d i s s e r t a t i o n s | 1 2 Jouni Heikkinen 3 | J o u Novel Applications of n i H Pseudomonas sp. Bacterial Fungal and bacterial diseases e i k Strains in Rainbow Trout cause remarkable losses in rainbow ki Jouni Heikkinen n trout aquaculture. In this work, e Aquaculture n applications of Pseudomonas sp. | N Novel Applications of strains M162 and M174 for the control o v e of Saprolegnia sp. infections during l A Pseudomonas sp. Bacterial p egg incubation and Flavobacterium p l i psychrophilum mortalities in rainbow c a Strains in Rainbow Trout t trout aquaculture has been assessed. io n The thesis provides new procedures s o Aquaculture f to prevent Saprolegnia infections on P s eggs and indicates the probiotic effect e u d and mode of action of M162 and M174 o m strains against F. psychrophilum in o n a vivo. s s p . B a c t e r i a l S t r a i n s in Publications of the University of Eastern Finland R a Dissertations in Forestry and Natural Sciences i n b o w T r o u t Publications of the University of Eastern Finland A q Dissertations in Forestry and Natural Sciences u a c u l t isbn 978-952-61-1234-3 ur e AUTHOR: JOUNI HEIKKINEN Novel applications of Pseudomonas sp. bacterial strains in rainbow trout aquaculture Publications of the University of Eastern Finland Dissertations in Forestry and Natural Sciences No 123 Academic Dissertation To be presented by permission of the Faculty of Science and Forestry for public examination in the Auditorium in Tietoteknia Building at the University of Eastern Finland, Kuopio, on October, 4, 2013, at 12 o’clock noon. Department of Biology Kopijyvä Oy Kuopio, 2013 Editors: Profs. Pertti Pasanen Pekka Kilpeläinen and Matti Vornanen Distribution: Eastern Finland University Library / Sales of publications [email protected] www.uef.fi/kirjasto ISBN: 978-952-61-1234-3 (nid.) ISBN: 978-952-61-1235-0 (PDF) ISSNL: 1798-5668 ISSN: 1798-5668 ISSN: 1798-5676 (PDF) Author’s address: University of Eastern Finland Department of Biology P.O. Box 1627 70211 KUOPIO FINLAND email: [email protected] Supervisors: Professor Atte von Wright, Ph.D. University of Eastern Finland School of Medicine, Institute of Public Health and Clinical Nutrition P.O. Box 1627 70211 KUOPIO FINLAND email: [email protected] Jouni Vielma, Ph.D. Finnish Game and Fisheries Research Institute Survontie 9, Jyväskylä FINLAND email: [email protected] Reviewers: Professor Kenneth Cain, Ph.D. University of Idaho Department of Fish and Wildlife Sciences 875 Perimeter Drive MS1136 Moscow ID83844-1136, UNITED STATES email: [email protected] Jose Luis Balcázar, Ph.D. Catalan Water research institute Emili Grahit 101, 17003, Girona SPAIN email: [email protected] Opponent: Dr. Daniel Merrifield University of Plymouth Aquatic Animal Nutrition and Health Research Group A406 Portland Square Drake Circus, Plymouth Devon PL4 8AA UNITED KINGDOM email: [email protected] ABSTRACT Fungal and bacterial diseases cause major losses in rainbow trout aquaculture. Therefore, formalin baths are currently commonly used to prevent infections by pathogens like Saprolegnia sp. during egg incubation. However, there are safety and environmental concerns related to the use of formalin in hatcheries. Rainbow trout fry syndrome (RTFS) and cold water disease, caused by Flavobacterium psychrophilum, are major problems during the fry and juvenile stage. There has been intense vaccine development against RTFS, but so far no commercial vaccine is available and hence antibiotics are currently the only method to treat this disease. The use of antibiotics in disease treatment causes an increased risk of the development of antibiotic resistant bacterial strains. Hence, there is an acute need for alternative disease prevention protocols. Probiotics are widely used in health promoting food products for humans, but also in feed of homoeothermic animals. In aquaculture, utilization of probiotics has been vastly studied during the last decade. In this thesis, sustainable methods to prevent infections caused by Saprolegnia sp. during rainbow trout egg incubation and by Flavobacterium psychrophilum during the fry and juvenile stage of rainbow trout aquaculture, were studied. Protective bacterial strains against Saprolegnia sp. infections on rainbow trout eggs were screened and tested under experimental conditions in vivo. Furthermore, supportive water treatment methods for bacterial culture utilization in rainbow trout egg incubation were developed and their efficiencies were evaluated. High dose (400 mWs/cm2) UV-irradiation of hatchery inlet water decreased rainbow trout egg mortality significantly at the eyed egg stage and utilization of protective bacterial cultures, Pseudomonas sp. M162, Pseudomonas sp. M174 and Janthinobacterium sp. M169 enhanced this effect. Supplementation of the protective cultures did not increase the mortality of the eggs. Feeding the rainbow trout fry with Pseudomonas sp. M174 reduced the mortality that occurred during the experimental Flavobacterium psychrophilum infection. The mode of probiotic action was to evoke immunostimulatory effects and siderophore production. Pseudomonas sp. M162 also decreased Flavobacterium psychrophilum related mortality, while the probiotic effect resulted mainly through immunostimulation. Both strains were found to be safe for the fish. As a conclusion, this thesis has demonstrated the remarkable potential of Pseudomonas sp. M162 and M174 strains and novel applications in which