Aquaculture Microbiology

Preface
Contents
Contributors
Part I: Isolation and Identification of Pathogens from Fishes
	Chapter 1: Isolation and Identification of Aeromonas sp. from Fishes
		1 Introduction
		2 Materials
			2.1 Isolation of Aeromonas from Infected  Fish
			2.2 Staining and Biochemical Methods
			2.3 Molecular Identification
		3 Methods
			3.1 Fish Sampling
			3.2 Isolation of Bacteria
			3.3 Identification of Aeromonas Bacteria
			3.4 Characterization of Aeromonas Bacteria
				3.4.1 Hemolytic Activity
				3.4.2 Proteolytic Activity
				3.4.3 Lipolytic Activity
				3.4.4 Nuclease Activity
				3.4.5 Congo Red Dye Uptake
				3.4.6 Molecular Identification
				3.4.7 Antimicrobial Susceptibility
		References
	Chapter 2: Isolation and Identification of Edwardsiellosis-Causing Microorganism
		1 Introduction
		2 Materials
			2.1 Isolation of  Edwardsiella from Infected Fish
			2.2 Staining and Biochemical Methods
			2.3 Molecular Identification
		3 Methods
			3.1 Isolation of the Edwardsiella spp.
				3.1.1 Isolation of E. tarda, E. piscicida, and E. hoshinae Species
				3.1.2 Isolation of Edwardsiella spp.
			3.2 Preliminary Identification of Isolates
				3.2.1 Gram Staining
				3.2.2 Biochemical Tests [12]
					Catalase  Test
					Oxidase  Test
					Triple Sugar Ion  Test
					Indole Production  Test
					Simmons´ Citrate  Test
					Lysine Decarboxylase  Test
				3.2.3 Rapid Identification Using Commercial API20E Kit
				3.2.4 Molecular Identification of Isolates
					DNA Isolation of Edwardsiella sp.
					Pathogen Strain from Fishes
					DNA Extraction Directly from Spleen
					Identification Using Conventional PCR Assay
					Identification Using Recombinase Polymerase Amplification (RPA) Assay
				3.2.5 Identification of Edwardsiella spp. Isolates by ELISA
					Extraction of Whole Cell Protein (WCP)
