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Jig UBRARY
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This is to certify that the
dissertation entitled

BACTERIAL KIDNEY DISEASE (BKD) IN MICHIGAN
SALMONIDS

presented by
Alaa-Eldin Abdel Mouty Mohamed Eissa

has been accepted towards fulfillment
of the requirements for the

Ph.D. degree in Pathology

Mom ,5 ”tango“

Major Professor’s Signature

We

ate

MSU is an Affirmative Action/Equal Opportunity Institution

PLACE IN RETURN Box to remove this checkout from your record.
10 AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2/05 c:/CiRC/DateDue.iMd-p.15‘

_, ._ . _____—__ ——

BACTERIAL KIDNEY DISEASE (BKD) IN MICHIGAN SALMONIDS
By

Alaa-Eldin Abdel Mouty Mohamed Eissa

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Pathobiology and Diagnostic Investigation

2005

ba

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WI

ABSTRACT

BACTERIAL KIDNEY DISEASE (BKD) IN MICHIGAN SALMONIDS

By

Alaa-Eldin Abdel Mouty Mohamed Eissa

Bacterial Kidney Disease (BKD) caused by a Gram-positive
bacterium, Renibacterium salmoninarum (R. salmoninarum) is a systemic
disease that threatens the expansion of both cultured and wild salmonines
worldwide. Historical records show that BKD affected Michigan’s brook
trout as early as 1955 (Allison 1958) and continued its spread to most of
other salmonid species such as coho salmon (Oncorhynchus kisutch)
spawners in the late sixties (MacLean and Yoder 1970) followed by a
potent epizootic among the Chinook salmon (Oncorhynchus tshawytscha)
populations in Lake Michigan in the late 1980's (Holey et al. 1998).
Despite the magnitude of the setback to fisheries conservation efforts in
Michigan, relatively little research has been done on R. salmoninarum,
primarily due to its slow-growing nature, which takes up to 12 weeks to
obtain growth upon primary isolation. The tissue processing protocol and
optimized culture technique developed in this study, has facilitated the
isolation of large number of R. salmoninarum and achieved a remarkably
shorter incubation time for primary isolation.

A sort of disagreement in results among the three used diagnostic

assays (nPCR, Culture, Q-ELISA) was recorded during BKD testing of the

teral
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role
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pre
BK

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se
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feral and captive salmonid stocks for BKD, which may reflect the different
phases of R. salmoninarum infection at the time of sampling. Further, the
testing results demonstrated the presence of six patterns, with each of the
patterns representing a probable stage along the course of R.
salmoninarum infection.

Data also demonstrated that male spawner salmon play the same
role played by female in transmission of R. salmoninarum by shedding of
the bacteria and its soluble antigens in milt. Moreover, results revealed
that Hinchenbrook coho strain is more susceptible to R. salmoninarum
than the Michigan adapted coho salmon strain. Data also, supported the
previous reports, which indicated that brook trout are highly susceptible to
BKD. Also, data showed that prevalence of R. salmoninarum in the
hatchery-raised brook trout is the same as wild strains residing in the
water source and that Iron River brook trout are more vulnerable to R.
salmoninarum infection than Assinica strain. Last, external parasites could
played a possible role in initiation, speeding up the R. salmoninarum
infection and BKD occurrence and mortalities is not related to changes in
seasons. Finally, findings indicated that the adult parasitic sea lamprey is
a new host range for R. salmoninarum where the bacteria were isolated

from the kidneys of a number of sea lampreys from Lake Ontario in 2003

and 2004.

Copyright by

ALAA-ELDIN ABDEL MOUTY MOHAMED EISSA
2005

DEDICATION

To my wife, children, and parents

More
wou:.

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ment

 

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and It

In

advls

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IOIQ

VaME

ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to my major advisor Dr.
Mohamed Faisal, for his sincere advising and endless support. This study
would not be possible without the integration of his profound experience in the
field of fish diseases.

Also, I wish to express my deep appreciation to my advisory committee
members Dr. Scot Fitzgerald, Dr. John Gerlach, Dr. Matti Kiupel and Dr.
Rocco Cipriano for their tremendous support, valuable discussions, patience
and fruitful collaboration.

Special appreciation goes to Dr. Ehab Elsayed, for his sincere, endless
advising and support. I also would like to thank all members of the Aquatic
Animal Health Laboratory at Michigan State University for their assistance.
My appreciation also, goes to Mr. Gary Whelan and all Michigan Department
of Natural Resources personnel who have facilitated sample collection.

I wish to express my appreciation to Dr. Clifford Starliper at the National
Fish Health Laboratory in Virginia for his valuable discussion and suggestions
for culture modifications. Also, I would like to thank Mr. John Hnath for his
valuable comments and suggestions during writing the dissertation.

I would like to thank the Egyptian government for giving me the
opportunity and means to study in the USA. Deep appreciation also goes to
the Great Lake Fisheries Trust (GLFT) for funding most of the research

described in this dissertation.

vi

 

grate—
sacrf

ace:

 

 

My deep gratitude goes to my beloved mother, father, brothers and sister
for their sincere love and support throughout my entire lifetime. Lastly, I am
grateful to my wife, Hend Nada and my kids for their eternal love, patience,
sacrifices, and sharing the responsibility that enabled me to successfully

accomplish my studies in the USA.

vii

 

 

usr or
LIST or
INTRO

cumr‘

I.I'IlSIOr
II. The p.
I) N
2) C
3) Is
4.: P
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IV. Epiz
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3H

TABLE OF CONTENTS

LIST OF TABLES ....................................................................... xi
LIST OF FIGURES ...................................................................... xiii
INTRODUCTION ......................................................................... 1
CHAPTER 1. LITERATURES REVIEW. .......................................... 4
l. Historical perspectives ............................................................... 4
II. The pathogen .......................................................................... 5
1) Nomenclature and current classification of the etiological agent. . .. 5
2) Cell morphology ......................................................................... 6
3) Isolation, culture and culture characteristics ............................. 6
4) Preservation of cultures ....................................................... 9
5) Biochemical characteristics .................................................. 1O
6) Antibiotic susceptibility ..................................................... 10
7) Antigenic characteristics and virulence factors ......................... 11
8) Molecular and genetic diversity ............................................ 12
Ill. The disease .......................................................................... 13
1) Disease Course ................................................................. 13
3) External signs ............................................................ 13
b) Internal Lesions ......................................................... 14
c) Histopathology .......................................................... 15
2) Susceptibility .................................................................... 16
3) Pathogenesis and Immunity ................................................ 17
a) Process of infection and pathogenesis ........................... 17
b) Effect of BKD on host immune response ........................ 19
0) Environmental factors ................................................. 20
Effect of diet ........................................................ 20
Effect of temperature ............................................. 21
Effect of estuarine and salt-water environment ............ 22
IV. Epizootiology ......................................................................... 23
1) Geographical Distribution ..................................................... 23
2) Host range ........................................................................ 24
3) Disease transmission .......................................................... 25
a) Source of infection ...................................................... 25
b) Horizontal transmission ............................................... 25
c) Vertical transmission ................................................... 26
d) Fish as possible vectors and carriers .............................. 27
e) Possible vectors other than fish ..................................... 28
f) Reservoirs ................................................................ 29

viii

 

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V. Diagnosis of BKD ..................................................................... 29

1) Isolation and bacteriological identification of the agent ............... 29

2) Antigen-antibody reactions ................................................... 30

a) Agglutination test ........................................................ 30

b) Immunofluorescence ................................................... 30

c) Enzyme linked immunosorbent assay (ELISA) .................. 31

d) lmmunohistochemistry (IHC) ......................................... 32

3) Polymerase chain reaction (PCR) .......................................... 33

VI. Differential diagnosis ............................................................... 34

VII. Control ................................................................................. 36
1) Chemotherapy .................................................................... 36 '

2) Adult segregation ................................................................ 38

3) Eradication ........................................................................ 39

4) Prophylaxis ........................................................................ 4O

1. Reducing the risk of BKD introduction .............................. 40

2. Vaccination ................................................................. 4O

Gap of BKD knowledge in Michigan before 2002 ................................ 42

CHAPTER 2. PRIMARY ISOLATION OF RENIBACTERIUM
SALMONINARUM FROM NATURALLY INFECTED SALMONINE
STOCKS IN MICHIGAN USING A MODIFIED TISSUE PROCESSING

PROTOCOL .............................................................................. 46
1. Abstract .............................................................................. 46
2. Introduction .......................................................................... 48
3. Material and Methods ............................................................ 51
4. Results ................................................................................ 58

1) Effect of sample processing and culture technique on the
primary isolation of R. salmoninarum ............................... 58
2) Prevalence of R. salmoninarum isolated from Michigan
salmonines .................................................................. 58
3) Confirmation of the retrieved isolates 60
5. Discussion ........................................................................... 62

CHAPTER 3. DIAGNOSTIC TESTING PATTERNS OF
RENIBACTERIUM SALMONINARUM INFECTION IN SOME FERAL

AND CAPTIVE SALMONINES ......................................................... 75
1. Abstract .............................................................................. 75
2. Introduction ......................................................................... 77
3. Material and Methods ............................................................ 8O
4. Results 84
5. Discussion ........................................................................... 86

ix

CHAPTER 4. CURRENT STATUS OF RENIBACTERIUM
SALMONINARUM INFECTION IN FERAL CHINOOK AND COHO

SALMON AT MICHIGAN WEIRS AND HATCHERIES ........................ 96
1. Abstract .............................................................................. 96
2. Introduction ......................................................................... 98
3. Material and Methods ............................................................ 102
4. Results ............................................................................... 106
5. Discussion ........................................................................... 113

CHAPTER 5. PREVALENCE, SHEDDING AND SPREAD OF
RENIBACTERIUM SALMONINARUM IN BROOK TROUT
(SALVELINUS FONTINALIS) WITH SPECIAL EMPHASIS ON THE

ASSOCIATED DISEASE EPIZOOTICS IN MICHIGAN ........................ 134
1 . Abstract .............................................................................. 1 34
2. Introduction ......................................................................... 136
3. Material and Methods ............................................................ 139
4. Results ............................................................................. 146
3) Prevalence of R. salmoninarum infections in hatcher

captive and wild brook trout .......................................... 146

b) BKD outbreaks (epizootics) among the hatchery raised
brook trout ................................................................ 148
5. Discussion ........................................................................... 152

CHAPTER 6. FIRST RECORD OF RENIBACTERIUM
SALMONINARUM IN SEA LAMPREY (PETROMYZON MARINUS)

FROM THE GREAT LAKES BASIN ................................................ 169
1 . Abstract .............................................................................. 169
2. Introduction .......................................................................... 170
3. Material and Methods ............................................................. 173
4. Results ................................................................................ 177
5. Discussion ........................................................................... 179
CHAPTER 7. CONCLUSION AND FUTURE RESEARCH ..................... 184
LIST OF REFERENCES ................................................................. 189

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LIST OF TABLES

Table 1. Summary of the morphological and biochemical characteristics
of Renibacterium salmoninarum .......................................................

Table 2. Summary of the metabolic, antigenic and pathogenic characters
of Renibacterium salmoninarum ......................................................

Table 3. Prevalence of Renibacterium salmoninarum in fall returning
chinook and coho salmon spawners from Lake Michigan and Lake Huron
watersheds .................................................................................

Table 4. Prevalence of Renibacterium salmoninarum in captive char and
trout broodstocks collected in fall 2002 from Michigan state fish
hatcheries ....................................................................................

Table 5. Prevalence of Renibacterium salmoninarum in 10-15-month-old
propagated fish. ...........................................................................

Table 6. Results of conventional biochemical and motility tests performed
on the 12 representative Renibacterium salmoninarum
isolates .......................................................................................

Table 7. Standard concentration and expected inhibition zones for each
used antibiotic disc ........................................................................

Table 8. Inhibition zones in mm produced by the 12 Renibacterium
salmoninarum isolates used in the antibiogram ...................................

Table 9. Frequencies of different diagnostic testing patterns of salmonid
feral spawners and captive broodstocks collected from different
geographical locations in Michigan during fall 2002 ..............................

Table 10. Details of samples collected from spawning chinook and coho
salmon returning to egg-take weirs throughout the period from 2001-

2005 ..........................................................................................

Table 11. Chinook salmon juvenile fingerlings collected from Michigan
state fish hatcheries in the period from 2002-2005 .............................

Table 12. Renibacterium salmoninarum prevalence and intensity in
ovarian fluid and milt of the 2004 chinook and coho salmon Spawners....
Table 13. Renibacterium salmoninarum prevalence in kidney and
gametes of the 2004 chinook salmon spawners ...................................

xi

44

45

67

68

69

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71

72

92

120

121

122

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Table 14. Renibacterium salmoninarum prevalence in Kidney and
gametes of the 2004 coho salmon spawners (Shedding agreements
possibilities between kidney and gametes) .........................................

Table 15. Renibacterium salmoninarum infection prevalence and intensity
among the Little Manistee River chinook salmon feral spawners and their
corresponding hatchery raised fingerlings throughout the period from

2001 to 2005 ................................................................................

Table 16. Renibacterium salmoninarum infection prevalence and intensity
among the Swan River Weir chinook salmon feral spawners and their
corresponding hatchery raised fingerlings throughout the period from
2001 to 2005 ...............................................................................

Table 17. Details of samples collected from brook trout broodstocks
throughout the period from 2001-2004 and pre-stocking 18-month-old
fingerlings collected throughout the period from 2002-2005 ....................

Table 18. Renibacterium salmoninarum infection prevalence and intensity
among brook trout broodstocks and their corresponding 18 months pre-
stockin'g fingerlings throughout the period from 2001 — 2005 ..................

Table 19. Prevalence of Renibacterium salmoninarum in 2003-2004 Lake
Ontario sea lampreys using nPCR, Q-ELISA and culture ......................

Table 20. Confirmation of R. salmoninarum isolates using some culture-
based assays, serological and molecular assays .................................

xii

124

125

127

159

160

181

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LIST OF FIGURES

Figure 1. Prevalence of Renibacterium salmoninarum in male and female
returning chinook salmon spawners from Lake Michigan and Lake Huron
watersheds .................................................................................. 73

Figure 2. Prevalence of Renibacterium salmoninarum in male and female
returning coho salmon spawners from Platte River Weir, Lake Michigan
watershed .................................................................................... 74

Figure 3. Frequencies of different diagnostic testing patterns of feral
chinook salmon spawners ............................................................... 93

Figure 4. Frequencies of different diagnostic testing patterns of feral coho
salmon spawners collected from the Platte River Weir .......................... 94

Figure 5 - 6. Frequencies of different diagnostic testing patterns of
captive hatchery broodstock ............................................................ 95

Figure 7. Prevalence of Renibacterium salmoninarum in feral chinook
salmon spawners collected from two of Michigan weirs between 2001

and 2005 ..................................................................................... 129

Figure 8. Renibacterium salmoninarum prevalences and intensities in
feral spawner coho salmon strains returning to Platte River Weir
throughout 2001-2004 .................................................................... 130

Figure 9. Prevalence of Renibacterium salmoninarum in feral Chinook
salmon spawners males and females collected from two of Michigan
weirs between 2001 and 2005 131

Figure 10. Renibacterium salmoninarum prevalence and intensity in
females and males of coho salmon spawners returning to Platte River
Weir throughout the period from 2002-2004 ....... . ................................ 132

Figure 11. Renibacterium salmoninarum prevalence and intensity of
infection in gamete donor feral spawner coho salmon strains versus their
corresponding 18-month juvenile fingerlings throughout 2001-2004 ......... 133

Figure 12. Prevalence and intensity of Renibacterium salmoninarum
among the adult brook trout collected through 2001-2003 ...................... 161

Figure 13. Renibacterium salmoninarum Prevalence and intensity in
kidneys and gametes of the Iron River brook trout broodstock in from

xiii

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Marquette State Fish Hatchery. ............................................ 162

Figure 14. An Iron River Brook trout fingerling with Bacterial Kidney
Disease. .................................................................................. 163

Figure 15. Kidney tissue of Iron River brook trout fingerling exhibiting
heavy Renibacterium salmoninarum infection .................................... 164

Figure16. Kidney tissue of Iron River brook trout fingerling exhibiting

heavy Renibacterium salmoninarum infection after enhanced antigen

retrieval procedures using Alkaline Phosphatase Red and goat anti-
Renibacterium salmoninarum antibody ............................................. 165

Figure 17. Hematoxylin and Eosin stained slide of kidney showing a
severe granulomatous reaction that is replacing kidney tissues of a 3
years old Assinica brook trout ......................................................... 166

Figure 18. An immunohistochemical stained Kidney tissue section with
chronic multi-focal granulomatous reaction from a 3 years old Assinica
brook trout with absence of bacterial cells or antigen from the affected
kidney tissues. ............................................................................ 167

Figure 19. An immunohistochemical stained Kidney tissue section with

chronic multi-focal granulomatous reaction from a 3 years old Assinica

brook trout exhibiting a mild chronic presence of bacterial cells or

antigens after enhanced antigen retrieval procedures ......................... 168

Figure 20. Nested PCR assay performed on isolates showing the
characteristic 320 bp band of R. salmoninarum isolates ......................... 183

xiv

 

 

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INTRODUCTION

Bacterial Kidney Disease (BKD), caused by the Gram-positive bacterium
Renibacterium salmoninarum (R. salmoninarum), is a systemic disease that
afflicts salmonid fish populations worldwide. R. salmoninarum is an obligate
intracellular pathogen that is transmitted both horizontally (Mitchum and
Sherman 1981; Bell et al 1984) and vertically (Evelyn et al. 1984, 1986). R.
salmoninarum pathogenicity relies upon a number of extracellular proteins
(ECP) that possess immunosuppressive (Turaga et al. 1987; Fredriksen et
al. 1997). The ECP contain a water-soluble, cell surface, 57-kDa protein
(p57) that has been demonstrated to be a major virulence factor of R.
salmoninarum (Getchell et al. 1985; reviewed in Wiens and Kaattari 1999).
BKD was originally known as the Dee Disease, due to its initial discovery in
Atlantic salmon (Salmo salar) from Aberdeenshire Dee and the River Spey in
Scotland in 1930 (Anonym 1933). Few years later the disease was first
reported in brook trout (Salve/inus fontinalis), brown trout (Salmo trutta) and
rainbow trout (Oncorhynchus mykiss) reared in a hatchery in Massachusetts,
USA (Belding and Merrill 1935). Since the appearance of this initial report, the

disease was reported in most of the cultured and wild salmonines from North
America.

