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42543039.Pdf ENVIRONMENTAL FACTORS AFFECTING THE PATHOGENESIS OF Edwardsiella ictaluri IN STRIPED CATFISH Pangasianodon hypophthalmus (Sauvage) THESIS SUBMITTED TO THE UNIVERSITY OF STIRLING FOR THE DEGREE OF DOCTOR OF PHILOSOPHY BY NGUYEN NGOC PHUOC 31 MARCH 2014 INSTITUTE OF AQUACULTURE Declaration I hereby declare that the work and results presented in this thesis has been composed in its entirety by me. Except where specifically acknowledged, the work described in this thesis has been conducted by me and has not been submitted for any other degree or qualification. Signature: ________________________________________ Signature of supervisor: ________________________________________ Date: ________________________________________ i ABSTRACT Bacillary Necrosis of Pangasius (BNP) caused by Edwardsiella ictaluri is considered to be the most serious disease occurring in farmed striped catfish (Pangasianodon hypophthalmus) in Vietnam. This disease has had an increasing impact over the last ten years and has been reported to cause 50-90% mortality of stocks during a single outbreak. Data obtained from natural outbreaks of E. ictaluri in striped catfish showed the role of environmental factors in the establishment and progression of this disease. At present, factors affecting the virulence and transmission of E. ictaluri in striped catfish are poorly understood. The central hypothesis of this thesis focuses on the complex picture of the environmental factors and infectivity of E. ictaluri in striped catfish. In this study, 80 isolates of E. ictaluri recovered from natural clinical disease outbreaks occurring in striped catfish farms between 2002 and 2011 located in 4 distinct geographical areas within Vietnam were characterised using a variety of methods. The biochemical profiles showed that E. ictaluri isolates from striped catfish in Vietnam have similar phenotypic characteristics to other E. ictaluri isolates from other infected fish species. These data showed high levels of phenotypic homogeneity between the E. ictaluri isolates investigated. The status of isolates recovered from natural infections over time and from geographically distinct farms was evaluated using pulsed-field gel electrophoresis (PFGE), plasmid profile identification and antibiotic sensitivity tests. The PFGE results showed 6 main groups with a similarity of 82% and the corresponding genotypes of the prevalent isolates illustrated annual differences. Three plasmid ii groups were identified distributed among the isolates investigated, in which high molecular weight plasmids of approximately 35 and 140 kb were found in two of the groups. Plasmid profiles of the present study did not show any trend of geographical region or year of isolation. The 140 kb plasmid has been considered as a multi- antibiotic resistance plasmid which confers resistance to tetracycline, trimethoprim and sulphonamides. All Vietnamese isolates showed a high level of resistance to Oxolinic acid, Sulfadimethoxine/Ormetoprim (Romet), Oxytetracycline and Amoxicillin. A reproducible bacterial immersion challenge model was developed and the LD60 estimated prior to performing subsequent experimental challenge studies. Fish were exposed to 107 cfu ml-1 of E. ictaluri by immersion for up to 30 seconds, resulting in a cumulative percentage mortality of 63%. Edwardsiella ictaluri was recovered and identified from all the dead and moribund fish during these experiments and affected fish showed similar clinical signs and pathology to those reported from natural E. ictaluri infections. The present study resulted in a successful experimental immersion challenge model for E. ictaluri infection in healthy striped catfish. Cohabitation challenges were also developed and produced 15-40% mortality, typical clinical signs and pathology, and successful recovery of the challenge organism demonstrating horizontal transmission of E. ictaluri in striped catfish. Experimental studies were then conducted to investigate the association between pH or salinity of water and susceptibility to E. ictaluri infection in striped catfish. The first experiments were performed in in vitro conditions in which E. ictaluri isolates were iii cultured in a variety of pH and salt concentrations. In vivo experiments were then designed where striped catfish were exposed to 107 cfu ml-1 of E. ictaluri for 30 seconds and then held at 4 different water pHs (5.5, 6.5, 7.5 and 8.5) or NaCl concentrations (0, 0.5, 1 and 1.5%). The results of in vitro experiments showed that a pH value between 5.5 to 6.5 and salt concentration between 0-0.5% were optimal for the growth of E. ictaluri. The in vivo experiments demonstrated that the cumulative mortality of striped catfish in water at pH 5 and pH 6 was significantly higher than that of fish maintained in more