(Qpcr) Assay for the Beta- Proteobacterium BK-BJC, and Its Application in Lake Trout (Salvelinus Namaycush) During an Epitheliocystis-Associated Mortality Event

Home , Neorickettsia, Paenalcaligenes, Thiomonas

Development of a Quantitative Real-Time PCR (qPCR) Assay for the Beta- Proteobacterium BK-BJC, and Its Application in Lake Trout (Salvelinus namaycush) During an Epitheliocystis-Associated Mortality Event

Doran Witherspoon Kirkbright

A Thesis presented to The University of Guelph

In partial fulfillment of the requirements for the degree of Doctor of Veterinary Science

Guelph, Ontario, Canada

Doran Witherspoon Kirkbright Advisors: University of Guelph, 2016 Dr. John Lumsden and Dr. Brandon Lillie

Epitheliocystis is a multi-etiologic gill condition in which a single cell is expanded by an intracytoplasmic inclusion filled with gram negative bacteria. My research focused on characterizing one purported agent, Blue Jay Creek Burkholderia (BK-BJC), and its relationship to epitheliocystis-associated annual mortality events and histopathologic gill lesions in economically valuable Ontario reared lake trout.

Based on the 1503 bp 16S rRNA gene sequence of BK-BJC identified by Contador et al. (2016), two primer sets were developed for detection of this bacterium. From the first primer set

(BKBJCV8F/R) with hydrolysis probe, a qPCR assay was developed and used to quantify BK-

BJC amounts in the gills of Blue Jay Creek Fish Culture Station lake trout and tank water during an epitheliocystis-associated mortality event in 2013 and a non-outbreak winter in 2015. A second primer set (BKBJCV3) was created to differentiate BK-BJC from ‘Candidatus

Branchiomonas cysticola’. In addition, BK-BJC-infected gills were streaked onto a variety of agar plates. Colony growth was assessed by Gram stain, MALDI-TOF mass spectrometry, and qPCR. The BKBJCV8 was found to be non-specific in silico and experimentally, potentially amplifying at least 33 non-target bacteria from GenBank. Conventional PCR with the BKBJCV3 confirmed that all BKBJCV8 qPCR positive samples were positive for BK-BJC. BK-BJC was present in 1

% of the samples collected from fish in 2015. There was a significant direct relationship

(p=0.035) between BK-BJC and BK-BJC-like, non-target bacterial loads and mortality rates, and there was an observable direct relationship with interlamellar hyperplasia and single cell necrosis of the gills. No relationship was discovered between epitheliocystis inclusions and bacteria loads in either 2013 or 2015 lake trout. Further, BK-BJC or BK-BJC-like bacteria were not found in the water samples, nor did they grow on any culture medium.

In conclusion, we developed two PCR assays and confirmed that BK-BJC was present in all lake trout from an EP outbreak at Blue Jay Creek in 2013. Future research should focus on the development of a more sensitive and specific, qPCR assay for the rapid diagnosis of BK-BJC and further investigations into the ability of BK-BJC to cause EP.

ACKNOWLEDGEMENTS

I would like to express my gratitude to Drs. John Lumsden and Brandon Lillie, and committee members Drs. Salvatore Frasca Jr and Niels Bols for all their invaluable advice, support, and effort in formulating and executing this DVSc project.

Deepest thanks also go to Drs. Foster, Caswell, Plattner, Hayes, Lillie, Smith, Turner, and Susta for the countless hours teaching me pathology at the microscope and on the post mortem floor, as well as supporting my (sometimes) wild etiologic diagnoses and helping me craft reports while maintaining my unique voice.

To all my pathology co-residents, I would like to say thank you for being great friends and colleagues. I am so grateful that our paths crossed and we went on this journey together. You will all remain my closest friends and confidants for life. You made living and working in

Canada a wonderful experience.

There are no words that can truly express how thankful I am to laboratory technicians—Leah

Read, Jutta Hammermueller, Paul Huber, and Pat Bell Rogers—and AHL histology department.

Thank you for your sagacious advice, patiently teaching me how laboratories work, and allowing me to run by with questions at all times of day. Your expertise and generosity is humbling.

I would also like to thank my semi-aquatic labmates—Elena, Ehab, Juan Ting, Maureen, Ryan,

Jaramar, Paige and Lowia for showing me new technologies and how to gracefully deal with the inevitable delays and set-backs that come with research.

This project would not have been possible without the fish collection and processing assistance of Paul Methner, Michael Burke, and the staff at BJC Fish Culture Station and Alma Research

Station. Also, thank you goes to Mykolas Kamaitis for taking the long journey to Manitoulin

Island to collect fish with me.

I would like to thank my funding sources—NSERC, OMNR, and the Ontario Veterinary College

(OVC)—with whom this project would not have been possible.

A personal debt of gratitude goes to Donna Kangas who always ensured that I stayed on the right side of registrar’s office and for our spontaneous chats that I will remember fondly.

Last, but not least, I thank my family and boyfriend, Alex Zaleznik, for their constant support and cheerleading. I could not have made it this far without you.

DECLARATION OF WORK PERFORMED

The majority of the work was performed by Doran Witherspoon Kirkbright under the supervision of Drs. John Lumsden, Brandon Lillie, and the advisory committee members of Dr.

Niels Bols and Dr. Salvatore Frasca Jr., except for the following:

The development of the initial primer set, BKBJCV8, and PCR optimization of that primer set was done by Elena Contador.

Collection and processing of 45 lake trout from Blue Jay Creek Fish Culture Station for DNA extraction and histopathologic examination was performed by Mykolas Kamaitis, a summer and veterinary student (University of Guelph, OVC).

All slides were prepared by Susan Lapos and the staff of the Animal Health Laboratory at the

University of Guelph.

TABLE OF CONTENTS

ABSTRACT ...... ii ACKNOWLEDGEMENTS ...... iv DECLARATION OF WORK PERFORMED ...... vi TABLE OF CONTENTS ...... vii LIST OF TABLES ...... xii LIST OF FIGURES ...... xiv LIST OF ABBREVIATIONS ...... xvi GENERAL INTRODUCTION ...... 1 1. LITERATURE REVIEW ...... 3 1.1. EPITHELIOCYSTIS ...... 3 1.2. CLINICAL SIGNS AND GROSS FINDINGS ...... 6 1.3.1. HISTOLOGY - SPECIAL STAINS ...... 8 1.4. DISEASE AND RELATIONSHIP WITH HOST DEATH ...... 10 1.5. CO-INFECTIONS ...... 12 1.6. ADVANCED DIAGNOSTICS ...... 14 1.6.1. TRANSMISSION ELECTRON MICROSCOPY ...... 14 1.6.2. BACTERIAL CULTURE ...... 16 1.6.3. POLYMERASE CHAIN REACTION ...... 19 1.6.4. In situ HYBRIDIZATION...... 20 1.6.5. IMMUNOHISTOCHEMISTRY ...... 22 1.7. BACTERIAL PATHOGENS ...... 24 1.7.1. CHLAMYDIAE ...... 24 1.7.2. BURKHOLDERIALES...... 26 1.7.3. ENDOZOICIMONAS ELYSICOLA ...... 30 1.8. TREATMENTS ...... 31 1.9. FURTHER RESEARCH ...... 32 2. RATIONALE, PURPOSE and HYPOTHESES ...... 34 2.1. RATIONALE ...... 34 2.2. PURPOSE ...... 35

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2.3. HYPOTHESES ...... 36 3. MATERIALS AND METHODS ...... 38 3.1. HISTORIC PCR IDENTIFICATION OF BK-BJC ...... 38 3.3. PCR VALIDATION AND OPTIMIZATION OF BKBJCV8 FORWARD AND REVERSE PRIMERS ...... 41 3.4. BKBJCV8 qPCR ...... 41 3.5. SAMPLING SITE ...... 44 3.6. 2012-13 SAMPLE COLLECTION AND PROCESSING...... 45 3.7. 2014-2015 SAMPLE COLLECTION AND PROCESSING ...... 45 3.8. DNA EXTRACTION FROM LT WHOLE GILLS ...... 46 3.9. FILTRATION AND DNA EXTRACTION FROM WATER SAMPLES ...... 46 3.10. 2012-2013 EP OUTBREAK AND MORTALITY STATISTICS ...... 47 3.11. SECOND PRIMER SET TO VALIDATE SPECIFICITY OF BKBJCV8 qPCR...... 49 3.12. BACTERIA PREPARATION FOR CULTURE ...... 51 3.13. AGAR MEDIA PREPARATION AND COLONY IDENTIFICATION ...... 51 4. RESULTS ...... 53 4.1. DEVELOPMENT OF NOVEL qPCR FOR BK-BJC DETECTION ...... 53 4.1.1. PCR IDENTIFICAITON OF BK-BJC ...... 53 4.1.2. DEVELOPMENT AND STANDARDIZATION OF THE BKBJCV8 ...... 53 4.1.3. PCR VALIDATION AND OPTIMIZATION OF THE BKBJCV8 ...... 59 4.1.4. BKBJCV8 qPCR EFFICIENCIES ...... 61 4.1.5. SPECIFICTY OF BKBJCV8 qPCR ...... 63 4.1.6. DEVELOPMENT OF OLIGONUCLEOTIDES AND BKBJCV8 qPCR ANALYSIS ...... 67 4.1.7. APPLYING BKBJCV8 qPCR TO BJC LT GILLS OF KNOWN BK-BJC STATUS 73 4.1.8. ESTABLISHING THE BKBJCV8 qPCR MINIMUM THRESHOLD ...... 75 4.2. UTILIZING THE NOVEL BK-BJC SPECIFIC qPCR TO ANALYZE HISTORIC SAMPLES FROM BJC LT INFECTED WITH EP ...... 75 4.2.1. UNIVERSAL PCR VERSUS BKBJCV8 qPCR RUN ON HISTORIC 2010-2013 LT GILLS FROM BJC ...... 75 4.3. UTILIZING THE NOVEL BK-BJC SPECIFIC qPCR TO ANALYZE AN EPITHELIOCYSTIS-ASSOCIATED MORTALITY EVENT IN BJC LT ...... 77

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4.3.1. EVENTS AT THE SAMPLING SITE, WINTERS 2012-3 AND 2014-15 ...... 77 4.3.2. 2012 EP OUTBREAK ...... 77 4.3.3. COMPARISON OF BBC QUANTIFICATION TO THE 2012-13 BJC LT MORTALITY EVENT...... 81 4.3.4. COMPARISON OF BBC QUANTIFICATION TO HISTOPATHOLOGIC GILL CHANGES ...... 84 4.3.5. MONITORING 2014-2015 BJC LT FOR EP AND BBC ...... 86 4.3.6. BKBJCV8 qPCR ANALYSIS OF BJC HATCHERY WATER SAMPLES ...... 88 4.4. DEVELOPMENT OF A SECOND PRIMER SET TO DISTINGUISH BK-BJC FROM ‘CA. B. CYSTICOLA’ ...... 88 4.4.1. CONVENTIONAL PCR VALIDATION OF THE BKBJCV3 ...... 95 4.4.2. BKBJCV3 PCR VERSUS BKBJCV8 qPCR ...... 97 4.5. GILL CULTURE ON AGAR PLATES ...... 97 5. DISCUSSION ...... 101 5.1. DEVELOPMENT AND THEORETICAL ASSESSMENT OF BKBJCV8 ...... 101 5.2. UTILIZING SYNTHETIC OLIGONUCLEOTIDES TO DETERMINE THE NUMBER OF NON-TARGET BACTERIA AMPLIFIED BY BKBJCV8 qPCR ...... 102 5.3. DEVELOPMENT OF BKBJCV3 TO DISTINGUISH BK-BJC FROM ‘Ca. B. CYSTICOLA’ ...... 104 5.4. BKBJCV8 qPCR ON 2013 BJC LT DURING AN EP-ASSOCIATED MORTALITY EVENT ...... 106 5.5. BBC CONCENTRATIONS COMPARED TO HISTOLOGIC GILL LESIONS ...... 106 5.6. BKBJCV8 qPCR ON 2015 BJC LT DURING A NORMAL YEAR ...... 108 5.7. BKBJCV8 qPCR ON BJC WATER SAMPLES ...... 109 5.8. CULTURE OF BBC ...... 110 6. CONCLUSION ...... 113 7. REFERENCES ...... 116 8. APPENDICES ...... 135 8.1. APPENDIX 1: PCR PRIMER SETS AND SEQUENCES...... 135 8.2. APPENDIX 2: PARTIAL SEQUENCE OF THE 16S rRNA GENE FROM BK-BJC.. . 136 8.3. APPENDIX 3: CLUSTAL ALIGNMENT OF BK-BJC AND ‘CA. B. CYSTICOLA’ CLONE GABI’S 16S rRNA GENE SEQUENCE WITH LOCATION OF THE BKBJCV3 AND BKBJCV8...... 137

8.4. APPENDIX 4: GBLOCK GENE FRAGMENT SEQUENCES...... 139 8.4.1. BK-BJC REGION 1257-1402 (250 bp)...... 139 8.4.2. BK-BJC REGION 1-750 bp...... 139 8.4.3. ‘CA. B. CYSTICOLA’ CLONE GABI REGION 1-750 BP...... 139 8.5. APPENDIX 5: BKBJCV8 AND BKBJCV3 qPCR AMPLICONS—LENGTH AND SEQUENCE...... 140 8.6. APPENDIX 6: REPRESENTATIVE SEQUENCES AND ORGANISMS OF THE 21 SEQUENCE GROUPS ≥95 % IDENTICAL TO THE BKBJCV8 qPCR AMPLICON...... 141 8.7. APPENDIX 7: ALIGNMENT OF BK-BJC AND EACH BACTERIAL SG WITH LOCATION OF THE BKBJCV8...... 143 8.8. APPENDIX 8: GEL SAMPLE INDICES FROM CONVENTIONAL PCR TEST OF BKBJCV8 PRIMERS...... 145 8.8.1 GEL 1 AND SAMPLE INDEX...... 145 8.8.2 GEL 2 AND SAMPLE INDEX...... 146 8.9. APPENDIX 9: INTRAASSAY VARIATION CALCULATED ACCORDING TO NCBI GUIDELINES...... 147 8.10. APPENDIX 10: BLASTN RESULTS OF FORWARD AND REVERSE SEQUENCES FROM THE BKBJCV8 qPCR PRODUCT OF SYNTHETIC BK-BJC STANDARD 1.02E8...... 148 8.10.1 BK-BJC 1.02E8 AMPLICON SEQUENCING RESULTS OF THE BKBJCV8 FORWARD SEQUENCE...... 148 8.10.2 BK-BJC 1.02E8 AMPLICON SEQUENCING RESULTS OF THE BKBJCV8 REVERSE SEQUENCE...... 148 8.11. APPENDIX 11: THE DIFFERENCES IN BP MISMATCHES BETWEEN BK-BJC AND THE NON-TARGET SEQUENCE GROUPS CATEGORIZED BY BKBJCV8 qPCR NEGATIVE AND POSITIVE STATUS...... 150 8.11.1 NEGATIVE SEQUENCE GROUPS BP MISMATCHES AND LOCATIONS. .... 150 8.11.2. POSITIVE SEQUENCE GROUPS BP MISMATCHES AND LOCATIONS...... 150 8.12. APPENDIX 12: GEL ELECTROPHORESIS RESULTS FROM THE TEMPERATURE GRADIENT PCR TO FIND THE OPTIMAL ANNEALING TEMPERATURE FOR BKBJCV3 PRIMERS...... 151 8.13. APPENDIX 13: GEL ELECTROPHORESIS RESULTS OF BKBJCV3 PCR ON BKBJCV8 qPCR POSITIVE SAMPLES FROM BJC IN 2013 AND 2015...... 152

8.14. APPENDIX 14: SEQUENCING RESULTS FROM BKBJCV8 qPCR FROM A Ct 35 SAMPLE (WEEK 1, TANK 1 FISH 14 FROM BJC)...... 154 8.15. APPENDIX 15: BK-BJC OUTBREAK 2012-13 ADJUSTED AND NON-MEDIAN AND UL AND LL...... 155 8.16. APPENDIX 16: NUMBER DEAD AND DAILY PERCENT MORTALITY OF BJC LT DURING AN EP ASSOCIATED MORTALITY EVENT IN 2013...... 156 8.17. APPENDIX 17: BLASTN MATCHES FOR THE BKBJCV8 qPCR PRODUCTS OF TWO POSITIVE LT (WEEK 1, TANK 1- FISH 1 AND 12)...... 160 8.18. APPENDIX 18: RATIONALE FOR EXCLUDING BKBJCV3’S TOP BLASTN MATCHES WITH ≥90 % NUCLEOTIDE IDENTITY FROM NON-TARGET AMPLIFICATION...... 161

LIST OF TABLES

Table 1. A brief list of special stains used to diagnose EP...... 9

Table 2. Reported comorbidities in fish with epitheliocystis...... 13

Table 3. Theoretical description of the BKBJCV8 primers and probe based on OligoAnalyzer and Primer-BLAST software...... 55

Table 4. BLASTn search matches to the BKBJCV8 probe sequence...... 57

Table 5. BLASTn alignment matches to the theoretical BKBJCV8 amplicon...... 58

Table 6. Calculated BKBJCV8 qPCR efficiency and variation within and between runs...... 62

Table 7. BLASTn results from the PCR product of synthetic BK-BJC standard (1.02E8) run on

BKBJCV8 qPCR...... 65

Table 8. BKBJCV8 qPCR results (Ct and concentration) for the 21 bacterial sequence groups based on BLAST matches ≥95 % similar to the BKBJCV8 amplicon...... 69

Table 9. BKBJCV8 qPCR positive, non-target oligonucleotide sequence groups and the bacteria they represent...... 71

Table 10. Comparison of universal PCR and BKBJCV8 qPCR assays run on identical groups of

LT...... 76

Table 11. Adjusted p-values from paired comparison of the log-transformed median BK-BJC concentrations on LT gills on each sampling date...... 80

Table 12. Daily percent mortality and BBC amounts in/on the gills of LT on each sampling date during the 2013 BJC EP-associated mortality event...... 82

Table 13. Description and assessment of BKBJCV3 primers based on Oligoanalyzer and Primer-

BLAST software analysis...... 89

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Table 14. BLASTn search of theoretical, ideal BKBJCV3 amplicon with ≥87 % nucleotide identity...... 92

Table 15. BLASTn search of the BKBJCV3 PCR consensus sequence from eight amplicons. .. 94

Table 16. Results of the growth on all agar culture plates at 15 and 22 °C for all BK-BJC positive and negative whole and homogenized gill tissues...... 99

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LIST OF FIGURES

Figure 1. Alignment and target sequences of the BKBJCV8 forward and reverse primers and probe with BK-BJC 16S rRNA gene sequence...... 40

Figure 2. Alignment and target sequences of BKBJCV3 forward and reverse primers with BK-

BJC 16S rRNA gene sequence...... 50

Figure 3A and B. 2 % TBE agarose gel electrophoresis results of standard PCR with BKBJCV8 primers run on BJC lake trout (LT) and various fish species of known and unknown BK-BJC status...... 60

Figure 4. Consensus sequence of forward and reverse primers on BKBJCV8 qPCR product. .... 64

Figure 5. Annotated BKBJCV8 qPCR results of the 21 oligonucleotide sequence groups ...... 68

Figure 6. Single qPCR product from BKBJCV8 qPCR on LT and RT samples on 2 % TBE agarose gel...... 74

Figure 7. Fluctuations in BK-BJC load in LT during the 2013 mortality event at BJC...... 78

Figure 8. Comparison of trends in percent daily mortality and concentration of BBC quantified by BKBJCV8 qPCR from LT gills randomly sampled from tank R8 during the 2013 EP- associated mortality event at BJC...... 83

Figure 9. Stacked comparison of median BBC concentrations (top) and semi-quantitative analysis of histopathologic changes in LT gills (bottom) during the 2013 BJC mortality event.. 85

Figure 10A-D. Histopathologic gill lesions in 2015 BJC LT...... 87

Figure 11. Clustal Omega software alignment of the BK-BJC 16S rRNA gene and three genetically related fish pathogens...... 91

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Figure 12. Alignment of eight sequenced BKBJCV3 amplicons, including LT samples and synthetic BK-BJC...... 93

Figure 13. 2 % TBE agarose gel of BKBJCV3 PCR products from synthetic ‘Ca. B. cysticola’, natural and synthetic BK-BJC, and negative control LT gill tissues...... 96

Figure 14A and B. Bacterial colony from a BK-BJC-positive gill homogenate grown on CA at 22

°C for 11 days...... 100

LIST OF ABBREVIATIONS

Alma Alma Aquaculture Research Station BA blood agar BBC BK-BJC complex: BK-BJC and/or BK-BJC-like bacteria BFCG agar made of blood, fetal bovine serum, cysteine, and glucose BJC Blue Jay Creek Fish Culture Station BK-BJC Blue Jay Creek Burkholderia BKBJCV3 secondary primer set targeting the V3 region of the 16S rRNA gene BKBJCV3F BKBJCV3 forward primer BKBJCV3R BKBJCV3 reverse primer BKBJCV8 initial primer set targetting the V8 region of the 16S rRNA gene BKBJCV8F BKBJCV8 forward primer BKBJCV8R BKBJCV8 reverse primer BLAST basic local alignment tool BLASTn basic local alignment tool- nucleotide bp basepair CA cytophaga agar CLO(s) Chlamydia-like organism(s) CPE cytopathic effect Ct crossing point value d day(s) DI deionized (water) DNA deoxyribonucleic acid EB elementary body EEDV epizootic epitheliotropic disease virus EIBS erythrocytic inclusion body syndrome EP epitheliocystis FFPE formalin-fixed paraffin-embedded FISH fluorescence in situ hybridization FPL fish pathology laboratory h hour(s) HE hematoxylin and eosin IB intermediate body ICC immunocytochemistry IHC immunohistochemistry ISH in situ hybridization LCM laser capture microdisstion LM light microscopy loc bp region or location on the gene LT lake trout MALDI-TOF matrix-assisted laser desorption ionization-time of flight min minute(s) N(s) undetermined nucleic acid(s) OMNR Ontario Ministry of Natural Resources

xvi

PBS phosphate buffered saline PCR polymerase chain reaction PGI proliferative gill inflammation PVK Pierce van der Kamp stain QC query coverage qPCR quantitative real-time PCR (q)PCR PCR and qPCR RB reticulate body rRNA ribosomal ribonucleic acid RLO(s) Rickettsia-like organism(s) RT rainbow trout RT-PCR reverse transcriptase PCR s seconds SG sequence groups SS suspended solids TBE tris-borate TEM transmission electron microscopy TSA trypticase soya agar

xvii

GENERAL INTRODUCTION

Epitheliocystis (EP) is a bacterial infection of the gills and/or, less frequently, the skin. It is aptly named for the characteristic swelling of affected cells by an intracytoplasmic inclusion filled with gram negative bacteria. There is inconsistent evidence as to whether EP is a disease, syndrome, or incidental finding in fish as the prevalence in affected regions and mortality can range from 20-100 % and 0-100 %, respectively (Katharios et al., 2008). Farmed species are the most studied and severely affected (Nowak and LaPatra, 2006); despite this, no association with population density has been determined (Katharios et al., 2008; Methner, Blue Jay Creek Fish

Culture Station manager, personal communication). The postulated etiologic agents originate from three classes for bacteria—Chlamydiae, γ-proteobacteria, and β-proteobacteria. To date, 51-

52 distantly related Chlamydia species, two γ-proteobacteria; and four β-proteobacterium have been posited as the causative agents of epitheliocystis. These bacteria have been identified by combinations of direct and indirect detection techniques, such as transmission electron microscopy (TEM), polymerase chain reaction (PCR), immunohistochemistry (IHC), and/or in situ hybridization (ISH) with varying degrees of success (Bradley et al., 1988; Langdon et al.,

1991; Groff et al., 1996; Crespo et al., 1999; Nowak and Clark, 1999; Draghi et al., 2004, 2007;

Meijer et al., 2006; Nowak and LaPatra, 2006; Karlsen et al., 2008; Toenshoff et al., 2012;

Camus et al., 2013; Mitchell et al., 2013; Seth-Smith et al., 2016). Despite numerous attempts, none of the bacterial agents of EP have been cultured to date; therefore, Koch’s postulates to establish a causal relationship have not been fulfilled. β-proteobacteria Blue Jay Creek

Burkholderia (BK-BJC), a member of the family Burkholderiales, was first discovered in Ontario

Canada in 2013 and has been associated by PCR with two geographically separate annual EP

1 outbreaks and mortality events (up to 42 % mortality) in aquaculture facilities containing lake trout (LT) (Salvelinus namaycush) and rainbow trout (RT) (Oncorhynchus mykiss) (Contador et al., 2016). These EP-associated annual mortality events are significant because remediation of wild LT populations through aquaculture sustains commercial and tourist activities that are a crucial source of income for Ontario (OMNR, 2013).

To date, one study has surveyed the water near fish infected with EP, but the chlamydial agent was not found (Draghi et al., 2010). However, observers of EP-associated mortality events in

Ontario note a correlation with increased suspended solids (SS) in the water column brought about by heavy rains or thaws (Methner, personal communication); this co-relationship between

EP and SS in the water column has been proposed elsewhere in the literature (Corsaro and

Venditti, 2004; Agamy, 2013). Water surveys have not previously screened for BK-BJC, but may be useful as analysis of water and sediment samples for other Burkholderia (E.g.

Burkholderia pseudomallei) (Minogue et al., 2015; Knappik et al., 2015).

The work of Contador et al. (2016) studying of EP outbreaks in Ontario hatchery has contributed to the paradigm shift away from Chlamydiales or Chlamydia-like organisms (CLOs) as the sole cause of EP. The discovery of a naturally occurring, potentially pathogenic Burkholderiales species such as BK-BJC in freshwater fish is a first.

1. LITERATURE REVIEW

1.1. EPITHELIOCYSTIS

Epitheliocystis is a syndrome associated with multiple etiologic agents in which a single gill or skin epithelial cell contains a variably sized vacuole filled with bacteria. The first incidence of this gill and skin disease, then called Mucophilosis cyprinid (or mucophilosis), was recorded in common carp (Cyprinus carpio) in 1920 and was thought to be caused by an alga or fungus

(Plehn, 1924). Several decades later, a similar condition in bluegill sunfish (Lepomis macrochirus) was recognized (Hoffman et al., 1969) and named epitheliocystis. Molnar and

Boros (1981) posited that epitheliocystis and mucophilosis were the same disease caused by

Chlamydia- or Rickettsia-like organisms (CLOs or RLOs). To date, numerous distantly related

Chlamydia species, two γ-proteobacteria named Endozoicimonas elysicola (Mendoza et al.,

2013) and ‘Candidatus Endozoicomonas cretensis’ (Katharios et al., 2015); and four β- proteobacterium ‘Ca. Ichthyocystis hellenicum’ and ‘Ca. I. sparus’ (Seth-Smith et al., 2016),

‘Ca. B. cysticola’ (Toenshoff et al., 2012) have been identified as additional causative agents of epitheliocystis. Confirmation of the association between Blue Jay Creek Burkholderia (BK-BJC) and EP is currently ongoing (Contador et al., 2016).

Epitheliocystis is reported in over 90 species of fish, in both teleosts and chondrichthyes, production and ornamental fish, freshwater and marine species, as well as juveniles and adults

(Stride et al., 2014), though it has been proposed to be most prevalent and severe in farmed fish

(Nowak and LaPatra, 2006). In certain fish species, such as African sharptooth catfish (Clarias gariepinus), striped trumpeter (Latris lineata), yellowtail kingfish (Seriola lalandi), sharpsnout

3 sea bream (Diplodus puntazzo), and winter flounder (Pleuronectes americanus), juveniles are more severely affected than adult conspecifics (Steigen et al., 2013; Stride et al., 2013; Katharios et al., 2008; MacLean, 1993). However, Nowak and Clark (1999) showed no correlation between age and disease prevalence in Atlantic salmon (Salmo salar). There are no documented cases of resistant species, but the infectious agents of epitheliocystis will often affect just one fish species within a body of water (Karlsen et al., 2008).

Epitheliocystis has a world-wide distribution. In affected regions its prevalence ranges from 20-

100 % (Katharios et al., 2008). The associated disease varies from benign, often clinically incidental, to 4-100 % morbidity and mortality (Nowak and LaPatra, 2006). From 2003 to 2005 epitheliocystis was the primary cause of mortalities in the Atlantic salmon in Ireland (Mitchell et al., 2010). The seasonality of the outbreaks also differs by region, but is consistent with respect to location. For example, in Atlantic salmon in Norway and Ireland, outbreaks occur in the spring (April through May); while in Canada in LT they are seen from late fall to winter

(Contador, 2013). However, it is not possible to separate seasonality from coincidental increases in temperature (Steinum et al., 2010), suspended particles, nutrient availability, and/or amoeba populations, which could also be responsible (at least in part) for EP outbreaks (Corsaro and

Venditti, 2004).

