DETECTION OF NOVEL MULTILOCUS SEQUENCE TYPING GENOTYPES OF FLAVOBACTERIUM PSYCHROPHILUM IN NORTH AMERICA By Christopher Kay Knupp Michigan State University in partial fulfillment of the requirements for the degree of Fisheries and Wildlife—Master of Science A THESIS Submitted to 2018 DETECTION OF NOVEL MULTILOCUS SEQUENCE TYPING GENOTYPES OF FLAVOBACTERIUM PSYCHROPHILUM IN NORTH AMERICA ABSTRACT By Christopher Kay Knupp The fish pathogen, Flavobacterium psychrophilum, has caused substantial economic losses, particularly to farm/hatchery reared salmonids on at least five continents. F. psychrophilum genetic diversity has been explored using several techniques, including multilocus sequence typing (MLST), which has proven to be superior to other molecular typing assays. Although isolates of F. psychrophilum have been MLST genotyped in Europe, Asia, and South America, the genetic diversity of North American (NA) F. psychrophilum is largely unexplored. To address this knowledge gap, I genotyped 314 F. psychrophilum isolates that were recovered from 20 US states and 1 Canadian province from 10 fish species over nearly 4 decades, which revealed 66 sequence types (STs), 47 of which were novel, and 7 novel clonal complexes (CCs). CC-ST10 was the predominant CC in NA and its genotypes were recovered from rainbow trout/steelhead (Oncorhynchus mykiss) from nearly every sampled state. Several other CCs were also discovered, some of which appeared host specific, whereas others appeared to be “generalists”. Recombination is hypothesized to have caused most variations among isolates of this study, although the mechanism by which this occurs remains unknown. To explore CC-ST10 pathogenicity in rainbow trout, I intramuscularly injected juvenile rainbow trout with several of its genotypes and compared them to the world’s second largest CC, CC- ST191, which resulted in significant mortality. Overall, both CC-ST10 and CC-ST191 were highly pathogenic to rainbow trout, yet significant differences in mean times to death were observed among the genotypes. TABLE OF CONTENTS LIST OF TABLES........................................................................................................................... v LIST OF FIGURES........................................................................................................................ vi KEY TO ABBREVIATIONS...................................................................................................... viii Introduction...................................................................................................................................... 1 1. Study Objectives................................................................................................................... 4 REFERENCES.................................................................................................................................5 Chapter 1. Literature Review........................................................................................................... 8 1. Bacterial Cold Water Disease................................................................................................ 9 1.1. Host range..................................................................................................................... 9 1.2. Geographic distribution.............................................................................................. 11 1.2.1. North America................................................................................................... 11 1.2.2. Europe................................................................................................................ 11 1.2.3. Other continents................................................................................................. 11 1.3. Clinical signs.............................................................................................................. 12 1.4. Losses and economic impact...................................................................................... 12 2. The Pathogen Flavobacterium psychrophilum.................................................................... 13 2.1. Taxonomy................................................................................................................... 13 2.2. Phenotypic characteristics.......................................................................................... 13 2.3. Bacterial culture conditions........................................................................................ 14 2.4. Diversity...................................................................................................................... 15 2.4.1. Phenotypic diversity.......................................................................................... 15 2.4.2. Serological diversity.......................................................................................... 16 2.4.3. Molecular and genetic diversity........................................................................ 17 2.4.3.1. Pulsed field gel electrophoresis............................................................. 17 2.4.3.2. Plasmid profiling................................................................................... 18 2.4.3.3. Randomly amplified polymorphic DNA analysis................................. 18 2.4.3.4. Restriction fragment length polymorphism analysis............................. 19 2.4.3.5. Ribotyping............................................................................................. 19 2.4.3.6. Multilocus sequence typing................................................................... 20 2.4.3.7. Whole genome multilocus sequence typing.......................................... 23 2.5. Flavobacterium psychrophilum and experimental challenge models........................ 23 2.5.1. Bacterial inoculum preparations........................................................................ 24 2.5.2. Immersion challenge studies.............................................................................. 25 2.5.3. Intraperitoneal injection studies......................................................................... 26 2.5.4. Intramuscular injection studies.......................................................................... 26 REFERENCES.............................................................................................................................. 28 Chapter 2. Novel MLST F. psychrophilum Genotypes Infecting North American Salmonids.... 38 1. Abstract................................................................................................................................ 39 iii 2. Introduction.......................................................................................................................... 40 3. Materials and Methods......................................................................................................... 42 3.1. Fish collection and isolation of F. psychrophilum...................................................... 42 3.2. Bacterial DNA extraction and F. psychrophilum identity confirmation.....................43 3.3. Multilocus sequence typing........................................................................................ 44 3.4. MLST data analysis.................................................................................................... 44 3.5. Bayesian inference...................................................................................................... 45 3.6. MLST genetic analyses............................................................................................... 46 4. Results.................................................................................................................................. 46 4.1. Identification of novel F. psychrophilum MLST genotypes in North America..........46 4.2. F. psychrophilum MLST genotypes detected in this study that were reported previously from North America and/or abroad.......................................................... 49 4.3. Bayesian inference of North American F. psychrophilum genotypes........................ 50 4.4. STs and geographic origin.......................................................................................... 51 4.5. F. psychrophilum genetic analyses............................................................................. 53 5. Discussion............................................................................................................................ 55 APPENDIX.................................................................................................................................... 64 REFERENCES.............................................................................................................................. 90 Chapter 3. MLST genotypes of F. psychrophilum vary in their virulence to naïve rainbow trout (Oncorhynchus mykiss)................................................................................................ 95 1. Abstract................................................................................................................................ 96 2. Introduction.......................................................................................................................... 97 3. Materials and Methods......................................................................................................... 99 3.1. F. psychrophilum isolates used in experimental challenge......................................... 99 3.2. Fish and rearing conditions......................................................................................... 99 3.3. Experimental challenge via intramuscular injection................................................. 100 3.4. Statistical analyses.................................................................................................... 101 4. Results................................................................................................................................ 102 4.1. Clinical signs............................................................................................................. 102 4.1.1. External signs................................................................................................. 102 4.1.2. Internal signs.................................................................................................. 103 4.2. Cumulative percent mortality................................................................................... 103 4.3. Mean days to death................................................................................................... 104 4.4. Reisolation of F. psychrophilum............................................................................... 105 5. Discussion.......................................................................................................................... 105 APPENDIX................................................................................................................................. 111 REFERENCES............................................................................................................................ 117 Chapter 4. Conclusions and Future Research.............................................................................. 122 1. Conclusions........................................................................................................................ 123 2. Future Research..................................................................................................................125 iv LIST OF TABLES Table 2.1a Complete information of 314 North American F. psychrophilum isolates presented in this study including year of isolation, location of isolation, host species, tissue of isolation, and host life stage. aAK = Alaska; BC = British Columbia; CA = California; CO = Colorado; ID = Idaho; IN = Indiana; MD = Maryland; MI = Michigan; MN = Minnesota; MT = Montana; NC = North Carolina; NM = New Mexico; OR = Oregon, PA = Pennsylvania; SD = South Dakota; UT = Utah; VA = Virginia; VT = Vermont; WA = Washington; WI = Wisconsin; WV = West Virginia; PF = private facility, SFH = state fish hatchery, WE = weir, WI = wild, Unk = Unknown. cHost was in captivity. wfHost was wild/feral. DIsolate was recovered from a fish experiencing BCWD. HIsolate was recovered from an apparently healthy fish. UFish disease status was unknown. OF = Ovarian Fluid .................................................................................... 65 Table 2.1b Multilocus sequence typing allelic profiles, sequence types (STs), and clonal complexes (CCs) of the 314 North American F. psychrophilum isolates. STs in bold are found in North America and abroad, whereas STs in italics are novel ....................................................... 73 Table 2.2 Allelic variations among 15 North American clonal complexes (CCs). aAncestral amino acid is listed first followed by SLV amino acid, Syn is a synonymous change ................ 81 Table 2.3 Summary of nucleotide sequence diversity among the 86 North American sequence types (STs). S, number of segregating sites. π, average pairwise nucleotide diversity per site. dN/dS, ratios of non-synonymous to synonymous mutations. Rmin, Hudson and Kaplan’s lower bound for the number of recombination ....................................................................................... 83 Table 3.1. Origin information for the 10 F. psychrophilum isolates that were selected for use in in vivo virulence experiments. All isolates were originally recovered from rainbow trout/steelhead. AIsolates denoted with D were recovered from a fish suffering from BCWD, whereas H indicates an isolate was recovered from an apparently healthy fish. *Sequence types denoted with 2016 were recovered and genotyped by Van Vliet et al. (2016) ............................. 112 Table 3.2 Statistical comparisons of mean days to death among 10 F. psychrophilum isolates, five sequence types (STs), and two clonal complexes (CCs). #Isolates with the same superscript letter (i.e., a, b, c), STs with the same superscript number (i.e., 1, 2) and CCs with the same symbol (i.e., *) are not significantly different. All significance testing was assessed at an alpha of 0.05. CFU/fish-1, colony forming units injected per fish; CPM, cumulative percent mortality; OD, optical density at 600nm; SD, standard deviation .............................................................. 113 Table 3.3 Percent of F. psychrophilum isolates, sequence types (STs), and clonal complex (CCs) presenting with gross external (i.e., skin erosion/hemorrhage/swelling/pallor, gill pallor/hemorrhage/erythema) and internal (i.e., hemorrhage, swelling, pallor) pathology ........ 114 v LIST OF FIGURES Figure 2.1 Map of the United States and Canada. Distribution of 314 F. psychrophilum isolates recovered from the current North American study. These isolates were recovered from 20 U.S. states and one province of Canada. AK = Alaska; BC = British Columbia; CA = California; CO = Colorado; ID = Idaho; IN = Indiana; MD = Maryland; MI = Michigan; MN = Minnesota; MT = Montana; NC = North Carolina; NM = New Mexico; OR = Oregon, PA = Pennsylvania; SD = South Dakota; UT = Utah; VA = Virginia; VT = Vermont; WA = Washington; WI = Wisconsin; WV = West Virginia ..................................................................................................................... 84 Figure 2.2 eBURST diagram of the 314 F. psychrophilum isolates typed in this study combined with all previously typed North American isolates (n = 107) in the F. psychrophilum MLST database. A dagger (†) denotes a sequence type (ST) that contains isolates typed in this study, whereas a double dagger (‡) denotes a ST containing previously typed isolates. Italicized numbers accompanying STs indicate the number of states that ST was found in (if no number, ST recovered from one state only). Clonal complexes (CC) are encircled and the founding sequence type (ST) for a CC is depicted as a number within a rectangle. Founding STs labeled with a star ((cid:2)(cid:1) indicate a newly described CC as a result of this study. The founding ST, or founder, is the ST with the highest number of single locus variants (SLVs). In instances where multiple STs have the same number of SLVs, the founder is named after the ST with the most isolates. If both STs have the same number of isolates, the CC is named for the earliest found ST ....................................................................................................................................................... 85 Figure 2.3 eBURST diagram of all 1,411 F. psychrophilum isolates. This includes the 1,097 isolates from the F. psychrophilum MLST database and the 314 newly typed isolates from this study. A dagger (†) indicates that ST is present in North America. A star ((cid:2)) indicates that ST was found in North America and abroad. The founding sequence type (ST), or founder, is the ST with the highest number of single locus variants (SLVs; depicted as gray circles). In instances where multiple STs have the same number of SLVs, the founder is named after the ST with the most isolates. If both STs have the same number of isolates, the clonal complex (CC) is named for the earliest found ST. The founding genotype of a CC is enclosed within a box. The size of each circle indicates how many isolates belong to that ST .......................................................... 86 Figure 2.4 Frequency and host of origin among 10 North American F. psychrophilum sequence types (STs). STs are displayed along the X-axis with four clonal complexes (CCs; e.g., CC- ST10, CC-ST9, CC-ST256, CC-ST276) and two singletons (e.g., ST253, ST258) being shown. The Y-axis displays the number of recovered isolates. All 421 currently typed North American F. psychrophilum isolates are presented here ............................................................................... 87 Figure 2.5 Phylogenetic reconstruction of the 86 F. psychrophilum STs currently described in North America using Bayesian inference. 15 clonal complexes (CCs) are in bold, 3 of which (in italics) are formed when the population structure of F. psychrophilum is evaluated in the context of the world. The fish host species each ST was recovered from is depicted as a symbol to the right of each ST and described in the key. Posterior probabilities ≥70% are displayed at branch nodes. The tree is drawn to scale and is unrooted ........................................................................ 88 vi Figure 3.1 External signs of BCWD in rainbow trout (Oncorhynchus mykiss) fingerlings that were intramuscularly injected with F. psychrophilum. A. Severe gill pallor with concurrent diffuse petechial hemorrhage. B. Multifocal to diffuse petechial hemorrhage on the trunk and congestion at the base of the pelvic fin. C. Ulceration of the skin and underlying musculature on the dorsum. D. Severe swelling and congestion at the injection site. E-G. Progression of experimentally induced BCWD, beginning with superficial ulceration (E) that extended (F) and deepened into the underlying muscle (G) as the infection progressed. Note also the yellowish discoloration overlying the lesions in C and E-G ....................................................................... 115 Figure 3.2 Internal signs of BCWD in rainbow trout (Oncorhynchus mykiss) fingerlings that were intramuscularly injected with F. psychrophilum. A. Hemorrhagic ovary. B. Diffuse petechial hemorrhage of the ventricle and bulbous arteriosus of the heart, as well as a moderately pale liver. C. Severely hemorrhagic liver accompanied by multifocal petechial hemorrhage of the adipose tissue overlying the pyloric cecae. D. Diffuse petechial hemorrhage of the ventricle of the heart accompanied by a severely pale liver with multifocal petechial hemorrhage and multifocal to diffuse petechial hemorrhage of the adipose tissue overlying the pyloric cecae .. 