Since, the first report of BKD in brook trout yearlings from Michigan in
1 955, the disease quickly spread to affect other salmonid species such as
echo and chinook salmon. BKD resulted in epizootics among coho salmon

spawners in 1967 (McLean and Yoder 1970), chinook salmon spawners

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during the 1980s (Holey et al. 1998) and recently harvested thousands of
hatchery raised brook trout between the period from 2002 to 2003 (Eissa
2005, this dissertation). These continuous eruptions of BKD outbreaks and
high prevalence of the R. salmoninarum among almost all the tested salmonid
populations throughout this study revealed a new fact that R. salmoninarum is
enzootic in Michigan salmonines and Great Lakes water basin.

Despite the aforementioned facts, the publications describing the status
of BKD in Michigan salmonines fish species are fairly scarce (Hnath and
Faisal 2005). This intensifies the needs to fill the gap between the rapid
progress of BKD and research performed on BKD in Michigan. This
submitted study is a trial to fill some of these gaps, by studying both the
causative agent and the affected salmonid fish populations in Michigan.

In brief, chapter, 2 in this study dealt with the development of tissue
processing protocol and culture technique that facilitated the isolation of large
number of R. salmoninarum isolates within relatively short incubation time.
The isolation of such large number of isolates from different salmonids, ages,
strains, and locations lead to a conclusion that BKD is enzootic in Michigan.

Chapter 3 describes the analysis of different diagnostic testing patterns
produced from different agreements and disagreements between results
obtained by three BKD diagnostic assays (Nested PCR, Quantitative ELISA,
and Culture) which allowed me to track the progress and different stages of
naturally occurring R. salmoninarum infection in some Michigan feral

salmonines. In chapter 4 the ability of both chinook and coho females and

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males to shed R. salmoninarum through the ovarian fluid and milt has been
discussed. These data shed the light on the possible role of male spawner
fish to spread the disease through shedding the pathogen via the milt. Thus,
both males and females should equally utilized for BKD testing and culling
procedures in weirs.

ln chapters 5, the retrieved data supported the previous reports, which
emphasized that brook trout are highly susceptible to R. salmoninarum
infection. Also, this study shed the light on the possible contribution of a
number of factors to development of BKD epizootics in Michigan hatcheries.
Further, the study suggested a number of efficacious control strategies for
stopping the BKD associated mortalities and minimizing the spread of BKD.
Finally, data in chapter six demonstrated the successful isolation of R.
salmoninarum from the kidneys of adult sea lamprey (Petromyzon man'nus)
but not from blood or any other organ, which indicated that sea lamprey, is a

possible non salmonid host range for R. salmoninarum infection.

CHAPTER ONE

LITERATURE REVIEWS

|. Historical perspectives

Bacterial Kidney Disease (BKD), caused by the Gram-positive bacterium
Renibacterium salmoninarum, is a systemic disease that afflicts salmonid fish
populations worldwide. The condition was originally described as the Dee
Disease because it was first observed among Atlantic salmon (Salmo salar)
from Aberdeenshire Dee and the River Spey in Scotland in 1930 (Anonym
1933 and Smith 1964) Other synonyms of the disease include Kidney
Disease, Corynebacterial Kidney Disease and Salmonid Kidney Disease
(Fryer and Sanders 1981 ). A few years later, Belding and Merrill (1935)
described a very similar infection that caused losses in brook trout (Salve/inus
fontinalis), brown trout (Salmo trutta) and rainbow trout (Oncorhynchus
mykiss) reared in a hatchery in Massachusetts, USA. By 1953, due to serious
outbreaks, BKD had become a limiting factor in rearing brook trout, brown
trout, rainbow trout, chinook salmon (Oncorhynchus tshawytscha), coho
salmon (Oncorhynchus kisutch) and sockeye salmon (Oncorhynchus nerka)
in many hatcheries in the State of Washington (Earp et al. 1953). In the
following year, the disease was found in the feral salmon in the same state
(Rucker et al. 1954). In 1955, BKD spread to the Great Lakes basin with the

introduction of salmonines and their products from the Pacific Northwest

 

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(Allison 1958). Reports from Canada linked the disease to mortalities in wild
salmonines from Nova Scotia (Pippy 1969; Paterson et al. 1979) to British
Columbia (Evelyn et al. 1973; 1981). By 1988, the disease became
widespread in Europe (England, France, Finland, Germany, Iceland, Italy,
Scotland, Spain, Turkey and Yugoslavia), North America (USA and Canada),
and Japan (reviewed in Bullock and Herman 1988; Fryer and Lannan 1993).
The disease continued its spread to Chile (Sanders and Barros 1986) and
there is a current consensus among fish health professionals that BKD is
virtually prevalent in all parts of the world where wild or cultured salmonines

exist (European Commission 1999).

II. The pathogen

1) Nomenclature and current classification of the etiological agent

Based on Gram stain properties, morphology, the causative bacterium
was suggested to be a member of the genus Corynebacterium by Ordal and
Earp (1956) and subsequently by Smith (1964). Sanders and Fryer (1980)
later refuted this classification based on the absence of mycolic acid, guanine
plus cytosine (G + C) content of DNA, cell wall sugar and amino acid
compositions of the peptidoglycan cell wall layer. The authors proposed that
this bacterium formed a single species in a new genus Renibacterium and

they identified the bacterium as Renibacterium salmoninarum (Sanders and

Fryer 1980). Sequencing of the 16S ribosomal ribonucleic acid (rRNA) from
R. salmoninarum (Gutenberger et al. 1991) and recent evaluation of G + C
content (Banner et al. 1991) placed the organism in the Gram-positive
eubacterial subdivision of actinomycetes. Arthrobacter and Micrococcus spp.

are the closest relatives to R. salmoninarum (Holt et al. 2001).

2) Cell morphology

Renibacterium salmoninarum is a short rod (0.3-1.0 by 1-1.5 um), Gram-
positive, non-sporulated, non-capsulated, non-motile, and non acid-fast
bacterium that is arranged in pairs (diplobacilli) and rarely as short chains
(Sanders and Fryer 1980). Renibacterium salmoninarum consists of two
regions; a central region filled with lightly stained filaments (represent DNA)

and a peripheral region filled with small, electron dense ribosomes (Young

and Chapman 1978).

3) Isolation, culture and cultural characteristics

Renibacterium salmoninarum is a slow growing organism (Sanders and
Fryer 1980). Earp et al. (1953) cultured the bacterium on an artificial medium
for the first time from infected kidney tissues on a medium that consisted of
fish extract, glucose, yeast extract and meat infusion in agar. The authors

achieved limited growth with first appearance of colonies after more than two

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weeks of incubation. When the same authors used minced chick embryo
tissues embedded in 1 % agar or Dorset’s Egg medium, they achieved better
growth. Addition of 0.05 to 0.1% L-cysteine to the Dorset’s Egg medium has
further enhanced the growth of R. salmoninarum upon primary isolation
(Ordal and Earp 1956). The authors noted that trypticase blood agar could be
used for secondary cultures and bacterial maintenance. Based on years of
research, Ordal and Earp (1956) formulated the Kidney Disease medium
(KDM1) which consisted of: tryptose 1.0 gm %, beef extract 0.3 gm %, NaCI
0.5 gm %, yeast extract 0.05 gm %, cysteine-hydrochloride 0.1 gm %, human
blood 20 v/v and agar 1.5 gm %. They designated this medium as “Cysteine
Blood Agar Medium”. While testing the in vitro sensitivity of R. salmoninarum
to a large number of therapeutics, Wolf and Dunbar (1959), achieved fair
growth on cysteine supplemented Mueller-Hinton medium (MH). This
modified MH medium became the medium of choice for the growth of R.
salmoninarum for several years (Bullock et al. 1974).

Evelyn (1977) modified Ordal and Earp’s KDM1 by replacing human
blood, tryptose and beef extract with 20 % fetal bovine serum and peptone
and designated the modified medium as KDM2. To reduce the time needed
for primary isolation, Evelyn et al. (1989) added 25ul of heavy inoculum of R.
salmoninarum culture (commonly known as a nurse culture) to the center of
KDM2 plates. The authors reported that this modification has accelerated
bacterial growth in primary cultures. Further, Evelyn et al. (1990) were able to

achieve more consistent growth of the primary culture by replacing the nurse

culture with 25pl of filter-sterilized R. salmoninarum spent medium. The major
drawbacks of KDM2 medium, however, were the high cost and presence of
serum proteins, which hampered the identification of proteins of bacterial
origin.

A number of serum-free media for R. salmoninarum growth have been
formulated. For example, Embley et al. (1982) described a serum-free, semi-
defined growth medium that supported secondary, but not primary, growth of
R. salmoninarum. Daly and Stevenson (1985) formulated the Charcoal Agar
Medium in which they substituted activated charcoal for serum. Starliper et
al. (1998) compared the performance of 13 serum-free media and 1 serum-
supplemented media for the growth of R. salmoninarum isolates and found
that there were no significant differences among the 14 medium formulations
used when mean cell counts were compared after 10, 20, 30 days incubation.
To control growth of other bacteria from fish lesions, Austin et al. (1983)
incorporated four antibiotics (Cycloheximide, D-cycloserine, Oxolinic acid and
Polymyxin B) to the KDM2 medium and reduced the volume of serum from 20
% to 10 % (designated Selective KDM or SKDM). By these modifications, the
authors significantly reduced bacterial contaminants, a matter that facilitated
the selected growth of R. salmoninarum from clinical and environmental
samples. Our current lab experience (Eissa 2005, Chapter 2) suggested that
the modification of Selective KDM (SKDM) by incorporating 1 °/o Spent

medium into the agar enhanced the growth of the R. salmoninarum colonies,

 

era"

R. 56
flocc
slew

(Sm'

4)l’r

 

Dmse
andfr
VVIHa

backs

shortened the period of incubation, and minimized the growth of
contaminating bacteria.

Renibacterium salmoninarum colonies are creamy (non-pigmented),
shiny, smooth, round, raised, entire, and 1-2mm in diameter on KDM2 after
incubation at 15 °C for 20 'days (Austin and Austin 1999). On cysteine
supplemented solid media, old colonies (i.e. 12 weeks) appeared extremely
granular due to crystallization of cysteine, while in broth culture media; some
R. salmoninarum strains produced a uniform turbidity whereas others
flocculated out of suspension (Austin and Austin 1999). The organism grows
slowly at 5°C, 22°C and optimally at 15 °C but there was no growth at 37 °C

(Smith 1964).

4) Preservation of cultures

Several methods have been used to preserve different species of
actinomycetes including Streptomyces, Actinomyces and Renibacterium
species. For long term preservation, methods such as lyophilization
(Hopwood and Ferguson 1969), storage under liquid nitrogen (Pridham and
Hesseltine 1975) were successfully used. Bacterial cells can also be
preserved in diluted glycerol (10-20 %-v/v) and frozen at -20 °C, but thawing,
and freezing cycles can affect cell stability and viability (Wellington and
Williams, 1979). To overcome this disadvantage Feltham et al. (1978) stored

bacteria on glass beads in 10 % (v/v) glycerol at — 76 °. The glass beads

ale

 

SIB

pate

resui
Thus

tand
Chara
5Lhen
The 0
Case”

Wham

 

UIIIIZe

 

allowed removal of small samples without thawing the entire culture, which
was advantageous for long-term preservation (Wellington and Williams 1979).
Preservation of small inocula of R. salmoninarum in KDM2 (Evelyn et al.
1977) or peptone saline (Starliper et al. 1997) and storage at -80 °C were

successfully used.

5) Biochemical characteristics

The organism is cytochrome oxidase negative, catalase positive,
proteolytic and cysteine HCl is required for its growth (Ordal and Earp 1956;
Smith 1964; Sanders and Fryer 1980). Interestingly, R. salmoninarum isolates
from different sources are homogeneous in their biochemical characteristics
(Austin et al.1983; Goodfellow et al. 1985; Bruno and Munro 1986 a) but the
result for a given test can vary depending upon the testing system used.
Thus, the organism is positive for the gelatinase and DNase reactions by
standard methods (Bruno and Munro 1986 a), but it was negative for these
characters by the APl-ZY M system (Goodfellow et al. 1985). The organism is
B-hemolytic on media supplemented with blood (Bruno and Munro 1986 a).
The organism can liquefy gelatin, degrade Tween (20—60), and hydrolyze
casein. The bacterium is negative for esculin hydrolysis, DNase, urease,
nitrate reduction, phosphatase, methyl red, indole test and carbohydrate

utilization test (table 1).

10

err."

and

enr:
aztr
D-cy

and

 

TIAJ

OIOU
al 19
salmg
posse
and F
CUHUn
mofia

IOpr

6) Antibiotic susceptibility

Renibacterium salmoninarum isolates are sensitive to chloramphenicol,
erythromycin, novobiocin, streptomycin, sulfamerazine, and tetracycline (Wolf
and Dunbar 1959; Austin and Rodgers 1980), carbenicillin, and cephaloridine
(Goodfellow et al. 1985). Renibacterium salmoninarum is also sensitive to
enrofloxacin (Hsu et al. 1994), tiamulin, cefazolin (Bandin et al. 1991) and
azithromycin (Rathbone et al. 1999). Furthermore, the organism is resistant to
D-cycloserine, oxolinic acid (4 pg / ml), polymyxin l3 and cycloheximide (Wolf

and Dunbar 1959; Goodfellow et al. 1985).

7) Antigenic characteristics and virulence factors

Renibacterium salmoninarum is an obligate intracellular pathogen that is
able to invade all types of fish cells particularly phagocytic cells (Gutenberger
et al. 1997; Ellis 1999). The ability of R. salmoninarum to invade phagocytes
or other cells depends upon certain virulence determinants (Gutenberger et
al. 1997; Ellis 1999; Piganelli et al. 1999). It was demonstrated that R.
salmoninarum secretes a number of extracellular products (ECP) that
possess proteolytic, hemolytic and DNA degradation activities in vitro (Austin
and Rodgers 1980; Bruno and Munro 1986 a). When crude or precipitated
culture supernatants were injected into Atlantic salmon fingerlings, 80-100%
mortalities were reported (Shieh 1988), but Bandin et al. (1991) were unable

to reproduce this finding using untreated culture supernatants. A 65-kDa R.

11

 

fish

sale
al. 1
hydr
viruli
p57.

aggt;

 

that

hndér

salm
strait
sai’rr,
the s
attrit
IFter
of R.
regio
differ

Michi

salmoninarum zinc metalloprotease-like protein has been extracted from R.
salmoninarum ECP that possesses hemolytic activities against a number of
fish and mammalian erythrocytes. The encoding gene of the R.
salmoninarum ECP with hemolytic activity was designated as hly (Grayson et
al. 1995). R. salmoninarum secretes a water-soluble, heat stable,
hydrophobic cell surface 57 kDa protein (p57) that is believed to be the major
virulence determinant of this bacterium (Getchell et al. 1985). In vitro, purified
p57 exhibited both hemolytic (Daly and Stevenson 1990) and leuco-
agglutinating (Wiens and Kaattari 1991) properties. Hamel (2001) reported
that R. salmoninarum isolates differed in their pathogenicity to salmonids, a
finding that correlated positively with the amount of surface associated p57.
Challenge of susceptible fish with non-auto-agglutinating strains of R.
salmoninarum caused significantly lower mortality than auto-agglutinating
strains (Daly and Stevenson 1990; O’ Farr'el et al. 2000). Soluble R.
salmoninarum surface proteins possess immunosuppressive action against
the salmonid specific antibody response (Turaga et al. 1987), which was
attributed not only to the p57 protein, but also to a 22-kDa surface protein
(Fredriksen et al. 1997). Starliper et al. (1997) compared a number of strains
of R. salmoninarum isolated from chinook and coho salmon from different
regions in North America for virulence. The authors found that virulence
differed among the used isolates and concluded that isolates retrieved from

Michigan weirs in the late 1980s were the most virulent.

12

8)I

 

 

 

hon
wet

highg

 

 

IH.T

1IDI:

deDGI

T978;

 

 

8) Molecular and genetic diversity

Although the biochemical uniformity (Bruno and Munro 1986a) and
phylogenetic homology of R. salmoninarum strains (Gutenberger et al. 1991 ),
a minimal molecular diversity was detected among strains isolated from
different parts in the world (Alexander et al. 2001). Alexander et al. (2001)
succeeded in differentiation between isolates of R. salmoninarum based on
PCR amplification and length polymorphism in the tRNA intergenic spacer
regions (tDNA ~lLPs). Moreover, a genetic diversity was detected among 40
North American isolates by using the multilocus enzyme electrophoresis
(MEE) assay with the highest genetic diversity detected in strains isolated
from chinook and coho salmon spawners returning to the Little Manistee river
weir in Michigan (Starliper 1996). Specially, Michigan isolates showed a

higher variation in succinate dehydrogenase and esterase loci.

Ill. The disease

1) Disease course

Despite the fact that BKD develops slowly, progress of the disease

depends on environmental factors such as water temperature (Sanders et al.

1978; Fryer and Sanders 1981; Bullock and Herman 1988), host factors

13

 

 

 

b) Int.

(Evenden et al.1993), and R. salmoninarum strain virulence (Starliper et al.