Experimental transmission of EP has been observed twice. The first transmission was achieved by exposing healthy bluegill sunfish to gills excised from bluegill sunfish with EP (Hoffman et al., 1969). Transmission was also observed in a second study where naïve RT were cohabitatedwith infected RT (Contador, personal communication). Horizontal transmission from

4 cohoused fish would explain the increased incidence in traditionally high density production systems. However, living in a high density habitat is not sufficient to explain the transmission of disease. As Katharios et al. (2008) demonstrated even in mesocosm-reared sea bream—a low density, high nutrient system—transmission can be high; therefore, transmission is likely multifactorial. Regardless of stocking densities, the infrequency of experimental transmission suggests either that optimization of experimental factors—such as tissue/vector type, density of bacterial inclusions, viability of bacteria in samples, and environmental or microenvironmental

(host) factors are difficult to achieve and/or that the agent(s) of EP are fastidious.

Additionally, it is possible that multiple species within a waterway are infected, but have variable clinical signs. For example, Schmidt-Posthaus et al. (2011) found wild brown trout with subclinical epitheliocystis are known to run by Atlantic salmon net pens. From this finding it is tempting to hypothesize that certain fish species may achieve a carrier state and act as reservoirs for the disease. Interspecific infection trials are needed to validate this theory.

One potential vector is free-living amoeba since 5 % of theses protists either carry or are infected by Chlamydiaceae (Horn, 2008), and Burkholderia spp. have been shown experimentally to live within the phagosome of Acanthamoeba spp. (Lamothe, Thyssen, and Valvano, 2004). Both bacterial families have been shown to cause epitheliocystis. The agents of epitheliocystis will be discussed further below. Nevertheless, it has yet to be determined if amoebae are vectors or contribute to virulence (Corsaro and Greub, 2006). General risk factors for epitheliocystis are host stress, immune compromise, co-infections, and environmental factors (such as

5 aforementioned in reference to seasonality) or man-made factors, such as crude and dispersed oil pollution, which is thought to increase the permeability of the gill epithelium (Agamy, 2013).

1.2. CLINICAL SIGNS AND GROSS FINDINGS

The clinical signs associated with epitheliocystis range from none to lethargy, decreased appetite that progresses to anorexia, swimming at the surface, pronounced opercular movement

(dyspnea), and dark pigmentation of the skin (Steigen et al., 2013). The wide range of clinical signs may be due to the wide variety of bacterial agents of EP, the tissue affected, and/or the different host-pathogen interplay during the various life stages of the bacteria.

Grossly, epitheliocystis is present on the gills, and rarely the pseudobranch and skin, occurring as pale tan to white, nodular to plaque-like, 1 mm regions affecting a few foci up to 80 % of the lamellae (Kim et al., 2005; Nowak and LaPatra, 2006). Concurrent melanization has also been noted in the gills of Atlantic salmon (Nylund et al., 1998). The distribution of the gill lesions is often irregular with some arches and filaments affected and others not. Frequently, there is an increased amount of mucus coating the gills consistent with non-specific irritation. In the vast majority of cases, lesions are restricted to the external surfaces, but there are rare reports of kidney and, less consistently, splenic lesions in bluestripe snapper (Lutjanus kasmira) (Work et al., 2003). In LT (Bradley et al., 1988) and Atlantic salmon (Nylund et al., 1998), presumptively secondary changes, such as swelling of the spleen, various nephropathies, and pale livers with petechia, have also been reported; however, in these cases the fish were co-infected with epizootic epitheliotropic disease (EEDV) and virus-like particles (presumptive erythrocytic inclusion body syndrome (EIBS)), respectively.

1.3. HISTOPATHOLOGIC FINDINGS

Histologic examination of hematoxylin and eosin (HE) stained gill tissue typically reveals variable numbers of individual or clustered lamellar epithelial cells expanded with a single intracytoplasmic inclusion of up to 150 µm composed of a discrete central, irregularly round, basophilic, granular to amorphous accumulation of indistinct bacteria surrounded by a clear halo.

According to Crespo et al. (1999), granular inclusions contain early stages of the pathogen

(infective); while the amorphous inclusions contain the later life stages (reproductive). There are several reports of distinct loose and dense inclusion morphologies, but Meijer et al. (2006) found they contain identical sequences and, therefore, the difference in appearance may constitute an artifactual change (Contador, 2013; Schmidt-Posthaus et al., 2011; Mitchell et al., 2010). Over time the inclusions grow and peripheralize the host cell’s cytoplasm and nucleus. Rarely and presumably in the chronic form, they are encapsulated by a two to three cells thick lining of elongate, spindle-shaped cells (Corsaro and Work, 2012; Crespo et al., 1999; Wolke et al., 1970;

Kumar et al., 2013). In a single report, the inclusion was seen to be invaded by inflammatory cells. Parasitized host cells were noted to dilate up to 400 µm and, in several cases, partially lose contact with the basement membrane (Groff et al., 1996). Epitheliocystis infects a variety of cell types: epithelial, mucus (Paperna and Alves de Matos, 1984), chloride (Crespo et al., 1999;

Paperna et al., 1981), pillar (Frances and Nowak, 1997), pavement (Lai et al., 2013), epithelial stem cells (Mitchell et al., 2010), and macrophages (Frances and Nowak, 1997). Often the cellular architecture is obscured by cytoplasmic compression or degeneration indicated by swelling and vacuolation of organelles, accumulation of fibrillar and hyaline deposits, or accumulation of tubules or mitochondria mimicking chloride cell cytoplasm (Paperna et al.,

1981; Groff et al., 1996; Nowak and LaPatra, 2006). The colonization of mitochondria-rich host

7 epithelial cells, such as chloride cells, may have evolved to take advantage of the energy rich microenvironment (Nowak and LaPatra, 2006).

As grossly noted, the distribution of epitheliocystis is random across the arch, but where present, the inclusions cluster (Karlsen et al., 2008). This has suggested to some researchers that the initial route of infection is via the water and then the spread is local (Karlsen et al., 2008).

Depending on the affected species or geographic location of the infection, there seems to be a site-specific predilection for different types of EP inclusions—such as the interlamellar spaces, middle of the filament, base of the secondary lamellae, or tBKBJCV8 of the primary lamellae

(Zachary and Paperna, 1977; Nowak and Clark 1999; Steigen et al., 2013; Nylund et al., 1998, respectively). Hyperinfections, consisting of hundreds of inclusions, have been seen in largemouth (Micropterus salmoides) and sea bass (Dicentrarchus labrax), sea bream, amberjack

(Seriola dumerili Risso), spotted eagle rays (Aetobatus narinari), yellowtail (Seriola mazatlana), and potentially in the first report of the disease in common carp (Nowak and LaPatra, 2006;

Camus et al., 2013; Molnar and Boros, 1981).

1.3.1. HISTOLOGY - SPECIAL STAINS

In addition to HE, numerous special stains have been applied to gills infected with epitheliocystis

(Table 1).

Table 1. A brief list of special stains used to diagnose EP throughout the literature.

Special stains References Hoffman et al., 1969; Wolke, Wyand, and Khairallah, 1970; Szakolczai, Vetési, and Pitz, 1999; Work et al., 2003; Giemsa Katharios et al., 2008; Katharios et al., 2015; Contador et al., 2016 Corsaro and Work, 2012; Groff et al., 1996; Draghi et al., Gimenez 2007 and 2010; Work et al., 2003 Pierce van der Kamp (PVK) Contador et al., 2016; LePage et al., 2015 Gram Most Crespo et al., 1999; Wolke, Wyand, and Khairallah, 1970; Macchiavello Groff et al., 1996; Draghi et al., 2007; Chang et al., 2016 Heidenhain's AZAN trichrome Crespo et al., 1999 Crespo et al., 1999; Wolke, Wyand, and Khairallah, 1980; Periodic acid–Schiff (PAS) Groff et al., 1996; Szakolczai, Vetési, and Pitz, 1999 Grocott's Methenamine Silver Wolke, Wyand, and Khairallah, 1970 Methyl green-pryonin Wolke, Wyand, and Khairallah, 1970 Ziehl-Neelsen Szakolczai, Vetési, and Pitz, 1999 Mallory's aniline blue collagen Hoffman et al., 1969

Gram, giemsa, gimenez, macchiavello, and PAS stains are the most commonly used stains for the diagnosis of EP and have fairly consistent results. The intracellular bacteria associated with

EP consistently stain gram and PAS negative; 4 out of 5 cases stain fuschin positive by giemsa, excluding inclusions in LT (Contador, 2013); 3 out of 4 stain positive by gimenez, excluding white sturgeon (Groff, 1996); and 4 out of 5 cases stain positive by macchiavello. In one case,

Crespo et al. (1999) found that the amorphous inclusions stain positive and the granular inclusions were negative. These results suggest in the majority of cases RLOs or CLOs are within the inclusion, but there is variation in the etiologic agent.

1.4. DISEASE AND RELATIONSHIP WITH HOST DEATH

Irrespective of the size, number, or distribution of the inclusions, the host reaction is variable putting into question the relevance of epitheliocystis as a disease process. In juvenile sea bream and cobia (Rachycentrum canadum), intraepithelial inclusions are found within the oral cavity and esophagus, and lead to anorexia and death in the sea bream (Mendoza et al., 2013; Katharios et al., 2008; Crespo et al., 1999). However, no other cases have such a neat pairing of lesions and clinical signs. The causal relationship is complicated by several observations: 1) the majority of the time there is no host reaction (as indicated by inflammation and gill histopathologic lesions);

2) when present reactive foci are often not near the inclusions; and 3) one host may react to only a portion of the inclusions. The closest demonstration of a causal relationship is transmission from co-habitated epitheliocystis-positive fish to naïve fish resulting in signs of hypoxemia and inappetence (Hoffman et al., 1969). One definitive indicator of host systemic effects or immunologic recognition of epitheliocystis is the significant increase in serum osmolality and lysozyme activity in Atlantic salmon with epitheliocystis (10 and 40 %, respectively) (Lai et al.,

2013). The alteration in serum osmolality is postulated to be due to a decreased number of chloride cells at the base of the gill filament near EP inclusions; however, this change is not seen in interlamellar areas (Lai et al., 2013). Additionally, it is not known whether these fish also had classic gill lesions, systemic manifestation of epitheliocystis and/or co-morbidities, or if other species would have had a similar reaction.

When present, gill inflammation reflects both the innate and adaptive immune responses with lymphocytes, macrophages, and/or eosinophilic granular cells. The classic gill lesions are single cell hypertrophy with an intracytoplasmic inclusion, interlamellar hyperplasia to lamellar fusion, goblet cell hyperplasia, and occasionally pressure necrosis of adjacent cells (Camus et al., 2013).

These gill changes also constitute four of five characteristic lesions of ‘proliferative gill inflammation’ (PGI) in salmonids with the exception of consistent vascular disturbances, such as filament hemorrhage and thrombosis (Steinum et al., 2009; 2010). However, there are a few reports of lamellar aneurysms, thrombi, and congestion associated with epitheliocystis (Draghi et al., 2007; Contador, 2013). PGI is a multifactorial disease and has been correlated with several

CLOs, microsporidians (Desmozoon lepeophtherii and Loma spp.), a presumptive poxvirus,

Atlantic salmon paramyxovirus, ciliated protozoa (Trichodina and Ichthyobodo spp.), and elevated water temperature (Matthews et al., 2009; Steinum et al., 2009; 2010). A definitive connection between epitheliocystis and PGI has yet to be made.

If and how epitheliocystis causes disease in the host is unknown, but may be ascribed to: 1) mechanical injury to the hypertrophic, colonized epithelial cells; 2) increased diffusion distances for oxygen exchange; and/or 3) induced dysfunction (Katharios et al., 2008; Mitchell et al.,

2010). The last theory is supported by the finding that mammalian Chlamydia, which can cause ciliary stasis in bronchial epithelial cells, promotes infection (Shemer-Avni and Lieberman,

1995). The importance of damage to the gill should not be underestimated since it is not only the site of osmoregulation and respiration, but also a physical and immunologic barrier to pathogens with the intersection of innate and adaptive immune systems and mucus containing antimicrobial peptides, complement factors, and immunoglobulins (Rebl et al., 2014; Magnadottir, 2010).

However, until the causative agent is cultured and Koch’s postulates fulfilled, the link between epitheliocystis and clinical disease is only correlational.

1.5. CO-INFECTIONS

Another complicating variable is that fish with epitheliocystis are often co-infected with a variety of viral, bacterial, and ectoparasitic pathogens (Table 2).

Table 2. Reported comorbidities in fish with epitheliocystis.

Co-infected with Fish Species References Myxozoans- A) Malacosporean A) Brown trout; A) Schmidt-Posthaus et al., Tetracapsuloides bryosalmonae; B) B) Striped 2011; B) Stride et al., 2013; Myxosporeans Kudoa neurophila, K. trumpter; Spotted Camus et al., 2013; Work et hemiscylli; unspecified. eagle rays; Taape al., 2003 Chondracanthid copepods Striped trumpter Stride et al., 2013 Camus et al., 2013; Spotted eagle rays; Mitchell et al., 2010; Atlantic Salmon; Steigen et al., 2013, Protozoans - Peritrichs; Trichophyra Catfish, Atlantic Steinum et al., 2010, sp.; Trichodina sp.; Ichthyobodo sp.; salmon, Pacu; Nylund et al., 1998, unspecified coccidia Catfish; Lake Szakolczai et al., 1999; Parasites trout; Taape Contador, 2013; Work et al., 2003 Sea bream, Padros and Crespo, 1995, Connecticut Wolke et al., 1970, striped bass, Polkinghorne et al., 2010, Monogeans - unspecified; Benedenia Leopard shark, Szakolczai et al., 1999, seriolae; Zeuxapta seriolae Pacu, Taape; Work et al., 2003; Stride et Yellowtail al., 2013; Montero et al., kingfish; 2004 Amberjack Microsporidian – Loma sp.; Steinum et al., 2010, Atlantic Salmon; Desmozoon lepeophtherii; Matthews et al., 2009; Taape unspecified Work et al., 2003 Steinum et al., 2010, Poxvirus Atlantic Salmon Karlsen et al., 2008 Steinum et al., 2010; Paramyxovirus Atlantic Salmon Kvellestad et al., 2003 Epizootic epitheliotropic disease virus Lake trout Bradley et al., 1988 Myxovirus-like virus Bluegill Wolke et al., 1970 Virus-like particles in epithelium, RBCs, and macrophages Atlantic Salmon Nylund et al., 1998 Viruses (presumptive EIBS)

Reovirus (Heart and skeletal muscle Atlantic Salmon Palacios et al., 2010 inflammation) Virus-like particles in epitheliocystis Valdron, Cone, and Cusac , Stickleback inclusions 1994 Lymphocystis Barramundi Mei er et al., 2006 Stickleback; Valdron, Cone, and Cusac , Bacteriophages Pacific white 1994; Jimenez et al., 2001 shrimp

The most intriguing comorbidities include: 1) virions were observed via TEM within inclusions and in the cell wall of Chlamydial organisms in penaeoid shrimp (Litopenaeus vannamei)

(Jimenez et al., 2001); and 2) chlamydial DNA that was co-localized via ISH to a site of lymphocystis, an iridovirus, infection in barramundi (Lates calcarifer) (Meijer et al., 2006). In addition, CLOs and shrimp cuticular epithelial necrosis virus have a reciprocal symbiosis in which the bacteria colonizes regions of virally induced necrosis, and the virus lives within the intracytoplasmic bacterial inclusion and in close association with the CLO’s cell wall (Jimenez et al., 2001).

1.6. ADVANCED DIAGNOSTICS

1.6.1. TRANSMISSION ELECTRON MICROSCOPY

Transmission electron microscopy has been used since the discovery of EP to characterize the agents of EP and the alterations to the host cell. TEM of the host cell shows a variably sized intracytoplasmic inclusion causing compression, displacement, and degeneration of the cytoplasm and nucleus. Lining of the inclusion is variably described as an eosinophilic hyaline membrane or a trilaminar membrane with a luminal surface that is occasionally adorned with 5

µm budding projections (Bradley et al., 1988; Groff et al., 1996). The inclusions of cultured pacu

(Piaractus mesopotamicus) do not have an inclusion membrane (Szakolczai et al., 1999); this may be artifactual from autolysis, poor fixation, or a degenerative change, or may point towards an RLO as the causative agent (versus CLOs which are enclosed in membranes) (Zachary and

Paperna, 1977). Although environmental shedding of the inclusion contents is likely the method of transmission, inclusions have only been captured twice in the act of rupturing microscopically;

14 and in one of these reports, the author states that the tissue was not adequately fixed (Szakolczai et al., 1999; Rourke et al., 1984).

The ultrastructure of the bacteria within the inclusions most often resembles CLOs (a a “novel

Chlamydiales”), or RLOs with Chlamydia-like developmental cycles (E.g. Ehrlichia,

Neorickettsia, Wolbachia, and Cowdria) (Crespo et al., 1999; Nylund et al., 1998). Briefly,

Chlamydia are pleomorphic, gram negative, obligate intracellular bacteria with a unique triphasic development. In the primary developmental cycle, the bacteria have three transitional phenotypes: elementary bodies (EBs), intermediate bodies (IBs), and reticulate bodies (RBs).

EBs are the infective stage, and RBs the metabolic and reproductive stage replicating by binary fission. Within a given inclusion or individual, the three forms are variably present which is thought to be due to the inclusion’s stage of maturation. Rarely, unique morphologies are also seen, such as head and tail cells in salmonids and crescent bodies in amoeba (Rourke et al., 1984;

Bradley et al., 1988; Draghi et al., 2004; Karlsen et al., 2008; Corsaro and Greub, 2006). It is not currently known whether these cells constitute an additional phenotype characteristic of certain

CLOs, or aberrant development of one of the more common bodies. Interestingly, recent work has shown that the distribution of the different bodies within the inclusion can indicate that different families of Chlamydiaceae are present. Namely, if the RBs are distributed throughout the inclusion this is attributed to ‘Candidatus Pisichlamydia salmonis’ (‘Ca. P. salmonis’), but if the RBs are at the periphery then it is more consistent with ‘Candidatus Clavochlamydia salmonicola’ (‘Ca. C. salmonicola’) (Schmidt-Posthaus et al., 2011). The Rickettsia-like developmental cycle is less frequently seen and has three stages: primary-long, intermediate- long, and small cells with or without vacuolation (Nowak and LaPatra, 2006). With no less than

15 six different phenotypes and variable host responses, microscopic identification of the bacterium is understandably challenging.

1.6.2. BACTERIAL CULTURE

Numerous unsuccessful attempts at culturing the organisms have been attempted to characterize the bacterial pathogens associated with EP and fulfill Koch’s postulates. The closest to growing an agent of EP is the culture of E. elysicola (strain MKT110T) from Japanese marine sea slugs on marine agar at 25 °C (Kurahashi and Yokota, 2007). This E. elysicola is 99 % genetically similar to the uncultured E. elysicola that causes EP in South American cobia larvae (Mendoza et al.,

2013). However, E. elysicola (strain MKT110T) and other member of the genus Endozoicomonas have been identified as symbionts of marine invertebrates; therefore, may have a different life- style from the fish pathogenic E. elysicola and, hence, would have different propensities to grow in culture. Further research is needed to determine if E. elysicola from cobia can be grown on marine agar or in cell culture. The inability to isolate and grow EP causing bacteria prevents recapitulating the disease through infection trials, and, therefore, prevents establishing the causal relationship between infection with epitheliocystis agents, development of disease, and death.

1.6.2.1 CELL CULTURE

The first report of attempted transmission of EP from infected fish material to fish cell lines was by Hoffman et al. (1969) who inoculated bluegill fry (BF-2), rainbow trout gonad (RTG-2) fibroblast, and fathead minnow (Pimephales promelas) (FHM) epitheliod cell lines with gritty white material from the caudal fin of a fish with EP. BF-2 and RTG-2 underwent fusiform cytopathic changes, but thin sections of the altered cells showed virions and not bacteria within

16 the cytoplasmic vacuoles (Hoffman et al., 1969). Therefore, transmission of the agent of EP remained elusive. Later, Kvellestad, Dannevig, and Falk (2003) attempted to grow the causative agent of EP from Atlantic salmon in RTgill cells and cytopathic effects (CPE) were seen in the monolayer. However, similar to Hoffman et al. (1969), instead of bacteria, a novel paramyxovirus, Atlantic salmon paramyxovirus, was found (Kvellestad, Dannevig, and Falk,

2003).

A later study demonstrated that three Chlamydia-like organisms—Waddlia chondrophila,

Estrella lausannensis, and Parachlamydia acanthamoebae—could be grown in Acanthamoeba castellanii [strain ATCC 30010] and then transmitted to fish cell culture lines epithelioma papulosum cyprini (EPC-175) from fathead minnow and RTG-2 producing intracellular inclusions and CPE (Kebbi-Beghdadi et al., 2011). This showed the ability of some Chlamydial organisms to grow in fish cell lines; however, none of these agents have been associated with EP.

Additionally, the different CLOs had greater or lesser affinities for the different cell lines

(Kebbi-Beghdadi et al., 2011) suggesting that non-ideal pairing due to species specificity or cellular tropism, for example, may be the cause of the inability to culture EP-associated bacteria.

Transmission of grass carp (Ctenopharyngodon idella) gill homogenate with ‘Candidatus

Piscichlamydia cyprinis’ to EPC-57, FHM-57, and common carp brain (CCβ816) cell lines was unsuccessfully attempted by Kumar et al. (2013). Most recently, ‘Ca. Ichthyocystis hellenicum’ and ‘Ca. I. sparus’ were unable to form an infection when inoculated onto EPC cells or

Acanthamoeba castellanii (ATCC 30010) (Seth-Smith et al., 2016).

1.6.2.2. AMOEBA CULTURE

Since Chlamydia are known to infect and/or be commensals of amoeba, like Parachlamydia acanthamoebae in Acanthamoeba spp., and Neochlamydia hartmanellae in Hartmannella vermiformis (Corsaro and Greub, 2006; Draghi et al., 2007), co-culturing is a promising diagnostic tool. In fact, Draghi et al. (2004) found that “Ca. P. salmonis” has the highest sequence similarity to two chlamydial endosymbionts of acanthamoeba (82 and 81 %).

Transmission of Chlamydia from amoeba to vertebrate hosts is documented in cases of human and feline ocular disease, as well as infections of herpetofauna (amphibians and reptiles)

(Corsaro and Venditti, 2004). Like Chlamydiales, the mammalian Burkholderia species are able to survive in free-living amoebae, Acanthamoeba spp., which is postulated to improve environmental survival and/or play a role in bacterial virulence (Inglis et al., 2000; Lamothe,

Thyssen, and Valvano, 2004; Wiersinga et al., 2006).

1.6.2.3. AGAR CULTURE

Like members of the order chlamydiales, certain Burkholderiales are difficult to culture; for example, Burkholderia cepacia and B. pseudomallei are known to enter a viable but nonculturable states (Oliver, 2010). However, likely due to the severity of disease, methods for culturing mammalian pathogenic Burkholderia—such as B. cepacia, B. mallei, and B. pseudomallei, are well established. Burkholderia spp. may be free-living, symbiotic, commensal, or intracellular pathogens (Olapade et al., 2005). They can be directly cultured on B. cepacia selective agar (B. cepacia), Ashdown’s agar, tryptic (trypticase) soy agar (TSA) (B. cepacia),

MacConkey, and blood agar (B. mallei/pseudomallei); co-cultured with Acanthamoeba polyphaga (Lamothe, Thyssen, and Valvano, 2004); and grown in cell culture (Brown and

Govan, 2007; Maravic et al., 2012; Hagen et al., 2011; Peacock et al., 2005). Attempts to grow the Burkholderiales BK-BJC from infected gills on sheep blood agar (BFCG), blood agar (BA), and TSA plates incubated at 10 and 21 °C have been unsuccessful (Contador, 2013). To date none of the agents of EP have grown in culture.

1.6.3. POLYMERASE CHAIN REACTION

The breakthrough in identifying the bacterial agents of epitheliocystis came through PCR assays using universal 16S and 23S rRNA and Chlamydiales-specific primers (16SIGF, 16SIGR,

806R), together with IHC and ISH. These direct and indirect detection techniques not only confirm the presence of a suspect bacterium within tissue in epitheliocystis, but can also be used to demonstrate co-localization of different agents within the intracellular bacterial inclusions.

There are inconsistencies in the literature, however, concerning the types of diagnostic tests used; most studies rely solely on indirect detection techniques such as PCR and sequencing, and non-specific techniques such as TEM. This does not fulfill Fredric s and Relman’s postulates of causation (or Koch’s molecular postulates) (Fredric and Relman, 1996). The characterization of

‘Ca. P. salmonis’ in Atlantic salmon is the best example of utilizing all available techniques to rigorously demonstrate the causal relationship between an agent of epitheliocystis and the EP inclusions (Draghi et al., 2004; Schmidt-Posthaus et al., 2011).

Identified using PCR assays, the following distantly-related Chlamydia have been discovered in relation to epitheliocystis: ‘Candidatus Actinochlamydia clariae’ (Steigen et al., 2013),

‘Candidatus Parilichlamydia carangidicola’ (Stride et al., 2013), Neochlamydia spp. (Draghi et al., 2007), ‘Candidatus Clavochlamydia salmonicola’ (Karlsen et al., 2008), ‘Candidatus

Piscichlamydia salmonis’ (Draghi et al., 2004), ‘Candidatus Renichlamydia lut ani’ (Corsaro and

Wor , 2012), ‘Candidatus Syngnamydia venezia’ (Fehr et al., 2013), and ‘Candidatus

Similichlamydia latridicola’ (Stride et al., 2013), ‘Ca. S. labri’ (Steigen et al., 2015), and unspeciated ‘Ca. S. sp.’ (Guevara Soto et al., 2016). These Chlamydiaceae and CLOs have been placed within the order Chlamydiales based on the percent nucleotide sequence similarity.

Organisms with >97 % nucleotide similarity are considered the same species, 95-97 % are within the same genus, 90-95 % are within the same family, and 80-90 % are a different family within the Chlamydiales order (Everett et al., 1999). CLOs have 80-90 % sequence similarity to

Chlamydiaceae, are gram negative, intracellular bacteria with the same biphasic developmental cycles (Everett et al., 1999; Stride et al., 2013). Compared with histology to detect EP, PCR is significantly more sensitive (Stride et al., 2013). Reverse transcriptase PCR (RT-PCR) can be used to demonstrate not only the presence, but also the viability of EP causing bacteria (Lleò et al., 2000); however, only a few researchers have employed this method (i.e., Mitchell et al.

(2010) and Steinum et al. (2009)). The challenges of PCR on gills with epitheliocystis are: 1) the uneven distribution of EP inclusions, 2) inclusions composed predominantly of EBs which have less DNA (Steinum et al., 2009); and 3) an excess of non-target bacteria (> 106 cells) (Hiney and

Smith, 1998). Therefore, histopathology, PCR with primers designed for specific EP agents, and direct forms of bacterial identification—ISH and IHC—are necessary for sensitive and accurate detection of the agents of EP.

1.6.4. In situ HYBRIDIZATION

ISH and fluorescence ISH (FISH) using PCR amplified, specific poly- or oligoprobes have been used to detect and localize in tissue bacterial sequences from E. elysicola, ‘Ca. I. hellenicum’

20 and ‘Ca. I. sparus’, and various Chlamydiales, including ‘Ca. P. salmonis’, ‘Ca. B. cysticola’, and Chlamydia-like organisms, (Draghi et al., 2004 and 2007; Meijer et al., 2006; Karlsen et al.,

2008; Toenshoff et al., 2012; Mendoza et al., 2013; Mitchell et al., 2013; Stride et al., 2013;

Seth-Smith et al., 2016). In numerous studies, ISH has clearly identified the PCR recognized bacteria within EP inclusions strongly demonstrating the presense of an agent (Draghi et al.,

2004 and 2007; Karlsen et al., 2008; Toenshoff et al., 2012; Mendoza et al., 2013; Mitchell et al.,

2013). ISH may resolve the EP associated agent when more than one is found via PCR—as is often the case when universal and Chlamydiales-specific primers are used. In studies by

Toenshoff et al. (2012), Atlantic salmon with EP were PCR positive for both ‘Ca. B. cysticola’ and ‘Ca. P. salmonis’, but the inclusions were only ISH positive for ‘Ca. B. cysticola’. This demonstrated that the causative agent was ‘Ca. B. cysticola’, and ‘Ca. P. salmonis’ was incidentally identified within the gill tissue external to the cyst (Toenshoff et al., 2012). ISH also demonstrates the distribution of the bacterial agent throughout the gill tissues; for example, E. elysiocola was localized within and occasionally living free adjacent to EP inclusions (Mendoza et al., 2013). Another advantage of ISH is that like PCR, it can detect infections before histologically apparent inclusions form (Karlsen et al., 2008).