116 vii AICc AK ANCOVA AT atpA BCWD BC bp C CA CC CFU CO CPM DLV DNA dnaK dN/dS fumC G g g KEY TO ABBREVIATIONS Corrected Akaike information criterion Alaska Analysis of covariance Allele type Adenosine triphosphate synthetase, α subunit Bacterial cold water disease British Columbia Base pair Celsius California or Cytophaga agar Clonal complex Colony forming unit Colorado Cumulative percent mortality Double locus variant Deoxyribonucleic acid Chaperone heat-shock protein 70 Ratio of non-synonymous to synonymous mutations Fumarate hydratase class II Gauge Gram Gravity viii gyrB H HSU ISA ID IM IN IP L MCMC MD mg MI min mL MLST MS-222 MN MT mTYES NA NC NaCl Topoisomerase II, β subunit Mean genetic diversity Hsu-Shotts medium Standardized index of association Idaho Intramuscular Indiana Intraperitoneal Liter Markov chain Monte Carlo Maryland Milligram Michigan Minute Milliliter Multilocus sequence typing Tricaine methanesulfonate Minnesota Montana Tryptone yeast extract agar supplemented with 5% horse serum and 0.02% Lewin and Lounsbery mineral solution North America North Carolina Sodium Chloride ix NM OD OF OR PA PF PCR PFGE PHI RAPD RFLP Rmin rpm RTFS rRNA S SD SFH SLV ST trpB tuf TYES New Mexico Optical density Ovarian fluid Oregon Pennsylvania Private facility Polymerase chain reaction Pulsed field gel electrophoresis Pairwise homoplasy index Randomly amplified polymorphic DNA Restriction fragment length polymorphism Hudson and Kaplan lower bound on the minimum number of recombination events Revolutions per minute Rainbow trout fry syndrome Ribosomal ribonucleic acid Segregating sites South Dakota or standard deviation State fish hatchery Single locus variant Sequence type Tryptophane synthetase, β subunit Elongation factor tu Tryptone yeast extract agar x UK Unk US USA UT VA vol VT WA WE WI WV µl π United Kingdom Unknown United States United States of America Utah Virginia Volume Vermont Washington Weir Wild or Wisconsin West Virginia Microliter Nucleotide diversity xi Introduction 1 Flavobacterium psychrophilum was first isolated during a mortality event among coho salmon (Oncorhynchus kisutch) in 1948 in the Pacific Norwest region of the United States (Borg 1948). Since then, F. psychrophilum has been recovered from numerous salmonids and in particular, rainbow trout (O. mykiss) and Atlantic salmon (Salmo salar; reviewed in Nematollahi et al. 2003). F. psychrophilum has been recovered from non-salmonid species as well, such as ayu (Plecoglossus altivelis) and common carp (Cyprinus carpio; reviewed in Nematollahi et al. 2003, Starliper 2011). Today, F. psychrophilum is known as the etiological agent of bacterial cold water disease (BCWD) and rainbow trout fry syndrome (RTFS) and is responsible for significant economic losses worldwide (reviewed in Loch & Faisal 2017). Since its initial isolation, researchers noticed phenotypic diversity within this species, specifically in the form of smooth and rough colonies (Bernardet & Kerouault 1989). As serological and molecular biological techniques progressed, additional researchers began to investigate F. psychrophilum heterogeneity further. For example, Pacha (1968) examined 10 F. psychrophilum isolates recovered from Chinook salmon from Washington (USA) and discovered them to be highly related using a serological technique. Likewise, Wakabayashi et al. (1994) described two F. psychrophilum serotypes (i.e., O-1, O-2) and found that isolates belonging to serotype O-1 were recovered predominantly from coho salmon, whereas O-2 was recovered from ayu and rainbow trout. As of 2017, at least seven serotypes have been described (reviewed in Loch and Faisal 2017), although variations in techniques have hindered inter-laboratory comparisons. Molecular biology has taken advantage of numerous genetically-based techniques for detecting F. psychrophilum diversity, including pulsed field gel electrophoresis (PFGE), plasmid profiling, ribotyping, randomly amplified polymorphic DNA (RAPD), and restriction fragment length polymorphism (RFLP). Most recently, a multilocus sequence typing (MLST) 2 scheme was developed for use with F. psychrophilum by Nicolas et al. (2008), which has proven to be a highly discriminatory technique for describing F. psychrophilum heterogeneity. Since their pioneering study, numerous researchers worldwide have applied this technique to F. psychrophilum to better understand how genetic diversity may correlate with virulence, geographic distribution, and host specificity. To date, the majority of MLST typed F. psychrophilum isolates have originated from Europe; however, isolates from Asia, South America, Oceania, and North America have been examined as well. MLST has been used to reveal certain sequence types (STs) and clonal complexes (CCs) as host specific (Fujiwara- Nagata et al. 2013, Nilsen et al. 2014), geographically isolated or widespread (Nicolas et al. 2008, Fujiwara-Nagata et al. 2013, Avendaño-Herrera et al. 2014) and virulent (Fujiwara-Nagata et al. 2012, 2013, Nilsen et al. 2014). All previously published research articles have discovered STs belonging to a common CC; namely CC-ST10. CC-ST10 is the largest CC worldwide and contains hundreds of isolates from different regions of the world (Van Vliet et al. 2016). Of note, the majority of these isolates have been recovered from diseased/systemically infected rainbow trout and so it is hypothesized that this CC is particularly adapted and highly virulent to rainbow trout (Nilsen et al. 2014, Van Vliet et al. 2016). In North America, few F. psychrophilum isolates have been genotyped using MLST. In fact, prior to 2016, only 10 F. psychrophilum isolates had been genotyped using this technique, which is surprising given the economic losses F. psychrophilum causes in North America. In 2016, Van Vliet et al. (2016) genotyped 95 new F. psychrophilum isolates although the majority of these came from Michigan (USA) and were recovered from three Oncorhynchus spp. (i.e., O. mykiss, O. kisutch, O. tshawytscha). Given the proportionally fewer F. psychrophilum isolates that have been MLST genotyped in North America in comparison to other regions of the world 3 that intensively rear salmonids (e.g., Europe), I opportunistically collected several hundred F. psychrophilum isolates from North America and evaluated their genetic diversity using MLST. 1. Study Objectives In Chapter 1, I thoroughly reviewed the literature on F. psychrophilum and bacterial cold water disease (BCWD). I provided specific information in regards to F. psychrophilum diversity, including its phenotypic, serological, and molecular diversity. I also reviewed previous experimental challenge studies, which used numerous routes of exposure. In Chapter 2, I reached out to fish health professionals across the US to gather 314 F. psychrophilum isolates, some of which were cryogenically preserved nearly four decades ago. These isolates originated from 10 fish host species, were recovered from 20 US states and 1 Canadian province, and I assessed their genetic diversity using MLST. I also evaluated the 7 MLST loci for recombination and mutation using multiple sequence and allelic profile based analyses in order to predict how F. psychrophilum achieved its genetic diversity. In Chapter 3, I examined the in vivo pathogenicity of several CC-ST10 genotypes as well as those from the world’s second largest MLST CC (i.e., CC-ST191) against their natural host, the rainbow trout. I hypothesized CC-ST10 genotypes were pathogenic to rainbow trout, isolates of the same genotype were similar in their pathogenicity, and the genotypes within and between the two largest CCs would differ in their pathogenicity. Chapter 4 provided overall conclusions based upon the findings of these studies and offered suggestions for future research that could contribute to the control and management of this important fish pathogen. 4 REFERENCES 5 REFERENCES Avendaño-Herrera, R., Houel, A., Irgang, R., Bernardet, J.F., Godoy, M., Nicolas, P., and E. Duchaud. 2014. Introduction, expansion and coexistence of epidemic Flavobacterium psychrophilum lineages in Chilean fish farms. Veterinary Microbiology 170:298-306. Bernardet, J.F., and B. Kerouault. 1989. Phenotypic and genomic studies of Cytophaga psychrophila isolated from diseased rainbow trout Oncorhynchus mykiss in France. Applied Environmental Microbiology 55:1796-1800. Borg, A.F. 1948. Ph.D. thesis. University of Washington, Seattle, Washington. Studies on myxobacteria associated with diseases in salmonid fishes. Duchaud, E., Rochat, T., Habib, C., Barbier, P., Loux, V., Guérin, C., Dalsgaard, I., Madsen, L., Nilsen, H., Sundell, K., Wiklund, T., Strepparava, N., Wahli, T., Caburlotto, G., Manfrin, A., Wiens, G.D., Fujiwara-Nagata, E., Avendaño-Herrera, R., Bernardet, J-F., and P. Nicolas. 2018. Genomic Diversity and Evolution of the Fish Pathogen Flavobacterium psychrophilum. Frontiers in Microbiology 9:138. Fujiwara-Nagata, E., Chantry-Darmon, C., Bernardet, J.F., M. Eguchi, M., Duchaud E., and P. Nicolas. 2013. Population structure of the fish pathogen Flavobacterium psychrophilum at a whole country and model river levels in Japan. Veterinary Research 44:34. Fujiwara-Nagata, E., Ikeda, J., Sugahara, K., and M. Eguchi. 2012. A novel genotyping technique for distinguishing between Flavobacterium psychrophilum isolates virulent and avirulent to ayu, Plecoglossus altivelis (Temminck & Schlegel). Journal of Fish Diseases 35:471–480. Loch T.P., M Faisal. 2017. Flavobacterium spp., p. 211–232. In Woo, PTK, Cipriano, RC (eds.), Fish Viruses and Bacteria: Pathobiology and Protection CABI. Nematollahi, A., Decostere, A., Pasmans, F., and F. Haesebrouck. 2003. Flavobacterium psychrophilum infections in salmonid fish. Journal of Fish Diseases 26:563-574. Nicolas, P., Mondot, S., Achaz, G., Bouchenot, C., Bernardet, J.F., and E. Duchaud. 2008. Population structure of the fish-pathogenic bacterium Flavobacterium psychrophilum. Applied and Environmental Microbiology 74:3702-3709. Nilsen, H., Sundell, K., Duchaud, E., Nicolas, P., Dalsgaard, I., Madsen, L., Aspán, A., Jansson, E., Colquhoun, D.J., and T. Wiklund. 2014. Multilocus sequence typing identifies epidemic clones of Flavobacterium psychrophilum in Nordic countries. Applied and Environmental Microbiology 80:2728-2736. Pacha, R.E. 1968. Characteristics of Cytophaga psychrophila (Borg) Isolated During Outbreaks of Bacterial Cold-Water Disease. Applied Microbiology 16:97-101. 6 Starliper, C.E. 2011. Bacterial coldwater disease of fishes caused by Flavobacterium psychrophilum. Journal of Advanced Research 2:97-108. Van Vliet, D., Wiens, G.D., Loch, T.P., Nicolas, P., and M. Faisal. 2016. Genetic Diversity of Flavobacterium psychrophilum Isolates from Three Oncorhynchus spp. in the United States, as Revealed by Multilocus Sequence Typing. Applied and Environmental Microbiology 82:3246- 3255. Wakabayashi, H., Toyama, T., and T. Iida. 1994. A study on serotyping of Cytophaga psychrophila isolated from fishes in Japan. Fish Pathology 29:101-104. 7 Chapter 1 Literature Review 8 The family Flavobacteriaceae contains a multitude of fish pathogens, the most notorious of which is Flavobacterium psychrophilum, the causative agent of bacterial cold water disease (BCWD) and rainbow trout fry syndrome (RTFS). In this chapter, I focused on bacterial cold water disease. 1. Bacterial Cold Water Disease F. psychrophilum is the etiological agent of bacterial cold water disease and rainbow trout fry syndrome (Madsen & Dalsgaard 1999). Disease signs consistent with what is now known as BCWD were first reported by Davis (1946), at which time he coined the syndrome “Peduncle Disease”. Affected rainbow trout had heavy loads of a bacterium associated with the affected tissues, yet the etiological agent could not be isolated at that time. However, in 1948, F. psychrophilum was isolated in the Pacific Northwest region of the United States after mass mortalities among coho salmon (Oncorhynchus kisutch; Borg 1948) occurred. 1.1. Host range Flavobacterium psychrophilum predominately affects fish within the family Salmonidae, including coho salmon (O. kisutch) and rainbow/steelhead trout (O. mykiss), which are considered to be particularly susceptible (reviewed in Nematollahi et al. 2003, reviewed in Cipriano & Holt 2005). F. psychrophilum has been recovered from many other salmonids species, including Atlantic salmon (Salmo salar), brown trout (S. trutta), Chinook salmon (O. tshawytscha), sockeye salmon (O. nerka), masou salmon (O. masou), chum salmon (O. keta), amago (O. rhodurus), cutthroat trout (O. clarkii), pink salmon (O. gorbuscha), brook trout (Salvelinus fontinalis), lake trout (S. namaycush), arctic char (S. alpinus), white spotted char (S. 9 leucomaenis), and grayling (Thymallus thymallus; Davis 1946, Rucker et al. 1953, Borg 1948, Schachte 1983, Holt 1987, Iida & Mizokami 1996, Ekman et al. 1999, and Madetoja et al. 2001, reviewed in Cipriano & Holt 2005, Fujiwara-Nagata et al. 2013). Additionally, F. psychrophilum can also infect non-salmonid fishes. For example, ayu (Plecoglossus altivelis), are particularly susceptible to infection with F. psychrophilum (Wakabayashi et al. 1994, Fujiwara-Nagata et al. 2013, Rochat et al. 2017), whereas isolation from other non-salmonid hosts, including European eel (Anguilla Anguilla), Japanese eel (A. japonica), forktongue goby (Chaenogobius urotaenia), common carp (Cyprinnus carpio), crucian carp (Carassius carassius), goldfish (C. auratus), Japanese crucian carp funbuna (C. auratus langsdorfii), Japanese dace (Trybolodon hakonensis), pale chub (Zacco platypus), lake goby (Rhinogobius brunneus), perch (Perca fluviatilis), roach (Rutilus rutilus), tench (Tinca tinca), Japanese smelt (Hypomesus nipponensis), far eastern brook lamprey (Lethenteron reissneri), sea lamprey (Petromyzon marinus L.) takahaya (Phoxinus jouyi), European flounder (Platichthys flesus), numachichibu (Tridentiger brevispinis), white sturgeon (Acipenser transmontanus), Indian catfish (Clarias batrachus), and three-spined stickleback (Gasteroosteus aculeatus) is reported infrequently (Lehmann et al. 1991, Amita et al. 2000, Iida & Mizokami 1996, Madetoja et al. 2002, reviewed in Starliper 2011, Fujiwara-Nagata et al. 2013, reviewed in Loch & Faisal 2017). F. psychrophilum has also been recovered from other aquatic/semi-aquatic hosts, such as freshwater leeches (Myzobdella lugubris; Schulz and Faisal 2010), newts (Brown et al. 1997), and even aquatic plants (e.g., algae; Amita et al. 2000) and insects (e.g., caddisfly; Fujiwara- Nagata et al. 2013). 10 1.2. Geographic distribution 1.2.1. North America Although F. psychrophilum was first described in the USA, its exact geographic distribution has not been determined. Nevertheless, F. psychrophilum is believed to be widespread across the USA (reviewed in Cipriano & Holt 2005, Starliper 2011, Loch & Faisal 2017) and has also been recovered from Canada (Allen et al. 2008, Hesami et al. 2008). 1.2.2. Europe F. psychrophilum is widespread among European countries. For example, F. psychrophilum has been isolated in the countries of Belgium (reviewed in Nematollahi et al. 2003), Denmark (Lorenzen et al. 1991, Nilsen et al. 2014), Estonia (Madetoja et al. 2001), Finland (Dalsgaard & Madsen 2000, Madetoja et al. 2001), France (Siekoula-Nguedia et al. 2012), Germany (Nilz et al. 2009), Italy (Sarti et al. 1992), Ireland (Lorenzen et al. 1997), Norway (Nilsen et al. 2014), Scotland (reviewed in Starliper 2011), Spain (Toranzo & Barja 1993), Sweden (Ekman et al. 1999), Switzerland (Strepparava et al. 2013), and the United Kingdom (Austin & Stobie 1991). 1.2.3. Other continents Compared to North America and Europe, reports of F. psychrophilum in Asia, South America, and Australia have been less frequent. The bacterium was first reported in Japan in 1987 (Wakabayashi et al. 1994) and has been regularly isolated since then (Fujiwara-Nagata et al. 2013). F. psychrophilum has also been reported in two other regions of Asia; namely Turkey (Kum et al. 2008) and the Korean Peninsula (Lee & Heo 1998). Within South America, most 11 reports of F. psychrophilum have come from Chile (Avendaño-Herrera et al. 2014), but F. psychrophilum has also been recovered in Peru (Leon et al. 2009). F. psychrophilum has been recovered in Australia (Schmidtke & Carson 1995). 1.3. Clinical signs The most characteristic external sign of BCWD is the formation of deep ulcers on the caudal peduncle, where extensive erosion of the skin and musculature has occurred (reviewed in Cipriano and Holt 2005). However, the severity and distribution of BCWD clinical signs are influenced by many factors (reviewed in Nematollahi et al. 2003). For example, in sac-fry suffering from BCWD, erosion of the yolk sac has been reported (Holt et al. 1993), whereas in fingerlings a unilateral dark pigmentation has been accompanied by fin erosion (Pacha & Ordal 1970). Additional external disease signs include exophthalmia, frayed fins, abdominal distention, pale and/or hemorrhagic gills, and erosion of skin on the snout and posterior portion of the head (reviewed in Nematollahi et al. 2003, Starliper 2011). Internally, the kidneys and liver appear pale (reviewed in Starliper 2011), whereas the spleen is enlarged or hypertrophied (Bernardet et al. 1988). Petechial hemorrhages on visceral organs also occur (reviewed in Loch & Faisal 2017). Histologically, necrotic myositis, scleritis, and cephalic osteochondritis are common lesions in fish experiencing BCWD (Lumsden et al. 1996, reviewed in Nematollahi et al. 2003). 1.4. Losses and economic impact F. psychrophilum causes millions of dollars in losses annually, much of which is due to mortality (Duchaud et al. 2018). For example, up to 70% mortality has been reported in F. 12 psychrophilum-infected fingerling rainbow trout (Avendaño-Herrera et al. 2014), whereas in older fish, losses < 20% are reported (Wood 1974). Fish that survive and recover from infection with F. psychrophilum often have spinal and ocular abnormalities (reviewed in Loch & Faisal 2017). 2. The Pathogen Flavobacterium psychrophilum 2.1. Taxonomy F. psychrophilum has undergone many taxonomic re-classifications since its initial isolation nearly 70 years ago. F. psychrophilum was originally placed within the genus Cytophaga by Borg (1948); due to its affinity for low temperatures, it was given the name Cytophaga psychrophila. Since this bacterium did not produce fruiting bodies or degrade polysaccharides, C. psychrophila was re-classified as Flexibacter psychrophilus (Bernardet & Grimont 1989), but subsequent analysis of DNA G+C content indicated that F. psychrophilus was more similar to bacteria within the genus Flavobacterium. In 1996, Bernardet et al. (1996) emended the description of the genus Flavobacterium and renamed Flexibacter psychrophilus as Flavobacterium psychrophilum, as it is known today. 2.2. Phenotypic characteristics F. psychrophilum is a weakly refractile (Pacha 1968) Gram-negative rod-shaped bacterium that is approximately 0.5 µm wide by 1-5 µm long (Bernardet & Kerouault 1989). This bacterium is strictly aerobic and produces yellow-pigmented (due to the presence of a flexirubin-type pigment) colonies with a raised center and smooth or thinly spreading edge (Bernardet & Kerouault 1989, Holt et al. 1993), although colony morphology can vary. F. 13 psychrophilum grows in the presence of up to 1.0% NaCl and at a pH range of 4.0 - 8.0 (Bernardet & Kerouault 1989). The bacterium hydrolyzes multiple substrates (e.g., gelatin, tributyrin, casein, tyrosine, lecithin, tween 20, and tween 80; Bernardet & Kerouault 1989) and also proteolyzes many host tissue constituents (e.g., collagen, fibrinogen, chondroitin sulphate, and fish muscle extract; Lorenzen et al. 1997). However, F. psychrophilum cannot hydrolyze xanthine, chitin, starch, agar, carboxymethylcellulose, or esculin or degrade simple or complex carbohydrates (Bernardet & Kerouault 1989, reviewed in Cipriano and Holt 2005). F. psychrophilum is weakly positive for both cytochrome oxidase and catalase, does not produce hydrogen sulfide, indole, arginine dihydrolase, lysine decarboxylase, or ornithine decarboxylase (Bernardet & Kerouault 1989, reviewed in Cipriano and Holt 2005). F. psychrophilum cannot absorb congo red nor reduce nitrate to nitrite (Bernardet & Kerouault 1989). 2.3. Bacterial culture conditions F. psychrophilum is a somewhat fastidious bacterium and as its name implies, grows well at cooler temperatures, particularly between 10°C - 20°C although some strains will grow at temperatures of up to 25°C (Bernardet & Kerouault 1989). F. psychrophilum has a generation time of 2 hours at 15°C, which is considered to be its optimal growth temperature (Holt et al. 1993, Barnes & Brown 2011). F. psychrophilum grows poorly or not at all on nutrient-rich media such as trypticase soy agar (TSA; Wiklund et al. 1994, Ekman et al. 1999) and therefore is cultured on nutrient deplete media such as Cytophaga agar (Anacker & Ordal 1959), modified Cytophaga agar (Wakabayashi & Egusa 1974), Hsu-Shotts (Bullock et al. 1986), or tryptone yeast extract agar (TYES; Holt et al. 1993). The overgrowth of non-target bacteria when attempting to culture F. psychrophilum can occur, so supplementing primary isolation media 14 with antibiotics has been shown to aide in bacterial recovery (reviewed in Nematollahi et al. 2003). The supplementation of primary media with 10% fetal calf serum (Obach & Laurencin 1991) or 5-10% horse serum (Michel et al. 1999) has been shown to improve recovery of F. psychrophilum. Likewise, the addition of trace elements at a concentration below 0.05% was found to improve F. psychrophilum recovery (Lewin & Lounsberry 1996, Michel et al. 1999). 2.4. Diversity 2.4.1. Phenotypic diversity Phenotypic variability within F. psychrophilum has been reported on numerous occasions. For example, variations in colony morphology (i.e., rough versus smooth; Bernardet & Kerouault 1989, Högfors-Rönnholm & Wiklund 2010) occur and are associated with differences in in vitro autoagglutination capacity and hydrophobicity. Högfors-Rönnholm & Wiklund (2010) provided evidence for phase variation (i.e., conversions between phenotypes) between the two phenotypes, whereby smooth phenotype colonies could convert to rough but not the other way around. Both phenotypes have proven equal in their abilities to cause mortality (Högfors-Rönnholm & Wiklund 2010). On the contrary, variability in elastase activity has been reported between F. psychrophilum strains (Madsen & Dalsgaard 1998), and this variability was associated with differential virulence (Europe). Likewise, variability in biofilm formation, enzymatic activity, and gliding motility have been reported (Madetoja et al. 2001, Hesami et al. 2008, Levipan & Avendaño-Herrera 2017). 15 2.4.2. Serological diversity The serological diversity of F. psychrophilum has been examined for decades (Pacha 1968, reviewed in Cipriano & Holt 2005). Serotyping has relied upon multiple assays, which makes comparisons between laboratories difficult but some trends among several serotypes have been reported. For instance, Wakabayashi et al. (1994) described two F. psychrophilum serotypes; namely O-1 and O-2, where O-1 was recovered exclusively from coho salmon in the US or Japan and O-2 was non-host specific but localized to Japan. Similarly, Izumi and Wakabayashi (1999) reported the presence of three serotypes O-1, O-2, and O-3 with O-1 being recovered from multiple host species (i.e., coho salmon, rainbow trout, carp, and grayling), O-2 being recovered exclusively from rainbow trout, while O-3 was almost exclusively recovered from rainbow trout. In 2003, Izumi et al. (2003) added serotype O-4, which was recovered from multiple host species, and described mixed serotypes O-2/4 and O-3/4 as well. Lorenzen and Olesen (1997) identified and described three serotypes (i.e., Th, Fd, FpT) using slide agglutination and found the majority of isolates in their study belonged to Th (with subtypes Th- 1 and Th-2), which were predominantly recovered from diseased rainbow trout. Likewise, many isolates identified as Fd were recovered from rainbow trout experiencing BCWD, whereas those identified as FpT were not recovered from BCWD outbreaks (Lorenzen & Olesen 1997). In agreement with this discovery, Madsen and Dalsgaard (2000) found that isolates belonging to serotype FpT did not appear to be highly virulent in comparison to other isolates. In 2002, Mata et al. (2002) described seven different serovars (i.e., 1, 2a, 2b, 3, 4, 5, 6, 7) using slide agglutination and an enzyme-linked immunosorbent assay (ELISA). These serovars appeared host specific as isolates recovered from rainbow trout belonged to serovars 2a, 2b, and 3, whereas coho salmon, European eel, common carp, tench, and ayu belonged to serovar 1, 4, 5, 6, 16 or 7, respectively. In summary, serotypes varied in their host specificities and virulence; however, many F. psychrophilum isolates were untypable with current protocols and serotyping techniques were not standardized across laboratories, which made comparisons difficult. 