1997).

a) External signs

Affected fishes manifest a wide range of external lesions as well as
behavioral changes that might vary according to the species, age of the fish
affected and the virulence of the R. salmoninarum strain (Fryer and Sanders
1981; Bullock and Herman 1988; Evenden et al. 1993). Erratic swimming
behavior, exophthalmia, superficial blebs of the skin, cavitations in muscles
and deep abscesses all over the body surface have been reported in affected
fish (Belding and Merrill 1935; Smith 1964; Fryer and Sanders 1981; Bullock
and Herman 1988). The blebs and cavitations might contain a white to
yellowish or hemorrhagic fluid (Bullock and Herman 1988). Ascitis and
peticheal hemorrhages in muscles and fins were also reported (Belding and
Merrill 1935; Earp et al. 1953; Evelyn 1993). In very rare cases, the external
signs of the disease in chinook and coho salmon might only be manifested by
exophthalmia with the accumulation of infective fluid containing large amount
of the bacteria, pus and necrotic tissue in the enlarged eyes (Bullock and

Herman 1988).

b) Internal lesions

14

Kidneys of affected fishes are usually swollen and exhibit white foci that
contain leucocytes, bacteria, and host cell debris (Fryer and Sanders 1981).
In advanced cases the kidneys appear mostly grayish in color, the spleen
may increase in size and the liver appears very pale in color (Woods and
Yasutake 1956; Fryer and Sanders 1981). The most typical clinical lesions
associated with BKD are the presence of scattered nodules of various sizes
over the surface of the kidneys, spleen and liver (Belding and Merrill 1935;
Snieszko and Griffin 1955; Klontz 1983). In some cases, peticheal
hemorrhages were noticed in the muscles lining the peritoneum with ascitic
fluid accumulation (Ferguson 1989). An opaque membrane
(pseudomembrane) that covers internal organs was reported, especially in
fish maintained at a temperature below 9 °C (Snieszko and Griffin 1955; Bell
1961; Fryer and Sanders 1981). The pseudomembrane consists of fibrin and
leucocytes (Smith 1964). Similar membranes occur in trout at higher
temperatures (12-13 °C) (Bullock and Herman 1988). Hemorrhages with
white or yellow viscous fluid in the hindgut and peticheal hemorrhages were

often found in the peritoneum of infected Atlantic salmon (Smith 1964).

c) Histopathology

The initial histopathological description by Belding and Merrill (1935)

indicated that the kidney as the major organ affected by the R. salmoninarum

infection. All infected brook trout and brown trout exhibited microscopic

15

lesions in the kidney, and to a lesser extent in the liver and spleen. Lesions
were chronic in nature with multiple granulomas that resemble those noticed
in mammalian tuberculosis (Snieszko and Griffin 1955; Wood and Yasutake
1956). Fibrotic lesions were also noticed in kidneys, spleen, liver and
intestines of the infected fish with proliferating fibroblasts forming distinct
nodules that coalesced to form large masses of affected tissues (Wood and
Yasutake 1956). The granulomatous lesions apparently arose in the
connective tissue stroma between the parenchymal cells of various organs
(Woods and Yasutake 1956; Jansson 2002). It is believed that the
granulomas are formed as a result of macrophages activation (Secombes
1985) followed by its adherence to each other forming epithelioid appearance
and then the fusion of a few number of these activated macrophages to form
giant cells (Secombes 1985). Both, giant cells and activated macrophages
release large amounts of Iytic enzymes into the surrounding tissues leading to
necrosis at the central part of the granuloma (Bruno 1986, Jansson 2002).
Interestingly, bacteria can occur intracellularly or extracellularly in the
granulomas or necrotic foci (Bruno 1986; Bullock and Herman 1988). The
hematopoietic tissue of the anterior kidney appeared to be affected firstly,
followed by extensive damage to the excretory part of the kidneys (Woods
and Yasutake 1956; Jansson 2002). Kidney pathology may include
hypercellularity of the glomeruli, occlusion of Bowman’s space by filamentous
or granular deposits (Sami et al. 1992) and presence of eosinophilic granules

in proximal tubules (Young and Chapman 1978). Massive myocarditis (Wood

16

8112

16C

 

 

for“

COT’

9V8.

 

 

and Yasutake 1956), meningitis, and encephalitis (Speare 1997) were
recorded in some salmonids. In the liver, histopathological changes take the
form of granulomatous nodules in the connective tissue stroma between the

cords of the hepatic cells (Woods and Yasutake 1956).

2) Susceptibility

There are a number of observations indicating that salmonid species and
even different strain of the same species can differ in their susceptibility to
BKD. For example, coho salmon of three different transferrin genotypes (AA,
AC and CC) differed in resistance to experimental infection with R.
salmoninarum (Suzumoto et al. 1977). Also, three populations of chinook
salmon from different rivers, showed various mortality rates to experimental
infection with R. salmoninarum (Beacham and Evelyn 1992). Winter et al.
(1980) reported similar results in coho salmon and steelhead trout
(Oncorhynchus mykiss). Further, Belding and Merrill (1935) reported that
brook trout was more susceptible to R. salmoninarum infection than the
rainbow trout when experimentally infected. Mitchum and Sherman (1981)
reported that brook trout were more susceptible to natural BKD infection than

rainbow trout and brown trout.

3) Pathogenesis and immunity

a) Process of infection and pathogenesis

17

 

the s
”70':
al. 1

110m

Tedu

(Gra

 

IWIe

neUtr

 

Renibacterium salmoninarum can induce uptake by non-phagocytic cells
and can survive ingestion, which provides a means of entry into the host via
the gills and the gastrointestinal tract (Evelyn 1996; Flafio et al. 1996; Balfry
et al. 1996), however a study demonstrated that R. salmoninarum can not be
internalized by healthy rainbow trout gills in vitro (McIntosh et al. 2000).
Renibacterium salmoninarum uptake by eggs is another possibility that result
in vertical transmission of the organism from parent to offspring (Evelyn et al.
1984; Evelyn et al. 1986a, b; Bruno and Munro 1986b).

Renibacterium salmoninarum is believed to spread through blood and also
through intracellular habitation and replication in macrophages (Gutenberger
et al. 1997; Ellis 1999). Although R. salmoninarum is a slow growing
organism, it can reach levels of 109 cells lg in spleen and kidney tissues
before initiation of fish mortality (Evelyn 1996).

Opsonization of the pathogen by antibody and /or complement increases
the success of R. salmoninarum to survive and replicate within phagocytes
more willingly than limit its activity as with most of other pathogens (Bandin et
al. 1995). To survive and replicate, R. salmoninarum must acquire nutrients
from the host. In the absence of iron, R. salmoninarum may produce iron
reductase, which makes bound iron more available for bacterial uptake
(Grayson et al. 1995).

Renibacterium salmoninarum produces large amounts of the p57 antigen
(Wiens and Kaattari 1989), both in serum and intracellularly. The quantity can

neutralize the vast majority of antibodies that may be evoked in response to

18

infection. These antibody-p57 complexes may remain in tissue and contribute
to tissue destructive hypersensitivity resulting in granulomas (Bruno 1986;
Sami et al. 1992).

The P57 has immunosuppressive and tissue destructive properties. The
p57 agglutinates salmon leukocytes (Wiens et al. 1991) and suppresses
antibody production against unrelated antigens in vitro (Turaga et al. 1987).
The p57 is a potent inhibitor of the phagocyte respiratory burst response
(Campos-Perez et al. 1997) and could decrease the bactericidal activity of
juvenile chinook macrophages against Aeromonas salmonicida (Siegel and
Congleton 1997).

Senson and Stevenson (1999) suggested that p57 and its breakdown
products might form a protective layer around R. salmoninarum cells.
Bacterial cells stripped of p57 induced stronger immune response than those
not stripped of p57 (Wood and Kaattari 1996). Cell surface associated p57
and its breakdown products may effectively block highly immunogenic areas
of the bacterial cell surface from detection by host defenses (Wiens and

Kaattari 1999).

b) Effect of BKD on host immune response

Grayson et al. (2002) studied the immunosuppressive effect of R.

salmoninarum in vitro and in vivo. Within an in vitro assay, macrophages

showed a rapid inflammatory response in which the expression of interleukin-

19

119, major histocompatibility complex class II, inducible cyclooxygenase, and
inducible nitric oxide synthase (iNOS) were enhanced, while tumor necrosis
factor-a (TNF-a) expression was greatly reduced initially and then increased.
In vivo study, intraperitoneal (i.p.) injection of R. salmoninarum DNA vaccine
constructs (msa) reduced the expression of lL-1B, Cox-2, and MHC II but
stimulated TNF-a. In this study, the authors concluded that p57 suppresses
the host immune response and hypothesized that the chronic granulomatous
reaction is due to prolonged stimulation of TNF-a. The p57 possess
immunosuppressive action against salmonid specific antibody response
(Turaga et al. 1987), tissue destructive properties (Bruno 1986) and capable
of agglutinating salmon leukocytes (Wiens and Kaattari 1999).

Aside from its opsonizing action, antibodies interact directly with free antigen
(p57), creating immune complexes that aggregate within the tissue and cause
hypersensitivity reactions, resulting in granulomas and tissue damage (Bruno
1986). Macrophage Activating Factor (MAF)-activated macrophages can
effectively kill R. salmoninarum cells (Hardie et al. 1996), but production of
MAP in immature helper T-cells may be suppressed at low temperature
(Siegel and Congleton 1997). The proliferation and action of T cells in
activating macrophages may be the primary successful immune response

against R. salmoninarum (Secombes 1985; Hardie et al.1996).

c) Environmental factors

20

Efl

88C

 

sev
dem
equ?
OI ir

SEW

BIen

Effr

Effect of diet

Studies suggested that the prevalence and severity of BKD might be partly
related to certain dietary and environmental factors. Diets formulated of gluten
as opposed to cottonseed meal have resulted in higher BKD prevalence in
several hatcheries in Washington (Wood 1974). Wedemeyer and Ross (1973)
demonstrated that BKD prevalence was similar in fish fed rations containing
equivalent amounts of either gluten or cottonseed, but the non-specific stress
of infection perhaps due to the increased ascorbate depletion was more
severe in the corn gluten group. Sakai et al. (1986) concluded that vitamins
had no effect on BKD prevalence. Woodall and LaRoche (1964) suggested
that iodine insufficiency was responsible for increased BKD incidence in
juvenile chinook salmon. Paterson et al. (1981) indicated that Vitamin A, zinc,
and iron levels are significantly reduced in BKD-infected fish and subsequent
feeding trials provided a lower incidence of BKD in fish fed diets high in trace

elements (Fe, Cu, Mn, Co, I and F) or low in calcium (0.2%).

Effects of temperature

Several authors reported that BKD could occur over a wide range of water
temperatures (Belding and Merrill 1935; Earp et al. 1953; Fryer and Sanders
1981; Bullock and Herman 1988). For example, at 15-20 °C, experimentally

infected juvenile salmon and trout died 21-34 days after inoculation, as

21

 

 

BK
an
m-
de:

NI-
IL:

 

pen

rate

TED:

opposed to 60-71 days post inoculation at 67°C (Sanders et al. 1978). Also,
Wood 1972 (cited in Fryer and Sanders 1981) reported that mortalities from
BKD occurred after 30-35 days post exposure at temperatures above 11 °C
and took 60-90 days at 7.2-10 °C. Sanders and Fryer (1981) indicated that
most of epizootics occurred during the autumn and winter, under conditions of
declining water temperatures; however the greatest mortality was associated
with periods of highest water temperatures. Also, it was noted that during

periods of low water temperatures the disease produced a slow steady death

rate.

Effect of estuarine and salt-water environments

Despite the fact that BKD occurs mainly in freshwater, significant
infections also occur in saltwater (Banner et al. 1983). Reports demonstrated
that deaths continued in chinook, coho and pink salmon stocks after
movement to salt water-rearing ponds (Earp et al. 1953; Bell 1961 ). Frantsi et
al. (1975) reported that R. salmoninarum impaired the ability of Atlantic
salmon smolts to acclimate to saltwater and caused a subsequent reduction
in ocean survival. Ellis et al. (1978) isolated the organism from juvenile
chinook salmon that had spent two winters in the ocean. Fryer and Sanders
(1981) indicated that BKD was thought to be the main cause of death among
coho salmon smolts released from Siletz hatchery in Oregon. The authors
reported that the majority of deaths occurred between two and four months

after the fish entered saltwater. They also concluded that fish infected with

22

 

IV. I

1)G

 

salrr
The
Ame
BUIII
wild
Mite
Ame
JOne
Fran;

Unite

BKD while in freshwater will continue to die from this disease, but at an
accelerated rate, after migration to saltwater. BKD infection can impair
acclimatization to seawater and cause death (Mesa et al. 1999). Further,
Price and Schreck (2003) experimentally assessed the effect of BKD on
saltwater preference of juvenile spring chinook salmon and concluded that

there is a significant negative relationship between mean infection level and

saltwater preference.

IV. Epizootiology

1) Geographical distribution

Bacterial Kidney Disease has been reported wherever susceptible
salmonid populations are present (Fryer and Sanders 1981; Klontz 1983).
The disease is commonly reported in cultured salmonid species from North
America, Europe, Japan and South America (Fryer and Sanders 1981;
Bullock and Herman 1988). BKD has also been observed in a wide range of
wild (Pippy 1969; Evelyn et al. 1973; Ellis et al. 1978; Paterson et al. 1979;
Mitchum and Sherman 1981) and feral salmonid populations from North
America (Elliot and Pascho 1991; Sanders et al. 1992; Holey et al. 1998 and
Jonas et al. 2002). The geographic range of BKD includes Canada, England,
France, Finland, Germany, Iceland, Italy, Japan, Scotland, Spain, Turkey,

United States, former Yugoslavia and Chile (Bullock and Herman 1988). BKD

23

W8

.9;
|

Syl‘

 

the
re;

20:

2)I

 

was presumptively diagnosed and reported in Australian Victoria in the early
19703 in farmed chinook salmon however further work identified the
syndrome to be nocardiosis (Humphrey et al. 1987). No evidence supporting
the presence of the disease in New Zealand, Russia. BKD was recently

reported in Denmark (Lorenzen et al. 1997) and Norway (Jansson et al.

2002)

2) Host range

Bacterial Kidney Disease has been reported in salmonids of the genera
Oncorhynchus, Salmo and Salvelinus (Fryer and Sanders 1981).
Renibacterium salmoninarum has also been detected in chinook salmon
(Holey et al. 1998), coho salmon (MacLean and Yoder1970), brown trout,
brook trout, rainbow trout (Belding and Merrill 1935; Mitchum et al. 1979),
Pacific salmon, Atlantic salmon, lake trout (Bullock and Herman 1988), pink
salmon (Bell 1961), Kokanee salmon (Awakura 1978), Grayling (Thymallus
thymallus) (Kettler et al. 1986), Lake Michigan Whitefish (Coregonus
clupeformis) and bloater (Coregonus hoyr) (Jonas et al. 2002) and whitefish
(Coregonus Iavretus) in Finland (Rimaila—Pamanen 2002) . The organism has
also been detected in absence of disease in few non-salmonid species such
as greenling (Heragrammos otakr), flathead (Platycephalus indicus) and
Pacific herring (G/upea pallasi pallasr) (Traxler and Bell 1988). Renibacterium

salmoninarum antigen has also been detected in Japanese sculpin (Cottus

24

 

 

 

 

 

Japonicus) and Japanese scallops (Patinopecten yessoensis) (Sakai and
Kobayashi 1992). Recently, the organism has been isolated for the first time
from the adult parasitic stage of Lake Ontario Sea Lamprey (Petromyzon

marinus) (Eissa et al.2004).

3) Disease transmission

a) Source of infection

Renibacterium salmoninarum is excreted in the feces of clinically diseased
trout and can survive for up to one week and two weeks in the feces and
sterile seawater respectively (Balfry et al. 1996). The organism can also
survive in non-sterile freshwater and pond sediments for up to 21 days
(Austin and Rayment 1985). Thus, the orofecal route of horizontal
transmission may contribute significantly to the increasing prevalence of BKD

in salmonids.

b. Horizontal transmission

Renibacterium salmoninarum possesses a powerful capability of

inducing uptake by tissue cells including the epithelial lining of the gastro-

intestinal tract (Bruno 1986; Evelyn 1996; Piano et al.1996). Infection is

thereby likely to occur where sufficient numbers of bacteria are present within

25

 

Fr,

inte

traI

 

TITO
d8v
hat:
hatc

dem

the immediate vicinity of aquatic environment. Oral-fecal route of infection
can also, occur in net pens by ingestion of contaminated feces (with up to
107 bacteria / gram of feces) during feeding (Balfry et al. 1996). Waterbome
infection may occur through gills, eyes, lesions, wounds and ingestion
(Evenden et al. 1993). The organism was also transmitted by feeding fish on
infected or inefficiently pasteurized fish offales or fish flesh (Wood 1974;
Fryer and Sanders 1981). Thus, uptake of R. salmoninarum through the
intestinal wall is a likely pathway of infection (Jansson 2002). Horizontal
transmission can also occur between wild and stocked hatchery trout in
natural systems (Mitchum and Sherman 1981). Long-term exposure (180
days) of healthy fish to highly infected or dying salmon resulted in the
infection and death of all exposed fish at an average water temperature of 10

C (Murray et al. 1992).

c. Vertical transmission

Numerous studies have been conducted in the last two decades in order
to study the possibility of vertical transmission of R. salmoninarum from
mother to offspring via eggs. Allison (1958) was the first to report the
development of BKD in offspring hatched from eggs transferred from a
hatchery where the disease had been endemic for many years to another
hatchery where it had never been detected. Bullock et al. (1978)

demonstrated transmission of R. salmoninarum from the broodstocks to their

26

 

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Do
the
tra'

the

be»;
WI".

ba:

salr
the
Yes

198.

