Transcriptome analysis and expression of immune related genes in megalobrama amblycephala after challenge with aeromonas hydrophila

分类号 密级 Huazhong Agricultural University Transcriptome Analysis and Expression of Immune-Related Genes in Megalobrama amblycephala after Challenge with Aeromonas hydrophila Name: Tran

HUAZHONG AGRICULTURAL UNIVERSITY

博 士 学 位 论 文

PhD DISSERTATION

TRANSCRIPTOME ANALYSIS AND EXPRESSION OF

IMMUNE-RELATED GENES IN MEGALOBRAMA AMBLYCEPHALA AFTER CHALLENGE WITH AEROMONAS HYDROPHILA

AS REVEALED BY MOLECULAR MARKERS

2015 年 6 月 JUNE，2015

分类号 密级

Huazhong Agricultural University

Transcriptome Analysis and Expression of Immune-Related Genes

in Megalobrama amblycephala after Challenge with Aeromonas

hydrophila

Name: Tran Ngoc Tuan

Advisor: Professor Wang Weimin

Degree: Doctor of Philosophy Date of receiving certificate: June 2015

College of Fisheries Huazhong Agricultural University

June 2015

华中农业大学学位论文独创性声明及使用授权书

摘 要 1

ABSTRACT 3

ABRBREVIATIONS 6

CHAPTER 1 Review of literature 7

1.1 General introduction 7

1.2 Introduction to blunt snout bream 9

1.2.1 Biological characteristics 9

1.2.2 Interests to research 10

1.3 Aeromonas hydrophila and its infection in fish 16

1.3.1 Classification and identification 16

1.3.2 Pathology 17

1.3.3 Clinical signs 17

1.3.4 Virulence factors 18

1.3.5 Infection of Aeromonas to blunt snout bream 18

1.4 Transcriptome study 19

CHAPTER 2 Pathogenicity of Aeromonas hydrophila to Megalobrama amblycephala 23

2.1 Introduction 23

2.2 Materials and Methods 23

2.2.1 Bacterial strain and bacterial suspension preparation 23

2.2.2 Fish for experiment 24

2.2.3 Pathogenicity test 24

2.2.4 Biochemical tests 25

2.2.5 Antibiotic susceptibility test 25

2.2.6 Molecular analysis 26

2.3 Results 26

2.3.1 Clinical signs 26

2.3.2 The cumulative mortality 27

2.3.3 Re-isolation and re-identification 27

2.3.4 16S rRNA sequences analysis 29

2.3.5 Antimicrobial susceptibility test 29

2.4 Discussion 30

CHAPTER 3 Transcriptome analysis of the Megalobrama amblycephala after challenge with Aeromonas hydrophila 34

3.1 Introduction 34

3.2 Materials and Methods 35

3.2.1 Fish and bacterial challenge 35

3.2.2 Sampling and RNA extraction 35

3.2.3 cDNA library preparation and Illumina sequencing 36

3.2.4 Sequence quality assessment and de novo assembly 37

3.2.5 Annotation 37

3.2.6 Differential gene expression analysis 37

3.2.7 Identification of immune-related genes 38

3.3 Results 38

3.3.1 Transcriptome sequencing and assembly 38

3.3.2 BLAST search analysis 40

3.3.3 Unigene functional annotation 41

3.3.4 Analysis of differentially expressed gene profiles 45

3.3.5 Identification of differentially expressed innate immune-related genes 46

3.3.6 Pathway analysis 47

3.4 Discussion 50

CHAPTER 4 Simple sequence repeat and single nucleotide polymorphism markers identified from Megalobrama amblycephala transcriptome profile 54

4.1 Introduction 54

4.2 Materials and Methods 55

4.2.1 SSR markers 55

4.2.2 SNP markers 55

4.3 Results 56

4.3.1 SSR markers 56

4.3.2 SNP markers 57

4.4 Discussion 63

CHAPTER 5 In silico characterization and homology modeling of proteins encoded by immune-related genes in Megalobrama amblycephala 65

5.1 Introduction 65

5.2 Materials and Methods 66

5.2.1 Sequence identification 66

5.2.2 Bioinformatics analysis 66

5.2.3 Protein structure prediction 67

5.3 Results 67

5.3.1 Sequence analysis 67

5.3.2 Physicochemical and functional characterization analysis 106

5.3.2 Protein structure prediction 110

5.4 Discussion 118

CHAPTER 6 Expression of immune-related genes in the Megalobrama amblycephala after Aeromonas hydrophila infection 124

6.1 Introduction 124

6.2 Materials and Methods 126

6.2.1Transcripts source 126

6.2.2 Fish and challenge experiment 126

6.2.3 Total RNA preparation and cDNA synthesis 126

6.2.4 Quantitative real-time PCR (qPCR) and statistics 127

6.3 Results 127

6.3.1 MaTLR5 128

6.3.2 MaNFKBIA 128

6.3.3 MaMyD88 129

6.3.4 MaTRAF6 129

6.3.5 MaC3 129

6.3.6 MaC7 130

6.3.7 MaMMP-9 130

6.3.8 MaCTSL 130

6.3.9 MaIL-8 130

6.3.10 MaIL-10 131

6.4 Discussion 133

6.4.1 Genes related to TLR pathway 134

6.4.2 Genes related to complement and coagulation cascades 135

6.4.3 Genes related to antigen processing and presentation and leukocyte transendothelial migration 136

6.4.4 Genes related to cytokine-cytokine receptor interaction 138

CHAPTER 7 Conclusion 140

REFERENCES 142

APPENDIX 1 Transcriptome profile database of Megalobrama amblycephala by using Solexa/Illumina technology 172

APPENDIX 2 Publications 173

APPENDIX 3 Awards and Funding 175

ACKNOWLEDMENTS 176

摘 要

团头鲂（Megalobrama amblycephala）是中国重要的淡水养殖草食性经济鱼类。

嗜水气单胞菌在团头鲂高密度养殖中的危害较为严重，可造成严重的经济损失。迄今为止，有关团头鲂感染嗜水气单胞菌的分子调控机理还未见报道，本研究通过注射嗜水气单胞菌对团头鲂进行攻毒，采用高通量测序技术对攻毒前后 6 个组织进行了转录组分析及免疫基因的鉴定，分析了这些基因的调控与表达，探讨了团头鲂对外源性病原体侵入的抵抗力和敏感性。主要内容如下：

（2）利用 RNA-seq 技术，对团头鲂嗜水气单胞菌感染前后的 6 个组织的混合样

本构建两个 cDNA 文库，然后进行 Illumina HiSeq2000 测序。通过 de novo 序列组装，

得到 155，052 个 unigene（平均长度为 692.8 bp），并对其进行了 KOG，KEGG 和

GO 注释。86% 的 unigene 与 NR 蛋白数据库比对上。30,482 个 unigene 注释到 GO分类上，25242 个 unigene 注释到“生物过程”，26096 个注释到“分子功能”，22778个注释到 “细胞组分”等三大生物类。在 KOG 和 KEGG 分析中，20909 个 unigene

分为 25 KOG 类别，28744 个 unigene 被注释到 315 个特定信号通路。238 个 unigene

（映射为 125 个基因）在两个文库中有显著差异表达，其中 101 个上调和 24 个下调。另外，303 个 unigene 识别为未知或新基因。在 125 个差异表达的基因中 53 个为免疫相关基因，其主要分布在 71 个信号通路中。

（3）从团头鲂转录组序列中获得了 10,877 个 SSR 分子标记，包括了二到五核苷酸重复序列，其中二核苷酸重复序列是最常见（7152，65.8％），其次是三核苷酸

（3172，29.2％），四核苷酸（549，5.05％）和五核苷酸重复序列（4，0.04％）。微卫星序列的长度在 12-25 bp 之间。可用于设计微卫星引物的序列为 5,077 个(46.7%)。

（4）利用计算机模拟分析方法对相应的免疫相关蛋白进行表征与同源建模分析，

包括 MaTLR5，MaNFKBIA，MaMyD88，MaTRAF6，MaC3，MaC7，MaCTSL， MaMMP-9，MaIL-8 和 MaIL-10 等，对其理化和功能特性进行了预测和分析，并应