they can be utilized as protective cultures and probiotics in rainbow trout aquaculture. Universal Decimal Classification: 591.2, 591.619, 597.552.51, 639.3.09 CAB Thesaurus: fish culture; hatcheries; fish diseases; disease prevention; pathogens; probiotics; Pseudomonas; Flavobacterium psychrophilum; Saprolegnia; rainbow trout; fish eggs; fry; ultraviolet radiation; siderophores; immunostimulation Yleinen suomalainen asiasanasto: kalanviljely; kalataudit; taudinaiheuttajat; sienitaudit; bakteeritaudit; probiootit; kirjolohi; mäti; ultraviolettisäteily Acknowledgements The work described in this thesis has taken almost a decade to accomplish and have seen the department changing its name from Institute of Applied Biotechnology to Department of Biosciences and finally Department of Biology. I would like to thank the staff at the Department for providing me a pleasant environment to work. I wish to express my sincere thanks to Professor Atte von Wright and Dr. Jouni Vielma, my supervisors, for their supportive and valuable comments during the preparation of manuscripts and writing the thesis I want to thank Professor Kenneth Cain and Dr. Jose Luis Balcazar, for kindly agreeing to review this thesis and giving thoughtful comments that significantly improved the thesis. I also express my warm thanks to my co-authors Dr. Marja Tiirola and Dr. Lotta-Riina Sundberg, Mrs. Päivi Eskelinen, Ms. Satu Mustonen, Ms. Dina Navia-Paldanius, Dr. Tiina Korkea- aho, Dr. Kim Thompson, Ms. Anna Papadopoulou, Professor Brian Austin and professor Alexandra Adams. I am particularly grateful to Dr. Paula Henttonen, for teaching me the secrets of aquaculture and fish biology and for her encouraging and constructive comments on thesis structure. I wish to express my sincere thanks to Dr. Hannu Mölsä, for hiring me into the ProBio AquaFeed-project and finally to working with Saprolegnia. My sincere thanks goes to the personnel of Fish Research Unit, Marko Kelo, Mikko Ikäheimo and Kauko Strengell. Without your technical knowledge and help, these experiments would have been, if not impossible, at least much more difficult. My warm thanks to Ms. Roseanna Avento for assistance in language issues and Dr. Jenny Makkonen for all instructive assistance you gave me. It was much easier to walk the same route a few months later than you. I am grateful to my colleagues at Food and Nutrition Biotechnology laboratory, Mirja, Elvi, Jenni, Outi, Kristiina, Kati, Riitta, Eeva-Liisa, Toni, and Paula. It was always nice to come to work in the lab. I want to thank my brothers and sisters in Claybay Skeleguins for giving me possibility to start ice-hockey at the age of 35. The energy and enthusiasm I have got from our practices at 7am helped me a lot during the writing process. I express my warmest thanks to my friends Heikki and Mikko. The annual hunting trips to Nurmes, Ilomantsi and Lieksa have been a necessary battery recharging for me. I really hope that “Operation Capercaillie” still continues. I thank my parents Martti and Pirkko and my sister Heidi for their support. Even though you probably did not always understand what I was doing, you always supported me in it. And last but not least I would like to thank my wife Tanja for her love and support during this challenging writing process and to my little son, Jesse, for being a boundless source of joy and getting my thoughts away from this thesis. Financial support for this study was provided by Kemira Grow- How Oy, Savon Taimen Oy and Ministry of Agriculture and Forestry, Finland. Kuopio, September 2013 Jouni Heikkinen LIST OF ABBREVIATIONS ARISA = Automated ribosomal intergenic spacer analysis ASA = atypical Aeromonas salmonicida ASS = furunculosis caused by Aeromonas salmonicida ssp. salmonicida BKD = Bacterial kidney disease CFU = colony forming unit CWD = Cold water disease ELISA= Enzyme-linked immunosorbent assay ERM = Enteric red mouth disease IHN = Infectious hematopoietic necrosis i.m. = intramuscular i.p. = intraperitoneal IPN = Infectious pancreatic necrosis PCA = Principle component analysis RTFS = Rainbow trout fry syndrome SD = Sleeping disease TBC = Total bacterial counts TSA =Tryptone soy agar TSB = Tryptone soy broth TYES =Tryptone yeast extract salts VHS = Viral haemorrhagic septicemia LIST OF ORIGINAL PUBLICATIONS This thesis is based on data presented in the following articles, referred to by the Roman numerals I-IV. I. Heikkinen, J, Mustonen, SM, Eskelinen, P, Sundberg, L-R, von Wright, A. Prevention of fungal infestation of rainbow trout (Oncorhynchus mykiss) eggs using UV irradiation of the hatching water. Aquacultural Engineering, 2013, 55, 9- 15. II. Heikkinen, J, Tiirola, M, Mustonen SM, Eskelinen P, Navia-Paldanius, D, von Wright, A. Ultraviolet irradiation of hatchery water and protective bacterial cultures as suppressor of Saprolegnia infections in rainbow trout (Oncorhynchus mykiss) eggs. Submitted manuscript III. Korkea-aho, T, Heikkinen, J, Thompson, K, von Wright, A, Austin, B. Pseudomonas sp. M174 inhibits the fish pathogen Flavobacterium psychrophilum. Journal of Applied Microbiology 2011, 111, 266-277. IV. Korkea-aho, TL, Papadopoulou, A, Heikkinen, J, von Wright, A, Adams, A, Austin, B, Thompson, KD. Pseudomonas M162 confers protection against rainbow trout fry syndrome by stimulating immunity. Journal of Applied Microbiology 2012, 113, 24–35. The original articles have been reproduced with the kind permission of the copyright holders.