					Production of Polyclonal Antibodies in Rabbit
					SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) Analysis
					Western Blotting of WCP Antigen
					Dot-ELISA for Species Identification
		References
	Chapter 3: Methods for Characterizing Flavobacterium in Fish
		1 Introduction
		2 Materials
			2.1 Isolation of Flavobacterium spp. from  Fish
			2.2 Staining and Biochemical  Test
			2.3 Molecular Identification
		3 Methods
			3.1 Isolation of Flavobacterium spp. from  Fish
		4 Identification
			4.1 Technical Count
				4.1.1 Total Bacterial Count
				4.1.2 Microscopy
			4.2 Gram Staining
			4.3 Motility  Test
			4.4 Gliding Motility  Test
			4.5 Biochemical  Test
			4.6 API 20E Commercial Identification  Kit
		5 Molecular Identification
			5.1 DNA Extraction
			5.2 Polymerase Chain Reaction (PCR)
			5.3 Product Purification Before Sequencing
			5.4 Sequencing
			5.5 Phylogenetic Analyzing
		References
	Chapter 4: Isolation and Identification of Citrobacter Species
		1 Introduction
		2 Materials
			2.1 Isolation of Citrobacter from Infected  Fish
			2.2 Staining and Biochemical Methods
			2.3 Molecular Identification
		3 Methods
			3.1 Fish Sampling
			3.2 Isolation of Citrobacter
			3.3 Identification of Citrobacter Bacteria
			3.4 Rapid Identification Using Commercial API20E Kit
		4 Molecular Identification of Citrobacter sp.
			4.1 DNA Isolation
			4.2 16S rRNA Sequencing
		5 Biochemical Characterization (Table 1)
		References
	Chapter 5: Isolation and Identification of Infectious Salmon Anemia Virus from Shrimp
		1 Introduction
		2 Materials
		3 Methods
			3.1 Dissection of Various Organs from shrimps infected with Infectious Salmon Anemia Virus
			3.2 Isolation of Infectious Salmon Anemia Virus from Cell Lines
				3.2.1 Sample Preparation
				3.2.2 Inoculation on Cell lines
			3.3 Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
				3.3.1 Extraction of  RNA
				3.3.2 cDNA Synthesis
				3.3.3 RT-PCR Assay
		References
	Chapter 6: Isolation and Identification of Betanodavirus from Shrimp
		1 Introduction
		2 Materials
		3 Methods
			3.1 Dissection of Various Organs from Shrimp
			3.2 Isolation of Betanodavirus Using Shrimp Cell Lines
				3.2.1 RNA Extraction
				3.2.2 cDNA Synthesis
			3.3 RT-PCR
		References
	Chapter 7: Isolation and Identification of Hemorrhagic Septicemia Virus from Shrimp
		1 Introduction
		2 Materials
		3 Methods
			3.1 Dissection of Organs from Hemorrhagic Septicemia Virus
			3.2 Isolation of Hemorrhagic Septicemia Virus Using Cell Line
			3.3 Inoculation of the Cell Monolayers
		4 Hemorrhagic Septicemia Virus Identification
			4.1 Real-Time Polymerase Chain Reaction (RT-PCR) Detection
				4.1.1 RNA Extraction
				4.1.2 cDNA Synthesis
				4.1.3 RT-PCR
		References
	Chapter 8: Isolation and Identification of Pathogens from Fish: Tilapia Lake Virus (TiLV)
		1 Introduction
			1.1 Tilapia Lake Virus
		2 Materials
			2.1 Maintenance of Fish and Reinfection
			2.2 RNA Extraction
			2.3 cDNA and RT-PCR
			2.4 Diagnosis of TiLV
			2.5 Morphological Examination of TiLV
		3 Methods
			3.1 Isolation of Tilapia tilapinevirus (TiLV)
			3.2 Preparation of TiLV Homogenate
			3.3 RNA Extraction and RT-PCR
			3.4 Diagnosis of TiLV
				3.4.1 Nested  PCR
				3.4.2 Semi-nested  PCR
				3.4.3 Quantitative Real-Time PCR for TiLV Diagnosis
				3.4.4 RT-LAMP
				3.4.5 In Situ Hybridization
			3.5 Morphological Examination of TiLV
				3.5.1 Clinical Observation
				3.5.2 Histology
				3.5.3 Transmission Electron Microscope
		References
	Chapter 9: Isolation and Identification of Pathogens from Shrimp: IHHNV
		1 Introduction
		2 Materials
			2.1 PCR Mixture
			2.2 Agarose Gel Preparation
			2.3 Lamp  PCR
			2.4 G.HCl Preparation
			2.5 TBE Buffer
		3 Extraction of IHHNV from Penaid Shrimp
			3.1 DNA Extraction
			3.2 Polymerase Chain Reaction (PCR) Amplification
			3.3 Post-PCR Analysis
			3.4 Sequence Comparison and Phylogenetic Analysis
			3.5 Real-Time  PCR
			3.6 Nested  PCR
			3.7 LAMP Reaction
			3.8 Virus Isolation by Cell Culture
			3.9 Histopathology
			3.10 In Situ Hybridization
			3.11 TEM Analysis
		References
	Chapter 10: Isolation and Identification of Ichthyophonus hoferi from Fishes
		1 Introduction
		2 Materials
		3 Methods
			3.1 Isolation of Ichthyophonus hoferi [4]
				3.1.1 Tissue Culture
				3.1.2 Mycological and Bacteriological Examination
					Bacteriological Examination
					Mycological Examination
			3.2 Identification of Ichthyophonus hoferi
				3.2.1 Microscopic Examination
			3.3 Polymerase Chain Reaction (PCR) Detection
				3.3.1 Extraction of DNA
				3.3.2 PCR Assay
		References
	Chapter 11: Isolation and Identification of Branchiomyces demigrans from Fishes
		1 Introduction
		2 Materials
		3 Methods
			3.1 Isolation of Branchiomyces demigrans
			3.2 Identification of Branchiomyces demigrans
				3.2.1 Mycological Examination
				3.2.2 Histopathological Examination
				3.2.3 Morphometry of Gills
				3.2.4 Molecular Detection Using Polymerase Chain Reaction (PCR)
					Extraction of  DNA
					PCR Assay
		References
	Chapter 12: Isolation and Identification of Microsporidian Parasites, Enterocytozoon hepatopenaei Infection of Penaeid Shrimp
		1 Introduction
		2 Materials and Methods
			2.1 Isolation of Microsporidian Spores from Shrimp Hepatopancreas and Fecal Matters
			2.2 Identification: Molecular Methods
				2.2.1 DNA Extraction
			2.3 Amplification of EHP Targeted Genome Will Perform in Thermal Cycler. The Standard Conventional PCR 20 μL Reaction (Karthik...