关键词：团头鲂，转录组，免疫相关基因，分子标记，基因表达

ABSTRACT

Blunt snout bream (BSB), Megalobrama amblycephala, is a herbivorous freshwater

fish native to China and a major aquaculture species in Chinese freshwater polyculture

systems Recently, the bacterium Aeromonas hydrophila has been reported to be its

pathogen causing hemorrhagic septicemia clinical signs and great great ecomomic losses

of farmed BSB This study used next generation sequencing technology to better comprehensive the transcriptome profiles of response-related genes in the BSB which will facilitate further research into the resistance and susceptibility of this fish species to exogenously invasive pathogens The data analysis of transcriptome profile and

expression of immune-related genes from BSB upon A hydrophila stimulation was also

described The main results are as follows:

1 As an initial step, the pathogenicity of A hydrophila to BSB was carried out to confirm whether A hydrophila is a main pathogen causing hemorrhagic septicemia in BSB

The fish was challenged with bacterial concentrations of 1.7×105, 1.7×106, 1.7×107 and 1.7×108 cfu/mL for 7 days post infection The results showed that mortality was bacterial-dose dependent, with 100% mortality observed at day 1 at the highest dose (1.7×108 cfu/mL) Control fish exhibited cumulative mortalities of 0% The median lethal dose (LD50) was 5×106 cfu/mL A total of 15 Aeromonas strains were re-isolated from

challenged fish and re-identified based on morphological characteristics, biochemical tests and genomic DNA gene sequencing No bacteria were isolated from the control group This

study results indicated that A hydrophila is capable of causing hemorrhagic septicemia in

BSB Moreover, antibiotic susceptibility test with two strains D4 and HU201301 was investigated; the results showed that both strains were sensitive to most of the tested antibiotic drugs

2 To understand the immune response of the BSB to A hydrophila infection, the

RNA-Seq technology was utilized to analyze the transcriptomic profile after artificial bacterial infection Two cDNA libraries synthesized from tissues collected from control

BSB or those injected with A hydrophila were sequenced by using Illumina HiSeq2000 After de novo assembly, 155,052 unigenes (average length 692.8 bp) were isolated All

unigenes were annotated using BLASTX relative to several public databases The sequence similarity (86%) of the assembled unigenes was to zebrafish based on the Nr database A

number of unigenes (n = 30,482) were assigned to three GO categories: biological processes (25,242 unigenes), molecular functions (26,096 unigenes), and cellular components (22,778 unigenes) 20,909 unigenes were classified into 25 KOG categories and 28,744 unigenes were assigned to 315 specific signaling pathways In total, 238 significantly differentially expressed unigenes (mapped to 125 genes) were identified: 101 upregulated genes and 24 downregulated genes Another 303 unigenes were mapped to unknown or novel genes Among the known expressed genes identified, 53 were immune-related genes and were distributed in 71 signaling pathways

3 Microsatellites (n = 10,877), including di- to pentanucleotide repeat motifs, were also identified in the BSB transcriptome profiles Dinucleotide repeats were most common (7,152, 65.8%), followed by tri- (3,172, 29.2%), tetra- (549, 5.05%), and pentanucleotide repeat motifs (4, 0.04%) The lengths of the SSRs ranged from 12 to 25 bp PCR primers were successfully designed for 5077 (46.7%) of the identified microsatellites Furthermore, a total of 36,326 putative SNPs were discovered from the transcripts The frequency of SNP was one SNP in 29.2 bp sequence length The transition and transversion mutation was 21,445 and 12,553 SNPs, respectively A ratio of transition to transversion was 1.71 Within those yielded SNPs, 10,812 SNPs identified from 2,421 unigenes could

be annotated to differential functionality in comparing to public database using BLASTX for GO, KOG and KEGG A number of SNPs (n = 7,727, 71.5%) found from 1,628 unigenes were assigned to three main GO categories: ‘cellular components’, ‘molecular function’ and ‘biology process’ In total, 5,812 (53.8%) SNPs identified from 1,324 unigenes were classified into 25 KOG categories 4,589 (42.4%) SNPs detected from 975 unigenes were assigned to 278 KEGG pathways Furthermore, a number of 600 SNPs

found from 111 unigenes were successfully annotated to the term ‘immune system’ via

KEGG classification The database could be useful for further genetic studies in BSB

4 In silico characterization and homology modeling of encoded proteins, including MaTLR5, MaNFKBIA, MaMyD88, MaTRAF6, MaC3, MaC7, MaCTSL, MaMMP-9, MaIL-8 and MaIL-10 in BSB were performed Physicochemical and functional

characteristics of proteins were analyzed The secondary and tertiary of the protein’s domains were constructed applying the comparative modeling method

5 Reverse transcription–quantitative PCR (RT–qPCR) was used to analyze the

expression of immune-related genes (MaTLR5, MaNFKBIA, MaMyD88, MaTRAF6,

MaC3, MaC7, MaCTSL, MaMMP-9, MaIL-8, and MaIL-10) in response to A hydrophila

The results showed that all investigated genes were highly up- or and downregulated in the liver, spleen and kidney during the challenge time from 4 to 120 hours post injection This research provides the important roles of these genes in the BSB’s innate immune system

The results provide significant valuable information of molecular data, which are useful for further study of the immunogenetics of BSB

Keywords: Megalobrama amblycephala, transcriptome, immune-related genes,

molecular markers, gene expression

ABRBREVIATIONS

CHAPTER 1 Review of literature

1.1 General introduction

Blunt snout bream (BSB) (Megalobrama amblycephala) or Wuchang fish or

bluntnose black bream is a herbivorous species and commonly distributes to the middle portion of Yangtze River, China (Fishbase, 2012) In China, BSB was known as a famous species contributing to Wuchang county and Ezhou city, Hubei province Histologically, the fish were a delicacy tribute in the Chinese feudal society Now, this is a major freshwater species and currently popularly cultured in China because of many valuable characteristics, including its comprising of high delicacy, high larval survival rate, natural foods feeding, fast growth, tender flesh, high disease resistance (Tsao, 1960; Zhou et al., 2008) Moreover, the fish is famous as a main ingredient for cooking many special traditional foods in China such as “Qingzheng Wuchang fish”, “Hongshao Wuchang fish” and “Youmen Wuchang fish” Because of high consumption in market, the development of BSB culture industry has been paid more attention, leading improving culture technique and other related subjects in culture procedure It was evidenced that since 1960s the BSB had started being a domestic culturing practice species A number of researches had been carried out, mainly focused on studying the improvement on cultured fish biomass, nutrient requirement and genetic variations in cultured and wild population In 1986, a project on selection of this fish species conducted using a population exploited from Yuni Lake, the project had been gained an improvement of fish growth up to 29% after 6 breeding generations (Li and Cai, 2000; Li and Cai, 2003) As a result, the production of BSB has gradually increased in recent years, for example, it was 552900 tons in 2005 increased up to

625789 tons in 2009, ranking the sixth in whole Chinese freshwater fish production in China (MAPRC, 2006; MAPRC, 2010)

In general, the higher intensification and commercialization in aquaculture caused the disease problems out-break and they are more popular and serious to cultured animals (Bondad-Reantaso et al., 2005) BSB was popularly cultured in pond culture system during summer time when the highest temperature period in a year may induce disease outbreak and high mortality in farmed fish (Ming et al., 2012) Among pathogens such as bacteria, parasite, fungi and virus were recorded commonly infecting to cultured aquatic animals, the

bacterium Aeromonas hydrophila has been reported as a main pathogen causing

hemorrhagic septicemia clinical signs and great losses of cultured BSB (Nielsen et al., 2001) On the fact that the prevention and control fish disease demand integrated approaches, comprising understanding the immunity improvement and the fields of study that related to pathology, physiology and genetic (Bachère, 2003) The previous studies demonstrated that seem not different between fish and terrestrial animals in immune systems; both innate and adaptive immunity was recorded exist in fish as a defense barrier for pathogen infection (Alvarez-Pellitero, 2008; Tort et al., 2003; Whyte, 2007) The innate immunity is as the first line to prevent the invasion of foreign agents infecting and causing diseases in fish Due to the important role of innate immune system, multiple previous studies on the increasing functions of innate immune system of different aquatic animals in respose to pathogens were performed and resulted in positive effectiveness in application for practice condition of BSB culture system Specifically, studies on the enhancement of immune response of BSB were conducted, for example, supplementary of substrates or

additives to BSB dietary enhanced the response ability to A hydrophila infection (Liu et al.,

2012a; Ming et al., 2012; Ming et al., 2011) Moreover, development and application of molecular biology in pathology-related studies grasping animals resistant to diseases are more essential, especially the expression of immune-related genes of host animals such as

rainbow trout (Oncorhynchus mykiss) (Evenhuis and Cleveland, 2012; Heinecke and Buchmann, 2013; Raida and Buchmann, 2009), common carp (Cyprinus carpio) (Huang et al., 2009), and giant freshwater shrimp (Macrobrachium rosenbergii) (Arockiaraj et al.,