2.4.3. Molecular and genetic diversity 2.4.3.1. Pulsed field gel electrophoresis Pulsed field gel electrophoresis (PFGE) has been used by multiple investigators to evaluate the genetic heterogeneity of F. psychrophilum isolates. For example, Arai et al. (2007) typed 80 F. psychrophilum isolates using PFGE, 64 of which were from ayu, and utilized the endonucleases BlnI and XhoI to identify 42 genotypes. Isolates recovered from ayu fell into three major clusters (i.e., XII, XVI, XVII), two of which (i.e., XII, XVII) contained isolates from multiple locations, whereas XVI contained isolates from a single location. Similarly, Chen et al. (2008) used PFGE to type 139 F. psychrophilum isolates recovered from salmonids to identify dozens of genotypes; however, the endonuclease used in this study was SacI. del Cerro et al. (2010) employed PFGE to genotype 25 F. psychrophilum isolates from Spain and revealed 17 PFGE patterns using the endonuclease StuI. Moreover, del Cerro et al. (2010) discovered that some F. psychrophilum isolates were specific to a particular aquaculture facility and the same genotypes were responsible for BCWD outbreaks over several months. Unfortunately, the lack of standardization in regard to the employed restriction enzyme impeded inter-laboratory comparisons. 17 2.4.3.2. Plasmid profiling The plasmid repertoire of F. psychrophilum was reported to be homogenous by numerous investigators. For instance, Holt (1987) evaluated the plasmid profiles of 20 F. psychrophilum isolates and found many isolates contained an identical plasmid. However, plasmid sizes have varied between laboratories, making comparisons between laboratories difficult. The discrepancy in plasmid size has been attributed to differences in laboratory techniques and protocols (Izumi 2004). For example, the type strain of F. psychrophilum, NCIMB1947, has been estimated to have a plasmid size of 2.8kb, 2.7kb, or 2.4kb, depending on the investigator (Lorenzen et al. 1997, Chakroun et al. 1998, Izumi 2004, Madsen & Dalsgaard 2000). Recently, Duchaud et al. (2018) identified 15 plasmids within 41 F. psychrophilum isolates using whole genome sequencing and provided evidence that gel electrophoresis cannot adequately distinguish between all F. psychrophilum plasmids. 2.4.3.3. Randomly amplified polymorphic DNA analysis Randomly amplified polymorphic DNA (RAPD) analysis has provided mixed results in regards to RAPD profile and fish host species (Chakroun et al. 1997, Valdebenito & Avendaño- Herrera 2009), whereby RAPD profiles appeared to differ among isolates recovered from ayu and tench but not among isolates recovered from Atlantic salmon and rainbow trout (Chakroun et al. 1997, Valdebenito & Avendaño-Herrera 2009). Chakroun et al. (1997) also noted that no clear demarcation between isolate and geographic origin could be detected among recovered RAPD profiles. 18 2.4.3.4. Restriction fragment length polymorphism analysis Restriction fragment length polymorphism (RFLP) analysis has been used to explore the genetic heterogeneity of F. psychrophilum isolates by utilizing the 16S rRNA and gyrB genes (Izumi et al. 2003). Restriction digest of gyrB using restriction enzyme HinfI yielded two distinct genotypes (i.e., genotypes A and B) and endonuclease RsaI generated another two genotypes (i.e., genotypes R and S) among 242 isolates of F. psychrophilum (Izumi et al. 2003). Isolates identified as genotype “A” were recovered exclusively from ayu, whereas genotype “B” isolates were non-host specific (Izumi et al. 2003). Furthermore, Izumi et al. (2003) found restriction digest of the 16S rRNA gene provided no distinct cleavage patterns among the examined isolates. Soule et al. (2005) identified two lineages of F. psychrophilum (i.e., lineages I and II), where lineage I was associated with an avirulent isolate of F. psychrophilum (i.e., ATCC 49418) and lineage II was associated with a virulent isolate (i.e., CSF 259-93). Furthermore, lineage II isolates had reduced susceptibility to tetracycline and were specific to trout, whereas lineage I was specific to salmon (Soule et al. 2005). Hesami et al. (2008) identified four RFLP variants; however, no host associations could be made. 2.4.3.5. Ribotyping Cipriano et al. (1996) described ribotyping as a useful tool for discriminating between closely related strains of F. psychrophilum. Two years later, Charkroun et al. (1998) genotyped 85 F. psychrophilum isolates and described 31 ribotypes when using endonuclease Hinc II and an additional 35 ribotypes when using endonuclease Pvu II. However, clear correlations between ribotype and host species could not be drawn (Chakroun et al. 1998). Madsen and Dalsgaard (2000) ribotyped 299 F. psychrophilum isolates by discovered remarkable homogeneity in which 19 85% of isolates belonged to the same ribotype. Madsen and Dalsgaard (2000) utilized the restriction endonuclease EcoRI and concluded that this restriction enzyme was not useful for epidemiologic investigations, underscoring the importance of endonuclease selection. 2.4.3.6. Multilocus sequence typing Multilocus sequence typing (MLST) was originally developed for the human pathogen Neisseria meningitidis (Maiden et al. 1998) and is a highly discriminatory genetic technique that is comparable among laboratories. This technique has since been successfully applied to numerous human, animal, plant, insect, and fish bacterial pathogens (Kotetishvili et al. 2005, Nemoy et al. 2005, Scally et al. 2005, Saunders & Holmes 2007, Clegg et al. 2016, Krongdang et al. 2017) and typically utilizes partial sequences of 6 - 8 highly conserved housekeeping genes to discriminate between isolates of the same species (Jolley & Maiden 2004). Each of the housekeeping genes are assigned an allele type (AT) based upon their nucleotide sequence. The specific combination of ATs (i.e., allelic profile), in turn, determines the sequence type (ST), which are then arranged into clonal complexes (CC) that are hypothesized to be closely related (Feil et al. 2004). In 2008, Nicolas et al. designed a MLST scheme for the genotyping of F. psychrophilum through the examination of 50 geographically diverse isolates from Asia, Europe, North America, and South America, which were recovered from 10 fish species. Out of the original 11 loci under investigation, 7 (i.e., trpB, gyrB, dnaK, tuf, fumC, murG, atpA) were selected to be used for future MLST investigations of F. psychrophilum (Nicolas et al. 2008). This study revealed the presence of 30 genotypes, some of which were more closely related to one another, or were correlated with a geographic location and/or host species. Following the initial study, Siekoula-Nguedia et al. (2012), typed 66 F. psychrophilum isolates collected from 20 farmed rainbow trout in France and identified 14 novel STs as well as previously identified ST2. Importantly, all ST2 isolates were recovered from rainbow trout experiencing episodes of BCWD. Moreover, they discovered an epidemic population structure in which a single clone (i.e., ST2) quickly gave rise to multiple new clones through recombination. Fujiwara-Nagata et al. (2013) analyzed 114 F. psychrophilum isolates recovered from 15 fish species in Japan. They discovered substantial F. psychrophilum genetic diversity within the country and revealed 32 STs unique to Japan. Additionally, the authors rediscovered 3 STs that had been previously reported in coho salmon and rainbow trout elsewhere in the world raising the possibility that the international trade of fish contributed to the spread this genotype. Strepparava et al. (2013) identified ST2 and its SLVs as the culprits of BCWD when 112 F. psychrophilum isolates were sampled from unhealthy rainbow trout, brown trout, and char in Swiss fish farms. Importantly, ST10, which is a SLV of ST2, was implicated in disease in these Swiss fish farms. ST10 had been previously recovered from diseased fish in other European countries, as well as from the state of Oregon in the United States. At this time, ST2 was designated the founding ST of a major clonal complex associated with an abundance of apparently virulent strains. However, this conclusion is now known to be inaccurate and was a product of insufficiently sampled F. psychrophilum isolates. In 2014, Nilsen et al. (2014) typed 560 F. psychrophilum isolates from 4 Nordic countries including, Denmark, Finland, Norway, and Sweden. This study identified numerous novel STs and CCs and it was at this time that ST10 was identified as the true founding ST of the major CC (i.e., CC-ST10) that has been repeatedly associated with BCWD outbreaks. Avendaño-Herrera et al. (2014) published a study on 91 F. psychrophilum isolates from Chile, which were recovered from Atlantic salmon, coho salmon, and rainbow trout – a majority of which presented with BCWD. Similar to previous studies, they identified STs 21 restricted to a particular geographic location as well as a few that were present elsewhere in the world (i.e., ST10, ST2, ST9, ST12, ST23). In 2016, Van Vliet et al. (2016) published the largest MLST study on 96 F. psychrophilum isolates from 3 Oncorhynchus spp. (e.g., rainbow/steelhead trout, chinook salmon, and coho salmon) and 9 states in the United States. However, the majority of these isolates (i.e., 50/96) were recovered from the state of Michigan, USA. Nevertheless, 34 F. psychrophilum STs, 28 of which were novel, were identified. Of note, novel ST78, was recovered from rainbow/steelhead trout experiencing acute mortality and belongs to CC-ST10. Furthermore, ST78 was recovered from 2 additional U.S. states, also experiencing BCWD mortalities. All ST10 and ST78 isolates were recovered exclusively from rainbow/steelhead trout, indicating this fish species may be the niche for these particular strains. This finding is in support of previous research, which has identified CC-ST10 isolates almost exclusively from rainbow trout. In comparison to the rest of the world, North America has had very few F. psychrophilum isolates evaluated using MLST, which is surprising considering the economic losses F. psychrophilum causes in US salmonid populations (reviewed in Cipriano & Holt 2005). Thus it remains unclear if the previous North American MLST studies on F. psychrophilum have adequately characterized the genetic heterogeneity in this area of the world and if the trends seen among Michigan F. psychrophilum isolates are also mirrored in other states, particularly those that intensively culture salmonids species. Furthermore, the contributions of recombination/mutation to the diversification of North American F. psychrophilum STs has not been evaluated previously. 22 2.4.3.7. Whole genome multilocus sequence typing Recently, a whole genome analysis was completed on 41 F. psychrophilum isolates, 30 of which had not been whole genome sequenced previously (Duchaud et al. 2018). Within this study, 1,549 single copy genes were considered for whole genome MLST, where up to 26 allele types were present per loci. ST10 was still considered the founding ST of CC-ST10 and the authors concluded that whole genome analysis largely reflected the conclusions of the original MLST protocol based on 7 genes (Duchaud et al. 2018). In particular, F. psychrophilum was noted to have low levels of nucleotide divergence (i.e., 0.285% inside CDSs in pairwise sequence comparisons) and high amounts recombination among alleles. Furthermore, the genome of F. psychrophilum was described to be an “open” pan-genome (i.e., as additional F. psychrophilum isolates are sequenced, new genes are discovered) and of the genes identified within CC-ST10 isolates, some appeared to encode the machinery for a type II secretion system, which may contribute to the apparent virulence of CC-ST10 isolates. In the same context, the tetX gene was present within CC-ST10 isolates and is known to contribute to enzymatically inactivate tetracycline (McArthur et al. 2013, Duchaud et al. 2018). 2.5. Flavobacterium psychrophilum and experimental challenge models Experimental challenge models are not only important for fulfilling Koch’s postulates, but also for studying microbial pathogenesis, virulence mechanisms, and vaccine efficacy. For F. psychrophilum, many routes of infection have been utilized including subcutaneous, intraperitoneal, and intramuscular injection (Holt 1987, Obach & Laurencin 1991, Fredriksen et al. 2013a), as well as oral, anal, cohabitation and bath immersion (Lorenzen et al. 1991, Decostere et al. 2000). However, finding a robust and reproducible F. psychrophilum challenge 23 model has been challenging. For example, the reproducibility of F. psychrophilum challenge models are influenced by host factors (e.g., fish size, species, and origin), pathogen factors (e.g., bacterial strain, inoculum size and method of preparation), and environmental factors (e.g., the number of fish in each tank and their stress prior to infection; Madsen & Dalsgaard 1999, Decostere et al. 2000, Long et al. 2013, Long et al. 2014). 2.5.1. Bacterial inoculum preparations Many different types of culture media have been utilized for growing F. psychrophilum in vitro, including TYES, iron-limited TYES, Shieh with or without iron, modified Cytophaga, Cytophaga supplemented with carbohydrates, and Cytophaga enriched with 5% horse serum (Madsen & Dalsgaard 1999, Decostere et al. 2000, Garcia et al. 2000, Madetoja et al. 2002, Aoki et al. 2005, Long et al. 2014). Likewise, different incubation temperatures (i.e., 15°C, 16°C, 17°C, 18°C), durations (i.e., 18, 24, 48, or 72 hours) with or without agitation, and the way in which inoculum size was estimated prior to infection (i.e., using incubation time to correlate with a particular growth phase or using optical density (OD) to correlate with viable cell counts) have been employed (Madsen & Dalsgaard 1999, Decostere et al. 2000, Garcia et al. 2000, Madetoja et al. 2002, Aoki et al. 2005, Long et al. 2014). Furthermore, Michel et al. (1999) provided evidence that the addition of 5% defibrinated horse serum, trace amounts of minerals (i.e., concentration < 0.05%), and stirring all improved the culturability of F. psychrophilum. The variability among laboratories in regards to inoculum preparation hinders reproducibility and comparability flavobacterial experimental challenge models. Furthermore, as Michel et al. (1999) provided evidence for, both the culture media and incubation environment can affect the accuracy of colony counts, which are used to determine the experimental inoculum size. 24 Therefore, a reliable and standardized approach to inoculum preparation must be adopted for inter-laboratory comparisons of results. 2.5.2. Immersion challenge studies Immersion studies have proven useful for mimicking a natural route of infection (reviewed in Nematollahi et al. 2003); however, reproducibility has been an issue without scarification or pre-treatment with therapeutic compounds (i.e., formalin, H2O2; Madsen & Dalsgaard 1999, Garcia et al. 2000, Madetoja et al. 2000, Henriksen et al. 2013, Long et al. 2013). For example, Decostere et al. (2000) immersed rainbow trout in several strains of F. psychrophilum at 3 different concentrations (i.e., 106, 107, 108 cfu/mL-1) for 45 minutes, which resulted in 0% mortality. Likewise, Decostere et al. (2000) was unable to produce mortalities in rainbow trout after skin abrasion with a 26 G needle or gill clipping followed by immersion in F. psychrophilum, again at varying concentrations (i.e., 106, 107, 108 cfu/mL-1). Madsen & Dalsgaard (1999) were able to slightly elevate mortalities (i.e., 27 - 31% to 41 - 66%) among rainbow trout pre-treated with 0.005% formalin for 30 prior to immersion in 107 - 108 cfu/mL-1 of F. psychrophilum in comparison to the control group that received immersion in 107 cfu/mL-1 of F. psychrophilum only. However, the difference between these two groups was not statistically significant and a formalin-treated control group was not included. Henriksen et al. (2013) found a significant difference in mortality among rainbow trout pre-treated with a 200 mg/L-1 H2O2 for 60 minutes then immersed in F. psychrophilum; however, a standardized approach was not established. 25 2.5.3. Intraperitoneal injection studies Intraperitoneal injection (IP) of F. psychrophilum has proven an effective means of eliciting BCWD among experimentally challenged fish. Decostere et al. (2000) produced similar mortality (i.e., 71% or 69%) among experimentally challenged rainbow trout challenged with either 4.5 x 105 cfu/fish-1 or 1.4 x 104 cfu/fish-1 and the observed difference was attributed to slightly different sizes of fish. Similarly, Decostere et al. (2001) IP-injected 1g (10 weeks old), 25g (20 weeks old), and 300g (15 months old) rainbow trout at a concentration of 106 cfu/fish-1 and was able to produce clinical signs and mortality among the 1g fish only. However, a drawback of this route of exposure is that it bypasses intrinsic mechanisms of host defense including physical barriers (e.g., skin, gills, mucus) and non-specific cells of the innate immune system (e.g., phagocytes, monocytes, cytotoxic cells; reviewed in Magnadóttir 2006). 2.5.4. Intramuscular injection studies Intramuscular injection (IM) is the preferred route of exposure for larger fish (i.e., >5-10 grams; Fredriksen et al. 2013b) and has consistently produced significant mortality among experimentally challenged fish. For example, ayu (mass of ~1.0 gram per fish) challenged IM (in the dorsal musculature) with a concentration 1.5 x 105 cfu/fish-1 of F. psychrophilum produced 88-90% mortality and acute signs of BCWD disease such as bulging, discolored skin with uncoagulated blood collecting underneath the injection site; however, no open lesions developed (Miwa & Nakayasu 2005). Garcia et al. (2000) found IM injection to be the preferred route of infection when experimentally challenging rainbow trout. In their study, rainbow trout (mass of ~3-6 grams per fish) injected with 4.2 x 107 cfu/fish-1 experienced cumulative mortality of 100% by 18 days post infection and exhibited necrosis of the skin and underlying musculature near the 26 site of injection (Garcia et al. 2000). Similarly, Fredriksen et al. (2013b) achieved 100% mortality among juvenile rainbow trout (mass of ~36.6 grams per fish) following IM challenge of 1.0 x 106 cfu/fish-1 of F. psychrophilum. Of note, Fredriksen et al. (2013b) was unable to produce mortalities among their rainbow trout using an intraperitoneal (IP) route of exposure, even though at least one group was challenged with a dose (i.e., 5.0 x 107 cfu/fish-1) higher than the one used for the IM challenge. Therefore, it was more advantageous to challenge their fish using IM injection since IP injection proved to be inadequate for reproducing BCWD. In the same context, Holt (1987) found that in order to produce comparable mortality in coho salmon, a much higher dose of F. psychrophilum was required for IP injection than IM injection. IM injection is an effective route of exposure for producing disease in fish of multiple sizes, whereas other routes of infection, such as immersion, are more appropriate for smaller fish (i.e., <5 grams; Fredriksen et al. 2013b). The reproducibility and consistently high mortality produced by IM injection makes this route of exposure useful not only for studying pathogenesis but also for testing vaccine efficacy. F. psychrophilum causes disease in numerous fish species around the world, which results in substantial economic losses. For decades, researchers have been examining the genetic diversity of F. psychrophilum with the hope of better understanding important aspects of this pathogen, such as its epidemiology and pathogenicity. 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Journal of the Japan Fisheries Society 40:751-757. 37 Chapter 2 Novel MLST F. psychrophilum Genotypes Infecting North American Salmonids 38 1. Abstract Flavobacterium psychrophilum is the etiological agent of Bacterial Coldwater Disease (BCWD) and Rainbow Trout Fry Syndrome (RTFS), which cause significant economic losses in salmonid aquaculture, particularly to farm-raised rainbow trout (Oncorhynchus mykiss). The genetic heterogeneity of this fish pathogen has been explored recently by numerous investigators, whereby isolates from Europe, Asia, South America, Australia, and most recently, North America, have been genotyped using multilocus sequencing typing (MLST). These analyses revealed North American F. psychrophilum isolates to be genetically heterogeneous; nevertheless, much remains to be elucidated in regards to the population structure of this bacterium in North America. In this study, MLST was used to genotype 314 North American F. psychrophilum isolates that were opportunistically obtained from 10 fish host species from 20 US states and one Canadian province over a period of nearly four decades. My analyses placed these isolates into 66 sequence types (STs), 47 of which were novel. Furthermore, seven novel North American clonal complexes (CCs) were discovered, which brings the total number of North American CCs to 12. Many of the identified CCs have only been detected in North America, whereas others were found in North America and abroad. These CCs were diverse and varied in terms of host association, distribution, and association with disease. The largest F. psychrophilum CC identified in this study was CC-ST10, within which 10 genotypes were discovered, most of which came from O. mykiss experiencing BCWD. ST275 of CC-ST10 was recovered from wild/feral adult steelhead and the recovery of CC-ST10 from feral/wild fish in North America has not been reported previously. Furthermore, the progeny of infected broodstock were found to harbor the same ST, suggesting some STs are capable of circumventing current egg disinfection practices. The diversification of F. psychrophilum in 39 North America was found to be driven four times more frequently by recombination than point mutation and may be an indication of how new phenotypes emerge within this pathogen. 