			2.4 Quantitative Polymerase Chain Reaction
			2.5 Identification: Microscopy Examination
				2.5.1 Hematoxylin and Eosin Stain
				2.5.2 Modified Trichrome Stain
				2.5.3 Fluorescent Staining: Chitin Staining
				2.5.4 Calcofluor White Stain
				2.5.5 Transmission Electron Microscopy (TEM)
		References
Part II: Isolation and Identification of Probiotic Bacteria in Aquaculture
	Chapter 13: Methods for Isolating and Identifying Probiotic Bacteria from Fishes
		1 Introduction
			1.1 Probiotics and Probiotic Bacteria
			1.2 Need for Probiotics in Fisheries
			1.3 Isolation of Probiotics
		2 Materials
			2.1 Media Composition
				2.1.1 Lactobacillus deMan, Rogosa, and Sharpe Agar (MRS):
				2.1.2 Tryptic Soy  Agar
				2.1.3 Nutrient  Agar
				2.1.4 Miller Luria Bertani Broth
				2.1.5 Zobell Marine  Agar
			2.2 Fine Chemicals
			2.3 Molecular Biology Reagents
		3 Methodology
			3.1 Sample Collection
			3.2 Isolation
				3.2.1 Serial Dilution and Plating (Fig. 1)
				3.2.2 Purification of Isolates
			3.3 Preliminary Identification Tests [30]
			3.4 Phenotypic Characterization
				3.4.1 Growth of Bacterial Isolates at Different Temperatures
				3.4.2 Growth of Bacterial Isolates at Different pH
				3.4.3 Growth of Bacterial Isolates at Different Salt Concentrations
				3.4.4 Sugar Fermentation  Test
			3.5 Molecular Identification
				3.5.1 Total DNA Isolation [35]
				3.5.2 Agarose Gel Electrophoresis
				3.5.3 Random Amplified Polymorphic DNA (RAPD-PCR)
				3.5.4 16S rRNA Gene Sequencing
		References
	Chapter 14: Isolation of Probiotic Bacteria from Gut of the Aquatic Animals
		1 Introduction
		2 Materials
			2.1 Collection of Sample
			2.2 Isolation of Intestinal Bacteria
			2.3 Antagonistic Effect of Isolated Bacteria
			2.4 Identification of Probiotic Bacteria
				2.4.1 Morphological and Biochemical Characteristics
				2.4.2 Molecular Identification
		3 Methods
			3.1 Collection of Sample
			3.2 Isolation of Intestinal Bacteria
			3.3 Antagonistic Effect of Isolated Bacteria
			3.4 Identification of Probiotic Bacteria
				3.4.1 Morphological and Biochemical Characteristic
				3.4.2 Molecular Identification
		References
	Chapter 15: Characterization of Probiotic Properties of Isolated Bacteria
		1 Introduction
		2 Materials
			2.1 Media Preparation
				2.1.1 Mueller Hinton  Agar
				2.1.2 Blood  Agar
				2.1.3 Peptone-Gelatin  Agar
				2.1.4 Starch  Agar
				2.1.5 Tributyrin  Agar
				2.1.6 Carboxymethyl Cellulose  Agar
			2.2 Chemicals and Reagents
		3 Methodology
			3.1 Safety Tests
				3.1.1 Hemolytic Activity
				3.1.2 Antibiotic Susceptibility  Test
			3.2 Probiotic Properties
				3.2.1 Antimicrobial Activity
				3.2.2 Tolerance to GIT Conditions
					Acid Tolerance
					Bile Tolerance
					Tolerance to Simulated Gastrointestinal Fluids
				3.2.3 Bile Salt Hydrolase (BSH) Assay
				3.2.4 Adhesion Assays
					Cell Surface Hydrophobicity Assay
					Auto-aggregation
					Co-aggregation
					In Vitro Cell Adhesion Assay
				3.2.5 Production of Extracellular Enzymes
		References