2012; Du et al., 2013) in resonse against invaded pathogens

To date, multiple studies relating to genetic aspect on BSB was performed, including study on the cloning, characterization and expression of genes isolated from BSB have been published Nevertheless, only few reports that related to immunogenetics especially to

expression of immune-related genes are certainly available In which, the occurrences of A hydrophila infection in the cultured BSB have been obviously observed, but available data

on the gene expression profile of the whole transcriptome or its response to aeromonas infection in blunt snout bream are still limited Therefore, performing a detailed study on the transcriptome profile analysis and expression of immune-related genes of BSB after

challenge with A hydrophila is necessary In this thesis, the transcriptome profile and expression of immune-related genes of BSB after challenge with A hydrophila were

profoundly investigated The study was also attempted in several aspects, including cDNA

library construction, transcriptomic profile analysis using next generation sequencing-

Solexa/Illumina technology, gene characterization employing in silico approach, and

investigation of gene expression in infected BSB compared with the controls as well

1.2 Introduction to blunt snout bream

1.2.1 Biological characteristics

BSB is a member of finfish Class of Actinopterygii, Order of Cypriniformes and Family of Cyprinidae (Fishbase, 2012) The morphological characteristics of BSB have been described as “the head is small, pointed and compressed; snout is short, eyes are large; and mouth is small, protractile, slightly oblique and arched The body compressed and high depth; ventral and dorsal profile arched; keel on the belly between pelvic fin origin and anal fin origin The scales are moderately cycloid, large; lateral line complete (L.l.:50-58); dorsal fin rays (III, 7); pectoral fin rays (1+14-19); pelvic fin rays (1+8); anal fin rays (3+27-32) Pectoral fin end is close to pelvic fin origin, the pelvic fin is at lower side of body; anal fin is base long; caudal fin is forked The dorsal is dark grayish, side and belly is silvery white, all fins are in grayish in color” (Shao, 2015) In morphology, it seems to differ between male and female, the male have longer pectoral fins, usually being observed nearby or end of the base of the ventral fin, while it certainly keep the distance in female; juvenile fish has longer pectoral fins and exceed the base of the ventral fin (Tsao, 1960)

In China, although the BSB was originally found in Newshan Lake and Yuli Lake (Fishbase, 2012; Zhou et al., 2008), the fish is widely distributed in the middle portion of Yangtze River (Tsao, 1960) The BSB prefers to be benthopelagic and habitat in deep freshwater pools (arrange of 5 to 20 m) or in lakes, impoundments and backwaters of rivers This is a hypoxia-sensitive fish species, the fish is able to alive with a short period of hypoxia (below 0.5 mg O2/L) at room temperature (Ouyang et al., 2001; Shen et al., 2010) Nutritionally, BSB is basically native herbivorous freshwater finfish (Ouyang et al.,

2001; Tsao, 1960) In wild condition, the fish mainly feed aquatic plants (Vallisneria natans with 68.3%, Hydrilla verticillata with 33.7%), algae and zooplankton (Tsao, 1960) The

protein requirement for fingerling was 35% and lipid 4-7% (Li et al., 2012b; Zhou et al., 1997) However, the protein demand is associated with temperature conditions, for example, the optimum protein consumption for fish are from 27 to 30.4% at 20oC and from

To reproduction and fecundity, the fish are initially matured after 2 years (Ouyang et al., 2001; Tsao, 1960) The highest maturity efficiency of the fish ovary is from May to June and the ovary is divided into six periods of development (I-VI) (Tsao, 1960) The average value of absolute fecundity is different in the different size of fish, generally in average of 7.8×104 eggs The average value of relative fecundity is 232 eggs/g and the ovary per unit contains average 1.320 eggs/g (Ouyang et al., 2001)

1.2.2 Interests to research

1.2.2.1 Physiological characteristics

The oxygen consumption of BSB is to be involving in the fish size and temperature of water, the low oxygen consumption is in bigger fish and low water temperature, for example, the fish weighs of 10.3 g consume 364 mg/kg/h oxygen, but it is only 193.88 mg/kg/h oxygen in 295 g fish (Ouyang et al., 2001) At the room temperature of 27oC just for a short time of hypoxia (below 0.5 mg O2/L) the fish may die (Ouyang et al., 2001) Although this is a benthopelagic species, dwelling in 5-20 m of water depth, but the oxygen consumption is relatively high The oxygen homeostasis maintain may be controled by the two genes of hypoxia- inducible factor (HIF-1α and HIF-2α), when HIF-2α was found to

be significantly overexpressed in liver and kidney during hypoxic treatment (Shen et al., 2010) Moreover, benzidine was reported as a stimuli affecting to the activation of two antioxidized enzymes of mitochondria (superoxide dismutase and glutathione peroxidase)

in the liver of BSB; both enzymes were recorded to be inhibited by the high concentration

of benzidine (1.5-2.0mg/mL) (Dai et al., 2003)

1.2.2.2 Nutrition and nutrient requirement

Albeit the requirement of protein and lipid for the optimum growth of different life

stages were seemly different in BSB, but the differences are not significantly large In fingerling stage, the protein and lipid consumption in the diet was 31% and 7%, respectively (Li et al., 2012b) This was approximately similar to yearling stage which was 30% (protein requirement) and 6% (lipid requirement) (Jiang et al., 2012) However, the protein consumption for fingerling fish still involves in water temperature, the optimum protein demand was from 27 to 30.4% at 20oC and its proportion increased up to

a range of 25.6 and 41.4% at 25 to 30oC (Shi et al., 1988) The mineral requirements are calcium (0.31-1.07%), phosphorus (0.38-0.72%), potassium (0.41-0.57%), sodium (0.14-0.15%), magnesium (0.04%) and iron (0.024-0.048%) (Shi et al., 1997)

By the feeding behavior of BSB, a non-animal ingredient was known suitable for digestibility The digestibility of BSB to non-animal ingredients in feed was highest (85.9%) in comparison to other ingredients, in which soybean was evaluated more effectively with 98.1% digestion (Zhou et al., 2008) As this study results, the non-animal ingredients in BSB’s diet with corn gluten meal or soybean meal showed the increasing of apparent digestibility, leading to decide using a lower cost diet with soybean meal as a main component in feed formulate (Jiang et al., 2012; Wu et al., 1995) On the other hand, some recent studies have proven that protein source from silkworm pupa or fish meal gives the best digestibility in feeding BSB (Cao et al., 2012; Jiang et al., 2012) The protein source from fish meal was the highest and followed by soybean meal, rapeseed meal and cottonseed meal (Cao et al., 2012) Actually, this difference in protein digestion may be explained by the difference in protein quality, amino acid compositions, and processing procedure of the feedstuff (Cao et al., 2012)

In addition, lipid ratio in the diet may be important because of its relationship to promote ratio growth and effectively utilize feed and protein; but the disadvantages of a high lipid ratio may cause rancidity and lowered flesh quality due to oxidation (Yilmaz and Genc, 2006) Actually, the utilization of lipid or fatty acids stills depends on the nutritional status of the fish, activities of digestive and metabolic enzymes, liver composition and plasma biochemical parameters, and other compositions in the diet (Jiang

et al., 2012; Li et al., 2012b; Liu et al., 1997; Pettersson, 2010; Zhou et al., 1997) For example, previous study showed that supplying lipid level from 40 to 70 g/kg in diet feeding BSB may promote the activities of digestive enzyme and reduce the proportion of protein catabolizing into energy (Li et al., 2012b) In which, fish oil was evaluated as an

important source in feed processing industry (Pettersson, 2010) The fish oil proportion of 4-6% in BSB diet was recommended (Zhou et al., 1997) Moreover, silver oil and soybean oil were also reported as good sources that compared with fish-liver oil, peanut oil, rape oil, menhaden oil, and lard in the diet of juvenile BSB, which promote growth and decrease feed coefficient ratio (Liu et al., 1997; Wei and Chen, 2003) However, the higher level of lipid in diet formulate may affect to the liver functions and non-immune responses of fingerling BSB to stresses (Li et al., 2012a)

Supplementing other extra components to fish feed is the best way to control the economic profits during culture time Experimentally, trifolium (add 20-40%) and alfalfa (add 10%) have been reported to be good in replacement for fish meal or soybean cake in the BSB diet (Hu et al., 2005; Jia et al., 1991) Furthermore, some other supplements such

as enzymolysis (Cao et al., 2012), emodin (60 mg/kg) and Vitamin C (700 mg/kg) (Ming et

al., 2012; Ming et al., 2011), or anthraquinone (0.1%) extracted from Rheum officinale

