SF) denotes two peaks exhibiting overlapping retention times with fatty acids in each of two elution profiles. Shorthand name Systematic name Trivial name Saturated fatty acids 12:00 Dodecanoic acid Lauric acid 14:00 Tetradecanoic acid Myristic acid 16:00 Hexadecanoic acid Palmitic acid 18:1?7cis cis-7-Octadecenoic acid Unknown SF* SF-2 14:0 3-OH 3-Hydroxytetradecanoic acid 3-Hydroxymyristic acid 16:1 iso I Hexadecenoic acid, isomer I Palmitoleic acid SF-3 15:0 iso 2-OH 2-Hydroxypentadecanoic acid Unknown 16:1 cis 7 cis-7-Hexadecenoic acid Palmitoleic acid 43 Table 2. List of sequences isolated from mutant strain AL09-71 N+R vs parent strain AL09-71 bacterial genome subtractive library. No. Clone no. Accessetion no. Gene Name Organism E-value Identity Insert size 1 A03 YP_855339 sigma-E factor regulatory protein RseB Aeromonas hydrophila 1.00E-38 100% 480 2 A06 YP_854878 TRAP transporter solute receptor TAXI family protein Aeromonas hydrophila 1.00E-47 84% 636 3 A07 YP_856271 queuine tRNA-ribosyltransferase Aeromonas hydrophila 1.00E-12 71% 916 4 A09 YP_855029 Mg2+ transporter Aeromonas hydrophila 2.00E-46 100% 435 5 A12 YP_854606 ubiquinone/menaquinone biosynthesis methyltransferase UbiE Aeromonas hydrophila 1.00E-31 97% 356 6 B03 YP_846969 ATPase central domain-containing protein Syntrophobacter fumaroxidans 5.00E-28 67% 225 7 B04 YP_002479020 AraC family transcriptional regulator Desulfovibrio desulfuricans 7.00E-77 67% 839 8 B08 YP_001141367 GGDEF domain-containing protein Aeromonas salmonicida 7.00E-103 99% 1014 9 B09 YP_858309 type 4 fimbrial assembly protein PilC Aeromonas hydrophila 6.00E-128 98% 583 10 B11 YP_857798 triosephosphate isomerase Aeromonas hydrophila 2.00E-47 100% 247 11 C02 YP_004393797 SNF2 family protein Aeromonas veronii 4.00E-156 98% 716 12 C04 ZP_08519501 two-component system response regulator, LuxR family protein Aeromonas caviae 8.00E-27 96% 225 13 C07 YP_855648 cyclopropane-fatty-acyl-phospholipid synthase Aeromonas hydrophila 7.00E-107 99% 482 14 C08 YP_854955 zinc protease Aeromonas hydrophila 4.00E-58 97% 431 15 C09 YP_857681 type IV pilus secretin PilQ Aeromonas hydrophila 4.00E-140 99% 752 16 D02 YP_857301 DNA-binding transcriptional activator UhpA Aeromonas hydrophila 2.00E-82 84% 1077 17 D04 ZP_07779650 phage integrase family protein Escherichia coli 1.00E-135 100% 1086 18 D05 YP_855854 4'-phosphopantetheinyltransferase family protein Aeromonas hydrophila 3.00E-83 95% 442 19 D06 YP_855273 diguanylate cyclase/phosphodiesterase Aeromonas hydrophila 4.00E-103 100% 843 20 D11 YP_001142237 ATP-dependent Clp protease, ATP-binding subunit ClpA Aeromonas salmonicida 8.00E-94 100% 442 21 E01 YP_855297 2-nitropropane dioxygenase family oxidoreductase Aeromonas hydrophila 3.00E-89 97% 801 22 E02 YP_855232 type IV pilus biogenesis protein Aeromonas hydrophila 2.00E-53 100% 264 23 E03 YP_001141849 Poly(hydroxyalcanoate) granule associated protein Aeromonas salmonicida 4.00E-31 95% 547 24 E05 YP_854909 histidine ammonia-lyase Aeromonas hydrophila 3.00E-37 100% 253 25 E06 YP_854569 sensory box/GGDEF family protein, putative Aeromonas hydrophila 4.00E-153 98% 888 26 F04 YP_856871 bifunctional fructose-specific PTS IIA/HPr protein Aeromonas hydrophila 1.00E-151 99% 784 27 F09 YP_856204 maltose transporter membrane protein Aeromonas hydrophila 2.00E-125 99% 579 28 F11 YP_854816 shikimate kinase Aeromonas hydrophila 2.00E-64 87% 1013 29 G04 YP_856398 recombination factor protein RarA Aeromonas hydrophila 0 99% 841 30 G08 ZP_08519441 DNA-binding transcriptional activator GcvA Aeromonas caviae 2.00E-72 99% 1045 31 G10 YP_857763 large-conductance mechanosensitive channel Aeromonas hydrophila 3.00E-82 99% 774 32 H01 YP_857314 putative transporter Aeromonas hydrophila 1.00E-19 98% 438 44 Table 3. Gene-specific primers used in PCR. Clone no. Forward primer (5??3?) Reverse primer (5??3?) A03 CAGTCCAGCCGTATTTTGCT GCCATCGAGATAGCTGAGGT A06 TCCGGAAATGGTCTATCACC AGCCGCGCTCTTTGTAGTAG A07 AGTGGGTGAGGCGAAAGAG GGTAGTTTTGATGCGGCAGT A09 ACGACTGATTGGTGGGACAT GATGGCGAGAAAGCAGTAGG A12 CCTCGACCGAGAAGGAAAC GAGCAGTCGATGGTGAAGC B03 GCCGATCAGTAAGCCAAGAG AGATAGACCGTGGGACAGGA B04 CATTTTCACTGCATCGTGCT GGCATCCTTGATTTTCCTCA B08 ATGGACGCCTCAATACTGCT CATAAAGGCCTGCAGGGTAA B09 GCGCTCTGGAGACCATCTAC AACTGGGGAATGACGAACAG B11 AGCAGGCACAAGAGGTTCAT GCCGCTTCAACAATTCCTAA C02 GAAGGACCACAGGTTCTGGA CAAGGAATCAACGCTTCACA C04 GCAGCCTCAACAAGCAGAT CGGTTCTTGACACCGAGTTT C07 CTTGCTGCTGGAGGACTACC AGCCACCGGGGAAGATATAG C08 AACAGGCTGTCGGTCAAACT GTTGAGCGCCTCTTCCAGAT C09 CCCTGTCTCTCAGGTTGAGG GAGATGGGTTTGCCCTGATA D02 ACTCATCTCGAACCCGACTG TGCACCGACAGCATCACTAC D04 AAACGAAATTTGGCAACCAG ATTTCTGCAGGCGACAAAGT D05 GCAGTTCAATCTCAGCCACA CGAGCAGCCACTCATATTCA D06 CCTTCTTCGACCATCTGAGC TCTCGTGGGTGATGTCGAT D11 GCTGGACGAGATCGAAAAAG ATCGTGGCTCATATCCTGCT E01 GGGCCATTCAACATCAACTT GGCAGTCCGAAATGAAAACT E02 CGCTACGTCTATCCCAGAGC GTTGTTGCTTGAGCGGGTAT E03 ACCGACAACCAACTCACCTC TCGAAGATTTTACCGCCTTC E05 TTTGCCGAGGATGAAGACAT CATGAAACCGGAGTTGACG E06 CAGACACTGCTGGTGGAAGA GAACAGCTGCTGGATCTTGG F04 GGCATCTGGTGAAGAACACC GCACCGGTCAGGATATTGAT F09 GCATCGCCTTCACCAACTAT ATTGGTCGTCCTGTTTCAGC F11 TGGCGGGTAAGAGCATTATC GTTCTTCTGCTCCAGGCTGT G04 CCGGGTCTATCTGCTCAAAC GCAGCTCCAGGTAGTTGAGG G08 ATCTTCAGCCACTCCACCAT TATCTTGCCCTGATCGGACT G10 CGGTCGGTATCATCATTGGT TCGATGATGGTCTGGATGAA H01 TGCCGATAGTTTATTTTTCCTGA CCATCACCTGCAACTTGATCT 45 Figure legends Figure 1. Colony morhology of Aeromonas hydrophila parent AL09-71 and mutant AL09-71 N+R strains. Quadruplicate plates for per strain were incubated overnight at 28?C. Figure 2. Growth curve of Areomonas hydrophila parent AL09-71 and mutant AL09-71 strains for 25 h. Four dilutions as follows: 1:40 (A), 1:80 (B), 1:160 (C), and 1:320 (D) with triplicate. Figure 3. Motility assay of Aeromonas hydrophila parent AL09-71 strain showed diffuse growth throughout the entire medium and mutant AL09-71 N+R strain grown only along the line of inoculation. Quadruplicate tubes were inoculated for per strain. Figure 4. Fatty acid composition (%) of total lipid from Aeromonas hydrophila parent AL09-71 and mutant AL09-71 N+R strains. Values are means of four replicates per strain. Significant deference between parent and mutant strain for the same fatty acid are labeled with a, b. Figure 5. The alignment results of part sequences from parent AL09-71 (P) and mutant AL09-71 N+R (N+R) strains. * Clone no. Figure 6. Chemotactic response of A. hydrophila AL09-71 and AL09-71 N+R to channel catfish mucus. Data were presented as mean ? standard deviation (S.D.) from four replicates. Significant difference (P<0.05) was marked by asterisk. Figure 7. In vitro invasion of A. hydrophila AL09-71 and AL09-71 N+R to G1B catfish gill cells. Data were presented as mean ? standard deviation (S.D.) from four replicates. Significant difference (P<0.05) was marked by asterisk. 46 Figure 1. 47 Figure 2. 48 Figure 3. Figure 4. 49 P_C07* 1 GAGATGATCGAAGCGGTGGGCCACGCCTTCCTGCCCGACTATTTCCGCCA 50 |||||||||||||||||||||||||||||||||||||||||||||||||| N+R_C07 1 GAGATGATCGAAGCGGTGGGCCACGCCTTCCTGCCCGACTATTTCCGCCA 50 P_C07 51 GCTGTCGCGGCTGCTCAAACCCGGTGGTCGCCTGCTCATTCAGGCCATCA 100 |||||||||||||||||||||||||||||||||||||||||||||||||| N+R_C07 51 GCTGTCGCGGCTGCTCAAACCCGGTGGTCGCCTGCTCATTCAGGCCATCA 100 P_C07 101 CCATCGCCGATCAACGCCATGCCCAGTATCTGCGCGGGGTGGATTTCATC 150 |||||||||||||||||||||||||||||||||||||||||||||||||| N+R_C07 101 CCATCGCCGATCAACGCCATGCCCAGTATCTGCGCGGGGTGGATTTCATC 150 P_C07 151 CAGCGCTATATCTTCCCCGGTGGCT 175 ||||||||||||||||||||||||| N+R_C07 151 CAGCGCTATATCTTCCCCGGTGGCT 175 P_E01* 1 CCGCTGGTTGCAGCGGCTGACGCCCTACTATGACGAATACGGCGTGCGCG 50 |||||||||||||||||||||||||||||||||||||||||||||||||| N+R_E01 1 CCGCTGGTTGCAGCGGCTGACGCCCTACTATGACGAATACGGCGTGCGCG 50 P_E01 51 ATGCTGCCGGCACGGCGGCTCCCAGTCGGGCCCCGTTCAATGCCGAGCAT 100 |||||||||||||||||||||||||||||||||||||||||||||||||| N+R_E01 51 ATGCTGCCGGCACGGCGGCTCCCAGTCGGGCCCCGTTCAATGCCGAGCAT 100 P_E01 101 GCCGCCATGGTGGCCGAGTTCAAACCGGCCGTGGTCAGTTTTCATTTCGG 150 |||||||||||||||||||||||||||||||||||||||||||||||||| N+R_E01 101 GCCGCCATGGTGGCCGAGTTCAAACCGGCCGTGGTCAGTTTTCATTTCGG 150 P_E06* 1 TGGAGAGCCGCAGCCACTTCGAGCTGTTGCTCGATGAGCGGCTGGCCAGC 50 |||||||||||||||||||||||||||||||||||||||||||||||||| N+R_E06 1 TGGAGAGCCGCAGCCACTTCGAGCTGTTGCTCGATGAGCGGCTGGCCAGC 50 P_E06 51 GAGAGCGGCAGCTTCAGCCTGCTGCAATTCAGCGTCGACCACAGGGCCAA 100 |||||||||||||||||||||||||||||||||||||||||||||||||| N+R_E06 51 GAGAGCGGCAGCTTCAGCCTGCTGCAATTCAGCGTCGACCACAGGGCCAA 100 P_E06 101 GATCCAGCAGCTGTTC 116 |||||||||||||||| N+R_E06 101 GATCCAGCAGCTGTTC 116 P_G04* 1 TCGATCAGGCGATGCAGGACGCTCGCGGGCTCAATGATCCGGCGCTGACC 50 |||||||||||||||||||||||||||||||||||||||||||||||||| N+R_G04 1 TCGATCAGGCGATGCAGGACGCTCGCGGGCTCAATGATCCGGCGCTGACC 50 P_G04 51 TTCGCCCCCGGGGTGAAGGAGGCGCTGGCCAAGGCGGTGGATGGGGACGG 100 |||||||||||||||||||||||||||||||||||||||||||||||||| N+R_G04 51 TTCGCCCCCGGGGTGAAGGAGGCGCTGGCCAAGGCGGTGGATGGGGACGG 100 P_G04 101 GCGCAAGTCCCTCAACTACCTGGAGCTGC 129 ||||||||||||||||||||||||||||| N+R_G04 101 GCGCAAGTCCCTCAACTACCTGGAGCTGC 129 Figure 5 50 Figure 6 51 Figure 7 52 III. TRANSCRITIONAL PROFILES OF MULTIPLE GENES IN THE ANTERIOR KIDNEY OF CHANNEL CATFISH VACCINATED WITH AN ATTENUATED AEROMONAS HYDROPHILA Introduction Aeromonas hydrophila, a Gram-negative motile bacillus widely distributed in aquatic environments, is a causative agent of motile aeromonad septicemia (MAS) (Harikrishnan et al., 2003). MAS is also known as epizootic ulcerative syndrome (EUS) (Mastan and Qureshi, 2001). The symptoms of A. hydrophila infections include swelling of tissues, dropsy, red sores, necrosis, ulceration, and hemorrhagic septicemia (Karunasagar et al., 1989; Azad et al., 2001). Fish species affected by MAS include tilapia (Abd-El-Rhman, 2009; Tellez-Ba?uelos et al., 2010), catfish (Majumdar et al., 2007; Ullal et al., 2008), goldfish (Irianto et al., 2003; Harikrishnan et al., 2009), common carp (Yin et al., 2009; Jeney et al., 2009), and eel (Esteve et al., 1994). Although usually considered as a secondary pathogen associated with disease outbreaks, A. hydrophila mayalso become a primary pathogen, causing outbreaks in fish farms with high mortality rates (Thore and Roberts, 1972; Nielsen et al., 2001; Fang et al., 2004). In West Alabama, a MAS disease outbreaks cause by A. hydrophila in 2009 alone led to an estimated loss of more than 3 million pounds of food size channel catfish (Pridgeon and Klesius, 2011a). Virulence studies have revealed that AL09-71 strain, a 2009 West Alabama isolate of A. hydrophila, is highly virulent to channel catfish, killing fish within 24 h post exposure (Pridgeon and Klesius, 2011b). 53 To control disease outbreaks caused by A. hydrophila, feeding infected fish with antibiotic-medicated feed is a general practice (DePaola et al., 1995). However, this practice is expensive and usually ineffective as sick fish tend to remain off feed. Furthermore, currently in the US, there are only three FDA approved antibiotics for use in aquaculture: oxytetracycline (Terramycin), sulfadimethoxine (Romet-30), and florfenicol (Aquaflor). Use of vaccine is an alternative control method to prevent MAS. The most extensively studied A. hydrophila vaccines are bacterins consisting of formalin or heat-killed bacteria of pathogenic A. hydrophila strains (Chandran et al., 2002; John et al., 2002). In addition, recombinant protein vaccines such as A. hydrophila outer membrane proteins and bacterial lysate have been demonstrated to elicit protection against A. hydrophila challenges (Khushiramani et al., 2007; Poobalane et al., 2010). Furthermore, live attenuated vaccines such as aroA mutant and transposon Tn916-generated mutant have been reported to confer significant protection against homologous A. hydrophila challenge (Hernanz Moral et al., 1998; Liu et al., 2007). However, it is well known that A. hydrophila is very heterogeneous biochemically and serologically, which is the obstacle in developing effective commercial vaccine against A. hydrophila (Khashe et al., 1996; Poobalane et al., 2010). To prevent disease outbreaks caused by the highly virulent West Alabama 2009 isolates of A. hydrophila, an attenuated vaccine AL09-71 N+R specifically targeting A. hydrophila AL09-71 strain was developed (Pridgeon and Klesius, 2011c). Several studies have demonstrated that protective immunity elicited by attenuated bacterial vaccines in channel catfish is largely mediated by cellular immune responses with humoral antibodies having a secondary role (Shoemaker and Klesius, 1997; Ellis, 1999). Similarly, it has been reported that the antibody titres of rainbow trout vaccinated with an attenuated A. hydrophila were not significantly different from that of control fish, although the 54 attenuated vaccine provided 64% protection against challenges by virulent A. hydrophila (Vivas, et al., 2004), suggesting that components of immunity other than antibody play an essential role in combating A. hydrophila. The objectives of this study are to: 1) identify up-regulated genes in channel catfish after vaccination with attenuated A. hydrophila; and 2) determine the transcriptional regulation of genes identified in response to vaccination or infection of A. hydrophila. We used two approaches in this study to identify up-regulated genes. Firstly, we used suppression subtractive cDNA hybridization (SSH) technique to identify up-regulated genes in the anterior kidney of vaccinated channel catfish without any preconception of their identities. Secondly, we screened channel catfish genes reported in literatures as responses to either attenuated bacterial vaccines or virulent bacterial infections to identify genes induced by the attenuated A. hydrophila. Materials and methods Bacteria source and growth conditions The AL09-71 isolate of A. hydrophila was obtained from diseased channel catfish in 2009 from West Alabama. The isolate has been confirmed to be A. hydrophila through biochemical and molecular identification (Pridgeon and Klesius, 2011a). The attenuated A. hydrophila AL09-71 N+R strain was obtained from the virulent parent strain of A. hydrohila AL09-71 through selection for resistance to both novobiocin and rifampicin (Pridgeon and Klesius, 2011c). Bacterial cultures were grown in tryptic soy broth (TSB) (Fisher Scientific, Pittsburgh, PA) for 24 h at 28 ?C. Experimental fish Channel catfish (4.6 ? 1.3 g) were obtained from stocks maintained at USDA-ARS, Aquatic Animal Health Research Laboratory (Auburn, AL, USA). All fish were maintained in 55 dechlorinated water in 340 L tanks. Prior to experiments, fish were acclimated in flow-through 57-L aquaria supplied with ~ 0.5 L h-1 dechlorinated water for 14 days. Experimental fish were confirmed to be culture negative for bacterial infection by culturing posterior kidney tissues from representative groups of fish on tryptic soy agar plates. A 12:12 hour light:dark period was maintained and supplemental aeration was supplied by air stones. Mean dissolved oxygen was ~5.6 mg L-1 at water temperature ~27? C, with pH ~ 7.1 and hardness ~ 100 mg L-1. Fish were fed ~3% body weight daily with commercial dry fish food. Sample collection from A. hydrophila vaccinated or infected fish Prior to vaccination or challenge, fish were moved to 57-L flow through aquaria and acclimated for 14 days. Vaccination dose of A. hydrophila AL09-71 N+R was 5?104 colony forming unit per fish (CFU/fish) based on published results (Pridgeon and Klesius, 2011c). Many studies have revealed that anterior kidney is an important immune organ involved in innate immunity against bacterial infections (Bao et al., 2005; Russo et al., 2009; Pridgeon et al., 2010a; Pridgeon and Klesius, 2010). Therefore, we decided to collect anterior kidney samples after vaccination with the attenuated A. hydrophila. At different time points (0 h, 3 h, 6 h, 12 h, 24 h, 48 h, and 14 days post vaccination,dpv), anterior kidney samples from five fish at each time point were collected and pooled together. Anterior kidney samples from five fish intraperitoneally (IP) injected with TSB at each time point were collected as control. The experiments were repeated three times. Using green fluorescent protein as a biomarker, the in vivo invasion pathway study of a virulent strain of A. hydrophila in Crucian carp (Carassius auratus gibelio) has revealed that the amount of bacteria in the kidney significantly increased at 12 h post challenge compared to that at 2 h post challenge (Chu and Lu, 2008). Therefore, we chose 12 h post vaccination as the time point to identify up-regulated genes induced by the 56 attenuated A. hydrophila. To understand whether infection by virulent A. hydrophila will have different effects on the transcription levels of genes at different time points (0 h, 3 h, 6 h, 12 h, 24 h, 48 h), the virulent parent strain of A. hydrophila AL09-71 was also IP injected to fish at a sublethal dose of 2?102 CFU/fish based on published virulence data of this strain (LD50 = 1.6?103 CFU/fish, (Pridgeon and Klesius, 2011b)). All anterior kidney tissues were flash frozen on dry ice during collection followed by storage at -80?C until RNA extraction. Total RNA extraction and cDNA synthesis Total RNA was isolated from anterior kidney tissues using TRIzol Reagent (Invitrogen, Carlsbad, CA) following the manufacturer?s protocol. All RNAs were treated with DNase provided by the DNA-free kit (Ambion, Austin, TX) and quantified on a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, Rockland, DE). The first strand cDNAs used for quantitative PCR were synthesized using 2 ?g of total RNA, AMV reverse transcriptase, and Oligo-dT primer provided by the cloned AMV first strand cDNA synthesis kit (Invitrogen, Carlsbad, CA). Construction of subtractive cDNA library For subtractive library construction, total RNAs were extracted from pooled anterior kidney samples of five fish either vaccinated with the attenuated A. hydrophila AL09-71 N+R or injected with the TSB control at the 12 h time point. cDNAs were then synthesized using PCR- select cDNA Subtraction Kit (Clontech, Palo Alto, CA). Two-step subtractive hybridizations were performed according to procedures described previously (Pridgeon et al., 2010a). Briefly, two primary hybridization reactions (A and B) were formed by adding excess amounts of unmodified TSB control cDNA (driver) to A. hydrophila AL09-71 infected cDNA (tester) samples at a 50:1 ratio. The samples were denatured for 2 min at 98 ?C and allowed to anneal for 57 8 h at 68 ?C. The remaining single-stranded, adaptor-ligated tester cDNAs were substantially enriched in each hybridization reaction for overexpressed sequences because non-target cDNAs present in the tester and driver could form hybrids. After filling in the adapter ends with DNA polymerase, over-expressed sequences (tester cDNA) had different annealing sites on their 3?