However, several studies have noted problems associated with ISH. First, a retrospective study of leafy sea dragons (Phycodurus eques), silver perch (Bidyanus bidyanus), and barramundi

(Lates calcarifer) found that though all were positive for PCR using primers specific for the

Chlamydiales 16S rRNA gene, they were variably positive for ISH using a Chlamydiales 16S rRNA specific oligoprobe (Meijer et al., 2006). The silver perch and barramundi were positive, but the leafy sea dragon was negative; this was attributed to the age of the sample—8-years old

(Mei er et al., 2006). Second, Karlsen et al. (2008) found that their specific probes for ‘Ca. C. salmonicola’ wea ly cross-reacted with ‘Ca. P. salmonis’ potentially due to conserved secondary structures in the 16S rRNA gene. The incongruity between PCR-detected bacterial sequences and ISH localization of the sequences to EP inclusions may be real, or may indicate: 1) blockage of the probe by secondary or tertiary structures; 2) below detection levels of bacterial 16S rRNA or rDNA; 3) dormant or metabolically inactive bacteria; or 4) sampling error due to paucity or irregular distribution of inclusions (Karlsen et al., 2008; Mitchell et al., 2013).

1.6.5. IMMUNOHISTOCHEMISTRY

Throughout the literature, there are reports that IHC inconsistently labels inclusions, but whether problems are due to technique and/or the diversity of the causative bacterium is currently unknown. For example, Camus et al. (2013) found that IHC with a polyclonal anti-chlamydial antibody (20-CR19) strongly labeled the gill inclusions in hyperinfected spotted eagle ray while the monoclonal (ACI) was negative. Since polyclonal antibodies can have lower specificity than monoclonals, this result may constitute a false positive (Camus et al., 2013). The aforementioned study by Meijer et al. (2006) demonstrates the inconsistency of IHC in that all of three fish tested are Chlamydiales-specific PCR positive and only one is IHC positive by both Chlamydiaceae specific anti-lipopolysaccharide (LPS) and Chlamydophila pneumoniae anti-membrane protein monoclonal antibodies. IHC based on Chlamydiaceae-specific or Chlamydia-genus LPS antibodies (e.g., 11B5) may be sporadically positive due to a lack of technical precision, diversity of the EP pathogen, multi-pathogen co-infections, large colony size (which stain less), or a late developmental stage where the inclusions mostly contain EBs which have less 16S rRNA (Nowak and LaPatra, 2006; Camus et al., 2013; Meijer et al., 2006; Groff et al., 1996;

Crespo et al., 1999). Occasionally, occasionally Chlamydiaceae-specific LPS antibody will label non-Chlamydial, resident membrane bound organelles in the cytoplasm of epithelial cells potentially based on cross-reactivity of trisaccharide-like molecules (Draghi et al., 2004).

Chlaymdia genus specific anti-LPS monoclonal antibodies were negative in LT and leafy sea dragons, but positive in white sturgeon (Bradley et al., 1988; Langdon et al., 1991; Groff et al.,

1996; Nowak and Clark, 1999). This likely demonstrates the diagnostic complexity of investigating a disease like EP that is caused by a number of distantly related bacteria.

The inconsistencies between PCR, and IHC or ISH suggest that Chlamydia may be an opportunistic commensal, or a co- or secondary pathogen that only sometimes forms EP inclusions. Chlamydia are not often cited as commensal gill bacteria. Steinum et al. (2009) found that gill bacterial populations are predominantly composed of six to seven taxa with one to two dominant ones. In marine and freshwater Atlantic salmon the dominant two are Burkholderia- like bacteria and Psychrobacter spp. (Family: Moraxellaceae) (Steinum et al., 2009). To a lesser extent, bacteria from the genera Cytophaga, Flavobacterium, Photobacterium, Aliivibrio,

Shewanella, Tenacibaculum, Pseudomonas, and rarely Francisella are present (Steinum et al.,

2009). On the other hand, though Draghi et al. (2004 and 2010) found ‘Ca. P. salmonis’ in EP inclusions in Atlantic salmon and Arctic charr (Salvelinus alpinus), Toenshoff et al. (2012) found

‘Ca. P. salmonis’ within gill tissues of Atlantic salmon, but not in association with any inclusions. The role of those scattered single or clustered, few bacteria was not evident.

However, this finding may be due to the different probe design (i.e. length and type of nucleic acid) and probe targets used in the two studies. Draghi et al. (2004 and 2010) used a 1.5 kb riboprobe targeting the 16S rRNA gene (DNA); whereas, Toenshoff et al (2012) used an 18 bp

23 oligonucleotide probe targeting the rRNA itself. Never-the-less, direct methods of bacterial detection reveal the complete picture about the relationship of PCR identified bacteria and EP inclusions; and, therefore, are necessary in conjunction to PCR to confirm a causative agent.

1.7. BACTERIAL PATHOGENS

1.7.1. CHLAMYDIAE

In the majority of cases, epitheliocystis is associated with a distantly-related cohort of

Chlamydiaceae families or novel CLOs. Of the 11 Chlamydiales families (including the

Candidatus families), seven have been linked to EP in fish—Actinochlamydiaceae (Steigen et al., 2013); Clavichlamydiaceae (Karlsen et al., 2008; Schmidt-Postahaus et al., 2012; Stride et al., 2014); Piscichlamydiaceae (Draghi et al., 2004; Nowak and LaPatra 2006; Kumar et al.,

2012; Camus et al., 2013); Parachlamydiaceae (Meijer et al., 2006; (Draghi et al., 2007);

Parilichlamydiaceae (Stride et al. 2013; Steigen et al., 2015); Simkaniaceae (Fehr et al. 2013;

Nylund et al., 2015; Steigen et al., 2013); and Rhabdochlamydiaceae (Meijer et al., 2006;

Corsaro and Work, 2012) (Pawlikowska-Warych and Deptuła, 2016).

The disparate evolutionary relationship between these bacteria may account for the variation in presentations of epitheliocystis from clinical signs to outcome of diagnostic techniques. For example, Atlantic salmon gill inclusions that contain ‘Ca. P. salmonis’ show local inflammation and lamellar hyperplasia; whereas, those containing ‘Ca. C. salmonicola’ do not (Draghi et al.,

2004; Schmidt-Posthaus et al., 2011). Additionally, the diversity of pathogenic Chlamydia has led some authors to speculate that epitheliocystis may be species-specific. If true, this might

24 explain why outbreaks of epitheliocystis often affect only one species in a waterway (Karlsen et al., 2008). However, ‘Ca. P. salmonis’ parasitizes the gills of Atlantic salmon, Artic charr, and brown trout (Salmo trutta) (Langdon et al., 1991). Additionally, one species of fish may be colonized with two different Chlamydiales as in Arctic charr and Altantic salmon (Draghi et al.,

2004, 2007, and 2010; Kalsen et al., 2008), or two different EP-causing agents as in Atlantic salmon with ‘Ca. Piscichlamydia salmonis’ and ‘Ca. B. cysticola’ (Draghi et al., 2004;

Toenshoff et al., 2012). Furthermore, a sequence specific riboprobe for Neochlamydia sp. found

Neochlamydia-like bacteria within EP inclusions that were >90 % similar to a Neochlamydia sp. that causes ocular disease in cats (Draghi et al., 2007). Also, the Simkania Chlamydia strain,

CRG20, which infects leafy sea dragons has high homology with the isopod endosymbiont

‘Candidatus Rhabdochlamydia porcellionis’ (Corsaro and Venditti, 2004). This demonstrates the ability of closely related Chlamydia to not only infect various fish species, but also mammals and crustaceans. Though incidences of zoonosis have not been documented, some Chlamydiales have this potential (e.g., psittacosis passes from birds to humans). Lastly, cell type specificity

(tropism) may be responsible for the varied host range and disease presentations. Cell specificity has not been extensively researched, likely due to the inclusions obscuring the identity of the infected cells.

Chlamydia-induced epitheliocystis remained the paradigm until 2007 when PCR with 16S rRNA universal primers and ISH discovered novel bacterial orders causing similar intraepithelial inclusions. These were: 1) ‘Ca. B. cysticola’ (Toenshoff et al., 2012); 2) BK-BJC (Contador et al., 2016); 3) Endozoicimonas elysicola (Mendoza et al., 2013); 4) ‘Candidatus Endozoicomonas

25 cretensis’ (Katharios et al., 2015), and 5 and 6) ‘Ca. Ichthyocystis hellenicum’ and ‘Ca. I. sparus’ (Seth-Smith et al., 2016).

1.7.2. BURKHOLDERIALES

1.7.2.1. ‘CANDIDATUS BRANCHIOMONAS CYSTICOLA’

In 2007, through the use of 16S universal PCR and FISH with both specific BraCy-129 and generic bacterial probes, gill inclusions of Norwegian and Irish Atlantic salmon were found to contain ‘Ca. B. cysticola’ (Toenshoff et al., 2012). ‘Ca. B. cysticola’ is a ß-proteobacterium belonging to the order Burkholderiales and is a pleomorphic, gram negative bacterium. It exhibits ultrastructural similarities to CLOs, namely IBs and RBs but not EBs (Toenshoff et al.,

2012); further, Burkholderia cenocepacia in pure culture have intermediate body-like morphologies as noted in Chiu et al. (2001). Interestingly, both B. cenocepacia and ‘Ca. B. cysticola’, found in infected cystic fibrosis (CF) patients, target epithelial cells that specialize in, or have abnormal, chloride regulation (Sajjan et al., 2006).

Toenshoff et al. (2012) noted the lac of EB forms in ‘Ca. B. cysticola’; however, EBs are not always seen in Chlamydia-containing inclusions, as is the case for ‘Ca. P. salmonis’ (Draghi et al., 2004). A lack of EBs may also indicate an immature EP inclusion. In studies by Toenshoff et al. (2012) ‘Ca. P. salmonis’ was identified in Norwegian and Irish Atlantic salmon by PCR, but it was not detected within inclusions by FISH (Toenshoff et al., 2012). This again demonstrates the importance of pairing histomorphology, PCR, and ISH to identify the causative agent. ‘Ca.

B. cysticola’ was identified as a gill commensal in Atlantic salmon (Steinum et al., 2009), and,

26 hence, may be an opportunistic pathogen responding to unknown local factors. How the gill microbiome is maintained is not well understood (Rebl et al., 2014). However, unlike

Chlamydia, there is a direct positive association between gill lesions and bacterial loads potentially suggesting a load-dependent pathogenicity (Toenshoff et al., 2012). Other members of the order Burkholderiales are facultative intracellular pathogens that may latently persist for years within an individual (Gan, 2005). Additionally, upon entering the cell, B. cepacia complex bacteria disrupt the glycocalyx and re-arrange the actin cytoskeleton (Saldias and Valvano,

2009); this behaviour may be homologous to the intracytoplasmic inclusion formation associated with most agents of epitheliocystis.

1.7.2.2. BLUE JAY CREEK BURKHOLDERIA

Recently, an additional novel Burkholderiales species, BK-BJC, was identified as a potential agent of yearly outbreaks of epitheliocystis in Ontario cultured LT and RT in two separate facilities: the Blue Jay Creek Fish Culture Station (BJC) and the Alma Aquaculture Research

Station (Alma), respectively (Contador et al., 2016). Using universal 16S PCR (primers U1,

U1R) and sequencing, BK-BJC was first identified on the whole gills of 56 (of 87) Ontario reared LT during an EP-associated mortality event at BJC in 2012-13 (Contador et al., 2016)

(Appendix 1). Universal 16S rRNA gene PCR coupled with sequencing was also used to confirm that BK-BJC was present in Alma RT during similar EP-associated mortality events (Contador,

2013). BK-BJC was further localized to intracellular inclusions within gill epithelial cells through laser capture microdissection (LCM) on historic formalin-fixed paraffin embedded

(FFPE) LT gills from BJC from 2006-2009 and subsequent targeted DNA extraction and PCR

(Contador et al., 2016). BK-BJC has 87 % nucleotide identity with the bacterial sequence

27 amplified using LCM and PCR; however, the author explained that this may indicate tissue degradation, formalin interference, or, as in Chlamydiales, different closely related, etiologic

Burkholderiales causing EP (Contador, 2013). These indirect methods of detection suggest an association between BK-BJC and EP in Ontario cultured LT and RT; however, direct diagnostic techniques have either failed to identify, or have not yet been developed for this novel bacterium to substantiate a relationship. BK-BJC has 92 % nucleotide identity with ‘Ca. B. cysticola’ clone

GABI and strain A1, and an unnamed β-proteobacterium, JN807444.1, found in an Atlantic salmon (Salmo salar) with proliferative gill disease. This percent similarity may place BK-BJC and ‘Ca. B. cysticola’ in the same unidentified family; however, bacterial strains within the same family can vary widely from 81.0-96.0 % similarity in non-chlamydial spp. (Kim et al., 2014).

In addition, Contador et al., (2016) reported that 10 different Chlamydiales were amplified by

Chlamydia-specific PCR, but the sequencing results were inconclusive since: 1) the PCR products were short (<300 bp); 2) the PCR results were not repeatable using more precise LCM and PCR; and 3) the colonies were IHC (using monoclonal antibodies against C. trachomatis), giemsa, and gimenez stains negative (Contador et al., 2016). Also, TEM of the inclusions showed variably sized rods (1.0-2.5 µm) and potentially cocci with central accumulations of electron-dense material and vesicles, not consistent with Chlamydia morphology (Contador,

2013). However, identification through ultrastructure can be misleading since RBs can appear coccoid or rod shaped (Horn, 2008). These findings suggest that multiple species of Chlamydia may be present around/on the gills, but not within the EP inclusions (Contador et al., 2016).

However, further validation by ISH to localize BK-BJC to the intraepithelial inclusions is necessary, because Burkholderia-like bacteria are the dominant gill commensal in freshwater

Atlantic salmon (Steinum et al., 2009) and Chlamydiales can be found on healthy BJC LT gills

(Contador et al., 2016). Future studies determining the etiologic agent of EP should demonstrate the bacterium(a) directly within the inclusion through ISH, as well as indirectly through PCR and sequencing.

BK-BJC is most closely related to the order Burkholderiales which is a vast group of bacteria and from which only dubious comparisons to BK-BJC can be made. In the order

Burkholderiales, unclassified Burkholderiales have been identified on the gills of European plaice (Pleuronectes platessa), and Comamonadaceae and Oxalobacteraceae have identified as normal intestinal microbiota in healthy midas cichlids (Amphilophus citrinellus) and RT, respectively (Wegner et al., 2012; Franchini, 2014; Ingerslev et al., 2014). Additionally, members of the genus Burkholderia (belonging to the family Burkholderiaceae) are associated with several fish species. B. cepacia is the most frequently documented in fish and has been isolated from the kidney, spleen, liver, and skin of RT (Kayis et a., 2009); the livers of moribund, cage-cultured red hybrid tilapia (Oreochromis niloticus) diagnosed with Streptococcus agalactiae (Najiah et al., 2012); the whole body homogenate of fingerling freshwater Atlantic salmon (Miranda and Zemelman, 2002); the oral cavity of tiger sharks (Galeocerdo cuvier)

(Interaminense et al., 2010); and the gut microbiota of juvenile grouper (Epinephelus coioides)

(Sun et al., 2009) and Nile tilapia (O. niloticus) (Filho et al., 2003). Several unidentified species of Burkholderia were found on the gills and heart of RT fry totaling 0.65 % and 1.92 % of the bacterial strains in or on the respective organs (Kapetanocic, Kurtovic, Teskeredzic, 2005). B. cenocepacia was identified in the intestinal tract of farmed yellow seahorse (Hippocampus kuda)

(Tanu et al., 2012) and its’ virulence mechanisms were studied in vitro in zebrafish embryos

(Mesureur and Vergunst, 2014). These experimental infections establish that B. cenocepacia are pathogenic in zebrafish, and introduce the possibility of Burkholderia being pathogenic in other fish species. In addition, several species of known pathogenic Burkholderia are saprophytic and live in aquatic environments—water and soil—including aquaculture facilities (Miranda and

Zemelman, 2002; Knappik et al., 2015; Minogue et al., 2015; Resende et al., 2015).

1.7.3. ENDOZOICIMONAS ELYSICOLA

In 2010-11 yet another non-Chlamydial agent of epitheliocystis was detected in 13- to 20-day old cobia larvae (Rachycentrum canadum) at a production facility in South America (Mendoza et al.,

2013). Three outbreaks of inappetence and hypoxia-related behaviors resulted in 100 % mortality in one hatchery. This prompted an investigation that found gill inclusions with intraepithelial, spherical, basophilic granular bodies that stained gram negative (Mendoza et al., 2013). There was slight lamellar hyperplasia, clubbing, and fusion explaining the clinical signs (Mendoza et al., 2013). A PCR assay revealed a novel γ-proteobacterium with 99 % homology to

Endozoicomonas elysicola (strain MKT110T) found in Japanese marine sea slugs (Elysia ornate)

(Kurahashi and Yokota, 2007). Until recently, members of the genus Endozoicomonas have been identified as symbionts of marine invertebrates; therefore, this is the first documented case of a pathogenic Endozoicomonas in fish (Katharios et al., 2015).

Also, ISH localized E. elysicola with variable intensity within inclusions and scattered along filaments (Mendoza et al., 2013). These results may indicate variable numbers or species of bacteria within the inclusions (Mendoza et al., 2013), or that E. elysicola has a cryptic developmental stage (like EBs in Chlamydia). Retrospective TEM could help answer these

30 questions. On the other hand, 57 % of normal gill bacteria are from the class of γ-proteobacteria

(Steinum et al., 2009), and E. elysicola is a documented commensal in 29 % of Atlantic salmon

(Steinum et al., 2009). As E. elysicola, li e ‘Ca. B. cysticola’, was localized in and outside of the inclusions; it is possible that E. elysicola is an opportunistic or facultative intracellular pathogen.

Interestingly, E. elysicola or an almost identical uncultured γ-proteobacterium has been associated with PGI and amoebic gill disease in Atlantic salmon (Steinum et al., 2009; Bowman and Nowak, 2004). Future research into isolation and challenge trials to re-capitulate disease is necessary to fulfill Koch’s postulates.

1.8. TREATMENTS

Treatments for epitheliocystis have been developed both with and without knowledge of the associated bacterium. The oldest insight comes from Hoffman et al. (1969) who found that the agent of epitheliocystis would not grow on a culture plate containing penicillin; however, this may be due to the organism’s fastidious nature. Later, Goodwin et al. (2005) found that a 25 ppm oxytetracycline bath twice daily for three days was sufficient to completely halt mortalities in hyperinfected largemouth bass. Hyperinfected spotted eagle rays were also treated with a 15 mg/kg daily intramuscular (IM) injection of oxytetracycline for 7 days and a 40 mg/kg IM injection of chloramphenicol every two days, but did not respond (Zachary and Paperna, 1977;

Goodwin et al., 2005; Camus et al., 2013). Additionally, in LT a dual antibiotic regime of tribrissen and oxytetracycline was used to limited effect (Contador, 2013). Lastly, enrofloxacin administration was not successful in infected leopard sharks (Polkinghorne et al., 2010).

Though it has not been therapeutically applied, Mitchell et al. (2010) noted that seawater transfer of salmon with epitheliocystis cleared the inclusions within 4- to 6-weeks. This 4- to 6-week convalescence is longer than expected based on the established 8-day gill epithelial turnover time in trout (Mitchell et al., 2010). The author postulated that this may be due to: 1) species variation in epithelial regrowth time; 2) reinfection; 3) infection of epithelial stem cells; or 4) delayed maturation or cell death (Mitchell et al., 2010). Furthermore, some Chlamydiaceae and

Burkholderiaceae have a type III secretion system that injects bacterial effector proteins into the host cell and disrupt host cell function—for example, leading to apoptosis or disrupt transcription

(Steigen et al., 2013; Vergunst et al., 2010). For extended or severe outbreaks, 4 to 6 weeks of salt water treatment may be an option, but in LT outbreaks self-resolve in 3 to 6 weeks

(Contador, 2013). Additionally, Miyaki et al. (1998) found that UV-irradiated water reduced the prevalence of disease in amberjack and coral grouper; while others found that merely more frequent water changes were more effective than formalin, salt, benzalkonium chloride, and potassium permanganate in treating epitheliocystis in juvenile tilapia (Somridhivej et al., 2009).

Cell culture would greatly advance in vitro testing of new treatment methods. Last, equally important to treatment is prevention through reduced host stress, good husbandry, and water quality.

1.9. FURTHER RESEARCH

The knowledge gaps concerning epitheliocystis range from the nature and validity of presumed causative agent(s) to their clinical significance and are fertile fields for future research. As novel bacterial pathogens are constantly being discovered in relation to EP, the progression of research for each agent ranges widely. In Ontario, the most critical research should be dedicated to

32 improving diagnostic and investigative techniques to identify and characterize BK-BJC and its relationship to EP in cultured LT and RT. For all EP agents further investigations are needed into reliable methods of culture and isolation. This is an essential step to fulfilling Koch’s postulates.

Once this is accomplished, transmission, risk factors, and progression of epitheliocystis may be studied, and the association between epitheliocystis and mortality events will be clarified.

2. RATIONALE, PURPOSE and HYPOTHESES

2.1. RATIONALE

Ontario aquaculture stocks the Great Lakes and lakes within the province with approximately 8 million fish yearly to balance population maintenance with public recreation, commercial fishing, and predation by invasive lampreys (OMNR, 2013). In doing so, Ontario’s aquaculture produces $60 million a year for the Ontario economy (OMNR, 2015). As of 2012, LT account for almost half (3,613,054) of the fish cultured and released throughout the province (OMNR,

2013). The Blue Jay Creek Fish Culture Station on Manitoulin Island in Lake Huron is a significant contributor to the overall LT stocks. Since 2011 this facility has experienced yearly late fall to early winter epizootics of epitheliocystis that resulted in mortalities of up to 42 %

(Contador et al., 2016). EP in BJC LT was once postulated to be a Chlamydial infection based on the breadth of literature implicating the chlamydia as the causative agent. However, Bradley et al. (1988) studied an EP outbreak with a 95 % mortality rate in cultured LT in the Great Lakes and came to the conclusion that it may have been caused by a non-Chlamydial bacterium, and, therefore, EP was likely a multi-etiologic disease. Diagnostic tests (IHC, histology, and TEM) suggested that the causative bacterium was either an evolutionarily distant Chlamydia, or a bacterium from another order, such as a γ- or β-proteobacteria (Bradley et al., 1988). However, the diagnosis was muddied when researchers found epizootic epitheliotropic disease virus

(EEDV) in the fish (Bradley et al., 1988). Later, the theory posed by Bradley et al. (1988) was revived when Contador (2013) discovered that epitheliocystis in BJC LT is potentially caused by a novel β-proteobacterium within the order Burkholderiales, BK-BJC. To my knowledge, this is

34 the second report of a Burkholderiales causing epitheliocystis and the first report of a potentially pathogenic Burkholderiales in freshwater fish.

2.2. PURPOSE

The first purpose of this study is to explore the association of BK-BJC and EP-linked mortality events through the development and implementation of a specific qPCR, in the absence of direct testing methods such as IHC, ISH, or culture. To do so, an initial primer set with hydrolysis probe (BKBJCV8) was designed based on the unique 1503 bp consensus sequence of BK-BJC’s

16S rRNA gene. The BKBJCV8 qPCR was run on frozen whole gill tissues of BJC LT from a mortality free winter in 2014-15, and a winter with a mortality event in 2012-13. The LT from

2013 were previously diagnosed as BK-BJC positive by conventional PCR with universal primers by Contador et al. (2016). The PCR results from the 2012-13 outbreak were then correlated with daily mortality percentages, and compared to the qPCR results of samples from

LT in the winter of 2014-15. Further, changes in BK-BJC quantity in/on LT gills were compared with histopathologic gill lesions in the same fish population. These results were meant to evaluate the temporal and biological relationship between BK-BJC concentration and severity of mortality in a population of production LT; as well as demonstrate the strength and consistency of this causal relationship according to Hill’s epidemiologic criteria for a causal association

(Fredricks and Relman, 1996). The development of a BK-BJC-specific qPCR will facilitate rapid, sensitive and specific detection of BK-BJC in the future.

The second purpose of this study was to use BKBJCV8 qPCR to identify and quantify BK-BJC in the water at BJC during winters 2013 and 2015, which were with and without an EP outbreak,

35 respectively. Though common species of Burkholderia are frequently isolated from water and sediment, BK-BJC has never previously been looked for. We expected to observe fluctuations in the amount of BK-BJC in the water column, because this phenomenon is well documented for other Burkholderia species (Olapade et al., 2005; Kenzaka et al., 1998). In research by Olapade et al. (2005), the levels of B. cepacia in the water column were found to increase in response to dissolved organic carbons and matter such as phenolic compounds and nitrates. This may explain the correlation between BK-BJC-associated epitheliocystis outbreaks and greater concentrations of SS in the water column from heavy rainfalls or thaws.

The third purpose of this study was to culture BK-BJC utilizing direct plating on a variety of agar media chosen based on the established techniques for culturing aquatic bacteria and mammalian pathogenic Burkholderia spp. (Brown and Govan, 2007). BKBJCV8 qPCR confirmed positive infected whole and homogenate gills will be grown on selective—Cepacia medium and MacConkey agar—and non-selective media (TSA and cytophaga agar (CA)).

Bacterial colonies will be analyzed by gram stain, matrix-assisted laser desorption ionization- time of flight (MALDI-TOF) mass spectrometry, and BKBJCV8 qPCR to determine if BK-BJC was successfully grown. If so, this will be the first successful attempt to grow BK-BJC or any bacterial agent of EP since its discovery in 1969.

2.3. HYPOTHESES

1. qPCR will demonstrate that BK-BJC is present in LT with epitheliocystis and is not

present in healthy LT from the BJC Fish Culture Station.

2. The quantity of BK-BJC in the gills of BJC LT will significantly increase during an EP-

associated mortality event, and decrease below the detectable limit at the conclusion of

the event.

3. BK-BJC concentrations in/on the gills of BJC LT have a significant direct correlation

with changes in the daily percent mortality, i.e. they will proportionally increase and

decrease simultaneously.

4. The burden of BK-BJC in/on the gills will increase the severity of all histopathologic

lesions over the course of an EP-associated mortality event.

5. BK-BJC will be present in the water column around the BJC Fish Culture Station at

higher levels during EP-associated mortality events.

6. BK-BJC can be cultured from infected RT gills using selective and non-selective media.

3. MATERIALS AND METHODS

3.1. HISTORIC PCR IDENTIFICATION OF BK-BJC

The BK-BJC status of individual LT from the winter EP-associated mortality events of 2011-

2013 was determined by standard PCR using universal primers [U1 and U1R (Relman, 1993)], for the 16S rRNA gene sequence by Contador et al. (2016) (Appendix 1). Briefly, the extracted

DNA from the 87 LT was amplified using universal primers, PCR assay was run and all were sent for sequencing (Contador et al., 2016). The sequenced products of 87 LT samples were aligned and 56 with 99 % nucleotide identity were used to construct an 800 bp consensus BK-

BJC sequence using Geneious software (Biomatters; New Zealand). From those 56 aligned samples, ten with 100 % nucleotide identity were rerun with standard PCR using universal primers 27f and U1492R (Weisburg et al., 1991), and the predetermined UNIRX3 protocol

(Appendix 1) (Contador, 2013). The resultant 1503 bp consensus sequence was a novel bacterium and designated Blue Jay Cree Bur holderia or “BK-BJC” by Contador et al. (2016)

(Appendix 2).

3.2. BKBJCV8 PROBE AND PRIMER DESIGN

BKBJCV8 qPCR probe and primers were constructed from the 1311-1391 or V8 region of the

BK-BJC 16S rRNA gene sequence using Geneious online software by Contador (unpublished).

The sequence of the forward primer (BKBJCV8F) is 21 base pairs [location (loc): 1311-1331]; reverse primer (BKBJCV8R) is 21 bp [loc: 1371-1391]; and the probe (BKBJCV8 probe) is 25 bp [loc: 1343-1367] (Figure 1 and Appendix 1 and 3). All were synthesized by The University of

Guelph’s Laboratory Services Division. A hydrolysis 5ꞌ 6FAM) and 3ꞌBlac Hole Quencher 1

(BHQ1) sequence probe was chosen to enhance the specificity of the qPCR.