2. Introduction Flavobacterium psychrophilum (Family Flavobacteriaceae; Phylum Bacteroidetes) is a Gram-negative, psychrophilic bacterium (reviewed in Nematollahi et al. 2003). It is the etiological agent of bacterial cold water disease (BCWD) and rainbow trout fry syndrome (RTFS), both of which principally impact salmonids species (Family Salmonidae; reviewed in Cipriano & Holt 2005). These diseases cause significant mortality in farmed/hatchery-reared salmonids and can result in substantial economic losses (reviewed in Loch & Faisal 2017, Duchaud et al. 2018). Since its initial isolation in 1948 in Washington, USA (Borg 1948), this bacterium has been reported from at least five continents (e.g., Asia, Europe, North America, Australia, and South America), giving it a nearly worldwide distribution, particularly where salmonids are present (Nematollahi et al. 2003). For decades, the degree of intraspecific genetic diversity within F. psychrophilum was unclear (Madsen & Dalsgaard 2000); however, advances in molecular typing techniques subsequently revealed considerable heterogeneity within this species. One such technique is multilocus sequence typing (MLST). Due to its discriminatory power, reproducibility, and repeatability (Cooper & Feil 2004), Nicolas et al. (2008) designed a MLST scheme for the genotyping of F. psychrophilum that is now being followed worldwide. In their original study, 50 F. psychrophilum isolates from Asia, Europe, North America, and South America, including 10 from North America (9 from the USA) were examined. Nicolas et al. (2008) revealed the presence of 30 genotypes, some of which were closely related or correlated to a geographic 40 location and/or host species. Since then, this genotyping technique has been applied to >1,000 F. psychrophilum isolates from different parts of the world, yielding nearly 200 distinct genotypes, which exhibited similar trends to those observed with the original 50 isolates tested (Siekoula- Nguedia et al. 2012, Fujiwara-Nagata et al. 2013, Strepparava et al. 2013, Avendaño-Herrera et al. 2014, Nilsen et al. 2014, Castillo et al. 2016, Van Vliet et al. 2016). To improve the comparison of all F. psychrophilum isolates, Jolley & Maiden (2010) developed a comprehensive and curated database (https://pubmlst.org/fpsychrophilum/) that is an invaluable resource for epidemiological studies of F. psychrophilum. In this context, as novel sequence types (STs; i.e., genotypes) were being discovered, some were found to be closely related, forming a clonal complex (CC), while others were genetically distinct from all other STs, described as a singleton. As the MLST database grew, a number of correlations became evident. For example, CC-ST10 encompasses hundreds of isolates and dozens of STs, yet almost all of them have been isolated from farm-raised rainbow trout (Oncorhynchus mykiss) on multiple continents. In the same context, members of CC-ST123 (12 isolates in 6 STs) have all been isolated from Atlantic salmon (Salmo salar) in Norway (Nilsen et al. 2014), whereas CC-ST52 and CC-ST48/CC-ST56 were recovered exclusively from non-salmonid species (Fujiwara- Nagata et al. 2013) and experimental challenge data provided evidence that these two CCs differ in their pathogenicity as well (Fujiwara-Nagata et al. 2012). Despite the economic losses F. psychrophilum causes in North American salmonid populations (reviewed in Cipriano & Holt 2005), only 10 isolates had been MLST genotyped as of 2016 (Nicolas et al. 2008). Van Vliet et al. (2016) characterized an additional 95 F. psychrophilum isolates from 9 US states, finding 34 F. psychrophilum STs, 28 of which were previously undescribed. These STs varied spatially, by host species, and in association with 41 mortality events similar to those recovered outside of North America (Van Vliet et al. 2016). This study focused on isolates from three Oncorhynchus spp. (e.g., O. mykiss, O. kisutch, and O. tshawytscha) and over half were recovered from one US state (i.e., Michigan). To better understand the genetic diversity of F. psychrophilum in North America, I undertook this study and MLST-genotyped 314 F. psychrophilum isolates recovered from 10 fish species, collected from 20 US states and one Canadian province. I took advantage of the presence of historical, cryopreserved F. psychrophilum isolates that were isolated over a period of four decades and included them in this study. This unique collection of isolates allowed for an evaluation of the genotypic relationships not only among North American isolates, but also among those in other parts of the world. An additional objective was to evaluate the role of point mutation and/or recombination in leading to the MLST diversity discovered in this study. 3. Materials and Methods 3.1. Fish collection and isolation of F. psychrophilum A total of 314 F. psychrophilum isolates recovered from 20 U.S. states and one Canadian province over nearly four decades (e.g., 1981 - 2018) were examined in this study (Table 2.1a). The isolates originated from Idaho (n = 104), Utah (n = 43), Michigan (n = 38), Pennsylvania (n = 29), North Carolina (n = 24), Washington (n = 19), Oregon (n = 18), West Virginia (n = 7), Montana (n = 6), California (n = 5), South Dakota (n = 4), Virginia (n = 4), Minnesota (n = 3), New Mexico (n = 2), Colorado (n = 2), Alaska (n = 1), Indiana (n = 1), Maryland (n= 1), Wisconsin (n = 1), and Vermont (n = 1) (Figure 2.1). Additionally, a single isolate from British Columbia was included (Figure 2.1). The isolates were recovered from four fish genera and 10 species, including rainbow trout/steelhead (O. mykiss; n = 260), coho salmon (O. kisutch; n = 42 25), Chinook salmon (O. tshawytscha; n = 11), cutthroat trout (O. clarkii; n = 1), sockeye salmon (O. nerka; n = 1), Atlantic salmon (S. salar; n = 6), brown trout (S. trutta; n = 6), lake trout (Salvelinus namaycush; n = 1), splake (S. fontinalis x S. namaycush; n = 1), and white sturgeon (Acipenser transmontanus; n = 2; Table 2.1a). The isolates were recovered from external and internal tissues of fish either showing gross signs of BCWD or that were apparently healthy (Table 2.1a). Recovery of the aforementioned F. psychrophilum isolates was completed by multiple investigators using flavobacterial-selective media, including cytophaga agar (CA; Anacker & Ordal 1959) and tryptone yeast extract agar (TYES; Holt 1987) with or without supplementation of 4 mg liter-1 of neomycin sulfate, or Hsu-Shotts medium (HSU; Bullock et al. 1986). F. psychrophilum isolates not collected in house were procured from project collaborators as actively growing cultures on solid media or as frozen cryo-stock and revived and subcultured on to TYES agar to verify isolate purity. A single colony was then inoculated into analagous broth media for generation of glycerol-supplemented cryo-stock (maintained at -80˚C) for further use. 3.2. Bacterial DNA extraction and F. psychrophilum identity confirmation F. psychrophilum isolates were revived from cryostocks into TYES broth and incubated for 72-96 hours at 15˚C, at which time bacterial cells were subcultured on to fresh TYES medium with agar to verify purity. Bacterial genomic DNA was extracted using the DNeasy Blood and Tissue kit (Qiagen, Inc., Valencia, California) according to the manufacure’s protocol for Gram-negative bacteria, quantified using a Qubit® Fluorometer (Life Technologies, Grand Island, New York), and then diluted to 20 ng/µl. Prior to MLST analyses, the identity of all 43 isolates was confirmed as F. psychrophilum using the conventional PCR assay of Toyama et al. (1994) as detailed previously (Van Vliet et al. 2015). 3.3. Multilocus sequence typing Partial sequences of seven housekeeping genes (trpB, gyrB, dnaK, fumC, murG, tuf, atpA), previously selected by Nicolas et al. (2008), were PCR amplified as previously detailed (Van Vliet et al. 2016). Five µl of amplicon was electrophoresed in a 1.5% agarose gel prepared with 1 X SYBR®Safe DNA gel stain for 45 min at 100V. The gel was then viewed under ultraviolet transillumination to confirm the presence of an appropriately sized band. One µl of PCR product was then combined with 0.25 µl of ExoSAP-IT™ (Applied Biosystems) and 3.0 µl of 1 X PCR buffer with MgCl2 (Sigma-Aldrich), incubating for 20 min at 37°C, followed by enzyme inactivation for 10 min at 95°C. The same primers employed for PCR amplification of each of the seven house-keeping genes were used to bidirectionally sequence purified PCR product at the Genomics Technology Support Facility (Michigan State University). 3.4. MLST data analysis Chromatograms were manually verified for quality prior to assignation of allele types (ATs) and STs via an in-house script (P. Nicolas, INRA). eBURST v3 (eburst.mlst.net; Feil et al. 2004, Spratt et al. 2004) was used to dichotomize STs into CCs or as singletons based on locus variations in their allelic profiles. STs were considered single-locus variants (SLVs) when they differed at one of the seven ATs, whereas STs that varied at two of the seven ATs were considered double-locus variants (DLVs). CC were comprised of STs on the basis of their shared SLVs and were named after the predicted founding ST (i.e., the ST with the highest 44 number of SLVs). STs that did not form part of a CC were referred to as singletons. All isolates within the publicly accessible MLST database for F. psychrophilum (n = 1,097; https://pubmlst.org/fpsychrophilum/; Jolley & Maiden 2010), along with the 314 isolates of this study, were included in the eBURST v3 (Feil et al. 2004, Spratt et al. 2004) analysis. All previously typed isolates from North America (n = 107; Nicolas et al. 2008, Castillo et al. 2016, Van Vliet et al. 2016) were included in the eBURST v3 analyses. 3.5. Bayesian inference Bayesian phylogenetic analysis was conducted using MrBayes v3.2.5 (Huelsenbeck & Ronquist 2001, Ronquist & Huelsenbeck 2003) after concatenating the seven MLST loci sequences for each North American ST. Automated model selection was implemented in PAUP* v4.0a (Build 162; Swofford 2002) and the most appropriate model of DNA substitution for each MLST locus was selected using the corrected Akaike information criterion (AICc) score. Subsequently, a partitioned data file, with one partition per MLST locus, was used for Markov chain Monte Carlo (MCMC) analysis. The analysis consisted of two independent runs, both of which used four Markov chains (one heated, three cold) and began with a random starting tree. This analysis sampled every thousandth tree for 520,000 generations, at which point the average standard deviation of split frequency was <0.05. Prior to phylogenetic reconstruction, a 25% burn-in period (i.e., the first 25% of trees were discarded and not used for phylogenetic reconstruction) was utilized. FigTree v1.4.3 (Rambaut 2009) was used to visualize and annotate the Bayesian dendrogram. 45 3.6. MLST genetic analyses Analysis of sequence variation among the seven housekeeping genes was implemented via multiple algorithms. DNAsp v6.10.01 (Rozas et al. 2017) was used to identify the number of alleles for each gene (i.e., allele types), the number of segregating sites (S), nucleotide diversity (π), and the Hudson and Kaplan lower bound on the minimum number of recombination events (Rmin; Hudson & Kaplan 1985). Utilizing SLV STs from CCs and a method described by Feil et al. (2000), the contributions of recombination or point mutations to clonal diversification was estimated. Recombination was also evaluated among the concatenated sequences of representative STs using the pairwise homoplasy index (PHI) executed in SplitsTree v4.14.6 (Huson & Bryant 2006). START2 (Jolley et al. 2001) was utilized to calculate the ratios of non- synonymous to synonymous substitutions (dN/dS) for each locus. LIAN 3.7 (Haubold & Hudson 2000) identified the extent of linkage disequilibrium (ISA) between the ATs using a Monte Carlo simulation, whereby loci were resampled 1,000 times without replacement and observed mismatch variance was compared to the expected mismatch variance. LIAN 3.7 was also used to measure mean genetic diversity (H). 4. Results 4.1. Identification of novel F. psychrophilum MLST genotypes in North America Out of the 314 North American F. psychrophilum isolates typed using MLST in the present study, 119 represented 47 novel MLST genotypes that have never been described anywhere in the world (Table 2.1b). Of these 47 genotypes, 11 formed four novel MLST clonal complexes (CC-ST288, CC-ST310, CC-ST281, CC-ST-287; Figure 2.2). CC-ST310 was recovered exclusively from juvenile rainbow trout (O. mykiss) suffering from BCWD at several 46 private facilities (Table 2.1a, Table 2.1b). Members of the CC-ST281were also recovered exclusively from steelhead trout (O. mykiss); however, 10/11 isolates belonging to this CC were from apparently healthy adult steelhead trout (O. mykiss), whereas the remaining isolate was recovered from recently hatched steelhead trout (O. mykiss) fry experiencing BCWD in a state fish hatchery (Table 2.1a, Table 2.1b). CC-ST288 was recovered exclusively from apparently healthy, adult Chinook salmon returning to spawn at a Michigan weir (i.e., MI-WE1). The fourth CC; CC-ST287, was recovered from both coho salmon and splake, both of which were being raised in two different state fish hatcheries (Table 2.1a, Table 2.1b). Of the remaining 36 novel MLST genotypes, four clustered with several previously described North American MLST genotypes to form three additional novel MLST CCs (Figure 2.2). More specifically, former singleton ST29 clustered with novel genotype ST308 to form CC-ST29, which contains F. psychrophilum isolates recovered exclusively from Chinook salmon (Nicolas et al. 2008, Van Vliet et al. 2016; Table 2.1a, Table 2.1b). Likewise, former singleton ST257, which was previously recovered from a diseased juvenile steelhead trout (O. mykiss) from a state fish hatchery in Michigan (Van Vliet et al. 2016), was rediscovered in the present study in apparently healthy adult steelhead trout (O. mykiss) returning to spawn, and clustered with novel genotype ST276 to form CC-ST276 (Table 2.1a, Table 2.1b; Figure 2.2). Like ST257, ST276 had been recovered from both apparently healthy and diseased adult and juvenile steelhead trout (O. mykiss); however, all isolates identified as ST276 were from steelhead trout (O. mykiss) at state fish hatcheries in Pennsylvania (Table 2.1a, Table 2.1b). The remaining novel CC, CC-ST296, was comprised of ST265, ST296, and ST299 (Figure 2.2). All three of these STs were recovered from different host species, whereby previously described ST265 was recovered from Chinook salmon (Van Vliet et al. 2016), novel genotype ST296 was recovered 47 from both rainbow trout (O. mykiss) and sockeye salmon, and novel genotype ST299 was recovered from steelhead trout (O. mykiss) (Table 2.1a, Table 2.1b). Of the remaining 32 novel MLST genotypes, 10 were placed into a single, preexisting North American CC (i.e., CC-ST10) that previously contained only six North American STs (Table 2.1a, Table 2.1b; Figure 2.2; https://pubmlst.org/fpsychrophilum/). Of these 10 novel genotypes, nine were recovered exclusively from O. mykiss, whereas the remaining genotype (i.e., ST306) was recovered from both rainbow trout (O. mykiss) and coho salmon (Table 2.1a, Table 2.1b). Additionally, 9/10 CC-ST10 genotypes were recovered from captive O. mykiss, whereas the remaining genotype (i.e., ST275) was recovered from both captive and wild/feral steelhead trout (O. mykiss; Table 2.1a, Table 2.1b). Furthermore, the wild/feral steelhead trout (O. mykiss) that novel genotype ST275 was recovered from was an apparently healthy adult, which is in contrast to all of the other nine genotypes as they were recovered from sub-adult O. mykiss that were displaying signs of BCWD disease (Table 2.1a, Table 2.1b). Nineteen of the remaining 22 novel genotypes were not placed into any previously or newly identified MLST CC (Figure 2.2) and thus were considered to be singleton genotypes, all of which were exclusive to a single fish host species (Table 2.1a, Table 2.1b). When the genotypes of this study were combined with all previously reported genotypes from other regions of the world (https://pubmlst.org/fpsychrophilum/), the remaining three novel MLST genotypes recovered in this study belonged to two additional CCs whose members were recovered from Europe (i.e., Denmark, Finland, Norway, Switzerland). In particular, ST301 grouped within preexisting CC-ST191 as a single locus variant (SLV) of ST191, whereas ST277 and ST283 clustered with former singleton ST232 to form CC-ST232 (Figure 2.3). 48 4.2. F. psychrophilum MLST genotypes detected in this study that were reported previously from North America and/or abroad The remaining 195 F. psychrophilum isolates of this study represented 19 MLST genotypes that have been previously reported in North America and/or abroad (Table 2.1a, Table 2.1b). Of these 19 genotypes, three (e.g., ST11, ST256, ST257) belonged to three CCs that are currently present only in North America. Of the remaining 16 previously described genotypes, seven belonged to one of three CCs that are present in both North America and/or Europe, Asia, and South America. For example, ST10, ST78, ST84, and ST86 were all recovered from diseased captive O. mykiss in the present study and belonged to CC-ST10, which is known to house numerous genotypes globally (Figure 2.3; https://pubmlst.org/fpsychrophilum/). Furthermore, ST10 was also recovered from coho salmon, Chinook salmon, and white sturgeon in this study and thus has the broadest host range of any North American genotype to date (Table 2.1a, Table 2.1b). Next, ST28 was recovered from cutthroat trout and belongs to CC-ST31, which is present not only in North America but Europe as well (Table 2.1a, Table 2.1b; Figure 2.2; https://pubmlst.org/fpsychrophilum/). Lastly, ST9 and ST13 were recovered herein from predominantly coho salmon; although one isolate belonging to ST9 was recovered from a rainbow trout (O. mykiss) as well. Nonetheless, these two genotypes form CC-ST9, which has been recovered predominantly from coho salmon in North America, Europe, Asia, and South America (Table 2.1a, Table 2.1b; Figure 2.2, Figure 2.4; http://pubmlst.org/fpsychrophilum). When the genotypes of this study were combined with all previously reported genotypes from other regions of the world, two genotypes that were recovered in this study (i.e., ST267, ST70) belonged to two CCs whose members are from Europe or both Europe and South America. For example, ST267 belonged to CC-ST191 and was recovered from apparently 49 healthy, wild/feral adult steelhead trout (O. mykiss), whereas ST70 belonged to CC-ST124, which was recovered from an Atlantic salmon (Table 2.1a, Table 2.1b; Figure 2.3). The remaining seven MLST genotypes were identified as singletons. Of these, five were recovered from a single fish host species, whereas the other two were recovered from multiple host species. In specific, ST27 and ST255 were recovered exclusively from steelhead trout (O. mykiss), both of which displayed signs of disease; ST30 and ST74 were recovered exclusively from coho salmon; and ST76 was recovered exclusively from Chinook salmon (Table 2.1a, Table 2.1b). Of the two singleton genotypes recovered from multiple host species, ST253 was recovered from an apparently healthy, captive adult rainbow trout (O. mykiss) and additionally from several brown trout, all of which displayed signs of BCWD disease. The final singleton genotype, ST258, was recovered predominantly from sub-adult, captive and diseased coho salmon but also from a single, apparently healthy adult steelhead trout (O. mykiss) returning to spawn (Table 2.1a, Table 2.1b). 4.3. Bayesian inference of North American F. psychrophilum genotypes Phylogenetic analysis of the 86 currently recognized North American STs yielded robust support (i.e., posterior probability >70%) for all 12 North American CCs identified by eBURST (Figure 2.5). In addition, the three CCs (i.e., CC-ST191, CC-ST232, CC-ST124) that contained North American STs, which were formed when all STs from other regions of the world were combined with those in North America, were also robustly supported (Figure 2.5). Of these 15 CCs, CC-ST10 was the most basal, whereas CC-ST9 was the most derived (Figure 2.5). Of note, the 16 STs of CC-ST10 present in North America were separated into four clades (i.e., CC- ST10a-d; Figure 2.5), three of which (e.g., CC-ST10a-c) were robustly supported (Figure 2.5). 50 Of the three well-supported clades, CC-ST10a was most basal and contained ST303 and ST305, CC-ST10b contained eight STs, two of which (i.e., ST304, ST306) formed a robustly supported sub-clade (Figure 2.5). Lastly, CC-ST10c was comprised of five STs and was the most derived (Figure 2.5). In addition to the five STs of CC-ST10c, another ST, ST27, was found to share the same most recent common ancestor as all others in this clade (Figure 2.5). The remaining 14 CCs were reconstructed as independent clades, some of which shared a most recent common ancestor with a singleton ST (Figure 2.5). Furthermore, the Bayesian phylogeny provided support for the formation of several other clades made up solely of singleton STs (Figure 2.5). One clade in particular contained F. psychrophilum isolates belonging to three genotypes (i.e., ST251, ST254, ST266), which were recovered exclusively from feral Michigan Chinook salmon. Similarly, another F. psychrophilum clade housed genotypes that had been recovered from feral Michigan Chinook salmon; however, this clade housed not only singleton STs but CC-ST288 as well (Figure 2.5). 