- and 5?- ends. The molecules were then subjected to suppression subtraction PCR. The PCR products were then cloned into pGEM-T easy vector (Promega, Madison, WI). Plasmids were transformed into One Shot? TOP10 competent cells (Invitrogen, Carlsbad, CA). Transformed cells were plated on Luria-Bertani (LB) plates containing ampicillin (100 ?g/ml) and X-Gal (5- bromo-4-chloro-3-indolyl- beta-D-galactopyranoside) (40 ?g/ml). DNA Sequencing From the library, a total of 192 colonies were subsequently picked to grow overnight in Lysogeny broth (LB) in the presence of ampicillin (100 ?g/ml) at 37?C and 235 rpm in InnovaTM 4000 Incubator Shaker (New Brunswick Scientific, Edison, NJ). Overnight cultures were then sent to USDA-ARS Mid South Genomics Laboratory in Stoneville, MS for plasmid DNA extraction and DNA sequencing with an ABI 3730 Genetic Analyzer (Applied Biosystems, Foster City, CA). Raw sequence base calling and trimming was conducted at the Mid South Genomics Laboratory by using Phred with a cut-off score of Q20. Vector and adaptor sequences were then manually trimmed. Trimmed cDNA sequences were then analyzed using the National Center for Biotechnology Information (NCBI) BLAST program to search for sequence homologies. Primer design and quantitative PCR Sequencing results of different clones were used to design gene-specific primers by using the Primer3 program (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). Quantitative 58 PCR (QPCR) was performed using Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA). For each cDNA sample, channel catfish18S ribosomal RNA primers were included as an internal control to normalize the variation in cDNA amount as published previously (Pridgeon et al., 2010a). All QPCR was performed using Platinum? SYBR? Green qPCR SuperMix-UDG with ROX (Invitrogen, Carlsbad, CA) in a total volume of 12.5 ?l. The QPCR mixture consisted of 1?l of cDNA (input RNA of 10 ng), 0.5 ?l of 5 ?M gene-specific forward primer, 0.5 ?l of 5 ?M gene-specific reverse primer and 10.5 ?l of 1? SYBR Green SuperMix. The QPCR thermal cycling parameters were 50?C for 2 min, 95?C for 10 min followed by 40 cycle of 95?C for 15s and 60?C for 1min. All QPCR was run in duplicate for each cDNA sample and three pooled cDNA samples were analyzed by QPCR. The fluorescence intensities of the control and treatment products for each gene, as measured by cycle threshold (Ct) values, were compared and converted to fold differences by the relative quantification method (Pfaffl, 2001) using the Relative Expression Software Tool 384 v. 1 (REST) and assuming 100% efficiencies. Expression differences between control and treatment groups were assessed for statistical significance using a randomization test in the REST software. The mRNA expression levels of all samples were normalized to the levels of 18S ribosomal RNA gene in the same sample. Expression levels of 18S were constant between all samples (<0.30 change in Ct). Each primer set amplified a single product as indicated by a single peak present for each gene during melting curve analysis. Genes reported in literatures screened in this study Previously we reported that the transcriptional levels of 43 channel catfish genes were induced by vaccination with attenuated Edwardsiella ictaluri vaccine (Pridgeon et al., 2010a). Similarly, the transcriptional levels of 28 genes have been reported to be up-regulated by 59 attenuated Flavobacterium columnare vaccine (Pridgeon and Klesius, 2010). In addition, the transcriptional levels of five toll-like receptors (TLRs) have been reported to be up-regulated by acute infection with E. ictaluri (Pridgeon et al., 2010b). Furthermore, the expression levels of tumor necrosis factor ? (TNF-?) (Zhang and Wang, 1998), interleukin (IL)-1 ? (Zhang and Wang, 1998), IL-10 (Zhang and Wang, 1998), chemokine CXCL10 (Baoprasertkul et al., 2004), hepcidin (Hu et al., 2007), bactericidal permeability-increasing protein (Xu et al., 2005), liver- expressed antimicrobial peptide-2 (Bao et al., 2006), transferrin (Elibol-Flemming et al., 2009), and cathepsin D (Feng et al., 2011) in channel catfish have been reported to be up-regulated by bacterial infections. In addition, three channel catfish NK-lysin genes have been reported to be expressed in the anterior kidney of uninfected fish (Wang et al., 2006). Therefore, primers for these total 88 selected genes were purchased from Sigma-Aldrich (St. Louis, MO) and used in this study to determine which genes were up-regulated by the A. hydrophila AL09-71 N+R vaccination. Vaccination of channel catfish followed by challenge with AL09-71 The attenuated AL09-71 N+R vaccine was cultured in TSB broth at 28?C with shaking at 125 rpm overnight before vaccination. Channel catfish were vaccinated with A. hydrophila AL09-71 N+R at dose of 5?104 CFU/fish in a total volume of 100?l by intraperitoneal injection. As sham-vaccination controls, 100?l of TSB were injected into each fish. A total of 60 fish were used in each treatment group (20 fish per tank, three replicates). At 14 days post vaccination (dpv), fish were challenged with the parent isolate of A. hydrophila AL09-71 at dose of approximately 5?104 CFU/fish through IP injection. Mortalities were recorded for 14 days post challenge. Results of challenge were presented as relative percent of survival (RPS) according to 60 the following formula as described previously (Amend et al., 1981): RPS = (1-(vaccinated mortality ? control mortality)) ? 100. Data analysis The relative transcriptional levels of different genes were determined by subtracting the cycle threshold (Ct) of the sample by that of the 18S rRNA, the calibrator or internal control, as per the formula: ?Ct = Ct (sample) ? Ct (calibrator). The relative expression level of a specific gene in TSB injected control fish or in bacteria injected fish were compared to that of average control fish by the formula 2-??Ct where ??Ct = ?Ct (infected) ? ?Ct (control) as described previously (Pridgeon et al., 2010a). The relative expression data of a specific gene in control or infected fish were examined by unpaired t-test using SigmaStat statistical analysis software (Systat Software, San Jose, CA) and the differences were considered significant when the P value was less than 0.05. Differences in antibody titre and mortality were analyzed with Student t-test and the significance level was defined as P < 0.05. Results Characteristics of the subtractive cDNA library A total of 192 clones were obtained from the subtractive library. Of the 192 clones, 149 contained inserts. Sequencing results revealed that these 149 clones represented 22 unique expressed sequence tags (ESTs) (Table 1). All ESTs listed in Table 1 have been deposited in the GenBank dbEST under accession numbers JK088411 to JK088432. Of the 22 unique ESTs identified from the subtractive library, 14 shared homology with deposited channel catfish (Ictalurus punctatus) proteins, six shared homology with zebrafish (Danio rerio) proteins, and one shared homology with deposited rainbow smelt (Osmerus mordax) and Atlantic salmon (Salmo salar) protein, respectively (Table 1). The biggest insert size was 549 bp (2E08) and the 61 smallest insert size was 135 bp (2B06). The average insert size of the 22 ESTs was 275 bp (Table 1). Expression of the 22 ESTs and the 88 known genes at 12 hpv To determine whether the expression levels of the 22 ESTs isolated from the subtractive library were up-regulated in A. hydrophila AL09-71 N+R vaccinated catfish, gene-specific primers for the 22 ESTs were designed (Table 2) for relative QPCR experiments. QPCR results revealed that, at 12 hpv, 6 ESTs were significantly (P<0.05) induced in the vaccinated fish compared to that in unvaccinated control fish. QPCR results also revealed that 14 of the 88 known genes were significantly (P<0.05) induced by the vaccination, including 7 genes that were reported to be induced by vaccination with attenuated E. ictaluri (Pridgeon et al., 2010a). The identities of the total 20 significantly induced genes are listed in Table 3. Expression kinetics of the six genes identified by SSH in this study To determine the expression kinetics of the six genes that were significantly induced by A. hydrophila vaccination at 12 hpv, QPCR analysis were performed using samples collected at different time points. To understand whether vaccination mimics infection, anterior kidney cDNA samples from infected fish were also included in the QPCR analysis. The expression kinetics of the six genes identified by SSH is summarized in Figure 1. Both vaccination and infection of A. hydrophila significantly (P<0.05) induced the transcriptional level of ADP/ATP translocase 2 at 12 h post treatment (Fig 1.A). Similarly, both vaccination and infection significantly (P<0.05) induced the transcriptional level of lymphokine-activated killer T-cell originated protein kinase at 12 h post treatment (Fig 1.B). In addition, vaccination also significantly induced its expression at 24 hpv (Fig 1.B). Of the six genes identified by SSH, lysozyme c at 24 h post vaccination was induced the most (Fig 1.C). For the remaining three 62 genes (motile sperm domain-containing protein 2, transcriptional regulator ATRX, and cadherin EGF LAG seven-pass G-type receptor 1), both vaccination and infection of A. hydrophila only induced their transcriptional levels at 12 h post treatment (Fig. 1D to 1F). Expression kinetics of the seven reported genes induced by E. ictaluri vaccination To determine the expression kinetics of the seven reported genes that were significantly induced by A. hydrophila vaccination at 12 hpv, QPCR analysis were performed using samples collected at different time points. Anterior kidney cDNA samples from infected fish were also included in the QPCR analysis. The expression kinetics of the seven reported genes is summarized in Figure 2. Both vaccination and infection of A. hydrophila significantly (P<0.05) induced the transcriptional level of Ring finger 144B at 12 h post treatment (Fig 2.A). Similarly, both vaccination and infection significantly (P<0.05) induced the transcriptional level of metacaspase-like protine at 12 h post treatment (Fig 2.B). The transcriptional level of complement C4a was significantly (P<0.05) induced at 6 h post treatment of both infection and vaccination (Fig 3.C). Of the seven reported genes, lysosomal-associated transmembrane protein 5 was induced the most by the infection at 6 h post treatment, followed by the vaccination or infection at 12 h post treatment (Fig 2.D). The transcriptional level of SET translocation B was only significantly induced at 6 hpi and 12 hpv (Fig 2.E). However, both vaccination and infection significantly induced the transcriptional level of uroporphyrinogen decarboxylase at 6 h post treatment (Fig 2.F). Of the seven reported genes, solute carrier family 25 member 3 isoform 3 was induced by both vaccination and infection as early as 3 h post treatment, with peaked level at 6 h post treatment (Fig 2.G). Expression kinetics of the other seven reported genes 63 The expression kinetics of the other seven reported genes that were found to be significantly induced by A. hydrophila vaccination at 12 hpv is summarized in Figure 3. Infection of A. hydrophila significantly (P<0.05) induced the transcriptional level of IL-1 ? at both 6h and12 h post treatment, whereas vaccination only significantly induced IL-1 ? at 12 hpv (Fig 3.A). Similarly, infection of A. hydrophila significantly (P<0.05) and dramatically induced the transcriptional level of IL-10 at 6h post treatment, followed by a reduced but significant induction at 12 hpi (Fig 3.B). IL-10 was slightly but significantly induced by the vaccination at 6 hpv, with peaked up-regulation at 12 hpv (Fig 3.B). Chemokine CXCL10 was significantly induced by the infection at 6 h post treatment, followed by peaked induction at 12 hpi (Fig 3.C). However, chemokine CXCL10 was significantly induced by the vaccination at 12 h post treatment, followed by peaked induction at 24 hpv (Fig 3.C). Infection of A. hydrophila significantly and dramatically induced TLR5 at 6 hpi, followed by a reduced but significant induction at 12 hpi (Fig 3.D). However, vaccination only slightly but significantly induced TLR5 at 6 hpv, followed by peaked induction and 12 hpv (Fig 3.D). Similarly, infection of A. hydrophila significantly and dramatically induced hepcidin at 6 hpi, followed by a reduced but significant induction at 12 hpi (Fig 3.E). Vaccination only slightly but significantly induced hepcidin at 6 hpv, followed by a peaked induction at 12 hpv (Fig 3.E). Both vaccination and infection significantly induced the transcriptional level of NK lysine type-2 and sodium/potassium-transporting ATPase alpha subunit at 12 h and 3h post treatment, respectively (Fig 3.F, G). Vaccination of channel catfish followed by challenge with AL09-71 When AL09-71 N+R vaccinated channel fish were challenged by its virulent parent AL09-71 at 14 dpv, no fish died. However, 40 to 50% fish died in the TSB-sham vaccination 64 group (Fig 4.). The relative percent of survival of vaccinated fish at 14dpv was 100%. At 14dpv, when channel catfish were challenged by virulent AL09-71, cumulative mortalities of AL09-71 N+R vaccinated fish at different time points were significantly (P<0.05) lower than that of TSB sham-vaccinated fish (Fig 4.), indicating that the vaccine provided significant protection against its virulent parent. Expression of the 20 identified genes at 14dpv The relative expression levels of the 20 genes in infected fish compared to that in vaccinated fish at 14dpv are summarized in Figure 5. At 14 dpv, only two genes (#17: TLR5; and #18: hepcidin) were significantly (P<0.05) up-regulated in vaccinated fish, with higher induction level of TLR5 compared to hepcidin (Fig 5.). Discussion Using the SSH technique, 22 unique ESTs were identified from a total of 192 clones. Of the 22 ESTs, only six were confirmed to be significantly up-regulated by the vaccination. The reasons for few genes were found to be significantly up-regulated may be due to the following.. First, only a small portion of the subtraction library was sequenced due to budget constraints. Although our library generated 192 clones, the library was generated from 3 ?l PCR products from a total of 25 ?l PCR reaction, which might have contributed to the limited discovery of well known genes related to immune response. Second, we used a pool of fish instead of single fish which might have eliminated the differences between individual fish. Third, we used total RNA instead of messenger RNA, which might have contributed to our limited discovery by the SSH. Fourth, only one set of specific primers were used to do the QPCR confirmation, which may have contributed to the unsuccessful confirmation of the SSH results. 