The BK-BJC primers and probe construction was analyzed with Primer-BLAST (Ye et al.,

2012), Oligoanalyzer [Integrated DNA Technologies (IDT); Coralville, USA], and UNAFold

[IDT] software analysis for non-target annealing, Tm, GC%, secondary structure formation, and self- and hetero-dimer scores. The specificity of the BKBJCV8 primers, probe, and amplicon

(Appendix 5) was theoretically assessed by inputting their individual and cumulative sequences in basic local alignment tool-nucleotide (BLASTn) online software and searching for non- specific matches in Genbank (Altschul et al., 1990).

The top 264 BLASTn matches of the BKBJCV8 amplicon with ≥95 % identity were grouped into 21 distinct sequence groups of 81 bp oligonucleotides by single nucleotide variations

(Appendices 6 and 7). A representative match was chosen for each group and the sequence of each 81 bp group was extended to 90 bp oligonucleotides which were manufactured by IDT. qPCR with the BKBJCV8 primer set was later run on these 21 sequence groups to rule-out non- target annealing.

Figure 1. Alignment and target sequences of the BKBJCV8 forward and reverse primers and probe with BK-BJC 16S rRNA gene sequence. A) location of primer and hydrolysis probe binding regions on the BK-BJC 16S gene B) 1290–1402 bp region of the BK-BJC 16S gene showing probe and primer sequences.

3.3. PCR VALIDATION AND OPTIMIZATION OF BKBJCV8 FORWARD AND REVERSE

PRIMERS

To test the BKBJCV8F and BKBJCV8R primers, five conventional PCRs were run with known and unknown BK-BJC status gills from fish of various species. 2 µg of DNA template was added to 25 µl 2x TopTaq Master Mix (QIAGEN), 2 µl of 15 µM BKBJCV8F/BKBJCV8R, and 16 µl water, for a total reaction volume of 50 µl. The optimized PCR protocol (BJCTest1) was set for one cycle of initial denaturation at 94 ˚C for 3 min, then 30 cycles of denaturation (94 ˚C for 30 s), annealing (58 ˚C for 30 s), and extension (72 ˚C for 1 min), and a final extension cycle at 72

˚C for 7 min. The PCR products were run on a 2 % w/v tris-borate (TBE) agarose gel with SYBR safe gel stain [Invitrogen; Burlington, ON], Quick-Load Purple 2-Log DNA ladder (0.1-10 kb)

[New England Biolab Inc., Ipswich MA], and Promega blue/orange loading dye [Madison, WI] and visualized by UV light. The resulting target bands (~80 bp) were excised and recovered from the gel using the Direct-Gel-SpinTM DNA Recovery Kit [Applied Biological Materials Inc.;

Richmond, BC] according to the manufacturer’s protocol and submitted to the University of

Guelph’s Laboratory Services, Agriculture and Food Laboratory [Guelph, ON] for direct sequencing on the 2720 Thermal Cycler with the ABI Prism BigDye® Terminator Cycle

Sequencing Ready Reaction kit v3.1 [Applied Biosystems, Foster City, CA].

3.4. BKBJCV8 qPCR

The total DNA concentrations were standardized to 100 ng/µl for gill samples and 50 ng/µl for water samples by diluting 10 µl of template DNA with a calculated quantity of PCR grade water.

Oligonucleotides were resuspended in TE to a concentration of 100 µM and then serially diluted to 10-6 or 100 picoM as per manufacturers protocol [IDT]. All tissue and oligonucleotide samples

41 were run twice and water samples once with LightCycler® 480 Probes Master (04707494001)

PCR reaction mix in accordance with the product insert (Roche Diagnostics/Applied Science;

Quebec, CANADA). The first run screened for positive and negative tissues and the second to quantify the absolute concentration of BK-BJC in or on the gill tissues by standard curve. The

PCR mix contained PCR grade water (3 µl), 15 µM forward and reverse primers (0.67 µl), 7.5

µM BK-BJC probe (0.67 µl), and 10 µl of 2x LightCycler® 480 Probes Master. 7.5 µl of this

PCR mix was combined with 2.5 µl of template DNA per well. For qPCR on the diluted synthetic oligonucleotides and water samples, 1 µl of the template was added to 9 µl of PCR Mix per well. All samples were loaded in duplicate onto 96-well plates with a single in-plate standard of synthetic BK-BJC 1.02E5, and run on the Lightcycler 480 instrument.

The BKBJCV8 primer set was used to specifically detect BK-BJC by the V8 region of the 16S rRNA gene. The probe assay detection format was set for a monocolor hydrolysis probe or

Universal ProbeLibrary assay detecting a FAM dye with excitation and detection wavelengths at

465 and 510 nm, respectively. The optimal thermal cycling protocol was determined to be a preincubation of 95 ˚C for 5 min at a ramp rate of 4.4 °C/s, then 40 cycles of amplification at 95

˚C (10 s at 4.4 °C/s) for polymerase activation and template DNA denaturation, 60 ˚C for annealing (30 s at 2.2 °C/s), and 72 ˚C for extension (1 s at 4.4. °C/s), and last a single cooling at

40 ˚C for 10 s at 2.2 °C/s. The PCR mix for water samples was augmented to omit sterile water, and was composed of 15 µM forward and reverse primers (0.6 µl), 7.5 µM BK-BJC probe (0.6

µl), and 8.2 µl of 2x LightCycler® 480 Probes Master. 5 µl of the PCR mix was added to 5 µl of template DNA (standardized to 50 ng/µl). The rest of the protocol was similar to tissue samples.

Primer and probe efficiency was established based on a 250 bp synthetic gBlocks gene fragment of the 1257-1402 bp region of the BK-BJC 16S rRNA gene sequence [IDT] (Appendix 4). The synthetic BK-BJC was 10-fold serially diluted from 1.02E9 to 1.02E1 gene fragment copies to make a standard curve spanning the range of potential sample concentrations. The quantitative valves for all samples were assigned based on a single in-plate BK-BJC standard of 1.02E5 copies that was corrected for plate variation by comparison to an external standard curve of the stock BK-BJC dilutions. BKBJCV8 qPCR efficiency and % difference of the run efficiencies was calculated from four standard curve runs and the equation:

Percent difference=(|ΔV|/(∑V/2))*100

From these same four runs the intra- and interassay coefficient of variation were calculated by

%CV=(Standard deviation of duplicate wells/ duplicate mean)*100 or

%CV=(Standard deviation of plate means/mean of plate means)*100, respectively.

To assess the validity of the results, the amplification and standard curves of the BK-BJC synthetic standards were analyzed for crossing point value (Ct) and sigmoidal shape. Positive samples were determined to be those with a Ct value between 1 and 35. Ct values of ≥35 corresponding to ≤1.02E1 copies were re-run in triplicate to confirm the values. Ct values that remained ≥35 were considered below the limit of detection for this assay and considered functionally negative for the purpose of these analyses. The qPCR was considered valid if the negative control tissue was Ct zero or ≥35, the negative template had a no CT value, and the

43 positive tissue control and extraction positive controls were Ct <35. Extraction positive tissue controls were either known-positive gill tissues diagnosed by conventional PCR with universal primers, or negative control gill tissue spiked with 3 µl known-positive, previously extracted LT

DNA.

3.5. SAMPLING SITE

All LT samples were acquired from the BJC Fish Culture Station on Manitoulin Island, Ontario,

Canada. The LT at the station are raised in a single pass, flow-through system where 6-11 ˚C spring fed, ground water is routed through multiple 6000 L indoor tanks on a 24 hr light cycle.

There are approximately 6400 LT yearly in each tank. RT and splake (Salvelinus namaycush X

Salvelinus fontinalis) also kept onsite are unaffected by or subclinical for EP.

The LT monitored during the 2012-13 mortality event were collected from tank R8. The tank was established on November 14th 2012 with the addition of 6444 LT fry. The following day,

November 15th, the fish were treated with a preventive formalin-bath as per standard operating procedure.

All RT tissues were acquired from the Alma Aquaculture Research Station in Alma, Ontario,

Canada. The RT at the station are raised in a single pass, flow-through system where 8.5 ˚C groundwater from an aquifer is routed through outdoor, concrete circular rearing tanks or fibreglass raceway tanks. Atlantic salmon and Arctic charr also kept onsite.

3.6. 2012-13 SAMPLE COLLECTION AND PROCESSING

Randomly sampled LT were collected from tank R8 at BJC Fish Culture Station. Samples were taken on December 3rd (n=5), January 3rd (n=5), 8th (n=8), and 18th (n=10), and March 21st (n=4) in 2013. The fish were euthanized onsite with a lethal overdose of benzocaine [Aqualife TMS,

Syndel Laboratories Ltd; BC], and decerebrated. The 1st gill arch was excised and placed in a labeled cassette in 10 % buffered formalin (ACT Chemicals Inc.; Saint-Léonard, Québec). The rest of the carcass was frozen and stored at -20 ˚C. At the University of Guelph’s Fish Pathology

Laboratory (FPL), the fish were defrosted, and 25-40 mg of gill tissue was excised and placed into a microcentrifuge tube with 20 µl ATL buffer for DNA extraction. To reduce cross contamination between fish, all surfaces and instruments were disinfected with bleach and 70 % ethanol, or flame sterilized between each fish. Two 500 ml water samples were collected from infected tanks (R1 and R8) and two 500 ml water samples collected from the bottom of S4 and mainline pipe. All water samples were frozen and stored at -20 ˚C.

3.7. 2014-2015 SAMPLE COLLECTION AND PROCESSING

All samples—fish and water—were collected biweekly over a 10 week period from December

29, 2014 to February 9th 2015. Fifteen random LT per tank were collected from tanks R5 and R9.

These tanks were chosen at random based on the haphazard distribution of mortality events in previous years. The fish were euthanized and processed for DNA extraction and histopathology using the protocol established in 2012-13. Five, clinically healthy RT were similarly sampled at the first sampling time point. At each time point, between 500 ml-1.0 L of water was collected from tanks R5 and R9, the hatchery outflow and inflow, and a reserve pond.

3.8. DNA EXTRACTION FROM LT WHOLE GILLS

Genomic DNA was extracted from frozen gill tissue using the DNeasy®Blood and Tissue kit and accompanying protocol from the DNeasy®Blood and Tissue Handbook [QIAGEN, 2003;

ON] as per the manufacturer’s instructions with inclusion of an overnight incubation with proteinase K.

3.9. FILTRATION AND DNA EXTRACTION FROM WATER SAMPLES

All water samples were processed according to Draghi et al. 2010 with the following alterations in protocol. The water was vacuum filtered through a 500 ml Nalgene™ Rapid-Flow™ Sterile

Disposable Filter Unit with 75 mm diameter polyethersulfone membrane with either a 0.1 µm pore size (2015 samples and 2013 positive control) or 0.2 µm pore size (2013 samples) due to the increase amount of particulate matter in the 2013 samples which clogged the 0.1 µm pores and blocked filtration [Nalgene Nunc International]. The filter membrane was sterilely excised from the apparatus using scalpel and forceps and cut into 5 pieces. The membrane was placed in a 50 ml Falcon™ conical centrifuge tube with 40 ml sterile 1x phosphate buffered saline (PBS) and vortexed for 3 min to release any bacteria from the membrane and to suspend it in the PBS.

The membrane was then removed from the PBS with sterile forceps. The 50 ml centrifuge tube with PBS and filtered material were then centrifuged at 9265 × g at 4 °C for 30 min. The supernatant was removed. The pellet was transferred to a 1.5 ml microcentrifuge tube. The pellet was digested in 180 µl QIAGEN ATL tissue lysis buffer and 50 µl proteinase K for 16 hr at 55

°C and agitated at 700 rpm. The samples were centrifuged at 1200 × g for 5 min at room temperature. All but 0.1 ml of the supernatant was decanted, the pellet was resuspended, and incubated at 100 °C with agitation at 300 rpm for 20 min. The resulting pellet was manually

46 broken up and resuspended in 200 µl of QIAGEN AL. The rest of the extraction followed the manufacturer’s protocol starting at step 3a (QIAGEN DNeasy®Blood and Tissue Handboo ), except that RNase A was not used. The positive control for the filtration was four crushed gill arches added to 500 ml deionized (DI) water and filtered. The positive DNA extraction control was DI water spiked with 5 µl of 1.02E9 synthetic BK-BJC (1257-1402 bp region of the BK-BJC

16S rRNA gene), and the negative control was DI water.

The concentration of DNA post-extraction was assessed using the Thermo Scientific NanoDrop

2000c (spectrophotometer) (Thermo Fisher Scientific; Wilmington, DE). For any samples with a total DNA concentration lower than 50 µg/ml for water or 100 µg/ml for gill tissue, a Thermo

Savant SPD1 1V SpeedVac concentrator (Thermo Fisher Scientific; Wilmington, DE) was utilized to evaporate off excess AE buffer and to concentrate the DNA to a minimum of 50 or

100 µg/ml. All extracted DNA was stored at -20˚C until used.

3.10. 2012-2013 EP OUTBREAK AND MORTALITY STATISTICS

The BBC quantities per LT from the five sampling dates during the 2012-13 EP outbreak were log transformed and analyzed for normality using four normality tests (Shapiro-Wilk,

Kolmogorov-Smirnov, Cramer-von Mises, and Anderson-Darling). Next, a one way ANOVA paired with a Tu ey’s range test (or HSD) was run to compare the median quantities of BBCs at each time point to all others through multiple single-step comparisons, and determine if they significantly differ.

Daily mortality was represented as a percent of dead LT over the number of fish in a static, single tank population that day. A logit transformation of the daily percent mortalities was done because this variable was a percent and a 0.25 bias correction term was added to the transformed values.

To correlate the logit transformed daily percent LT mortality to the log-transformed median concentration of BK-BJC at each sampling time point, an odds ratio and p-value were calculated using one-tailed Pearson and Spearman’s correlation coefficient tests to demonstrate the correlation between BK-BJC loads and daily percent mortality. Both tests were run as a point of comparison since the data was relatively normal, but was difficult to assess due to having a sample number (n) of 5. A one-tailed assessment was chosen because of the expectation of a direct relationship between increased BK-BJC concentration in the gills and fish mortality rates.

The power was not calculated due to the lack of accepted difference of interest.

Concentrations of BBC on the gills of LT from 2013 were compared to semi-quantitative histological lesion scores of lamellar epithelial hyperplasia, EP inclusions, and single cell necrosis by Contador et al. (2016); however, a statistical correlation between these two variables was not able to be determined due to insufficient data points.

Neither the winter 2014-15 LT nor the water samples from either year assayed by BKBJCV8 qPCR required statistical analysis.

3.11. SECOND PRIMER SET TO VALIDATE SPECIFICITY OF BKBJCV8 qPCR

To address the lack of specificity of the BKBJCV8, a second set of primers (BKBJCV3) was developed and validated as described above and rigorously assessed before commencing testing our samples. This set of primers differentiates BK-BJC from ‘Ca. B. cysticola’ clone GABI

[JQ723599.1], which is 100 % identical to BK-BJC over 99 % of the target region of the

BKBJCV8 primers (Appendices 3 and 4). The 397-612 bp region or the V3 region of the 16S rRNA gene was selected as it had the greatest variation between the two bacterial 16S rRNA gene sequences. The sequence of the forward primer (BKBJCV3F) is 22 base pairs [loc: 397-

418]; reverse primer (BKBJCV3R) is 22 bp [loc: 591-612] (Figure 2 and Appendix 1). All were synthesized by Sigma-Aldrich [Oakville, ON]. The BKBJCV3 primers were theoretically analyzed and validated according to the same guidelines as BKBJCV8 primers before implementation.

Figure 2. Alignment and target sequences of BKBJCV3 forward and reverse primers with BK-

BJC 16S rRNA gene sequence. A) Complete BK-BJC sequence with primer annealing locations.

B) Enhanced, elided view of the 390–421 and 590-614 bp regions with primer locations.

To test the BKBJCV3 forward and reverse primers, regular PCR was run with synthetic gBlock gene fragments of BK-BJC (region 1-750 bp) and ‘Ca. B. cysticola’ clone GABI (JQ723599.1)

[IDT] (Appendix 4), BK-BJC-positive LT gills from BJC 2013, and BBC-negative LT gills from

BJC 2015. For all runs, 1 µg of DNA template was added to 25 µl 2x TopTaq Master Mix

(QIAGEN), 1 µl of 10 µM BKBJCV3 forward and reverse primers, and 21 µl NF water. The

BKBJCV3 PCR protocol was one cycle of initial denaturation at 94 ˚C for 3 min, then 30 amplification cycles of denaturation (94 ˚C for 30 s), annealing (56 ˚C for 30 s), and extension

(72 ˚C for 1 min), and a final extension cycle at 72 ˚C for 7 min.

3.12. BACTERIA PREPARATION FOR CULTURE

BK-BJC and BK-BJC-like bacteria complex (BBC) positive tissues were acquired from naturally infected RT from Alma Aquaculture Research Station on September 24, 2012, case number

B139-12, previously processed by FPL staff and stored for at -20 ˚C. BBC negative tissues were acquired from an infection trial of RT with Flavobacterium psychrophilum at the FPL in 2015.

Gill arches (½ to 1) from all fish were pre-screened for BBC by BKBJCV8 qPCR. One to four gill arches (5.23 g) from eight BK-BJC-positive and 10 negative RT were homogenized separately in glass tissue grinders with 5 ml sterile 1x PBS. The resulting 8 ml were frozen at -20

˚C. The BBC status of the respective gill homogenates was again assayed by BKBJCV8 qPCR.

3.13. AGAR MEDIA PREPARATION AND COLONY IDENTIFICATION

Cytophaga agar (CA) and MacConkey agar were purchased as ready-made plates from the

University of Guelph’s Animal Health Laboratory (AHL). Cepacia medium plates were purchased from BD Canada [Mississauga, ON]. Trypic soy agar (TSA) was made in the FPL

51 according to the manufacturer’s instructions [BD Difco™; Mississauga, ON]. Eight plates of each type of media were streaked with either concentrated BK-BJC-positive gill homogenate

(from B139-12), BK-BJC-positive whole RT gills (from B139-12), BK-BJC-negative whole RT gills (from B139-12), or a negative control RT gill homogenate. Four plates were incubated at room temperature (22 °C) and four at 15 °C. All colonies were gram stained. All bacterial colonies grown on TSA and CA agars underwent DNA extraction as per the manufacturer’s instructions (Protocol C, 2006 QIAGEN handbook) for isolation of genomic DNA from bacterial culture, and the DNA analyzed by BKBJCV8 qPCR, previously described for genomic DNA extractions. Colonies of interest were sent to AHL for identification by MALDI-TOF mass spectrometry.

4. RESULTS

4.1. DEVELOPMENT OF NOVEL qPCR FOR BK-BJC DETECTION

4.1.1. PCR IDENTIFICAITON OF BK-BJC

Through the work of Contador et al. (2016), BK-BJC was discovered using universal PCR

(primers 27f and U1492R) (Appendix 1), which amplified a novel 1503 bp sequence of the 16S rRNA gene from 56 of 87 individual LT during gill disease outbreaks from 2010-13. The consensus sequence was designated as the partial 16S rRNA gene sequence of BK-BJC and deposited into Genbank with the accession number KM504995.1 (Contador et al., 2016)

(Appendix 2).

4.1.2. DEVELOPMENT AND STANDARDIZATION OF THE BKBJCV8

Contador et al. (2016) designed the BKBJCV8 probe and primers, but in silico assessment of their construction was done by this thesis’ author after use of the primer set. OligoAnalyzer software assessment of BKBJCV8 primers and probe is shown in Table 3. The primers were considered acceptable by Oligoanalyzer and BLAST-primer software because 1) the hairpin Tm was less than the reaction Tm, 2) most of the homo- and heterodimer scores were greater than -9 kcal/mole, 3) the GC % was between 40-60 %, and 4) the melting temperatures was within the ideal range and the forward and reverse primers were within 3 °C (Thornton and Basu, 2011)

(Table 3). The probe had a delta G of -11.52 kcal/mole, but since the probe fit the other criteria, the initial design was retained and monitored for inefficiencies.

The assessment of specificity of the BKBJCV8 found that the forward and reverse primers matched with 443 and 897 unique bacterial sequences with 100 % identity and query coverage

(QC), respectively. The probe had two alignment matches with 100 % identity and QC: BK-BJC and ‘Ca. B. cysticola’ clone GABI_14 16S ribosomal RNA gene, partial sequence [JQ723599.1].

The subsequent 268 matches have 100 % identity over 88 % of the QC, and have bp mismatches within the last 3 bases at 5ꞌ end (Table 4).

The BK-BJC 81 bp BKBJCV8 amplicon sequence similarly aligns 100 % with ‘Ca. B. cysticola’ clone GABI over 98 % of the QC, and has 99 % identity and 98 % QC with ‘Ca. B. cysticola’ strain A1-483-L1 [JN968376.1] and an uncultured β-proteobacterium [JN807444.1] found in

Atlantic salmon with proliferative gill disease (Table 5). ‘Ca. B. cysticola’ is an established bacterial agent of EP. The next closest match is a β-proteobacteria [AB690756.1], with a 96 % identity and 97 % QC, was identified in environmental samples from Chinese wetlands (Table

5). BLAST matches 6 through 264 are 95 % identical with QC ranging from 97-100 % (Table 5).

Table 3. Theoretical description of the BKBJCV8 primers and probe based on OligoAnalyzer and Primer-BLAST software.

Self-Dimer Score Hetero-Dimer Hairpin Tm Tm [°C +/- 1.4 GC% [Max. Delta G Score [Max. Delta (°C) (Allawi, 1997)] (kcal/mole)] G (kcal/mole)] Forward 25.2 and 33.8 57.67 47.62 -6.3 -7.96 F to R primer (F) Reverse 37.7 59.8 52.38 -4.41 -7.96 R to F primer (R) -5.02 P to F Probe (P) 33-41.7 65.68 56 -11.52 -6.36 P to R

Alignments of the ≥95 % matches with BKBJCV8’s 81 bp amplicon revealed 21 distinct sequence groups with single nucleotide variations throughout (Appendices 6 and 7). The notable patterns of nucleotide mismatches for each sequence group are as follows. SGs 4 and 5 in the alignment correspond to ‘Ca. B. cysticola’ clone GABI [JQ723599.1] (P4), and ‘Ca. B. cysticola’ strain A1-483-L1 [JN968376.1], and an uncultured β-proteobacterium [JN807444.1]

(P5), respectively, and differ from BK-BJC by one or two bp mismatches. SG 4 and 5 are identical to the BK-BJC amplicon in the forward primer region (green); SG 4 is identical in the probe region; and SGs 1, 9, 18, 20, and 21 are identical over the reverse primer region. The SG most similar to BK-BJC is SG 4 or ‘Ca. B. cysticola’ clone GABI which has a single nucleotide difference from at the 5ꞌ end of the reverse primer. The only SG that has a nucleotide difference in the non-primer and probe region is SG 1.

The other known agents of epitheliocystis were searched for among the top 20,000 BLAST matches of the BKBJCV8 qPCR amplicon. The search revealed no alignment with any

Chlamydial species, Endozoicimonas elysicola, Piscirickettsia salmonis, or ‘Ca. Ichthyocystis hellenicum’ or ‘Ca. I. sparus’; however, within the matches with ≥95 % identity there were four

Burkholderia and 16 unnamed β-proteobacteria.

Table 4. BLASTn search matches to the BKBJCV8 probe sequence. Note: the E value is the calculated measure of significance of the BLAST match, and the exactness of the BLAST match is assessed by taking the E value, QC, and identity into account. BK-BJC is highlighted in yellow.

BLAST hits for the BKBJCV8 probe Max Total QC E value Identity [Genbank accession #] score score (%) (%) Uncultured bacterium clone BK-BJC 16S ribosomal RNA gene, partial sequence 50.1 50.1 100 5.00E-04 100 [KM504995.1] Candidatus Branchiomonas cysticola clone GABI_14 16S ribosomal RNA gene, partial 50.1 50.1 100 5.00E-04 100 sequence [JQ723599.1] Thiomonas sp. Dg-D5 partial 16S rRNA gene, isolate Dg-D5 [LN864652.1] 44.1 44.1 88 0.03 100 268 additional bacteria 44.1 44.1 88 0.03 100

Table 5. BLASTn alignment matches to the theoretical BKBJCV8 amplicon (i.e., the V8 or

1311-1391 bp region of BK-BJC’s 16S rRNA gene). BK-BJC is highlighted in yellow.

BKBJCV8 amplicon BLAST hits with ≥95% Max Total QC Identity E value identity [Genbank accession #] score score (%) (%) Uncultured bacterium clone BK-BJC 16S ribosomal RNA gene, partial sequence 150 150 100 2.00E-33 100 [KM504995.1] Candidatus Branchiomonas cysticola clone GABI_14 16S ribosomal RNA gene, partial 148 148 98 9.00E-33 100 sequence [JQ723599.1] Candidatus Branchiomonas cysticola strain A1- 483-L1 16S ribosomal RNA gene, complete 143 143 98 4.00E-31 99 sequence [JN968376.1] Uncultured beta proteobacterium clone T200910 16S ribosomal RNA gene, partial sequence 143 143 98 4.00E-31 99 [JN807444.1] Uncultured beta proteobacterium gene for 16S rRNA, partial sequence, isolate: sd-jx66 130 130 97 3.00E-27 96 [AB690756.1] Matches 6-143 128 128 100 1.00E-26 95 Matches 144-152 126 126 98 4.00E-26 95 Matches 153-264 124 124 97 1.00E-25 95

4.1.3. PCR VALIDATION AND OPTIMIZATION OF THE BKBJCV8

The specificity of the BKBJCV8F and BKBJCV8R primers were experimentally assessed by running conventional PCR on gills from of variety of fish species of BK-BJC status predetermined by PCR with universal 16S primers by Contador et al. (2016). Positive status was assigned to samples that were ≥90 % similar to ‘Ca. B. cysticola’ or had 99 % percent alignment with the BK-BJC consensus sequence (Contador, 2013). Seven of eight LT had two bands: one at at ~190 bp and one at ~80 bp (Gel A lanes 6, 7, 12, and 13; and gel B lanes 7, 8, 14, and 15)

(Figure 3A and B). The ~80 bp band was the correct size to be the target BKBJCV8 amplicon and was excised, sequenced, and confirmed to be similar to the partial sequence of the BK-BJC

16S rRNA gene. The ~190 bp band was similarly seen on a gel containing the results of a SYBR green qPCR with BKBJCV8 primers (not shown), and was gel purified and sequenced. The 190 bp band consensus sequence was searched on BLASTn and found to have 96 % nucleotide similarity with 89 % QC with three clones of an unpublished partial mRNA sequence for a T-cell receptor beta chain in Oncorhynchus mykiss (RT) [AJ517919.1]. Brook trout (Salvelinus fontinalis) gill tissue samples produced a 1000 bp band, but it was not sequenced (Figure 3A lanes 9, 10). See Appendix 8 for the sample case numbers and descriptors.

Figure 3A and B. 2 % TBE agarose gel electrophoresis results of standard PCR with BKBJCV8 primers run on BJC LT and various fish species of known and unknown BK-BJC status (Gels A and B). The same positive and negative controls were used for both PCR runs and gels (Gel A

Lane 12=Gel B Lane 14; Gel A Lane 13=Gel B Lane 15), which were LT B002-13 3R8 (+) and

B037-10 m3-1 (-), respectively.

4.1.4. BKBJCV8 qPCR EFFICIENCIES

To validate the repeatability of the BKBJCV8 qPCR assay, four BKBJCV8 qPCRs were run with the same dilution as the standard curve of synthetic BK-BJC (i.e., E1-E9). In 3 of 4 runs, the standards were run in duplicate and in the 4th run in quintuplicate. The concentration range E2-E8 was chosen because it was the most consistent and spanned the PCR product concentration range in BJC LT samples. Also, only two of the five E1 wells were positive. The amplification factors and percent efficiencies for all runs ranged from 1.853-1.863 and 85.33-86.32 %, respectively

(online qPCR efficiency calculator, ThermoFisher Scientific) (Table 6A). The percent differences between run efficiencies are all ≤0.861 %, below the 1% standard maximum

(Vaerman et al., 2004) (Table 6B). The Ct values of all duplicate and most quintuplicate wells were within 0.5 cycle of one another, except for E2 run in quintuplicate. The difference between the highest and lowest Ct values of the five E2 wells was 0.77. The percent coefficients of variation (%CV) for intraplate assay were all below 10 %, and the interplate assays were all below 20 % with all but 1.02E3 being below 10 % (Tables 6C and D). These intra- and interassay %CVs are well below the acceptable %CV set by Iversen et al. (2012). An alternate intraassay variation was calculated by NCBI guidelines which take sample number into account and are reported in Appendix 9 (Iversen et al., 2012). The intra-plate assay mean %CV calculation based on the NCBI guidelines showed smaller mean %CVs ranging from 3.108-4.073 %.