(Liu et al., 2012a) could be good in enhancing the digestibility to feedstuffs, feed conversion rate, fish growth, and non-specific immunity of BSB

1.2.2.3 Genetics and breeding

BSB is an only species which commonly distribute in the Yangtze River (China) Since

1955 this fish species has gradually became one of the major cultured objects in China (Zhao et al., 2009) In 1986, first selected generation achieved according to the fish origin from Lake Yuni (the middle of Yangtze River, China) (Li and Cai, 2000; Li and Cai, 2003) Surprisingly, the cultured BSB were recorded with earlier maturity, growth rate depression; thin and longer body and disease susceptibility (Gao et al., 2012; Li and Cai, 2003) These consequences have been explained by the effects of inbreeding depression during artificial selection (Li and Cai, 2003) However, the lower level of genetic diversity, obtaining 5.4% per five generations was also reported, leading to lower response in size selection of fish even though the growth rate of BSB was improved up to 29% (Li and Cai, 2003) The chromosomal number and karyotype formula of BSB was defied as 2n=48 and 26m+18sm+4st, respectively (Yin et al., 1995)

The aspiration of generating polyploidy in BSB was considered to improving meat quality and ecological safety This was the main purpose of producting fast growing sterile triploids by interploid hybridization and producting relatively large fish by higher heterozygosity (Zou et al., 2004) Basically, thermal shock was introduced as the most

common method (Strunjak-Perovic et al., 2003) applied to reproduce tetraploids of BSB which reproduced the highest rate of tetraploid (6.3%) by combination of 40oC (at 1.40 to

or 2.17 to) for 2 minutes (Zou et al., 2004) Moreover, cross preproduction of using 4n♀×4n♂ yielded the hatching and tetraploidy rate of offspring BSB increasing up to 60.8% and 93.2%, respectively (Zou et al., 2004) In additional to the observation in the erythrocyte morphology of polyploidy fish may indicates a positive or negative response to the cumulative mortality of cultured fish when the morphological parameters of erythrocyte and nuclei of 2n compared with autotetraploid and interploid 3n (Zou et al., 2005) The interploid hybrids produced by matting allo-tetraploid males (an interspecific

tetraploidy of BSB (M amblycephala) ♀ × black bream (M terminalis) ♂) with BSB

females were high fertilization rate, stable survival after 60 days culture and increased in daily growth rate (7.5% when it was compared with diploid BSBs during 180-day culture) (Zou et al., 2011)

Molecular markers development in genetic researches to blunt snout bream

DNA markers were used as a powerful assist to animal breeding in defining the genetic makeup and predict the performance of an animal (Beuzen et al., 2000) To BSB, utilization

of DNA markers was popularly applied and developed (Gao et al., 2012; Li, 2008; Li, 2010; Tang et al., 2009; Zhang, 2001)

Mitochondrial DNA (mtDNA): mtDNA was known with small molecules, simple

structure, maternal inheritance and fast evolution, especially, the evolution rate of the mtDNA control region (D-loop) is five times that of the other regions Thus, it has become

an important marker of fish genetic evolution, molecular ecology, biological genetic diversity and conservation research (Tang et al., 2011) Previously, mtDNA were used as a marker to estimate for the genetic diversity and structure of different (wild and cultured) populations of BSB (Bian et al., 2007; Li, 2008; Tang et al., 2011) The results showed that three variable sites and five haplotypes were detected among the collected samples (Li, 2008) Also, the restriction sites were observed by nine enzymes, polymorphism and two haplotypes were identified (Bian et al., 2007) Genetic diversity analysis in mentioned experiments was low level of population diversity among tested populations (Bian et al., 2007; Li, 2008) Moreover, differences in variability and differentiation of genetics between types of genetic-ecological populations was observed by the use of combination nucleotide sequences of control region and CO I gene of mtDNA (Tang et al., 2011)

Random amplified polymorphic DNA (RAPD): RAPD was also used in studying

genetic diversity between BSB populations from Yunihu and Liangzi lakes (Zhang, 2001) Besides, the genetic relation between three common freshwater fishes such as common carp, bighead carp and BSB was analyzed by using RAPD markers (Zhang et al., 2008) In addition, RAPD was evaluated as molecular marker in comparison for the differences in heredity of gynogenetic fry (G1) and non-gynogenetic fry (F6) of BSB with 1100 bp band amplified by primer S8 and 860 bp band amplified by primer S18 which was only found in F6 group and G1 group, respectively (Zou et al., 2001)

Microsatellite/Simple sequence repeats (SSR): this is a marker considered as useful

tool for discovering genetic variation levels of BSB (Li et al., 2007; Li, 2010; Tang et al., 2009; Tang et al., 2014) SSRs were used to predict the highest hybrid vigor of different geographical BSB populations of the sire from Liangzi Lake population and the dam from Yuni Lake population where indicated the highest hybrid vigor ratio (Zeng et al., 2012) From the transcriptopme profile of BSB, a total of 4,952 microsatellites, including from di-

to hexa-nucleotide repeats were identified from 100,477 unique sequences (Gao et al., 2012)

Single-nucleotide polymorphism (SNP): SNP is considered to be a good molecular

marker which is commonly applied in genetic-related studies in aquatic animals In BSB,

a number of 25,697 SNPs, including 17,272 transitions and 8,425 transversions, and a total

of 23,287 filtered indels (with one indel per 392 bp of the transcribed sequences) were identified from the BSB’s transcriptomic profile These SNPs were expected to developing genome analysis in gene-based linkage map and large-scale expression analysis of bream

or comparative genome analysis for the genus Megalobrama fish species (Gao et al., 2012) Sequence-related amplified polymorphism (SRAP): the genetic linkage maps of BSB

was constructed by using SRAP markers which comprised 99 markers attributed to 14 linkage groups in sire population and 100 markers attributed to 16 linkage group in dam population (Li, 2010)

1.2.2.4 Disease infection and immune-related approaches

The innate immune system was known as the first line in protecting fish responding to the invading of various pathogenic organisms In fact, the immune-related genes were released in response to the presence of pathogens in the host These pathogens stimulate and allow communication between the host’s cells to promote the responses of immune

system that protect the host by attacking In general, studies on the innate immune system

in aquatic animals have been usually focused on the constitutive expression of various immune-related genes; these gave data on the molecular mechanisms implicated in animal that susceptibility/resistance to disease infection Multiple studies on expressions of immune-related genes have been carried out in various aquatic animal species (Baoprasertkul et al., 2007; Sritunyalucksana et al., 2001) To date, a number of scientific papers have been carried out focusing on cloning, characterization, and expression of genes such as thyrotropin beta (Qu et al., 2006), gonadotropin (GtH I, II, IIβ) (Qu et al., 2008b; Qu et al., 2010; Qu et al., 2007), glycoprotein hormone alpha (Qu et al., 2008a), Hoxb3α (He et al., 2009), HoxBlb (Pu et al., 2011), heat shock proteins (HSP70, HSC70, HSP90α/β) (Ding et al., 2013; Ming et al., 2009; Ming et al., 2010), hypoxia-inducible factor (Shen et al., 2010), peroxisome proliferator-activated receptor gamma (Li et al., 2013b), major histocompatibility complex (MHC I, IIa, IIb) (Luo et al., 2014b) of BSB in China However, only few reports that involving in immune-related genes of BSB are certainly available (Ding et al., 2013; Luo et al., 2014b; Ma et al., 2011; Su et al., 2009; Zhao et al., 2011) Although, a limit data and non-empirical approaches of immune related genes expression level have been reported, but they seem to provide useful information on the more major studies later on the immune-related gene expression of this fish species For example, among investigated immune-related genes, toll-like receptors (TLRs) play important roles in sensitivity with microbial infection and stimulation of inflammatory and immune responses TLR3 and TLR4 were recorded high expressed in various tissues

of BSB (Su et al., 2009; Zhang et al., 2009), in which TLR3 was found highest expressed

in hepatopancreas and heart, whereas lowest expressed in muscle (Su et al., 2009) Furthermore, anti-viral galectin-like protein was isolated from BSB serum and evaluated as

an innate immune factor against invasive infections (He et al., 2003b; He et al., 2003c) Moreover, the vaccination application was also gradually studied and developed In