65 Of the six up-regulated genes confirmed by QPCR, two of which (ADP/ATP translocase 2 and lysozyme c) have been previously reported to be up-regulated in fish infected by Aeromonas. ADP/APT translocase 2 is one of the four genes identified (from 4131 known genes by microarray) to be up-regulated in Atlantic salmon (Salmo salar) in response to Aeromonas salmonicida infection (Tsoi et al., 2003). Recently, significant transcriptional up-regulation of lysozyme c has been reported in the kidney of ray-finned fish (Puntius sarana) following A. hydrophila infection (Das et al., 2011). Gene expression profiles at different early time points revealed that the pattern of gene up-regulation in vaccinated fish was similar to that in infected fish, confirming that vaccination of attenuated bacteria mimics live infection at molecular level. However, the extent of gene induction by the infection differed from that by the vaccination. For example, at 3 hpv, Na+/K+ ATPase ? subunit was up-regulated less than 50 fold. However, the infection by the virulent A. hydrophila AL09-71 strain resulted in more than 200 fold of up-regulation. Other genes at other time points also showed this trend. For example, at 6 hpv, IL-1? was induced less than 10 fold by the vaccination, whereas the infection resulted in more than 100 fold up-regulation. Similarly, vaccination induced hepcidin less than 50 fold at 6 hpv, whereas infection up-regulated hepcidin more than 200 fold, suggesting that their regulation may be associated with severity of the infection. It has been reported that the induction of human ?-defensin 3 (HBD-3) is associated with the severity of Staphylococcus aureus skin infection (Zanger et al., 2010). For example, immune genes such as IL-1?, TNF?1, serum amyloid A, and interferon-? in rainbow trout (Oncorhynchus mykiss) are all significantly up-regulated at 24 h post infection by Yersinia ruckeri (the causative agent of enteric red mouth disease) at lethal doses injected, but not at sublethal doses (Wiens and Vallejo, 2010). 66 Time course studies revealed that Na+/K+ ATPase ? subunit was highly and significantly up-regulated at 3 hpv or 3 hpi, indicating that it might play an early role in fish immune response to Aeromonas infection. Up-regulation of Na+/K+ ATPase has been reported as an early response in channel catfish fry at 10 min post vaccination with attenuated F. columnare (Pridgeon and Klesius, 2010). In addition, significant up-regulation of Na+/K+ ATPase ? subunit has been reported in Atlantic salmon (Salmo salar) as an early response to Aeromonas salmonicida infection (Tsoi et al., 2003). Time course studies also revealed that solute carrier family 25 member 3 isoform 3 was significantly up-regulated at 3, 6, and 12 hpv. Significant up-regulation of solute carrier family 25 members 3 isoform 3 has been reported in channel catfish at 48h post immersion vaccination of attenuated E. ictaluri (Pridgeon et al., 2010a). Taken together, these results suggest that Na+/K+ ATPase ? subunit and solute carrier family 25 member 3 isoform 3 may play important roles in the early immune response to Aeromonas infection. Toll-like receptors (TLRs) are evolutionarily conserved receptors that function in innate immunity through recognition of the conserved pathogen-associated molecular patterns (PAMPs) of an invading pathogen and eliciting inflammatory immune responses (Medzhitov and Janeway, 2010). The best characterized ligand that TLRs recognize include: (1) lipoproteins by TLR2; (2) dsRNA by TLR3; (3) lipopolysaccharide (LPS) by TLR4; and (4) bacterial flagellin by TLR5 (Baoprasertkul et al., 2007). In mammals, inflammation will result in a cytokine cascade whereby tumor necrosis factor ? (TNF?) is released, followed by IL-1? and IL-6. After the release of these cytokines, chemokines are released to serve as potent chemoattractants to induce migration of neutrophils and macrophages to the site of infection (Secombes et al., 2001). It has been reported that TLR5 in channel catfish is significantly up-regulated at 4 and 6 hpi after acute infection of E. ictaluri (Pridgeon et al., 2010a). Similarly, after infection of A. hydrophila, the 67 expression level of TLR5 increased at 3 hpi and significantly peaked at 6 hpi, suggesting that TLR5 plays an essential role in recognizing this flagellated bacteria. It has been demonstrated that TLR5 also functions as an endocytic receptor to enhance flagellin-specific adaptive immunity (Letran et al., 2011). Significant up-regulation of IL-1? has been reported in the kidney of ray-finned fish (Puntius sarana) at 1, 3, and 6 h post A. hydrophila infection (Das et al., 2011). In consistent with that report, our results also revealed that IL-1? was significantly up- regulated at 3 and 6 hpi, confirming that IL-1? plays an important role in the immune response to Aeromonas infection. In addition to IL-1?, IL-10 was also significantly up-regulated at 6 and 12 hpi. Since IL-10 was not significantly up-regulated at 3 hpi, whereas IL-1? was significantly up- regulated at 3 hpi, suggesting that IL-10 is the downstream cytokine of IL-1?. From 6 hpi to 12 hpi, the induced level of IL-10 was reduced from 40 fold to 11 fold, whereas the induced level of chemokine CXCL10 was increased from 3 fold to 5 fold, suggesting that CXCL10 is a downstream chemokine followed by the release of IL-10. Antimicrobial peptides (AMPs) are evolutionarily ancient defensive weapons against bacteria, fungi and viruses (Zasloff et al., 2002). In channel catfish, overexpression of hepcidin (an AMP) after E. ictaluri infection has been reported at 1 to 3 dpi (Bao et al., 2005). Similarly, significant up-regulation of hepcidin in channel catfish has been reported at 4, 24, and 48 hpi of E. icataluri (Hu et al., 2007). In consistent with previous reports, our time course studies also revealed that hepcidin was significantly up-regulated at 6, 12, 24, and 48 hpi of A. hydrophila. In addition to hepcidin, NK-lysin-type 2 antimicrobial peptide was also significantly up-regulated at 6 and 12 hpi, further confirming that AMPs play important roles in host defense against bacterial infections. In addition to AMPs, lysozyme is also an important parameter in the immune defense of both invertebrates and vertebrates. Lysozyme works by hydrolysing the glycoside bonds of 68 bacterial cell wall, therefore resulting in the lysis of bacteria (Magnad?ttir, 2006). In this study, lysozyme c and lysomal-associated transmembrane protein 5 were both found to be significantly up-regulated at 6, 12, and 24 hpv or hpi.. Transcriptional profile studies revealed that the transcriptional levels of TLR 5 and hepcidin at 14 dpv were significantly up-regulated in vaccinated fish. Efficacy studies revealed that the vaccination with the attenuated A. hydrohila provided 100% protection against challenges by the virulent parent at 14 dpv. In summary, a total of 22 ESTs were identified from channel catfish anterior kidney subtractive cDNA library at 12 h post vaccination with an attenuated A. hydrophila (AL09-71 N+R). Of the 22 ESTs, six were confirmed to be significantly (P<0.05) induced by the vaccination. Of the selected 88 channel catfish genes reported in literatures, 14 were found to be significantly (P<0.05) up- regulated by the vaccination. The transcriptional levels the total 20 genes induced by the vaccination were then compared to that by the virulent parent A. hydrophila (AL09-71) at different time points. At 3 hpv or hpi, Na+/K+ ATPase ? subunit was up-regulated the most. At 6 and 12 hpv or hpi, hepcidin and interleukin-1? were induced the most. At 24 hpv or hpi, hepcidin was up-regulated the most, followed by lysozyme c. At 48 hpi, lysozyme c and hepcidin were significantly induced. When vaccinated fish were challenged by AL09-71, relative percent of survival of vaccinated fish were 100% at 14 dpv. 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Res. 59, 717-721. 77 Table 1. List of the 22 genes isolated from the Aeromonas hydrophila AL09-71 N+R vaccinated vs non-vaccinated channel catfish anterior kidney subtractive cDNA library Clone Blast Search Homology (Putative Gene Name) Accession No. Organism Identities (%) E value Insert (bp) 2B02 60S acidic ribosomal protein P0 NP_001187030 Ictalurus punctatus 99% 7.00E-79 456 2B03 40S ribosomal protein S12 NP_001187076 Ictalurus punctatus 100% 9.00E-31 195 2B06 ADP/ATP translocase 2 NP_001188059 Ictalurus punctatus 96% 2.00E-18 135 2D08 40S ribosomal protein SA NP_001187066 Ictalurus punctatus 99% 1.00E-40 243 2E08 Lymphokine-activated killer T-cell-originated protein kinase NP_001187345 Ictalurus punctatus 100% 5.00E-88 549 2G12 Transcriptional regulator ATRX NP_956947 Danio rerio 73% 4.00E-06 198 2H10 Ribosomal protein L13a AAK95140 Ictalurus punctatus 99% 8.00E-48 285 3F04 Retrotransposable element Tf2 155 kDa protein type 1-like XP_003199901 Danio rerio 45% 1.00E-31 432 3H03 60S ribosomal protein L7a NP_001187036 Ictalurus punctatus 99% 2.00E-80 492 4A02 40S ribosomal protein S25 NP_001187221 Ictalurus punctatus 100% 3.00E-22 198 4A09 Cadherin EGF LAG seven-pass G-type receptor 1 XP_001920772 Danio rerio 91% 4.00E-32 231 4B04 Lysozyme c NP_001187718 Ictalurus punctatus 98% 5.00E-70 369 4C04 Sorbin and SH3 domain containing 2 isoform 1 BX537102 Danio rerio 88% 4.00E-21 144 4C06 Hemoglobin-beta NP_001187115 Ictalurus punctatus 100% 2.00E-24 165 4E12 Cytochrome c oxidase subunit III YP_004300503 Osmerus mordax 93% 2.00E-27 204 4G01 Ras-related protein Rab-35-like XP_001339327 Danio rerio 93% 7.00E-42 270 4G11 motile sperm domain-containing protein 2 NP_001007294 Danio rerio 61% 4.00E-21 297 4H09 60S ribosomal protein L10a NP_001187211 Ictalurus punctatus 98% 4.00E-26 183 5D09 Elongation factor 2 ACN58590 Salmo salar 96% 2.00E-70 408 5F08 40S ribosomal protein S16 NP_001187219 Ictalurus punctatus 100% 1.00E-19 144 5F09 Proteasome activator complex subunit 1 ADO29299 Ictalurus punctatus 100% 3.00E-33 213 6E03 60S ribosomal protein L19 ADO29213 Ictalurus punctatus 100% 5.00E-40 249 78 Table 2. Gene-specific primers used in qPCR Clone No. Forward Primer (5? to 3?) Reverse Primer (5? to 3?) 2B02 CTTCCAGGCTTTGGGTATCA TCACACCCTCCAGGAATCTC 2B03 CGAGCATCAAATCAACCTCA CTCCTCGATGACATCCTTGG 2B06 ACCATCGACTGCTGGAAGAA ATACAGGACCAGCACGAAGG 2D08 ATCAGATTCAGGCTGCCTTC AGGGGATGGCAATATCAACA 2E08 CTTCTGCACGGAGACATGAA GTGAGCCCATACGCAAAGAT 2G12 CAAACGGGGCAAAGTTAAAA GTTCAAGCCGTTTGTCGTCT 2H10 TCTGGAGAGGCTGAAGGTGT CCTGTGATGGCCTGGTACTT 3F04 AATGGGAACAGGAAGGGAAC CAAGAGCGTAATCTGCCACA 3H03 ATGGGTGTCCCATACTGCAT TGGGACCCATGATGTTACCT 4A02 TGGTGTCCGAGAGACTGAAG TTTCTCTGGTGCTCCCTCAT 4A09 CCGTGACGGTGTCTTCATC GCGGTTCAGGTAGATTTGCT 4B04 TCTGGCTAACTGGGTTTGCT TGCCCTGCTGTCTCACTATG 4C04 CCCTCTCTCATCCCTCTCCT CCAAACTCCAGCTCTGCAA 4C06 CAGCAACTTCACGCTTCTTG GGAACTTCTGCCAAGTCTCG 4E12 GTCATCATCGGCTCAACCTT CCTCATCAGTAAATAGAGACA 4G01 ATCCCGAGTCGTTTGTGAAC ACGCCATGAACATCTCTTCC 4G11 TCTCGAGCCTCCGAAATCTA GCCATCAGGACTGAAACCAT 4H09 GCTGCAGATCAGCTTGAAGA AGAATTTGGGACGTGGAGTG 5D09 CTTCTCTGGCTGTGTGTCCA GGTCCCAGTCTTCACCAAAA 5F08 CCTGCAGTCTGTCCAGGTCT CAGTCCATTCCCTCGCTTAC 5F09 GGATGGGAACAACTTTGGTG CGTGAACAAGCTGCCTGTAA 6E03 GCTGTGGCAAAAAGAAGGTC TCTCTTACCGATGCCCATGT 79 Table 3. List of the 20 genes identified to be significantly upregulated at 12h post vaccination in the anterior kidney of channel catfish Code No. Blast Search Homology (Putative Gene Name) Gene Source 1 ADP/ATP translocase 2 SSH library sequencing results of this study 2 Lymphokine-activated killer t-cell-originated protein kinase-like SSH library sequencing results of this study 3 Lysozyme c SSH library sequencing results of this study 4 Motile sperm domain-containing protein 2 SSH library sequencing results of this study 5 Transcriptional regulator ATRX SSH library sequencing results of this study 6 Cadherin EGF LAG seven-pass G-type receptor 1 SSH library sequencing results of this study 7 Ring finger 144B Pridgeon et al. 2010a 8 Metacaspase-like protein Pridgeon et al. 2010a 9 Complement C4a Pridgeon et al. 2010a 10 Lysosomal-associated transmembrane protein 5 Pridgeon et al. 2010a 11 SET translocation B Pridgeon et al. 2010a 12 Uroporphyrinogen decarboxylase Pridgeon et al. 2010a 13 Solute carrier family 25, member 3 isoform 3 Pridgeon et al. 2010a 14 Interleukin-1? Zhang and Wang 1998 15 Interleukin-10 Zhang and Wang 1998 16 Chemokine CXCL10 Baoprasertkul et al. 2004 17 Toll-like receptor 5 Pridgeon et al. 2010c 18 Hepcidin Bao et al. 2005 19 NK lysine type-2 Wang et al. 2006 20 Sodium/potassium-transporting ATPase, alpha subunit Pridgeon et al. 2010b 80 Figure legends Figure 1. Effect of Aeromonas hydrophila treatment on the transcriptional kinetics of the six genes identified by suppression subtractive hybridization. A: ADP/ATP translocase 2; B: Lymphokine-activated killer T-cell originated protein kinase; C: ADP/ATP translocase 2; C: Lysozyme c; D: Motile sperm domain-containing protein 2; E: Transcriptional regulator ATRX; F: Cadherin EGP LAG seven-pass G-type receptor 1. Data are presented as means ? S.D. from three replicates. Figure 2. Effect of Aeromonas hydrophila treatment on the transcriptional kinetics of the seven genes induced by attenuated Edwardsiella ictaluri. A: Ring finger 144B; B: Metacaspase-like protein; C: Complement C4a; D: Lysosomal-associated transmembrane protein 5; E: SET translocation B; F: Uroporphyrinogen decarboxylase; G: Solute carrier family 25 member 3 isoform 3. Data are presented as means ? S.D. from three replicates. Figure 3. Effect of Aeromonas hydrophila treatment on the transcriptional kinetics of the other seven genes selected from literature. A: Interleukin-1?; B: Interleukin-10; C: Chemokine CXCL10; D: Toll-like receptor 5; E: Hepcidin; F: NK lysine type 2; G: Sodium/potassium- transporting ATPase alpha subunit. Data are presented as means ? S.D. from three replicates. Figure 4. Cumulative mortality of channel catfish intraperitoneally vaccinated with or without the Aeromonas hydrophila AL09-71 N+R and challenged with their respective virulent parent isolates of A. hydrophila through intraperitoneal injection at 14 days post vaccination. Data are presented as mean ? S.D. from three trials. 81 Figure 5. Relative transcriptional levels of the 20 genes in the anterior kidney of channel catfish at 14 days post vaccination. Data are presented as means ? S.D. from three replicates. Differences were considered statistically significant between vaccinated or infected and control fish when P value < 0.05. Significant difference is marked by an asterisk. TSB: tryptic soy broth; N+R: A. hydrophila AL09-71 N+R; parent: A. hydrophila AL09-71. 82 Figure 1 83 Figure 2 84 Figure 3 85 86 IV. USING SSH TO IDENTIFY GENES OVEREXPRESSED IN CHANNEL CATFISH AFTER SECONDARY EXPOSURE TO AEROMONAS HYDROPHILA COMPARED TO PRIMARY EXPOSURE Introduction: Aeromonas hydrophila is the causative agent of motile aeromonad septicemia (MAS) in channel catfish (Ictalurus punctatus). In 2009 and 2010, A. hydrophila is responsible for estimated loss of more than 3 million pounds of food size channel catfish (Pridgeon and Klesius, 2011a; Hemstreet, 2010). Virulence studies have revealed that AL09-71, a 2009 West Alabama strain of A. hydrophila, is highly virulent to channel catfish, killing fish within 24 h post exposure (Pridgeon and Klesius, 2011b). In fish, the anterior kidney plays an important function in immune defense (Tort et al., 2003). The anterior kidney is responsible for phagocytosis (Danneving et al., 1994), antigen processing (Brattgjerd et al., 1996; Kaattari et al., 1985), formation of IgM and immune memory through melanomacrophagic centres (Herraez et al., 1986; Tsujii et al., 1990). It is also an important endocrine organ, homologous to mammalian adrenal glands, releasing corticosteroids and other hormones. Thus, the anterior kidney is an important organ that plays key regulatory functions for immune-endocrine interactions and neuroimmunoendocrine connections (Tort et al., 2003). The main white blood cells found in the anterior kidney are macrophages, which aggregate into structures called melanomacrophage centers, and lymphoid cells, which are found at all developmental stages and exist mostly as Ig+ cells (B cells) (Press et al., 1994). 