Table 6. Calculated BKBJCV8 qPCR efficiency and variation within and between runs. A)

Shows the amplification factor or efficiency of four qPCR runs on a synthetic BK-BJC standard curve. B) Compares the percent difference of efficiencies of each of the runs. C) and D) show the calculated intra- and interplate assay %CV on synthetic BK-BJC standards 1.02E8-1.02E2.

A. Variation of the amplification factor Amplification factor (1.02E8-1.02E2) Run 1 Run 2 Run 3 Run 4 85.33 % 85.52 % 85.76 % 86.32 %

B. Comparison of amplification factor between four runs % Difference in run efficiencies Run 1 vs 2 Run 1 vs 3 Run 1 vs 4 Run 2 vs 3 Run 2 vs 4 Run 3 vs 4 0.161 0.376 0.861 0.215 0.700 0.485

C. Intraassay variation Intraplate assay of standards 1.02E8 thru 1.02E2 by run qPCR Run (# wells/sample) Mean %CV (Concentration) 1 (dupl) 5.139 2 (dupl) 4.686 3 (dupl) 5.760 4 (quint) 6.949

D. Interassay variation Interplate assay of standards 1.02E8-1.02E2 from four runs Standard Concentration Mean %CV (Concentration) 1.02E8 7.707 1.02E7 5.208 1.02E6 6.252 1.02E5 6.632 1.02E4 9.160 1.02E3 18.084 1.02E2 1.945 Avg. Interplate Mean 7.855

4.1.5. SPECIFICTY OF BKBJCV8 qPCR

To determine if the predicted PCR product was produced, the 81 bp amplicon resulting from

BKBJCV8 qPCR amplification of synthetic BK-BJC standard 1.02E8 was sent for sequencing.

Consensus sequence from the edited forward and reverse sequences overlapped by 3-4 bp

(Figure 4), and the BLAST results showed several non-target bacteria ranked above BK-BJC; however, BK-BJC had the highest combined % nucleotide identity and QC at 92 % and 78 %, respectively (Table 7). The difficulties with sequencing were attributed to a small amplicon size and 19 potential secondary structures with up to 6 hairpins made by the amplicon.

BKBJCV8 forward and reverse sequences were also run through BLASTn separately (Appendix

10). The forward sequence was most similar to BK-BJC with a 61 % QC and 97 % nucleotide similarity. ‘Ca. B. cysticola’, clones GABI and A1, and JN807444.1 were the next matches at 62

% QC and 95 % nucleotide similarity. The reverse sequence results were less specific than the forward; however, BK-BJC had the highest combined QC and identity (93 % and 92 %, respectively).

Figure 4. Consensus sequence of forward and reverse primers on BKBJCV8 qPCR product

[constructed using Geneious software].

Table 7. BLASTn results from the PCR product of synthetic BK-BJC standard (1.02E8) run on

BKBJCV8 qPCR. BK-BJC is highlighted in yellow.

Description of BLAST match Max Total QC E value Identity [Genbank accession #] score score (%) (%) Uncultured bacterium partial 16S rRNA gene, 62.6 62.6 43 1.00E-06 91 clone D3M-23 [AM238185.1] Uncultured bacterium clone plate116a11 16S 60.8 60.8 43 4.00E-06 91 ribosomal RNA gene, partial sequence [KT450223.1] Carnobacterium sp. SN_83 16S ribosomal RNA 60.8 60.8 42 4.00E-06 91 gene, partial sequence [KR088662.1] Burkholderia sp. UFSM-B33 16S ribosomal RNA 60.8 60.8 39 4.00E-06 93 gene, partial sequence [KJ532452.1] Uncultured bacterium clone 4783190 16S 60.8 60.8 43 4.00E-06 91 ribosomal RNA gene, partial sequence [JQ696406.1] Staphylococcus arlettae strain BTMT04 16S 60.8 60.8 43 4.00E-06 91 ribosomal RNA gene, partial sequence [JN228201.1] Uncultured bacterium clone 16slp69-01f05.p1k 60.8 60.8 32 4.00E-06 100 16S ribosomal RNA gene, partial sequence [FJ508610.1] Uncultured Firmicutes bacterium clone 60.8 60.8 42 4.00E-06 91 7mos_10s_E4 16S ribosomal RNA gene, partial sequence [GQ261869.1] Uncultured alpha proteobacterium clone 60.8 60.8 43 4.00E-06 91 AEGEAN_233 16S ribosomal RNA gene, partial sequence [AF406547.1] Uncultured Fibrobacter/Acidobacteria group 60.8 60.8 43 4.00E-06 91 bacterium clone AEGEAN_225 16S ribosomal RNA gene, partial sequence [AF406544.1] Lactobacillus paracasei strain LI3 16S ribosomal 59 59 43 1.00E-05 89 RNA gene, partial sequence [KR265315.1] Rhodocyclaceae bacterium ICHIOC6 gene for 59 59 38 1.00E-05 93 16S ribosomal RNA, partial sequence [LC132803.1] Staphylococcus sciuri strain Bp-2 16S ribosomal 59 59 41 1.00E-05 91 RNA gene, partial sequence [KJ888130.1] Staphylococcus sciuri strain RCB550 16S 59 59 41 1.00E-05 91 ribosomal RNA gene, partial sequence [KT260762.1] Ralstonia pickettii strain LT7-MRL 16S 59 59 38 1.00E-05 93

65 ribosomal RNA gene, partial sequence [KP318034.1] Uncultured bacterium clone BK-BJC 16S 59 117 78 1.00E-05 92 ribosomal RNA gene, partial sequence [KM504995.1] Paenibacillus polymyxa CR1, complete genome 59 648 41 1.00E-05 91 [CP006941.2]

4.1.6. DEVELOPMENT OF OLIGONUCLEOTIDES AND BKBJCV8 qPCR ANALYSIS

The BLASTn results of the 81 bp BKBJCV8 amplicon revealed 264 different bacterial sequences with ≥95 % identity. As as described above, to rule-out the amplification of the non- target bacteria, the 264 bacterial sequences were categorized into 21 groups and synthetic oligonucleotides representing each were ordered. BKBJCV8 qPCR was run twice on the 21 oligonucleotides with three positive controls 1) synthetic BK-BJC 1.02E6 gBlock, 2) previously positive LT gill tissue, and 3) a synthetic 90 bp oligonucleotide of BK-BJC [KM504995.1] (P3); and two negative controls 1) known negative LT gill tissue and 2) no template added wells

(Figure 5 and Table 8).

Figure 5. Annotated BKBJCV8 qPCR results of the 21 oligonucleotide sequence groups (SG#).

The sigmoidal amplification curves represent positive samples and are labeled on the right of the figure or just below the curve. BK-BJC 1.02E6=synthetic gBlock of BK-BJC at 1.02E6 copies

(in-plate standard); POS Tissue=LT gill tissue positive control; and SG 3=synthetic 90 bp oligonucleotide of BK-BJC positive control.

Table 8. BKBJCV8 qPCR results (Ct and concentration) for the 21 bacterial sequence groups based on BLAST matches ≥95 % similar to the BKBJCV8 amplicon. SG #=sequence group #. Ct

≥35 is considered negative.

Mean Samples Mean Ct Mean Std Concentration Mean Std SG 1 26.47 0.21 3.50E+03 5.05E+02 SG 2 - - - - SG 3 BK-BJC 14.96 0.01 1.01E+07 6.68E+04 SG 4 ‘Ca. B. cysticola’ clone GABI 14.73 0.05 1.19E+07 3.75E+05 SG 5 ‘Ca. B. cysticola’ strain A1 17.44 0.2 1.83E+06 2.54E+05 SG 6 30.21 0.18 2.62E+02 3.33E+01 SG 7 23.76 0.02 2.29E+04 2.56E+02 SG 8 - - - - SG 9* 18.79* 7.16E+05* SG 10 21.6 0.21 1.03E+05 1.50E+04 SG 11 19.85 0 3.43E+05 5.79E+02 SG 12 20.57 0.08 2.09E+05 1.13E+04 SG 13 19.35 0.23 4.89E+05 7.84E+04 SG 14 17.44 0.33 1.85E+06 4.24E+05 SG 15 - - - - SG 16 - - - - SG 17 - - - - SG 18 - - - - SG 19 - - - - SG 20 - - - - SG 21 16.24 1.01 4.70E+06 3.05E+06 Standard synthetic BK- BJC 1.02E6 copies 18.28 0.09 1.02EE+06 6.67E+04 POS Control Tissue 31.58 0.13 1.01E+02 8.84E+00 NEG Control Tissue 35 9.43E+00 NEG Control PCR - - - - Master Mix Dashes indicate a negative sample—e.g., SG 2, 8, and 15-20. *1 of the 4 SG 9 wells was positive and considered contaminated.

69 qPCR with BKBJCV8 primers and probe revealed 11 of 21 sequence groups were positive

(Figure 5 and Table 8). The positive groups were SG 1, 3-7, 10-14, and 21, and had Ct values between 13.7 and 30.3. The most consistent difference between the qPCR negative and positive samples is that all the negatives had three single bp mismatches at nucleotide 3, 6, and 13

(counting from the 5ꞌ end). The positives also had mismatches in the probe location either 1, 2, 3 or 6 mismatches in various locations including some with mismatches in positions 3, 6, or 13, but none had all three mismatches (Appendix 11). In total these groups represent 33 different

16S rRNA bacterial sequences in Genbank (Table 9).

SGs 4 and 5—which are ‘Ca. B. cysticola’ clones, GABI and A1, and an uncultured β- proteobacterium clone T200910 associated with gill disease in Atlantic salmon [JN807444.1]— are the only bacteria on the list that are known fish pathogens (Table 9). All 33 non-target bacteria have been found within environmental samples often associated with aquatic sediment or sludge.

Table 9. BKBJCV8 qPCR positive, non-target oligonucleotide sequence groups and the bacteria they represent. All bacteria within the group had identical genomic sequences that were complementary to the BKBJCV8 primers and probe.

Sequence Bacterial members of each sequence group [Genbank accession #] Group Uncultured bacterium clone Kas156B 16S ribosomal RNA gene, partial sequence [EF203198.1] SG 1 Uncultured bacterium clone Kas146B 16S ribosomal RNA gene, partial sequence [EF203191.1] Candidatus Branchiomonas cysticola clone GABI_14 16S ribosomal RNA gene, SG 4 partial sequence [JQ723599.1] Candidatus Branchiomonas cysticola strain A1-483-L1 16S ribosomal RNA gene, complete sequence [JN968376.1] SG 5 Uncultured beta proteobacterium clone T200910 16S ribosomal RNA gene, partial sequence [JN807444.1] SG 6 Uncultured bacterium partial 16S rRNA gene, clone H2SRC225 [FM213005.1] Uncultured bacterium clone 335-16 16S ribosomal RNA gene, partial sequence [KT386383.1] Paenalcaligenes suwonensis strain ABC02-12 16S ribosomal RNA, partial sequence [NR_133804.1 and JX217748.1] Alcaligenes faecalis strain Hin10.1 16S ribosomal RNA gene, partial sequence [KP962332.1] Paenalcaligenes sp. UN24 16S ribosomal RNA gene, partial sequence SG 7 [KP277115.1] Alcaligenes sp. ASS-1 16S ribosomal RNA gene, partial sequence [KM243661.1] Alcaligenes faecalis strain HCB8 16S ribosomal RNA gene, partial sequence [KF534477.1] Uncultured Alcaligenes sp. clone OTU-18-ABB 16S ribosomal RNA gene, partial sequence [JQ624326.1] Alcaligenes faecalis strain KH-37 16S ribosomal RNA gene, partial sequence [JQ612522.1] Uncultured beta proteobacterium partial 16S rRNA gene, clone O:RM-E3 [HE974836.1] SG 10 Uncultured bacterium clone 164 16S ribosomal RNA gene, partial sequence [EU223963.1] Uncultured beta proteobacterium gene for 16S rRNA, partial sequence, isolate: SG 11 sd-jx66 [AB690756.1] Uncultured bacterium clone WRPbac17 16S ribosomal RNA gene, partial SG 12 sequence [KT166993.1] Uncultured beta proteobacterium clone H2-OTU10 16S ribosomal RNA gene,

partial sequence [KM016252.1] Uncultured bacterium clone PAE-43 16S ribosomal RNA gene, partial sequence [JX875903.1] Uncultured bacterium isolate DGGE gel band RB1-20 16S ribosomal RNA gene, partial sequen [KT835490.1] Uncultured bacterium clone Wu-B146 16S ribosomal RNA gene, partial sequence [KJ782987.1] Uncultured beta proteobacterium clone CatInokulum034 16S ribosomal RNA gene, partial seq [KJ600259.1] Uncultured bacterium clone a57 16S ribosomal RNA gene, partial sequence [KJ568494.1] Uncultured bacterium clone ambient_alkaline-48 16S ribosomal RNA gene, partial sequence [GU455031.1] Uncultured bacterium clone ambient_uncontrolled-76 16S ribosomal RNA gene, SG 13 partial seque [GU454937.1] Uncultured prokaryote clone Td2-3 16S ribosomal RNA gene, partial sequence [GU208374.1] Uncultured bacterium gene for 16S ribosomal RNA, partial sequence, clone: K26J1-29 [AB504547.1] Uncultured bacterium clone Feb-eub12.2 16S ribosomal RNA gene, partial sequence [EF110598.1] Uncultured bacterium clone ISS-46 16S ribosomal RNA gene, partial sequence [EF095026.1] Uncultured bacterium clone E1d2 16S ribosomal RNA gene, partial sequence [DQ676764.1] Uncultured bacterium clone AAN22 16S ribosomal RNA gene, partial sequence SG 14 [KF428038.1] Uncultured Burkholderiales bacterium clone clone LW-3C919b 16S ribosomal SG 21 RNA gene, par [JQ254420.1]

4.1.7. APPLYING BKBJCV8 qPCR TO BJC LT GILLS OF KNOWN BK-BJC STATUS

Prior to completely validating the BKBJCV8 primers, BKBJCV8 qPCR was run on BJC

Hatchery LT gill tissues of known (2013 samples) and unknown BK-BJC status (2015 samples)

(Figure 6). Unlike the positive LT samples from conventional PCR with the BKBJCV8, the gel of BKBJCV8 qPCR products showed only one band at ~80 bp per positive sample (Figure 6).

The ~80 bp band varies in intensity with the Ct value of the sample (Figure 6). Lanes 2-5, 8 and

9 have dark bands corresponding to Ct values <35. Of those, lanes 8 and 9 are known-positive

LT gill tissue used as extraction positive tissue controls. Lanes 10 and 11 have a faint band corresponding to qPCR Ct ≥35 the minimum limit of detection and despite the band were considered functionally negative (See below). Last, fish derived samples that have no Ct value have no bands demonstrating that the BKBJCV8 probe eliminated the ~190 bp band seen on conventional PCR (Lanes 5 and 12).

Figure 6. Single qPCR product from BKBJCV8 qPCR on LT and RT samples on 2 % TBE agarose gel. The Ct values at the end of the lane correspond to the BKBJCV8 qPCR value for each sample. The known BK-BJC positive control LT from BJC in 2013 was diagnosed as positive by conventional PCR using universal 16S primers (Lanes 8 and 9).

Lanes Sample description 2,3 2015 RT, unknown BK-BJC status 4-6, 10-12 2015 LT, unknown BK-BJC status 7 Negative Control PCR Master Mix 8,9 2013 LT, known BK-BJC positive status

4.1.8. ESTABLISHING THE BKBJCV8 qPCR MINIMUM THRESHOLD

Based on the BKBJCV8 qPCR Ct values, gel electrophoresis and sequencing results, samples with Ct values ≥35 or ≤1.02E1 copies of BK-BJC have reduced amplification efficiency and consistency between the duplicate wells. Specifically, this was chosen as the limit of detection due to1) 40 % of known positive samples of synthetic BK-BJC at 1.02E1 when run in quintuplicate were positive by BKBJCV8 qPCR which is below the OIE recommended 50 %

(Caraguel et al., 2011), and 2) the last five cycles are prone to probe degradation, cross contamination, or amplification of background nucleic acids (Burns and Valdivia, 2008). The qPCR product of one Ct <35 and one Ct 35 sample were sent for sequencing. The sequencing results were slightly more variable for the Ct 35 sample than for the sample with lower Ct value; however, both were consistent with BK-BJC 16S rRNA gene sequence (Appendices 14 and 17).

This justifies considering all samples registering Ct 35 and above as functionally negative.

4.2. UTILIZING THE NOVEL BK-BJC SPECIFIC qPCR TO ANALYZE HISTORIC

SAMPLES FROM BJC LT INFECTED WITH EP

4.2.1. UNIVERSAL PCR VERSUS BKBJCV8 qPCR RUN ON HISTORIC 2010-2013 LT

GILLS FROM BJC

BKBJCV8 qPCR and standard PCR with universal primers (U1 and U1R) were used with the same extracted genomic DNA from BJC LT gills from 2010-2013 (Table 10). When comparing the two assays, the coefficient of concordance or correlation between universal PCR and

BKBJCV8 qPCR was 0.74 (max=1) with a 0.21 and 0.94 confidence interval. This suggests that there is a good degree of agreement despite having only 11 samples. Interestingly, neither PCR

Table 10. Comparison of universal PCR and BKBJCV8 qPCR assays run on identical groups of

LT. Universal PCR results were from Contador thesis (2013). Positive samples are denoted by

POS. Based on optimization, qPCR positive values were samples with Ct <35, and universal

PCR positive sample were ≥90 % similar to ‘Ca. B. cysticola’ (Contador, 2013). The total number of positive BKBJCV8 qPCR samples includes split well samples.

BKBJCV8 qPCR qPCR split well BJC LT PCR Results Results samples (Cp 35 (case #-yr) (#POS/total) (#POS/total) and POS) B037-10 0/6 0/6 — B047-10 0/8 0/8 — B034-11 4/6 4/5 1 - 35/POS B042-11 8/8 8/8 — B214-11 6/6 5/5 — B016-12 January 10/10 3/10 — B016-12 February 4/10 4/10 1 - 35/POS B002-13 Dec 3rd 5/5 5/5 — B002-13 Jan 3rd 5/5 5/5 — B002-13 Jan 8th 7/10 8/8 — B002-13 Jan 18th 7/10 10/10 —

76 nor qPCR consistently detects more positive samples and neither is considered the gold standard

(Table 10).

4.3. UTILIZING THE NOVEL BK-BJC SPECIFIC qPCR TO ANALYZE AN

EPITHELIOCYSTIS-ASSOCIATED MORTALITY EVENT IN BJC LT

4.3.1. EVENTS AT THE SAMPLING SITE, WINTERS 2012-3 AND 2014-15

During the monitoring and sample collecting period of 2012-13, the BJC Fish Culture Station experienced heavy rains, melting snow, and several days of cloudiness from December 16-19th

2012. From January 3 to February 5, 2013, the LT in tank R8 had a daily percent mortality above the acceptable range (>0.10 %). There were no notable weather or mortality events during the winter of 2015.

4.3.2. 2012 EP OUTBREAK

The BKBJCV8 qPCR was used to quantify the numbers of BK-BJC 16S rRNA gene partial sequences that are on/in the gills of LT from tank R8 at BJC Hatchery during an EP-associated mortality event from December 3, 2012 to March 21, 2013. However, we later learned that the

BKBJCV8 primers were not specific for BK-BJC, and so BK-BJC and non-target bacteria amplified by BKBJCV8 qPCR were designated the BK-BJC-like Bacteria Complex (BBC).

Figure 7 graphs the quantities of BBC in individual fish at different time points with the group median (□).

Figure 7. Fluctuations in BK-BJC load in LT during the 2013 mortality event at BJC. Note the □ denotes the group median BBC concentrations, and the colored dots represent BBC concentrations on the gills of individual fish. Dec 3rd (1), Jan 3rd (2), Jan 8th (3), Jan 18th (4), and

Mar 21st (5) are the dates sampled and the significant differences are indicted by superscript numbers (p<0.05).

Over the course of the sampling period, there was a significant increase in the amount of BBC

DNA found in or on the gills from January 3rd to January 8th (Figure 7). The copy numbers of

BBCs peaked on January 8th, and then decreased on January 18th (not significantly) and March

21st (significantly). The qPCR results from this time period were normal and symmetrically distributed. The median amount and standard deviation of BBC DNA on the LT gills sampled per date were calculated and based on post-hoc analysis. All adjusted and non-adjusted data time-points significantly differed except the first two and middle two time points: December 3rd and January 3rd, and January 8th and 18th, respectively (Table 11 and Appendix 15). The widest range of BBC loads was seen at the resolution of the mortality event on March 21st. Amounts of

BBC per fish were most similar at the time of peak mortality on January 18th.

Table 11. Adjusted p-values from paired comparison of the log-transformed median BK-BJC concentrations on LT gills on each sampling date.

Comparison of Sampling Dates 3-Dec-12 3-Jan-13 8-Jan-13 18-Jan-13 21-Mar-13 3-Dec-12 0.9244 <.0001 <.0001 0.0002 3-Jan-13 <.0001 <.0001 0.0015 8-Jan-13 0.4194 <.0001 18-Jan-13 <.0001 21-Mar-13

4.3.3. COMPARISON OF BBC QUANTIFICATION TO THE 2012-13 BJC LT MORTALITY

EVENT

The gill concentrations of BBCs were statistically correlated to the daily percent mortality during the 2013 outbreak in tank R8 at BJC Hatchery (Figure 8). Daily mortality numbers from BJC recorded from December 1, 2012 to March 31, 2013 encompass the mortality event. On day 1

(December 1st) tank R8 contained 6436 LT and on day 121 (March 31st) 3876 remained

(Appendix 16). The peak mortality occurred from January 12th to the 29st; the highest rate of mortality was on January 19th which had 4.12 % mortality or 205 deaths. The number of LT dead on each sampling day is listed in Table 12 and the trend clearly showes a gradual increase in mortality until January 18th when it peaked, and then no mortalities were seen on March 21st.

This trend in sampling dates mirrored the general daily mortality numbers trend during this time

(Figure 8 and Appendix 16).

The curve of the daily percent mortality throughout the EP-associated mortality event is similar in shape to plotted median BBC concentrations from static time points: December 3rd, January

3rd, 8th, and 18th, and March 21st (Figure 8). Both daily percent mortality and BBC concentrations hover just above zero in the months before January 3rd (the beginning of the EP-associated outbreak) and from the end of February on. However, the BBC load curve was shifted to the left of the daily percent mortality curve. The peak BBC load was on January 8th and the peak daily percent mortality was January 19th. However, the BBC concentrations at January 8th and 18th were not statistically significantly different.

Table 12. Daily percent mortality and BBC amounts in/on the gills of LT on each sampling date during the 2013 BJC EP-associated mortality event.

Daily Daily total Date Daily percent Sampled Median BBC concentration mortality in the tank (2012-13) mortality (%) LT (#) (Confidence interval) (# of LT) (# of LT) 3669.21 Dec 3rd 1 6435 0.01554 5 (1822.22 - 7388.31) 2144.61 Jan 3rd 7 6406 0.10927 5 (1065.06 - 4318.38) 110863 Jan 8th 18 6348 0.28355 8 (63749.37 - 192796.15) 61997.6 Jan 18th 157 5177 3.03264 10 (36796.55 - 104458.16) 187.68 Mar 21st 0 3878 0 4 (85.82 - 410.47)

The calculated one-tailed p-values from the Pearson and Spearman correlation coefficient tests were 0.035 (=0.071/2) and 0.067 (=0.133/2), respectively. Since the data were relatively normal for having only 5 points, use of the Pearson correlation coefficient was justified and was significant at p=0.035. This p-valve suggests a significant relationship between the concentration of BBCs on/in the gills and LT mortality. A predictive mortality equation was formulated to estimate the fold increase in the odds of mortality with a particular fold increase of BK-BJC concentration: logit=-13.3219+ 1.8609*log10(dup)+e. The calculated predictive mortality equation demonstrated that, for example, if the BBC concentration increases 10-fold, then the odds of LT mortality increase 6-fold (6.430).

4.3.4. COMPARISON OF BBC QUANTIFICATION TO HISTOPATHOLOGIC GILL

CHANGES

The gills from the same LT analyzed by BKBJCV8 qPCR were given semi-quantitative histological lesion scores by Contador et al. (2016). A statistical correlation was not possible between gill qPCR concentrations of BBCs and histopathologic lesions since there were not sufficient data points. However, analysis of the general trends showed that lamellar epithelial hyperplasia mirrored the changes in quantity of BBCs in/on the gills (Figure 9). Single cell necrosis did not significantly alter during the 2013 outbreak. Intracellular bacterial inclusions (or epitheliocystis inclusions) generally followed the fluctions in BBC quantities over the entire

2013 winter; however, paradoxically the number of EP inclusions decreased just as BBC quantities were at their peak (Figure 9).

Figure 9. Stacked comparison of median BBC concentrations (top) and semi-quantitative analysis of histopathologic changes in LT gills (bottom) during the 2013 BJC mortality event.

The scoring of the gill lesions came from the Contador et al. (2016). Note the top graph data points correspond to the median BBC concentrations in or on LT gills at each sampling time point. On the bottom graph, the orange line represents lamellar epithelial hyperplasia, the red line single cell necrosis (karyorrhexis), and the green line EP inclusions.

4.3.5. MONITORING 2014-2015 BJC LT FOR EP AND BBC

Neither an increase in concentration of BBCs in/on the LT gills, nor daily % mortality was seen at the BJC hatchery during the 2015 sampling period. Over the 10-week sampling period

(December 29, 2014 to February 9, 2015), two of 150 LT tested were qPCR positive for BBCs.

The positive LT, randomly designated fish 1 and fish 12, were both collected on December 29th from tank 1. Fish #1 had a BBC concentration of 10,200 (Ct 26.84) and fish #12 of 2,090 (Ct

29.29) copies. The qPCR amplicons for both fish were sent for sequencing and the results queried using BLASTn. The sequencing results of both fish were nearly identical to one another.

The forward sequence was 100 % identical with 100 % QC to the partial 16S rRNA gene sequence of uncultured bacterium clone BK-BJC, and the reverse sequence was 97 % identical with 97 % QC (Appendix 17). Histologic assessment of all fish revealed one fish (#7) from tank

2 on week 5 had a single epitheliocystis colony in the interlamellar space (Figure 10A). This fish was negative for BBC by BKBJCV8 qPCR.

LT from all weeks had a very mild, diffuse lymphocytic branchitis and multifocal random interlamellar hyperplasia with superficial squamous metaplasia, and rare synechiae and karyorrhexis (single cell necrosis) (Figure 10C and D). Two BK-BJC-negative fish had severe lesions—fibrin thrombi and fibrinoid vascular necrosis of unknown origin (Figure 10B).

Figure 10A-D. Histopathologic gill lesions in 2015 BJC LT. A) Epitheliocystis inclusion in the gill (black arrow) 100x; B) Fibrin thrombus at the tip of one secondary lamella (black arrow)

40x; C) One focus of interlamellar hyperplasia and fusion with superficial squamous metaplasia

(epithelialization) 40x; and D) karyorrhectic debris (black arrow heads), lamellar fusion, and epithelialization (black arrow) 60x. Scale bar on Figure A is 10 µm; and scale bars on Figures B,

C, D are 20 µm.

4.3.6. BKBJCV8 qPCR ANALYSIS OF BJC HATCHERY WATER SAMPLES

All water samples were negative for BBCs by qPCR; however, the filtration positive control and tank R8 and R1 water samples from 2013 were at the limit of detection (or functionally negative) at Ct 35. The DNA extraction positives were positive. This result points towards a potentially flawed filtration protocol. Therefore, the BK-BJC status of the water cannot be assessed.

4.4. DEVELOPMENT OF A SECOND PRIMER SET TO DISTINGUISH BK-BJC FROM ‘CA.

B. CYSTICOLA’

Since the BKBJCV8 primers could not distinguish between BK-BJC and the most genetically similar bacteria that cause fish gill disease, BKBJCV3 primers were developed to target the more divergent 397-612 bp or V3 region of BK-BJC’s 16S rRNA gene sequence (Figure 11 and

Appendix 3). In silico assessement of the BKBJCV3 primer set met all the recommended guidelines and was considered valid (Table 13).

Table 13. Description and assessment of BKBJCV3 primers based on Oligoanalyzer and Primer-

BLAST software analysis.