2011 and 2012, gene transfer vector pFastBacTM Dual was used to construct for the recombinant as DNA vaccine against grass carp reovirus (GCRV) in BSB (Liu et al., 2012b;

Xu et al., 2011) A recombinant of pFastBac-β-VP71-VP72 was constructed from double GCRV outer capsid protein VP7 genes (0.9 kb) and ß-actin promoter (0.56 kb) from BSB which were amplified and cloned into gene transfer vector pFastBacTM Dual This recombinant can be used as DNA vaccine against GCRV due to it benefits in controling the

VP7 gene expression by β-actin promoter, increasing of titers of antibody (reaching a peak

in 21 day after infection), and increasing the survival rate, showing 100%, 100% and 95%

in injection with 10 µg, 30 µg and 60 µg vaccine after after 49 day challenged with GCRV (Xu et al., 2011)

Also, DNA vaccine vector pFastBac-FA-VP6-ph-VP6 was evaluated as effective vaccine that promotes fish survival after 28 days challenged with GCRV (Liu et al., 2012b) Therefore, the recombinant of gene vector of the gene transfer vector pFastBacTMDual, pathogen genes, and β-actin promoter of the host can be used as the DNA vaccine for resistance to pathogen invasion

1.3 Aeromonas hydrophila and its infection in fish

1.3.1 Classification and identification

A hydrophila belongs to the genus of Aeromonas which is refered as synonyms with aquatic environments Aeromonas spp are ubiquitously isolated from aquatic ecosystems,

coastal waters, freshwater, coastal water, sewage and a variety of foods (Krovacek et al.,

1994; Rahman et al., 2002) Aeromonas was known playing a part of natural bio-filters in

freshwater reservoirs and promotes self-purification of water body They are necessarily present in normal microflora of hydrobionts inhabiting freshwater reservoirs (Kompanets

et al., 1992) Otherwise, Aeromonas is also recorded as main pathogens infected to man

and animals (Jayavignesh et al., 2011)

Motile aeromonads can widely distribute in any conductivity, turbidity, pH, salinity and temperature (Jayavignesh et al., 2011), especially, they can habitat in extreme environment conditions such as saline, thermal springs, and polluted waters (Janda and

Abbott, 2010) The optimum temperature for Aeromonas growth was from 25 °C to 35 °C

(Janda and Abbott, 2010) Therefore, previous records were recognized that more epizootics among warm water fishes in the south eastern United States (Jayavignesh et al.,

2011) However, A hydrophila isolated from a diseased catfish was able to grow well at

the optimum temperature of 37 ºC (they can grow at 4 ºC and up to 41 ºC) (Jayavignesh et al., 2011) This report was a similarity to previous description (Aberoum and Jooyandeh,

2010), the psychrophilic strains of A hydrophilia have an optimum growth temperature of

between 15 and 20°C and may grow at temperatures as low as 0 oC to 5 °C The thermal

death point of A hydrophila may be ranged from 40 ºC to 50 ºC and the optimum pH

value for growing of the A hydrophilia isolated from a diseased catfish was 9, but it also

able to grow at pH: 5 (Jayavignesh et al., 2011)

Preliminary aeromonads were identified base on the morphological characteristics, Gram staining and biochemical characteristics tests which include indole, catalase test, oxidase test, voges prosakur, citrate utilization, carbohydrate utilisation lactose, glucose, trehalose, starch, gelatine hydrolysis, triple sugar iron, methyl red and urease test (Jayavignesh et al., 2011) Moreover, the identity of isolates with histamine-forming have been asserted by amplifying and sequencing approximately 1.5 kbp of the 16S ribosomal DNA (rDNA) (Hossain, 2008)

1.3.2 Pathology

The motile Aeromonas was isolated and defined distributed widely in fresh and

estuarine water conditions Generally, the bacterium was recognized as pathogens causing diseases in man and animals (Janda and Abbott, 2010; Jayavignesh et al., 2011; Krovacek

et al., 1994; Rahman et al., 2002) Aeromonas are revealed related to many diseases in

different both cultured and feral fish species, which were the most common infected bacteria in cultured warm water fish species and they recently defined involving in diseases of pond fish (Cipriano et al., 1984; Hossain, 2008) The pathologic conditions could be acute, chronic, and covert infections, depending on other factors such as bacterial virulence, the kind and degree of stress impacted on a fish population, the host’s physiological possibility, as well as the genetic resistance inherent within fish populations

(Cipriano et al., 1984) However, Aeromonas was also known as an opportunistic pathogen

in freshwater aquatic habitats with heavily organic load and in the stress condition (Austin

and Adams, 1996; Citarasu et al., 2011; Peters et al., 1988) Many aeromonads species, A hydrophila, A sobria and A caviae have been reported associated with disease

occurrence in fish species (Wahli et al., 2005)

1.3.3 Clinical signs

In fish, A hydrophila has been commonly associated with skin lesions, tail and fin rot,

and hemorrhagic septicemia over the body and tissue destruction, epizootic ulceration and

necrosis in the liver and kidney (Austin and Adams, 1996; Janda and Abbott, 2010) A hydrophila were recorded widely infecting to freshwater fish and marine fish species (Austin and Adams, 1996; Doukas et al., 1998; Duc et al., 2013) In carp (C carpio) after

infected by A hydrophila, numerous hemorrhagic and ulcerative lesions in the skin, on

body surface including wings, mouth, gills and eye have been observed (Chirila et al.,

2008) The similar clinical signs were also recorded in goldfish (Carassius auratus) and Koi (C carpio koi) (Citarasu et al., 2011), rainbow trout (Salmo gairdneri) (Peters et al., 1988), walking catfish (Clarias batrachus) (Angka, 1990), goldfish (C auratus) (Iqbal et

al., 1999), Nile tilapia (Oreochromis niloticus) (Ibrahem et al., 2008), and snakehead fish (Channa striata) (Duc et al., 2013)

1.3.4 Virulence factors

Many extracellular proteins such as amylase, chitinase, elastase, aerolysin, nuclease, gelatinase, lecithinase, lipase and protease have been previously reported excreting by the

species of Aeromonas These proteins are known as virulence factors that caused diseases

in humans and fish (Aberoum and Jooyandeh, 2010) Among these proteins, aerolysin

was a representative virulence factor of Aeromonas; its functions were similar to

hemolysins and cytolytic enterotoxins (Aberoum and Jooyandeh, 2010) The virulence

factors can be produced by strains of Aeromonas isolated from different sources, but it

seems to be similar from strains isolated from the cases of infection with respect to the prevalence of virulence factors (Krovacek et al., 1994; Rahman et al., 2002) However, the

higher virulence of Aeromonas strains were evaluated when compared with those from

isolated from pond water (De Figueiredo and Plumb, 1977)

1.3.5 Infection of Aeromonas to blunt snout bream

During the past culture time, BSB culture industry as discussed above has suffered problems related to degrading pond environments through commercialization and intensification in culture system, which resulted in the odd of stress-induced diseases (Nielsen et al., 2001) It is apparently similar to BSB cultured in Zhejiang Province (China)

where the first time A hydrophila was determined as pathogen (Nielsen et al., 2001) The A hydrophila infected to BSB was described seem to not differ from other fishes in clinical

signs such as hemorrhagic septicemia on the fish body (the mouth, eyes, opercula, at the base of fins, and around the anus), erosions and ulcers in the skin, histopathological changes (for example, inflammatory edema, necrosis in the dermis, and hemorrhage in the dermis and subcutis) and the lower number of red blood cells and higher number of white blood cells in haematological physiological indexes (He et al., 2006)