87 The immune system in fish is divided into two parts: the innate (non-specific) and the adaptive (specific) immune systems. The innate immune system, which is characterized as being non-specific and therefore not depend on previous recognition of the surface structures of the pathogen, is of prime importance in the immune defense of fish (Tort et al., 2003). It is the first and basic defense line in fish, which is is generally subdivided into 3 parts: the epithelial/mucosal barrier, humoraland the cellular components. The innate immune systerm is composed of germ-line encoded, relatively non-specific recognition parameters, showing instant action but of short duration (Magnad?ttir, 2010). The adaptive immune system is composed of T- and B-lymphocyte, random and highly diverse receptors, which encoded by recombinant activation genes, and contribute to a more specific and efficient immune response against infections (Medzhitov, 2007; Alvarez-Pellitero, 2008). The innate and adaptive immune systems are connected in different ways and both form part of an integrated and efficient immune system (Fearon and Locksley, 1996; Medzhitov and Janeway, 1997; Dixon and Stet, 2001; Medzhitov, 2007; Alvarez-Pellitero, 2008; Magnad?ttir, 2010). The immune response mechanisms against A. hydrophila have been studied in several fish species (Sahu et al., 2007; Rodr?guez et al., 2008; Poobalane et al., 2010; Reyes et al., 2010; Harikrishnan et al., 2010; Mu et al., 2010; Pridgeon et al., 2011; Reyes-Becerril et al., 2011). The objectives of this study are to: 1) identify up-regulated genes in channel catfish after secondary infection compared with primary infection; and 2) determine the transcriptional profiles of genes identified in response to primary and secondary infections at different time points. Materials and methods Bacteria source and growth conditions 88 The AL09-71 strain of A. hydrophila was obtained from diseased channel catfish in 2009 from West Alabama and has been confirmed to be A. hydrophila through biochemical and molecular identification (Pridgeon and Klesius, 2011a). The isolate was grown in tryptic soy broth (TSB) (Fisher Scientific, Pittsburgh, PA) for 18 h at 28 ?C before challenge. Experimental fish Channel catfish (21.2?3.3 g) were obtained from stocks maintained at USDA-ARS, Aquatic Animal Health Research Laboratory (Auburn, AL, USA). All fish were maintained in dechlorinated water in 340 L tanks. Prior to experiments, fish were acclimated in flowthrough 57 L aquaria supplied with ~2 L h-1 dechlorinated water for 14 days. Experimental fish were confirmed to be not infected by bacteria by culturing posterior kidney tissues from representative groups of fish on tryptic soy agar plates. A 12:12 h light:dark period was maintained and supplemental aeration was supplied by air stones. Mean dissolved oxygen was ~5.6 mg L-1 at water temperature ~27 ?C, with pH ~7.1 and hardness ~100 mg L-1. Fish were fed ~3% body weight daily with commercial dry fish food. Sample collection from primary and secondary infected fish The infection dose of A. hydrophila AL09-71 was 1x105 colony forming unit per fish (CFU/fish) based on previous challenge experiment result (LD50=1x105 CFU/fish). For the primary challenge experiment, five hundred channel catfish were infected by intra-peritoneal (ip.) injection (1x106 CFU/fish in 100 ?l TSB). And, two hundred and fifty non-infected control fish were injected with 100?l sterile TSB. At different time points (0 h, 3 h, 6 h, 12 h, 24 h, 2 days, and 7 days post infection), anterior kidney samples from 15 infected fish at each time point were collected and each five were pooled together. Anterior kidney samples from 15 fish intraperitoneally (IP) injected with TSB at each time point were also collected and each five 89 were pooled together as control. In the primary challenge experiment the infected fish received an ip. injection and were observed for 28 days. In the secondary challenge experiment, the surviving fish from the primary challenge received an additional injection of (1x106 CFU/fish in 100 ?l TSB) bacteria 28 days after the primary infection. The non-infected control fish were also reinjected with sterile TBS at day 28 days post-primary injection. In secondary challenge experiment, anterior kidney samples were collected from infected fish and non-infected fish as the same as we did in primary challenge experiment at each time point. All anterior kidney tissues were flash frozen on dry ice during collection followed by storage at -80 ?C until RNA extraction. Total RNA extraction and cDNA synthesis Total RNA was extracted from anterior kidney tissues using TRIzol Reagent (Invitrogen, Carlsbad, CA) following the manufacturer?s protocol. All RNAs were quantified on a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, Rockland, DE) and analyzed on 1% agarose gel by electrophoresis to confirm the integrity. For each pooled sample, 2 mg of total RNA was used to synthesize the first strand cDNAs. The first strand cDNAs used for quantitative PCR were synthesized by using the cloned AMV first strand cDNA synthesis kit (Invitrogen, Carlsbad, CA) following the protocol. Construction of subtractive cDNA library Our previous experiment results have shown that some immune related genes up- regulated at 6h post-infection (Mu et al., 2011). Therefore, in this study, we chose 6 h post infection as the time point to identify up-regulated genes after secondary infection compared with primary infection. For subtractive library construction, total RNAs were extracted from pooled anterior kidney samples of five fish either after primary infection or secondary infection 90 with A. hydrophila AL09-71 at the 6 h post infection. cDNAs were then synthesized using PCR- select cDNA Subtraction Kit (Clontech, Palo Alto, CA). Two-step subtractive hybridizations were performed according to procedures described previously (Mu et al., 2011). cDNAs from secondary infection experiment were used as tester and cDNAs from primary infection experiment were used as driver. According to the manufacturer?s instructions, cDNAs from both tester and driver were digested by RsaI at 37 ?C overnight. The digested tester cDNAs were purified, subdivided equally and ligated with two different adaptors (adaptor 1 and adaptor 2R supplied by the kit) respectively. Two hybridizations were performed. In the first, an excess of driver was added to each adaptor-ligated tester sample followed by 98?C for 1.5 min, 63?C for 6 h. In the second hybridization, the denatured driver was added into two primary hybridization samples which were not denatured followed by 63?C overnight. Finally, the ratio of driver DNA:tester DNA sample was 50:1. After filling in the adapter ends with DNA polymerase, the entire population of molecules was then subjected to PCR to amplify the tester-specific sequences as described in the manual. The secondary PCR amplification product was cloned into pGEM-T Easy vector (Promega, Madison, WI, USA) following manufacturer?s instructions and transformed into one Shot? TOP10 chemically competent E. coli (Invitrogen, Carlsbad, CA) according to the manual. Transformed cells were then plated on Luria?Bertani (LB) plates containing ampicillin (100 ?g/ml) and X-Gal (5-bromo-4-chloro-3-indolyl-beta-D- galactopyranoside) (40 ?g/ml). Plasmid DNA isolation and sequencing From the library, a total of 96 colonies were subsequently picked to grow overnight in LB broth in the presence of ampicillin (100 ug/ml) at 37 ?C and 235 rpm in InnovaTM 4000 Incubator Shaker (New Brunswick Scientific, Edison, NJ). Overnight cultures were then sent to USDA- 91 ARS Mid South Genomic Laboratories in Stoneville, MS for plasmid DNA extraction and DNA sequencing with an ABI 3730 Genetic Analyzer (Applied Biosystems, Foster City, CA). Vector and adaptor sequences were then manually trimmed. Trimmed sequences were analyzed using the national Center for Biotechnology Information (NCBI) BLAST program to search for sequence homologies. Primer design and quantitative PCR Sequencing results of different clones were used to design gene-specific primers by using Primer3 program (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). Quantitative PCR (QPCR) was performed using Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA). For each cDNA sample, channel catfish 18S ribosomal RNA primers were included as an internal control to normalize the variation in cDNA amount as published previously (Pridgeon et al., 2010a). All QPCR was performed using Platinum? SYBR? Green qPCR SuperMix-UDG with ROX (Invitrogen, Carlsbad, CA) in a total volume of 12.5 ul. The QPCR mixture consisted of 1 ?l of cDNA (input RNA of 10 ng), 0.5 ?l of 5 ?M gene- specific forward primer, 0.5 ?l of 5 uM gene-specific reverse primer and 10.5 ?l of 1xSYBR Green SuperMix. The QPCR thermal cycling parameters were 50 ?C for 2 min, 95 ?C for 10 min followed by 40 cycles of 95 ?C for 15 s and 60 ?C for 1 min. All QPCR was run in duplicate for each cDNA sample and three pooled cDNA samples were analyzed by QPCR. The fluorescence intensities of the control and treatment products for each gene, as measured by cycle threshold (Ct) values, were compared and converted to fold differences by the relative quantification method (Pfaffl, 2001) using the Relative Expression Software Tool 384 v. 1 (REST) and assuming 100% efficiencies. Expression differences between control and treatment groups were assessed for statistical significance using a randomization test in the REST software. The mRNA 92 expression levels of all genes were normalized to the levels of 18S ribosomal RNA gene in the same sample. Expression levels of 18S were constant between all samples (<0.30 change in Ct). Each primer set amplified a single product as indicated by a single peak present for each gene during melting curve analysis. Data analysis The relative transcriptional levels of different genes were determined by subtracting the cycle threshold (Ct) of the sample by that of the 18S rRNA, the calibrator or internal control, as per the formula: ?Ct = Ct (sample) ? Ct (calibrator). The relative expression level of a specific gene in control fish or in infected fish were compared to that of average control fish by the formula 2-??Ct where ??Ct = ?Ct (infected) ? ?Ct (control) as described previously (Pridgeon et al., 2010a). The relative expression data of a specific gene in control or infected fish were examined by unpaired t-test using SigmaStat statistical analysis software (Systat Software, San Jose, CA) and the differences were considered significant when the P value was less than 0.05. Results Characteristics of the subtractive cDNA library A total of 96 clones were obtained from the subtractive library using the samples from secondary infected fish as tester and samples from primary infected fish as driver. All 96 clones were subjected to sequencing. Of the 96 clones, 94 contained inserts. The Blastx and Blastn results revealed that a total of 28 unique expressed sequence tags (ESTs) (30%) were obtained from 94 sequences (Table1). The insert sizes of the 28 unique ESTs ranged in size from 124 bp to 1054bp. The average insert size was 452 bp (Table 1). According to the Blastx and Blastn results, 26 (93%) of 28 unique ESTs were found had an E-value lower than 10-20(Table 1). 18 of 93 the 28 sequences shared high homologies with channel catfish. Six shared homology with zebrafish (Danio rerio), the other four ESTs shared with other four organisms. Expression of the 28 ESTs at 6 h post-infection For relative QPCR experiments, 28 pairs ESTs-specific primers for the 28 ESTs were designed in order to determine whether the expression levels of the 28 ESTs isolated from the subtractive library were up-regulated in secondary infected channel catfish comparing with that in primary infected fish (Table 2). QPCR results revealed that 8 ESTs were significantly (P<0.05) induced in secondary infected fish compared to that in primary infected fish at 6 hpi (Figure 1). In the 8 upregulated ESTs, two (Ictalurus punctatus strain Auburn XbaI element 5 and Ictalurus punctatus strain Stuttgart XbaI element 7) in secondary infection were up-regulated greater than 3-fold compared to primary infection (Figure 1). The other 6 were up-regulated around 2-fold including TLR20-1 and TLR20-2 genes, inward rectifier potassium channel 13, unknown gene on immunoglobulin heavy chain locus, prolactin, NADH dehydrogenase subunit 2, reverse transcriptase-like protein (Figure 1). Expression kinetics of the 8 genes at different time points To determine whether the expression levels of these 8 ESTs in secondary infection were also up-regulated at the other time points compared to that in primary infection, the relative QPCR analysis were also conducted on anterior kidney cDNA samples from other time points using uninfected fish (IP injected with TSB) as control. The expression kinetics of the 8 ESTs identified by SSH is summarized in Fig. 2. All ESTs have higher expression level in primary infection than that in secondary infection at 3 hpi, 12 hpi, 24 hpi, 2 dpi and 7dpi except 6 hpi. All ESTs? expression level were significantly (P<0.05) increased and rapidly reached the first and highest peak at 3hpi. Of 8 ESTs, transcriptional levels of the two XbaI elements were 94 significantly increased over 100 folds in both primary and secondary infections at 3 hpi compared to un-infected controls (Figure 2). Following the primary infection, these two ESTs were induced all most 300 folds at 3 hpi. After 3 hpi, expressions of all 8 ESTs in both primary and secondary infections dropped rapidly. The transcriptional levels of six ESTs in primary experiment reached another peak at 24 hpi, but another two genes (prolactin and NADH dehydrogenase subunit 2) reached the second peak earlier at 12 hpi. The second peaks were much lower than the first peak. With the advancement of time, the transcriptional levels of 8 ESTs in primary infection almostly dropped to the same level as uninfected controls at 2 dpi. But, at 7 dpi, the third significantly up-regulation was detected in 4 genes including: TLR20-1 and TLR20-2 genes, unknown gene on the immunoglobulin heavy chain, prolactin, NADH dehydrogenase subunit 2. For the transcripts of 8 ESTs in secondary infection, there was no peak after 3 hpi. Only slightly up-regulations following secondary infection after 3 hpi were detected at 24 hpi (inward rectifier potassium channel 1 and reverse transcriptase-like protein) and at 7 dpi (immunoglobulin heavy chain gene locus, prolactin and NADH dehydrogenase subunit 2). Discussion Using SSH technique, 28 unique ESTs were identified from a total of 96 clones, only 8 were confirmed to be significantly up-regulated following secondary infection compared with those following primary infection at 6 hpi. This result may suggest that these 8 ESTs might play a more important role in the adaptive immune response against A. hydrophila infection than the innate immune response. However, the expression kinetics results showed that, at the other time points (at 3 hpi, 12 hpi, 24 hpi, 2 dpi and 7dpi), these 8 ESTs have higher expression levels following primary infection compared with that following secondary infection. Our results also revealed that the transcriptional levels of all 8 ESTs had 3 peaks after primary infection, while 95 only one peak at 3 hpi was detected following secondary infection. Similarly, the low transcriptional levels of immune genes following the secondary infection were also found in rainbow trout (Oncorhynchus mykiss) infected with Yersinia ruckeri (Raida and Buchmann, 2008). EST A08 shew a higher homology (E-value 1.00E-143, identity 95%) with channel catfish TLR20-1 and TLR20-2 genes complete cds submitted by Quiniou. (2010). This EST was significantly induced up to 60-fold following primary infection and up to almost 30-fold following secondary infection at 3 hpi. Our results suggest it may play an important role in the innate and adaptive immune response against A. hydrophila infection. The dynamic expression levels of TLR20-1 and TLR20-2 genes were first reported. In addition, TLR20a, which was specific in teleost, were identified in zebrafish (Danio rerio) (Meijer et al., 2004) and channel catfish (Baoprasertkul et al., 2007). Our previous results showed that expression level of TLR20a was significantly induced by Edwardsiella ictaluri infection at 6 hpi in