 

Cal)

 

We 'r

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progeny via the eggs. Interestingly, the organism has been transmitted even
after the surface disinfection of eggs which likely due to the fact that the
pathogen was located within the perivitteline membrane of the egg away from
the reach of the disinfectant (Evelyn 1993). The intra-ovum route of
transmission has now been firmly established (Evelyn et al. 1986 a, b) where
the pathogen is located in the yolk and is protected from surface disinfectants
(Evelyn et al. 1986a,b; Bruno and Munro 1986 c). Infected coelomic fluid has
been shown to be an important source of infection for the egg (Evelyn 1993)
where the organism found its way to the yolk via the micropyle due to high
bacterial counts in coelomic fluid. There are some instances that intra-ovum

infections can also occur prior to ovulation and directly from the ovarian tissue

(Evelyn 1993).

d. Fish as possible vectors and carriers

Although there are enough satisfactory data indicating that R.
salmoninarum is an obligate intracellular pathogen of salmonid fishes and that
the reservoir and carrier of infection are other infected salmonid (Woods and
Yasutake 1956; Fryer and Sanders 1981; Klontz 1983; Bullock and Herman
1988), yet there are few existing data about the possibility that non salmonids
can act as a reservoir or vector for the organism. Few non-salmonid species
were able to contract the infection naturally or experimentally and in turn they

might become accidental carriers and play an important role in transmission

27

 

I
I

I

- I.

I PI .
I.

III

that

pos-

Spe:

bee

salr

So"

sa/rr

 

OCCE

of the disease to salmonid species by cohabitation. For example, Pacific
herring (Clupea harengus pallasr) living in net pens with R. salmoninarum
infected coho salmon have been reported as infected (Paclibare et al.1988).
Also, Pacific herring (Traxler and Bell, 1988), sablefish (Anoplopoma fumbria)
(Bell et al. 1990), Common shiner (Notropis comutus) (Hicks et al. 1986), and
the fathead minnow (Pimephales promelas) (Hicks et al. 1986) were able to
contract infection by i.p. injection of R. salmoninarum. The organism was also
detected in moribund Pacific hakes (Mefluccius productus) (Kent et al. 1998).
In addition, Greenlings (Hexagrammos otakir) and flathead (Platycephalus

indicus) were also reported as possible vectors for the disease (Sakai and

Kobayashi 1992).

e. Possible vectors other than fish

A limited number of studies have been conducted in the last two decades
that have lead to the assumption that animals other than fish can act as
possible vectors for the transmission of R. salmoninarum to salmonid fish
species. For example, the Japanese scallop (Patinopecten yessoensis) has
been reported as a possible vector for R. salmoninarum transmission to coho
salmon pen-raised in the neighboring seawater (Sakai and Kobayashi 1992).

Some blood-sucking ectoparasites, like salmon lice (Lepeophteirus
salmonis), can act as vectors for the pathogen. Although, salmon lice can

occasionally harbor the pathogen, no record of active transmission of R.

28

88

He

88:

an:

vis:

1) Is

ISOIe
Cyst

Sele

 

COIT‘;

38/0-

salmoninarum between sea lice infected and non-infected fish exists

(Richards et al. 1985; Frerichs and Roberts 1989).

f. Reservoirs

Clinically infected, subclinically infected or latent carrier salmonids are the
main reservoir of infection (Klontz 1983; Richards et al. 1985, Bullock and
Herman 1988). Bacterial laden-feces and R. salmoninarum rich pond
sediment can also act as a reservoir of infection (Balfry et al. 1996, Austin
and Rayment 1985). In addition, inefficiently pasteurized infected salmon

viscera are a confirmed reservoir of infection (Woods 1974).

V. Diagnosis of BKD

1) Isolation and bacteriological identification of the agent.

A number of culture media have been successfully used for the primary
isolation of R. salmoninarum from clinically infected fish. Among these media
cysteine blood agar (Ordal and Earp 1956), KDM2 (Evelyn et al. 1977),
selective KDM (Austin et al. 1983) and charcoal agar medium (Daly and
Stevenson 1985) were used with varying degrees of success. The most
Common drawback of bacterial culture is the slow growing nature of R.

Sc’zllmoninarum, which requires up to 12 weeks to achieve bacterial growth.

29

OF

 

Ct

2)

cu!

 

dia;
5136
C06

SUC

use

Date

the i

 

The optimal incubation temperature for the isolation of R. salmoninarum
on culture media is 15 °C (Sanders and Fryer 1980). The organism is
differentiated from other Gram-positive bacteria using the morpho-

chemotaxonomic features described by Sanders and Fryer (1980).

2) Antigen-antibody reactions

a. Agglutination test

Although easy and rapid to perform, the test requires that bacteria are first
cultured which conveys no advantage if Compared with that of other
diagnostic methods. Kimura and Yoshimizu (1981) used staphylococci
specifically sensitized with antibody against R. salmoninarum to develop a
coagglutination test to detect R. salmoninarum in kidney tissues with limited

success.

b. lmmunofluorescence

Direct and indirect fluorescent antibody tests (FAT) have commonly been

used to detect R. salmoninarum in infected tissues including fixed and

Paraffin embedded tissues. Bullock and Stuckey (1975) were first to describe

the indirect fluorescent antibody technique (IFAT) to visualize the R.

30

38

 

 

FA

Ch

I'IU

DTE

 

Au.
ma
tel

381
the

inf

C.E

 

salmoninarum cells in tissues of infected fish. They concluded that IFAT is
more sensitive than Gram stain and can detect the bacteria in subclinical
infections. Several methods to quantify R. salmoninarum utilizing FAT tests
have been used, including a subjective scoring of fluorescence intensity (1+
to 4+) in tissue smears (Bullock et al. 1980). In a later procedure, bacteria are
immobilized on filter-paper grids and titers expressed as cells per unit of
tissue or ovarian fluid (Elliot and Barila 1987).

Elliot and McKibben (1997) compared two fluorescent antibody
techniques (FATs) (membrane filtration FAT or MF-FAT and Smear-FAT or S-
FAT) for detection of R. salmoninarum in ovarian fluid from naturally infected
chinook salmon. They reported greater sensitivity of MF-FAT compared to the
S-FAT and concluded that MF-FAT was preferable for detection of low
numbers of bacteria. Cross reactivity of other bacterial species with antisera
prepared against R. salmoninarum have been reported (Bullock et al. 1980;
Austin et al. 1985; Brown et al. 1995), thus the inclusion of any FAT of control
material from R. salmoninarum-positive fish is necessary for comparison of
cell morphology and staining properties of bacteria in test and control
samples (Elliot and McKibben 1997). Inter-laboratory comparisons revealed
that FAT reproducibility is poor when used in detection of very low levels of

infection (Armstrong et al. 1989).

c. Enzyme linked immunosorbent assay (ELISA)

31

EL

 

 

 

d.|

PA
sat ,
fro z

em:

Hsu et al. (1991) developed an improved monoclonal antibody based
ELISA assay for detection of the p57 protein of R. salmoninarum. The assay
was both specific and sensitive for detection of soluble R. salmoninarum
antigen at concentrations as low as 50-100 ng /ml.

A double antibody sandwich ELISA, is also known as quantitative ELISA
(Q-ELISA), provides accurate indication about the real prevalence of BKD in
the tested fish population because it determines both prevalence and intensity
of the infection (Pascho et al. 1998). The procedures are fairly standardized
by the studies of Pascho and Mulcahy (1987) and Pascho et al. (1991). A
positive threshold has been computed and proposed for Q-ELISA results
interpretation (Meyers et al. 1993; Pascho et al. 1998). The positive—negative
cutoff absorbance for the kidney homogenate was determined as 0.10.
Pascho et al. 1998 assigned the following antigen level categories for tested
positive kidney samples: low (0.10 to 0.19), medium (0.20-0.99) and high

(1.000 or more).

d. lmmunohistochemistry (IHC)

Hoffmann et al. (1989) compared various staining techniques (Gram,
PAS, IFAT and indirect peroxidase procedures) for their ability to detect R.
salmoninarum in the tissues of rainbow trout fixed by various methods (Fresh
frozen tissue, frozen formalin- fixed tissue, formalin or Bouin’s fixed paraffin-

embedded tissue) and concluded that only the indirect peroxidase technique

32

 

3)I

salr

SBI“.

 

 

 

frag
frag.
Ierr

COTT

gave satisfactory results regardless of the fixation method used.
lmmunohistochemistry has the advantage of visualizing R. salmoninarum and
the tissue alteration they cause simultaneously (Jansson et al. 1991; Evensen
et al. 1994). lmmunohistochemistry has been used to detect BKD natural and
experimental infections. For example, using in situ lHC, Lorenzen et al.
(1997) reported the first demonstration of BKD in rainbow trout in Denmark.
Evensen et al. (1994) detected the organism in situ by using IHC in paraffin
embedded tissue specimens from Atlantic salmon and they reported the use
of monoclonal antibodies specific for the R. salmoninarum p57 protein.
However, it has been reported that prolonged preservation of tissue samples
in formalin has very deleterious effect on the antigen detection and retrieval in

immunohistochemical assays (Evensen et al. 1994).

3) Polymerase chain reaction (PCR)

Polymerase chain reaction (PCR) has been successfully used to detect R.
salmoninarum DNA within individual chinook salmon eggs with a detection
sensitivity of 2 bacterial cells / egg (Brown et al. 1994). A nested PCR (nPCR)
has been developed by Chase and Pascho (1998) to amplify a 320 bp
fragment of the gene encoding the p57 protein and they recorded no specific
fragments amplification when other fish bacterial pathogens were used as
templates for nPCR. The sensitivity of the method increased one hundredfold

Compared to a conventional PCR method (Pascho et al. 1998). The authors

33

VI.

the

pre

dise

 

(Cal

compared the sensitivities of nPCR, ELISA and FAT assays to detection R.
salmoninarum in kidneys of infected chinook salmon and concluded that
nPCR showed the highest sensitivity (61 %) followed by ELISA (47 %) then
FAT (43 %). Pascho et al. (1998) reported that nPCR detected R.
salmoninarum in 100 % of the tested ovarian fluid samples and thereby
concluded that nPCR was the most accurate and sensitive method for
detection of R. salmoninarum. Hong et al. (2002) designed a pair of specific
primer for nested amplification of 501 bp and 314 bp DNA fragments of the
sequence coding p57 of R. salmoninarum respectively and they also recorded
no specific fragments amplification when other principal fish bacterial
pathogens were used as templates in PCR and nested-PCR tests. However,
Miriam et al. (1997) have cautioned that PCR positive samples may contain
some proportion of dead R. salmoninarum with detectable level of DNA. This
means that kidney tissues containing non-culturable R. salmoninarum can be

falsely positive when tested with nPCR.

VI. Differential diagnosis

External manifestations of BKD are non-pathognomonic, but the course of
the disease and the granulomatous nature of the kidney lesions may provide
presumptive indications. The disease can be differentiated from other kidney
diseases of chronic progression including pseudo-kidney disease

(Camobacterium piscicola) (Ross and Toth 1974), nephrocalcinosis (calcium

34

deposits) (Peddie 2004) and proliferative kidney disease (lymphoid
hyperplasia in response to myxozoan parasite Tetracapsula bryosalmonae)
(Clifton-Hadley et al. 1984). Differentiation is mainly based on observation
and detection of the organism or its antigens using immunofluorescence, IHC,
ELISA, PCR. In case of nephrocalcinosis, differentiation is mainly based on
bacteriological assessment to rule out the presence of the bacterium,
however on-farm examination of lesion consistency can help to discriminate
between these conditions as BKD lesions are soft whilst those caused by
nephrocalcinosis have a gritty texture (Peddie 2004).

Renibacterium salmoninarum can be differentiated from coryneforrn group
of bacteria, which includes the genera of Listeria, Erysipelothrix,
Corynebacterium, Actinomyces, Celullomonas, Curtobacterium, Arthrobacter
and Brevibacten'um by cell wall composition and G+C contents of DNA (Stuart
and Welshimer 1974; Sanders and Fryer 1980).

Although R. salmoninarum share certain characteristics with Actinomyces
pyogenes (formerly Corynebactefium pyogenes), they differ in a number of
other characteristics. A. pyogenes is facultatively anaerobic, catalase
negative and produce acid from carbohydrates (Holt et al. 2001). The genus
Renibacterium can be separated from pathogenic Corynebacten'a and genus
Caseobacter by the presence of lysine in the cell wall and the absence of
mycolic acids. The genus Caseobacter is further differentiated by a mol % G

+ C of 60-67 (Crombach 1978). Genus Celullomonas contains the diamino

35

acid omithine in its cell wall peptidoglycan and has a mol % G + C ranging

from 65-72.

Interestingly some of the coryneforrn groups of bacteria have an
overlapping characteristics and phylogenetic homology. Among this group of
bacteria a cell wall peptidoglycan containing lysine occurs primarily in
Arthrobacter and Brevibacterium (Holt et al.2001). DNA homology studies
showed close relationship between several species in these two genera.
However, these bacteria have usually been isolated from the environment,
are chemoorganotrophic, show a progression of morphological changes
during the growth cycle and have a mol % G + C above 60 (Holt et al.2001).
Interestingly, all these characteristics are distinctly different from that of

Renibacterium.

VII. Control

1) Chemotherapy

Since the early 19505 a relatively large number of chemotherapeutics
have been intensively tested in vivo and in vitro for efficacy in treating BKD.
Rucker et al. (1951) was first to use antimicrobial agents against clinical BKD
and their results showed a definite decrease in mortalities when sulfadiazine
was incorporated into fish diets. Although treatment did not completely cure

clinically sick fish, sulfamerazine reduced BKD mortalities alone and

36

 

z».
0 5
>\

He

I6»

Slit

er;

Ints

 

60'

combined with sulfaguanidine and sulfadiazine (Allison 1958). Wolf and
Dunbar (1959) tested 34 therapeutic agents including erythromycin
thiocyanate and sulfamerazine on 16 strains of R. salmoninarum using the
disk method for drug sensitivity screening followed by in vivo feeding trials
with experimentally infected fish. They concluded that erythromycin fed at the
rate of 100 mg per kg of fish for 5 consecutive days gave the best results.
Generally, due to the occurrence of the bacterium intracellularly as well as
extracellularly, these treatments only suppressed the systemic spread of the
organism and induced partial relief (Amos 1977). Intramuscular (i.m.) and
lntraperitoneal (i.p.) administration of sulfonamide drugs significantly reduced
prespawning mortality among chinook salmon broodstocks being hold prior to
spawning (Amend and Fryer 1968). However, sulfonamides administered by
i.m. or i.p. routes often produced sterile abscesses at the injection site in
adults and induced mortalities and teratogenicity with their progeny (Amos
1977)

In an attempt to reduce or prevent vertical transmission of BKD, salmon
eggs were water hardened for 1hour in 2 ppm erythromycin (Amos 1977).
However, erythromycin was rapidly eliminated and dropped below detectable
level within 24 hours after water hardening (Evelyn et al. 1986a). Monthly
subcutaneous (S. C.) injection of adult female Pacific salmon with 11mg/kg
erythromycin reduced pre-spawning mortality due to BKD (Klontz 1983).

.lnterestingly, erythromycin remains in the eggs of injected females for up to

50 days before spawning (Evelyn et al. 1986; Moffitt 1991). It is believed that

37

v
I
p

“mt

em;

min.

dlSlr

the]

2) A<

erythromycin residues in the eggs assist in preventing vertical transmission of
R. salmoninarum from parents to their offspring (Lee and Evelyn, 1994).
Detectable amounts of erythromycin often remain in the perfused tissues of
both juvenile and adult salmon long after they are no longer detected in the
plasma and muscle (Moffitt 1991; Haukenes and Moffitt 1999) and this
possibly contributes to the efficacy of erythromycin against the slow growing
R. salmoninarum. Feeding erythromycin can efficiently reduce mortalities of
infected hatchery raised salmonids (Wolf and Dunbar 1959; Austin 1985;
Moffitt and Bjornn 1989). A dose of 200 mg/ kg body weight for 21 days was
most effective (Moffitt, 1992). Erythromycin is only available as an
lnvestigational New Animal Drug (INAD) through the Food and Drug
Administration (FDA) (Moffitt 1992).

Austin (1985) tested more than 70 antimicrobial compounds both in vivo
and in vitro and found that clindamycin, erythromycin, kitasamycin, penicillin
G and spiramycin were useful for combating early clinical BKD cases while
cephradine, lincomycin and rifampicin were effective prophylactically but had
limited use therapeutically. Hsu et al. (1994) tested the efficacy of
enrofloxacin in treating BKD in vitro and in vivo and they concluded that low
minimal inhibition concentrations (Mle), high bioavailability and large volume
distribution of the antibiotic make it good candidate for use as effective

therapeutic against BKD.

2) Adult segregation

38

 

us.

\‘l' E

3)!

erac
man

sa/n

their

 

1992

dish

 

Broodstock segregation is a more practical method for reducing the
prevalence and levels of R. salmoninarum in hatchery-reared salmon (Pascho
et al. 1991) and for increasing survival during their downriver migration and
entry into seawater (Pascho et al. 1993; Elliot et al. 1995). This procedure
aims to interrupt vertical transmission of R. salmoninarum by isolating or
destroying eggs from brood fish that exhibit clinical signs of BKD or test
positive, with a high titer, against R. salmoninarum antigens. The method is
used successfully in a number of US states and Canadian provinces such as

Washington, Idaho, Michigan, Wisconsin, and Ontario.

3) Eradication

Due to the complicated nature of BKD and its obvious threats to fisheries,
Hoskins et al. (1976) recommended complete destruction of the infected
stocks and disinfection of the holding facilities to achieve complete
eradication of the disease. However, this procedure is considered by fisheries
managers as impractical due to the widespread occurrence of R.
salmoninarum (Sanders and Fryer 1980).