1.4 Transcriptome study

Transcriptome implies a part of genes from the genome that are functionally active in

a selected tissue of organisms (Velculescu et al., 1997; Xia et al., 2011) As previously described, the transcriptome is initial product of genome expression, which is the collection of all RNA molecular, comprising mRNA, rRNA, tRNA and other non-coding RNA transcribed in one cell or various cells (Brown, 2002) The transcriptome is complex which consists of copies of hundreds, if not thousands, of different mRNAs (Brown, 2002; Forrest and Carninci, 2009) The collection of RNA molecular generates the important role of transcriptome in cells by in the case of containing the coding RNAs that specify the composition of the proteome and determine the biochemical capacity of the cell The RNA sequence shows the sequence of its transcribed DNA (Brown, 2002) Based on analyzing the entire collection of RNAs in a cell facilitates determining the beginning and ending of

a gene in the cells and tissues of an organism Moreover, to the whole genome database of species that has not yet shown the transcriptome analysis facilitates to investigate global gene expression, discover new genes, assembly of full-length genes and to discovery single nucleotide polymorphism (Emrich et al., 2007; Huang et al., 2011; Vera et al., 2008; Wang et al., 2012a) Further, transcriptome analysis is also utilized to study multiple genome features comprising genome duplication (Wang et al., 2012b) Usually, it possibly counts the number of transcripts in order to define the amount of gene activity, which is known as gene expression, in a cell or tissue The different cells show different patterns of gene expression profile, even though the same genes were found in every cell The differences in gene expression are associated with the differences in types of properties and behaviors within various cells and tissues Analyzing the different transcriptome profiles of different types of cells provides a more comprehensive on the compositions of a specific cell type, the list of tissues in which a gene is expressed, the normally specific functions and the levels of gene expression in a type of investigated cells (NIH, 2012) Using expression arrays in genome-wide transcriptome analysis was concernced in oder to better understanding on molecular classification, disease pathogenesis, and biomarker identification (Woroniecka et al., 2011) Analysis of genome-wide differential RNA expression provides better comprehensives on the biological pathways and molecular mechanisms involving in the fate, development, and disease progression of cells

There are two general methods of inferring transcriptomes have been developed One method is aligning sequence reads to an existing reference genome from the organism itself or a closely related species and then assembling the remaining unmapped reads The

other method, a de novo assembly is firstly employed and the genome is then utilized to

improve the transcript assembly (Feldmesser et al., 2014; Martin and Wang, 2011) On the other hand, the difficulties of transcriptome analysis were usually observed due to the lacks or incomplete database of the genome used as reference sources The major advance

of transcriptome assembly analyses have been employed and gained worthy in various species such as bacteria (Høvik et al., 2012; Mazin et al., 2014), mouse (Sharov et al., 2003) and human (Gupta et al., 2014; Peffers et al., 2015)

Studies of the transcriptome profile of aquatic animal

Whole-transcriptome analysis is more important in comprehensive how altered expression of genetic variants which contributes to complex traits of an organism Transcriptomic profiling is used to analyze the gene expression and signaling pathways in specific tissues or cells Recently, the quick evolution of next-generation sequencing (NGS) technologies, the Solexa/Illumina technology included, offers great advantages in analyzing the functional complexity of the entire transcriptome (Wang et al., 2009) NGS based on the RNA sequencing (RNA-seq) approach is a means of accurate characterization in quality and quantity of the expressed element from the genome (Montgomery et al., 2010; t Hoen et al., 2008) NGS techniques have been used for transcriptome analyses in order to simultaneously provide the data on sequence polymorphisms and the levels of gene expression involved in cellular development, cancer, and the immune responses (Czesny et al., 2012; Xiang et al., 2010) In fact, transcriptome studies have been applied to many aquatic animal species, including

Thalassophryne nattereri (Magalhães et al., 2006), Lates calcarifer (Xia and Yue, 2010; Xia et al., 2011; Xia et al., 2013), Pseudosciaena crocea (Mu et al., 2010), C auratus (Liao et al., 2013), Crassostrea gigas (de Lorgeril et al., 2011), Epinephelus coioides (Huang et al., 2011), M rosenbergii (Jung et al., 2011), O mykiss (Aquilino et al., 2014; Pérez-Sánchez et al., 2011), Salmo salar (Tadiso et al., 2011), Apostichopus japonicus (Du et al., 2012), Hypophthalmichthys molitrix (Fu and He, 2012), Ctenopharyngodon idella (Chen et al., 2012a), C carpio (Wang et al., 2012b), M amblycephala (Gao et al., 2012), Sebastiscus marmoratus (Sun et al., 2012), Sepiella maindroni (Song et al., 2012),

Fenneropenaeus chinensis (Li et al., 2013c), Eriocheir sinensis (Li et al., 2013d), Misgurnus anguillicaudatus (Long et al., 2013), Portunus trituberculatus (Lv et al., 2014),

O niloticus (Tao et al., 2013; Zhang et al., 2013a), Ictalurus furcatus (Li et al., 2013a), Mizuhopecten yessoensis (Meng et al., 2013), Sinonovacula constricta (Niu et al., 2013b), Bombina maxima (Zhao et al., 2014), Crassostrea virginica (Zhang et al., 2014), Evechinus chloroticus (Gillard et al., 2014)

Numerous studies have been reported on the analyses of transcriptome profile which are associated with the immune-related field of study Actually, analysis of the transcriptome of an organism in response to invasive infecting pathogens can provide information on the immunological mechanisms and cellular pathways that are in the process of initial infection, disease progression and ultimately active disease (Nalpas et al., 2013) These studies have contributed to the biological data across the species and provided the bases for studies on functional genomics (Xia et al., 2011) Some interesting results have been obtained For example, a non-normalized cDNA library of head kidney

was constructed from grass carp (Ct idella) before and after GCRV infection (Chen et al.,

2012a) The results obtained 3,027 high-quality unigenes by using Solexa sequencing method From this study, a total of 22,144 tags were determined differentially expressed (>2-fold in infected groups compered with uninfected one) Among these obtained unigenes, 42 were discovered as novol genes of unknown function having no matches to available sequences in the databases (Chen et al., 2012a) Until now, only one study has used transcriptome profiling in the blunt snout bream to analyze growth-related traits was performed (Gao et al., 2012) Understanding the transcription profiles of response-related genes in the BSB will facilitate further research on the resistance and susceptibility of this fish species against exogenous invasive pathogens The main objective of this study is to study the expression of the important immune-related genes in the larger context of the

BSB transcriptome following A hydrophila infection, and then confirm the expression

pattern of selected genes of interest by quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR) The significance of this study is the first time the expression of immune-related genes in the bacterial challenged fish compared with that in control fish at the transcriptome level This result may be applied for further fish selection

of disease resistance generations

The thesis describes the transcriptome analysis and expression of immune-related

genes in BSB (M amblycephala) after challenged with A hydrophila It includes seven

chapters In Chapter 1, general introduction and literature review of BSB, bacterium A hydrophila and transcriptome study are described In Chapter 2, pathogenicity of A

hydrophila to M amblycephala was carried out The challenge test was conducted to confirm the pathogenic of A hydrophila to BSB Biological, physiological characteristics,

antibiotic susceptibility and molecular analysis for the tested bacterial strain were also

studied profoundly In Chapter 3, transcriptome analysis of the BSB after challenge with A hydrophila was attempted In Chapter 4, microsatellites (SSRs) and single nucleotide

polymorphisms (SNPs) were identified from the transcriptome profile Obtained SNPs

were successfully annotated In Chapter 5, characterization and homology modeling of

proteins, including MaTLR5, MaNFKBIA, MaMyD88, MaTRAF6, MaC3, MaC7, MaMMP-9, MaCTSL, MaIL-8, and MaIL-10 encoded by immune-related genes in M

amblycephala was studied using in silico approach In Chapter 6, expression of

immune-related genes (MaTLR5, MaNFKBIA, MaMyD88, MaTRAF6, MaC3, MaC7, MaMMP-9, MaCTSL, MaIL-8, and MaIL-10) in BSB after challenge with A hydrophila

was examined In Chapter 7 is a conclusion of this thesis

have been particularly infected by epidemics of A hydrophila which has been reported

causing hemorrhagic septicemia clinical signs and histopathological changes, and causing

a great loss in cultured BSB (He et al., 2006; Nielsen et al., 2001)