channel catfish and the transcriptional level was higher than those of TLR2, TLR3 or TLR21 (Pridgeon et al., 2010a). However, EST A08 did not show any homology with TLR20a in channel catfish. Of all 8 ESTs, EST A10 and B04 were up-regulated the most after primary infection (up to 300-fold) and secondary infection (up to 200-fold). Both of them shared higher identities with XbaI element. EST A10 shared 99% identity with channel catfish strain Auburn XbaI element 5, complete sequence. EST B04 shared 96% identity with channel catfish strain Stuttgart XbaI element 7. In channel catfish, the XbaI elements were firstly characterized as a kind of A/T-rich tandem repetitive DNA sequence with four copies of the ATTA repeat and eight copies of (A)3?6 GT/TG motifs (Liu et al., 1998). They may be useful for the identification of strains by polymerase chain reaction analysis based on their polymorphism (Liu et al., 1998). Though it is 96 unknown whether RNA transcripts of these tandem repetitive DNA sequence are produced merely by "accident" due to failure of transcription termination or whether their production serves some biological function during the developmental stage at which they are synthesized, substantial information has been gathered for repetitive sequences in eukaryotic DNA concerning their structures and their associations with protein encoding sequences both in DNA and RNA transcripts (Jelinek and Schmid, 1982). Some repetitive DNA sequence can be transcribed to nuclear RNA and form RNA-RNA duplexes which might regulate the production of messenger RNA of some genes (Davidson and Britten, 1979). Therefore, the higher transcription levels of these two XbaI elements suggest that they may play an important role in the immune response through regulate the production of messenger RNA of some immune genes. EST C11 was characterized to share 99% identity with protaclin (PRL) in channel catfish. As we know, PRL, a kind of peptide hormone, shares many properties with cytokines such as homologous receptor structure, similar signal transduction pathway, and immunomodulatory action (Kooijman et al., 1996; Yu-Lee, 2002). PRL is also known to enhance immune functions in fish as in mammals (Balm, 1997; Clark, 1997; Harris et al., 2000; Olavarr?a et al., 2010). In innate immune response, PRL can enhance the mitotic activity of leukocytes of the chum salmon (Oncorhynchus keta) (Sakai et al., 1996) and stimulate the phagocytic activity of fish leukocytes. These results indicate that there is a kind of cross-talk between the PRL and the TLR-induced pathway for activation of leukocytes (Balm, 1997; Clark, 1997; Harris et al., 2000). PRL is also known to be necessary to maintain circulating levels of IgM in the rainbow trout (O. mykiss) (Yada et al., 1999). Our results revealed that PRL was rapidly induced up to almost 60-fold after primary infection and over 20-fold after secondary infection at 3 hpi. The expression pattern is 97 similar to those of TLR20-1 and TLR20-2 genes and unknown gene on the immunoglobulin heavy chain locus. Our results suggest that PRL may has a positive relation with TLR on the activation of leukocytes and PRL may also play an important role in maintaining circulating levels of IgM in channel catfish. Moreover, to the best of our knowledge, PLR was first found to be expressed in the anterior kidney of channel catfish. EST C09 was identified to show lower e-value (6.00E-102) and share 89% with unknown gene on the immunoglobulin (Ig) heavy (H) chain gene locus of channel catfish. As reported, the catfish Ig heavy chain locus is a translocon-type locus with three Ig? genes linked to an Ig? gene or pseudogene, which is estimated to contain approximately 200 variable region genes representing 13 families as well as at least three diversity and 11 joining genes (Bengt?n et al., 2006). The unknown gene was induced rapidly to over 25 folds following primary infection and almost 10 folds following secondary infection at 3 hpi. However, the expression level dropped also more rapidly after primary infection compared that after secondary infection, which leaded to a relative higher expression level at 6 hpi after secondary infection. EST C12 was up-regulated greater than 10-fold following primary infection and 6-fold after secondary infection at 3 hpi respectively. EST C12 shared 94% identity with NADH dehydrogenase subunit 2 of channel catfish at protein level. The NADH dehydrogenase complex, also called NADH coenzyme Q oxidoreductase or Complex I, is an enzyme located in the inner mitochondrial membrane that catalyzes the transfer of electrons from NADH to coenzyme Q. Since energy production is more efficient with the NADH dehydrogenase pathway than with the succinate ubiquinone reductase pathway (the second complex of the electron chain which uses FADH2) (Marchand et al., 2006), the up-regulation of this gene might indicate an optimization of energy production in the immune response. In addition, NADH dehydrogenase subunit 3 and 98 NADH dehydrogenase 1 alpha subcomplex 4 were induced by modified live Edwardsiella ictaluri vaccination in the anterior kidney of channel catfish (Pridgeon et al., 2010b). Further more, in Atlantic salmon (Salmo salar), NADH dehydrogenase chain 1 were also found to be up- regulated in anterior kidney and liver of fish infected by A. salmonicida (Tsoi et al., 2004). Taken together, our results suggest that NADH dehydrogenase complex may play an important role in the immune response against infection of A. hydrophila through increasing the energy production, which may be used to support the immune activity and increasing respiration of host after infection. EST C04 was up-regulated greater than 50-fold in primary infection and 20-fold in secondary infection at 3 hpi respectively. EST C04 shared 74% with inward rectifier potassium channel (Kir) 13 of zebra fish at protein level (E-value=3.00E-104). Earlier studies have shown that macrophages express two types of K+ channels, inwardly rectifying K+ channels and voltage-gated K+ channels (Mackenzie et al., 2003; Vicente et al., 2003). These earlier research have also revealed that the potassium channels activity can affect the macrophage antimicrobial functions. It has been found that the activation of potassium channels is an early step in the transmembrane signal transduction in macrophage and the perturbation of potassium channels would significantly impair antimicrobial functions of macrophages in goldfish (Carassius auratus) (Stafford et al., 2002). A recent study has found that increase in the activity of macrophage K+ channels is necessary but not sufficient to induce macrophage to release IL-1? (Olavarr?a et al., 2010). Therefore, the significant up-regulation of Kir 13 may induce the expression level of IL-1?, which, in turn, activates the functions of macrophages. EST E05 was up-regulated greater than 50-fold after primary infection and 5-fold after secondary infection at 3 hpi respectively. EST E05 shared 45% with reverse transcriptase-like 99 protein of Takifugu rubripesat (E-value= 1.00E-21). This reverse transcriptase-like protein has also been found to be up-reglated in spleen of mandarin fish Siniperca chuatsi infected with infectious spleen and kidney necrosis virus at 4 dpi (He et al., 2006). However, the reverse transcriptase-like protein was found to be down-regulated after vaccination with an attenuated E. ictaluri at 2 dpi (Pridgeon et al., 2012). In conclusion, 8 ESTs were identified and confirmed to significantly (P < 0.05) induced by secondary infection compared to those induced by primary infection in anterior kidney at 6 h post challenge. All 8 ESTs were significantly induced at 3 hpi by both primary and secondary infections, suggesting their important roles in earlier immune response. For most of ESTs, other significant up-regulations were also detected out later at 12 hpi, 24 hpi or 7dpi after primary infection, which suggests that these ESTs might take part in the development of adaptive immunity or other functions. 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Elsevier, Amsterdam, pp. 111?122. 106 Table 1. List of genes isolated from the secondary infected versus primary infected catfish subtractive cDNA library. Clone no. Accessetion no. Homology gene name or protein name Organism E-value Identies Insert size A01 GU329038 Voucher INHS 47559 12S ribosomal RNA gene, partial sequence; mitochondrial Ictalurus punctatus 7.00E-52 100% 498 A03 GQ465835 Clone BS2 18S ribosomal RNA gene, partial sequence Ictalurus punctatus 0 100% 424 A04 XM_002663870 C-Jun-amino-terminal kinase-interacting protein 4-like (LOC100333761), mRNA Danio rerio 9.00E-25 75% 813 A05 JC1348 Hypothetical 18K protein - goldfish mitochondrion Carassius auratus 2.00E-29 54% 812 A07 AF482987 Ictalurus punctatus mitochondrion, complete genome Ictalurus punctatus 0 99% 389 A08 HQ677723 Ictalurus punctatus TLR20-1 and TLR20-2 genes, complete cds Ictalurus punctatus 1.00E-143 95% 400 A09 GU385707 voucher OP-10-CG-NBFGR-LKO 28S ribosomal RNA gene, partial sequence Ompok pabda 0 100% 549 A10 AF112197 Ictalurus punctatus strain Auburn XbaI element 5, complete sequence Ictalurus punctatus 2.00E-89 99% 192 B03 AY458870 12S ribosomal RNA gene, partial sequence Ictalurus punctatus 8.00E-123 100% 428 B04 AF112199 Ictalurus punctatus strain Stuttgart XbaI element 7, complete sequence Ictalurus punctatus 4.00E-83 96% 625 B06 AF112195 Ictalurus punctatus strain Kansas XbaI element 3, complete sequence Ictalurus punctatus 3.00E-40 96% 184 B10 NP_612135 NADH dehydrogenase subunit 5 Ictalurus punctatus 1.00E-131 97% 1054 B11 GQ465835 Ictalurus punctatus clone BS2 18S ribosomal RNA gene, partial sequence Ictalurus punctatus 5.00E-145 99% 293 C02 XR_117934 PREDICTED: Danio rerio hypothetical LOC100537045 , miscRNA Danio rerio 2.00E-65 99% 146 C04 NP_001039014 Inward rectifier potassium channel 13 Danio rerio 3.00E-104 74% 735 C09 DQ400445 Immunoglobulin heavy chain gene locus, partial sequence and unknown gene Ictalurus punctatus 6.00E-102 89% 308 C10 AF401559 Ribosomal protein L7 mRNA, partial cds Ictalurus punctatus 1.00E-110 100% 316 C11 AF267990 Prolactin gene, complete cds Ictalurus punctatus 8.00E-145 99% 296 C12 NP_612126 NADH dehydrogenase subunit 2 Ictalurus punctatus 1.00E-07 94% 157 107 Table 1. Continue Clone no. Accessetion no. Homology gene name or protein name Organism E-value Identies Insert size D01 NP_612128 Cytochrome c oxidase subunit II Ictalurus punctatus 7.00E-42 96% 543 D12 CU861890 DNA sequence from clone CH1073-21K6 in linkage group 20 Danio rerio 3.00E-18 81% 596 E05 AAD19348 Reverse transcriptase-like protein Takifugu rubripes 1.00E-21 45% 816 E06 NP_001038715 Rho GTPase-activating protein 10 Danio rerio 7.00E-42 88% 273 E07 NP_001187074 40S ribosomal protein S9 Ictalurus punctatus 5.00E-79 100% 373 F01 NM_001200275 Leukocyte DNA binding receptor (LOC100305038), mRNA, complete cds Ictalurus punctatus 7.00E-29 94% 438 F08 AY049812 Internal transcribed spacer 2 Callorhinchus milii 6.00E-93 96% 218 G08 NM_131310 Heat shock protein 90 Danio rerio 0 86% 624 H04 JN872359 Isolate IP-P-03 control region, complete sequence; mitochondrial Ictalurus punctatus 0 99% 481 108 Table 2. Gene-specific primers used in qPCR. No. Clone no. Forward primer Reverse primer 1 A01 CCCACCTAGAGGAGCCTGTT GGCGGGATAAACAAGAAGTG 2 A03 CGGCGTCCAACTTCTTAGAG ATCAACGCGAGCTTATGACC 3 A04 TGACAAGCCAACAGCAGACT TTAACCATGACCTGCCACAC 4 A05 CCTCGCCTGTTTACCAAAAA ACAGTTAAGCCCTCGTTCCA 5 A07 CCTCGCCTGTTTACCAAAAA ACAGTTAAGCCCTCGTTCCA 6 A08 CATCCAACCTGATGGAGCTT GCACCTTTGGCACCAATTAC 7 A09 TGGGTTTTAAGCAGGAGGTG ACGCTTGGTGAATTCTGCTT 8 A10 TCGCATTCATCCTGTGTCTC GAGTAGGTCGGGCAACTGAT 9 B03 AGGACACAACCTCACCAGGA TTGTAGTCTGCCGGAGCTTT 10 B04 TTGTGCTCTTTATCCGCTCA TTGGACTACGTTGTGCACTTG 11 B06 TGTTTCTGACCCAAAATATTACGA TTTTTCAACAACTTTTGCATTTT 12 B10 ACGTCTAGCCTGAGGAAGCA GGAAAAGTTGTGGGGAGTCA 13 B11 TTTGATCGCTCCACACGTTA CGGCTCGAGGTTATCAAGAG 14 C02 CCAGAGGAAACTCTGGTGGA TCGGAGGGAACCAGCTACTA 15 C04 ACCTTGTCCCCTCTTCCTGT GCTGCCATATTGGATCTCGT 16 C09 TCGAGTCAGTGCAAAAACCTT TGAGCATCTTGCAGAACCAG 17 C10 CGAAAGCCTCCTGAAGAAGA GTAGATCAGGTTCCGGGTGA 18 C11 AGAGCGAGCTCCTGTCTCTG GTCCTGCAGCTCTCTGGTCT 19 C12 ACCATCCTATTTGCCAGCAC GGGGCTAGTCCTACTTTTAATGC 20 D01 GTCCCAGGACGACTAAACCA CAAAGTGCTCTAGGGGAACG 21 D12 GGATCCTGAAAAAGGACTAGAGC TCTTCAGAACCTCCGTCTGC 22 E05 GCTACACGCTCCACTCAAAA TGGTTTGACGCCATTTACAA 23 E06 GCCATCATGGACCTCAAGTT GCGTGCCTGCTTCTTAGACT 24 E07 CGTCGTCCCTTTGAGAAGTC CTTCATCCAGCACACCAATG 25 F01 ATGCTCCCTGGGATAGGC TCCATCCATCTTCTACTACTTACTCC 26 F08 GCCGAAACGATCTCAACCTA GTAACCCGGCTTTCGGTTC 27 G08 TCATTCCCAACAAGCATGAA AGAAACCCACACCAAACTGC 28 H04 GAGGGTCACACAACTTGCAC TGTTTGTTGATTGCCTGCAT 109 Figure legends Figure 1. Effect of Aeromonas hydrophila treatment on the transcriptional levels of the 8 ESTs at 6 h post-infection including primary, secondary infections and TSB control. Data are presented as means ?S.D. from three replicates. Figure 2. Effect of Aeromonas hydrophila treatment on expression kinetics of the 8 genes at different time points post infection including primary, secondary infections and TSB control. Data are presented as means ?S.D. from three replicates. 110 111 Figure 2 112 V. TRANSCRIPTIONAL PROFILES OF MULTIPLE KNOWN GENES IN CHANNEL CATFISH AFTER SECONDARY EXPOSURE TO AEROMONAS HYDROPHILA COMPARED TO PRIMARY EXPOSURE Introduction Aeromonas hydrophila, a Gram-negative motile bacillus widely distributed in aquatic environments, is a causative agent of motile aeromonad septicemia (MAS) (Harikrishnan et al., 2003). Although usually considered as a secondary pathogen associated with disease outbreaks, A. hydrophila could also become a primary pathogen, causing outbreaks in fish farms with high mortality rates (Thore and Roberts, 1972; Nielsen et al., 2001; Fang et al., 2004). In West Alabama, MAS disease outbreaks caused by A. hydrophila in 2009 alone led to an estimated loss of more than 3 million pounds of food size channel catfish (Pridgeon and Klesius, 2011a). Virulence studies have revealed that AL09-71, a 2009 West Alabama strain of A. hydrophila, is highly virulent to channel catfish (Ictalurus punctatus), killing fish within 24h post exposure (Pridgeon and Klesius, 2011b). In 2010, Aeromonas disease outbreaks were common (Hemstreet, 2010). To prevent future disease outbreaks caused by the highly virulent West Alabama 2009 isolate, an attenuated vaccine specifically targeting A. hydrophila AL09-71 was developed through selection for resistance to both novobiocin and rifampicin (named AL09-71 N+R, Pridgeon and Klesius, 2011c). Recently, transcriptions of some genes encoding both innate and adaptive immune parameters in the anterior kidney following vaccination with AL09-71 N+R 113 and infection with AL09-71 have been described. Knowledge about the immune defence of channel catfish against A. hydrophila is important in terms of control and prevention. Previously we reported that the transcriptional levels of 43 channel catfish genes were induced by vaccination with attenuated Edwardsiella ictaluri vaccine (Pridgeon et al., 2010b). Similarly, the transcriptional levels of 28 genes have been reported to be up-regulated by attenuated Flavobacterium columnare vaccine in channel catfish (Pridgeon and Klesius, 2010). In addition, the transcriptional levels of five toll-like receptors (TLRs) have been reported to be induced by acute infection with E. ictaluri (Pridgeon et al., 2010a). The bacterial infections can aslo induce the expression levels of tumor necrosis factor (Zhang and Wang, 1998), interleukin-1? (Zhang et al., 1998; Mu et al., 2011), interleukin-10 (Zhang et al., 1998; Mu et al., 2011), chemokine CXCL10 (Baoprasertkul et al., 2004; Mu et al., 2011) in channel catfish according to the ealier reports. Furthermore, 8 antimicrobial peptides (AMPs) genes (NK-lysin type 1, NK-lysin type 2, NK-lysin type3, bactericidal permeability-increasing protein, cathepsin D, liver-expressed AMP 2, hepcidin and transferrin) were up-regulated in the anterior kidney of channel catfish in response to the infection of A. hydrophila or E. ictaluri (Liu et al., 2010; Mu et al., 2011; Pridgeon et al., 2012). Recently, Mu et al. (2011) have reported the expression kinetics of six channel catfish genes, which were identified by SSH, in response to the primary exposure to A. hydrophila vaccine. Although those genes are up-regulated by infection after primary exposure to either vaccine or infection, little is known whether they might be up-regulated by secondary exposure. Therefore, the objective of this study is to analyze the transcription levels of these genes in channel catfish after secondary exposure compared to primary exposure. In fish, the anterior kidney is the central organ, which plays the main function in immune defence. This organ works as the principal immune organ (Tort et al., 2003) responsible for 114 phagocytosis (Danneving et al., 1994), antigen processing (Brattgjerd et al., 1996; Kaattari et al., 1985), formation of IgM and immune memory through melanomacrophagic centres (Herraez et al., 1986; Tsujii et al., 1990). Due to the central role of the anterior kidney in immune response, our study focus on analyzing the expression kinetics of genes encoding toll-like receptors (TLRs), antimicrobial peptides (AMPs), cytokines, lysozyme C and other reported genes in the anterior kidney. At the same time, the present study also analyzed the activity of lysozyme in serum following primary and secondary exposures. In the present study, we analyzed the transcriptional profiles of 22 genes, selected from 94 reported immune related genes, at different time points and compared their transcriptional patterns after primary and secondary infections. Materials and methods Bacteria source and growth conditions The AL09-71 isolate of A. hydrophila was obtained from diseased channel catfish in 2009 from West Alabama and has been confirmed to be A. hydrophila through biochemical and molecular identification (Pridgeon and Klesius, 2011a). The isolate was grown in tryptic soy broth (TSB) (Fisher Scientific, Pittsburgh, PA) for 18 h at 28 ?C. Experimental fish Channel catfish (21.2 ? 