Eradication can be of value in single fish farms or hatcheries that receive
their water supply from specific pathogen free source (European Commission
1999). Eradication procedures should be followed by standard, cleaning and

disinfection procedures. Although some trials have been made to eradicate

39

 

0f

 

 

 

8C

 

BKD from fish farmed in open waters (e.g. sea and lake cages) or from farms
and hatcheries with water supply from rivers, results were very discouraging.
After eradication procedures have been applied in the fish farm and
hatcheries, restocking should only utilize certified BKD-free stocks.
Restocking should be followed by two inspections and laboratory
examinations per year for a total period of two years before the facility can be

designated as “BKD-free” (European Commission 1999).

4) Prophylaxis

1. Reducing the risk of BKD introduction

Special attention should be paid to prevent the introduction of infected fish
or their gametes (Evelyn et al. 1984; Yoshimizu 1996). This can only be
achieved through prior examination and quarantine. Special requirements of
water supply, wild birds and amphibians’ control. In addition, restriction of
movement of vehicle, visitors as well as utensils from infected into free areas
is equally important. Repopulation must be accompanied with certificate
issued by the competent authority certifying that the fish or eggs are specific

pathogen free.

2. Vaccination

4O

 

Sd

 

9X

the

In the last two decades, vaccination against BKD has achieved different
levels of success. Paterson et al. (1981) reported that an inactivated
suspension of R. salmoninarum mixed 1:1 with Freunds adjuvant (FCA)
administered by i.p. injection, reduced the level of infection of R.
salmoninarum in yearling salmon but, did not completely eliminate the
infection. Sakai et al. (1993; 1995) found that although vaccination evoked
specific antibodies, these antibodies did not endow with a protection.
Piganelli et al. (1999) demonstrated that oral administration of R.
salmoninarum expressing low levels of cell associated p57, resulted in an
extension of the mean time to death after challenge and they concluded that
the protection was not due to humoral antibody. This conclusion supported
earlier histopathological indications of an involvement of the cell mediated
immune response in recovery, due to intracellular survival and the
composition of inflammatory cells in connection with signs of regression
(Munro and Bruno 1988). Rhodes et al. (2004) presented DNA adjuvants and
whole bacterial cell vaccines against R. salmoninarum that were tested in
chinook salmon fingerlings. These authors concluded that whole cell vaccines
of either a nonpathogenic Arthrobacter spp. or an attenuated R.
salmoninarum strain produced limited protection against acute intraperitoneal
challenge with virulent R. salmoninarum. They also concluded that the
addition of either synthetic oligodeoxynucleotides or purified R. salmoninarum
genomic DNA as adjuvants did not increase protection, however a

combination of both whole cell vaccines significantly increased survival

41

among fish naturally infected with R. salmoninarum. Also, the surviving fish
treated with the combination vaccine exhibited reduced levels of bacterial

antigens in the kidney.

Gap of BKD knowledge in Michigan before 2002

Although BKD was virtually reported wherever salmonid fishes are present
(Fryer and Sanders 1981; Bullock and Herman 1988, Klontz 1983),
publications describing the status of BKD in Michigan salmonids fish species
are rather scarce (Hnath and Faisal 2005). Historical records showed that
BKD affected Michigan brook trout as early as 1955 (Allison 1958). Like in
other parts of the USA and Europe, BKD continued its spread to the Coho
salmon (Oncorhynchus kisutch) spawners in the late sixties (MacLean and
Yoder 1970) and caused epizootics of chinook salmon (Oncorhynchus
tshawytscha) in Lake Michigan in the late 1980’s (Holey et al. 1998; Johnson
and Hnath 1991). Despite these reports of BKD in Michigan, only two studies
have dealt with R. salmoninarum isolation from infected Michigan fish.
Starliper (1996) and Starliper et al. (1997) isolated and identified R.
salmoninarum isolates from chinook salmon collected from Lake Michigan
weirs in Michigan and Wisconsin. Additional investigations showed that R.
salmoninarum affects more salmonid fish species than originally thought. For
example, Jonas et al. 2002 detected R. salmoninarum antigens in Lake

whitefish (Coregonus clupeformis) and bloaters (C. hoyr) collected from Lake

42

Michigan in 1997-1999. Hnath and Faisal (2005); Wright and Faisal (2005)
emphasized the fact that R. salmoninarum is endemic to the Great Lakes
basin and effective control measures have to be seriously pursued. In order
to develop sound management strategies to control BKD in Michigan, several
gaps of knowledge have to be filled. This submitted study is a trial to fill some

of these gaps.

43

Test Criteria

Gmmsmm +
PAS (Periodic Acid Schiff) +
stain

Zeihl-Nielsen ( Acid Fast) Non acid fast
stain

nine
Bile solubil
r

utilization
Casein
Catalase
rome oxidase

DNase APl-ZYM'
Escufin

Esterase

Gelatin l - APl-ZYM*

Hemolytic activity Complete clearance
zone around bacteria

lndole test
M Red
Nitrate reduction

Tween-20, 40 and 60

Tween-80
Urease

 

Table 1. Summary of the morphological and biochemical characteristics

of Renibacterium salmoninarum.

ABl-ZYM" is a bacterial enzymes based assay used for the specific identification of different

bacteria.

44

 

 

 

 

 

 

 

Criteria Description
Motility Non motile
Growth at 15 “C +

Growth at 5, 22 °C +

Growth at O, 30, 37 °C No growth
Mol % G+C 53

Mycolic acid Absent

 

Cell wall peptidoglycan
amino acids

Glutamic, lysine , alanine and glycine

 

Cell wall sugars

Glucose, arabinose, mannose, and

 

Requirement for cysteine

Required

 

 

 

Survival Obligate intarcellular
Major antigenic factor 57 kDa protein (P57)
Antigenicicity Strains appears antigenically homologous

 

Leucoagglutinins

Exist

 

Leucocytolysins

Exist

 

Metalloproteinase

Exist

 

Immune-complexes

Occurs in kidney, liver, spleen

 

Biochemical and genetic

Biochemically uniform with very minimal

 

diversity genetic differences
Phylogeny Phylogentically related to actinomycetes and

closely related to Arthrobacter spp and
Brevibacterium

 

Survival in river water

Up to 1 week

 

Survival in feaces and
mud

Up to 21 days

 

Transmission

 

 

Horizontal (wounds, orofecal, gills, contact)
and vertical (intraovum or egg shell
contamination)

 

 

Table 2. Summary of different metabolic, antigenic and pathogenic

characters of Renibacterium salmoninarum

45

 

in La‘
not 6
Sam;

fishe

 

 

 

R. sa
wmd
Gems

Magi
shor

rel/8‘7

Spafi

CHAPTER TWO

Primary Isolation of Renibacterium salmoninarum from naturally infected
salmonine stocks in Michigan using a modified tissue processing

protocol

ABSTRACT

Throughout the 1980s, massive dieoffs involving chinook salmon occurred
in Lake Michigan. These well-publicized epizootics were attributed primarily, but
not entirely, to Bacterial Kidney Disease (BKD) caused by Renibacterium
salmoninarum (R. salmoninarum). Despite the magnitude of the setback to
fisheries conservation efforts in Michigan, relatively little research was done on
R. salmoninarum, primarily due to the slow-growing nature of this bacterium
which takes up to four weeks to obtain a primary isolation. In this study, I
developed a tissue processing protocol that collected the whole kidney of an
infected fish and exposed it to high sheer stomaching. This significantly
shortened the incubation time and allowed R. salmoninarum to be cultured with
relative ease.

A total of 566 R. salmoninarum isolates have been retrieved from the
kidneys of wild, feral, and captive fish assayed between 2002-2004 from

spawning weirs and state fish hatcheries at various locations in Michigan.

46

 

exar
with

gee.

simil
Antll
enro

erylj

 

 

These isolates constitute a unique resource for future studies of R.
salmoninarum and BKD in the Great Lakes.

Findings also demonstrated that R. salmoninarum is ubiquitous in Michigan
because the pathogen was isolated from each population and lot of fish
examined. Prevalence of R. salmoninarum and the clinical lesions associated
with infection varied among species, strains of the same species, and
geographic locations.

Renibacterium salmoninarum isolates were biochemically homogenous and
similar to those of other R. salmoninarum strains isolated worldwide.
Antibiogram revealed high sensitivity of R. salmoninarum isolates to
enrofioxacin and ciprofloxacin. Interestingly, two isolates were resistant to

erythromycin, which is the antibiotic of choice for treatment of BKD.

47

 

 

modéf

lime.

 

 

 

INTRODUCTION

Due to its slow-growing nature, Renibacterium salmoninarum Sanders and
Fryer 1980, the causative agent of bacterial kidney disease (BKD), is difficult to
isolate. Over the last six decades, scientists have utilized a number of bacterial
media for this bacterium such as Minced Chick Embryo medium, Dorset Egg
Medium, Cysteine Blood Agar (KDM1) (Ordal and Earp, 1956), KDM2, which
contains 20 % fetal bovine serum (Evelyn, 1977), Charcoal Agar Medium (Daly
and Stevenson, 1985) and Selective Kidney Disease Medium (SKDM)
supplemented with the four antibiotics; cycloheximide, D-cycloserine, polymyxin
B, and oxolinic acid (Austin et al., 1983). Incubation periods of up to 12 weeks
have been necessary to isolate R. salmoninarum.

To shorten the incubation time, Evelyn, et al. (1989) added a nurse R.
salmoninarum culture to the center of the KDM2 culture plates. This
modification both accelerated growth of the bacterium and shortened incubation
time. The authors attributed the accelerated growth to the metabolites secreted
by the initial nurse culture. Further, Evelyn et al. (1990) replaced the nurse
culture with 25 ul of filter- sterilized broth that has previously been used to grow
R. salmoninarum (referred to as spent broth) and continued to observe the
accelerated growth. The authors attributed the accelerated growth to the
presence of metabolites produced by the pathogen during its growth. Using the
same approach, Teska (1993) and Starliper et al. (1998) incorporated R.

salmoninarum spent medium into the KDM2 medium’s constituents (1% WV,

48

designated as KDM 2+M) and reported a shortened incubation time and
profuse bacterial growth. Most of the previously mentioned studies have been
performed on secondary bacterial cultures. However, primary isolation,
particularly from carrier fish with negligible tissue bacterial concentration,
continues to be a challenge for accurate epizootiological studies (European
commission, 1999).

In the State of Michigan, it has been documented that BKD has exist since
the early 19508 (Allison, 1958). BKD has been incriminated to cause mass
mortalities in the Great Lakes and in hatchery-propagated fish (Holey et al,
1998; Johnson and Hnath, 1991). Despite the potential association between R.
salmoninarum and clinical cases of BKD in Michigan’s salmonines, a relatively
limited number of R. salmoninarum isolates were retrieved from Michigan and
Wisconsin feral spawner salmon in fall of 1991 (Teska, 1994; Starliper 1996).
Among the retrieved isolates, those from the Michigan side of Lake Michigan
were the most genetically diverse (Starliper, 1996) and of higher virulence
(Starliper et al., 1997).

Integral to the host-pathogen interaction of R. salmoninarum is the
formation of granulomatous host reaction in which bacteria are sequestered
(Bruno, 1986; Sami et al., 1992). The presence of such granulomas can
impede bacterial isolation using standard bacterial loops and thereby lead to
inconsistent isolation results. In this study we combined a modified tissue
processing procedure with the use of KDM2+M medium supplemented with

antibiotics to select and enhance the primary isolation of R. salmoninarum. The

49

modified protocol was then used to determine R. salmoninarum prevalence

among representative propagated and feral salmonine populations in Michigan.

50

 

i

grow
befc
Brief
Cystj

wale

 

"uni
aut0<
lollov.
slerl
Wflv)
AH)
Lou

LEn

9909

andc

MATERIAL AND METHODS

Bacterial growth medium. The medium used throughout this study
combined the addition of 10% fetal calf serum recommended by Evelyn et al.
(1990) into the KDM2 medium, the four antibiotics recommended by Austin et
al. (1983) in the SKDM, and the 1% (v/v) R. salmoninarum spent medium used
by Teska et al. (1993) and Starliper et al. (1998) in the KDM2+M. The bacterial
growth medium used in this study, that combined the modifications listed
before, will be referred to as MKDM (modified Kidney Disease Medium).

Briefly, MKDM consists of peptone (1 % w/v), yeast extract (0.05 % w/v), L-
cysteine HCl (0.1 % WM and Cycloheximide (0.005 % w/v) dissolved in distilled
water. The medium’s pH is adjusted to 6.8. Agar (1.5 % w/v) is added
immediately after adjusting the pH of the medium. The medium is sterilized by
autoclaving at 121 °C for 15 minutes and left to cool down to 48 °C then the
following ingredients are added: new born calf serum (10 % v/v), 0.22 pm filter-
sterilized R. salmoninarum spent broth (1 % v/v), Oxolinic acid (0.00025 %
w/v), Polymyxin B sulfate (0.0025 % w/v) and D-cycloserine (0.00125 % w/v).
All MKDM ingredients were purchased from sigma (Sigma Chemical Co, St.
Louis, MO, USA) with the exception of agar, which was from Remel (Remel,

Lenexa, Kansas, USA).

Fish and sample processing. Details regarding host, age, and
geographic origins are found in tables 3-5. In the fall of 2002, a total of 515 feral

and captive spawning salmonids were collected from Michigan weirs and state

51

 

Hm?

bmc:

(Salt

and 1

fish h;

 

OSFF
mum;
”WE:
eXDOS
FOlloy
lildivic

COlleC

WEre C(

beOkt

fish hatcheries. Fish included 150 returning chinook salmon (Oncorhynchus
tshawytscha) collected from the Little Manistee River Weir (LMRW), Manistee
county, Michigan (Lake Michigan watershed), Swan River Weir (SRW) at
Rogers City, Presque Isle county, Michigan (Lake Huron watershed) and Platte
River Weir (PRW) at Beulah, Michigan (Lake Michigan watershed). An
additional 165 Michigan-adapted coho (Oncorhynchus kisutch) and 56
Hinchenbrook coho salmon were collected from the Platte River Weir. Captive
brood stock included 60 brook trout (Salvelinus fontinalis), 60 lake trout
(Salvelinus namaycush) that were kept in raceways that receive water from
Cherry Creek (Lake Superior watershed) at the Marquette State Fish Hatchery
in Michigan’s Upper Peninsula. Further, a total of 12 brown trout (Salmo trutta)
and 12 rainbow trout (Oncorhynchus mykiss) were collected from Oden State
fish hatchery (OSFH) at Oden, Alanson, Michigan (Lake Michigan watershed).
OSF H is the brown and rainbow trout broodstock station and is a major rearing
facility for those two species in Michigan. Males and females were equally
represented among samples. The sacrifice of the feral spawners entailed
exposing the fish to carbon dioxide-laden water, followed by a blow to the head.
Following gamete collection, the abdominal cavity was cut open to examine
individual internal organs for signs associated with BKD, followed by the

collection of kidney tissue samples.

In the spring of 2003, a total of 480 hatchery-reared pre-stocking fingerlings
were collected from a number of state fish hatcheries. In brief, a total of 120

brook trout and 120 lake trout fingerlings were collected from MSFH. Additional

52

180 brown trout and 60 rainbow trout fingerlings were collected from OSFH. In
August 2004, a total of 15 brook trout fingerlings were sampled from

Cedarbrook Trout Farm at Harrisonville, Michigan.

Cross contamination was avoided by replacing dissecting tools with sterile
tools following the dissection of each fish. Attempts to isolate R. salmoninarum

from kidney tissues were performed by each of the following procedures:

1. streaking a 10 pl loopful of kidney tissue onto MKDM plates,
2. harvesting as much kidney tissue as possible, mincing the
tissue in a sterile, plastic Petri dish with scissors, suspending the
minced tissue in Hank’s balanced salt solution (HBSS, 1:4 w/v,
Sigma), and then streaking one hundred microliters (ul) inoculum
of the suspension onto MKDM, or
3. transferring the homogenate to 7.5 cm x 18.5 cm Whirl-Pak®
bags (Nasco, Fort Atkinson, WI), suspending in HBSS (1 :4 w/v),
then crushing the suspension in a Biomaster Stomacher—80 (Wolf
Laboratories Limited) at the high speed setting for 120 seconds.
One hundred pl of the suspension were added to one end of an
MKDM plate and then spread over the surface using a sterile
bacteriological loop.
Inoculated plates were incubated at 15 °C for up to 30 days and were
checked periodically for growth using an inverted dissecting microscope, thus

allowing the detection of early colonial growth.

53

Confirmation of isolates. All colonies were investigated for their
conformance with colonial and bacterial morphological criteria of R.
salmoninarum, as previously detailed in Sander and Fryer (1980) and Austin
and Austin (1999). A number of biochemical tests were performed including
motility, using motility test medium (DIFCO- BD and Company Sparks, MD,
USA), cytochrome oxidase with Pathotec strips (Remel), catalase test with 3 %
hydrogen peroxide, hydrolysis of esculin using bile esculin agar (Remel), and
DNAse test using DNAse test medium (Remel). Carbohydrate utilization was
performed using basal media (DIFCO-BD). The basal medium was prepared
according to manufacturer instructions prior to the addition of individual sugars.
Ten ml of filter sterilized (0.45 pm) 10 % sugar solution was added to
autoclaved and cooled (48 °C) basal media to obtain a final concentration of 1
% with the exception of salicin which was made as 5 % solution to reach 0.5 %
final concentration. Each one of the following sugars was added individually to
the basal medium to test for the utilization of each sugar: arabinose, glucose,
lactose, maltose, rhamnose, salicin, sucrose, sorbitol, xylose. All sugars were
from Sigma. Results of biochemical tests were matched against standard R.

salmoninarum biochemical characters described by Bruno and Munro (1986).