It has been known for decades that Aeromonas plays a causative agent role in fish

diseases The wide-spread distribution in aquatic ecology systems indicates that

interactions of Aeromonas species with fish are continuous and unavoidable, facilitating their opportunistic pathogenicity (Hu et al., 2012; Ottaviani et al., 2011) A hydrophila

were recorded widely infecting freshwater fish and marine fish species associated with skin lesions, tail and fin rot, haemorrhagic septicaemia over the body and tissue destruction, epizootic ulceration and necrosis in the liver and kidney of fish (Austin and Adams, 1996; Doukas et al., 1998; Janda and Abbott, 2010) It was considered as a significant economic

problem in China and India (Citarasu et al., 2011) The existence and pathogenicity of A hydrophila have been reported in a variety of freshwater species such as S gairdneri (Peters et al., 1988), C batrachus (Angka, 1990), tilapia (Liu et al., 1999), C auratus (Citarasu et al., 2011; Iqbal et al., 1999), C carpio (Chirila et al., 2008; Citarasu et al., 2011), O niloticus (Ibrahem et al., 2008), and C striata (Duc et al., 2013) Nevertheless, to

date the infection information of BSB in China is still limited The aim of this chapter is to

confirm whether A hydrophila is really pathogenic to BSB or not

2.2 Materials and Methods

2.2.1 Bacterial strain and bacterial suspension preparation

A hydrophila D4 used in this experiment was isolated from BSB with clinical signs of

hemorrhagic septicemia cultured in Dongxihu, Hubei, China in 2012 and maintained in the College of Fisheries, Huazhong Agricultural University (HZAU) The bacterium was cultured on the agar medium (including 3 g beef extract, 10 g peptone, 5 g NaCl, and 15 g agar in 1,000 mL distilled water) and incubated at 28 oC for 24 hours In order to harvest bacterial cells, sterile physiological saline (0.85% NaCl) was added into the bacterial incubated test tubes, and the surface colonies were crushed by using a sterile loop to dilute bacterial suspension The desired densities of bacteria (1.7×105, 1.7×106, 1.7×107 and 1.7×108 cfu/mL) were determined by counting on a neubauer hemocytometer and prepared

by ten-fold dilution method To confirm the inocula density of bacterial suspension, a method for plate count by serial dilution was conducted

2.2.2 Fish for experiment

Healthy BSB weighing 27.1±8.3 g were collected in the fish hatchery in Huanggang, Hubei, China in November 2013 and transferred to the laboratory of the College of Fisheries, HZAU All tested fish were acclimatized in 4m3-glass tanks (maintained at 28 oC and pH of 7.5) for 2 week before the artificial injection experiment During the

acclimatization period, fish were fed ad libitum with pelleted feed (Haid Company, China)

2.2.3 Pathogenicity test

Fifty fish without clinical signs by naked eye observation were used for intraperitoneal injection experiment The experiment included 5 groups (n=10 for each), comprising the negative control (physiological saline injection) and four concentrations of bacterial suspension Before infection experiment, fish were slightly anesthetized by tricaine methanesulfonate (MS-222, Sigma, USA) Inocula were set up as intraperitoneal injections at 0.1 mL/fish of physiological saline or desirable densities of bacterial suspension The experiment was conducted in 50 L aquarium with aeration at 28 oC for 7 days No feed was fed during the experiment

The fish were monitored daily and mortality was recorded The cumulative mortality was calculated after challenge test Moribund fish was observed for the external and internal clinical signs Bacteria were re-isolated and identified by biochemical tests and

sequencing on 16S rRNA gene The median lethal dose (LD50) was finally determined

(Reed and Muech, 1938): logLD 50 = alogb + c, where a=(The mortality higher than

50%-50%)÷(The mortality higher than 50%-The mortality lower than 50%); b=dilution

rate; c= the log of minimum dilution rate, when the mortality higher than 50%

growth on A hydrophila specific-medium (AHM) were investigated Arabinose, aesculin,

mannitol, salicin were carried out by microbial biochemical identification tubes (Hangzhou Microbial Reagent Co., Ltd) following to manufacture’s instruction Two strains (D4 and HU201301) were used in tolerance of to salt environment at 28 oC for 24 hours Tolerance

to salt was measured by culturing bacterial colonies into sterile nutrient broth (2 mL) with 0.0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0 and 7.0% (w/v) NaCl added The growth bacterium in each test tube was measured by OD610

2.2.5 Antibiotic susceptibility test

The antibiotic susceptibility test was conducted on the basis of the method of disk diffusion previously described by Bauer et al (1966) A total 20 antimicrobial agents (Hangzhou Microbial Reagent Co., Ltd) including amoxicillin (10 µg/disc), florfenicol (75 µg/disc), levofloxacin (5 µg/disc), neomycin (30 µg/disc), tetracycline (30 µg/disc), novobiocin (30 µg/disc), doxycycline (30 µg/disc), gentamicin (10 µg/disc), norfloxacin (10 µg/disc), aztreonan (30 µg/disc), fortum (30 µg/disc), kanamycin (30 µg/disc), ceftriaxone sodium (30 µg/disc), ofloxacin (5 µg/disc), piperacillin (100 µg/disc), clindamycin (2 µg/disc), rifampicin (5 µg/disc), imipenem (10 µg/disc), erythromycin (15 µg/disc) and chloramphenicol (30 µg/disc) were used for investigation Two strains of bacteria, D4 and HU201301, were used in antibiotic susceptibility test A bacterial suspension (100 µL) was spread onto nutrient agar plates and then chemotherapeutic agent discs were placed on The plates were incubated at 28 oC for 24 h The diameter of each inhibition zone (including the diameter of the disc) was measured and recorded in mm The responses were illustrated, which is resistant (R), intermediate (I) or sensitive (S) according

to manufacturer’s recommendation

2000 (Thermo Scientific, USA) DNA was stored at -20oC until use Polymerase Chain Reaction (PCR) was performed on all strains Two universal primers 27F (forward: 5’-agagtttgatcctggctcag-3’) and 1492R (reverse: 5’-tacggctaccttgttacgactt-3’) (Sangon

Biotech (Shanghai) Co., Ltd.), were used to amplify 16S rRNA gene The PCR

amplifications were performed in a final volume of 10 µL containing 5 U Taq DNA polymerase, 1.0 µL 10× buffer (with Mg2+) for Taq DNA polymerase, 0.25 µL dNTP, 0.25

µL each primer and 50 ng genomic DNA A negative control (without template DNA) was also carried out The PCR amplification program was set with initial denaturation at 94 °C for 5 min, 30 cycles at 94 °C for 30 s, 58 °C for 45 s, 72 °C for 1.5 min, and followed by final extension step at 72 °C for 10 min The PCR products were analyzed by using electrophoresis in 1% agarose gels The PCR products were sequenced in Sangon Biotech (Shanghai) Co Ltd by the Sanger’s sequencing method using ABI 3730 The identity of

16S rRNA gene sequences was obtained based on BLAST search in GeneBank database

clinical signs of fish challenged to A hydrophila were similar to those of the naturally

infected fish recorded (Fig 2-1)

Fig 2-1 Clinical signs of BSB intraperitoneally challenged to A hydrophila

(1.7×10 8 cfu/mL) (A) Haemorrhage at the site of injection and ventral surface; (B) Red odiferous fluid, swollen and colour-deep spleen (arrow)

2.3.2 The cumulative mortality

The cumulative mortality of BSB challenged with A hydrophila at 7 days

post-inoculation was showed in Fig 2-2 The different mortalities were found in different bacterial suspension densities The mortality was recorded as 10, 10, 70 and 100% after bacterial intraperitoneal injection with 1.7×105, 1.7×106, 1.7×107 and 1.7×108 cfu/mL, respectively Non-died fish was recorded in negative control group during experiment carried out The median lethal dose (LD50) was 5×106 cfu/mL in intraperitoneal injection

of A hydrophila to BSB

Fig 2-2 Cumulative mortality of BSB challenged with A hydrophila

2.3.3 Re-isolation and re-identification

A total of 15 bacterial strains were re-isolated from spleen, liver and kidney of moribund artificially injected fish with clinical signs No evident external lesions or died fish in control group were recorded and no bacteria were isolated from either the control experimental fish

A similarity was found in biochemical characteristics between experimental original strain (D4) and re-isolated strains (HU201301, HU201302 and HU201303) shown in Table 2-1 For the morphological characteristics, these bacterial strains grew on nutrient agar at

28 oC for 24 hours with mucoid yellow color colonies All can growth on AHM medium at

28 oC Microscopic observation exhibited a phonotypical homogenization of short rod-shaped All were gram-negative, motile, oxidase-positive and catalase-negative Biochemical testing resulted in positive reaction of all tested bacterial strains for production of indole and H2S, utilization of citrate, hydrolysis of starch and esculin, fermentation of glucose, lactose, arabinose and mannitol and Methyl Red/Voges-Proskauer Nevertheless, negative reaction was shown for fermentation of sucrose and salicine in all tested strains

Table 2-1 Biochemical profile of D4 and re-isolated strains of A hydrophila

(+) positive reaction; (-) negative reaction

The effect of different NaCl concentrations on the growth of experimental original

strain (D4) and re-isolated strain (HU201301) of A hydrophila was checked and is shown

in Fig 2-3 The tested bacteria were able to tolerate up to 3% NaCl, but no growth of

bacteria was observed in nutrient medium containing 4-7% NaCl The optimum concentration of NaCl for growth of tested organisms was 1%

