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Vibrio Harveyi Isolates 1 Detection and characterisation of Vibrio harveyi isolates Katarina S. Themptander BSc Biomedical Sciences May 2005 School of Biological Sciences Dublin Institute of Technology, Kevin Street, Dublin 8 2 ABSTRACT Aim Because of the major problems that certain Vibrio specie, especially Vibrio harveyi, can cause the aquaculture industries a rapid method to identify Vibrio isolates is required. Early diagnosis of a V. harveyi infection could facilitate disease surveillance, treatment and prevention in cultured marine animals. Therefore, the use of PCR to aid in the identification of Vibrio is increasing and a way of extracting DNA in a cheap, fast and easy way is also of an important requirement to facilitate rapid diagnosis. Methods This report comprises biochemical profiling and PCR methods in the characterisation of four isolates of V. harveyi and single isolates of V. tubiashii, V. alginolyticus, V. anguillarum, V. splendidus, V. tapetis and V. parahaemolyticus. Strains were examined for adherence to a Hep-2 cell line. Four different DNA extraction methods were evaluated and compared. The detection limits and the analytical limits of two PCR methods for Vibrio were determined. Results The overall findings were that the use of a greater range of biochemical substrates than are in the API 20E is necessary to identify Vibrio strains, and that none of the strains tested adhered to Hep-2 cells. All extraction methods successfully produced DNA with the kit method giving the purest samples. RNA was a contaminant of the other techniques but this could be overcome by treating extracts with RNase. The rapid microwave extraction method gave appropriate PCR amplicons when tested. Conclusion PCR determination of the VH-sequence in combination with VHA and a distinguishable colonial morphology may be a good choice for the identifying of Vibrio harveyi. 3 TABLE OF CONTENTS 1.0 INTRODUCTION PAGE No. 1.1: Vibrionaceae 1 1.2. Reservoir and transmission 2 1.3: Vibrio species as fish pathogens 2 1.4: Clinical features of Vibrio infection in aquatic animals 4 1.5: Virulence factors of Vibrio species 4 1.6: Treatment 6 1.7: Isolation and identification of Vibrio species 6 2.0 MATERIAL AND METHODS 2.1 Material 2.1.1: Bacterial isolates 9 2.1.2: Growth media 10 2.1.3: Apparatus 11 2.1.4: Equipment 11 2.1.5: Biochemical identification of bacterial isolates 12 2.1.6: DNA extraction 12 2.1.6.i: Phenol: chloroform extraction 12 2.1.6.ii: High-Pure PCR Template Preparation Kit 13 2.1.7: Agarose Gel Electrophoresis 13 2.1.8: PCR (polymerase chain reaction) 13 2.2 Methods 2.2.1: Biochemical characterisation 15 4 2.2.1.i: General biochemical characterisation 15 2.2.1.ii: Comparison of Vibrio growth on four different media 15 2.2.1.iii: API 20E, biochemical characterisation 15 2.2.2: Tissue adherence test 17 2.2.3: DNA extraction 18 2.2.3.i: Phenol: chloroform extraction according to Montes et al., 2003 18 2.2.3.ii: High-Pure PCR Template Preparation Kit 18 2.2.3.iii: Boiling 19 2.2.3.iv: Microwaving 20 2.2.4: DNA quantification 20 2.2.5: Agarose Gel Electrophoresis 20 2.2.5.i: Visualisation of extracted DNA 20 2.2.5.ii: Visualisation of PCR-products 21 2.2.6: PCR (polymerase chain reaction) 21 2.2.6.i: [MgCl2] optimization 22 2.2.6.ii: 16S rDNA sequence determination 22 2.2.6.iii: VH Amplification 23 2.2.6.iv: Diagnostic Detection Limit 23 2.2.6.v: Analytical Sensitivity 23 3.0 RESULTS 3.1: Phenotypic identification 25 3.1.1: Morphology 25 3.1.3: Tissue adherence test 28 3.2: Biochemical characteristics 29 5 3.2.1: General characteristics 29 3.2.2: API E20, biochemical characterisation 29 3.3: DNA extraction 31 3.3.1: Visualisation of genomic DNA from the four different extracting 32 methods using gel electrophoresis 3.4: 16S rDNA gene determination 34 3.4.1: [MgCl2] optimization 34 3.4.2: Amplicon determination using the 16S rDNA-primers 35 3.5: PCR using V. harveyi VH-1 and VH-2 primers 36 3.5.1: [MgCl2] optimization 36 3.5.2: Amplicon determination using VH-primers 38 3.6 Diagnostic Detection Limit 40 3.7 Analytical Sensitivity 41 4.0 DISCUSSION 43 5.0 CONCLUSION 53 6.0 REFERENCES 54 7.0 APPENDIX 57 6 ACKNOWLEDGEMENTS I would like to extend my appreciation to the following people, who all played their part in the realisation of this project. I would first like to thank my supervisor Mr Patrick McHale for all his support and patience and for guiding me through this project. Thank you for proofreading and being so much help. I would also like to thank you Mr Ted Doody for ordering all the reagents I needed and for being so helpful. And thank you Fergus Ryan for the help with the molecular work. A special thanks to Leanne Harris; thank you for all the help and encouragements. Thank you for lending me reagents and for good advices. And also big thank you to the rest of the people in the lab for being so helpful and making me feel welcome. Last but not least I would like to thank my parents, Jan and Maggie, and my brother Christofer, for their continued support, patience and endless encouragements throughout my education. 