Nested PCR. Single bacterial colonies were identified using highly specific
oligonucleotide primers designed by Pascho et al. (1998), which amplify a
region of the gene encoding the R. salmoninarum p57 antigen in a nested

polymerase chain reaction (nPCR). The nPCR assay using these primers is

54

considered the method of choice to confirm R. salmoninarum isolates (OlE
2003, Pascho and Elliott, 2004). Briefly, DNA was extracted from each isolate
using DNeasy Extraction Tissue Kit (Qiagen Inc., Valencia, CA) according to
the manufacturer’s protocol with minor modifications. Bacterial pellets were
obtained by centrifugation at 6000 g for 20 minutes at 4 °C and subsequently
incubated with lysozyme buffer that consisted of 180 pl of 20 mg lysozyme
(Sigma) , 20 mM Tris-HCI, pH 8.0; 2 mM EDTA (Sigma) and 1.2 % (v/v) Triton
X100 (Sigma) at 37 °C for 1 hour. The nPCR method and the primers
recommended by Pascho et al. (1998) were used with slight modification to the
volume of utilized DNA (5 pl for first round and 2 pl for second round PCR
reaction), water, and master mixes (45 pl for first round and 48 pl for second
round nPCR reaction). The controls were composed of a PCR mixture
containing no DNA template (reagent negative control), a positive R.
salmoninarum, and a positive tissue control. A volume of 10 pl of the nPCR
product and controls were mixed with 2 pl of 6X loading dye (Sigma) and used
to load a gel consists of 2 % agarose (Invitrogen Life Technologies, Carlsbad,
CA). Each electrophoresis gel included a 1 k bp (with 100 bp increments) DNA
ladder (Invitrogen). Gels were run at 100 volt for 30 minutes in 1 X Tris Acetate
Buffer (1 X TAE) (Sigma). Gels were visualized with the KODAK EDAS Camera
System and UV Trans-illuminator. Suspect colonies were considered positive

for R. salmoninarum when a 320 bp band was detected.

55

Antibiogram. Two media were used to test the isolates for sensitivity to
antibiotics using a modified Kirby-Bauer disc diffusion method (Bauer et al.,
1966)

1. Antibiotic free MKDM agar medium

2. Modified Muller’s Hinton agar medium (MMHA): Muller’s Hinton Medium
(MHA) is used in standard antibiotic sensitivity testing. L-cysteine was added to
MHA to support the growth of R. salmoninarum. The MMHA was prepared by
adding 1 g L-cysteine HCI (Sigma) to 0.5 g yeast extract (Sigma), 100 ml fetal
bovine serum (Sigma), and 38 g of dehydrated MH agar medium (Remel), and
then suspending the mixture in double distilled water to obtain a 1000 ml media
volume.

The antibiogram was performed on 14 representative R. salmoninarum
isolates. Five-day-old colonies were suspended in sterile saline to obtain
turbidity equivalent to a 0.5 McFarland standards (Remel). From each bacterial
suspension 200 pl was spread onto antibiotic free MKDM and MMHA plates
(one plate each/isolate). Cultures were left for a few minutes to allow the
bacteria to adsorb to the agar surface. Using an automatic dispenser (Remel),
antibiotic discs (5 mm in diameter) were placed on the culture plate surface.
The plates were then inverted and incubated at 15°C for 5 days in a
subambient temperature incubator (Fisher Scientific Company L.L.C. Hanover
Park, IL). Culture plates were observed and results recorded by measuring the
diameter of the zone of inhibition in millimeters around each disc using a

calibrated ruler.

56

 

ant
trlr.’
Ka'

oil:

iso'
La':
fro."

the'

 

The following antibiotic discs (all from Remel) were used in the
antibiogram: chloramphenicol (C), terramycin (TE), sulmethoxazole -
trimethoprim (SXT), carbenicillin (CAR), erythromycin (E), azithromycin (AZM),
Kanamycin (K), clindamycin (DA), polymyxin B sulfate (PB), novobiocin (NV),

ofloxacin (OFX), ciprofloxacin (CIP), enrofloxacin (ENO) and norfloxacin (NOR).

Preservation and storage of isolates. Identified R. salmoninarum
isolates were cryoperserved and deposited at the Aquatic Animal Health
Laboratory, Michigan State University. Bacterial suspensions were prepared
from 5 day-old cultures in MKDM broth (not supplemented with antibiotics) and

then stored at —80 °C.

57

RESULTS

Effects of sample processing and culture technique on primary
isolation of Renibacterium salmoninarum. When MKDM was streaked with
a loopful (10 pl) of infected kidney tissue, colonies were evident 15-20 days
post incubation. Using minced kidney of the same tissue and increasing the
inoculum to 100 pl shortened the incubation time to 10-15 days. However,
when kidney tissue samples were stomached for 120 seconds, profuse growth
was achieved within a relatively short period (5-10 days). Renibacterium
salmoninarum colonies grew on and around streaked tissues and were creamy,
glistening, smooth, convex and 1-2 mm in diameter. Representative colonies
were individually picked and their identities confirmed using nested PCR assay.
Because of the astounding success in shortening the incubation period and the
absence of contaminants, stomaching of minced tissues and inoculating MKDM
plates with 100 pl of tissue homogenate became the routine tissue processing
procedure for the primary isolation of R. salmoninarum from tissues in this and

subsequent studies.

Prevalence of R. salmoninarum isolated from Michigan salmonines
1. Feral salmon population. Tables (3-5) show the numbers of fish examined,
prevalence, and numbers of fish exhibiting BKD clinical signs. Most of the
examined fish looked healthy and exhibited healthy-looking kidneys and

internal organs. A number of fish in each of the species and lots examined

58

(IS
an
tha‘
exa

BKI

 

2. P
saln
brov
Dem

sloc

3. P.
sal/x
high
addi:
sa/m
Simul

 

displayed whitish abscesses like nodules that sometimes coalesce to form
patches in the kidneys’ parenchyma. The prevalence of the pathogen was
highest in chinook salmon from the Platte River Weir chinook (96.3%), followed
by Little Manistee weir chinook (82.5%), and Swan River weir chinook
(48.3%)(table 3). As shown in figure 1 and 2, prevalence seems not to differ
among males and females. Coho salmon spawners showed prevalence rates
that reached up to 100% in the Hinchenbrook strain, with more than half of the
examined fish showing clinical BKD signs (table 3). As depicted in Figure (2),

BKD lesions were more apparent in females coho salmon than males.

2. Prevalence in captive broodstocks. Results indicated that R.
salmoninarum is also widespread among captive broodstocks (table 4), with
brown trout (the Wild Rose strain) being the highest in both prevalence and
percent of clinical cases, followed by rainbow trout, brook trout, and Lake trout

stocks.

3. Prevalence in propagated offspring fish. The prevalence of R.
salmoninarum in propagated Iron River Brook trout fingerlings were remarkably
high (83 %), with 50 % of the fish presenting with lesions typical of BKD. In
addition, propagated Assinica BKT fingerlings also showed relatively high R.
salmoninarum prevalence (42 %), with 16.66% showing clinical signs.
Similarly, the R. salmoninarum prevalence and percentage of fish (60 %) with

clinical signs were very high in BKT obtained from a fish farm (table 5).

59

fOL
wt'
dlpl

dEit

 

Rs

II. E

 

F ingerlings of lake trout, three strains of brown trout, and rainbow trout showed
relatively lower prevalences (<25%) and lesser numbers of fish with clinical

signs (table 5).

Confirmation of the retrieved R. salmoninarum isolates. A total of 566
R. salmoninarum isolates were retrieved from infected fish tissues over a two-
year period. All colonies were creamy-whitish, glistening, 1-2 mm in diameter,
rounded, and smooth. Old colonies (over 40 days incubation) showed granular
white or crystalline appearance. Gram staining demonstrated Gram-positive
diplo- or coccobacilli. No capsules, metachromatic granules, or bipolarity were

detected in the stained slides of all isolates.

I. Molecular Confirmation. Nested PCR performed on all isolates exhibited the

R. salmoninarum characteristic 320 bp band.

ll. Biochemical Reactions. Results of the conventional biochemical testing of
12 representative isolates demonstrated isolate uniformity. Isolates were non-
motile, catalase positive, cytochrome oxidase negative, esculin hydrolysis

negative, DNAse negative and carbohydrate utilization negative (table 6).

III. Antibiogram of the R. salmoninarum Isolates. Tables 7 and 8 show the
details of the R. salmoninarum antibiogram results. Results indicate that, after
5-10 days incubation, the inhibition zones obtained from isolates cultured on

antibiotic-free MKDM medium were sharper and more obvious than those

60

obtained by culture on MMHA medium. However, both media yielded relatively
similar results. All 12 isolates were highly sensitive to enrofloxacin and
ciprofloxacin with a 27mm and 23 mm average inhibition zone diameters
respectively). Ten isolates were markedly sensitive to terramycin (21 mm
average inhibition zone). Interestingly, two of the isolates retrieved from captive
brown trout broodstock were resistant to erythromycin and azithromycin, while
the remaining 10 isolates were sensitive or intermediately sensitive to
erythromycin, azithromycin and sulfamethoxazole —trimethoprim, with average
inhibition zones of 17mm, 15.5mm and 18mm, respectively. Moreover, all
isolates were resistant to polymyxin B and clindamycin (average inhibition zone
diameter of 0, 4mm respectively). In addition, most of the isolates were
resistant to kanamycin; with an average inhibition zone diameter of 9mm.
Isolates were sensitive to chloramphenicol, novobiocin, and carbenicillin
showing average inhibition zones diameters of 20 mm, 14mm and 16mm

respectively.

61

DISCUSSION

Results from this study demonstrated that the combined use of MKDM
medium and stomaching minced tissues diluted in HBSS was optimal for
primary isolation of R. salmoninarum. The antibiotics Austin et al. (1983)
incorporated into the medium combined with a shorter incubation period
minimized the growth of contaminating bacteria and fungi. There are a number
of factors that may have led to the improved growth of R. salmoninamm that
was observed. First, thorough mincing and homogenization may have caused
release of R. salmoninarum from granulomas and fibrous layers. Second, the
relatively aggressive tissue processing may have led to a massive release of
bacterial metabolites that are known to boost bacterial growth in vitro (Evelyn et
al.1989; 1990). Third, a major part of the kidneys (posterior and anterior) were
used in homogenization, a matter that increases the likelihood of isolating
bacteria even if present in low numbers, as in the case of carrier fish. Pascho
et al. (1987) were able to double the likelihood of isolating R. salmoninarum
from infected fish by combining samples taken from three different spots in the
kidneys of individually tested fish. Fourth, a heavy tissue inoculum (100 pl) was
used, a matter which enhances the likelihood of bacterial isolation when
compared to using a loopful (~ 10 pl only). Fifth, mixing kidney tissues with four
times their weight of HBSS may have diluted inhibitory molecules present in
tissue that Evelyn et al. (1981); Daly and Stevenson 1988 and Olsen et al.
(1992) believed to lower the likelihood of isolating R. salmoninarum from

homogenized kidney tissues. Last, the unique formula of HBSS with its rich

62

inorganic ions and electrolyte content might have played a role in adjusting the
pH and osmotic balance. Our improved R. salmoninarum growth may be a
result of an additive effect from some or all of the aforementioned points.
Findings of the current study clearly demonstrate the advantages of this
modified tissue processing in the improvement of primary isolation of R.
salmoninarum from infected tissues, even if infection intensity is relatively low.
Conventional biochemical tests revealed that R. salmoninarum isolates tested
in this study were non-motile, catalase positive, cytochrome oxidase negative,
esculin hydrolysis negative, DNAse negative and carbohydrate utilization
negative. These findings coincide with the standard biochemical criteria of R.
salmoninarum described by Smith (1964); Sanders and Fryer (1980); Bruno
and Munro (1986) and indicate the biochemical uniformity of Michigan isolates
among themselves and among other R. salmoninarum strains isolated
worldwide. Our identifications were further confirmed with nPCR results as all
R. salmoninarum isolates had the 320 bp band characteristic for R.
salmoninarum (Pascho et al., 1998).

The antibiogram performed on 12 R. salmoninarum representative isolates
indicated that all tested isolates were highly sensitive to enrofloxacin, which is
in agreement with the report of Hsu et al. (1994). In the study of Hsu et al.
(1994), the authors performed an in vitro sensitivity test using the standard MIC
(minimal inhibition concentration) method and an in vivo efficacy trial using
enrofloxacin medicated food to treat juvenile rainbow trout experimentally

infected with R. salmoninarum. The authors concluded that R. salmoninarum is

63

highly sensitive to enrofloxacin and suggested that the low Mle, high
bioavailability, and large volume distribution of enrofloxacin make it a good
candidate for treatment of BKD. Results of the present study showed that all
isolates were highly sensitive to ciprofloxacin and intermediate sensitive to
ofloxacin and norfloxacin. Moreover, the sensitivity of the isolates to
terramycin, chloramphenicol, novobiocin, sulfamethaxozole - trimethoprim,
carbenicillin and the resistance to polymyxin B coincide with previous reports
(Austin, 1985; Wolf and Dunbar, 1959; Goodfellow et al., 1985).

An unexpected result was the presence of two erythromycin and azithromycin
resistant isolates (Oden BNT-BS-02 and Oden BNT-BS1-02), since the “wild —
type” R. salmoninarum strains are known to be sensitive to macrolide
antibiotics (Austin, 1985). However, this finding corroborates with Bell et al.
(1988), who were able to develop erythromycin-resistant R. salmoninarum
strains in vitro but were unable to describe the mechanism behind this
resistance. The brown trout stock from which these two isolates were retrieved
has never been treated with erythromycin. Resistance to erythromycin and
other macrolide antibiotics is believed to be associated with mutational (Tanaka
et al., 1968) or plasmid —mediated blockage (e.g. methylation) (Weisblum et al.,
1971) of the binding sites on the 23 S RNA. Whether erythromycin-resistance
observed in the brown trout strain has developed as a result of any of the
mechanisms described for other Gram-positive bacteria is currently unknown

and deserves further investigation.

64

From the relatively limited survey performed in this study, a number of
interesting results were demonstrated that would vastly improve our current
understandings of the status of BKD in Michigan, as well as help shape the
design of future epidemiological studies. First, it is quite clear that R.
salmoninarum is widespread in Michigan, as it has been isolated from every
fish population and lot that was tested (tables 3-5). Second, infected fish are
not only carriers, but some also developed clinical kidney lesions consistent
with BKD. Third, vertical transmission seems to play a major role in BKD
transmission in brown and rainbow trout, as fingerlings were kept throughout
their life in raceways supplied with well water. Fourth, results also suggested
that R. salmoninarum prevalence varies among the species examined, with
brook trout and coho salmon being the highest in both prevalence and
presence of kidney lesions. Fifth, even within the same species, fish strain
seems to play a role in susceptibility to R. salmoninarum. For example, the
Hinchenbrook coho salmon strain showed higher prevalence than that of the
Michigan adapted coho salmon strain collected from Platte River. Also, the
Hinchenbrook coho salmon strain showed relatively higher percent of kidney
lesions when compared to Michigan-adapted coho salmon strain. This
increased susceptibility could be due to the fact that the Hinchenbrook coho
salmon strain was recently introduced to Michigan (in the 19903), while the
other coho salmon strain has adapted to the Great Lakes basin since it’s
introduction in the 1960s (Michigan Department of Natural Resources (MDNR).

This also applies for the brook trout Iron River strain, which has recently been

65

 

 

inf

cc

 

 

 

hi.

{ES

 

adopted for propagation purposes, as opposed to the Assinica strain, which has
been domesticated for five decades. On the contrary, there have been no
noticeable differences in R. salmoninamm prevalence among three strains of
brown trout. Variation in resistance to BKD among strains within the same
species has also been reported by Winter et al. (1980) who made a similar
observation with steelhead trout (Oncorhynchus mykiss). Last, in the case of
chinook salmon, results suggest that R. salmoninarum prevalence varied
among sites to which spawning runs return. For example, adult Platte River
chinook salmon coming from Lake Michigan showed higher R. salmoninarum
prevalence (96 %) and higher percent of clinical cases (11 %) when compared
to Swan River chinook salmon (48 %, 5 % respectively) coming from Lake
Huron. The same trend was observed in chinook salmon from the Little
Manistee Weir, which exhibited a higher R. salmoninarum prevalence than
those found at the Swan River weir. Spatial distribution and population density
could play a role in elevating the prevalence of BKD among fish populations
(Reno, 1998).

In conclusion, this study has shed light on the widespread R. salmoninarum
infections occurring in Michigan’s salmonids. The tissue processing protocol,
combined with the modified medium, has facilitated the isolation of several
hundred R. salmoninarum isolates. These isolates constitute an important
resource for scientists studying R. salmoninarum and BKD in the United States

in general and the Great Lakes in particular.

66

 

 

 

 

 

 

 

 

 

 

Host Weir # of Prevalence (positive/total)
{lessrted Apparently Fish with Total
healthy fish gross
kidney
Lesions *
Chinook Little 63 47/ 63 5/63 52/63
salmon Manistee (74.60 %) (7.93 %) (82.53 %)
River
Swan 60 26/60 3/60 29/60
River (43.33 %) (5 %) (48.33 %)
Platte 27 23/27 3/27 26/27
River (85.18 %) (11.11 %) (96.29 %)
Michigan - Platte 165 112/165 30/165 142/165
adapted coho River (67.87 %) (18.18 %) (86 %)
salmon
Hinchenbrook 56 26/56 30/56 56/56
coho salmon (46.42 %) (53.57 %) (100 %)
Total 371 234/371 71/371 305/371
(63 %) (19.1%) (82.2 %)

 

 

 

 

 

 

 

Table 3. Prevalence of Renibacterium salmoninarum in fall returning

chinook and coho salmon spawners from Lake Michigan and Lake Huron

watersheds.