4.4. STs and geographic origin Due to the inconsistent number of isolates recovered from each state, no statistical comparisons of STs in association with their geographical origin were conducted. Nevertheless, several observations were made. For example, the three most abundant STs (e.g., ST10, ST78, ST275), all of which belong to CC-ST10, also had the widest geographic distribution in our dataset (Table 2.1a, Table 2.1b; Figure 2.2). In specific, ST10, ST78, and ST275 were detected in 13, nine, and five states, respectively (Table 2.1a, Table 2.1b; Figure 2.2). The next most abundant STs (e.g., ST9, ST85, ST86, ST258, ST291, ST296, ST300, ST301, ST306) were recovered from two to four states, whereas the remaining 54 STs were recovered exclusively 51 from one state (Table 2.1a, Table 2.1b). In the state of Idaho, where 104/314 study isolates originated, 61/104 isolates were identified as ST10 and were detected in 9 distinct locations (Table 2.1a, Table 2.1b). The remaining 43 isolates fell into 16 additional STs, the bulk of which (12/16 STs) were recovered from a single location (Table 2.1a, Table 2.1b). Most isolates recovered from Idaho (n = 91/104 isolates) were affiliated with a ST that belonged to CC-ST10 (e.g., ST10, ST78, ST300, ST303, ST304, ST305, ST306, ST316). It appeared CC-ST10 had existed in Idaho for at least three decades since two of its genotypes (e.g., ST10, ST306) were recovered from this state in the early 1990s (Table 2.1a, Table 2.1b). In the state of Utah, most isolates were identified as ST10, ST78, or ST86, all of which belong to CC-ST10. Of these, ST10 was recovered most frequently (n = 37/43 isolates) followed by ST86 (n = 3 isolates), and then ST78 (n = 2 isolates). Of note, ST10 was recovered from 10 distinct locations between the years of 2004 and 2015 (Table 2.1a, Table 2.1b). The remaining isolate from Utah was identified as ST9 (Table 2.1a, Table 2.1b). In Michigan (38 isolates), 17 STs were identified among seven distinct locations between the years of 2010 and 2018 (Table 2.1a, Table 2.1b). Of these, most of the isolates were identified as ST253 (n = 6 isolates), ST256 (n = 5 isolates), or ST258 (n = 5 isolates), whereas the remaining 14 STs contained ≤ four isolates (Table 2.1a, Table 2.1b). In contrast to the isolates of Idaho and Utah, nearly half of the isolates recovered from Michigan (n = 16/38) were identified as singletons (e.g., ST253, ST258, ST278, ST282, ST286, ST290), whereas the remaining 22 isolates belonged to CC-ST10 (n = 5 isolates), CC- ST256 (n = 5 isolates), CC-ST288 (n = 4 isolates), CC-ST232 (n = 3 isolates), CC-ST191 (n = 2 isolates), CC-ST276 (n = 2 isolates), or CC-ST287 (n = 1 isolate; Table 2.1a, Table 2.1b). The geographical origins and genotypes for the remaining isolates are presented in Tables 2.1a and 2.1b. 52 4.5. F. psychrophilum genetic analyses The mean genetic diversity (H) of all typed F. psychrophilum isolates recovered in North America (n = 421) to date was calculated to be 0.68 ± 0.04. H was also calculated for all other continents were F. psychrophilum isolates have been genotyped using MLST and ranged from 0.00 in Australia, 0.48 in South America, 0.59 in Europe, and 0.66 in Asia (https://pubmlst.org/fpsychrophilum/; Haubold & Hudson 2000). H was calculated to be 0.48 ± 0.06, 0.74 ± 0.05, and 0.90 ±0.01 for F. psychrophilum isolates recovered from North American O. mykiss (n = 318 isolates), O. kisutch (n = 44 isolates), and O. tshawytscha (n = 38 isolates), respectively. Mean genetic diversity among all other species (i.e., S. salar, n = 8; S. trutta, n = 6; A. transmontanus, n = 2; O. clarkii, n = 2; O. nerka, n = 1; S. namaycush, n = 1; S. namaycush x S. fontinalis, n = 1) was found to be 0.85 ± 0.02. The ISA value for all F. psychrophilum isolates recovered in North America was estimated to be 0.66 and differed significantly from 0 (P < 0.001), indicating linkage disequilibrium (i.e., nonrandom association between ATs). As multiple isolates belong to the same ST, sampling bias may alter the overall mean genetic diversity among the North American F. psychrophilum isolates (Nilsen et al. 2014). As such, mean genetic diversity was calculated between the 86 currently identified F. psychrophilum STs in North America and found to be 0.92 ± 0.02. The ISA value was closer to 0 (ISA = 0.2648) among the 86 representative STs; however, it still differed significantly from 0 (P < 0.001), indicating linkage disequilibrium. The 12 North American CCs presented in Figure 2.2 were combined with the three CCs in Figure 2.3 containing STs from North America (i.e., CC-ST124, CC-ST191, CC-ST232) and 30 SLVs were discovered among the 15 founding STs (Table 2.2). The CC with the highest 53 number of SLVs was CC-ST10 (n = 11), whereas the other 14 CCs had one to two SLVs. The loci tuf and trpB each accounted for the highest number of SLVs (n =7 SLVs per locus) followed by gyrB (n = 6 SLVs), murG (n = 4 SLVs), atpA (n =2 SLVs), dnaK (n = 2 SLVs), and fumC (n = 2 SLVs). Among the 30 SLVs, 20 were the result of multiple nucleotide changes and thus were likely the result of recombination. Additionally, another four SLVs were likely the result of recombination because even though their variant AT differed from their ancestral AT by only one nucleotide, that variant AT was also present in another CC (Table 2.2). The remaining six SLVs differed from their ancestral AT by a single nucleotide and were not found elsewhere in the dataset, thereby suggesting those variant ATs likely arose from point mutation (Table 2.2). If 24/30 SLVs resulted from recombination and the remaining 6/30 SLVs resulted from point mutation, then North American F. psychrophilum loci are estimated to be four times more likely to diversify through recombination than by point mutation. The potential for amino acid shifts resulting from these mutational events was also analyzed, revealing that 25 mutations were synonymous and five were non-synonymous. Of note is murG1 (i.e., locus murG designated with AT 1), which produced three distinct amino acid changes (e.g., proline to histidine, glutamic acid to lysine, and isoleucine to valine) resulting from seven nucleotide polymorphisms (Table 2.2). This contrasts with all other non- synonymous substitutions, which only produced a single amino acid change. In fact, of the five non-synonymous substitutions, four were due to variations at the murG locus and these four variations were the only ones found at this locus among the 15 CCs (Table 2.2). When the seven loci used in the MLST scheme for F. psychrophilum were concatenated for each of the 86 representative STs in North America, a single stretch of DNA that was 5,799 54 base pairs (bp) long was produced. Among these STs, 139 SNPs were identified, meaning the nucleotide positions of the concatenated loci differed overall by 2.40% (Table 2.3). Pairwise comparisons among loci yielded a nucleotide diversity (π) ranging from 0.29% at fumC to 1.25% at atpA and the number of unique ATs varied from eight at fumC to 41 at gyrB (Table 2.3). The ratio of non-synonymous to synonymous mutations ranged from 0.0020 at gyrB to 0.2751 at murG, which indicates that non-synonymous mutations are rare and these loci are under purifying selection (dN/dS < 1). In addition, recombination was detected among all loci (Rmin > 1) with fumC having the lowest Rmin value (n = 2) and atpA having the highest (Rmin = 10). The cumulative minimum number of recombination events among the seven loci was 38, whereas Rmin for the concatenated sequences was 44, indicating additional recombination between the loci as well (Table 2.3). A PHI test conducted on the concatenated sequences revealed statistically significant evidence (P = < 0.001) of recombination between the 86 representative STs. 5. Discussion Results presented herein reveal substantial genetic diversity within F. psychrophilum recovered from North America. In fact, combining data from this study with those of Nicolas et al. (2008) and Van Vliet et al. (2016) revealed that the mean genetic diversity of MLST-typed F. psychrophilum in North America is higher than all other studied continents (i.e., 0.68 in North America vs. 0.00 – 0.66 elsewhere). In this context, a recent study provided evidence that F. psychrophilum may have originated in North America, from where at least one successful genotype (e.g., ST10) was distributed to other regions of the world through the international trade of fish (Duchaud et al. 2018). If F. psychrophilum did indeed originate from North America, the bacterium would have had more time to diversify in this region. However, the 55 observed diversity in this study may be attributable, at least in part, to the life-history of some of the sampled fish host populations. For example, just 30 F. psychrophilum isolates were recovered from wild/feral fish but accounted for 15 STs (i.e., an average of two isolates for every ST), whereas 243 isolates were recovered from captive fish but belonged to 40 STs (i.e., >6 isolates for every ST). In this context, F. psychrophilum isolates from other regions of the world, such as Europe and South America, have been recovered primarily from captive host species (Siekoula-Nguedia et al. 2012, Strepparava et al. 2013, Avendaño-Herrera et al. 2014), which may have led to lower levels of diversity. For example, in Europe, Siekoula-Nguedia et al. (2012) discovered their 66 F. psychrophilum isolates from France belonged to 15 STs (i.e., >4 isolates for every ST) and in Switzerland, Strepparava et al. (2013) identified 27 STs among 112 F. psychrophilum isolates, equating to >4 isolates for every ST. Likewise, in South America, Avendaño-Herrera et al. (2014) utilized 94 F. psychrophilum isolates from Chile and discovered 15 STs (i.e., >6 isolates for every ST). On the other hand, in Asia, the majority of isolates originated from fish within a single river and of the 120 F. psychrophilum isolates typed from 15 different fish species, 35 STs were discovered (i.e., <3 isolates for every ST; Nicolas et al. 2008, Fujiwara-Nagata et al. 2013). In addition to revealing substantial genetic diversity, MLST genotyping also revealed some important aspects of F. psychrophilum epidemiology in North America. Of the 31 currently described F. psychrophilum CCs (Figure 2.3), nearly a third (seven novel, two previously described) have, to date, been exclusively found in the USA, where they differ in their geographic distribution (Table 2.1a, Table 2.1b; Figure 2.2). Although six US-specific CCs were recovered from multiple states (i.e., CC-ST11, CC-ST29, CC-ST276, CC-ST287, CC-ST310), the exact distribution pattern and F. psychrophilum transmission dynamic in North America 56 requires additional typing of isolates originating from different regions and fish species. It is strongly believed that the common practice of transferring live fish and their gametes for aquaculture and stock enhancement purposes in North America contributes to pathogen dissemination (Parker 1989, Faisal 2007). In this context, it may not be a coincidence that Michigan Chinook salmon harbor members of CC-ST29, which in our study could not be found except in Oregon Chinook salmon, where the Chinook salmon that were transferred to Michigan originated (Hansen & Holey 2002). Likewise, it was evident that most North American CCs that originated from aquaculture facilities and hatcheries were found in multiple states, whereas those confined to one state were commonly associated with free roaming adult fish, underscoring the potential of pathogen transmission through trade. This study revealed the diverse host ranges some of these US-specific CCs display, whereby some were collected from a single fish species (e.g., CC-ST29, CC-ST281, CC-ST288, CC-ST310) and others from multiple fish species (e.g., CC-ST11, CC-ST256, CC-ST276, CC- ST287, CC-ST296), which may be an indication that some North American CCs are adapted to a particular host species, whereas others are non-specific “generalists”. The North American CC, CC-ST10, has been reported outside of North America as well, where its isolates were also predominantly recovered from farmed rainbow trout experiencing BCWD (Siekoula-Nguedia et al. 2012, Strepparava et al. 2013, Nilsen et al. 2014, Van Vliet et al. 2016). These previous reports correlate with our findings, whereby CC-ST10 was predominantly recovered from rainbow and steelhead trout (O. mykiss) associated with systemic infections/BCWD epizootics. However, two genotypes belonging to CC-ST10 (i.e., ST10, ST306) were recovered from fish species other than O. mykiss (i.e., Chinook salmon, coho salmon, white sturgeon) in this study and it remains to be determined if these genotypes have a broader host range or if the hosts these 57 isolates were recovered from were in proximity to O. mykiss. The relevance of CC-ST10 in North America and the world makes its genotypes targets for BCWD vaccine development. Within North America, CC-ST10 was the most widespread among currently recognized CCs where it has been detected in 16 out of the 20 states in the Northwest, West, Southwest, Midwest, Southeast, and Northeast regions of the USA that were examined in this study. Van Vliet et al. (2016) also found CC-ST10 to be the most widespread in the 9 states that were investigated therein. That study, along with Nilsen et al. (2014) and Duchaud et al. (2018), suggested that ST10, the predicted CC founder strain (Nilsen et al. 2014), along with other CC- ST10 STs, have been disseminated around the world as a result of the international salmonid egg trade, where they are also now widespread (Duchaud et al. 2018). One novel genotype of CC-ST10 (i.e., ST275) was recovered from apparently healthy steelhead trout (O. mykiss) at a weir in Michigan and is to date the only ST of CC-ST10 reported to have been recovered from a wild/feral host in North America (Table 2.1a, Table 2.1b). Furthermore, the eggs from this steelhead trout (O. mykiss) were then reared at a Michigan state fish hatchery that later had an outbreak of BCWD, causing mass mortality among the resultant juvenile steelhead trout (O. mykiss). When the recovered F. psychrophilum isolates were genotyped, they were discovered to be ST275 (isolates US86 & US87; Table 2.1a, Table 2.1b), a ST not previously detected at this facility despite repeated MLST investigations. The original source of ST275 in the hatchery facility remains to be determined, but the possibility for vertical transmission and thus circumvention of modern egg disinfection strategies (i.e., iodophore disinfection) cannot be discounted. Indeed, F. psychrophilum has been shown to resist surface disinfection with povidone iodine in previous studies (Cipriano et al. 1995, Brown et al. 1997, Kumagai et al. 1998) and F. psychrophilum is believed to reside within the perivitellin space 58 (Kumagai et al. 2000), where it can resist salmonid egg lysozyme (reviewed in Cipriano & Holt 2005). However, other explanations for the occurrence of ST275 both at the weir and in the hatchery environment are possible, including improper disinfection of facility equipment at the weir that may have allowed for the transportation of ST275 to the hatchery facility, where it was introduced to the newly fertilized eggs. In any case, strict biosecurity protocols are needed to prevent the spread of F. psychrophilum and future research exploring additional egg and equipment disinfection protocols are required. Although not nearly as widespread as CC-ST10, some STs within CC-ST191 (e.g., ST267, ST301) were also detected in multiple regions of the USA (e.g., Idaho, Michigan, Pennsylvania, West Virginia). CC-ST191 appeared to be confined to Europe (e.g., Denmark, Finland, France, Norway, Switzerland; Nicolas et al. 2008, Siekoula-Nguedia et al. 2012, Strepparava et al. 2013, Nilsen et al. 2014) until Van Vliet et al. (2016) detected it in Michigan, USA. The detection of this CC from the US on multiple occasions is of interest, because the founding ST and almost all of its SLVs were exclusively detected in Europe, suggesting this CC may have originated from Europe and subsequently was introduced to North America. Likewise, all STs within CC-ST124, which is the third largest currently described F. psychrophilum CC in the world and predominately recovered from S. salar, are found only within Europe except ST70, which was found in Chile by Avendaño et al. (2014) following the Atlantic salmon trade between Chile and Norway in the 1990’s (Storebakken 2002). In addition, ST70 was discovered in the present study and by Nilsen et al. (2014), and in both instances from North American Atlantic salmon; however, any connection between the ST70 isolates from North America and those from Europe or Chile remains to be determined. 59 Thirty-one North America F. psychrophilum singletons were also detected in this study, bringing the total number of recognized North American singletons to 44 (Figure 2.2; Nicolas et al. 2008, Van Vliet et al. 2016). Based upon current data, most F. psychrophilum singletons appear to be geographically limited, where they were recovered from one specific aquaculture facility or site (Table 2.1a, Table 2.1b; Van Vliet et al. 2016). Although this may not be surprising, it was striking that some singletons were found repeatedly at the same facility over the years (e.g., ST253, 2010, 2013, 2017; Table 2.1a, Table 2.1b), emphasizing that some unique strains may find a niche and perpetuate themselves within aquaculture facilities. By combining the 314 North America F. psychrophilum isolates presented herein and all previously typed North America F. psychrophilum isolates, it is evident that some North American STs have persisted longer than others. For example, some of the earliest recovered STs in North America (e.g., ST6, ST9, ST10, ST11, ST27, ST28, ST29, ST30, ST70, ST74, ST78, ST297, ST306, ST307, ST308, 1981-1996; Nicolas et al. 2008, Van Vliet et al. 2016) have not been rediscovered in the last 20 years (e.g., ST6, ST11, ST28, ST70, ST297, ST306, ST307, ST308), whereas others are still reappearing decades later (e.g., ST10, ST27, ST29, ST78), suggesting these variants have adapted mechanisms that promote their longevity even though great strides in biosecurity and disease control (i.e., antimicrobials) have been made. Interestingly, ST10 appears to be capable of producing equally successful and persistent clones. For example, ST10 was first recognized in North America (e.g., Oregon, USA) in 1990 (Table 2.1a, Table 2.1b), then its SLV, ST78, was recovered later in Colorado in 1996 (Van Vliet et al. 2016), and over the next 15 years, ST78 has been recovered from two additional US states (e.g., West Virginia, Michigan; Van Vliet et al. 2016) and one province of Canada (e.g., British Columbia; Castillo et al. 2016). Furthermore, ST78 has produced three of its own clones (e.g., 60 ST84, ST316, ST317), which have been recovered from three additional US states (i.e., Idaho, North Carolina, Washington) where ST78 has yet to be recovered from, suggesting ST78 may be more geographically widespread than is currently recognized. The phylogenetic relationships among the 86 North American STs were evaluated using eBURST v3, a program used in all previous MLST studies on F. psychrophilum (Nicolas et al. 2008, Siekoula-Nguedia 2012, Strepparava et al. 2013, Fujiwara-Nagata et al. 2013, Avendaño- Herrera et al. 2014, Nilsen et al. 2014, Van Vliet et al. 2016), and Bayesian inference. The Bayesian analysis, which was based upon the concatenated MLST loci, largely corroborated the results of the eBURST analysis, whereby all CCs established by eBURST were reconstructed with robust support (Figure 2.2, Figure 2.5); however, a few differences were noted. For example, ST27 and ST6 were placed within CC-ST10 via Bayesian analysis but not by eBURST (Figure 2.2, Figure 2.5); however, ST27 and ST6 share 5/7 MLST loci with several genotypes of CC-ST10 (i.e., DLV; data not shown) and would be allocated to CC-ST10 in a less stringent eBURST analysis. In this context, the hypothesized phylogenetic relationships depicted by eBURST are more conservative than those portrayed by Bayesian inference. The conservativeness of eBURST has been levied as a strength in that evolutionary relationships are not forced among genotypes (eburst.mlst.net; Feil et al. 2004, Spratt et al. 2004). Furthermore, Duchaud et al. (2018) recently suggested that recombination within F. psychrophilum may disrupt the relationships between F. psychrophilum genotypes. Therefore, inferences on the genealogical history of F. psychrophilum should be made with caution when using traditional phylogenetic analyses. Nonetheless, it is interesting that Bayesian inference robustly supported the eBURST CCs given the apparent frequency of recombination throughout the genome of F. psychrophilum (Duchaud et al. 2018). Furthermore, the Bayesian phylogeny provided support 61 for the formation of several other clades, which appeared to be rooted biologically (i.e., by host species) and geographically. In this study, the contribution of recombination/mutation to the diversification among North America F. psychrophilum STs was best explained best by recombination, whereby 24/30 SLVs were hypothesized to have resulted from recombination and the remaining 6/30 SLVs resulted from mutation (i.e., ratio of 4:1), which is similar to what was reported initially by Nicolas et al. (2008; i.e., ratio of 4.5:1). This estimation, originally described by Feil et al. (2000), relies upon a visual inspection of gene sequences to estimate the mechanism of diversification. To validate this estimation, additional sequence-based computational tests (e.g., PHI, Rmin) were utilized and both tests revealed evidence for recombination either within and/or between the MLST loci. Interestingly, all polymorphisms at the locus murG, which encodes for glycosyltransferase murein G, were non-synonymous. The exact function of this enzyme in F. psychrophilum has not been examined; however, in Escherichia coli, murG is required for biosynthesis of the peptidoglycan layer (Mohammadi et al. 2007) and therefore may confer an environmental advantage to epidemic clones of F. psychrophilum. The ratio of non-synonymous to synonymous mutations within murG however, is < 1 (i.e., dN/dS = 0.2751), and therefore accumulated non-synonymous mutations are selected against and are projected to be lost over time (Nilsen et al. 2014) but may create short-lived advantages during BCWD outbreaks. Although the natural mechanism(s) for recombination (e.g., transduction, transformation, conjugation) among F. psychrophilum isolates remain to be determined, the intermingling of isolates naturally and during the trade and introduction of fish and eggs to different facilities, bodies of water, or states provides a means for interaction of different F. psychrophilum genotypes. Indeed, multiple STs have been reported from a single infected host on multiple 62 occasions (Fujiwara-Nagata et al. 2013, Nilsen et al. 2014, Van Vliet et al. 2016). Even though recombination appears to be a driving force for diversity among F. psychrophilum isolates, the results of our analysis on linkage disequilibrium revealed statistically significant evidence for clonal populations (i.e., ISA value differs significantly from 0). Therefore, it would appear even in the presence of recombination, well adapted F. psychrophilum STs are capable of producing epidemic clones, some of which have persisted long enough to produce clones of their own (i.e., ST78, ST306). Nilsen et al. (2014) described an identical discovery among their 560 F. psychrophilum isolates, which were recovered from several Nordic countries. In summation, MLST was used to reveal substantial diversity among 314 newly typed North American F. psychrophilum isolates, whereby 66 STs were identified, 47 of which were novel and recovered in this study for the first time. These isolates were recovered from 20 US states and one Canadian province over a period of four decades, which is the most comprehensive collection of F. psychrophilum isolates to be genetically analyzed in North America to date. These results expanded our knowledge on the population structure of F. psychrophilum in North America through the addition of seven novel CCs. These CCs have diverse host ranges and vary in their virulence. Additionally, for the first time, recombination was implicated as the driving force of F. psychrophilum diversification in North America, which may also be the mechanism for phenotypic changes, such as antimicrobial resistance, within this species, and warrants further investigation. 