Fig 2-3 Effect of NaCl concentrations (%) on growth of D4 and HU201301

2.3.4 16S rRNA sequences analysis

The identification of A hydrophila based on morphological and biochemical

characteristics were carried out Subsequently, a number of colonies of strains incubated for

24 hours were chosen for PCR amplification PCR products of 16S rRNA gene of D4 (RID: BFNCE4D201R) and HU201301 (RID: BFP1K17601R) were sequenced to validate for A hydrophila identification A similarity of identity percentage (98%) with the nucleotide sequences of 16S rRNA gene of two strains was showed; and a high similarity (99% identity) to other available A hydrophila sequences in the GenBank database by using

BLAST network search

2.3.5 Antimicrobial susceptibility test

The pattern of antibiotic susceptibility of two strains of A hydrophila (D4 and

HU201301) is shown in Table 2-2 This present study found the relevant similarities between the original bacterial strain (D4) used in conducting challenge experiment and the re-isolated strain (HU201301) Both strains showed resistance to amoxicillin, and clindamycin and sensitive to novobiocin, florfenicol, levofloxacin, tetracylin, neomycin, doxycylin, gentamicin, norfloxacin, aztreonan, fortum, kanamycin, ceftriaxone sodium, ofloxacin, piperacillin, rifampicin, and chloramphenicol Interestingly, the D4 strain showed resistance to imipenem and sensitive to erythromycin, while HU201301 showed intermediate to those of antibiotic agents

Table 2-2 Antimicrobial susceptibility testing of experimental original strain (D4) and

re-isolated strain of A hydrophila HU201301

In this study, a challenge test with A hydrophila demonstrated similarities in the

clinical signs recorded by artificially challenged and naturally infected BSB The observed symptoms in the present study resembled those reported in other fish species (Duc et al., 2013; He et al., 2006; Nielsen et al., 2001; Topic et al., 2000) Three among fifteen strains re-isolated from tested fish were used to conduct experiment on biochemical tests Based

on biochemical characteristics, it was determined that the four strains (D4, HU201301, HU201302 and HU201303) of bacteria were homogenous, which is comparable to previous reports (Abbott et al., 2003; Lee et al., 2000) All four strains grew on the previously described AH medium (Kaper et al., 1979), used for species identification,

confirming that these bacterial strains are A hydrophila

The optimum concentration of NaCl for growth of D4 and HU201301 strains was close

to 1% and these strains were able to grow in concentrations up to 3% NaCl, classifying the strains as halotolerant (Marquez et al., 1987) Previously, it was reported that some strains

of A hydrophila were able to grow in concentrations up 4% NaCl (Delamare et al., 2000;

Palumbo et al., 1985) Thus, these strains can still inhabit a high salinity freshwater, as well

as brackish water

Molecular method for detecting of A hydrophila was introduced and applied in

numerous previous studies (Nielsen et al., 2001; Swaminathan et al., 2004; Yogananth et al.,

2009) The 16S rRNA was the coding region of ribosomal RNA, which was used for

phylogenetic studies (Weisburg et al., 1991) as it is highly conserved in bacteria and archaea species (Coenye and Vandamme, 2003) and as important taxonomic tools (Grimont

and Grimont, 1986) such as taxonomy of the genera Aeromonas (Chen et al., 2012b; Lee et

al., 2002; Liu and Li, 2012; Sarkar et al., 2012) Two universal primers, 27F and 1492R,

were developed in amplification of 16S rRNA genes and had allowed discriminating of

identification up to the species level and typing of other bacteria (Jiang et al., 2006; Sarkar

et al., 2012) It has been indicated previously that levels of similarity between 16S rRNA

gene sequences higher than 97% suggest that the strains in question belong to the same species (Stackebrandt and Goebel, 1994), so a 98% identity of sequences of the D4 and HU201301 strains implies that they belong to the same species This challenge test experiment did fulfill Koch’s postulates (Walker et al., 2006)

The high cumulative mortality (100%) of BSB (challenged with 1.7×108 cfu/mL by intraperitoneal injection method) in this study was comparable to the results reported

(Mostafa et al., 2008) The authors have challenged Heteropneustes fossilis with A hydrophila at a dose of 9.6×107 cfu/fish using intraperitoneal and intramuscular injection, which also resulted in 100% mortality of the tested fish within 1 -9 days Similarly, the reported mortality of some other fish species challenged by intraperitoneal injection with

lower bacterial density, such as walking catfish C batrachus with 106 cfu/fish (Angka,

1990) and snakehead C striata with 106 cfu/fish (Duc et al., 2013) was 100% Also,

several fish species (Labeo rohita, Catla catla and Cirrhinus cirrhosus) challenged by

intramuscular method at a dose of 2×106 cfu/fish had 80 to 100% mortality, while H

(Sabur, 2006) (cited by Sarkar and Rashid (2012)) The LD50 value in this study was 5×105

cfu/fish, similar to the LD50 of A hydrophila challenged to snakehead fingerling (Duc et al., 2013), and lower than for some other fish species, such as Anguilla anguilla (106.2 to

107.4 cfu/fish) (Esteve et al., 2004), Channa punctatus (3.42×107 cfu/fish) (Yesmin et al.,

2004) and Brycon amazonicus (6.66×1011 cfu/fish) (de Oliveira et al., 2011), indicating that isolates used in challenge test were strongly virulent to BSB Surprisingly, a previous study has reported higher LD50 for BSB after artificial infection with A hydrophila (Luo et al., 2014b) This can be explained by the fact that the infection of A hydrophila in fish varies

depending on resistance ability (Schlotfeldt and Alderman, 1995) and the size of the fish (de Oliveira et al., 2011) Indeed, compared to the weight of the fish used in this study (27.1±8.3 g), compared with previous study has used larger BSB specimens (150-200 g) (Luo et al., 2014b)

Chemotherapeutic agents were previously introduced to treat A hydrophila infection

in cultured fish (Saitanu and Chularak, 1983) In this study, the antibiotic sensitivity patterns of two strains (D4 and HU201301) were evaluated and confirmed to be mostly similar This test was conducted in order to confirm the resemblance of re-isolated strain (HU201301) and experimental inoculated bacterial strain (D4) in sensitive ability to antimicrobials, and also to determine the suitable therapeutical medicines for controlling

Aeromonas infection in BSB Both strains were sensitive to most tested drugs, except for

amoxicillin and clindamycin D4 was also resistant to imipenem, while HU201301 was

only intermediately resistant to this drug In previous studies, A hydrophila was reported to

be sensitive to chloramphenicol, erythromycin, kanamycin, neomycin (Boonyaratpalin, 1989) and resistant to amoxicillin and clindamycin (Adanir and Turutoglu, 2007; Belém-Costa and Cyrino, 2006; Jayavignesh et al., 2011) The results from the present study were similar to these, but different from the results reported (Son et al., 1997;

Vivekanandhan et al., 2002), A hydrophila was resistant to chloramphenicol, erythromycin,

kanamycin, tetracycline, rifampicin and novobiocin

The susceptibility of the experimental bacterial strains (D4 and HU201301) to some beta-lactam antibiotics was also investigated In comparison to a previous report (Morita et al., 1994), the results showed the similarity in susceptibility to aztreonam and imipenem, and difference in resistance to piperacillin and ceftriaxone These differences may be related to the source of bacterial isolates and the source of antibiotic agents used in the experiment Moreover, it is known that the bacteria can receive and transmit antibiotic

resistant genes from and to other gram-negative bacteria (Marchandin et al., 2003) Thus, aeromonad-infection in BSB may still be controlled by using correct drugs Nevertheless, decreasing the use of chemicals in aquaculture industry is considered a good management practice Some types of chemicals, especially antibiotics, if used inappropriately, not only cause damages to animals and the environment, but also increase production costs and adverse consequences (Tonguthai, 2000) Furthermore, the observation of disease outbreak usually correlates with anorexia (Duc et al., 2013); hence the utilization of antibiotics at the disease outbreak time may not be effective Also, the extensive use of antibiotic and other chemotherapeutics to prevent and treat fish diseases may cause drug-resistance phenomenon in cultured fish (Son et al., 1997) Vaccination treatment was recommended for use in disease prevention in cultured fish (Bakopoulos et al., 1995;

Sommerset et al., 2005) and herbal concoction was used in the therapy of A hydrophila

infection in goldfish (Harikrishnan et al., 2009)

In conclusion, in this study it was demonstrated A hydrophila as a potential pathogen,

which can cause hemorrhagic septicemia clinical signs in BSB In the antimicrobial susceptibility test, both tested bacterial strains showed sensitivity to most used antibiotic drugs