* White-creamy patches in kidneys

67

 

Tal
Iro

hal

 

 

 

 

 

 

 

 

 

 

Host Number of Prevalence
2::mined Apparently Fish with Total
healthL kidney lesions
Brook trout 60 22 10 32
(36.66 %) (16.66 %) (53.33 %)
Lake Trout 60 40 3 43
(66.66 %) (5 %) (71.66 %)
Brown Trout 12 8 4 12
(66.66 %) (33.33 %) (100 %)
Rainbow trout 12 9 (75 %) 2 11
(16.66 %) (91.66 %)
Total 144 79/144 19/144 98/144
(54.9%) (13.2%) (68%)

 

 

 

 

 

 

Table 4. Prevalence of Renibacterium salmoninarum in captive char and
trout broodstocks collected in the fall 2002 from Michigan state fish

hatcheries.

68

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Fish Species Number of Prevalence
fish tested
Apparently Fish with Total
healthy kidney lesion *

Brook trout 60 15 10 25
Assinica (25 %) (16.7 %) (41.7 %)
Brook trout Iron 60 20 30 50
River (33.3 %) (50 %) (83.3 %)
Brook trout 15 0 9 9
Cedarbrook (0 %) (60 %) (60 %)
Lake Trout 120 22 0 22

(18.3 %) (0 %) (18.3 %)
Brown trout 60 8 2 10
(Wild Rose) (13.3 %) (3.3 %) (16.7 %)
Brown trout 60 14 1 15
Seeforellen (23.3 %) (1.7 %) (25 %)
Brown trout 60 12 3 15
Gilchrist (20 %) (5 %) (25 %)
Rainbow trout 60 8 2 10

(13.3 %) (3.3 %) (16.7 %)
Total 495 99/495 57/495 1 56/495

(20%) (11.5 %) (31.5 %)

 

Table 5. Prevalence of Renibacterium salmoninarum in 10-15 month-old

propagated fish. All samples were collected in January 2003 except those

from Cedarbrook, which were collected in August 2004.

69

 

Ta

Ofl

esl

 

 

 

 

Isolate ID Conventional biochemical tests Motility

 

C O E D CHO

 

Marquette BKT-BS-02 + - - - - -
Marquette LT-02-A

Wild Cherry Creek BKT44-
02

Oden BNT-BS-02
Marquette LT-02-B

Oden RBT-02

Marquette LT 24-02

Platte River CH-02

Oden BNT-BS1-02

Platte River HB-02

Swan River OHS-02

Platte River CO-02

 

+
I
I
I
I
I

 

+
I
I
I
I
u

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

+++++++++
I
I
I
I
I

 

Table 6. Results of conventional biochemical and motility tests performed
on the 12 representative Renibacterium salmoninarum isolates.
Conventional biochemical tests used in this study are catalase (C), oxidase (O),

esculin hydrolysis (E), DNAse (D), and carbohydrate utilization tests (CHO).

7O

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Antibiotic Inhibition zone diameter standards in mm

Name Code Disk Resistant Intermediate Sensitive
potency

Terramycin TE 30 pg No zone <15 2 5
Chloramphenicol C 30 pg No zone 13-17 21 8
Trimethoprim - SXT 25 pg No zone <15 25 5
Sulfamethoxazole
Erythromycin E 15 pg No zone <15 215
Azithromycin AZM 15 pg No zone <15 2 5
Clindamycin DA 241g No zone <15 35
Ciprofloxacin CIP 5 pg No zone <15 35
Enrofloxacin EN0 5 pg No zone <15 21 5
Ofloxacin OFX 5 fl No zone 13-15 36
Norfloxacin NOR 10 pg No zone 14-16 217
Kanamycin K 30 pg No zone 14—17 218
Carbenicillin CAR 100 pg No zone <15 27
Novobiocin NV 30 pg No zone <10 20
Polymyxin B PB 300 U No zone 9-11 22
Sulfate

 

 

 

 

 

 

 

Table 7. Standard concentration and expected inhibition zones for each

used antibiotic disc.

71

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Isolate ID 1 2 3 4 5 6 7 8 9 10 11 12 Mean SE SD
Antibiotic

TE 25 20 20 8 25 27 25 24 8 26 25 22 21 .25 1 .89 6.50
SXT 20 18 19 18 20 22 18 14 12 18 19 18 18.00 0.76 2.70
C 25 22 12 20 24 18 17 20 20 27 18 16 20.00 1.20 4.16
CAR 17 18 7 18 15 14 12 14 10 18 18 8 14.00 1.16 4.03
E 19 22 13 7 10 20 20 23 9 20 17 20 16.70 1.57 5.46
AZM 17 20 12 8 12 17 18 22 8 17 19 16 15.50 1.30 4.52
K 10 8 12 10 9 14 10 10 6 8 10 6 9.40 0.65 2.27
DA 6 0 0 0 8 10 0 6 0 6 8 0 3.66 1.15 3.98
PB 0 0 0 0 0 0 0 0 0 0 0 0 0.00 0.00 0.00
NV 15 20 10 14 17 14 13 15 17 18 18 16 15.60 0.77 2.67
OFX 20 18 20 19 20 20 19 18 16 20 19 18 19.00 0.35 1.24
CIP 27 23 22 25 22 28 20 19 17 22 22 25 22.70 0.92 3.20
ENO 30 32 28 27 31 32 25 18 19 28 30 29 27.40 1.33 4.64
NOR 19 20 18 16 18 20 17 15 16 18 19 20 18.00 0.49 1.70

 

Table 8. Inhibition zones in millimeters (mm) produced by the 12

Renibacterium salmoninarum isolates used in the antibiogram.

Note: 1 (Marquette BKT-BS-02), 2 (Marquette LT-02-A), 3 (Cherry Creek BKT44-02), 4 (Oden
BNT-BS-02), 5 (Marquette LT-02-B), 6 (Oden RBT-02), 7 (Marquette LT 24-02), 8 (Platte River

CH—02), 9 (Oden BNT-BS1-02), 1o (Platte River HB-02), 11 (Swan River CH5-02), 12(Platte River

00-02)

72

 

El % Non clinical
I% Clinical

 

 

LMCH LMCH SRCH SRCH
female male female male

Figure 1. Prevalence of Renibacterium salmoninarum in male and
female returning chinook salmon spawners from Lake Michigan and
Lake Huron watersheds. Prevalence was determined by calculating %
positive Renibacterium salmoninarum culture retrieved from fish with clinical

or without clinical lesions.

* LMCH: Little Manistee River Weir chinook salmon
* SRCH: Swan River Weir chinook salmon.

73

100%-
90%-
80%-
70%-
60%7
50°/o‘
40°/o‘
30%'
20°/o‘
10%‘

0%

0% non Clinical
I% Clinical

 

 

 

J;

Ml Coho Coho HB Coho HB Coho
female male female male

Figure 2. Prevalence of Renibacterium salmoninarum in male and
female returning coho salmon spawners from Platte River Weir, Lake
Michigan watershed. Prevalence was determined by calculating % positive
Renibacterium salmoninarum culture retrieved from fish with clinical or

without clinical lesions.

* Ml Coho: Platte River Weir Michigan adapted coho salmon
" HB Coho: Platte River Weir Hinchenbrook coho salmon.

74

CHAPTER THREE

DIAGNSOTIC TESTING PATTERNS OF RENIBACTERIUM
SALMONINARUM INFECTION IN SOME FERAL AND CAPTIVE

SALMONINES

ABSTRACT

Bacterial kidney disease, caused by Renibacterium salmoninarum (R.
salmoninarum), is a slowly progressing disease that hampers salmon
conservation and restoration programs in North America. The purpose of this
study was to track the progress of naturally occurring R. salmoninarum
infection in some of Michigan’s salmonid stocks using nested polymerase
chain reaction (nPCR), quantitative enzyme linked immunosorbent assay (Q-
ELISA), and culture. The Q-ELISA test detected 67.6 % infection
prevalence, which is lower than culture (77.2 %) or nPCR (94.2 %), yet it
provided semi-quantitative data on infection intensity. The disagreement in
results among the three assays may reflect the different phases of R.
salmoninarum infection at the time of sampling. The testing results
demonstrated the presence of six patterns, with each of the patterns

representing a probable stage along the course of R. salmoninarum infection.

75

Findings also suggested that fish stocks tested in this study are not uniform

in the distribution of the diagnostic patterns.

76

INTRODUCTION

Bacterial Kidney Disease (BKD) of salmonines, caused by
Renibacterium salmoninarum, is a slowly progressing systemic infection,
which often causes high losses among infected fish (Fryer and Sanders
1981). In addition to horizontal transmission, R. salmoninarum can be
transmitted vertically (Evelyn et al. 1986 a,b). Therefore, BKD is a major
concern for salmonine conservation and restoration programs worldwide in

general, and in the Great Lakes basin, in particular (Faisal and Hnath 2005).

Since the first report of Kidney Disease in 1930, a number of diagnostic
assays have been developed to determine the presence of R. salmoninarum
in infected fish tissues. Culture on selective media, fluorescent antibody
technique, quantitative ELISA (Q-ELISA), and nested polymerase chain
reaction (nPCR) are currently the most common diagnostic techniques used
in the detection of R. salmoninarum (Pascho and Elliott 2004; OIE 2003).
When multiple diagnostic tests were performed on the same sample,
numerous discrepancies among findings were observed (Cipriano et al.
1985; Sakai et al. 1989; White et al. 1995; Jansson et al. 1996; Miriam et al.
1997; Pascho et al. 1998). While these inconsistencies were often difficult
to interpret, most authors concentrated on these discrepancies to merely
evaluate the sensitivities of various detection assays (Jansson et al. 1996;
Chase and Pascho 1998; Pascho et al. 1998). For example, it has been

estimated that the nested PCR assay, developed by Chase and Pascho

77

(1998), which uses primers specific for conserved regions of the major
soluble antigen (msa) gene, has enabled the detection of as little as 10
bacterial cells/gram of kidney tissue (Pascho et al. 1998). Culture, on the
other hand, requires the presence of 40-100 bacterial cells per gram tissue to
ensure bacterial isolation (Lee 1989; Miriam et al. 1997). The quantitative
ELISA assay primarily targets bacteria-secreted soluble proteins (Pascho
and Mulcahy 1987; Meyers et al. 1993; Pascho et al. 1998). It has been
estimated that Q-ELISA requires a minimal bacterial concentration of 1.3x
104 bacterial cells/ml of ovarian fluid and 103-cells/g kidney tissues are

needed to produce consistent positive results (Pascho et al. 1998).

Mechanisms involved in the initiation of infection, progression of the
disease, death, and/or recovery from natural R. salmoninarum infections are
currently unknown (reviewed in Evenden et al. 1993; Wiens and Kaattari
1999). It is well documented that R. salmoninarum can be transmitted
vertically from parents to offspring (Evelyn et al. 1984; 1986). Horizontal
transmission is believed to occur primarily through the oral-fecal route (Balfry
et al. 1996), and to some extent through the gills (Flano et al. 1996) and skin
lesions (Evenden et al. 1993). Renibacterium salmoninarum infections can
persist in fish lacking clinical signs, while in others, the infection may
progress, causing clinical signs with bacterial numbers reaching up to 109
colony forming units/ g of kidney tissues prior to death (Bruno 1986; Evelyn

et al. 1996).

78

Renibacterium salmoninarum secretes a number of soluble proteins that
seem to play a role in pathogenicity (Austin and Rodgers 1980; Bruno and
Munro 1986; Wiens and Kaattari 1991; Hamel 2001). These bacterial
proteins form complexes with fish antibodies that are deposited in kidney
glomeruli and are then slowly eliminated (Sami et al. 1992). Therefore, the
aim of this study is to utilize the limits of detection for each assay, what each
assay detects, and the collective results of these assays to shed some light

on the kinetics and course of infection within certain fish population.

79

MATERIALS AND METHODS

Fish. Between October-November 2002, a total of 364 feral and captive
spawning salmonids were collected from Michigan weirs and state fish
hatcheries. Fish included 100 returning chinook salmon (Oncorhynchus
tshawytscha) collected from the Little Manistee River Weir (LMRW),
Manistee county, Michigan (Lake Michigan watershed) and Swan River Weir
(SRW) at Rogers City, Presque Isle county, Michigan (Lake Huron
watershed). An additional 131 Michigan-adapted coho (Oncorhynchus
kisutch) and 53 Hinchenbrook coho salmon were collected from the Platte
River Weir (PRW) at Beulah, Michigan (Lake Michigan watershed). Captive
brood stock included 41 brook trout (Salvelinus fontinalis) and 39 lake trout
(Salvelinus namaycush). The captive stocks were kept in raceways that
receive water from Cherry Creek (Lake Superior watershed) at the Marquette
State Fish Hatchery in Michigan’s Upper Peninsula. Males and females were
equally represented among samples. The sacrifice of the feral spawners
entailed exposing the fish to carbon dioxide-laden water, followed by a blow
to the head. Following gamete collection, the abdominal cavity was cut open
to examine individual internal organs for signs associated with BKD, followed
by the collection of approximately one gram of tissue from anterior, posterior
and middle kidney sections. Cross contamination was avoided by replacing

dissecting tools with sterile tools following the dissection of each fish.

80

Sampling and sample processing: Samples from fish were analyzed
individually unless othenlvise indicated. Kidney samples representing the
anterior, posterior, and middle sections of the kidney were transferred in
sterile 7.5 cm x 18.5 cm Whirl Pak® bags (Nasco, Forte Atkinson, and WI),
kept on ice, and were softened as much as possible through multiple cycles
of physical pressure. The homogenized kidney tissues were diluted in 1:4
(w/v) Hank’s Balanced Salt Solution (HBSS, Sigma Chemical Co, St. Louis,
MO, USA) and then stomached for 2 minutes at high-speed using the
Biomaster Stomacher-80 (Wolf Laboratories Limited, Pocklington, York, UK).
In the case of coelomic fluid, 1 ml of each sample was diluted 1:2 (v/v) in

HBSS for Q-ELISA testing. In the case of the milt, 1ml from each sample
was diluted 1:2 (v/v) in HBSS for Q-ELISA testing.

Culture. Isolation of R. salmoninarum was performed as described in
Chapter 2 in this thesis. Briefly, 100 pl aliquots of stomached kidney tissues
were spread onto MKDM (Eissa 2005, chapter 2). Culture plates were
incubated for a period of 20 days at 15 °C. Inoculated plates were checked
every 5 days for bacterial growth. Identification of the isolates was done
according to the standard morphological criteria of R. salmoninarum
(Sanders and Fryer 1980; Austin and Austin 1999). Molecular confirmation of

the isolates was done using the nPCR method described by Pascho et al.

(1998).

81

 

Measurements of Renibacterium salmoninarum antigen using the
Quantitative Enzyme-linked Immunosorbent Assay (Q-ELISA). Aliquots
(250 pl) of each sample were transferred into 1.5 ml safe lock microfuge
tubes, to which an equal volume of 0.01 M Phospate Buffered Saline Tween
20 (0.05 %) (PBS-T20) (Sigma) with 5 % natural goat serum (Sigma) (Olea
et al. 1993) and 50 pl CitriSoIv solution (Fisher Chemicals, Fairlawn, New
Jersey, USA) (Gudmunsdottir et al. 1993) were added. The solution was then
thoroughly mixed via vortexing, incubated at 100 °C on heat blocks with a
rotary shaker for 15 minutes, followed by 2 hours of incubation at 4 °C. After
incubation, the mixture was centrifuged at 60009 for 15 minutes at 4 °C.

The aqueous supernatant of each sample was carefully collected and then
transferred to a 1.5 ml microfuge tube for Q-ELISA testing. The Q-ELISA
method used in this study is that described in detail by Pascho and Mulcahy
(1987) as well as Alcorn and Pascho (2000). The positive-negative threshold
absorbances were calculated according to the method described by Meyers
et al. (1993) .The positive—negative cutoff absorbance for the kidney
homogenate was 0.10. The tested positive samples were assigned the
following antigen level categories: low (0.10 to 0.19), medium (0.20-0.99) and

high (1.000 or more) (Pascho et al. 1998).
Nested PCR. Bacterial DNA was extracted using the DNeasy tissue

extraction kit (Qiagen-Valencia, CA, USA). DNA was extracted from 100 pl

aliquots of kidney tissue homogenates according to manufacturer’s

82

 

instructions and the method described by Pascho et al. (1998) with minor
modifications. The tissue pellets were obtained by centrifugation at 6000 g
for 20 minutes at 4 °C and then incubated with lysozyme buffer consisting of
180 pl of 20 mg lysozyme (Sigma-Aldrich, Inc. St. Louis, MO), 20mM Tris-
HCI (pH 8.0), 2 mM EDTA (Sigma) and 1.2 % (v/v) Triton X 100 (Sigma) at
37 °C for 1 hour. The nPCR method used primers recommended by Pascho
et al. (1998) with slight modifications to the volume of DNA (5pl for the first
round and 2p| for the second round nPCR) and master mixes (45pl for the
first round and 48pl for the second round nPCR). The controls were
composed of a PCR mixture containing no DNA template (reagent negative
control), positive R. salmoninarum and positive tissue control. A volume of 10
pl of the nPCR product and controls were mixed with 2 pl of 6X loading dye
(Sigma) and used on a 2 % agarose gel (Invitrogen Life Technologies,
Carlsbad, CA). Each electrophoresis gel included a 1kbp DNA ladder with
100 bp increments (Invitrogen). Gels were run in 1 X Tris Acetate (1 X TAE)
Buffer (Sigma). Gels were visualized under the KODAK EDAS Camera

System and UV Trans-illuminator. Samples were considered positive when a

320 bp band was detected.