63 APPENDIX 64 Table 2.1a Complete information of 314 North American F. psychrophilum isolates presented in this study including year of isolation, location of isolation, host species, tissue of isolation, and host life stage. aAK = Alaska; BC = British Columbia; CA = California; CO = Colorado; ID = Idaho; IN = Indiana; MD = Maryland; MI = Michigan; MN = Minnesota; MT = Montana; NC = North Carolina; NM = New Mexico; OR = Oregon, PA = Pennsylvania; SD = South Dakota; UT = Utah; VA = Virginia; VT = Vermont; WA = Washington; WI = Wisconsin; WV = West Virginia; PF = private facility, SFH = state fish hatchery, WE = weir, WI = wild, Unk = Unknown. cHost was in captivity. wfHost was wild/feral. DIsolate was recovered from a fish experiencing BCWD. HIsolate was recovered from an apparently healthy fish. UFish disease status was unknown. OF = Ovarian Fluid. Isolation tissue Unk Unk Unk Unk Unk OF Unk Spleen Unk Unk OF Kidney Unk Kidney Spleen Spleen Spleen Brain Lesion Spleen Spleen Spleen Spleen Spleen Brain Spleen Unk Lesion Spleen Spleen Spleen Spleen Spleen Caudal Fin Life stageD,H,U UnkU UnkU UnkU UnkU UnkU UnkU UnkU UnkU UnkU UnkU UnkU UnkU BroodstockD FingerlingD FingerlingD FingerlingD FingerlingD FingerlingD FingerlingD FingerlingD FingerlingD FingerlingD FingerlingD FingerlingD FingerlingD FingerlingD FingerlingD FingerlingD FingerlingD FingerlingD FingerlingD FingerlingD FingerlingD FingerlingD Isolate ID Locationa Year OR-Unk 1989 US159 OR-Unk 1990 US160 OR-Unk 1990 US161 BC-Unk 1984 US165 1990 OR-Unk US202 US248 2000 WA-SFH2 US256 Unk WA-PF2 US254 Unk UT-SFH1 US154 Unk WA-Unk US155 Unk WA-Unk US250 2000 WA-PF3 1998 WA-SFH1 US246 2014 US284 ID-PF7 NC-PF3 US121 2013 UT-SFH2 2011 US123 UT-SFH1 2011 US125 UT-SFH1 2011 US126 UT-SFH1 US127 2011 UT-SFH2 2011 US128 UT-SFH2 2011 US129 UT-SFH2 US130 2011 UT-SFH1 2012 US135 UT-SFH1 2012 US136 UT-SFH1 2012 US137 NC-PF3 US140 2013 2012 US143 UT-SFH1 2016 MD-SFH1 US188 US221 2011 UT-SFH2 NC-PF3 2013 US226 UT-PF2 2012 US227 UT-PF2 2012 US229 US236 2011 UT-SFH2 UT-SFH1 2011 US238 US240 2012 UT-PF2 O. tshawytscha Speciesc,wf O. kisutch O. kisutch O. kisutch O. kisutch O. kisutch O. kisutch O. kisutch O. mykiss O. kisutch O. kisutch O. kisutch O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc 65 Table 2.1a (cont’d) Isolate Locationa Year NC-PF3 2013 UT-SFH1 2011 UT-SFH3 2005 2015 UT-SFH6 2010 MT-SFH2 2010 MT-SFH2 UT-SFH1 2011 2012 UT-PF2 UT-SFH2 2009 UT-PF1 2004 UT-SFH7 2013 2013 UT-SFH2 UT-SFH7 2013 UT-SFH7 2011 UT-SFH6 2011 2011 UT-SFH1 UT-SFH5 2009 UT-SFH5 2009 2011 ID-PF4 ID-PF4 2011 ID-PF4 2011 ID-PF4 2011 2011 ID-PF4 2014 ID-PF5 ID-PF2 2015 2015 ID-PF2 ID-PF1 2016 ID-PF1 2011 ID-PF1 2013 2015 ID-PF6 ID-PF3 2015 ID-PF3 2014 2016 ID-PF3 ID-PF2 2016 2014 ID-PF1 2011 2013 2013 2011 2011 2011 2014 ID-PF3 ID-PF2 ID-PF4 ID-PF4 ID-PF4 ID-PF1 ID-PF-Unk ID US243 US293 US298 US299 US311 US312 US315 US316 US295 US297 US301 US302 US303 US304 US305 US306 US366 US369 US058 US114 US124 US146 US147 US169 US182 US183 US184 US193 US195 US206 US207 US209 US210 US211 US213 US222 US228 US230 US234 US235 US237 US262 Isolation tissue Kidney Spleen Unk Spleen Kidney Kidney Spleen Spleen Spleen Unk Unk Spleen Spleen Spleen Spleen Spleen Spleen Spleen Unk Spleen Spleen Kidney Spleen Spleen Spleen Kidney Spleen Unk Kidney Kidney Spleen Spleen Spleen Spleen Kidney Unk Spleen Spleen Spleen Kidney Spleen Spleen Speciesc,wf O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc 66 Life stageD,H,U FingerlingD FingerlingD FingerlingD FingerlingD FingerlingD FingerlingD FingerlingD FingerlingD FryD FryD FryD FryD FryD FryD FryD FryD FryD FryD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD Table 2.1a (cont’d) Locationa ID-PF1 ID-PF5 ID-PF-Unk Isolate ID Year 2014 US263 2014 US264 2014 US267 2014 US268 2014 US269 2015 US271 2015 US272 2015 US275 2015 US279 2015 US280 2016 US281 2014 US286 2014 US288 2012 US319 2012 US320 2012 US322 2014 US326 2014 US327 2015 US330 2014 US333 2014 US334 2014 US336 2014 US342 2014 US345 2015 US346 2015 US347 2015 US348 2015 US350 2015 US351 2012 US353 2012 US132 2012 US133 2012 US134 US144 2012 US145 Unk US150 Unk US162 1990 2014 US173 1994 US175 US179 2015 US186 2014 US253 Unk Isolation tissue Kidney Kidney Spleen Spleen Spleen Spleen Spleen Kidney Kidney Unk Spleen Kidney Kidney Spleen Spleen Spleen Spleen Spleen Spleen Spleen Spleen Spleen Kidney Kidney Kidney Spleen Kidney Kidney Spleen Spleen Head swab Pool tissue Pool tissue Pool tissue Unk Spleen Unk Spleen Unk Spleen Spleen Unk Speciesc,wf O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykiss O. mykiss O. mykissc O. mykissc O. mykissc O. mykissc O. mykiss 67 Life stageD,H,U JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD UnkD UnkD UnkD UnkD UnkU UnkU UnkU UnkD UnkD UnkD UnkD UnkU ID-PF-Unk ID-PF5 ID-PF5 ID-PF6 ID-PF2 ID-PF6 ID-PF3 ID-PF1 ID-PF3 ID-PF2 ID-PF4 ID-PF2 ID-PF6 ID-PF5 ID-PF5 ID-PF3 ID-PF1 ID-PF2 ID-PF1 ID-PF-Unk ID-PF5 ID-PF3 ID-PF5 ID-PF2 ID-PF6 ID-PF6 ID-PF2 SD-SFH1 SD-SFH2 SD-SFH1 SD-SFH1 UT-SFH4 UT-SFH4 OR-Unk ID-SFH1 ID-SFH1 ID-SFH1 ID-SFH1 CO-Unk Table 2.1a (cont’d) Isolate ID ID-PF-Unk Year Locationa 2014 ID-SFH1 2014 ID-SFH1 2004 UT-SFH2 2008 UT-SFH2 2015 UT-Unk 2002 NM-SFH1 PA-SFH2 2014 2016 PA-SFH2 PA-SFH2 2016 2015 PA-SFH1 2010 MI-SFH4 CA-SFH2 CA-Unk NC-PF1 NC-PF2 UT-SFH2 UT-SFH1 ID-PF6 US291 US292 US294 US296 US307 US368 US059 US075 US079 US180 US352 US148 Unk Unk 518 2013 US118 2013 US138 US365 2011 2008 US367 2013 US120 US196 2012 2014 US204 2015 US208 2014 US212 US241 2013 2014 US266 2014 US285 US287 2014 2013 US289 2012 US317 ID-PF6 2012 US318 ID-PF1 US321 2012 ID-PF1 2014 US335 2015 US349 ID-PF5 2002 NM-SFH1 US251 US252 Unk CO-Unk 2014 WA-PF1 US172 2014 WA-PF1 US177 US337 2014 WA-PF1 2014 WA-PF1 US338 2009 US167 NC-PF3 US189 2016 CA-SFH1 2016 US190 CA-SFH1 US364 2014 WA-PF1 ID-PF6 ID-PF1 ID-PF2 ID-PF6 ID-PF3 ID-PF5 ID-PF1 ID-PF-Unk ID-PF-Unk A. transmontanus A. transmontanus Speciesc,wf O. mykissc O. mykissc O. mykissc O. mykissc O. mykiss O. mykiss O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykiss O. mykiss O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc 68 Isolation tissue Spleen Spleen Spleen Spleen Unk Kidney Kidney Kidney Kidney Unk Kidney Kidney Kidney Unk Spleen Kidney Spleen Spleen Unk Unk Spleen Spleen Unk Kidney Gill Spleen Spleen Unk Spleen Spleen Kidney Kidney Kidney Kidney Kidney Skin Kidney Spleen Spleen Spleen Heart Side lesion UnkD UnkD UnkD UnkU UnkU UnkU FingerlingD BroodstockH BroodstockH FingerlingD UnkD UnkU UnkD FingerlingD FingerlingD FingerlingD Life stageD,H,U FryD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD UnkU UnkU YearlingD YearlingD YearlingD YearlingD FingerlingD UnkD UnkD YearlingD Table 2.1a (cont’d) Isolate Locationa Year ID VT-Unk 2007 551 UT-SFH4 2008 US073 UT-SFH6 2010 US074 2010 UT-SFH4 US300 2015 WA-PF1 US354 2016 MI-WE1 US104 NC-PF3 2011 US115 2013 NC-PF1 US116 2013 WV-PF1 US117 2013 WV-PF1 US119 NC-PF3 2011 US131 2014 VA-PF1 US170 2014 US171 VA-PF1 2014 US185 VA-PF1 VA-PF1 2014 US192 2011 US194 NC-PF3 NC-PF1 2013 US224 NC-PF1 2013 US242 2014 PA-SFH5 US057 2016 MI-SFH4 US085 2016 MI-SFH4 US086 US087 2016 MI-SFH4 US151 Unk WA-PF2 2011 MT-SFH1 US313 2011 MT-SFH1 US314 US223 2011 ID-PF4 ID-PF4 2013 US233 ID-PF4 2011 US239 ID-PF1 2015 US274 US276 2015 ID-PF6 ID-PF2 2016 US282 ID-PF5 2014 US290 US332 2014 ID-PF1 ID-SFH1 2015 US178 2013 US197 ID-PF-Unk ID-PF1 2013 US323 US205 2015 ID-PF5 ID-PF4 2011 US245 OR-Unk 1990 US258 ID-Unk US259 1990 2015 WA-PF1 US355 US356 2016 ID-PF2 Speciesc,wf O. mykiss O. mykissc O. mykissc O. mykissc O. mykissc O. mykisswf O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykiss O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. kisutch O. mykiss O. mykissc O. mykissc 69 Isolation tissue Unk Spleen Spleen Spleen Skin Kidney Brain Spleen Spleen Spleen Kidney Spleen Spleen Spleen Spleen Kidney Spleen Spleen Kidney Kidney Ascites Ext. lesion Unk Kidney Kidney Kidney Spleen Kidney Kidney Kidney Kidney Spleen Spleen Spleen Spleen Spleen Kidney Kidney Unk Unk Unk Spleen Life stageD,H,U UnkD FryD FryD FryD YearlingD AdultH FingerlingD FingerlingD FingerlingD FingerlingD FingerlingD FingerlingD FingerlingD FingerlingD FingerlingD FingerlingD FingerlingD FingerlingD FingerlingD FingerlingD FingerlingD FingerlingD UnkU FingerlingD FingerlingD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD UnkD JuvenileD JuvenileD JuvenileD JuvenileD UnkU UnkU YearlingD JuvenileD Table 2.1a (cont’d) Isolate ID Year 1991 1984 1981 1986 Locationa OR-Unk US157 OR-Unk US163 OR-Unk US261 US164 OR-Unk US149 Unk WA-Unk 2017 MI-WE1 US215 2017 MI-WE1 US216 US339 2014 WV-PF2 2014 WV-PF2 US340 2014 WV-PF2 US341 2014 WV-PF2 US343 US344 2014 WV-PF2 ID-PF1 2015 US277 2015 US181 PA-SFH1 2012 MI-WE2 US062 US064 2012 MI-WE2 2016 MI-WE2 US084 2014 MI-WE1 US097 US101 2014 MI-WE1 2017 MI-WE1 US217 2017 MI-WE1 US219 2017 MI-WE1 US220 US095 2016 MI-WE1 2016 MI-WE1 US105 PA-SFH3 2014 US061 US076 2016 PA-SFH2 PA-SFH2 2016 US077 PA-SFH2 2016 US078 PA-SFH2 2016 US080 US111 2017 PA-SFH2 PA-SFH2 2017 US112 PA-SFH2 2017 US113 US199 2016 PA-SFH2 PA-SFH2 2016 US200 PA-SFH2 2016 US201 PA-WI1 2016 US065 US068 2016 PA-WI1 PA-WI1 2016 US070 PA-WI1 2016 US071 US072 2016 PA-WI1 PA-SFH4 2016 US067 US069 2016 PA-WI1 O. tshawytschawf O. tshawytschawf Speciesc,wf S. salar O. mykiss O. tshawytscha O. clarkii S. salar O. mykisswf O. mykisswf O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc S. salarwf S. salarwf S. salarwf O. mykisswf O. mykisswf O. mykisswf O. mykisswf O. mykisswf O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykisswf O. mykisswf O. mykisswf O. mykisswf O. mykisswf O. mykissc O. mykisswf Isolation tissue Unk Unk Unk Unk Unk Kidney Spleen Unk Unk Unk Caudal Fin Spleen Spleen Unk Ext. lesion Kidney Kidney Kidney Kidney Kidney Kidney Kidney Kidney Kidney Kidney Kidney Ulcer Kidney Eye Kidney Kidney Kidney Ulcer Kidney Kidney Kidney Kidney Kidney Kidney Kidney Kidney Ext. lesion Life stageD,H,U UnkU UnkU UnkU UnkU UnkU AdultH AdultH FingerlingD FingerlingD FingerlingD FingerlingD FingerlingD JuvenileD UnkD AdultD AdultH AdultH AdultH AdultH AdultH AdultH AdultH AdultH AdultH FingerlingD BroodstockH BroodstockD BroodstockH BroodstockD FryD FryD FryD BroodstockD BroodstockD BroodstockD AdultH AdultH AdultH AdultH AdultH FryD AdultH 70 Table 2.1a (cont’d) Isolate ID Locationa Year PA-WI1 2017 US379 PA-WI1 2017 US380 PA-WI1 2017 US381 2017 PA-WI1 US382 2016 WI-SFH1 US066 2013 MI-SFH3 US096 2016 MI-WE1 US106 2016 MI-WE1 US107 2016 MI-WE1 US108 2016 MI-WE3 US109 2014 ID-SFH1 US174 US153 Unk WA-PF2 2014 WA-PF1 US176 NC-PF3 2011 US122 US198 2011 NC-PF3 ID-PF1 2013 US231 ID-PF1 2015 US273 US331 2014 ID-PF1 ID-PF6 2015 US270 ID-PF4 2014 US329 ID-PF2 2015 US278 US328 2014 ID-PF2 CA-SFH1 2016 US191 OR-Unk 1981 US156 OR-Unk 1990 US203 US247 1998 OR-SFH1 OR-Unk 1981 US257 OR-Unk 1981 US370 US255 1981 OR-SFH2 2000 WA-PF3 US249 2013 MI-SFH2 US094 2013 MI-SFH2 US098 US102 2013 MI-SFH2 2013 MI-SFH2 US103 2010 MI-SFH2 US060 US093 2017 MI-SFH2 2018 MI-SFH4 US383 2016 MI-SFH3 US081 2016 MI-SFH3 US082 US099 2013 MI-SFH3 2013 MI-SFH3 US100 US214 2017 IN-SFH1 Speciesc,wf O. mykisswf O. mykisswf O. mykisswf O. mykisswf S. namaycush x S. fontinalisc O. kisutchc O. tshawytschawf O. tshawytschawf O. tshawytschawf O. tshawytschawf O. mykissc O. nerka O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. kisutch O. kisutch O. kisutch O. kisutch O. kisutch O. kisutch S. truttac S. truttac S. truttac S. truttac S. truttac O. mykissc O. mykissc O. kisutchc O. kisutchc O. kisutchc O. kisutchc O. kisutchc O. tshawytscha Isolation tissue Kidney Kidney Kidney Kidney Unk Life stageD,H,U AdultH AdultH AdultH AdultH UnkU Dorsal Fin Kidney Kidney Kidney Kidney Spleen Unk Kidney Brain Unk Spleen Kidney Kidney Kidney Kidney Kidney Spleen Spleen Unk Unk Kidney Unk Unk Unk OF Kidney Spleen Kidney Kidney Ascites Kidney Kidney Kidney Gill Gill Ulcer Kidney YearlingD AdultH AdultH AdultH AdultH UnkD UnkU YearlingD FingerlingD FingerlingD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD JuvenileD UnkD UnkU UnkU UnkU UnkU UnkU UnkU UnkU FingerlingD FingerlingD FingerlingD FingerlingD AdultD BroodstockH FingerlingD FingerlingD FingerlingD YearlingD YearlingD FryD 71 Isolation tissue Kidney Fin Eye Lesion Fin Kidney Kidney Kidney Kidney Spleen Kidney Spleen Spleen Kidney Spleen Brain Unk Unk Kidney Spleen Unk Kidney Spleen lesion Kidney Spleen Spleen Kidney Life stageD,H,U AdultH FingerlingD AdultD AdultD BroodstockD BroodstockH BroodstockH BroodstockH AdultH FingerlingD FingerlingD FingerlingD FingerlingD FingerlingD FingerlingD FingerlingD JuvenileD UnkU UnkU UnkD UnkU JuvenileD JuvenileD FingerlingD FingerlingD FryD FryD FingerlingD Table 2.1a (cont’d) Isolate ID Year Locationa 2017 MI-WE1 US218 2012 MI-SFH1 US063 2016 MI-WE2 US083 2016 PA-PF1 US088 2016 MN-SFH1 US089 2016 MN-SFH1 US090 2016 MN-SFH1 US091 2017 MI-SFH2 US092 2016 MI-WE3 US110 NC-PF1 2013 US139 2011 US141 NC-PF3 2011 NC-PF3 US166 NC-PF1 2013 US225 NC-PF3 2011 US232 2011 US244 NC-PF3 US142 2011 NC-PF3 US152 Unk WA-PF2 OR-Unk US158 1989 US168 2014 ID-PF5 ID-SFH1 US187 2014 OR-Unk US260 1985 US265 2014 ID-PF5 2014 US283 ID-PF5 2010 MT-SFH1 US309 2010 MT-SFH1 US310 US324 2014 NC-PF2 NC-PF2 2014 US325 US372 2011 AK-PF1 O. tshawytschawf O. tshawytscha Speciesc,wf O. mykisswf S. namaycushc S. salarwf S. truttac O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. kisutch O. mykissc O. mykissc O. mykiss O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. mykissc O. kisutchc 72 Table 2.1b Multilocus sequence typing allelic profiles, sequence types (STs), and clonal complexes (CCs) of the 314 North American F. psychrophilum isolates. STs in bold are found in North America and abroad, whereas STs in italics are novel. Allelic profile fumC murG tuf 5 5 5 5 5 5 5 5 8 8 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 atpA 4 4 4 4 4 4 4 4 4 4 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 CC ST ST9 CC-ST9 ST9 CC-ST9 CC-ST9 ST9 ST9 CC-ST9 ST9 CC-ST9 ST9 CC-ST9 CC-ST9 ST9 ST9 CC-ST9 CC-ST9 ST13 ST13 CC-ST9 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 6 6 6 6 6 6 6 6 6 6 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Isolate ID US159 US160 US161 US165 US202 US248 US256 US254 US154 US155 US250 US246 US284 US121 US123 US125 US126 US127 US128 US129 US130 US135 US136 US137 US140 US143 US188 US221 US226 US227 US229 US236 US238 US240 US243 US293 US298 US299 US311 US312 trpB 4 4 4 4 4 4 4 4 4 4 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 gyrB 7 7 7 7 7 7 7 7 7 7 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 dnaK 6 6 6 6 6 6 6 6 6 6 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 5 5 5 5 5 5 5 5 5 5 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 73 Table 2.1b (cont’d) Allelic profile fumC murG tuf 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 atpA 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 CC ST ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 74 trpB 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 gyrB 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 dnaK 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Isolate ID US315 US316 US295 US297 US301 US302 US303 US304 US305 US306 US366 US369 US058 US114 US124 US146 US147 US169 US182 US183 US184 US193 US195 US206 US207 US209 US210 US211 US213 US222 US228 US230 US234 US235 US237 US262 US263 US264 US267 US268 Table 2.1b (cont’d) Allelic profile fumC murG tuf 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 atpA 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 CC ST ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 75 trpB 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 gyrB 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 dnaK 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Isolate ID US269 US271 US272 US275 US279 US280 US281 US286 US288 US319 US320 US322 US326 US327 US330 US333 US334 US336 US342 US345 US346 US347 US348 US350 US351 US353 US132 US133 US134 US144 US145 US150 US162 US173 US175 US179 US186 US253 US291 US292 Table 2.1b (cont’d) Allelic profile fumC murG tuf 2 2 2 2 2 2 2 2 2 2 2 41 41 41 41 41 41 41 41 41 41 41 41 41 41 41 41 41 41 41 41 41 41 41 41 41 41 46 46 46 atpA 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 CC ST ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST10 CC-ST10 ST78 CC-ST10 ST78 CC-ST10 ST78 CC-ST10 ST78 CC-ST10 ST78 CC-ST10 ST78 CC-ST10 ST78 CC-ST10 ST78 CC-ST10 ST78 CC-ST10 ST78 CC-ST10 ST78 CC-ST10 ST78 CC-ST10 ST78 CC-ST10 ST78 CC-ST10 ST78 CC-ST10 ST78 CC-ST10 ST78 CC-ST10 ST78 CC-ST10 ST78 CC-ST10 ST78 CC-ST10 ST78 CC-ST10 ST78 CC-ST10 ST78 CC-ST10 ST78 CC-ST10 ST78 CC-ST10 ST84 CC-ST10 ST85 CC-ST10 ST85 CC-ST10 ST85 CC-ST10 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 76 trpB 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 gyrB 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 41 8 8 8 dnaK 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Isolate ID US294 US296 US307 US368 US059 US075 US079 US180 US352 US148 518 US118 US138 US365 US367 US120 US196 US204 US208 US212 US241 US266 US285 US287 US289 US317 US318 US321 US335 US349 US251 US252 US172 US177 US337 US338 US167 US189 US190 US364 Table 2.1b (cont’d) Allelic profile fumC murG tuf 46 2 2 2 2 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 atpA 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 72 2 2 2 CC ST ST85 CC-ST10 ST86 CC-ST10 ST86 CC-ST10 ST86 CC-ST10 ST86 CC-ST10 ST275 CC-ST10 ST275 CC-ST10 ST275 CC-ST10 ST275 CC-ST10 ST275 CC-ST10 ST275 CC-ST10 ST275 CC-ST10 ST275 CC-ST10 ST275 CC-ST10 ST275 CC-ST10 ST275 CC-ST10 ST275 CC-ST10 ST275 CC-ST10 ST275 CC-ST10 ST275 CC-ST10 ST275 CC-ST10 ST275 CC-ST10 ST294 CC-ST10 ST300 CC-ST10 ST300 CC-ST10 ST300 CC-ST10 ST300 CC-ST10 ST300 CC-ST10 ST300 CC-ST10 ST300 CC-ST10 ST300 CC-ST10 ST300 CC-ST10 ST300 CC-ST10 ST300 CC-ST10 ST303 CC-ST10 ST303 CC-ST10 ST304 CC-ST10 ST305 CC-ST10 ST306 CC-ST10 ST306 CC-ST10 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 2 1 1 77 trpB 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 45 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 gyrB 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 81 81 8 13 8 8 dnaK 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 34 34 34 34 34 34 34 34 34 34 34 2 2 2 2 2 2 2 3 3 3 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Isolate ID 551 US073 US074 US300 US354 US104 US115 US116 US117 US119 US131 US170 US171 US185 US192 US194 US224 US242 US057 US085 US086 US087 US151 US313 US314 US223 US233 US239 US274 US276 US282 US290 US332 US178 US197 US323 US205 US245 US258 US259 Table 2.1b (cont’d) Allelic profile fumC murG trpB 2 2 6 6 15 3 1 4 4 46 46 46 46 46 46 46 28 28 8 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 6 6 6 gyrB 8 8 9 9 17 16 13 73 73 3 3 3 3 3 3 3 75 75 62 28 28 28 28 28 2 2 2 2 2 2 2 2 2 2 2 2 2 9 9 9 dnaK 2 2 7 7 14 13 15 22 22 22 22 22 22 22 22 22 15 15 15 4 4 4 4 4 2 2 2 2 2 2 2 2 2 2 2 2 2 7 7 7 Isolate ID US355 US356 US157 US163 US261 US164 US149 US215 US216 US339 US340 US341 US343 US344 US277 US181 US062 US064 US084 US097 US101 US217 US219 US220 US095 US105 US061 US076 US077 US078 US080 US111 US112 US113 US199 US200 US201 US065 US068 US070 tuf 41 41 5 5 17 16 7 3 3 3 3 3 3 3 3 3 1 1 1 25 25 25 25 25 12 12 12 12 12 12 12 12 12 12 12 12 12 5 5 5 atpA 2 2 7 7 16 15 1 3 3 3 3 3 3 3 3 3 54 54 54 59 59 59 59 59 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 ST CC ST316 CC-ST10 ST317 CC-ST10 ST11 CC-ST11 CC-ST11 ST11 ST308 CC-ST29 ST28 CC-ST31 ST70 CC-ST124 ST267 CC-ST191 ST267 CC-ST191 ST301 CC-ST191 ST301 CC-ST191 ST301 CC-ST191 ST301 CC-ST191 ST301 CC-ST191 ST301 CC-ST191 ST301 CC-ST191 ST277 CC-ST232 ST277 CC-ST232 ST283 CC-ST232 ST256 CC-ST256 ST256 CC-ST256 ST256 CC-ST256 ST256 CC-ST256 ST256 CC-ST256 ST257 CC-ST276 ST257 CC-ST276 ST276 CC-ST276 ST276 CC-ST276 ST276 CC-ST276 ST276 CC-ST276 ST276 CC-ST276 ST276 CC-ST276 ST276 CC-ST276 ST276 CC-ST276 ST276 CC-ST276 ST276 CC-ST276 ST276 CC-ST276 ST279 CC-ST281 ST279 CC-ST281 ST279 CC-ST281 3 1 3 3 9 9 14 3 3 3 3 3 3 3 3 3 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 2 2 2 2 2 7 7 13 12 1 3 3 3 3 3 3 3 3 3 25 25 25 25 25 25 25 25 46 46 25 25 25 25 25 25 25 25 25 25 25 49 49 49 78 Table 2.1b (cont’d) Allelic profile fumC murG tuf 5 5 63 63 63 63 63 63 3 3 12 12 12 12 3 3 3 17 17 17 17 17 17 17 17 17 15 18 18 18 18 18 38 40 1 1 1 1 1 1 atpA 3 3 3 3 75 75 75 75 11 11 57 57 57 57 68 68 68 10 10 10 10 10 10 10 10 10 2 17 17 17 17 17 37 39 1 1 1 1 1 1 CC ST ST279 CC-ST281 ST279 CC-ST281 ST281 CC-ST281 ST281 CC-ST281 ST331 CC-ST281 ST332 CC-ST281 ST332 CC-ST281 ST332 CC-ST281 ST280 CC-ST287 ST287 CC-ST287 ST288 CC-ST288 ST288 CC-ST288 ST288 CC-ST288 ST289 CC-ST288 ST296 CC-ST296 ST296 CC-ST296 ST299 CC-ST296 ST291 CC-ST310 ST291 CC-ST310 ST291 CC-ST310 ST291 CC-ST310 ST291 CC-ST310 ST310 CC-ST310 ST310 CC-ST310 ST311 CC-ST310 ST311 CC-ST310 ST27 ST30 ST30 ST30 ST30 ST30 ST74 ST76 ST253 ST253 ST253 ST253 ST253 ST253 49 49 49 49 49 55 55 55 3 3 50 50 50 50 13 13 13 1 1 1 1 1 1 1 1 1 11 14 14 14 14 14 25 29 1 1 1 1 1 1 79 trpB 6 6 6 6 6 6 6 6 4 4 43 43 43 44 18 18 18 11 11 11 11 11 11 11 2 2 2 11 11 11 11 11 8 8 1 1 1 1 1 1 gyrB 9 9 9 9 9 9 9 9 2 2 69 69 69 69 47 47 79 13 13 13 13 13 13 13 13 13 8 18 18 18 18 18 38 19 13 13 13 13 13 13 dnaK 7 7 7 7 7 7 7 7 22 15 15 15 15 15 8 8 8 15 15 15 15 15 15 15 15 15 2 7 7 7 7 7 8 7 8 8 8 8 8 8 2 2 2 2 2 2 2 2 3 3 10 10 10 10 3 3 3 2 2 2 2 2 1 1 1 1 2 5 5 5 5 5 2 1 1 1 1 1 1 1 Isolate ID US071 US072 US067 US069 US379 US380 US381 US382 US066 US096 US106 US107 US108 US109 US174 US153 US176 US122 US198 US231 US273 US331 US270 US329 US278 US328 US191 US156 US203 US247 US257 US370 US255 US249 US094 US098 US102 US103 US060 US093 Table 2.1b (cont’d) Allelic profile fumC murG tuf 18 8 8 8 8 8 8 4 3 12 64 64 64 65 12 66 66 66 66 66 66 39 67 61 68 69 39 6 6 5 5 7 36 71 atpA 14 14 14 14 14 14 14 14 11 32 14 14 14 64 26 65 65 65 65 65 65 66 67 69 70 71 73 21 21 7 7 1 12 74 ST ST255 ST258 ST258 ST258 ST258 ST258 ST258 ST278 ST282 ST284 ST285 ST285 ST285 ST286 ST290 ST292 ST292 ST292 ST292 ST292 ST292 ST293 ST295 ST297 ST298 ST302 ST307 ST309 ST312 ST313 ST313 ST314 ST315 ST320 CC 12 47 47 47 47 47 47 10 13 8 51 51 51 1 52 36 36 36 36 36 36 23 36 17 33 20 25 10 2 49 49 14 53 43 Isolate ID US383 US081 US082 US099 US100 US214 US218 US063 US083 US088 US089 US090 US091 US092 US110 US139 US141 US166 US225 US232 US244 US142 US152 US158 US168 US187 US260 US265 US283 US309 US310 US324 US325 US372 trpB 41 42 42 42 42 42 42 2 18 8 2 2 2 1 13 2 2 2 2 2 2 11 8 8 29 26 18 2 30 18 18 42 43 47 gyrB 68 18 18 18 18 18 18 28 2 18 14 14 14 76 69 8 8 8 8 8 8 77 78 28 12 80 28 82 29 8 8 3 83 85 dnaK 10 26 26 26 26 26 26 8 8 8 16 16 16 9 15 2 2 2 2 2 2 8 8 33 8 4 35 36 36 7 7 11 2 4 9 5 5 5 5 5 5 14 3 10 10 10 10 5 5 3 3 3 3 3 3 10 7 3 10 5 3 9 5 3 3 5 7 2 80 Table 2.2 Allelic variations among 15 North American clonal complexes (CCs). aAncestral amino acid is listed first followed by SLV amino acid, Syn is a synonymous change Variant locus of SLV tuf tuf tuf tuf tuf fumC No. of nucleotide differences 5 5 6 1 8 2 Clonal ancestor AT 5 2 2 2 2 2 ST of clonal ancestor 9 10 10 10 10 10 ST of SLV 13 78 81 82 85 86 CC CC-ST9 CC-ST10 CC-ST10 CC-ST10 CC-ST10 CC-ST10 SLV AT 8 41 43 44 46 3 CC-ST10 CC-ST10 CC-ST10 CC-ST10 CC-ST10 CC-ST10 10 10 10 10 10 10 CC-ST11 11 CC-ST29 29 CC-ST31 31 CC-ST124 124 CC-ST191 191 CC-ST191 191 CC-ST232 232 275 294 300 303 305 tuf trpB dnaK gyrB gyrB 306 murG 262 murG trpB 308 gyrB 28 trpB 70 267 301 277 gyrB trpB gyrB 2 2 2 8 8 2 7 8 19 2 3 4 62 62 45 34 81 13 1 49 15 16 1 73 46 75 1 1 1 4 3 7 1 1 9 3 10 1 3 81 None None None None CC-ST124; CC- ST310 CC-ST124; CC- ST310 CC-ST281 None None CC-ST10; CC- ST256; CC-ST276 None None None Other CC(s) containing SLV None None None None None CC-ST10; CC-ST11; CC-ST191; CC- ST287; CC-ST296; CC-ST276 Other singletons containing SLV ST258 None None None None ST254; ST261; ST264; ST266; ST282; ST292; ST297; ST307; ST313 None None None None ST253 ST253; ST286 ST264; ST313 None None ST6; ST83; ST253; ST286 None None None Amino acid changea Syn Syn Syn P-S Syn Syn Syn Syn Syn Syn Syn P-H E-K I-V T-A Syn Syn Syn Syn Syn Syn Table 2.2 (cont’d) ST of clonal ancestor CC CC-ST256 256 CC-ST276 276 CC-ST281 281 CC-ST281 281 CC-ST287 287 CC-ST288 288 Variant ST locus of of SLV SLV 252 murG 257 murG 279 331 280 289 tuf atpA dnaK trpB Clonal ancestor AT 25 25 63 3 15 43 CC-ST296 296 CC-ST296 296 CC-ST310 310 265 299 291 atpA gyrB fumC 68 47 1 SLV AT 16 46 5 75 22 44 62 79 2 CC-ST310 310 311 trpB 11 2 No. of nucleotide differences 2 6 9 4 2 2 1 4 1 1 Other singletons Other CC(s) containing SLV containing SLV ST250 None None None ST266; ST313 CC-ST9; CC-ST11 None None CC-ST10; CC-ST191 None None None None None ST27; ST74; ST77; ST83; ST320 None None CC-ST10; CC- ST124; CC-ST232; CC-ST256; CC- ST281; CC-ST310 CC-ST10; CC-ST124 ST27; ST278; ST285; ST292; ST309 Amino acid changea H-P K-E Syn Syn Syn Syn Syn Syn Syn Syn 82 Table 2.3 Summary of nucleotide sequence diversity among the 86 North American sequence types (STs). S, number of segregating sites. π, average pairwise nucleotide diversity per site. dN/dS, ratios of non-synonymous to synonymous mutations. Rmin, Hudson and Kaplan’s lower bound for the number of recombination. π dN/dS Rmin 0.0042 0.0068 0.0057 0.0029 0.0066 0.0074 0.0125 Average 0.0055 0.0079 0.0020 0.0586 0.0422 0.2751 0.0027 0.0162 0.0578 4 7 3 2 6 6 10 38 44 Locus trpB gyrB dnaK fumC murG tuf atpA Sum Concatenation Fragment size (bp) 789 1077 873 750 681 795 834 5,799 No. of alleles 24 41 20 8 32 36 38 S 13 22 29 5 16 26 28 139 139 83 Figure 2.1 Map of the United States and Canada. Distribution of 314 F. psychrophilum isolates recovered from the current North American study. These isolates were recovered from 20 U.S. states and one province of Canada. AK = Alaska; BC = British Columbia; CA = California; CO = Colorado; ID = Idaho; IN = Indiana; MD = Maryland; MI = Michigan; MN = Minnesota; MT = Montana; NC = North Carolina; NM = New Mexico; OR = Oregon, PA = Pennsylvania; SD = South Dakota; UT = Utah; VA = Virginia; VT = Vermont; WA = Washington; WI = Wisconsin; WV = West Virginia. 