3.3 g) were obtained from stocks maintained at USDA-ARS, Aquatic Animal Health Research Laboratory (Auburn, AL, USA). All fish were maintained in dechlorinated water in 340 L tanks. Prior to experiments, fish were acclimated in flow through 57 L aquaria supplied with ~2 L h-1 dechlorinated water for 14 days. Experimental fish were confirmed to be not infected by bacteria by culturing posterior kidney tissues from representative groups of fish on tryptic soy agar plates. A12:12 h light:dark period was maintained and 115 supplemental aeration was supplied by air stones. Mean dissolved oxygen was ~5.6 mg L-1 at water temperature ~27 ?C, with pH ~7.1 and hardness ~100 mg L-1. Fish were fed ~3% body weight daily with commercial dry fish food. Sample collection from primary and secondary infected fish The infection dose of A. hydrophila AL09-71 was 1x105 colony forming unit per fish (CFU/fish) based on previous challenge experiment result (LD50=1x105 CFU/fish). For the primary challenge experiment, five hundred channel catfish were infected by intraperitoneal (ip.) injection (1x106 CFU/fish in 100 ?l TSB). And, two hundred and fifty non-infected control fish were injected with 100 ?l sterile TSB. At different time points (0 h, 3 h, 6 h, 12 h, 24 h, 2 days, and 7 days post infection), anterior kidney samples from 15 infected fish at each time point were collected and each five were pooled together. Anterior kidney samples from 15 fish ip. injected with TSB at each time point were also collected and each five were pooled together as control. In the primary challenge experiment the infected fish received an ip. injection and were observed for 28 days. In the secondary challenge experiment the surviving fish from the primary challenge received an additional injection of (1x106 CFU/fish in 100 ?l TSB) bacteria 28 days after the primary infection. The non-infected control fish were also reinjected with sterile TBS at day 28 days post-primary injection. In secondary challenge experiment, anterior kidney samples were collected from infected fish and non-infected fish as the same as we did in primary challenge experiment at each time point. All anterior kidney tissues were flash frozen on dry ice during collection followed by storage at -80 ?C until RNA extraction. Total RNA extraction and cDNA synthesis Total RNA was extracted from anterior kidney tissues using TRIzol Reagent (Invitrogen, Carlsbad, CA) following the manufacturer?s protocol. All RNAs were quantified on a Nanodrop 116 ND-1000 spectrophotometer (Nanodrop Technologies, Rockland, DE) and analyzed on 1% agarose gel by electrophoresis to confirm the integrity. For each pooled sample, 2 mg of total RNA was used to synthesize the first strand cDNAs. The first strand cDNAs used for quantitative PCR were synthesized by using the cloned AMV first strand cDNA synthesis kit (Invitrogen, Carlsbad, CA) following the protocol. Genes reported in literatures screened in this study and primer design Primers of 94 reported genes were purchased from SigmaeAldrich (St. Louis, MO) and used in this study to determine which genes were up-regulated by secondary infection. Of 94 reported genes, 22 genes were determined to be up-regulated at 3hpi following secondary infection. For each cDNA sample, channel catfish 18S ribosomal RNA primers were included as an internal control to normalize the variation in cDNA amount as published previously (Pridgeon et al., 2010a). Primers used for amplification of 22 genes listed in Table 1. The transcriptional levels of five toll-like receptors (TLRs) genes (TLR2, TLR3, TLR5, TLR20a and TLR21) have been reported to be up-regulated by acute infection with E. ictaluri and A. hydrophila (Pridgeon et al., 2010a; Pridgeon et al., 2010b; Mu et al., 2011). The expression levels of 8 antimicrobial peptides (AMPs) genes (NK-lysin type 1, NK-lysin type 2, NK-lysin type3, bactericidal permeability-increasing protein, cathepsin D, liver-expressed AMP 2, hepcidin and transferrin) were studied in the anterior kidney of channel catfish in response to the infection of A. hydrophila and E. ictaluri (Liu et al., 2010; Mu et al., 2011; Pridgeon et al., 2012). Furthermore, the expression of four cytokine genes, interleukin-1 beta (Zhang et al., 1998; Mu et al., 2011), interleukin-10 (Zhang et al., 1998; Mu et al., 2011), chemokine CXCL10 (Baoprasertkul et al., 2004; Mu et al., 2011), tumor necrosis factor alpha, alpha-induced protein 2 (TNFAIP2) (Pridgeon et al., 2010b), and some other reported genes such as lysozyme C (Mu et al., 2011); 117 cadherin EGF LAG seven-pass G-type receptor 1 (CELSR1) (Mu et al., 2011), lymphokine- activated killer T-cell-originated protein kinase-like (TOPK) (Mu et al., 2011), protein-arginine deiminasae type II-like (PADII) (Pridgeon et al., 2010b), very large inducible GTPase 1 (VLIG) (Pridgeon et al., 2010b) were investigated in this study. Sequencing results of different clones were used to design gene-specific primers by using Primer3 program (http://frodo.wi.mit.edu/cgibin/primer3/-primer3_www.cgi). Primers for these total 22 selected genes were purchased from SigmaeAldrich (St. Louis, MO) and used in this study to determine which genes were up-regulated by the A. hydrophila AL09-71. Quantitative PCR All QPCR was performed using Platinum? SYBR? Green qPCR SuperMix-UDG with ROX (Invitrogen, Carlsbad, CA) in a total volume of 12.5 ?l. The QPCR mixture consisted of 1 ?l of cDNA (input RNA of 10 ng), 0.5 ?l of 5 ?M gene-specific forward primer, 0.5 ?l of 5 ?M gene-specific reverse primer and 10.5 ?l of 1xSYBR Green SuperMix. The QPCR thermal cycling parameters were 50 ?C for 2 min, 95 ?C for 10 min followed by 40 cycles of 95 ?C for 15 s and 60 ?C for 1 min. All QPCR was run in duplicate for each cDNA sample and three pooled cDNA samples were analyzed by QPCR. The fluorescence intensities of the control and treatment products for each gene, as measured by cycle threshold (Ct) values, were compared and converted to fold differences by the relative quantification method (Pfaffl, 2001) using the Relative Expression Software Tool 384 v. 1 (REST) and assuming 100% efficiencies. Expression differences between control and treatment groups were assessed for statistical significance using a randomization test in the REST software. The mRNA expression levels of all genes were normalized to the levels of 18S ribosomal RNA gene in the same sample. Expression levels of 118 18S were constant between all samples (<0.30 change in Ct). Each primer set amplified a single product as indicated by a single peak present for each gene during melting curve analysis. Lysozyme assay Plasma lysozyme activity was measured spectrophotometrically according to the method as described by Sankaran and Gurnani (1972). The lysozyme substrate was a 0.025% (w/v) suspension of Micrococcus lysodeikticus (Sigma) was prepared freshly with sodium phosphate buffer (0.04 M, pH 6.0). Serum (20 ?l/well in duplicate) from 5 fish/group was placed in a microtiter plate and 250 ?l of bacterial cell suspension was added to each well. Hen egg white lysozyme was used as an external standard. The initial and final (25 min incubation at 30?C) absorbances of the samples were measured at 450 nm. The rate of reduction in absorbance of samples was converted to lysozyme concentration (?g/ml) using a standard curve. The results were expressed as ?g/ml equivalent of hen egg white enzyme activity. Data analysis The relative transcriptional levels of different genes were determined by subtracting the cycle threshold (Ct) of the sample by that of the 18S rRNA, the calibrator or internal control, as per the formula: ?Ct = Ct (sample) ? Ct (calibrator). The relative expression level of a specific gene in control fish or in infected fish were compared to that of average control fish by the formula 2-??Ct where ??Ct = ?Ct (infected) ? ?Ct (control) as described previously (Pridgeon et al., 2010a). The relative expression data of a specific gene and lysozyme activity in control or infected fish were examined by unpaired t-test using SigmaStat statistical analysis software (Systat Software, San Jose, CA) and the differences were considered significant when the P value was less than 0.05. 119 Results Expression kinetics of five TLRs in the anterior kidney after infection Using quantitative PCR (QPCR), the relative transcriptional levels of five TLRs (TLR2, TLR3, TLR5, TLR20 and TLR21) were studied in the anterior kidney of channel catfish under TSB control, primary infection and secondary infection at different time points (3 hpi, 6 hpi, 12 hpi, 24 hpi, 2 dpi and 7 dpi). The results of time course studies revealed that all these 5 genes have a similar transcriptional pattern following primary and secondary infection (Fig. 1). All five TLRs were significantly (P < 0.05) up-regulated at 3 hpi, declined to the control level at 6 hpi and significantly (P < 0.05) induced again at 7 dpi following both primary and secondary infection (Fig. 1). At 12 hpi, most genes remained lower expression level like that at 6 hpi (Fig. 1B, C, D, E) except TLR2 which was significantly induced in primary infection (Fig. 1A). The expression levels of TLR2, TLR5 and TLR20 were stable from 12 hpi to 2 dpi (Fig. 1A, C, D). But, TLR3 and TLR21were rapidly induced from 12 hpi to 24 hpi (Fig. 1B, E) after primary infection. From 24 hpi to 2 dpi, the expression level of TLR21 remained stable (Fig. 1E), whereas the expression level of TLR3 dropt rapidly after primary infection (Fig. 1B). At 3 hpi and 24 hpi, the expression levels of TLR3, TLR20 and TLR21 in the primary experiment were significantly (P < 0.05) higher than those in the secondary experiment (Fig. 1B, D, E). And, at 7 dpi, the expressions of all 5 genes under primary infection were significantly (P < 0.05) induced much more highly than those under secondary infection (Fig. 1). However, at 6 hpi, the expression levels of TLR2, TLR3 and TLR5 under secondary infection were significantly (P < 0.05) higher than those under primary infection (Fig. 1A, B, C). Of the five TLRs genes, TLR5 was induced the most by the infection at 3 hpi, while induced slightly at 7 dpi as compared to the other 4 TLRs (Fig. 1C). 120 Expression kinetics of eight AMPs in the anterior kidney after infection The expression kinetics of the eight reported AMPs genes were summarized in Fig. 2. The transcriptional patterns of NK-lysin type 1 (NKL-1), NK-lysin type 2 (NKL-2), NK-lysin type 3 (NKL-3), Bactericidal permeability-increasing protein (BPI), hepcidin and transferrin were found to be similar under both primary and secondary infection (Fig. 2. A, B, C, E, G, H). Under primary infection, the transcriptional profiles of these genes showed 3 peaks at 3 hpi (except NKL-2, transferrin (first peak at 6 hpi)), 24 hpi (except BPI and transferrin) and 7 dpi respectively (the highest peak) respectively (Fig. 2. A, B, C, E, G, H). But, under secondary infection, only tow peaks were found in most genes except hepcidin, which still has three peaks like under primary infection, among the six genes with similar transcriptional pattern (Fig. 2.A, B, C, E, G, H). It was notable that the transcriptional levels of NKL-2, BPI and transferrin after secondary infection were significantly higher than that after primary infection at 6 hpi (Fig. 2.B, E, H). Of the eight AMPs, hepcidin was upreglated most up to 3800-fold and 1500-fold under primary and secondary infection respectively at 7 dpi (Fig. 2.G). As shown in Fig. 2.H, transferrin was significantly induced (around 40-fold) at 6 hpi, and, thereafter, was up-regulated again (around 500-fold) at 7 dpi by both primary and secondary infections. Interestingly, at these two time points, the expressional levels of tranferrin were significantly (P < 0.05) higher under secondary infection as compared to that under primary infection. All three types NKL genes were induced over 25-fold at 7 dpi (Fig. 2.A, B, C). The other two genes (cathepsin D and liver- expressed AMP 2) have lower expression levels compared with the other six genes described above (Fig. 2.D, F). Remarkably, the expression levels of NKL-1 and NKL-2 after secondary exposure were more significantly (P < 0.05) higher than those after primary exposure at 7 dpi (Fig. 2.A, B). 121 Expression kinetics of four cytokines in the anterior kidney after infection The expression kinetics of four cytokine genes encoding the interleukin-1 beta (IL-1?), interleukin-10 (IL-10), chemokine CXCL10 (IP10) and tumor necrosis factor alpha, alpha- induced protein 2 (TNFAIP2) were determined in the anterior kidney of channel catfish. Both primary and secondary infections significantly (P < 0.05) induced all four cytokine at 3 hpi. However, expression levels (around 20-fold) under primary infection were much higher than those under secondary infection (around 10-fold) (Fig. 3.). Another two expression peaks (around 20-fold) of IL-1? were detected at 24 hpi and 7 dpi respectively following primary infection (Fig. 3.A). While, the expression level of IL-10 touched the peak (60-fold) at 12 hpi following primary infection (Fig. 3.B). The transcriptional patterns of IP10 and TNFAIP2 were similar after primary infection with highest peak (around 40-fold) at 24 hpi (Fig. 3. C, D). The only difference happened at 7 dpi, where the expression of IP10 was up-regulated to more than 20-fold, while the expression of TNFAIP2 was stable from 2 dpi to 7 dpi under primary infection (Fig. 3. C, D). The expression patterns of all four cytokine genes were similar following secondary infection with significant up-regulations at early stage of infection (3 hpi and 6 hpi), but lower than those of primary infection (Fig. 3. A, B, C, D). Expression kinetics of five reported genes induced by A. hydrophila infection As shown in Fig. 4, the expression kinetics of five reported genes were summarized, lysozyme C; cadherin EGF LAG seven-pass G-type receptor 1 (CELSR1), lymphokine-activated killer T-cell-originated protein kinase-like (TOPK), protein-arginine deiminasae type II-like (PADII), very large inducible GTPase 1 (VLIG). The transcription level of lysozyme C peaked at 24 hpi (around 16-fold) under primary infection, while, in secondary infection, it peaked ealier at 12 hpi (around 5-fold) (Fig. 4. A). CELSR1 was significantly induced over 15-fold at 3 hpi, 24 122 hpi and 7 dpi respectively (Fig. 4. B). The expression of TOPK peaks at 12 hpi around 8-fold, and, thereafter, it declined to lower than control at 2 dpi and 7 dpi (Fig. 4. C). The expression levels of PADII and VLIG were rapidly up-regualted at 3 hpi by primary infection (over 15-fold) and secondary infection (over 5-fold) (Fig. 4. D, E). With the advancement of time, the expressions of these two genes after secondary exposure gradually declined to normal level. However, their expressions after primary exposure were rapidly induced again (over 10-fold) at 12 hpi (Fig. 4. D, E). Lysozyme activity Plasma lysozyme activity was increased 3.3-fold under primary