Self dimer, Heterodimer, Hairpin Tm Primers Sequence Length GC% Delta G Delta G Tm (ºC) (ºC) (kcal/mole) (kcal/mole) AAGGACTC Forward TTTCTAAC 22 32.9-21.8 56.83 45.45 -3.54 -7.74 AGGGAG CCTCTGCC Reverse CTACTCAA 22 14.2-31.4 56.97 45.45 -3.17 -7.74 GATAAA

Figure 11 shows the sequence differences in BK-BJC, ‘Ca. B. cysticola’ clone GABI

[JQ723599.1], Ca. B. cysticola’ strain A1-483-L1 [JN968376.1], uncultured beta proteobacterium clone T200910 [JN807444.1] in the region of the BKBJCV3 primers, and uncultured beta proteobacterium partial 16S ribosomal RNA, isolate DGGE band 8

[FM165651.1]. The primers were designed to have two and three basepair mismatches at the 3ꞌ end of the reverse and forward primers, respectively, between BK-BJC and ‘Ca. B. cysticola’, and to have ≥4 bp mismatches over both the forward and reverse primer hybridization regions.

The location and/or number of these bp mismatches have been shown to cause PCR failure and, therefore, BKBJCV3 PCR should not cross-react with any known causes of EP or proliferative gill disease. BLASTn search of the BKBJCV3 amplicon (Appendix 5) confirmed that after BK-

BJC the next closest match was ‘Ca. B. cysticola’ clone GABI at 91 % identical over 100 % of the query (Table 14).

Seven 216 bp theoretical and experimental PCR products of the BKBJCV3 primers were compared and determined to be nearly identical (Figure 12). The BLASTn search of the consensus sequence was 100 % identical to BK-BJC over 99% of the query (Table 15). The next closest match was ‘Ca. B. cysticola’ clone GABI at 91 % identical over 99 % of the query. The theoretical PCR product consensus sequences were appropriately similar. There were 12 bacteria with >90 % similarity to the BKBJCV3 amplicon and all were either theoretically and/or experimentally excluded from being amplified with BKBJCV3 PCR for having ≥4 bp mismatches in the regions of both the forward and reverse primers (Appendix 18).

Figure 11. Clustal Omega software (Sievers et al., 2011) alignment of the BK-BJC 16S rRNA gene and three genetically related fish pathogens. The BKBJCV3 forward and reverse primers are highlighted in green. Note that BranchA1=‘Ca. B. cysticola’ strain A1;

JN807444.1=Uncultured beta proteobacterium clone T200910; DGGE8=Uncultured beta proteobacterium, isolate DGGE band 8; and BranchGABI= ‘Ca. B. cysticola’ clone GABI.

*=basepair agreement and blank space=base pair differences.

BJCBK AAGGACTCTTTCTAACAGGGAGAGGCGATGACGGTACCTAATGAATAAGCACCGGCTAAC BranchA1 AAGGACTGATTCTAACAGGATCAGGAGATGACGGTACCTGAGGAATAAGCACCGGCTAAC JN807444.1 AAGGACTGATTCTAACAGGATCAGGAGATGACGGTACCTGAGGAATAAGCACCGGCTAAC BranchGABI AAGGACTGATTCTAACAGGATCAGGAGATAACGGTACCTGAGGAATAAGCACCGGCTAAC ******* ********** *** *** ********* * ******************

BJCBK TACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTAATCGGATTTACTGGGCGT BranchA1 TACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTAATCGGATTTACTGGGCGT JN807444.1 TACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTAATCGGATTTACTGGGCGT BranchGABI TACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTAATCGGATTTACTGGGCGT ********************************** *************************

BJCBK AAAGGGTGTGTAGGCGGATAGATAAGTTAGATGTGAAATACCTGGGCTTAACCTAGGGAT BranchA1 AAAGGGTGTGTAGGCGGATATTTAAGTTGGATGTGAAATACCTGGGCTTAACCGAGGAAT JN807444.1 AAAGGGTGTGTAGGCGGATATTTAAGTTGGATGTGAAATACCTGGGCTTAACCGAGGAAT BranchGABI AAAGGGTGTGTAGGCGGATATTTAAGTTGGATGTGAAATACCTGGGCTTAACCGAGGAAT ******************** ****** ************************ *** **

BJCBK TGCATTTAAAACTGTTTATCTTGAGTAGGGCAGAGG BranchA1 TGCATTTGAAACTGGATATCTAGAGTAAGGCAGAGG JN807444.1 TGCATTTGAAACTGGATATCTAGAGTAAGGCAGAGG BranchGABI TGCATTTGAAACTGGATATCTAGAGTAAGGCAGAGG ******* ****** ***** ***** ********

Table 14. BLASTn search of theoretical, ideal BKBJCV3 amplicon with ≥87 % nucleotide identity. BK-BJC is highlighted in yellow.

Description of BLAST match [Genbank Max Total QC Identity E value accession #] score score (%) (%) Uncultured bacterium clone BK-BJC 16S 399 399 100 8.00E-108 100 ribosomal RNA gene, partial sequence [KM504995.1] Candidatus Branchiomonas cysticola strain 294 294 100 4.00E-76 91 A1-483-L1 16S ribosomal RNA gene, complete sequence [JN968376.1] Uncultured beta proteobacterium clone 294 294 100 4.00E-76 91 T200910 16S ribosomal RNA gene, partial sequence [JN807444.1] Uncultured beta proteobacterium partial 16S 294 294 100 4.00E-76 91 ribosomal RNA, isolate DGGE band 8 [FM165651.1] Candidatus Branchiomonas cysticola clone 289 289 100 2.00E-74 91 GABI_14 16S ribosomal RNA gene, partial sequence [JQ723599.1] Uncultured bacterium partial 16S rRNA 250 250 87 9.00E-63 91 gene, isolate Mineral.top.1.6.1.4_322869 [LN538534.1] Uncultured bacterium partial 16S rRNA 244 244 87 4.00E-61 90 gene, isolate Mineral.top.2.1.6_343230 [LN549245.1] Uncultured Neisseriaceae bacterium clone 243 243 84 1.00E-60 91 1FG05 16S ribosomal RNA gene, partial sequence [JF460980.1] Uncultured bacterium partial 16S rRNA 241 241 87 5.00E-60 90 gene, isolate Mineral.top.1.6.1.4_230900 [LN536665.1] Comamonadaceae bacterium 71L 16S 239 239 100 2.00E-59 87 ribosomal RNA gene, partial sequence [KM020962.1]

Figure 12. Alignment of eight sequenced BKBJCV3 amplicons, including LT samples (Identities

1-7) and synthetic BK-BJC (Identity 8).

Table 15. BLASTn search of the BKBJCV3 PCR consensus sequence from eight amplicons.

BK-BJC is highlighted in yellow.

Description of BLAST match Max Total QC Identity E value [Genbank accession #] score score (%) (%) Uncultured bacterium clone BK-BJC 16S 387 387 99 6.00E-104 100 ribosomal RNA gene, partial sequence [KM504995.1] Candidatus Branchiomonas cysticola strain 300 300 99 7.00E-78 91 A1-483-L1 16S ribosomal RNA gene, complete sequence [JN968376.1] Uncultured beta proteobacterium clone 300 300 99 7.00E-78 91 T200910 16S ribosomal RNA gene, partial sequence [JN807444.1] Uncultured beta proteobacterium partial 16S 300 300 99 7.00E-78 91 ribosomal RNA, isolate DGGE band 8 [FM165651.1] Candidatus Branchiomonas cysticola clone 297 297 99 8.00E-77 91 GABI_14 16S ribosomal RNA gene, partial sequence [JQ723599.1] Uncultured bacterium partial 16S rRNA 255 255 87 3.00E-64 90 gene, isolate Mineral.top.1.6.1.4_322869 [LN538534.1] Uncultured bacterium partial 16S rRNA 255 255 87 3.00E-64 90 gene, isolate Mineral.top.2.1.6_343230 [LN549245.1] Comamonadaceae bacterium 71L 16S 253 253 99 9.00E-64 86 ribosomal RNA gene, partial sequence [KM020962.1]

4.4.1. CONVENTIONAL PCR VALIDATION OF THE BKBJCV3

The PCR assay with thermal gradient program run on BKBJCV3 primers determined the optimal range of annealing temperatures to be 52.5-62.0 °C with the most intense band at 52.5 °C

(Appendix 12). The test of the BKBJCV3 demonstrated the ability of these primers to distinguish synthetic and naturally occurring BK-BJC 16S rRNA gene (region 1-750 bp) from ‘Ca. B. cysticola’ clone GABI, as well as strain A1 and an uncultured β-proteobacterium JN807444.1 since all are identical at the primer binding sites (Figure 13).

Figure 13. 2 % TBE agarose gel of BKBJCV3 PCR products from synthetic ‘Ca. B. cysticola’, natural and synthetic BK-BJC, and negative control LT gill tissues. This gel demonstrates the ability to differentiate between BK-BJC and synthetic ‘Ca. B. cysticola’ clone GABI, and not cross-react with LT tissues. The legend below describes the samples in the gel lanes.

Lanes Sample 1,2 synthetic ‘Ca. Branchiomonas cysticola’ (10 ng/µl) 3,4 synthetic BK-BJC (10 ng/µl) 5,6 BJC LT 2013 B002-13 Dec 3 Fish #2 7,8 Negative Tissue BJC LT 2015 W1T1 Fish #11 9,10 Negative Template

4.4.2. BKBJCV3 PCR VERSUS BKBJCV8 qPCR

Applying the BKBJCV3 primers to all known status samples from BJC 2013 and 2015 LT demonstrated that all known BBC-positive samples were BK-BJC positive (Appendix 11- gel images 1-5).

4.5. GILL CULTURE ON AGAR PLATES

All negative control RT gill tissues and the homogenates made from them were confirmed to be

BK-BJC-like Bacteria Complex (BBC), including BK-BJC, negative by BKBJCV8 qPCR. The

RT gills (B139-12 fish #6-13) selected for the experiment were from an EP outbreak at Alma in

2013 and were diagnosed BBC positive by universal PCR and BKBJCV8 qPCR, as was the homogenate. The positive fish gills had Ct values and BBC copy numbers ranging from 23.24-

31.92 and 2,150-425,000, respectively.

Table 16 shows the bacterial colonies that grew categorized by agar type, incubation temperature, sample origin, and gram stain and morphologic description. BK-BJC negative

(control) RT gill tissues grew numerous colonies of various morphologies on TSA, CA, and

MacConkey agar at 15 °C and 22 °C as early as day 2 (Table 16). Yellow, tan, and orange colonies were between 1-10 in number, circular to oval, raised, and glistening with entire to undulate margins. The pink-tan colonies were dull, wrinkled, centrally pointed and had subtly undulate margins. The microscopic appearance of bacteria from the negative control gills was not consistent with the gram negative, bipolar bacilli or coccobacilli as would be expected of a

Burkholderia, and by extension BK-BJC.

The negative whole gills from 2013 grew a single, round, smooth, and glistening yellow colony of predominantly gram positive cocci with rare diplococci on CA media at 15 °C. No bacteria grew from the BK-BJC positive whole gills. No bacteria grew on cepacia agar from any gill materials and at any temperature.

One, white, rhizoid, umbonate colony with undulate margins grew on day 11 on CA agar at 22

°C from the whole BK-BJC positive gills (Table 16 and Figure 14A). Gram stain of the bacteria forming this colony showed gram positive bipolar rods (Figure 14B). The morphology of this colony was dissimilar to any other colonies grown from any gill samples. A subculture of this bacterium was cultivated on CA and submitted to AHL for speciation by MALDI-TOF mass spectrometry. It was identified as a bacterium of the genus Bacillus. All bacterial colonies from

TSA and CA plates were BKBJCV8 qPCR negative.

Table 16. Results of the growth on all agar culture plates at 15 and 22 °C for all BK-BJC positive and negative whole and homogenized gill tissues.

Temp Colony Gram Media Sample Morphology (°C) color Stain Yellow, translucent POS small rods White-tan NEG medium length, plump rods NEG Control 15 Homogenate NEG, (2015) 10% Pink POS chains of small cocci TSA long, thin rods with Orange POS alternating dark and light bars Pink NEG medium length, plump rods NEG Control angular rods alternating dark 22 Homogenate Orange A POS and light bars (2015) large cocci and var. length Orange B NEG rods Yellow NEG plump coccobacilli NEG Control thin, long rods of variable Homogenate Orange POS length 15 (2015) White-tan NEG plump, medium length rods NEG Whole variably staining cocci, rare Gill (2013) Yellow POS diplococci Tan NEG plump, medium length rods CA plump, medium length rods NEG Control with alternating dark and Homogenate Orange POS light bars (2015) 22 Yellow A POS small cocci Yellow B NEG plump, medium rods POS Homogenate (2013) White POS medium length, bipolar rods NEG Contol 15 Homogenate (2015) Light pink NEG plump, short rods MacConkey NEG Control Light pink NEG plump, medium length rods 22 Homogenate medium-long, bulb shaped, (2015) rods with alternating dark and Dark pink NEG light bars 15 & BD cepacia No growth 22

Figure 14A and B. Bacterial colony from a BK-BJC-positive gill homogenate grown on CA at 22

°C for 11 days. Macroscopic and microscopic images of the cells gram stained.

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5. DISCUSSION

The first purpose of this study was to demonstrate the association of BK-BJC and EP-linked mortality events in Ontario cultured LT through the development and use of a specific qPCR.

The second was to grow BK-BJC on selective or non-selective culture media.

5.1. DEVELOPMENT AND THEORETICAL ASSESSMENT OF BKBJCV8

The BKBJCV8 primer set was developed from the 16S rRNA gene of BK-BJC, (Contador et al.,

2016) and Geneious software was used to select the location. However, the primers and hydrolysis probe chosen were found to be not entirely specific. For example, the BKBJCV8 probe was a perfect match for BK-BJC and ‘Ca. B. cysticola’ clone GABI_14 [JQ723599.1] 16S rRNA gene, and the subsequent 268 BLAST matches differed at the terminus of the 5ꞌ end.

Basepair mismatches in the 5ꞌ location are unli ely to have an adverse effect on the PCR, and it is postulated that they may even increase PCR efficiency (Lefever et al., 2013). In fact, the synthetic oligonucleotide representing ‘Ca. B. cysticola’ clone GABI, which differs from BK-

BJC by one bp mismatch in the 5ꞌ end of the reverse primer (G for A), had an equivalent Ct value to BK-BJC using the BKBJCV8 qPCR assay.

The large number of BLAST matches for the BKBJCV8 probe suggests that this was not an ideal location for primer design. Primer design—which in turn affects primer-template association and dissociation kinetics, formation of secondary structures, and primer-template complementarity— determines the specificity, sensitivity, and efficiency of a PCR reaction and, therefore,

101 inappropriate primer and probe designs can lead to skewed results or PCR failure (Stadhouders et al., 2010; Wang and Qian, 2009).

The entire 81 bp BKBJCV8 amplicon had approximately twenty thousand different bacterial

16S rRNA sequences in Genban with ≥90 % nucleotide identity and 264 with ≥95 % similarity.

The ≥95 % identical sequences had between one and seven, randomly distributed bp mismatches

(though never at the 3’ end) in the regions of the primers and probe. Most commonly 2-4 of the mismatches were in the center to 5ꞌ end of the probe (Appendix 11). The effect of these particular mismatches to qPCR efficiency and Ct values has not been studied. However, generally, bp mismatches will decrease the thermal stability of the primer-template duplex and, hence, decrease the efficiency and specificity of the qPCR (Stadhouders et al., 2010). Lefever et al.

(2013) found that ≥4 bp mismatches in one primer, or 3 mismatches in one primer and 2 mismatches in the other led to PCR failure. When location of the mismatches was taken into account, bp mismatches greater than 5 bp from the 3′ end of the primer moderately effect qPCR amplification and did not cause PCR failure (Lefever et al., 2013).

5.2. UTILIZING SYNTHETIC OLIGONUCLEOTIDES TO DETERMINE THE NUMBER OF

NON-TARGET BACTERIA AMPLIFIED BY BKBJCV8 qPCR

When the BKBJCV8 qPCR was run on the synthetic oligonucleotides representing the 21 sequence groups (SGs), 11 SGs (1, 4-7, 10-14, and 21) were detected. These 11 SGs corresponded to at least 33 different bacterial sequences in Genbank. The slopes and heights of amplification curves of the positive oligonucleotides were typically more gradual and lower than

BK-BJC (P3), except SG 6 and SG 4 (‘Ca. B. cysticola’ clone GABI). This reduced reaction

102 efficiency may be due to the reduced efficiency of primer/probe hybridization to non-target samples with bp mismatches (Wu, Hong, and Liu, 2009). Further research should be conducted to see if it is possible to discriminate between positive samples by the slope of the amplification curves and, therefore, to determine the bacteria (or sequence group) present in an unknown sample. SG 4 and SG 5 were the synthetic 90 bp oligonucleotides for ‘Ca. B. cysticola’. As they differed from BK-BJC at only one to two nucleotide residues at the 5ꞌ end of the reverse

BKBJCV8 primer and the center of the probe, they were theoretically expected to be positive by

BKBJCV8 qPCR. This non-target amplification was experimentally demonstrated and ‘Ca. B. cysticola’ clone GABI had identical amplification efficiency when compared to BK-BJC.

The common pattern of bp mismatches that all negative or unrecognized sequence groups possessed was three bp mismatches at locations 3, 6, and 13, or, generally, in the central to 5ꞌ end of the 25 bp probe (Appendix 11). This finding was in contrast to the ABI Primer Express software v2.0 guidelines for designing probes to discriminate between alleles, which recommends orienting the SNP(s) in the center third of the probe, or, if not, then nearer to the 3ꞌ end to differentiate alleles (Malkki and Petersdorf, 2012). This suggests that non-target SNPs in the center to 3ꞌ of the probe are less likely to be recognized than at other locations. However,

Chen and Sullivan (2003) acknowledge that despite guidelines, the only way to know the ability of a primer set to differentiate between sequences with SNPs is empirical testing and optimization. Greater numbers of mismatches in other locations on the probe did not result in a negative sample, e.g. P21.

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Additionally, the pattern of primer mismatches appeared to be irrelevant. Based on the mismatch criteria to block PCR amplification enumerated by Lefever et al. (2013), this may be due to the fact that mismatches in the primers were never ≥4, nor were there more than 3 mismatches in one primer and 2 mismatches in the other. Therefore, there are possibly more than 33 cross-reacting bacteria within the uninvestigated, <95 % identical bacteria. The known cross-reacting bacteria are environmental often from aquatic sediments, and, therefore, their presence in/on the LT gills or contamination of the DNA extraction cannot be ruled-out. However, it has been demonstrated that the bacterial composition of the gills is dramatically different from that of the environment

(water) (Trust, 1975; Wang et al., 2010).

5.3. DEVELOPMENT OF BKBJCV3 TO DISTINGUISH BK-BJC FROM ‘Ca. B.

CYSTICOLA’

To differentiate BK-BJC from ‘Ca. B. cysticola’ clone GABI [JQ723599.1], ‘Ca. B. cysticola’ strain A1-483-L1 [JN968376.1], and an uncultured β-proteobacterium [JN807444.1], a second primer set, BKBJCV3, was developed over a region where their 16S rRNA sequences most diverge. Fortuitously, this design also eliminated amplification of the uncultured beta proteobacterium partial 16S ribosomal RNA, isolate DGGE band 8 [FM165651.1]. BKBJCV3 primers were constructed with three and two 3ꞌ bp mismatches in forward and reverse primers, respectively, to disrupt the polymerase binding and extension site, and minimize that likelihood of amplifying the four aforementioned non-target bacteria (Stadhouders et al., 2010). According to Stadhouders et al. (2010), the terminus at the 3ꞌ end of the primer is the most deleterious location for a mismatch and may result in 2-fold decrease in qPCR concentrations or complete

104 failure. This is partially dependent on the length of the primers with the effect decreasing in severity as the primer length extends from 20 to 30 bp (Simsek and Adnan, 2000).

BLAST matches of BKBJCV3 216 bp amplicon have 12 bacteria with >90 % identity. All were theoretically and experimentally excluded from non-target amplification with BKBJCV3 PCR due to having ≥4 bp mismatches in both the region of the forward and reverse primer (Appendix

18). However, this should be experminentally verified.

PCR using the BKBJCV3 was applied to all BKBJCV8 qPCR positives and one negative sample. 100 % of the samples indentified as BBC positive by BKBJCV8 qPCR were BK-BJC positive by BKBJCV3 PCR (Appendix 13- gel images 1-5). This confirms that BK-BJC is part of the BK-BJC-like bacteria complex and is present in all BKBJCV8 qPCR positive BJC LT gills. The LT gill tissue negative control was negative. Further demonstrating the presence of

BK-BJC in the LT gills with epitheliocystis, the samples diagnosed positive by universal 16S

PCR had a 0.74 coefficient of concordance (confidence interval: 0.21-0.94) when compared to

BKBJCV8 qPCR (Table 10). This level of concordance is considered a good positive agreement though not perfectly correlated (Kwiecien et al., 2011). Some degree of discordance is expected when comparing two different diagnostic tests with biological materials (William Sears, personal communication, March 2016). Without a gold standard or a consistent trend in the difference between the results and with 11 groups of samples to compare, it is only possible to declare that the two PCRs are in agreement, and not that either are correct (Kwiecien et al., 2011). The lack of agreement between the assays may be due to the use of different primer target regions,

105 different PCR efficiencies or thresholds of detection, or differential deterioration of the samples over the years in storage.

5.4. BKBJCV8 qPCR ON 2013 BJC LT DURING AN EP-ASSOCIATED MORTALITY

EVENT

Despite issues with specificity, the BKBJCV8 qPCR proved to be repeatable and highly sensitive

(10 copies of BBC in 250 ng/µl of genomic DNA) and was used to assess the gills of LT from a mortality event during winter 2012-13. That winter BJC Hatchery LT experienced an EP- associated mortality event and all LT sampled were positive for BBC. The differences in BBC quantification between the time-points, excluding December 3rd vs January 3rd, and January 8th vs 18th, demonstrate significant changes in amount of BBCs over the course of the mortality event. Though there was a significant relationship (p=0.0354) between the concentration of

BBCs on/in the gills and LT mortality, the exact amount of BBC that causes death, and which bacterium(a), if any, from the BBC is responsible for the increase risk have yet to be determined.

5.5. BBC CONCENTRATIONS COMPARED TO HISTOLOGIC GILL LESIONS

A statistical correlation between qPCR and histology was not possible due to the paucity of data points (n=5). Lamellar hyperplasia and single cell necrosis are commonly seen in EP and PGI

(Steinum et al., 2009; 2010; Camus et al., 2013; Mendoza et al., 2013), they are non-specific histologic changes that may result from a variety of respiratory insults. Additionally, in the literature there is no consistent relationship between EP and host gill reactions. It is postulated that gill pathology in response to EP inclusions may depend on the bacterial agent involved. For

106 example, ‘Ca. P. salmonis’ induces lamellar hyperplasia but ‘Ca. C. salmonicola’ does not

(Schmidt-Posthaus et al., 2011).

There was a decrease in the number of EP inclusions at peak BBC concentrations on January 8th; however, EP inclusions were most numerous on January 18th when BBC concentrations were the second highest and LT deaths were reaching their peak (on January 19th). Nearing the end of the mortality event, the last sampling time point (March 21st) showed both the number of EP inclusions and the amount of BBC approaching zero. The relationship between detectable EP inclusions and BBC loads on the gill is not straightforward and difficult to assess with only five time points in 2013 and no information from other years. Furthermore, in 2015 a single EP inclusion was found in an BKBJCV8 qPCR negative LT further demonstrating little relationship between the two assay results, and potentially the BBC bacteria and EP (Figure 10A).

However, the lack of relationship between the number of EP inclusions and qPCR positivity of affected fish is supported by the literature (Steinum et al., 2010; Seth-Smith et al., 2016; Guevara

Soto et al., 2016). Guevara Soto et al. (2016) hypothesized that this may be due to either EP- associated pathogens infecting the host but only occasionally forming EP inclusions, or different sampling sites on the gill or uneven distribution of the EP inclusions resulting in inconsistent histologic and qPCR findings. This inverse relationship could also be explained by high environmental or microenvironmental (host body surface) concentrations from recent inclusion rupture. Greater numbers of samples collected at more frequent time periods would determine if the inverse relationship between EP inclusions and BBC load is at some time points an artifact.

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5.6. BKBJCV8 qPCR ON 2015 BJC LT DURING A NORMAL YEAR

In the LT samples collected at BJC Hatchery from 2014-15, two of 150 fish were positive with

BBC copy numbers 10,200 and 2,090. These fish were from the same tank and collected on the first week of the sampling period: December 29, 2014. The BKBJCV8 qPCR products from both fish were sent for sequencing and the results were queried on BLASTn. Both were identical to

BK-BJC in the forward sequence and similar to BK-BJC in the reverse (97 % nucleotide similarity and QC) (Appendix 17). These two BBC positive fish are significant because they neither have detectable EP inclusions, nor are associated with increased mortality at BJC. As previously stated, this lack of relationship between EP inclusion numbers and BBC load quantification is well documented (Steinum et al., 2010; Seth-Smith et al., 2016; Guevara Soto et al., 2016); however, Mitchell et al. (2013) found a significant correlation (gamma=0.62) between numbers of EP inclusions and ‘Ca. B. cysticola’ concentrations on the gills of Atlantic salmon.

Many hypotheses have been proposed to explain the lack of relationship between EP inclusions and BBC loads. They range from sampling error to alterations in EP pathogens’ mechanisms of infection; however, the lack of correlation may suggest that BK-BJC is an incidentally discovered, non-pathogenic gill bacteria. Recently, symbiotic intracellular β-proteobacteria have been identified in clusters in common carp gill epithelial cells (van Kessel et al., 2016). These bacteria are thought to be involved in ammonia oxidizing and nitrogenous waste excretion in the gills (van Kessel et al., 2016). Interestingly, common carp in Hungary, Israel, Portugal, and

South Korea have been documented with EP associated with CLOs and/or RLOs (Molnar and

Boros, 1981; Paperna and Alves de Matos, 1984; Kim et al., 2005). The difference between these bacterial clusters and EP inclusions is a single versus a double membrane lining the inclusion, respectively (van Kessel et al., 2016; Toenshoff et al., 2012). However, the visibility of the

108 inclusion membrane varies greatly with the preservation of the tissue samples. Further research is needed to determine whether BK-BJC causes LT gill intracellular inclusions, and whether those inclusions are related to EP infection or co-habitating ammonium-excreting symbionts.

When comparing BBC load to LT mortality events, typically 40-100% of the fish tested are positive, not merely two (Contador, 2013). Conversely, in 2010, a sample year when the mortality rates were low, zero of 14 fish were positive. Therefore, it is unusual for BJC LT to have PCR positive fish in a year with no mortality event; however, subclinical EP positive fish are well documented in studies on chlyamdial agents of EP (Schmidt-Posthaus et al., 2011;

Stride et al., 2013; Guevara Soto et al., 2016). It is also possible that these two positive LT were found because of the much larger sampling numbers in 2015.

5.7. BKBJCV8 qPCR ON BJC WATER SAMPLES

In our study, no environmental BBCs were found in the water taken from various locations in the

BJC hatchery during the 2012 mortality event and winter of 2015 as all samples were functionally negative with a Ct of zero or ≥35. The inability to find our EP-associated bacterium in the water column is consistent with the literature (Draghi et al., 2010; Katharios et al., 2015).

Draghi et al. (2010), from whom our protocol was based, did not find any EP-associated

Chlamydia despite identifying 54 Chlamydiales 16S rRNA gene sequences by PCR with

Chlamydiales-specific primers. There are several reasons for not identifying BBC in the water column, such as low or transient amounts of BBCs in the water column (Draghi et al. 2010), or lack of a waterborne life stage.

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However, the filter positive control, which consisted of BK-BJC positive homogenized gills in

500 ml of sterile water, was also functionally negative. Therefore, our finding may be attributable to a suboptimal filtration protocol or below threshold concentrations of BBC. Since

BK-BJC is a ~0.5–2.5 µm rod shaped bacterium (Contador et al., 2016), it is unlikely that it passed through the 0.2 pore sized filters used in 2013; however, the qPCR negative, positive control was filtered through 0.1 pore size filter. It is also possible that the cells in the gill samples were degraded or lysed which allowed cellular DNA to slip through the filter. Future studies should utilize DNA isolation kits specific for water or soil samples.

Free living BK-BJC in the water column is of particular interest, because several pathogenic

Burkholderia are commonly found in the water column including around aquaculture facilities, and this may suggest an ecological niche and mode of transmission (Miranda and Zemelman,

2002; Minogue et al., 2015; Knappik et al., 2015; Kenzaka et al., 1998; Resende et al., 2015).