84 Figure 2.2 eBURST diagram of the 314 F. psychrophilum isolates typed in this study combined with all previously typed North American isolates (n = 107) in the F. psychrophilum MLST database. A dagger (†) denotes a sequence type (ST) that contains isolates typed in this study, whereas a double dagger (‡) denotes a ST containing previously typed isolates. Italicized numbers accompanying STs indicate the number of states that ST was found in (if no number, ST recovered from one state only). Clonal complexes (CC) are encircled and the founding sequence type (ST) for a CC is depicted as a number within a rectangle. Founding STs labeled with a star ((cid:2)(cid:1) indicate a newly described CC as a result of this study. The founding ST, or founder, is the ST with the highest number of single locus variants (SLVs). In instances where multiple STs have the same number of SLVs, the founder is named after the ST with the most isolates. If both STs have the same number of isolates, the CC is named for the earliest found ST. 85 Figure 2.3 eBURST diagram of all 1,411 F. psychrophilum isolates. This includes the 1,097 isolates from the F. psychrophilum MLST database and the 314 newly typed isolates from this study. A dagger (†) indicates that ST is present in North America. A star ((cid:2)) indicates that ST was found in North America and abroad. The founding sequence type (ST), or founder, is the ST with the highest number of single locus variants (SLVs; depicted as gray circles). In instances where multiple STs have the same number of SLVs, the founder is named after the ST with the most isolates. If both STs have the same number of isolates, the clonal complex (CC) is named for the earliest found ST. The founding genotype of a CC is enclosed within a box. The size of each circle indicates how many isolates belong to that ST. 86 Figure 2.4 Frequency and host of origin among 10 North American F. psychrophilum sequence types (STs). STs are displayed along the X-axis with four clonal complexes (CCs; e.g., CC-ST10, CC-ST9, CC-ST256, CC-ST276) and two singletons (e.g., ST253, ST258) being shown. The Y-axis displays the number of recovered isolates. All 421 currently typed North American F. psychrophilum isolates are presented here. 87 Figure 2.5 Phylogenetic reconstruction of the 86 F. psychrophilum sequence types (STs) currently described in North America using Bayesian inference. 15 clonal complexes (CCs) are in bold, 3 of which (in italics) are formed when the population structure of F. psychrophilum is evaluated in the context of the world. 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Journal of Aquatic Animal Health 27:192-202. 94 Chapter 3 MLST genotypes of F. psychrophilum vary in their pathogenicity to naïve rainbow trout (Oncorhynchus mykiss) 95 1. Abstract Flavobacterium psychrophilum is the etiological agent of rainbow trout fry syndrome (RTFS) and bacterial cold water disease (BCWD) in salmonids. Multilocus sequence typing (MLST) has demonstrated that F. psychrophilum isolates are genetically diverse and comprise multiple MLST clonal complexes (CC). Among the largest currently recognized CCs are CC- ST10 and CC-ST191, both of which primarily affect rainbow trout (Oncorhynchus mykiss); however, many studies have only highlighted the importance of CC-ST10. In the USA, 16 CC- ST10 genotypes have been identified and cause remarkable economic losses, particularly in farmed rainbow trout. Conversely, only two genotypes exist within CC-ST191 in the USA but also devastate farmed rainbow trout. In this study, differences in pathogenicity within and between the two main MLST CCs affecting rainbow trout were investigated. A series of intramuscular experimental infection studies were performed in triplicates using laboratory raised seven-month old rainbow trout. Each fish received ~107 - 108 cfu/fish-1 of each of 10 F. psychrophilum strains belonging to three STs of CC-ST10 (i.e., ST10, ST78, ST275) and two STs of CC-ST191 (i.e., ST267, ST301). All but one strain (i.e., US181) caused 100% mortality in their host; however, significant differences were found in mean days to death between isolates, including those of the same genotype. Overall, all strains appeared nearly equal in their ability to cause BCWD in rainbow trout and there were no significant differences between mean days to death for CC-ST10 and CC-ST191. Findings of this study underscore the ability of F. psychrophilum isolates of these two clonal complexes to cause significant mortalities in their host and suggest that multiple MLST CCs are highly virulent to rainbow trout. 96 2. Introduction Flavobacterium psychrophilum is the etiological agent of bacterial cold water disease (BCWD) and rainbow trout fry syndrome (RTSF), which are responsible for significant economic losses in farmed fish worldwide (reviewed in Loch & Faisal 2017). This bacterial pathogen primarily infects trout and salmon (Family Salmonidae), including rainbow trout/steelhead (Oncorhynchus mykiss), coho salmon (O. kisutch), Chinook salmon (O. tshawytscha), and Atlantic salmon (Salmo salar; reviewed in Nematollahi et al. 2003, Loch & Faisal 2017). Multilocus sequence typing (MLST) has revealed significant genetic heterogeneity within F. psychrophilum in the form of >240 sequence types (STs) that form >30 clonal complexes (CCs), some of which are present on multiple continents and appear highly virulent (Nicolas et al. 2008, Siekoula-Nguedia et al. 2012, Fujiwara-Nagata et al. 2013, Strepparava et al. 2013, Avendaño-Herrera et al. 2014, Nilsen et al. 2014, Van Vliet et al. 2016; Chapter 2). One such CC is CC-ST10, which has been reported on five continents (e.g., Asia, Europe, North America, Australia, South America) and is predominantly recovered from rainbow trout/steelhead suffering from BCWD. Likewise, previous research (i.e., Nicolas et al. 2008, Van Vliet et al. 2016), as well as Chapter 2 results, has shown that CC-ST10 isolates comprise a majority of those recovered from disease epizootics in the USA. Although these isolates appeared highly pathogenic and some have highlighted CC-ST10 as virulent (Nilsen et al. 2014), particularly to rainbow trout/steelhead, the relationship of F. psychrophilum MLST genotype to virulence has not been adequately addressed. In fact, very few isolates from CC-ST10 have been used in experimental challenge studies but at least one has been used many times before; namely, CSF 259-93. CSF 259-93 was identified as ST10 in 2008 97 (Nicolas et al. 2008) and has proven to be highly virulent in both injection and immersion studies (LaFrentz et al. 2003, Long et al. 2014, Wagner & Oplinger 2014). It remains unclear; however, if this single ST10 isolate reflects the pathogenicity of other ST10 isolates, if this genotype’s pathogenicity is mirrored by its single locus variants (SLVs), and if CC-ST10 is truly more virulent than other F. psychrophilum CCs. The second largest currently recognized F. psychrophilum CC worldwide is CC-ST191. Similar to CC-ST10, the majority of CC-ST191 genotypes have been recovered from diseased rainbow trout/steelhead (Nicolas et al. 2008, Siekoula-Nguedia et al. 2012, Strepparava et al. 2013, Nilsen et al. 2014). Although most CC-ST191 genotypes have been reported in Europe, two (i.e., ST267, ST301) have now been reported in the US (Chapter 2). Furthermore, isolates belonging to these two genotypes were recovered from rainbow trout/steelhead, many of which were also diseased. However, there are no published reports of isolates recognized as CC-ST191 being evaluated for their virulence. Although the comparative virulence of F. psychrophilum MLST genotypes has not been studied in salmonids, Fujiwara-Nagata et al. (2012, 2013), assessed the in vivo virulence of 40 F. psychrophilum isolates belonging to two different CCs (e.g., CC-ST52, CC-ST48/CC-ST56) in ayu (Plecoglossus altivelis) that were exclusively recovered from non-salmonids. Interestingly, the isolates belonging to CC-ST48/CC-ST56 caused BCWD-associated mortality, whereas CC- ST52 caused little to no mortality, thereby suggesting MLST CCs vary in their virulence. To examine the correlation between F. psychrophilum pathogenicity and MLST genotype in rainbow trout, 10 F. psychrophilum isolates that were recently genotyped using MLST were selected for in vivo virulence experiments. The isolates included in my experiments belonged to ST10, ST78, and ST275 (CC-ST10) and ST267 and ST301 (CC-ST191). My first objective was 98 to confirm that multiple variants within CC-ST10 were indeed virulent to their natural host, the rainbow trout. My second objective was to determine if in vivo virulence varies between isolates and genotypes in the same CC, and my third objective was to compare the virulence of the two largest MLST CCs primarily affecting rainbow trout/steelhead in the USA. 3. Materials and Methods 3.1. F. psychrophilum isolates used in experimental challenge Ten F. psychrophilum isolates that were genotyped using MLST and determined to belong to five STs within the two largest CCs were selected for experimental use (Table 3.1). These isolates were recovered from rainbow trout/steelhead that either displayed gross signs of BCWD or were apparently healthy and were recovered from three US states (e.g. Michigan, Pennsylvania, West Virginia) between the years of 2010 and 2017 (Table 3.1). Each of the two isolates within the five STs were either recovered from independent episodes of BCWD or during routine disease health surveillance (Table 3.1). 3.2. Fish and rearing conditions Embryonated rainbow trout eggs (i.e., eyed eggs) were obtained from Trout Lodge (Bonney Lake, WA) and maintained in a vertical incubator supplied with dechlorinated pathogen-free water (12ºC ± 1ºC; ~19 L/min) at the Michigan State University (MSU) – University Research Containment Facility until hatching. After most of the yolk-sac was absorbed, sac-fry were then transferred into aerated flow-through tanks (~40 L; ~34 L/min; 12-h photoperiod) supplied with dechlorinated pathogen-free water (12ºC ± 1ºC) until exogenous feeding began, at which point fry were fed a commercial trout diet of appropriately sized food 99 via an automatic feeder. After several weeks, fish were fed by hand twice daily until satiation and the water volume was increased to ~400L (~19L/min; 12-h photoperiod). Upon commencement of feeding, all tanks were siphoned once to twice daily to remove detritus and uneaten food. 3.3. Experimental challenge via intramuscular injection Intramuscular (IM) injection was selected as the route of infection because it has been established as a highly reproducible F. psychrophilum exposure route (Garcia et al. 2000). The 10 F. psychrophilum isolates (Table 3.1) were passed a single time through naïve rainbow trout via IM injection and re-isolated on tryptone yeast extract agar (TYES; Holt 1987) supplemented with 5% horse serum and 0.02% Lewin and Lounsbery mineral solution (referred to hereafter as mTYES; Lewin & Lounsbery 1996, Michel et al. 1999). After cultures were verified for purity, a single colony of each isolate was cryopreserved in mTYES supplemented with glycerol (20% [vol/vol]) for use in IM challenges. For the experimental challenge, the 10 F. psychrophilum isolates were revived from cryo- stock on mTYES agar and incubated at 15ºC for 72 hours, after which, a single colony was sub- cultured on to fresh mTYES agar and incubated for 72 hours at 15ºC to verify cryo-stock purity. Next, an isolated colony of each isolate was inoculated into 400 mL of mTYES broth and incubated at 15ºC with constant shaking at a speed of 100 rpm for 72 hours. Bacterial pellets were harvested from mTYES broth via centrifugation (2,571 x g, 15 min; Thermo Scientific™ Sorvall™ Legend™ X1R centrifuge). The supernatant was decanted and the bacterial cell pellet was suspended in sterile 0.65% sodium chloride (NaCl) and adjusted to an optical density at 600- nm (OD600) corresponding to a target of ~1 x 109 colony forming units (CFU) per mL using a 100 Biowave CO8000 Cell Density meter (WPA Inc., Cambridge, UK). To verify challenge dose, each challenge isolate suspension was serially diluted in ten-fold increments from 10-1 to 10-8 and 10 µl of each dilution was plated in duplicate on mTYES agar, incubated at 15ºC for 7 days, and final colony counts performed. For the IM challenge, fish (n = 10 per isolate, in triplicate) were randomly selected, anesthetized in sodium bicarbonate-buffered tricaine methanesulfonate (MS-222) at a concentration of 100mg L-1, and then IM-injected with 100 µl of the bacterial suspension (corresponding to 5.00 x 107 – 4.20 x 108 cfu/fish-1; Table 3.2) proximal to the anterior portion of the dorsal fin on the left side using a 26 G needle. Control fish (n = 10, in triplicate) were anesthetized as described above and sham-inoculated with 100 µl of sterile 0.65% NaCl. After IM-injection, fish were immediately transferred into aerated flow-through glass tanks (37.85L; 12-h photoperiod; randomly selected for each isolate prior to challenge) supplied with dechlorinated pathogen-free water (12ºC ± 1). Fish were monitored daily for 28-days and cared for as described above; mortalities were necropsied, clinically examined, and multiple tissues (e.g., brain, gills, heart, kidney, liver, skin, and/or spleen) were bacteriologically analyzed. In addition, a representative of each challenge isolate recovered from a challenged fish was extracted and confirmed as F. psychrophilum using an endpoint PCR assay as detailed in Chapter 2. All challenge experiments were conducted in accordance with the MSU-Institutional Animal Care and Use Committee. 3.4. Statistical analyses The cumulative percent mortality (CPM) and mean days to death were calculated for all challenge isolates. A one-way analysis of covariance (ANCOVA) was conducted to determine if 101 a statistically significant difference between F. psychrophilum isolates on the mean days to death while controlling for the continuous covariate, fish mass, was present. Testing was not conducted on CPM observations as all isolates caused ≥96.7% CPM. Fish mass was chosen to be a covariate in the testing of mean days to death because it influences fish mortality during experimental challenge with F. psychrophilum (Madsen & Dalsgaard 1999). Fish mass (i.e., the covariate) was rescaled to have a mean of zero and a standard deviation of one to standardize observations for analysis. For the experiment and ANCOVA analysis, fish tanks (n = 3 tanks per isolate) composed the experimental units, whereas individual fish were observational units (n = 10 fish per tank). Differences in least squares mean days to death between each F. psychrophilum isolate were tested with the Tukey-Kramer method used to adjust P-values for the pairwise tests. Differences between STs and CCs were tested using linear contrasts of means, where the elements of the contrast statement were based on which ST and CC each isolate belonged (see earlier description of isolates). Furthermore, to determine if the inoculum size influenced the mean days to death, a correlation analysis was conducted. P-values were considered statistically significant at an alpha level of 0.05. All statistical analyses were computed using SAS® software, Version 9.4 for Windows. 4. Results 4.1. Clinical signs 4.1.1. External signs Within 24-hours post infection, all fish challenged with F. psychrophilum were lethargic and inappetent, except for a single fish inoculated with strain US181. All challenged fish showed a combination of external signs consistent with BCWD (Figure 3.1), including 102 superficial ulceration that deepened and extended into the underlying muscle as infection progressed (Figure 3.1c, e-g). Ulcerations were often accompanied by a prominent yellowish discoloration of the affected tissues (Figure 3.1c, e-g). In some instances, ulcerations became focally extensive and would spread either cranially or caudally (Figure 3.1f, g). Additional disease signs included hemorrhage within the eye, isthmus, and fins, and severe gill pallor (Figure 3.1a, b) accompanied by either erythema or diffuse petechial hemorrhage (Figure 3.1b). In most challenged fish, a focal area of hemorrhage and/or pallor occurred near the site of injection (Figure 3.1d). The percent of fish showing lesions in the gills, skin or fins are shown in Table 3.3. 4.1.2. Internal signs Gross internal disease signs in rainbow trout infected with F. psychrophilum included renal, hepatic, and cardiac pallor; splenic swelling and/or enlargement; hemorrhage within the ovaries, serosal surface of the swim bladder, adipose tissue overlying the pyloric cecae, ventricle of the heart, and the liver (Figure 3.2a-d). The percent of fish showing lesions in the heart, kidney, liver, spleen, ovary, and/or swim bladder are shown in Table 3.3. 4.2. Cumulative percent mortality Cumulative percent mortality (CPM) was 100% for all tested isolates, except for US181, in which a single fish survived for the duration of the study (96.7% CPM). Given the high CPM among all isolate groups, no statistical comparisons were made. All negative control fish survived for the duration of the study (Table 3.2). 103 4.3. Mean days to death Overall, the mean days to death and the covariate, fish mass, were significantly different among all isolates (data not shown). Pairwise comparisons of the mean days to death for each combination of isolates revealed these differences further (Table 3.2). For example, although isolates CC10-US352 (i.e., isolate US352 belongs to CC-ST10), CC10-US26, and CC191- US215 were not significantly different from one another (P > 0.99), they were significantly different from all other isolates and overall had the highest mean days to death (P ≤ 0.0083; Table 3.2). Isolates CC10-US75, CC10-US57, CC10-US87, CC191-US54, CC191-US181, and CC191-US343 did not differ significantly in their mean days to death either (P > 0.17; Table 3.2). Of these six isolates, five (i.e., CC10-US75, CC10-US57, CC10-US87, CC191-US54, CC191-US181) did not differ significantly from CC10-US53 (P > 0.14), whereas one (i.e., CC191-US343) did (P < .05; Table 3.2). On average, fish infected with isolate CC10-US53 died the quickest (i.e., 2.0 ± 1.4 days), whereas those infected with CC191-US215 survived the longest (i.e., 6.8 ± 3.8 days; Table 3.2). The mean days to death among STs were found to differ significantly from one another too. In specific, CC10-ST10, CC10-ST78, and CC191-ST267 were not significantly different from one another in their mean days to death (P > 0.13), but were significantly different from CC10-ST275 and CC191-ST301 (P ≤ 0.0072). On average, fish inoculated with CC10-ST10, CC10-ST78, or CC191-ST267 survived longer than those infected with CC10-ST275 or CC191- ST301 (Table 3.2). Likewise, CC10-ST275 and CC191-ST301 were not found to be significantly different from one another and had nearly identical mean days to death (i.e., 3.3 ± 2.0 versus 3.2 ± 1.7, respectively; P = 0.8957). On average, fish infected with a CC10-ST10 isolate survived the longest (i.e., 5.1 ± 3.2 days), whereas those infected with a CC191-ST301 104 isolate died the quickest (i.e., 3.2 ± 1.7). No significant difference in mean days to death was discovered between the two CCs (i.e., CC-ST10, CC-ST191; P = 0.3414). The correlation analysis revealed no statistically significant relationship between isolate inoculum size (i.e., the dose) and the mean days to death (P = 0.5020). In fact, a positive correlation was seen between inoculum size and mean days to death (i.e., as inoculum size increases, fish survive longer; correlation coefficient = 0.2412). This positive relationship, however, was influenced by isolate US352 (ST10), which had the highest dose of F. psychrophilum (i.e., 4.20 x 108 cfu/fish-1) as well as one of the longest mean days to death (i.e., 6.4 ± 3.9). When this isolate was removed from the analysis, a negative correlation between dose and mean days to death (correlation coefficient = -0.1486) that was not statistically significant (P = .7027) was discovered. 