	Chapter 16: Antibacterial Activity of Probiotic Bacteria from Aquaculture
		1 Introduction
		2 Materials
		3 Methodology
			3.1 Sample Collection and Processing
			3.2 Agar Overlay Assay
				3.2.1 Method-I
				3.2.2 Method-II
			3.3 Cell-Free Supernatant (CFS) Preparation
			3.4 Antibacterial Activity by Agar Well Diffusion Assay
				3.4.1 Determination of Bacteriocin Activity
			3.5 Growth Dynamics and Bacteriocin Production
			3.6 Broth Microdilution Assay
			3.7 Time-Kill Assay
			3.8 Time-Kill Co-culture Assay
			3.9 Scanning Electron Microscopy
			3.10 Purification of Bacteriocin
			3.11 Characterization of Bacteriocin
				3.11.1 Determination of Molecular Weight
				3.11.2 Sensitivity of Probiotic-Generated Antibacterials to Microenvironments
		References
Part III: Methods for Treating Infections in Fishes and Shrimps
	Chapter 17: Preparation of Marine Algal (Seaweed) Extracts and Quantification of Phytocompounds
		1 Introduction
		2 Materials
			2.1 Reagents and Raw Materials Required
			2.2 Collection of Seaweeds
		3 Methods
			3.1 Processing of Seaweed Extract
			3.2 Extract Preparation (Conventional Method)
			3.3 Phytochemical Detection in Seaweed Extracts
				3.3.1 Alkaloids
				3.3.2 Flavonoids
				3.3.3 Carbohydrates
				3.3.4 Test for Quinine
				3.3.5 Test for Glycosides
				3.3.6 Test for Triterpenes
				3.3.7 Phenolics
				3.3.8 Proteins
				3.3.9 Phytosteroids and Steroids
				3.3.10 Phlobatannins
				3.3.11 Anthraquinones
		References
	Chapter 18: Treating Bacterial Infections in Fishes and Shrimps Using Seaweed Extracts
		1 Introduction
		2 Materials
			2.1 Collection of Experimental Fishes [6]
			2.2 Isolation of Bacterial Pathogen
			2.3 Preparation of Bacterial Inoculum
		3 Methods
			3.1 Maintenance of Experimental Animals [7]
			3.2 Collection and Processing of Seaweeds
			3.3 Antibacterial Activity
			3.4 Pathogenicity and Treatment of Seaweed Extracts
		References
	Chapter 19: Preparation and Treatment of Seaweed Encapsulated Pellet Feed in Fisheries Aquaculture
		1 Introduction
		2 Materials
			2.1 Collection of Seaweeds and Experimental Fishes [11]
			2.2 Extraction of Active Compounds
		3 Methods
			3.1 Preparation of Bioactive Compounds
			3.2 Encapsulation of Active Compounds
			3.3 Feed Treatment
		References
	Chapter 20: Treatment Using Seaweeds in Fishes and Shrimp by In Vivo Method
		1 Introduction
		2 Materials
			2.1 Collection of Seaweeds
			2.2 Preparation of Seaweed Extract
				2.2.1 In Vitro Antibacterial Activity
			2.3 Treatment Using Seaweed Extract
				2.3.1 Bioassay
			2.4 Treatment Using Pellet  Feed
				2.4.1 Preparation of Seaweed  Diet
				2.4.2 Feeding Trail
			2.5 Determination of Immune Parameters After Bioactive Compound Administration
				2.5.1 Blood Sample Analysis
		3 Methods
			3.1 Collection of Seaweeds
			3.2 Preparation of Seaweed Extract
			3.3 In Vitro Antibacterial Activity
			3.4 Treatment Using Seaweed Extract
				3.4.1 Bioassay
				3.4.2 GC-MS Analysis of Seaweed Extract for Identification of Bioactive Compounds
				3.4.3 FTIR Analysis for Identification of a Functional Group