infection and 3.0-fold under secondary infection at 12 hpi as compared to the activity level under control (Fig. 5). After 12 hpi, the lysozyme activity started decreasing. At 2 dpi, the lysozyme activity after secondary infection declined to lower than control. At 7 dpi, the lysozyme activity following primary infection was also detected to be lower than control level. It was notable that the lysozyme activity after primary exposure was significantly higher than that after secondary exposure at almost all time points except 12 hpi. Discussion TLRs are transmembrane proteins, which are essential in recognizing the highly conserved pathogen-associated molecular patterns (PAMPs) and activating immunostimulatory and immunomodulatory cell signalling pathways, which are essential for both innate immune and adaptive immune responses (Janeway and Medzhitov, 2002). TLRs are able to elicit the inflammatory and immune responses in vertebrate (Medzhitov and Janeway, 2000). The best characterized ligand that TLRs recognize include: (1) lipoteichoic acid and lipoproteins by TLR2; (2) dsRNA by TLR3; (3) lipopolysaccharide (LPS) by TLR4; (4) bacterial flagellin by 123 TLR5, (5) single stranded RNA (ssRNA) by TLR7, and (6) dsDNA by TLR9 (Baoprasertkul et al., 2007b; Iwasaki and Medzhitov, 2010). Additionally, TLRs were also reported to have multi- functions and act together in pathogen recognition and signaling (Ishii et al., 2005; Baoprasertkul et al., 2007a). For example, even though TLR2 is best known as a receptor recognising conserved components of Gram-positive bacteria such as lipoteichoic acid and lipoproteins, TLR2 is also able to interact with a wide range of additional ligand types such as zymosan, derived from yeast, glycosylphosphatidylinositols (GPIs) from protozoan parasites, LPS of Gram-negative bacterium which is generally considered to be the ligand of TLR4 (Baoprasertkul et al., 2007a). The previous studies have shown that TLR2 may function together with TLR4 (Hadley et al., 2005) or independently in this role (O?Connell et al., 2006). Furthermore, TLR3 is well known to also responsible for virus detection with dsRNA as its ligand. However, the successful stimulation of TLR3 with bacterial PAMPs has also been detected in zebrafish and channel catfish after infection with the Gram-negative E. tarda and E.ictulari, respectively (Bilodeau et al., 2005; Bilodeau and Waldbieser, 2005). Recently, Pridgeon et al. (2010a) reported that both TLR2 and TLR 3 in the anterior kidney were significantly induced by the infection of E. ictulari and touched peaks at 6 hpi, while, thereafter, the transcriptional levels of two genes declined. In addition, TLR2 was most significantly (P < 0.05) induced at 12 hpi in the kidney of Indian major carp, mrigal (Cirrhinus mrigala), infected by A. hydrophila, and, after that, the TLR2 expression level declined (Basu et al., 2012a). Similarly, in our study, TLR2 and TLR3 were also found to be up-regulated at 3hpi, suggesting that TLR2 and TLR3 play important roles in the host defense of channel catfish against infection of A. hydrophila at the earlier infection time course. 124 TLR5 in channel catfish was found to be significantly up-regulated at 4 and 6 hpi after acute infection of E. ictaluri (Pridgeon et al., 2010a). Recently, Mu et al. (2011) reported that, after infection of A. hydrophila, the expression level of TLR5 in the anterior kidney of channel catfish increased at 3 hpi and significantly peaked at 6 hpi. In addition, the expressions of TLR5 in the kidney were found to touch the peaks at 6 hpi (around 17-fold) and decline to around 3 fold at 24 hpi in Indian major carp (Cirrhinus mrigala) infected by A. hydrophila (Basu et al., 2012b). Similarly, the present study reveals that, of the five TLRs, TLR5 was up-regulated most highly (around 50-fold) at 3 hpi by both primary and secondary infections, which further confirms the essential role of TLR5 in recognizing flagellated bacteria in both innate and adaptive immunity. The channel catfish TLR20 and TLR21 were characterized by Baoprasertkul et al. (2007b), which belong to so-called ?fish-specific? TLR family and were not identified in mammalian (Rebl et al., 2010). Although they appear to branch with the murine TLR11, 12 and 13 in phylogenetic analyses, they form distinct branches (Baoprasertkul et al., 2007b; Palti, 2011). In this study, TLR20 and TLR21 in the anterior kidney were induced significantly at 3 hpi by both primary and secondary infections. In addition, TLR20 and TLR21 in the anterior kidney were significantly up-regulated at 6 hpi in channel catfish infected by Edwardsiella ictaluri (Pridgeon et al., 2010a). Therefore, TLR20 and TLR21 may play important roles in channel catfish immune against bacterial infection. However, no direct evidence of ligand specificity has been shown for TLR20 and TLR21 (Palti, 2011). The function of TLR20 and TLR21 are unknown in channel catfish. Future studies investigating the ligand specificity of the TLR20 and TLR21 genes will provide a better understanding of the function of these two genes. 125 Interestingly, a significant up-regulation was detected out at 7 dpi in all five TLRs after both primary and secondary infections, suggesting the TLRs may have some special functions at 7 dpi in both innate and adaptive immune repons. Especially, the primary infection induced the highest expressions of TLR2, TLR3, TLR20 and TLR21 at 7 dpi, which were much higher than those induced by secondary infection. Kawai and Akira (2011) reported that TLRs play important roles in initiation of adaptive immunity, inflammation and tissue repair through activating different downstream signaling pathways. Thereafter, we speculate the up-regulation of TLRs at 7 dpi may be related to the activation of adaptive immunity, inflammation or tissue repair. Fish secrete different kinds of antimicrobial peptides (AMPs), which are positively charged short amino-acid-chain molecules involved in host defense mechanisms. An increasing number of AMPs have been isolated from fish and with their abundance in many tissues, they may represent the most important innate defense in fish (Noga et al., 2011). In channel catfish, eight AMPs (NK-lysin type 1, NK-lysin type 2, NK-lysin type 3, bactericidal permeability- increasing protein (BPI), cathepsin D, hepcidin, liver-expressed AMP 2 (LEAP2), and transferrin) have been reported. Of the eight AMPs, hepcidin and transferrin were induced mostly by both primary and secondary infections. Recently, hepcidin has been found to act as an important iron regulator by binding to ferroportin (a key iron exporter on macrophages), thereby decreasing iron transfer into blood plasma from macrophages and inducing ferroportin-mediated endocytosis and proteolysis in mammalian (Ganz, 2011; Kroot et al., 2012; Zhu et al., 2012). Transferrin also has a critical role in iron metabolism, maintaining low levels of extracellular free iron and transporting iron to tissues as required, which also participates in a wide variety of metabolic processes, including immune regulation, antimicrobial and antioxidant activity, DNA 126 synthesis, cytoprotection, and electron transport (Stafford et al., 2003; Ong et al., 2006). The dramatical and significant up-regulations of hepcidin and transferrin were observed in our study, suggesting that both hepcidin and transferrin play very important roles in immune against A. hydrophila through decreasing the ion level in extracelluar environment. Recent studies show that the expression of hepcidin is regulated by a transferrin in the zebrafish embryo (Fraenkel et al., 2009) and transferrin is a major determinant of hepcidin expression in hypotransferrinemic mice (Bartnikas et al., 2011), suggesting that there is a kind of synergy between transferring and hepcidin. Our time course study reveals that they have a similar expression pattern following primary and secondary infections, indicating the existence of the cooperation of them in innate and adaptive immunity against A. hydrophila infection. Remarkably, the expression level of transferrin under secondary infection was significantly (P < 0.05) higher than that under primary infection at 6 hpi and 7 dpi (Fig. 2.H), which suggests that transferrin may play a more important role in adaptive immunity than that in innate immunity. In addition, the striking inductions of hepcidin were also detected at 6 hpi in A. hydrophila and E. ictaluri infected channel catfish (Mu et al., 2011; Pridgeon et al., 2012). Three distinct NK-lysin transcripts have been cloned in channel catfish and a higher expression level was detected at 7 dpi in the anterior kidney in channel catfish infected by E. ictaluri (Wang et al., 2006 a, b), which is in agreement with our result in this study (Fig. 2.A, B, C). We found that NK-lysin type 1 and 2 may function heavier at 7 dpi after secondary infection, since they have higher expression levels following secondary infection (Fig. 2.A, B). Whereas, the express pattern of NK-lysin3 is different in this study, suggesting a difference in function. BPI was identified and detected to be up-regulated at 7 dpi in the anterior kidney of E. ictaluri infected channel catfish (Xu et al., 2005). Our study also revealed a strong expressional induce of 127 BPI at 7 dpi following both primary and secondary infections (Fig. 2. E). In addition, cathepsin D and LEAP2 were also found to be up-regulated by primary and secondary infections, but not as highly as the other six AMPs. Furthermore, recent study shows that five AMPs (NKL-1, NKL-3, BPI, cathepsin D and hepcidin) were significantly up-regulated within 48 hpi in the anterior kidney of channel catfish infected by E. ictaluri (Pridgeon et al., 2012). The higher and similar temporal expression profiles were found in NKL-1, NKL-2, NKL-3, BPI, transferrin and hepcidin, which suggest these six AMPs might cooperate and play major roles in the immune defense against A. hydrophila. Furthermore, NKL-2, BPI and tranferrin may play more important roles in the adaptive immunity at 6 hpi, since the expression levels of these three AMPs following secondary infection were significantly higher than in primary infection at 6 hpi. Interleukin-1 ? (IL-1?) is an important early response pro-inflammatory cytokine that mediates immune regulation in both innate and adaptive immunity (Bird et al., 2002; Huising et al., 2004; Wang et al., 2006c). However, interleukin-10 (IL-10) is regarded as an anti- inflammatory cytokine and plays a crucial role in the regulation of inflammation in mammalian by downregulating expression of other cytokines such as IL-1?, primarily at the transcriptional level (Aste-Amezaga et al., 1998). Significant up-regulation of IL-1 ? has been reported in the kidney of ray-finned fish (P. sarana) at 1, 3, and 6 h (Das et al., 2011) and in the anterior kidney of channel catfish at 3 and 6h post A. hydrophila infection (Mu et al., 2011). In consistent with these reports, our results also revealed that IL-1 ? was significantly up-regulated over 20-fold at 3 hpi, 6 hpi, 24 hpi and 7 dpi under primary infection, further confirming that IL-1 ? plays an important role in the pro-inflammatory response to Aeromonas infection. Additionally, in this experiment, a clear association was observed between higher expression (around 60-fold) of IL- 10 and a corresponding lower expression (around 10-fold) of IL-1 ? at 12 hpi under primary 128 infection, indicating a suppressive role of IL-10 on the transcriptional level of the pro- inflammatory cytokine IL-10. This association suggests that IL-10 might have anti-inflammatory activity in channel catfish. Similar report can be found in Atlantic cod (Gadus morhua) (Seppola et al., 2008). On the other hand, the relative slightly up-regulation of IL-10 at 7 dpi and 3 hpi may reflect the processes of anterior kidney rebuilding and the limitation of potentially harmful inflammatory responses which start immediately after induction of the inflammatory reaction caused by IL-1 ?, respectively. Chemokine CXCL10 was firstly identified and reported to be up- reglated at 4 hpi and peaked at 24 hpi in the anterior kidney of E. ictaluri infected channel catfish (Baoprasertkul et al., 2004). Recently, a significantly up-regulation was detected at 3 hpi and peaked at 12 hpi in A. hydrophila infected catfish (Mu et al., 2011). Similarly, in this study, we found that Chemokine CXCL10 was pronounced up-regulated at 3 hpi and peaks at 24 hpi, which suggests CXCL10 may play an important role in recruiting monocytes, neutrophils, lymphocytes and other effector cells to the inflammatory sites to eliminate A. hdrophila in the early stages of infection. TNFAIP2 is also a TNFa-regulated gene that is first found expressed in human endothelial cells (Sarma et al., 1992). Although the role of TNFAIP2 is still unclear, it is an important gene involved in apoptosis (Liu et al., 2011). TNFAIP2 may also play an important role in activating effector cells to eliminate A. hdrophila since its expression pattern is similar with that of Chemokine CXCL10. Lysozyme C is an important molecule of innate immune system for the defense against bacterial infections. G-type and c-type lysozymes have been identified in fish and other vertebrates (Ye et al., 2010). The completed cDNA sequences of g-type and c-type lysozymes in channel catfish were reported by Chen et al. (2010). Recently, we reported that the expression of lysozyme C was significantly induced at 12 h post challenged with A. hydrophila in the anterior 129 kidney of channel catfish, and thereafter it touched the peak at 24 hpi (Mu et al., 2011). In this study, a same expression pattern of lysozyme C was shown in the primary infection (Fig. 4. A). The secondary response was significantly induced earlier at 3 hpi and reached a peak at 12 hpi (Fig. 4. A). The expression level of lysozyme C touched the peak later as compared to other up- regulated genes (at 3 hpi) in this study, which suggests it may be the down-stream gene of other genes. Lysozyme is effective when the outer cell wall of Gram-negative bacteria is disrupted due to the action of complement and other enzymes (Saurab et al., 2008). The results (Fig. 5.) of plasma lysozyme activity reflect the transcriptional profiles (Fig. 4.A) after both primary and secondary infections, suggesting that up-regulation of lysozyme genes might be responsible for the rise in plasma lysozyme activity of catfish following A. hydrophila infection. Furthermore, the lysozyme activity and expression level under primary infection are significantly higher than those under secondary infection except at 12 hpi. This result may indicate lysozyme is more important in innate immune defense against infection of A. hydrophila. Very large inducible GTPase 1 (VLIG) can be induced by interferons (IFNs) in humans and mice and play a critical role in preventing microbial infections, while its function is not well understood (MacMicking et al., 2004; Martens and Howard, 2006; Li et al., 2009). VLIG was firstly reported to be up-regulated at 48h post vaccination in the anterior kidney of channel catfish vaccinated with attenuated E. ictaluri (Pridgeon et al., 2010b). Our study showed that VLIG was also significantly induced at 3 hpi, 12 hpi and 7 dpi in the anterior kidney of A. hydrophila infected channel catfish (Fig. 4.B). For the remaining 3 genes, cadherin EGF LAG seven-pass G-type receptor 1 (CELSR1), and lymphokine-activated killer T-cell-originated protein kinase-like (TOPK), they were all significantly induced by the infection of A. hydrophila. Our results indicate they may all function in the immune response against A. hydrophila 130 infection in channel catfish. For all these 5 five genes, it is notable that the expressions under secondary exposure were much lower as compared to those under primary exposure except at 6 hpi, suggesting these genes may be more important in innate immunity than that in adaptive immuntiy. In conclusion, we determined the expression kinetics of 22 genes, selected from 94 reported immune related genes, at different time points and compared after primary and secondary infections. Of 22 genes, hepcidin and tranferrin were detected to be the most highly induced two genes. Though literatures indicate their function in immune response, further study might need to focus on their correlation and interaction based on their associated immune functions. We also found that some genes were up-regulated earlier at 3 hpi or 6 hpi compared to our previous study at 6 hpi or 12 hpi (Mu et al., 2011). This may be due to the higher challenge dose (1x106 CFU/fish) (Pridgeon et al., 2011d) and bigger fish (21.2?3.3 g) used in this study. Furthermore, even though the transcriptional levels of most of 22 genes following primary infection are higher than those following secondary infection except at 6 hpi, we found several genes that had higher transcriptional levels after secondary infection such as transferrin, NKL-1, and NKL-2 except at 6 hpi. The results suggest that these 3 genes may be more important in the adaptive immune response compared with that in innate immune response. Our results also revealed that a lot of genes have a higher expression level at 6 hpi after secondary exposure than that after primary exposure such as TLR2, TLR3, TLR5, NKL-2 and so on, which is caused by the slighter drop of expression level after 3 hpi under secondary infection. Most interestingly, it was noteworthy the lower expression levels of investigated genes in re-infected channel catfish. This corresponds to the transcriptional data of the immune response in rainbow trout reacting to Yersinia ruckeri where, following full recovery from the primary infection, secondary infection 131 did not elicit higher transcription levels (Raida and Buchmann, 2008). Raida and Buchmann (2008) reported that one explanation of the lower expression levels induced by secondary infection is that the pathogen is killed very fast in re-injected fish, whereby the associated expression of genes is kept at a minimum. 