Several epitheliocystis researchers have postulated an increased risk of EP after high levels of SS in the water column (Corsaro and Venditti, 2004; Agamy, 2013; Olapade et al., 2005). BJC experienced heavy rains and abrupt snow melts leading to turbid water several days before the onset of an EP-associated mortality event in 2012 and not in 2015 (Contador, 2013). The niches of BK-BJC need further investigation to better understand the ecology, environmental distribution, prevalence of the bacterium, and to prevent exposure of susceptible LT.

5.8. CULTURE OF BBC

None of the 54 different EP-associated bacteria detected by PCR were successfully grown on agar or in cell culture. This inability to culture the agent has been the largest impediment to

110 understanding and combatting this syndrome and the agents associated with it. Without the ability to culture the bacteria associated with EP, recapitulation of the disease in experimental studies cannot be demonstrated. Furthermore, since natural occurrence of the disease is relied upon to acquire the bacteria, there is no consistent source of live EP-associated bacteria to study.

Consequently, little is known about their physiology, environmental niches, or roles in incidental and pathologic forms of EP. This knowledge is crucial to predict, prevent, and formulate treatment strategies for EP. From a medical perspective, Bhattacharya, Vijayalakshmi, and Parija

(2002) point out that culture is essential to determine antibiotic sensitivities, to produce antigens for testing, and to formulate vaccines.

Attempts to use agar culture to grow and isolate BK-BJC from whole and homogenized qPCR

BK-BJC positive gills collected from Alma Aquaculture Research Station in 2012 was unsuccessful. In fact, only one Bacillus sp. colony was grown from BK-BJC-positive homogenized gills, and no bacteria grew from BK-BJC-positive whole gills. Furthermore, the whole gills from BK-BJC-negative Alma RT sampled on that day grew one bacterial colony composed of gram positive cocci. In all, two colonies were grown from the gill tissues collected at Alma in 2012. This lack of growth compared to myriad of colonies grown from the negative control RT gills from 2016 indicates that there were few viable bacteria on/in the 2012 gills as virtually sterile gills are not found in nature. The gills are home to large populations of bacteria from numerous genera (Cahill, 1990). Therefore, the sterility of the gills may be due to sample processing such as the long term frozen storage at -20 °C, a suboptimal temperature, or thawing events over the 3 years storage. It is standard practice to maintain tissue samples at -80 °C

(Cuthbertson et al., 2015), and not doing so may have compromised the viability of the bacteria

111 within the tissue. Interestingly, Cuthbertson et al. (2015) found that multiple freeze–thaw cycles did not alter the overall bacterial diversity of a sample detected by pyrosequencing, but did disproportionately, adversely affect the rarer bacteria species after 3 freeze-thaw cycles.

However, inappropriate culture conditions—such as suboptimal incubation temperatures, omission of essential nutrients, or requirement for an intermediate life stage within amoeba— cannot be discounted. For example, recent studies of Burkholderia cepacia and B. pseudomallei demonstrated that both are capable of intra-amoebic survival and suggest that amoebas may play a role in the diseases these bacteria are associated with (Marolda et al., 1999 and Inglis et al.,

2000, respectively). Additionally, amoebic co-culture with rainbow trout gill cells (RTgill) in vitro facilitates transmission of Neoparamoeba pemaquidensis (Lee et al., 2006). Therefore, it is possible that BK-BJC requires interaction with amoeba to complete its life cycle.

Growth of a Bacillus sp. from the gill homogenate is not unusual as they are routinely found in fresh water and at a high incidence in the intestinal tracks of reared RT (Gonzalez et al., 1999).

Therefore, the Bacillus sp. is likely a hardy external contaminant, and potentially the only viable bacteria in the tissue because it is a sporeformer (Nicholson et al., 2000). Future studies should use fresh or rapidly frozen to -80 °C EP-positive gill samples and should consider immersing the gills in 70 % ethanol for 5 min before culture to reduce likelihood of growing gill surface or contaminating bacteria (Zouache et al., 2009).

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6. CONCLUSION

The development of a BK-BJC-specific qPCR is crucial for the study of BK-BJC, a presently unculturable intracellular bacterium, repeatedly identified from BJC LT gills during epitheliocystis-associated die-offs. The recently discovered BK-BJC is minimally characterized and attempts to cultivate it on various types of culture media and in fish cell lines have been unsuccessful to date (Contador, 2013 and personal unpublished research). In lieu of ISH or IHC to localize and detect BK-BJC, qPCR is currently the best assay available to study, diagnose, track, and correlate BK-BJC concentrations with epitheliocystis inclusions and LT mortality in

Ontario.

Our hypotheses were

1. qPCR will demonstrate that BK-BJC is present in LT with epitheliocystis and is not

present in healthy LT from BJC Fish Culture Station.

2. BK-BJC quantity in the gills of BJC LT will significantly increase during an EP-

associated mortality event, and decrease below threshold at the conclusion of the event.

3. BK-BJC concentrations in/on the gills of BJC LT have a significant direct correlation

with changes in the daily percent mortality, i.e. they will proportionally increase and

decrease simultaneously.

4. The burden of BK-BJC in/on the gills will increase with severity of all histopathologic

lesions over the course of an EP-associated mortality event.

5. BK-BJC will be present in the water column around BJC Fish Culture Station at higher

levels during EP-associated mortality events.

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6. BK-BJC can be cultured from infected RT gills using selective and non-selective media.

At the conclusion of our study, the absolute quantifications from BKBJCV8 qPCR were proven not valid due to non-specific cross-reaction with a minimum of 33 different BBC 16S rRNA gene sequences. So we could neither confirm hypotheses 1 to 4, nor demonstrate a causal association between BK-BJC and EP or LT mortality events. However, it was possible to demonstrate that BBC, including BK-BJC, was present in LT during an EP-associated mortality event and in 1 % of healthy LT during a non-outbreak year. Moreover, the number of BBCs significantly changed during the course of an EP outbreak, but did not drop below the threshold of detection at the end of the outbreak. BBC quantities significantly fluctuated with the daily percent mortality, and, though no statistical correlation could be made, mirrored the changes in gill lamellar hyperplasia during outbreak winter 2013. No relationship was found between BBC concentrations and number of EP inclusions identified histologically either in winter 2013 or

2015.

BBC, including BK-BJC, was not found (at the limit of detection) in water samples collected from multiple sites around BJC in 2013 and 2015. However, this was likely due to a suboptimal filtration protocol. Therefore, the hypothesis that BK-BJC can be found in the water around BJC

Fish Culture Station at higher levels during mortality events cannot be commented on. Future research should utilize alternate methods to extract bacterial DNA from water and aquatic sediment.

Finally, hypothesis 6, based on research done here, was neither refuted nor confirmed.

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Future studies should focus on developing the BKBJCV3 into a qPCR specific for BK-BJC and retesting of the BJC 2013 and 2015 samples should clarify BK-BJCs role in the EP-associated mortality events in Ontario LT. Once validated, the BKBJCV3 should replace the BKBJCV8 primers for BK-BJC diagnostics as they have been proven to be more specific. Additionally, future studies should focus on more robust, year round sampling at regular intervals from the two hatcheries with annual EP outbrea s. ‘Ca. B. cysticola’ specific primers should also be developed to rule out the presence of the two strains and β-proteobacterium JN807444.1 in BJC

LT. Last, to crystalize the causal relationship of BK-BJC, EP, and mortality, direct methods of

BK-BJC detection, such as IHC, ISH, and cell culture, are a critical next step.

Research into BK-BJC should continue because little is known about this elusive bacterium, and its links to EP and significant annual die-offs of reared LT and RT in Ontario. As potentially the first fresh water pathogenic Burkholderiales in fish, knowledge of BK-BJC’s physiology, ecological niche, virulence strategies, and antibiotic sensitivities will be instrumental to help

Ontario aquaculture facilities prevent devastating future outbreaks.

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8. APPENDICES

8.1. APPENDIX 1: PCR PRIMER SETS AND SEQUENCES.

Forward Primers Sequence (5ꞌ-3ꞌ) Reference Reverse U1 ACGCGTCGACAGAGTTTGATCCTGGCT Relman, 1993 U1R GGACTACCAGGGTATCTAAT Universal 27f AGAGTTTGATCMTGGCTCAG Weisburg et al., 1991 U1492R GGTTACCTTGTTACGACTT BKBJCV8F TGAATACGTTCCCAGGTCTTG BKBJCV8R TGCGGTTAGACTACCTGCTTC unpublished BK-BJC BKBJCV8 specific probe GTCACACCATGGAAGTGGGGTTGAC BKBJCV3F AAGGACTCTTTCTAACAGGGAG unpublished BKBJCV3R CCTCTGCCCTACTCAAGATAAA

135

8.2. APPENDIX 2: PARTIAL SEQUENCE OF THE 16S rRNA GENE FROM BK-BJC (1503 BP). This sequence was deposited into GenBank and assigned the accession number KM504995.1.

1 TAGAGTTTGATCATGGCTCAGATTGAACGCTGGCGGCATGCTTTACACATGCAAGTCGAA 61 CGGCAGCGGTATATATTTTCGGATATATAGCCGGCGAGTGGCGGACGGGTGAGTAATACA 121 TCGGAACATGACCATTAGTCTGGGATAACTTAGCGAAAGATAGGCTAATACCGGATGAGC 181 TTGAGAAAGGAAAGTCTGGGACCTGAGAGGGCCAGATGCTAAAGGGGTGGCTGATGACTG 241 ATTAGCTAGTTGGTGTGGTAAAAGCGCACCAAGGCGACGATCGGTAGCTGGTCTGAGAGG 301 ATGACCAGCCACACTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGG 361 AATATTGGACAATGGGGGAAACCCTGATCCAGCGATGCCGCGTGAGTGAAGAAGGCTTTA 421 GGGTTGTAAAGCTCTTTTATTAGGGAAGAAAGGACTCTTTCTAACAGGGAGAGGCGATGA 481 CGGTACCTAATGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGG 541 TGCAAGCGTTAATCGGATTTACTGGGCGTAAAGGGTGTGTAGGCGGATAGATAAGTTAGA 601 TGTGAAATACCTGGGCTTAACCTAGGGATTGCATTTAAAACTGTTTATCTTGAGTAGGGC 661 AGAGGGAGGTAGAATTCCATGTGTAGCAGTGAAATGCGTAGATATATGGAGGAATACCAA 721 TGGCGAAGGCAGCCTCCTGGGCCACTACTGACGCTGAGGCACGAAAGCGTGGGGAGCAAA 781 CAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCGACTAGTTGTTGGGGTGAA 841 ATACCTTAGTAACGAAGCTAACGCGTGAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGT 901 TAAAACTCAAAGGAATTGACGGGGACCTGCACAAGCAGTGGATGATGTGGATTAATTCGA 961 TGCAACGCGAAGAACCTTACCTACCCTTGACATGTATGGAATCCTGAAGAGATTTGGGAG 1021 TGCCCGCAAGGGAACCATAACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGA 1081 TGTTGGGTTAAGTCCCGCAACGAGCGTAACCCTTGTTGTCAGTTGCTACGAAAGAGCACT 1141 CTGGCGAGACTGCCGGTGACAAATCGGAGGAAGGTGGGGACGACGTCAAGTCCTCATGGC 1201 CCTTATGGGTAGGGCTTCACACGTCATACAATGGCTGGTACAGAGGGGAGCGAAACCGCG 1261 AGGTGGAGCGAATCCCAGAAAGCCAGTCGTAGTCCGGATCGCAGTCTGCAATTCGACTGC 1321 GTGAAGTCGGAATTGCTAGTAATCGCGGATCAGAATGCCGCGGTGAATACGTTCCCAGGT 1381 CTTGTACACACCGCCCGTCACACCATGGAAGTGGGGTTGACCAGAAGCAGGTAGTCTAAC 1441 CGCAAGGAGGGCGCCTGCCACGGTGAGCTTCATGACTGGGGTGAAGTCGTAACAAGGTAA 1501 CCA

136

8.3. APPENDIX 3: CLUSTAL ALIGNMENT OF BK-BJC AND ‘CA. B. CYSTICOLA’ CLONE GABI’S 16S rRNA GENE SEQUENCE WITH LOCATION OF THE BKBJCV3 AND BKBJCV8.

partialBK-BJC ------AGTCGAACGGCAGCGGTATATATT-- 24 Bcystseq agattgaacgctggcggcatgctttacacatgcaagttgaacggcaacgcgacatagtgc 60 *** ********.** * *** *

partialBK-BJC TTCGGATATATAGCCGGCGAGTGGCGGACGGGTGAGTAATACATCGGAACATGACCATTA 84 Bcystseq ttgcactatgttggcggcgagtagcggacgggtgagtaatacatcggaacgtgtctagtg 120 ** ..***.*:* ********.***************************.**:* * *.

partialBK-BJC GTCTGGGATAACTTAGCGAAAGATAGGCTAATACCGGATGAGCTTGAGAAAGGAAAGTCT 144 Bcystseq gtgggggataacttagcgaaagttaagctaataccgcataagcttgggagaggaaagcct 180 ** ******************:**.********** **.******.**.******* **

partialBK-BJC GGGACCTGAGAGGGCCAGATGCTAAAGGGGTGGCTGATGACTGATTAGCTAGTTGGTGTG 204 Bcystseq gggaccgag—aggccaggcgccacatgagcggctgatggctgattagctagttggtagg 238 ****** .. .******. ** *.* *.* ********.*****************. *

partialBK-BJC GTAAAAGCGCACCAAGGCGACGATCGGTAGCTGGTCTGAGAGGATGACCAGCCACACTGG 264 Bcystseq gtaagagcctaccaaggctacgatcagtagctggtctgagaggatgaccagccacactgg 298 ****.*** ******** ******.**********************************

partialBK-BJC GACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGG 324 Bcystseq gactgagatacggcccagactcctacgggaggcagcagtggggaatattggacaatgggg 358 ************************************************************

partialBK-BJC GAAACCCTGATCCAGCGATGCCGCGTGAGTGAAGAAGGCTTTAGGGTTGTAAAGCTCTTT 384 Bcystseq gcaaccctgatccagcgatgccgcgtgagtgaagaaggctttagggttgtaaagctcttt 418 *.**********************************************************

partialBK-BJC TATTAGGGAAGAAAGGACTCTTTCTAACAGGGAGAGGCGATGACGGTACCTAATGAATAA 444 Bcystseq tatcaggggcgaaaggactgattctaacaggatcaggagataacggtacctgaggaataa 478 *** ****..********* :**********.: ***.***.*********.* ******

partialBK-BJC GCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTAATCGGA 504 Bcystseq gcaccggctaactacgtgccagcagccgcggtaatacgtagggtgcgagcgttaatcgga 538 **********************************************.*************

partialBK-BJC TTTACTGGGCGTAAAGGGTGTGTAGGCGGATAGATAAGTTAGATGTGAAATACCTGGGCT 564 Bcystseq tttactgggcgtaaagggtgtgtaggcggatatttaagttggatgtgaaatacctgggct 598 ******************************** :******.*******************

partialBK-BJC TAACCTAGGGATTGCATTTAAAACTGTTTATCTTGAGTAGGGCAGAGGGAGGTAGAATTC 624 Bcystseq taaccgaggaattgcatttgaaactggatatctagagtaaggcagaggggggtggaattc 658 ***** ***.*********.****** :*****:*****.*********.***.******

partialBK-BJC CATGTGTAGCAGTGAAATGCGTAGATATATGGAGGAATACCAATGGCGAAGGCAGCCTCC 684 Bcystseq catgtgtagcagtgaaatgcgtagagatatggaggaacaccgatggcgaaggcagccccc 718 ************************* *********** ***.*************** **

partialBK-BJC TGGGCCACTACTGACGCTGAGGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTG 744 Bcystseq tgggttattactgacgctgaggcacgaaagcgtggggagcaaacaggattagataccctg 778 **** * ****************************************************

partialBK-BJC GTAGTCCACGCCCTAAACGATGTCGACTAGTTGTTGGGGTGAAATACCTTAGTAACGAAG 804 Bcystseq gtagtccacgctgtaaacgatgtcgactagttgttggggcaaatagccttagtaacgaag 838 *********** ************************** .**::.**************

partialBK-BJC CTAACGCGTGAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAAGGAATT 864 Bcystseq caaacgcgtgaagtcgaccgcctggggagtacggccgcaaggttaaaactcaaaggaatt 898 *:**********************************************************

partialBK-BJC GACGGGGACCTGCACAAGCAGTGGATGATGTGGATTAATTCGATGCAACGCGAAGAACCT 924 Bcystseq gacggggacctgcacaagcagtggatgatgtggattaattcgatgcaacgcgaagaacct 958 ************************************************************

137

partialBK-BJC TACCTACCCTTGACATGTATGGAATCCTGAAGAGATTTGGGAGTGCCCGCAAGGGAACCA 984 Bcystseq tacctacccttgacatgtgcggaagctgtaagagattacggcgtgcctgagaaggagccg 1018 ******************. **** * ********: **.***** *..*.***.**. partialBK-BJC TAACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCG 1044 Bcystseq caacacaggtgctgcatggctgtcgtcagctcgtgtcgtgagatgttgggttaagtcccg 1078 *********************************************************** partialBK-BJC CAACGAGCGTAACCCTTGTTGTCAGTTGCTACGAAAGAGCACTCTGGCGAGACTGCCGGT 1104 Bcystseq caacgagcggaacccttgttgccagttgctacgtaagagcactttggcgatactgccggt 1138 ********* *********** ***********:********* ****** ********* partialBK-BJC GACAAATCGGAGGAAGGTGGGGACGACGTCAAGTCCTCATGGCCCTTATGGGTAGGGCTT 1164 Bcystseq gacaaatcggaggaaggtggggacgacgtcaagtcctcatggcccttatgggtagggctt 1198 ************************************************************ partialBK-BJC CACACGTCATACAATGGCTGGTACAGAGGGGAGCGAAACCGCGAGGTGGAGCGAATCCCA 1224 Bcystseq cacacgtcatacaatggctagtacagagggcagcgaaaccgcgaggtggagcgaatctca 1258 *******************.********** ************************** ** partialBK-BJC GAAAGCCAGTCGTAGTCCGGATCGCAGTCTGCAATTCGACTGCGTGAAGTCGGAATTGCT 1284 Bcystseq gaaagctagtcgtagtccggattgcagtctgaaactcgactgcatgaagtcggaattgct 1318 ****** *************** ********.** ********.**************** partialBK-BJC AGTAATCGCGGATCAGAATGCCGCGGTGAATACGTTCCCAGGTCTTGTACACACCGCCCG 1344 Bcystseq agtaatcgcggatcagaatgccgcggtgaatacgttcccaggtcttgtacacaccgcccg 1378 ************************************************************ partialBK-BJC TCACACCATGGAAGTGGGGTTGACCAGAAGCAGGTAGTCTAACCGCAAGGAGGGCGCC-- 1402 Bcystseq tcacaccatggaagtggggttgaccagaagcaggtagtctaaccgcgaggagggcgcctg 1438 **********************************************.*********** partialBK-BJC ------1402 Bcystseq ccacggtgagcttcatgactggggtg 1464

Legend BKBJCV8 primers and probe BKBJCV3 primers Sequence differences

138

8.4. APPENDIX 4: GBLOCK GENE FRAGMENT SEQUENCES.

8.4.1. BK-BJC REGION 1257-1402 (250 bp). AATTCGACTGCGTGAAGTCGGAATTGCTAGTAATCGCGGATCAGAATGCCGCGGTGAATACGTT CCCAGGTCTTGTACACACCGCCCGTCACACCATGGAAGTGGGGTTGACCAGAAGCAGGTAGTCT AACCGCAAGGAGGGCGCCTGCCACGGTGAGCTTCATGACTGGGGTGAAGTCGTAACAAGGTAAC CAATCACGAATTCTGGATCCGATACGTAACGCGTCTGCAGCATGCGTGGTACCGAGCT

8.4.2. BK-BJC REGION 1-750 bp. AGTCGAACGGCAGCGGTATATATTTTCGGATATATAGCCGGCGAGTGGCGGACGGGTGAGTAAT ACATCGGAACATGACCATTAGTCTGGGATAACTTAGCGAAAGATAGGCTAATACCGGATGAGCT TGAGAAAGGAAAGTCTGGGACCTGAGAGGGCCAGATGCTAAAGGGGTGGCTGATGACTGATTAG CTAGTTGGTGTGGTAAAAGCGCACCAAGGCGACGATCGGTAGCTGGTCTGAGAGGATGACCAGC CACACTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAAT GGGGGAAACCCTGATCCAGCGATGCCGCGTGAGTGAAGAAGGCTTTAGGGTTGTAAAGCTCTTT TATTAGGGAAGAAAGGACTCTTTCTAACAGGGAGAGGCGATGACGGTACCTAATGAATAAGCAC CGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTAATCGGATTTACTGG GCGTAAAGGGTGTGTAGGCGGATAGATAAGTTAGATGTGAAATACCTGGGCTTAACCTAGGGAT TGCATTTAAAACTGTTTATCTTGAGTAGGGCAGAGGGAGGTAGAATTCCATGTGTAGCAGTGAA ATGCGTAGATATATGGAGGAATACCAATGGCGAAGGCAGCCTCCTGGGCCACTACTGACGCTGA GGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTC

8.4.3. ‘CA. B. CYSTICOLA’ CLONE GABI REGION 1-750 BP. AGATTGAACGCTGGCGGCATGCTTTACACATGCAAGTTGAACGGCAACGCGACATAGTGCTTGC ACTATGTTGGCGGCGAGTAGCGGACGGGTGAGTAATACATCGGAACGTGTCTAGTGGTGGGGGA TAACTTAGCGAAAGTTAAGCTAATACCGCATAAGCTTGGGAGAGGAAAGCCTGGGACCGAGAGG CCAGGCGCCACATGAGCGGCTGATGGCTGATTAGCTAGTTGGTAGGGTAAGAGCCTACCAAGGC TACGATCAGTAGCTGGTCTGAGAGGATGACCAGCCACACTGGGACTGAGATACGGCCCAGACTC CTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGGGCAACCCTGATCCAGCGATGCCGCGT GAGTGAAGAAGGCTTTAGGGTTGTAAAGCTCTTTTATCAGGGGCGAAAGGACTGATTCTAACAG GATCAGGAGATAACGGTACCTGAGGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAA TACGTAGGGTGCGAGCGTTAATCGGATTTACTGGGCGTAAAGGGTGTGTAGGCGGATATTTAAG TTGGATGTGAAATACCTGGGCTTAACCGAGGAATTGCATTTGAAACTGGATATCTAGAGTAAGG CAGAGGGGGGTGGAATTCCATGTGTAGCAGTGAAATGCGTAGAGATATGGAGGAACACCGATGG CGAAGGCAGCCCCCTGGGTTATTACTGACGCTGAGGCACGAAAGCG

139

8.5. APPENDIX 5: BKBJCV8 AND BKBJCV3 qPCR AMPLICONS—LENGTH AND SEQUENCE.

Amplicon Length Sequence (5ꞌ-3ꞌ) primer set (bp) BKBJCV TGAATACGTTCCCAGGTCTTGTACACACCGCCCGTCACACCATGGAA 81 8 GTGGGGTTGACCAGAAGCAGGTAGTCTAACCGCA AAGGACTCTTTCTAACAGGGAGAGGCGATGACGGTACCTAATGAATA AGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGC BKBJCV 216 AAGCGTTAATCGGATTTACTGGGCGTAAAGGGTGTGTAGGCGGATAG 3 ATAAGTTAGATGTGAAATACCTGGGCTTAACCTAGGGATTGCATTTA AAACTGTTTATCTTGAGTAGGGCAGAGG

140

8.6. APPENDIX 6: REPRESENTATIVE SEQUENCES AND ORGANISMS [BY GENBANK ACCESSION NUMBER] OF THE 21 SEQUENCE GROUPS ≥95 % IDENTICAL TO THE BKBJCV8 qPCR AMPLICON.

Representative BLAST SG match Sequence [Genbank accession #] TGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCAC 1 EF203198.1 ACCATGGAAGTGGGTTGCACCAGAAGCAGGTAGTCTAA CCGCA TGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCAC 2 HG917718.1 ACCATGGGAGTGGGTTTCACCAGAAGCAGGTAGTCTAA CCGTA TGAATACGTTCCCAGGTCTTGTACACACCGCCCGTCAC 3 KM504995.1 BK-BJC ACCATGGAAGTGGGGTTGACCAGAAGCAGGTAGTCTAA CCGCA TGAATACGTTCCCAGGTCTTGTACACACCGCCCGTCAC JQ723599.1 ‘Ca. B. 4 ACCATGGAAGTGGGGTTGACCAGAAGCAGGTAGTCTAA cysticola’ clone GABI CCGCG JN968376.1 ‘Ca. B. TGAATACGTTCCCAGGTCTTGTACACACCGCCCGTCAC 5 cysticola’ strain A1 and ACCATGGGAGTGGGGTTGACCAGAAGCAGGTAGTCTAA JN807444.1 CCGCG TGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCAC 6 FM213005.1 ACCATGGAAGTGGGGTTTACCAGAAGTAGGTAGCCTAA CCGTA TGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCAC 7 KT386383.1 ACCATGGAAGTGGGTTTCACCAGAAGTAGGTAGTCTAA CCGTA TGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCAC 8 AB818471.1 ACCATGGGAGTGGGCTTCACCAGAAGCAGGTAGTCTAA CCGTA TGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCAC 9 KC836771.1 ACCATGGGAGTGGGCTTCACCAGAAGCAGGTAGTCTAA CCGCA TGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCAC 10 HE974836.1 ACCATGGGAGTGGGGTTCACCAGAAGCAGGTAGGCTAA CCGCA TGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCAC 11 AB690756.1 ACCATGGGAGTGGGGTTCACCAGAAGCAGGTAGTCTAA CCGTA TGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCAC 12 KT166993.1 ACCATGGGAGTGGGGTTTACCAGAAGCAGGTAGCCTAA CCGCG TGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCAC 13 KT835490.1 ACCATGGGAGTGGGGTTTACCAGAAGCAGGTAGCCTAA CCGCA

141

TGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCAC 14 KF428038.1 ACCATGGGAGTGGGGTTTACCAGAAGCAGGTAGGCTAA CCGCA TGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCAC 15 LC126443.1 ACCATGGGAGTGGGTTTCACCAGAAGCAGGTAGTCTAA CCGAA TGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCAC 16 AJ000344.1 ACCATGGGAGTGGGTTTCACCAGAAGCAGGTAGTCTAA CCGNA TGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCAC 17 KM210534.1 ACCATGGGAGTGGGTTTCACCAGAAGCAGGTAGTCTAA CCGCG TGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCAC 18 HQ221925.1 ACCATGGGAGTGGGTTTCACCAGAAGCAGGTAGTCTAA CCGCA TGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCAC 19 JQ254453.1 ACCATGGGAGTGGGTTTTACCAGAAGCAGGTAGTCTAA CCGCG TGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCAC 20 JQ254134.1 ACCATGGGAGTGGGTTTTACCAGAAGCAGGTAGTCTAA CCGCA TGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCAC 21 JQ254420.1 ACCATGGGAAGTGGGTTTTACCAGAAGCAGGTAGTCTA ACCGCA

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8.7. APPENDIX 7: ALIGNMENT OF BK-BJC AND EACH BACTERIAL SG WITH LOCATION OF THE BKBJCV8.