			3.5 Treatment Using Pellet  Feed
				3.5.1 Preparation of Seaweed  Diet
				3.5.2 Feeding Trail
				3.5.3 Growth Performance
			3.6 Determination of Immune Parameters After Bioactive Compound Administration
				3.6.1 Blood Sample Analysis
				3.6.2 RT-PCR Analysis
				3.6.3 Histological Analysis
		References
Part IV: Treatment Using Medicinal Plants
	Chapter 21: Treatment Using Medicinal Plants in Fish and Shrimp
		1 Background
		2 Soxhlet Method
			2.1 Materials
			2.2 Methods
		3 Digestion
			3.1 Materials
			3.2 Methods
		4 Plant Dye Extraction
			4.1 Materials
			4.2 Methods
			4.3 Source of Medicinal Plant
			4.4 Preparation of Fish Feed Formulations
			4.5 Administration of Medicinal Plants in Aquaculture
			4.6 Bacterial Diseases of Fish and Shrimp
		5 Materials and Methods
			5.1 Preparation of Herb Extracts
			5.2 Antibacterial Tests
			5.3 Efficiency of Herbs
			5.4 Viral Diseases of Fish and Shrimp
				5.4.1 Preparation of Viral Inoculum
				5.4.2 Preparation of Plant Extracts
				5.4.3 Determination of Antiviral Activity
		References
	Chapter 22: Preparation of Feed and Characterization of Feed Supplemented with Phytocompounds
		1 Introduction
		2 Materials
		3 Methods
			3.1 Preparation of Feed Supplemented with Phytocompounds
			3.2 Aqua Feed Characterization
		4 Conclusion
		References
Part V: Aquatic Actinomycetes and Copepods
	Chapter 23: Isolation and Identification of Actinomycetes
		1 Introduction
		2 Materials
			2.1 Isolation of Actinomycetes
		3 Actinomycetes Identification
			3.1 Staining Methods
			3.2 Biochemical Tests
			3.3 Molecular Identification
		4 Methods
			4.1 Pretreatment of Marine Sediment Samples
			4.2 Isolation of Actinomycetes from Marine Sediments/Soil
			4.3 Identification of Actinomycetes
				4.3.1 Staining Methods and Biochemical Tests
				4.3.2 Scanning Electron Microscopy (SEM) Analysis
				4.3.3 Molecular Identification
		References
	Chapter 24: Assay of Hemolytic Activity
		1 Introduction
		2 Materials
		3 Methods
			3.1 Hemolytic Assay [7]
			3.2 In Vitro Hemolytic Assay
		References
	Chapter 25: Cytotoxicity Assay
		1 Introduction
		2 Materials
		3 Methods
			3.1 Assay of Cytotoxicity
		References
	Chapter 26: Antibacterial Activity and Extraction of Bioactive Compound from Actinomycetes
		1 Introduction
		2 Materials
		3 Methods
			3.1 In Vitro Antibacterial Activity
				3.1.1 Primary Screening
				3.1.2 Secondary Screening
			3.2 Agar Well Diffusion Method
			3.3 Extraction of the Bioactive Compound
		References
	Chapter 27: Isolation and Identification of Harpacticoid Copepod
		1 Introduction
		2 Materials
			2.1 Collection of Zooplankton Sample
			2.2 Isolation of Harpacticoid Copepod
			2.3 Identification of Harpacticoid Copepods
				2.3.1 Morphology
				2.3.2 Camera Lucida Drawings
				2.3.3 SEM Analysis
		3 Methods
			3.1 Collection of Zooplankton Sample
			3.2 Isolation of Harpacticoid Copepod
			3.3 Identification of Isolated Harpacticoid Copepod
				3.3.1 Morphology
				3.3.2 Camera Lucida Drawings
				3.3.3 SEM Analysis
		References
Index