7 1.0 INTRODUCTION 1.1 Vibrionaceae Vibrionaceae are a family of Gram-negative rod-shaped bacteria. They are facultative anaerobes and have a fermentative metabolism. They are separated from the family Enterobacteriaceae because of a positive oxidase reaction and the presence of polar flagella. The taxonomy of the group is in the process of revision due to increasing data obtained with modern molecular biology techniques, where different genes are examined or where the whole genome is inspected. Special emphasis has been paid to the 16S rRNA gene, although other genes, such as the 23S rRNA and gyrB genes, have also been employed (Dorsch et al., 1992; Gomez-Gil et al., 2004). The sequencing of the 16S rRNA gene is considered the most reliable tool for the allocation of genera, species, and strains into the family Vibrionaceae. Following this approach the former three genera (Vibrio, Aeromonas and Plesiomonas) in the family (Murray et al., 2002) have been replaced by eight genera, including Vibrio, in the outline of Bergey’s Manual of Systematic Bacteriology (Garrity and Holt, 2000). To facilitate further studies of vibrios Thompson proposed spliting the Vibrionaceae into three new families (Thompson et al., 2004). The newly proposed family Vibrionaceae comprises only the genus Vibrio, with 63 species. The number of Vibrio species is increasing with new species being described every year (Thompson et al., 2004). The family is heterogeneous and may require further splitting. 8 1.2 Reservoir and transmission Vibrios are ubiquitous in aquatic environments and in association with eukaryotes. They appear at particularly high densities in and/or on marine organisms including corals, fish, molluscs, seagrass, sponges, shrimp and zooplankton (Thompson et al., 2004). The exact route by which Vibrio infect fish is unknown but oral transmission is suspected. The organisms may, in certain circumstances, be able to cross the intestinal wall and cause systemic disease. During outbreaks the numbers of infectious particles in the environment will increase dramatically increasing the likelihood that fish will become contaminated. 1.3 Vibrio species as fish pathogens The first known Vibrio species, V. cholerae, was discovered in 1854 by the Italian physician Filippo Pacini while he was studying outbreaks of cholera in Florence. V. cholerae, Vibrio parahaemolyticus and Vibrio vulnificus are the most serious human pathogens and, in all, twelve Vibrio species known today are involved in human diseases (Murray et al., 2002). Many Vibrio species have also been implicated in aquaculture infections. They are the most important pathogens for reared aquatic organisms such as penaeid shrimps and for several fish species and molluscs, as well as for corals (Gomez-Gil et al., 2004). The species associated with disease in fish and shellfish include Vibrio harveyi, Vibrio alginolyticus, Vibrio splendidus 1 and 2, Vibrio tapetis, Vibrio tubiashii, and Vibrio anguillarum. Different species infect different types of host animals. In marine fish diseases the main pathogens are V. anguillarum, Vibrio salmonicida and V. vulnificus (Thompson et al., 2004). The closely related species V. harveyi and V. campbellii have caused disease in 9 shrimp larvae while Vibrio penaeicida and V. parahaemolyticus have infected juveniles and adults. V. harveyi is one of the most commonly isolated marine Vibrio species, and can easily be found both as free-living organisms or associated with the normal intestinal microflora of marine animals (Hernandez et al., 2004). Moreover, it is the dominant heterotrophic species in western Mediterranean seawater and marine bivalves during the warm season. V. harveyi has been recognised as pathogenic for several crustacean larvae, particularly, Penaeus species, and is considered responsible for mass mortalities of the bivalve Pinctada maxima. It has been related to several opportunistic infections of ornamental or edible cultured fish in the last decade, and recent reports confirm the virulence of some strains for gilthead sea bream, silver mullet, salmon and seahorse (Pujalte et al., 2003). Although some strains of V. harveyi are highly pathogenic other strains may be considered as opportunistic pathogens. Those that do cause disease cause pose a severe threat to the multi-billion dollar aquaculture industry (Oakey et al., 2003). V. carchariae was first reported as a pathogen in aquaculture in 1984 after it had been isolated from a brown shark found dead in captivity.
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