alkaline water (p<0.05). By contrast, the cumulative mortality of the striped catfish maintained in 0.5% salt concentration was significantly lower than those kept in 0%, 1% and 1.5% salt concentration (p<0.05). Clinical signs, lesions and histopathological changes in the affected fish were consistent with those reported in natural infections. This study highlighted the use of pH 8.5 and salinity of 0.5% NaCl as a means of decreasing the susceptibility of striped catfish to E. ictaluri. In conclusion, this study used a variety of methods in order to enhance the understanding of the biochemical, biophysical characteristics, plasmid profile and antibiotic resistance as well as the relatedness of E. ictaluri isolates recovered from farmed striped catfish in Vietnam. This study provided two reliable and reproducible bacterial challenge models (immersion and cohabitation) and emphasised the link between pH and salinity with the infectivity and pathogenicity of E. ictaluri in striped catfish. iv ACKNOWLEDGEMENTS I would like to thank a number of people whom without which I am certain I would have been unable to have completed this research. Firstly and foremostly, I would like to thank my supervisors Dr. Mags Crumlish and Prof. Randolph Richards for contributing many hours of guidance, support, and encouragements throughout this project, some of which were entirely unrelated to academic endeavours. I would like to thanks the Vietnamese Government and Hue University of Agriculture and Forestry, Vietnam for funding my PhD. Thank you to the Fisheries Society of the Bristish Isles (UK) and University of Ghent (Belgium) for the travel grant which allowed me to take up the opportunity to present my work at international conferences. Special thanks to the bacteriology group and in particular Mrs. Gillian Dreczkowski, Mr. Niall Auchinachie, Mrs. Cathryn Dickson, Mrs. Debbie Faichney, Mrs. Fiona Muir for advises on all matters technical. Also thanks to Dr. John Taggart, Mrs. Ireland Jacquies, Dr. Kerry Bartie for their excellent help and technical assistance in my molecular study. I also would like to thanks to Ho Chi Minh International University, Vietnam for supporting my field work in Vietnam. My gratitude also goes to all my friends past and present at Stirling who made some of the darker days just that little bit brighter (you all know who you are!!). Finally, I’d like to dedicate this thesis to my family whose love and support throughout has been boundless. My special thanks to my incrediably supportive Moms Le Thi Lien and Nguyen Thi Bac, without them, I would not be where I am today. v TABLE OF CONTENTS Declaration ..................................................................................................................................... i ABSTRACT ...................................................................................................................................... ii ACKNOWLEDGEMENTS ................................................................................................................. v TABLE OF CONTENTS .................................................................................................................... vi LIST OF FIGURES ............................................................................................................................ x LIST OF TABLES ........................................................................................................................... xiii LIST OF ABBREVIATIONS.............................................................................................................. xv Chapter 1. GENERAL INTRODUCTION AND LITERATURE REVIEW. ................................................ 1 Edwardsiella ictaluri, A MAJOR SIGNIFICANT PATHOGEN IN STRIPED CATFISH Pangasianodon hypophthalmus (Sauvage 1878) .................................................................................................... 1 1.1. Edwardsiella ictaluri as a bacterial pathogen. ....................................................................... 1 1.1.1. Bacterial identification and characterization. ............................................................. 2 1.1.2. Geographic distribution and host species of E. ictaluri. ............................................. 5 1.1.3. Pathology .................................................................................................................... 7 1.1.4. Transmission ............................................................................................................... 8 1.1.5. Virulence and pathogenicity ....................................................................................... 9 1.1.6. Diagnosis ..................................................................................................................
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