4.4. Reisolation of F. psychrophilum A total of 242 F. psychrophilum isolates were recovered from the 115 fish that were sampled. Of these, 99 isolates were recovered from the kidney, followed by 68 from the skin near the injection site, 30 from the gills, 20 from the spleen, 13 from the liver, six from the brain, two from the dorsal fin, two from the heart, one from the adipose tissue overlying the pyloric cecae, and one from the musculature at the site of injection. All representative isolates were PCR confirmed as F. psychrophilum. 5. Discussion This study examined the pathogenicity of 10 North American F. psychrophilum strains belonging to five STs within two distinct MLST CCs (i.e., CC-ST10, CC-ST191), both of which 105 are widespread globally. Indeed, CC-ST10 is regarded as a putatively virulent CC (Nilsen et al. 2014), but despite CC-ST191 being recovered repeatedly from BCWD epizootics in farmed rainbow trout on multiple continents (Nilsen et al. 2014, Van Vliet et al. 2016), no mention of its virulence has been made. Although similarities between these two CCs exist (i.e., both are predominantly recovered from diseased rainbow trout and found in overlapping geographic locations), their housekeeping genes are genetically distinct from one another (Chapter 2) and previous studies have shown that F. psychrophilum strains vary in their virulence (Madsen & Dalsgaard 1998, Madsen & Dalsgaard 2000, Fujiwara-Nagata et al. 2012, Fujiwara-Nagata et al. 2013). This encouraged me to explore F. psychrophilum MLST genetic diversity in the context of pathogenicity, particularly because heterogeneity in the core (i.e., conserved) genome may equate to differences in the accessory genome, which is more variable and associated with virulence (Segerman 2012). Despite their genetic distinctness, representative strains within CC- ST10 and CC-ST191 proved to be highly pathogenic, causing fulminant mortality (i.e., ≥96.7%) in a short amount of time (i.e., ~two to seven days; Table 3.2). Correlation analysis provided evidence that the observed mean days to death was influenced by the isolate and not the inoculum size, thereby indicating that all utilized CC-ST10 and CC-ST191 isolates were highly pathogenic irrespective of the challenge dose. Still, the rapid mortality caused by these strains was unexpected. Garcia et al. (2000) used a similar challenged dose (i.e., 4.2 x 107 cfu/fish-1) when IM-injecting rainbow trout of a smaller size (i.e., average mass of ~three to six grams per fish), and yet 100% CPM was not reached until 18-days post infection compared to two to seven days, on average, in the current study. However, the MLST genotype of the F. psychrophilum isolate (i.e., JIP 02-97) used by Garcia et al. (2000) is unknown and may partially explain the observed differences in time to death. 106 The significant differences in mean days to death in rainbow trout challenged with F. psychrophilum isolates of the same MLST genotype suggests that MLST genotype may not entirely reflect an isolate’s pathogenicity, which is determined in part by its virulence factors, of which F. psychrophilum has many (reviewed in Nematollahi et al. 2003, Starliper 2011, Castillo et al. 2016, Duchaud et al. 2018). Although CC-ST10 isolates that have been genomically characterized possess multiple factors that are believed to be important for virulence (e.g., putative hemolysin D transporter, type II secretion system, metalloproteases, Por secretion system; Castillo et al. 2016, Duchaud et al. 2018), the factors important for CC-ST191 virulence and whether these overlap or differ from those of CC-ST10 are unknown and warrant further investigation. With this in mind, previous research has shown that F. psychrophilum isolates belonging to different genotypes of the same CC share the majority of their proteins encoding genes, yet differences exist. For example, the protein encoding genes of two F. psychrophilum isolates belonging to CC-ST9 (i.e., OSU THC02-90 identified as ST9, NCMB 1947T identified as ST13; Nicolas et al. 2008) differed by <100 genes according to whole genome sequencing (Wu et al. 2015, Rochat et al. 2017). In a similar context, recent research has shown that dozens of gene families within F. psychrophilum genotypes of the same CC are shared and are not present in other CCs or stand-alone genotypes (i.e., singletons). For example, within CC-ST10, multiple isolates belonging to several different genotypes (i.e., ST2, ST10, ST20, ST21, ST78) have been examined using whole genome sequencing (Duchaud et al. 2007, Wu et al. 2015, Castillo et al. 2016, Duchaud et al. 2018), and all isolates that were analyzed shared >80 gene families not present in other CCs (Duchaud et al. 2018). Interestingly, genes putatively linked to virulence, such as tetX and those encoding for a type II secretion system, were discovered among these gene families specific to CC-ST10. Furthermore, isolates of the same genotype 107 (i.e., ST2, ST12) have been found to differ in their gene families, which may have differential consequences for pathogenicity. For example, F. psychrophilum isolates 950106-1/1 and VQ50 share an identical MLST genotype (i.e., ST2); however, 950106-1/1 contains bacteriophage 6H, which is known to contain putative virulence factors that may aid in pathogenicity (Castillo et al. 2014), whereas VQ50 does not (Castillo et al. 2014, Duchaud et al. 2018). In this context, comparative studies linking the differential expression of genes linked to F. psychrophilum virulence in multiple MLST CCs and how these relate to in vivo virulence will greatly clarify the molecular pathogenesis of this fish-pathogenic bacterium and guide the development of future BCWD control strategies. Previous research has shown that some F. psychrophilum genotypes, including those investigated in this study, are widely distributed across multiple continents (Avendaño-Herrera et al. 2014, Nilsen et al. 2014, Van Vliet et al. 2016) and some have implicated the trade of salmonids and their eggs as a factor in global flavobacterial dissemination (Nicolas et al. 2008, Avendaño-Herrera et al. 2014, Duchaud et al. 2018). Indeed, millions of eyed rainbow trout eggs are distributed every year both intra- and trans-continentally (Adeli & Baghaei 2013), which increases the likelihood of disseminating F. psychrophilum, a non-reportable pathogen that is transmitted vertically and can survive routinely employed iodophore egg-surface disinfection practices (Cipriano et al. 1995, Brown et al. 1997, Kumagai et al. 1998, Kumagai & Nawata 2010, reviewed in Loch & Faisal 2017). Furthermore, F. psychrophilum is transmitted horizontally through direct (i.e., fish to fish) and indirect contact (i.e., through the water column; Madetoja et al. 2000). Both transmission routes raise the possibility for multiple F. psychrophilum genotypes to intermingle in the fish farming environment and may help to 108 partially explain the relatively high recombination rates (Chapter 2, Nicolas et al. 2008, Duchaud et al. 2018) within the species. In addition to potentially facilitating flavobacterial dissemination, it is possible that current aquaculture practices may also select for highly pathogenic F. psychrophilum strains, including those that were the focus of this study. Indeed, previous studies on Flavobacterium columnare, the causative agent of columnaris disease (reviewed in Loch & Faisal 2017), have suggested that intensive fish farming in combination with biological factors of the pathogen (i.e., F. columnare sheds at a rapid rate from dead fish and can survive for extended periods outside of its host; Kunttu et al. 2009) have contributed to its increased virulence over both long and short time scales (Pulkkinen et al. 2010, Sundberg et al. 2016). In this context, the numerous susceptible hosts that are reared in artificially high densities in aquaculture facilities likely facilitates the transmission of virulent F. psychrophilum isolates, whereby fish infected with and killed by F. psychrophilum shed the bacterium at substantially higher numbers and for longer periods of time compared to live infected fish (Madetoja et al. 2000). Moreover, the abundance of nutrients that are typically present in hatchery rearing units from sources such as uneaten food, may create an energetically inexpensive environment for F. psychrophilum, allowing strains to retain their virulence making subsequent infections during an outbreak easier, which has been documented for F. columnare (Penttinen et al. 2016). As such, the rapid rate at which CC-ST10 and CC-ST191 strains killed juvenile rainbow trout in this study could be advantageous for F. psychrophilum in aquaculture facilities, where successive transmission may occur before a treatment strategy is implemented. From a practical perspective, this highlights the need to remove dead fish during BCWD epizootics, as well as keep organic material (i.e., uneaten food, feces) to a minimum. 109 In the current study, I selected isolates from two predominating MLST CCs (i.e., CC- ST10, CC-ST191) that primarily affect rainbow trout/steelhead, which comprise the majority of trout species raised in US aquaculture (USDA, 2009). Although CC-ST10 and CC-ST191 are highly virulent in rainbow trout/steelhead, it remains to be determined if they are differentially virulent in other salmonid species. In this context, F. psychrophilum genotypes belonging to CC-ST9, which have been predominantly recovered from coho salmon (Nicolas et al. 2008, Avendaño-Herrera et al. 2014, Nilsen et al. 2014, Van Vliet et al. 2016, Chapter 2), induced minimal mortality (i.e., 12% CPM) in experimentally infected rainbow trout (Madsen & Dalsgaard, 2000), possibly indicating a lack of adaptation(s) (i.e., virulence factors) necessary to cause disease in rainbow trout. In conclusion, results herein have highlighted the pathogenic capacity of two of the largest and most widespread F. psychrophilum CCs (e.g., CC-ST10, CC-ST191) in their natural host, the rainbow trout. Their highly pathogenic nature, host association, and global distribution highlights their importance in the development of future targeted prevention and control strategies, including the development of autogenous and/or multivalent vaccine preparations. However, much remains to be explored, not only within these CCs, but in other F. psychrophilum CCs that have different host specificities. Until an effective control strategy is discovered, strict biosecurity and fish husbandry practices at aquaculture and fish farming facilities around the world are needed to prevent the spread of this devastating fish pathogen. 110 APPENDIX 111 Table 3.1. Origin information for the 10 F. psychrophilum isolates that were selected for use in in vivo virulence experiments. All isolates were originally recovered from rainbow trout/steelhead. AIsolates denoted with D were recovered from a fish suffering from BCWD, whereas H indicates an isolate was recovered from an apparently healthy fish. *Sequence types denoted with 2016 were recovered and genotyped by Van Vliet et al. (2016). Isolate IDA State of isolation MI US352D PA US75H MI US53D US26D MI PA US57D MI US87D US215H MI MI US54D PA US181D US343D WV Location of isolation SFH4 SFH2 SFH4 SFH4 SFH4 SFH4 WE1 SFH4 SFH1 PF2 Clonal complex CC-ST10 CC-ST10 CC-ST10 CC-ST10 CC-ST10 CC-ST10 CC-ST191 CC-ST191 CC-ST191 CC-ST191 Year of isolation 2010 2016 2011 2010 2014 2016 2017 2013 2015 2014 Sequence type* ST10 ST10 ST782016 ST782016 ST275 ST275 ST267 ST2672016 ST301 ST301 112 Table 3.2 Statistical comparisons of mean days to death among 10 F. psychrophilum isolates, five sequence types (STs), and two clonal complexes (CCs). #Isolates with the same superscript letter (i.e., a, b, c), STs with the same superscript number (i.e., 1, 2) and CCs with the same symbol (i.e., *) are not significantly different. All significance testing was assessed at an alpha of 0.05. CFU/fish-1, colony forming units injected per fish; CPM, cumulative percent mortality; OD, optical density at 600nm; SD, standard deviation. Challenge dose (CFU/fish-1) 4.20 x 108 5.00 x 107 2.55 x 108 1.90 x 108 2.10 x 108 1.45 x 108 5.50 x 107 5.50 x 107 1.05 x 108 1.45 x 108 2.35 x 108 2.23 x 108 1.78 x 108 5.50 x 107 1.25 x 108 2.87 x 108 9.00 x 107 CPM ± SD 0.00 100 ± 0 100 ± 0 100 ± 0 100 ± 0 100 ± 0 100 ± 0 100 ± 0 100 ± 0 96.7 ± 4.7 100 ± 0 100 ± 0 100 ± 0 100 ± 0 100 ± 0 98.9 ± 3.7 100 ± 0 99.5 ± 3.1 Mean days to death ± SD# 6.4 ± 3.9a 3.7 ± 2.5b,c 2.0 ± 1.4c 6.9 ± 2.1a 3.3 ± 1.7b,c 3.3 ± 2.2b,c 6.8 ± 3.8a 2.8 ± 1.3b,c 2.4 ± 2.0b,c 4.0 ± 1.4b 5.1 ± 3.21 4.6 ± 1.81 3.3 ± 2.02 4.8 ± 2.61 3.2 ± 1.72 4.3 ± 2.3* 4.0 ± 2.2* CC ST Isolate ID Sham- inoculated US352 ST10 CC-ST10 US75 ST10 US53 ST78 ST78 US26 ST275 US57 ST275 US87 CC-ST191 ST267 US215 ST267 US54 ST301 US181 ST301 US343 ST10 CC-ST10 ST78 ST275 CC-ST191 ST267 ST301 CC-ST10 CC-ST191 Mean body weight (g) 16.6 14.6 14.6 15.7 15.7 12.7 14.5 14.9 13.0 14.1 15.3 14.6 15.7 13.6 14.0 14.7 14.6 14.4 Total no. of fish 30 30 30 30 30 30 30 30 30 30 30 60 60 60 60 60 180 120 113 Table 3.3 Percent of F. psychrophilum isolates, sequence types (STs), and clonal complex (CCs) presenting with gross external (i.e., skin erosion/hemorrhage/swelling/pallor, gill pallor/hemorrhage/erythema) and internal (i.e., hemorrhage, swelling, pallor) pathology. ID US352 US75 US53 US26 US57 US87 External Skin or Fin Gill 66.7% 96.7% 80.0% 100% 73.3% 100% 83.3% 83.3% 73.3% 100% 56.7% 100% US215 70.0% 93.3% US54 US181 US343 ST10 ST78 ST275 ST267 ST301 53.3% 100% 53.3% 83.3% 90.0% 100% 73.4% 98.4% 78.6% 91.7% 65.0% 100% 61.7% 96.7% 71.7% 91.7% CC-ST10 72.3% 96.7% CC-ST191 66.7% 94.2% Heart 96.7% 100% 100% 100% 100% 93.3% 100% 100% 83.3% 100% 98.4% 100% 96.7% 100% 91.7% 98.4% 95.9% Internal Kidney Liver 96.7% Spleen Ovary 100% 16.7% 3.3% Swim Bladder 3.3% 96.7% 100% 6.7% 0.0% 0.0% 100% 100% 6.7% 0.0% 0.0% 100% 100% 16.7% 3.3% 6.7% 100% 100% 6.7% 0.0% 0.0% 93.3% 93.3% 20% 0.0% 3.3% 93.3% 96.7% 3.3% 0.0% 0.0% 100% 96.7% 6.7% 0.0% 3.3% 83.3% 83.3% 13.3% 0.0% 0.0% 100% 100% 3.3% 0.0% 0.0% 96.7% 100% 11.7% 1.7% 1.7% 100% 100% 11.7% 1.7% 3.4% 96.7% 96.7% 13.4% 0.0% 1.7% 96.7% 96.7% 5.0% 0.0% 1.7% 91.7% 91.7% 8.3% 0.0% 0.0% 97.8% 98.9% 12.3% 1.1% 2.3% 94.2% 94.2% 6.7% 0.0% 0.9% 114 D E F B A Figure 3.1 External signs of BCWD in rainbow trout (Oncorhynchus mykiss) fingerlings that were intramuscularly injected with F. psychrophilum. A. Severe gill pallor with concurrent diffuse petechial hemorrhage. B. Multifocal to diffuse petechial hemorrhage on the trunk and congestion at the base of the pelvic fin. C. Ulceration of the skin and underlying musculature on the dorsum. D. Severe swelling and congestion at the injection site. E-G. Progression of experimentally induced BCWD, beginning with superficial ulceration (E) that extended (F) and deepened into the underlying muscle (G) as the infection progressed. Note also the yellowish discoloration overlying the lesions in C and E-G. G C 115 A C B Figure 3.2 Internal signs of BCWD in rainbow trout (Oncorhynchus mykiss) fingerlings that were intramuscularly injected with F. psychrophilum. A. Hemorrhagic ovary. B. Diffuse petechial hemorrhage of the ventricle and bulbous arteriosus of the heart, as well as a moderately pale liver. C. Severely hemorrhagic liver accompanied by multifocal petechial hemorrhage of the adipose tissue overlying the pyloric cecae. D. 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Conclusions Prior to these studies, very little was known about the genetic diversity of Flavobacterium psychrophilum in North America and how this diversity was related to fish host species, geographic distribution, and virulence. Furthermore, it was unclear how F. psychrophilum was acquiring this diversity and if this was a product of recombination or mutation. Likewise, the most prevalent F. psychrophilum MLST genotypes, all of which belonged to CC-ST10, had never had their pathogenicity confirmed or compared either within or between clonal complexes. The answers to these question may improve the control and spread of this serious pathogen of fish. In Chapter 2, I examined the most comprehensive set of F. psychrophilum isolates in North America to date using MLST. This study revealed North American F. psychrophilum isolates to be more genetically diverse than anywhere else in the world (H = 0.68 ± 0.04). This information supports a recent notion that F. psychrophilum originated in North America and subsequently spread to other regions of the world through the international trade of fish and eggs. In addition, I identified several genotypes within North America that have been reported in other locations around the world and interestingly, the majority of those isolates came from identical fish species. Although it currently remains to be proven, it would appear that the transportation of eggs and fish within North America has led to the spread of many genotypes as well. In this context, we recovered CC-ST29 from Chinook salmon in Oregon, USA, a state that distributed Chinook to Michigan, USA (a state where Chinook salmon are non-native) and it would appear F. psychrophilum isolates belonging to this CC were transported along with these fish as CC- ST29 has been detected among Chinook salmon in Michigan as well. In the same context, we discovered strong evidence that at least one genotype, ST275, was transported from an egg 123 collection facility to a state fish hatchery that later had a severe outbreak of BCWD in the resultant progeny due to ST275. This scenario, although unfortunate, is enlightening because even though strict biosecurity measures are implemented in order to limit the spread different pathogens, F. psychrophilum appears to be circumventing our current prevention strategies. Indeed, it is unknown whether this genotype was transported in or on the surface of the egg, and/or on equipment, nevertheless, this is cause for alarm. With this unique collection of F. psychrophilum isolates, we were able to utilize strains recovered as early as 1981. Interestingly, it appears that some genotypes (e.g., ST10, ST27, ST29, ST78) have existed in North America for decades and thus, are likely established within the salmonid population, which may be due to evolutionary adaptations to their hosts; however, this remains to be determined. Furthermore, multiple sequence analyses indicated recombination is responsible for the diversification of F. psychrophilum in North America, even though clonal populations (i.e., clonal complexes) persist. Although it remains to be determined, clonal populations of F. psychrophilum established in a particular state or facility may contain genes related to virulence or antimicrobial resistance. In a situation where biosecurity fails, this may lead to a virulent complex coming into contact with a less or non-virulent complex with recombination permitting the less or non-virulent population to become virulent. In Chapter 3, I examined the pathogenicity of the most widespread genotypes in North America, which belonged to CC-ST10, a CC known for its apparent virulence, and compared them to one another, as well as to two other genotypes, which belong to the second largest CC (i.e., CC- ST191) but does not receive notoriety for its apparent virulence. Not surprisingly, the genotypes of CC-ST10 were very pathogenic towards rainbow trout; however, not all genotypes or strains within the same genotype were equal in their pathogenicity. Interestingly, ST10 did not appear 124 to be the most virulent genotype, ST10 actually took the longest to cause death in its host. However, this may be an adaptive mechanism that allows for a longer-lasting infection and thereby allows ST10 more time to colonize its next host. Furthermore, CC-ST191 was nearly identical to CC-ST10 in terms of pathogenicity towards rainbow trout and raises the question of how pathogenic other CCs may be toward their respective hosts. Overall, findings of this study pave the way for multiple research directions including the development of effective vaccines that encompass most genotypes, host-pathogens interactions, and evolution of genetic diversity among F. psychrophilum strains. 2. Future Research My studies have provided a framework upon which future studies can be developed. I was able to genotype over 300 isolates of F. psychrophilum, none of which have ever had their genetic diversity explored previously. Despite the uniqueness of these isolates, in that they were recovered from multiple fish species and geographic areas; some facilities, fish species, and states were represented more than others, a matter that did not allow for the application of rigorous statistical analyses. As such, there is a need for more isolates that have been recovered from salmonids and from all locations in which they exist. The isolates used in this study included all but one isolate from the USA and therefore, there is a need to obtain more isolates (fresh and archived) from Canada and Mexico so that we can better understand the genetic diversity and pathogen transmission dynamics of this serious pathogen over a larger spatial and temporal landscape in North America. Integrated into that is the need to better understand the degree of host specificity and temporal persistence of F. psychrophilum genotypes. This is of paramount importance because previous attempts to use a single strain for vaccine production 125 have been unsuccessful. Therefore, there is a need for a polyvalent vaccine against F. psychrophilum, one for that is effective for each salmonid species, in particular those that are intensively reared. Future studies should also examine how F. psychrophilum MLST genotypes relate to flavobacterial mechanisms of virulence (i.e., pathogen-host interactions), as well as mechanisms for environmental persistence, especially given my findings that some strains seem to persist in aquaculture facilities over extended periods of time/rearing cycles. The results of Chapter 3 suggest that those five genotypes are highly pathogenic to rainbow trout. Future experiments should explore their pathogenicity in other salmonids species in order to evaluate whether they are truly adapted to a rainbow trout or are equally pathogenic to multiple salmonid species. Furthermore, it remains unclear how well these genotypes can reproduce BCWD through a more natural route of exposure (i.e., immersion). However, immersion studies remain to be standardized, a matter that should must be addressed first and foremost. 126