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Gene Name Accession no Forward primer Reverse Primer Gene Source 18s rRNA BE469353 ATGGCCGTTCTTAGTTGGTG TAGGTAGCACACGCTGATCG Pridgeon et al., 2010a TLR2 DQ372072 GCGTGGTTAAGAGCGAAAAG GGAAGGAAGTCTCGCTTGTG Baoprasertkul et al., 2007a; Pridgeon et al., 2010a TLR3 DQ423776 TTGCACCTGTGAGAGCATTC AGTGCACCAGGAAGGCTAGA Bilodeau & Waldbieser 2005; Pridgeon et al., 2010a; TLR5 GO898818 CAGAAACAGCTTTGCACCTG CTCATTCCCCTGTCCATCAC Baoprasertkul et al., 2007b; Mu et al., 2011 TLR20 DQ529275 CACCTCTCTGGGACTGGTGT GCTCATCTTTCCCGCAGTAG Baoprasertkul et al., 2007b; Pridgeon et al., 2010a; TLR21 DQ529276 TTCCTCTGCAGTGAGTGGTG TGTGTCCAGAACAGCTCCTG Baoprasertkul et al., 2007b; Pridgeon et al., 2010a; NKL-1 AY934592 GGGCCATGAAGAAAGTGAAA GCTTGGAACAATTCCAGCAT Wang et al., 2006; Pridgeon et al., 2012 NKL-2 DQ153186 TGTAAGTGGGCCATGAACAA TCCTCCACCAAGGTATCCAA Wang et al., 2006; Pridgeon et al., 2012 NKL-3 DQ153187 GGCTGTGACAAACTCCCAGT GGATCAATTCCCACATGTCC Wang et al., 2006; Pridgeon et al., 2012 BPI AY816351 TGTTGGCTTTGCTCTCCTTT TGCCTATGGGAGACACCTTC Xu et al., 2005; Pridgeon et al., 2012 Cathepsin D GU588646 CTGGGAGGGAAAGTGTTCAA GGTGTAGAAACGGCCCATAA Cho et al., 2002; Pridgeon et al., 2012 Hepcidin AY834209 TGCAGCTTTACCATCTGAGG AGGTGACTCTGACGCTTCGT Bao et al., 2005; Mu et al., 2011 LEAP2 AY845141 TTGGAAGCGCTACAAATCCT ACCCGGAGGTTGAATAATCC Bao et al., 2006; Pridgeon et al., 2012 Transferrin FJ176740 AAACAAATGTGACGCATGGA CAGATTGCACTTTCCAGCAA Liu et al., 2010 TNFAIP2 GO898786 TCATGTATGACCCAGCCTCA CTTGATGGGGTGCATAGACA Pridgeon et al., 2010b IL-1? DQ157743 CAGTCACCTCCAGCTGTTCA CAGAAAGTTTTCGGGAGCTG Wang et al., 2006; Mu et al., 2011 IL-10 FD020902 TGCTGACTGTTCTGCTGCTT AGGTGTCCAGGTCATCCTTG Liu, 2008; Mu et al., 2011 IP10 AY335951 GCCAGGACCAGTGTAAGGAG TTCAGATTCCGGATTCAAGC Baoprasertkul et al., 2004; Mu et al., 2011 Lysozyme C JK088422 TCTGGCTAACTGGGTTTGCT TGCCCTGCTGTCTCACTATG Mu et al., 2011 CELG JK088421 CCGTGACGGTGTCTTCATC GCGGTTCAGGTAGATTTGCT Mu et al., 2011 TOPK JK088415 CTTCTGCACGGAGACATGAA GTGAGCCCATACGCAAAGAT Mu et al., 2011 PADT GO898811 TGAGACGTGCTCTTTGCTTG TCTCACAGCTCAAGGTTCCA Pridgeon et al., 2010b IGTP GO898801 TCCATGAGCACAGTGGAGAG AAGGCTCATCTTGGGGTTTT Pridgeon et al., 2010b 142 Figure legends Figure 1. Transcription profiles of 5 TLRs in anterior kidney of channel catfish at different time points including primary, secondary infections and TSB control. Data are presented as means ?S.D. from three replicates. Figure 2. Transcription profiles of 8 AMPs in anterior kidney of channel catfish at different time points including primary, secondary infections and TSB control. Data are presented as means ?S.D. from three replicates. Figure 3. Transcription profiles of 4 cytokines in anterior kidney of channel catfish at different time points including primary, secondary infections and TSB control. Data are presented as means ?S.D. from three replicates. Figure 4. Transcription profiles of 5 reported genes in anterior kidney of channel catfish at different time points including primary, secondary infections and TSB control. Data are presented as means ?S.D. from three replicates. Figure 5. Plasma lysozyme activity in primary, secondary infected and TSB control fish. Data are presented as means ?S.D. from three replicates. 143 Figure 1 144 Figure 2 145 Figure 3 146 Figure 4 147 Figure 5 148 VI. OVERALL RESULTS AND FUTURE DIRECTIONS Overall results and discussion To characterize an attenuated Aeromonas hydrophila strain (AL09-71 N+R) compared with its parent strain (AL09-71), the growth rate, motility, chemotactic response, invasion ability and whole cellular fatty acid profile were investigated and compared between parent (AL09-71) and mutant (AL09-71 N+R) strain. Our results indicate that the attenuated vaccine strain A. hydrophila AL09-71 N+R has smaller colony size and slower growth rate compared to its parent AL09-71. In vitro motility assay revealed that AL09-71 N+R was immotile whereas AL09- 71was motile. The chemotactic response of AL09-71 N+R to channel catfish mucus was significantly lower than that of AL09-71. The ability of AL09-71 N+R to invade catfish gill cells was significantly lower than that of AL09-71. Furthermore, significantly different cellular fatty acid profiles were detected between the vaccine strain and its virulent parent strain. However, at genomic DNA level, the vaccine strain and its virulent parent strain appeared to be similar to each other. To understand the molecular mechanisms of protection elicited by the attenuated AL09- 71 N+R vaccine strain in catfish, suppression subtractive hybridization (SSH) was used to identify genes up-regulated by the vaccine. A total of 22 unique genes were identified at 12 h post vaccination. Of the 22, six were confirmed to be significantly induced by vaccination. In addition, 88 channel catfish genes that were reported to be associated with host immunity were included in the expression analysis. Of the 88 genes, 14 were found to be significantly up- 149 regulated by the vaccination at12 hour post infection. Expression profiles of the 20 genes at different time points showed that the pattern of gene up-regulation in vaccinated fish was similar to that in infected fish, confirming that vaccination of attenuated bacteria mimics infection by live bacteria at molecular level. However, the extent of gene induction by the infection differed from that by the vaccination. For example, at 6 hpv, IL-1? was induced less than 10 fold by the vaccination, whereas the infection resulted in more than 100 fold up-regulation. Similarly, vaccination induced hepcidin less than 50 fold at 6 hpv, whereas infection up-regulated hepcidin more than 200 fold, suggesting that their regulation might be associated with severity of the infection. Time course studies revealed that Na+/K+ ATPase subunit was highly and significantly up-regulated at 3 hpv or 3 hpi, indicating that it might play an early role in fish immune response to Aeromonas infection. After infection of A. hydrophila, the expression level of TLR5 increased at 3 hpi and significantly peaked at 6 hpi, suggesting that TLR5 plays an essential role in recognizing flagellated bacteria. In addition, our results also revealed that IL-1? was significantly up-regulated at 3 and 6 hpi, confirming that IL-1? plays an important role in the early immune response to Aeromonas infection. In addition to IL-1?, IL-10 was also significantly up-regulated at 6 and 12 hpi. Since IL-10 was not significantly up-regulated at 3 hpi, whereas IL-1? was significantly up-regulated at 3 hpi, suggesting that IL-10 is the downstream cytokine of IL-1?. From 6 hpi to 12 hpi, the induced level of IL-10 was reduced from 40 fold to 11 fold, whereas the induced level of chemokine CXCL10 was increased from 3 fold to 5 fold, suggesting that CXCL10 is a downstream chemokine followed by the release of IL-10. Moreover, our time course studies also revealed that hepcidin was significantly up- regulated at 6, 12, 24, and 48 hpi of A. hydrophila. In addition to hepcidin, NK-lysin-type 2 antimicrobial peptide was also significantly up-regulated at 6 and 12 hpi, further confirming that 150 AMPs play important roles in host defense against bacterial infections. Furthermore, in this study, lysozyme c and lysomal-associated transmembrane protein 5 were both found to be significantly up-regulated at 6, 12, and 24 hpv or hpi, further confirming the important roles they play in the immune response against bacterial infection. Taken together, our results suggest that vaccination with attenuated A. hydrophila mimics infection with live bacteria, inducing multiple immune genes in channel catfish. To understand whether channel catfish response to secondary infection is similar to primary infection, SSH was used to identify genes up-regulated by secondary infection. Of the 28 unique genes identified by the library, eight were confirmed to be significantly induced by secondary infection compared to that by primary infection at 6 hpi. All 8 ESTs were confirmed significantly up-regulated at 6 hpi following secondary infection compared those following primary infection, indicating that these 8 ESTs might play a more important role in the adaptive immune response against A. hydrophila infection. However, the expression kinetics results showed that, at the other time points (at 3 hpi, 12 hpi, 24 hpi, 2 dpi and 7dpi), these 8 ESTs have higher expression levels following primary infection compared with that following secondary infection. The results also showed that the transcriptional levels of all 8 ESTs had 3 peaks under primary exposure, while only one peak at 3 hpi was detected out under secondary expoure. EST A08 shows a higher homology (E-value 1.00E-143, identity 95%) with channel catfish TLR20-1 and TLR20-2 genes complete cds, which was significantly induced up to 60-fold following primary infection and up to almost 30-fold following secondary infection at 3 hpi. Of all ESTs identified, EST A10 and B04 were up-regulated the most (up to 300-fold) after primary infection and (up to 200-fold) after secondary infection. Both of them shared the higher identity with XbaI element. EST A10 and B04 shared 99% identity with channel catfish strain Auburn XbaI 151 element 5, complete sequence and 96% identity with channel catfish strain Stuttgart XbaI element 7 respectively. The higher transcription levels of these two XbaI elements indicate they may play an important role in the immune response against the infection through regulating the expression of immune related genes. EST C11 was characterized to share 99% identity with protaclin (PRL) in channel catfish. Our results on the expression kinetics of PRL after primary and secondary infection suggests that PRL may has a positive relation with TLR on the activation of leukocytes and may also play an important role in maintaining circulating levels of IgM in channel catfish. Moreover, to the best of our knowledge, the expression of PLR was first found in the anterior kidney of channel catfish. EST C12 was up-regulated greater than 10-fold under primary exposure and 6-fold under secondary exposure at 3 hpi respectively. EST C12 shares 94% identity with NADH dehydrogenase subunit 2 of channel catfish at protein level. Our results indicate that NADH dehydrogenase complex may play an important role in the immune response against infection of A. hydrophila through increasing the energy production, which can be used to support the immune activity. EST C04 which shares 74% with inward rectifier potassium channel (Kir) 13 of zebra fish was up-regulated greater than 50-fold in primary infection and 20-fold in secondary infection at 3 hpi respectively, suggesting that it might play an important role in immune response through increasing the IL-1? level and activating antimicrobial functions of macrophages. In addition to the eight genes identified by SSH, 94 genes known to be associated with host immunity were also subjected to expression analysis. Of the 94 genes, 22 were identified to be induced and differential regulated at different time points. Of the 22 genes, hepcidin and transferrin were induced most under both primary and secondary infections. Our time course study reveals that they have a similar expression pattern following primary and secondary 152 infections, indicating the existence of the cooperation of them in innate and adaptive immunity against A. hydrophila infection. Remarkably, the expression level of transferrin under secondary exposure was significantly (P < 0.05) higher than that under primary exposure at 6 hpi, which suggests that transferring may play a more important role in adaptive immunity than that in innate immunity. Our present study reveals that, of the five TLRs, TLR5 was up-regulated most highly (around 50-fold) at 3 hpi by primary and secondary infection, which further confirms the essential role of TLR5 in recognizing flagellated bacteria and suggests that TLR5 is not only necessary in the innate immunity but also essential in the adaptive immunity against A. hydrophila infection. Interestingly, a significantly up-regulation was detected at 7 dpi in all five TLRs under both primary and secondary infections, suggesting the TLRs has some special function at 7 dpi in innate and adaptive immune. All investigated eight AMPs may play important roles in the innate and adaptive immune defense against A. hydrophila infection. The higher and similar temporal expression profiles were found in NKL-1, NKL-2, NKL-3, BPI, transferrin and hepcidin, which suggest these six AMPs might cooperate and play major roles in the immune defense. our results also revealed that IL-1 ? was significantly up-regulated over 20- fold at 3 hpi, 6 hpi, 24 hpi and 7 dpi under primary infection, further confirming that IL-1 ? plays an important role in the pro-inflammatory response to Aeromonas infection. Additionally, in this experiment, a clear association was observed between higher expression (around 60-fold) of IL- 10 and a corresponding lower expression (around 10-fold) of IL-1 ? at 12 hpi under primary exposure, indicating a suppressive role for IL-10 on the transcriptional level of the pro- inflammatory cytokine IL-10. This association suggests that IL-10 might have anti-inflammatory activity in channel catfish. We also found that some genes were up-regulated earlier at 3 hpi or 6 hpi compared to our previous study at 6 hpi or 12 hpi (Mu et al., 2011). This may be due to the 153 use of higher challenge dose (1x106 CFU/fish) and bigger fish (21.2?3.3 g) in this study. Even though the transcriptional levels of most of 22 genes following primary infection are higher than those following secondary infection, we found several genes that had higher transcriptional levels after secondary infection such as transferrin, NKL-1, and NKL-2. The results suggest that these 3 genes may be more important in adaptive immune response as compared to that in innate immune response. Taken together, we believe that the study of the expression kinetics of immune related genes following secondary infection is helpful to identify more valuable immune response related genes. Future directions For characterizing the attenuated A. hydrophila strain, more characters need to be investigated in future study such as chemotaxis assay, adhesion abilities, invasion abilities, whole protein profile and total lipopolysaccharide. Next generation sequencing technology might be used to for further investigation on the genomic difference between parent and mutant strains. The mutant on the transcriptional level also needs to be determined by SSH or next generation sequencing technology. For the transcriptional analysis of multiple immune response genes, hepcidin and transferrin were found to be the most up-regulated two genes after infection. Though literatures indicate their function in immune response, further study might need to focus on their correlation and interaction based on their associated immune functions. 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