BK-BJC vs SG 1 TGAATACGTTCCCAGGTCTTGTACACACCGCCCGTCACACCATGGAAGTGGGGTTGACC-AGAAGCAGGTAGTCTAACCGCA TGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGAAGTGGG-TTGCACCAGAAGCAGGTAGTCTAACCGCA ************* ** *********************************** *** * ********************** BK-BJC vs. SG 2 TGAATACGTTCCCAGGTCTTGTACACACCGCCCGTCACACCATGGAAGTGGGGTTGACCAGAAGCAGGTAGTCTAACCGCA TGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTTCACCAGAAGCAGGTAGTCTAACCGTA ************* ******************************* ****** ** *********************** * BK-BJC vs. SG 4 (‘Ca. B. cysticola’ clone GABI) TGAATACGTTCCCAGGTCTTGTACACACCGCCCGTCACACCATGGAAGTGGGGTTGACCAGAAGCAGGTAGTCTAACCGCA TGAATACGTTCCCAGGTCTTGTACACACCGCCCGTCACACCATGGAAGTGGGGTTGACCAGAAGCAGGTAGTCTAACCGCG ******************************************************************************** BK-BJC vs. SG 5 TGAATACGTTCCCAGGTCTTGTACACACCGCCCGTCACACCATGGAAGTGGGGTTGACCAGAAGCAGGTAGTCTAACCGCA TGAATACGTTCCCAGGTCTTGTACACACCGCCCGTCACACCATGGGAGTGGGGTTGACCAGAAGCAGGTAGTCTAACCGCG ********************************************* ********************************** BK-BJC vs. SG 6 TGAATACGTTCCCAGGTCTTGTACACACCGCCCGTCACACCATGGAAGTGGGGTTGACCAGAAGCAGGTAGTCTAACCGCA TGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGAAGTGGGGTTTACCAGAAGTAGGTAGCCTAACCGTA ************* ***************************************** ******** ****** ******* * BK-BJC vs. SG 7 TGAATACGTTCCCAGGTCTTGTACACACCGCCCGTCACACCATGGAAGTGGGGTTGACCAGAAGCAGGTAGTCTAACCGCA TGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGAAGTGGGTTTCACCAGAAGTAGGTAGTCTAACCGTA ************* ************************************** ** ******** ************** * BK-BJC vs. SG 8 TGAATACGTTCCCAGGTCTTGTACACACCGCCCGTCACACCATGGAAGTGGGGTTGACCAGAAGCAGGTAGTCTAACCGCA TGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTGGGCTTCACCAGAAGCAGGTAGTCTAACCGTA ************* ******************************* ****** ** *********************** * BK-BJC vs. SG 9 TGAATACGTTCCCAGGTCTTGTACACACCGCCCGTCACACCATGGAAGTGGGGTTGACCAGAAGCAGGTAGTCTAACCGCA TGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTGGGCTTCACCAGAAGCAGGTAGTCTAACCGCA ************* ******************************* ****** ** ************************* BK-BJC vs. SG 10 TGAATACGTTCCCAGGTCTTGTACACACCGCCCGTCACACCATGGAAGTGGGGTTGACCAGAAGCAGGTAGTCTAACCGCA TGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTGGGGTTCACCAGAAGCAGGTAGGCTAACCGCA ************* ******************************* ********* *************** ********* BK-BJC vs. SG 11 TGAATACGTTCCCAGGTCTTGTACACACCGCCCGTCACACCATGGAAGTGGGGTTGACCAGAAGCAGGTAGTCTAACCGCA TGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTGGGGTTCACCAGAAGCAGGTAGTCTAACCGTA ************* ******************************* ********* *********************** * BK-BJC vs. SG 12 TGAATACGTTCCCAGGTCTTGTACACACCGCCCGTCACACCATGGAAGTGGGGTTGACCAGAAGCAGGTAGTCTAACCGCA TGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTGGGGTTTACCAGAAGCAGGTAGCCTAACCGCG ************* ******************************* ********* *************** ******** BK-BJC vs. SG 13 TGAATACGTTCCCAGGTCTTGTACACACCGCCCGTCACACCATGGAAGTGGGGTTGACCAGAAGCAGGTAGTCTAACCGCA TGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTGGGGTTTACCAGAAGCAGGTAGCCTAACCGCA ************* ******************************* ********* *************** ********* BK-BJC vs. SG 14 TGAATACGTTCCCAGGTCTTGTACACACCGCCCGTCACACCATGGAAGTGGGGTTGACCAGAAGCAGGTAGTCTAACCGCA TGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTGGGGTTTACCAGAAGCAGGTAGGCTAACCGCA ************* ******************************* ********* *************** *********

143

BK-BJC vs. SG 15 TGAATACGTTCCCAGGTCTTGTACACACCGCCCGTCACACCATGGAAGTGGGGTTGACCAGAAGCAGGTAGTCTAACCGCA TGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTTCACCAGAAGCAGGTAGTCTAACCGAA ************* ******************************* ****** ** *********************** * BK-BJC vs. SG 16 TGAATACGTTCCCAGGTCTTGTACACACCGCCCGTCACACCATGGAAGTGGGGTTGACCAGAAGCAGGTAGTCTAACCGCA TGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTTCACCAGAAGCAGGTAGTCTAACCGNA ************* ******************************* ****** ** *********************** * BK-BJC vs. SG 17 TGAATACGTTCCCAGGTCTTGTACACACCGCCCGTCACACCATGGAAGTGGGGTTGACCAGAAGCAGGTAGTCTAACCGCA TGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTTCACCAGAAGCAGGTAGTCTAACCGCG ************* ******************************* ****** ** ************************ BK-BJC vs. SG 18 TGAATACGTTCCCAGGTCTTGTACACACCGCCCGTCACACCATGGAAGTGGGGTTGACCAGAAGCAGGTAGTCTAACCGCA TGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTTCACCAGAAGCAGGTAGTCTAACCGCA ************* ******************************* ****** ** ************************* BK-BJC vs. SG 19 TGAATACGTTCCCAGGTCTTGTACACACCGCCCGTCACACCATGGAAGTGGGGTTGACCAGAAGCAGGTAGTCTAACCGCA TGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTTTACCAGAAGCAGGTAGTCTAACCGCG ************* ******************************* ****** ** ************************ BK-BJC vs. SG 20 TGAATACGTTCCCAGGTCTTGTACACACCGCCCGTCACACCATGGAAGTGGGGTTGACCAGAAGCAGGTAGTCTAACCGCA TGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTTTACCAGAAGCAGGTAGTCTAACCGCA ************* ******************************* ****** ** ************************* BK-BJC vs. SG 21 TGAATACGTTCCCAGGTCTTGTACACACCGCCCGTCACACCATGGAAGT-GGGGTTGACCAGAAGCAGGTAGTCTAACCGCA TGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAAGTGGGTTTTACCAGAAGCAGGTAGTCTAACCGCA ************* ******************************* * *** ** *************************

Legend Forward primer Probe Reverse primer

144

8.8. APPENDIX 8: GEL SAMPLE INDICES FROM CONVENTIONAL PCR TEST OF BKBJCV8 PRIMERS. This work was done in conjunction with Contador (2013).

8.8.1 GEL 1 AND SAMPLE INDEX. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

PCR Extraction Gel Band Results/ Description Tube # ID lane extracted? 1 D5 12_1_14_2 2 No Positive/ RT CAF 2 D5 12_1_14_3 3 No Positive/ RT CAF 3 D5 12_1_14_4 4 No Positive/ RT CAF 4 D5 12_1_14_5 5 No Negative/ F#2 March (lost) 5 D5 12_1_14_6 6 No Positive/ B002-13 LT-BJC-Jan 8 F#3 (Known +) 6 D5 12_1_14_7 7 No Positive/ B002-13 LT-BJC Jan 8 F#4 (Known -) 7 D5 12_3_14_1 8 No Negative/ Sturgeon 8 D5 12_3_14_2 9 No Positive/ Brook trout 9 D5 12_3_14_3 10 No Positive/ Brook trout 10D5 12_3_14_4 11 No Negative/ Control CAF MST toxic (?) May 11 2013 (RT) (Known -) 25D5 *Negative 12 No Neg/ B037-10 m3-1 LT (Known -) 26D5 *Positive 13 No Positive/ B002-13 3R8 LT (Known +) 21D5 12_3_14_1 14 No Neg/ From Alma randomly chosen May 13 2013 7 22D5 12_3_14_1 15 No Neg/ From Alma randomly chosen May 13 2013 8 * These controls should be used in both gels

145

8.8.2 GEL 2 AND SAMPLE INDEX. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

PCR Extraction Gel Band Results/ Description Tube ID lane extracted? # 11D5 12_3_14_5 2 No Neg/ CAF MST toxic. May 11 2013 (Known -) 12 D5 12_3_14_6 3 No Neg/ B5 and B6 # 8 (Known +) 13 D5 12_3_14_7 4 No Neg/ B5 and B6 #9 14 D5 12_3_14_8 5 No Neg/ B5 and B6 #2 15 D5 12_3_14_9 6 No Neg/ B5 and B6 #6 16 D5 12_3_14_10 7 No Neg/ B047-14 F1 17 D5 12_3_14_11 8 No Neg/ B047-14 F1 (LT from BJC) 18 D5 12_3_14_12 9 No Neg/ B147-13 Cold water fisheries 19 D5 12_3_14_13 10 No Positive/ Tilapia #6 Jan 15 2013 (Chlamydia +) 20D5 12_3_14_14 11 No Positive/ Tilapia # 5 Jan 15 2013 (Chlamydia +) 23D5 12_3_14_17 12 No Neg/ RT CLO Alma randomly chosen May 13 2013 24D5 12_3_14_18 13 No Neg/ RT CLO Alma randomly chosen May 13 2013 25D5 Negative 14 No Neg/ B037-10 m3-1 (Known -) 26D5 Positive 15 No Positive/ B002-13 3R8 (Known +)

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8.9. APPENDIX 9: INTRAASSAY VARIATION CALCULATED ACCORDING TO NCBI GUIDELINES. (Iversen et al., 2012).

Intraplate Assay of Standards 1.02E8 thru 1.02E2 by Run qPCR Run (# wells/sample) Mean %CV (Concentration) 2 (dupl) 3.634 3 (dupl) 3.314 4 (dupl) 4.073 5 (quint) 3.108

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8.10. APPENDIX 10: BLASTN RESULTS OF FORWARD A) AND REVERSE B) SEQUENCES FROM THE BKBJCV8 qPCR PRODUCT OF SYNTHETIC BK-BJC STANDARD 1.02E8. BK-BJC is highlighted in yellow.

8.10.1 BK-BJC 1.02E8 AMPLICON SEQUENCING RESULTS OF THE BKBJCV8 FORWARD SEQUENCE. Description of BLAST match Max Total QC Identity E value [Genbank accession #] score score (%) (%) Uncultured bacterium clone BK-BJC 16S 59 59 61 5.00E-06 97 ribosomal RNA gene, partial sequence [KM504995.1] Candidatus Branchiomonas cysticola strain A1- 57.2 57.2 62 2.00E-05 95 483-L1 16S ribosomal RNA gene, complete sequence [JN968376.1] Uncultured beta proteobacterium clone T200910 57.2 57.2 62 2.00E-05 95 16S ribosomal RNA gene, partial sequence [JN807444.1] Candidatus Branchiomonas cysticola clone 55.4 55.4 61 7.00E-05 95 GABI_14 16S ribosomal RNA gene, partial sequence [JQ723599.1] Uncultured bacterium clone JJ-85 16S ribosomal 53.6 53.6 62 2.00E-04 92 RNA gene, partial sequence [KJ782257.1]

8.10.2 BK-BJC 1.02E8 AMPLICON SEQUENCING RESULTS OF THE BKBJCV8 REVERSE SEQUENCE. Description of BLAST match Max Total QC Identity E value [Genbank accession #] score score (%) (%) Uncultured bacterium partial 16S rRNA gene, 62.6 62.6 93 3.00E-07 91 clone D3M-23 [AM238185.1] Uncultured bacterium clone plate116a11 16S 60.8 60.8 93 1.00E-06 91 ribosomal RNA gene, partial sequence [KT450223.1] Carnobacterium sp. SN_83 16S ribosomal RNA 60.8 60.8 91 1.00E-06 91 gene, partial sequence [KR088662.1] Burkholderia sp. UFSM-B33 16S ribosomal 60.8 60.8 85 1.00E-06 93 RNA gene, partial sequence [KJ532452.1] Uncultured bacterium clone 4783190 16S 60.8 60.8 93 1.00E-06 91 ribosomal RNA gene, partial sequence [JQ696406.1] Staphylococcus arlettae strain BTMT04 16S 60.8 60.8 93 1.00E-06 91 ribosomal RNA gene, partial sequence [JN228201.1] Uncultured bacterium clone 16slp69-01f05.p1k 60.8 60.8 68 1.00E-06 100 16S ribosomal RNA gene, partial sequence

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[FJ508610.1] Uncultured Firmicutes bacterium clone 60.8 60.8 91 1.00E-06 91 7mos_10s_E4 16S ribosomal RNA gene, partial sequence [GQ261869.1] Uncultured alpha proteobacterium clone 60.8 60.8 93 1.00E-06 91 AEGEAN_233 16S ribosomal RNA gene, partial sequence [AF406547.1] Uncultured Fibrobacter/Acidobacteria group 60.8 60.8 93 1.00E-06 91 bacterium clone AEGEAN_225 16S ribosomal RNA gene, partial sequence [AF406544.1] Lactobacillus paracasei strain LI3 16S 59 59 93 3.00E-06 89 ribosomal RNA gene, partial sequence [KR265315.1] Rhodocyclaceae bacterium ICHIOC6 gene for 59 59 83 3.00E-06 93 16S ribosomal RNA, partial sequence [LC132803.1] Staphylococcus warneri strain RA18 16S 59 59 89 3.00E-06 91 ribosomal RNA gene, partial sequence [KU588403.1] Staphylococcus sciuri strain Bp-2 16S ribosomal 59 59 89 3.00E-06 91 RNA gene, partial sequence [KJ888130.1] Staphylococcus sciuri strain RCB550 16S 59 59 89 3.00E-06 91 ribosomal RNA gene, partial sequence [KT260762.1] Ralstonia pickettii strain LT7-MRL 16S 59 59 83 3.00E-06 93 ribosomal RNA gene, partial sequence [KP318034.1] Uncultured bacterium clone BK-BJC 16S 59 59 93 3.00E-06 92 ribosomal RNA gene, partial sequence [KM504995.1]

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8.11. APPENDIX 11: THE DIFFERENCES IN BP MISMATCHES BETWEEN BK-BJC AND THE NON-TARGET SEQUENCE GROUPS CATEGORIZED BY BKBJCV8 qPCR NEGATIVE AND POSITIVE STATUS. (-)=this sample has BLASTn imposed breaks within the sequence to optimize the alignment.

8.11.1 NEGATIVE SEQUENCE GROUPS BP MISMATCHES AND LOCATIONS. NEG Mismatch Position Mismatch Position Mismatch Position SGs # from the 5' # from the 5' # from the 5'

Primer F Probe Primer R SG2 1 14 3 3,6,13 1 2 SG8 1 14 3 3,6,13 1 2 SG9 1 14 3 3,6,13 0 SG15 1 14 3 3,6,13 1 2 SG16 1 14 3 3,6,13 1 2 SG17 1 14 3 3,6,13 1 1 SG18 1 14 3 3,6,13 0 SG19 1 14 3 3,6,13 1 1 SG20 1 14 3 3,6,13 0

8.11.2. POSITIVE SEQUENCE GROUPS BP MISMATCHES AND LOCATIONS. POS SGs Position Position Mismatch Mismatch Position from Mismatch ranked from the from the # # the 5' # by Ct 5' 5' value Primer F Probe Primer R SG4 0 0 1 1 SG21 1 14 6 3,6,10-12,14 0 SG14 1 14 2 3,13 1 10 SG5 0 1 13 1 1 SG13 1 14 2 3,13 1 10 SG11 1 14 2 3,13 1 2 SG12 1 14 2 3,13 2 1,10 SG10 1 14 2 3,13 1 10 SG7 1 14 2 3,6 2 2,17 SG1 2 14,17 3 1,2,6 (-) 0 SG6 1 14 1 3 3 2,10,17

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8.12. APPENDIX 12: GEL ELECTROPHORESIS RESULTS FROM THE TEMPERATURE GRADIENT PCR TO FIND THE OPTIMAL ANNEALING TEMPERATURE FOR BKBJCV3 PRIMERS.

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8.13. APPENDIX 13: GEL ELECTROPHORESIS RESULTS OF BKBJCV3 PCR ON BKBJCV8 qPCR POSITIVE SAMPLES FROM BJC IN 2013 AND 2015.

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8.14. APPENDIX 14: SEQUENCING RESULTS FROM BKBJCV8 qPCR FROM A Ct 35 SAMPLE (WEEK 1, TANK 1 FISH 14 FROM BJC). BK-BJC is highlighted in yellow.

BLAST match description Max Total QC E value Identity [Genbank accession #] score score (%) (%) Uncultured bacterium clone BK-BJC 51.8 51.8 100 9.00E-05 94 16S ribosomal RNA gene, partial sequence [KM504995.1] Uncultured bacterium clone KD7-78 51.8 51.8 100 9.00E-05 94 16S ribosomal RNA gene, partial sequence [AY218714.1] Candidatus Branchiomonas cysticola 48.2 48.2 93 0.001 93 clone GABI_14 16S ribosomal RNA gene, partial sequence [JQ723599.1] BK-BJC Candidatus Branchiomonas cysticola 48.2 48.2 93 0.001 93 Forward strain A1-483-L1 16S ribosomal Primers RNA gene, complete sequence [JN968376.1] Uncultured beta proteobacterium 48.2 48.2 93 0.001 93 clone T200910 16S ribosomal RNA gene, partial sequence [JN807444.1] Uncultured bacterium clone 16S- 44.6 44.6 100 0.014 91 27Fand1492R-C12-clone3 16S ribosomal RNA gene, partial sequence [KX348536.1] Uncultured bacterium isolate 59 59 100 6.00E-07 100 1112864242229 16S ribosomal RNA gene, partial sequence [HQ120304.1] Uncultured bacterium partial 16S 59 59 100 6.00E-07 100 rRNA gene, clone M6A-304 [AM991224.1] Uncultured bacterium clone 59 59 100 6.00E-07 100 BK-BJC Ca_094970_1830_3106 16S Reverse ribosomal RNA gene, partial Primers sequence [EF345105.1] Uncultured bacterium clone 59 59 100 6.00E-07 100 Ca_052683_1864_2027 16S ribosomal RNA gene, partial sequence [EF325921.1] Uncultured bacterium clone BK-BJC 57.2 57.2 96 2.00E-06 100 16S ribosomal RNA gene, partial sequence [KM504995.1]

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8.15. APPENDIX 15: BK-BJC OUTBREAK 2012-13 ADJUSTED AND NON-MEDIAN AND UL AND LL.

Sample Date Untransformed Log transformed (number of LT sampled) LL Median UL LLlog LogMean ULlog 3-Dec-12 (n=5) 775.37 1616.77 3371.23 2.88951 3.20865 3.52779 3-Jan-13 (n=5) 513.68 1071.11 2233.44 2.71069 3.02983 3.34897 8-Jan-13 (n=8) 28197.85 50410.26 90120.14 4.45022 4.70252 4.95482 18-Jan-13 (n=10) 15394.7 25884.38 43521.56 4.18737 4.41304 4.6387 21-Mar-13 (n=4) 45.29 103.01 234.24 1.65605 2.01286 2.36967

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8.16. APPENDIX 16: NUMBER DEAD AND DAILY PERCENT MORTALITY OF BJC LT DURING AN EP ASSOCIATED MORTALITY EVENT IN 2013.

Day of mortality Daily Number Daily percent Date event (#) dead (#) remaining in tank mortality (%) December 1, 2012 1 0 6436 0.00 December 2, 2012 2 0 6436 0.00 December 3, 2012 3 1 6435 0.02 December 4, 2012 4 2 6433 0.03 December 5, 2012 5 0 6433 0.00 December 6, 2012 6 0 6433 0.00 December 7, 2012 7 0 6433 0.00 December 8, 2012 8 1 6432 0.02 December 9, 2012 9 0 6432 0.00 December 10, 2012 10 1 6431 0.02 December 11, 2012 11 0 6431 0.00 December 12, 2012 12 0 6431 0.00 December 13, 2012 13 0 6431 0.00 December 14, 2012 14 0 6431 0.00 December 15, 2012 15 2 6429 0.03 December 16, 2012 16 0 6429 0.00 December 17, 2012 17 2 6427 0.03 December 18, 2012 18 0 6427 0.00 December 19, 2012 19 1 6426 0.02 December 20, 2012 20 0 6426 0.00 December 21, 2012 21 0 6426 0.00 December 22, 2012 22 0 6426 0.00 December 23, 2012 23 1 6425 0.02 December 24, 2012 24 0 6425 0.00 December 25, 2012 25 0 6425 0.00 December 26, 2012 26 1 6424 0.02 December 27, 2012 27 1 6423 0.02 December 28, 2012 28 0 6423 0.00 December 29, 2012 29 2 6421 0.03 December 30, 2012 30 4 6417 0.06 December 31, 2012 31 0 6417 0.00 January 1, 2013 32 2 6415 0.03 January 2, 2013 33 2 6413 0.03 January 3, 2013 34 7 6406 0.11

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January 4, 2013 35 7 6399 0.11 January 5, 2013 36 8 6391 0.13 January 6, 2013 37 12 6379 0.19 January 7, 2013 38 13 6366 0.20 January 8, 2013 39 18 6348 0.28 January 9, 2013 40 20 6328 0.32 January 10, 2013 41 41 6287 0.65 January 11, 2013 42 57 6230 0.91 January 12, 2013 43 106 6124 1.73 January 13, 2013 44 124 6000 2.07 January 14, 2013 45 115 5885 1.95 January 15, 2013 46 165 5720 2.88 January 16, 2013 47 185 5535 3.34 January 17, 2013 48 201 5334 3.77 January 18, 2013 49 157 5177 3.03 January 19, 2013 50 205 4972 4.12 January 20, 2013 51 138 4834 2.85 January 21, 2013 52 130 4704 2.76 January 22, 2013 53 152 4552 3.34 January 23, 2013 54 118 4434 2.66 January 24, 2013 55 106 4328 2.45 January 25, 2013 56 83 4245 1.96 January 26, 2013 57 60 4185 1.43 January 27, 2013 58 58 4127 1.41 January 28, 2013 59 64 4063 1.58 January 29, 2013 60 48 4015 1.20 January 30, 2013 61 30 3985 0.75 January 31, 2013 62 29 3956 0.73 February 1, 2013 63 9 3947 0.23 February 2, 2013 64 12 3935 0.30 February 3, 2013 65 9 3926 0.23 February 4, 2013 66 4 3922 0.10 February 5, 2013 67 4 3918 0.10 February 6, 2013 68 2 3916 0.05 February 7, 2013 69 3 3913 0.08 February 8, 2013 70 0 3913 0.00 February 9, 2013 71 1 3912 0.03 February 10, 2013 72 1 3911 0.03 February 11, 2013 73 1 3910 0.03

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February 12, 2013 74 1 3909 0.03 February 13, 2013 75 2 3907 0.05 February 14, 2013 76 2 3905 0.05 February 15, 2013 77 0 3905 0.00 February 16, 2013 78 0 3905 0.00 February 17, 2013 79 0 3905 0.00 February 18, 2013 80 0 3905 0.00 February 19, 2013 81 0 3905 0.00 February 20, 2013 82 2 3903 0.05 February 21, 2013 83 1 3902 0.03 February 22, 2013 84 0 3902 0.00 February 23, 2013 85 1 3901 0.03 February 24, 2013 86 2 3899 0.05 February 25, 2013 87 1 3898 0.03 February 26, 2013 88 1 3897 0.03 February 27, 2013 89 1 3896 0.03 February 28, 2013 90 0 3896 0.00 March 1, 2013 91 1 3895 0.03 March 2, 2013 92 0 3895 0.00 March 3, 2013 93 0 3895 0.00 March 4, 2013 94 0 3895 0.00 March 5, 2013 95 0 3895 0.00 March 6, 2013 96 0 3895 0.00 March 7, 2013 97 0 3895 0.00 March 8, 2013 98 0 3895 0.00 March 9, 2013 99 0 3895 0.00 March 10, 2013 100 0 3895 0.00 March 11, 2013 101 0 3895 0.00 March 12, 2013 102 0 3895 0.00 March 13, 2013 103 6 3889 0.15 March 14, 2013 104 3 3886 0.08 March 15, 2013 105 3 3883 0.08 March 16, 2013 106 0 3883 0.00 March 17, 2013 107 2 3881 0.05 March 18, 2013 108 0 3881 0.00 March 19, 2013 109 2 3879 0.05 March 20, 2013 110 1 3878 0.03 March 21, 2013 111 0 3878 0.00 March 22, 2013 112 0 3878 0.00

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March 23, 2013 113 0 3878 0.00 March 24, 2013 114 2 3876 0.05 March 25, 2013 115 0 3876 0.00 March 26, 2013 116 0 3876 0.00 March 27, 2013 117 0 3876 0.00 March 28, 2013 118 0 3876 0.00 March 29, 2013 119 0 3876 0.00 March 30, 2013 120 0 3876 0.00 March 31, 2013 121 0 3876 0.00 Note: yellow highlighting indicates a LT sampling day.

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8.17. APPENDIX 17: BLASTN MATCHES FOR THE BKBJCV8 qPCR PRODUCTS OF TWO POSITIVE LT (WEEK 1, TANK 1- FISH 1 AND 12). BK-BJC is highlighted in yellow.

BLASTn match description Max Total QC Identity E value [Genbank accession #] score score (%) (%) Uncultured bacterium clone BK- 66.2 66.2 100 7.00E-09 100 BJC 16S ribosomal RNA gene, partial sequence [KM504995.1] Candidatus Branchiomonas 62.6 62.6 94 9.00E-08 100 cysticola clone GABI_14 16S ribosomal RNA gene, partial sequence [JQ723599.1] Candidatus Branchiomonas 62.6 62.6 94 9.00E-08 100 BK-BJC cysticola strain A1-483-L1 16S Forward ribosomal RNA gene, complete Primer sequence [JN968376.1] Uncultured beta proteobacterium 62.6 62.6 94 9.00E-08 100 clone T200910 16S ribosomal RNA gene, partial sequence [JN807444.1] Uncultured candidate division 57.2 57.2 100 4.00E-06 94 NC10 bacterium clone 6a 16S ribosomal RNA gene, partial sequence [KU933978.1] Uncultured bacterium isolate 66.2 66.2 100 1.00E-08 97 1112864242229 16S ribosomal RNA gene, partial sequence [HQ120304.1] Uncultured bacterium partial 16S 66.2 66.2 100 1.00E-08 97 rRNA gene, clone M6A-304 [AM991224.1] Uncultured bacterium clone 66.2 66.2 100 1.00E-08 97 BK-BJC Ca_094970_1830_3106 16S Reverse ribosomal RNA gene, partial Primer sequence [EF345105.1] Uncultured bacterium clone 66.2 66.2 100 1.00E-08 97 Ca_052683_1864_2027 16S ribosomal RNA gene, partial sequence [EF325921.1] Uncultured bacterium clone BK- 64.4 64.4 97 3.00E-08 97 BJC 16S ribosomal RNA gene, partial sequence [KM504995.1]

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8.18. APPENDIX 18: RATIONALE FOR EXCLUDING BKBJCV3’S TOP BLASTN MATCHES WITH ≥90 % NUCLEOTIDE IDENTITY FROM NON-TARGET AMPLIFICATION.

Experim- BKBJCV3 BLASTn matches with Reasons for predicted exclusion from entally >90 % nucleotide identity cross-reactivity (Lefever et al., 2013) Validated Candidatus Branchiomonas cysticola strain A1-483-L1 16S ribosomal RNA Yes gene, complete sequence Uncultured beta proteobacterium Forward- 5 bp mismatches, including clone T200910 16S ribosomal RNA position 3 at the 3’end Yes gene, partial sequence Uncultured beta proteobacterium Reverse- 4 bp mismatches, including partial 16S ribosomal RNA, isolate position 2 at the 3’ end Yes DGGE band 8 Candidatus Branchiomonas cysticola clone GABI_14 16S ribosomal RNA Yes gene, partial sequence Forward- 9 bp mismatches, including Uncultured bacterium partial 16S positions 2, 3, 4, 5 at the 3’ end rRNA gene, isolate No Reverse- 6 bp mismatches, including Mineral.top.1.6.1.4_322869 positions 2, 4, 5 at the 3’ end Forward- 11 bp mismatches, including Uncultured bacterium partial 16S positions 2, 3, 4, 5 at the 3’ end rRNA gene, isolate No Reverse- 6 bp mismatches, including Mineral.top.2.1.6_343230 positions 2, 4, 5 at the 3’ end Forward- 12 bp mismatches, including Uncultured Neisseriaceae bacterium positions 3, 4 at the 3’ end clone 1FG05 16S ribosomal RNA No Reverse- 6 bp mismatches, including gene, partial sequence positions 2, 4 at the 3’ end Forward- 14 bp mismatches, including Uncultured bacterium partial 16S positions 1-5 at the 3’ end rRNA gene, isolate No Reverse- 6 bp mismatches, including Mineral.top.1.6.1.4_230900 positions 2, 4, 5 at the 3’ end Uncultured Enterobacteriaceae Forward- 15 bp mismatches, including bacterium clone HPM_066L 16S positions 1-5 at the 3’ end No ribosomal RNA gene, partial sequence Reverse- 5 bp mismatches, including

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Uncultured Enterobacteriaceae position 1 at the 3' end bacterium clone HPM_024L 16S No ribosomal RNA gene, partial sequence

Uncultured Enterobacteriaceae bacterium clone XDE_077L 16S No ribosomal RNA gene, partial sequence Forward- 13 bp mismatches, including Uncultured bacterium gene for 16S positions 1-4 at the 3’ end rRNA, partial sequence, clone: No Reverse- 7 bp mismatches, including oytB017 positions 2, 4 at the 3' end

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