83

 

RESULTS

Among the 364 fish examined, 343 fish (94.2%) were positive in the
nPCR assay, 281 (77.2%) were culture positive and 246 (67.6%) were
positive with the Q-ELISA. Over half (53.3%) of the fish used in this study
gave positive results by all three diagnostic assays (table 9). The
consistency among findings was highest in the case of Hinchenbrook coho
(81.1%), followed by brook trout (63.4%). Only 7 fish (1.9%) were negative
by all three diagnostic assays. As displayed in table (9), most Q-ELISA
positive fish possessed low R. salmoninarum antigen concentrations
(193/231, 83.5%), the highest proportions of which were in the LMRW
Manistee chinook salmon and PRW Michigan-adapted coho salmon (64.3 %,
67.9 % respectively). Fish with medium and high R. salmoninarum antigen
concentrations were found primarily in Hinchenbrook coho salmon (45.2%)
and brook trout (29.2%) stocks.

Combining the results of the three diagnostic assays performed on the
same samples, 6 patterns were recognized. Pattern 1 represents fish that
were positive with the nPCR only (24/364, 6.7%). Pattern 2 represents fish
positive with both nPCR and culture assays (87/364, 23.9%). The majority of
fish (194/364, 53.3%) were in Pattern 3, with positive results in all three
diagnostic techniques. Pattern 4 represents fish positive with nPCR and Q-
ELISA (38/364, 10.4%). Pattern 5 represents fish that were barely positive in

the Q-ELISA assay (14/364, 3.8%). Pattern 6 was the least represented,

84

 

with only 7 fish (1.9%) that had negative results with all three assays used in

this study.

The distribution of fish representing each of the patterns differed among
the fish stocks tested. For example, the majority of LMRW chinook salmon
were in patterns 2 and 3 (Figure 3A), while SRW chinook salmon showed a
wider distribution consisting of over half of the fish in patterns 1 and 2 (Figure
3B). In the case of PRW Michigan-adapted coho, almost all fish were in
patterns 2-4 (Figure 4A). On the other hand, 75% of the Hinchenbrook coho
salmon strain belonged to pattern 3 (Figure 48). Similarly, the two captive
Salvelinus spp. broodstocks, though kept in the same hatchery, exhibited

different diagnostic testing patterns (Figures 5 - 6), with over 60% of the

brook trout belonging to Pattern 3.

85

DISCUSSION

Findings clearIy suggested that R. salmoninarum infection is widespread
in adult fish of the stocks tested in this study. Only 7 fish were negative out
of 364 when tested by the three diagnostic assays. While these figures are
staggering, one should not be surprised since R. salmoninarum has existed
in Michigan salmonines for at least 50 years (Allison 1958) and was involved
in the massive chinook salmon die offs of the 19808 in Lake Michigan
(Johnson and Hnath 1991; Holey et al. 1998). However, it should also be
emphasized that fish tested in this study were spawning adults (>4 years
old); a matter that increases the likelihood of exposure to R. salmoninarum
and allows time for the slow progression of this infection. Moreover,
sampling took place at the peak of the spawning season, meaning that the
fish were subjected to multiple stressors, such as starvation, hormonal
changes, and physical pressure on internal organs due to distension of the
gonads. Therefore, infection rates obtained in this study should not be
considered representative of the overall R. salmoninarum prevalence at the
population level. Regardless of these factors, data strongly suggested that
R. salmoninarum continues to be enzootic in Michigan’s salmon and char
species.

In other areas of the world where R. salmoninarum is enzootic,

prevalence of infection in feral and wild fish species have reached up to

100%. For example, in Iceland, arctic char and brown trout reached infection

86

 

levels of 100% and 81%, respectively (J6nsd6ttir et al. 1998). In North
America, R. salmoninarum infection rates of 83% in brook trout from
Wyoming, USA (Mitchum et al. 1979), and 35% in returning Atlantic salmon
in the Margaree River, Halifax, Canada (Paterson et al. 1979) were reported.

The findings also suggested that nPCR performed with primers targeting
the msa gene is superior to culture and Q-ELISA methods in detecting R.
salmoninarum infection. Most other studies comparing diagnostic assays
concur with the supreme specificity and sensitivity of the nPCR technique
developed by Chase and Pascho (1998). However, despite its high
specificity and sensitivity, one cannot determine infection intensity based
exclusively on nPCR results. The isolation of R. salmoninarum from infected
tissues, in conjunction with the confirmation of representative colonies via
nPCR has also been effective in identifying 76.4% of the infected fish in this
study, although culturing is lower in sensitivity than nPCR alone. Retrieving
R. salmoninarum from tissues by culture alone indicated a presence of at
least 40-100 live bacterial cells/g, but did provide an estimate for the intensity
of infection (Lee 1989; Miriam et al. 1997).

Q-ELISA yielded 67.6% R. salmoninarum prevalence, which was lower
than either nPCR (94.2%) or the culture assays (77.2%). Previous studies
estimated that relatively high numbers of bacterial cells (1.3 x 104 bacteria/ml
ovarian fluid, Pascho et al. 1998; and 103 cells/g kidney tissue, Jansson et al.
1996) are necessary for the detection of R. salmoninarum excreted proteins

via the Q-ELISA assay. This lower sensitivity of Q-ELISA may not be due to

87

 

the assay or reagents themselves, but rather to the metabolic activities of R.
salmoninarum at the time of testing, which influences the amount of bacterial
antigens secreted. It is known that R. salmoninarum can live within fish
tissues for a relatively long period in low numbers and in a quiescent state
(Bruno 1986), and that R. salmoninarum activation and the secretion of
extracellular proteins do not take place in infected fish all of the time. The
inherent advantage of Q-ELISA is that this technique allowed us to recognize
that the majority Q-ELISA positive fish had relatively low concentrations of R.
salmoninarum antigen (83.5%, table 1). Only a minority of fish exhibited
medium (6.5%) or high (10%) antigen concentrations. Low R. salmoninarum
antigen concentrations indicated the presence of relatively lower numbers of
R. salmoninarum in a less active metabolic status when compared to fish
with higher R. salmoninarum antigen concentration, a matter that could be
associated with either early or late stages of infection (Sami et al. 1992;
J6nsdéttir et al. 1998).

The lack of agreement in results of the three assays is difficult to explain,
however, not surprising since the fish were naturally, rather than
experimentally infected. Naturally infected fish of this study were likely at
different phases of R. salmoninarum infection at the time samples were
collected. This factor may have contributed to the appearance of diverse
diagnostic patterns that ranged from full agreement among the three
diagnostic assays (e.g., Patterns 3 and 6) to a more unexpected pattern

(e.g., Pattern 5). Other factors that likely have contributed to the diversity of

88

diagnostic patterns include differences in the R. salmoninarum dose to which
the fish were exposed, pathogenicity of specific R. salmoninarum strains, fish
immunologic status, and individual genetic susceptibility.

Careful examination of the six patterns reveals what appears to “be a
logical progression of infection, with each of the patterns representing a
probable stage along the course of R. salmoninarum infection. Pattern 1 is
most likely the initial stage of infection establishment within the kidney, with a
minimal number of bacteria localized in tissues. Pattern 2 indicates that the
infection has been established and bacterial numbers are high enough to be
isolated on the MKDM medium. Pattern 3 is the most common, with R.
salmoninarum antigens exceeding the detection limit of Q-ELISA. In the fish
in which the infection has progressed, R. salmoninarum antigen
concentrations increase from low to medium to high. While high R.
salmoninarum antigen concentrations are a strong indicator of active, well-
established infections that may lead to clinical cases with mortalities, it does
not necessarily indicate the presence of the characteristic clinical and
pathological manifestations of BKD, including granuloma formation (Miriam et
al. 1997; Jensdottir et al. 1998). This is most likely due to the fact that R.
salmoninarum soluble antigens suppress a number of fish immune defense
mechanisms and thereby host reactions to infection may be lacking (Turaga

et al. 1987; Wiens and Kaattari, 1991; Fredricksen et al. 1997, Densmore et

al., 1998, Jonstttir et al., 1998; Grayson et al. 2002).

89

Pattern 4 may represents fish that appear to be recovering from R.
salmoninarum infection, as viable bacteria present in their tissues were not
plentiful enough to be isolated, yet bacterial DNA and R. salmoninarum
antigens continue to be present. Fish in Pattern 5 are possibly in an
advanced stage of recovery, with only minute traces of R. salmoninarum
antigens remaining. Indeed, all 14 fish in this pattern exhibited Q-ELISA
values that neared those of the negative control. Renibacterium
salmoninarum antigens form immune complexes that deposit in the kidney
glomeruli and are eliminated slowly through the kidneys (Sami et al. 1992).
Fish in Pattern 6 were either never exposed to R. salmoninarum, refractory to
infection, or were infected and then totally eliminated R. salmoninarum and
its antigen from their systems. Since R. salmoninarum is widespread in
Michigan waters, it is more likely that fish in this group have been exposed to

R. salmoninarum before testing. Further restoration and conservation efforts

should focus on increasing the proportion of this pattern in salmonid

populations.
Findings suggest that fish stocks tested in this study are not uniform in

the distribution of patterns (Figures 6-9). For example, most of the tested
LMRW chinook salmon and PRW MI-adapted coho salmon (both returning
from Lake Michigan) were either in pattern 2 (>25%) or 3 (>50%), albeit with
low antigen concentrations. It is likely that R. salmoninarum infection in
these two Lake Michigan stocks seldom progress. Patterns of SRW chinook

salmon and captive lake trout were evenly distributed, indicating an ongoing

9O

infection with many fish recovering. In the case of Hinchenbrook coho
salmon and captive brook trout, both prevalence and intensity were high, with
very few fish in patterns 46 Indeed, in both these stocks, overt clinical signs
of BKD and mortalities (in the case of brook trout) are often observed
(unpublished observations). Brook trout are known for their high
susceptibility to R. salmoninarum infection (Snieszko and Griffin 1955;
Mitchum et al. 1979), Hinchenbrook coho salmon strain were introduced to
the Great Lakes basin from New York State relatively recently (G. Whelan,
Michigan Department of Natural Resources, personal communication) and
have proven to be more susceptible to R. salmoninarum infection when
compared to the Michigan-adapted coho salmon strain that was introduced to
the Great Lakes basin in 1967 (Borgeson 1970; Keller et al. 1990).

While the explanations provided herein may logically illustrate the course
of R. salmoninarum natural infection, it should be emphasized that the data
of this study were generated using kidney tissues only. Kidneys are the
primary targets of R. salmoninarum (Fryer and Sanders, 1981); however,
other organs should also be assessed in future studies to better understand
BKD pathogenesis, particularly in natural infections. So far, the relatively few
studies addressing BKD course and progression of infection relied upon
experimental infection (Flafio et al 1996, White et al. 1995). Moreover, further
correlation of diagnostic testing patterns with clinical observations and tissue

alterations in stained sections are needed to better evaluate impacts of R.

salmoninarum infection at the population level.

91

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Fish & # fish Frequencies of different diagnostic testing patterns"
source“ tested
Pattern 1 Pattern 2 Pattern 3 Pattern 4 Pattern 5 Pattern 6
LM-CH 42 0 1 1/42 25/42 1/42 5/42 0
(0 %) (26 %) (59.5 %) (2.4 %) (12 %) (0 %)
SR-CH 58 1 5/58 1 5/58 12/58 8/58 5/58 2/58
(26 %) (26%) (20.7 %) (13.8 %) (8.6 %) (3.4 %)
PR-CO 131 3/131 34/131 75/131 18/131 0 2/131
(2.3 %) (26%) (57.2 %) (13.7%) (0 %) (1.5 %)
PR-HB 53 1/53 8/53 43/53 1/53 0 0
(1.9 %) (15%) (81.1 %) (1.9 %) (0 %) (0 %)
MBKT 41 4/41 2/41 26/41 8/41 1/41 0
(9.8 %) (4.9 %) (63.4 %) (19.5 %) (2.4 %) (0 %)
MLT 39 1/39 17/39 13/39 2/39 3/39 3/39
(2.6 %) (43.5 %) (30.8 %) (5 %) (7.7 %) (7.7 %)
Total 364 24 87 1 94 38 14 7
(6.7 %) (23.9 %) (53.3 %) (10.4 %) (3.8 %) (1.9 %)

 

 

 

Table 9. Frequencies of different diagnostic testing patterns of salmonid
feral spawners and captive broodstocks collected from different
geographical locations in Michigan during fall 2002.

* Diagnostic testing patterns: Refer to different possible combinations of nPCR, Q-ELISA

and Culture results:

Pattern 1:
Pattern 2:
Pattern 3:
Pattern 4:
Pattern 5:
Pattern 6:

FOR (+) Q-ELISA (-) Culture (-)

pCR (+) Q-ELISA (-) Culture (+)
pCR (+) Q-ELISA (+) Culture (+)
pCR (+) Q-ELISA (+) Culture (-)
PCR (-) Q-ELISA (+) Culture (-)
PCR (-) Q-ELISA (-) Culture H

** Fish & source:

LM-CH: Chinook salmon collected from Little Manistee Weir
SR-CH: Chinook salmon collected from Swan River Weir
PR-CO: Michigan adapted coho salmon collected from Platt River Weir
PR-HB: Hinchenbrook coho salmon collected from Platt River Weir
MBKT: Brook trout collected from Marquette State Fish Hatchery

MLT: Lake trout collected from Marquette State Fish Hatchery

92

 

[- Low I Medium I High [:1 patterns other than 3]

 

 

 

100x.- _
sow Flgure (3A)
G)
o
c:
2
9
(D
t-
a.
100%
90%, Diagnostic Testing Patterns
30%-
8 70%‘I
e 333
T; 40%: Flgure(3B)
ID
L—
o.

 

 

Diagnostic Testing Patterns

Figure 3. Frequencies of different diagnostic testing patterns of feral
chinook salmon spawners.

Note: 3A: Chinook salmon from Little Manistee Weir

3B: Chinook salmon from Swan River Weir

Diagnostic testing patterns: P (nPCR), Q (Q-ELISA) and C (Culture).

Clear columns represent any diagnostic testing pattern other than pattern (3)

Colored column represents pattern (3) with three colors referring to different 0-
ELISA intensities in this pattern. Intensity was considered high if the antigen
concentration 21; medium if the antigen concentration = 0.20-0.99; and Low if the antigen
concentration = 0.10 to 0.19.

This figure Is In color

93

 

Ll Low IMedIum Ingh Deny pattern other than (3)]

100% '
90% ‘
80% "
70% ‘
00% ‘
509p FIQUI’O (4A)
40% ‘
30% '
20* ‘
10% ‘

0% ‘

 

prevalence

 

Dlag nostlc Testing Patterns

100% -
90% "
80% r
70% -
00% ~
50% ‘
40% ..
3096 r
20% .
10% ..

0% ‘

   

Figure (4B)

prevalence

 

Dlag nostlc Testlng Patterns

Figure 4. Frequencies of different diagnostic testing patterns of feral
coho salmon spawners collected from the Platte River Weir

Note: 4A: Michigan adapted coho salmon

48: Hinchenbrook coho salmon

Diagnostic testing patterns: P (nPCR), Q (Q-ELISA) and C (Culture)

Clear columns: represent any diagnostic testing pattern other than pattern (3)

Colored column: represents pattern (3) with three colors referring to different 0-
ELISA intensities in this pattern. Intensity was considered high if the antigen
concentration 21; medium if the antigen concentration = 0.20-0.99; and Low if the antigen
concentration = 0.10 to 0.19.

This figure color

94

 

II Low II Medium ll High Cl any pattern other than (3)]

100%-
90% ‘
30%-
709%
60%-
sow
40%
sow
20%
10%

0%-

Figure (5)

   
 
 

prevalence

 

Diagnostic Testing Patterns

100%" Figure (6)
90% .
nox-
70%
cow
50%-
40%-
30%“
20%-
109%

ow

prevalence

 

 

Diagnostic Testing Patterns

Figure 5 - 6. Frequencies of different diagnostic testing patterns of
captive hatchery broodstock.

Note: Figure 5: Brook trout

Figure 6: Lake trout

Diagnostic testing patterns: P (nPCR), Q (Q-ELISA) and C (Culture)

Clear columns: represent any diagnostic testing pattern other than pattern (3)

Colored column: represents pattern (3) with three colors referring to different 0-
ELISA intensities in this pattern. Intensity was considered high if the antigen
concentration 21; medium if the antigen concentration = 0.20-0.99; and Low if the antigen
concentration = 0.10 to 0.19.

This figure is In color

95

CHAPTER FOUR

CURRENT STATUS OF RENIBACTERIUM SALMONINARUM INFECTION IN
CHINOOK (ONCORHYNCHUS TSHAWYTSCHA) AND COHO
(ONCORHYNCHUS KISUTCH) SALMON AT MICHIGAN WEIRS AND

HATCH ERIES
ABSTRACT

Since the recent introduction of chinook and coho salmon to Lake Michigan
in the late 19603, number of Bacterial Kidney Disease (BKD) epizootics has
resulted in mass dieoffs among the feral spawner stage of these fish. The first
epizootic occurred among the coho salmon spawners in 1967 while the latest
occurred among the same stage of chinook salmon throughout the period
between late 19803 and early 19903. Management of the disease by hatchery
personnel and natural resources managers attempting to interrupt the cycle of
infection and focus on two life stages; namely, the gamete-collection stage and
the fry/fingerling stage while reared at state fish hatcheries prior to their release.

Data generated in this study provide additional evidence that (Renibacterium
salmoninarum) R. salmoninarum infection is enzootic in Michigan and
widespread in feral stocks of chinook and coho salmon in both lakes Michigan
and Huron. Lake Michigan’s salmon tend to have an overall higher prevalence

than that observed in Lake Huron’s fish. Renibacterium salmoninarum

96

prevalence in returning feral chinook salmon seem to fluctuate among years,
although a definitive decrease can be observed since the start of this study in
2001. However, both strains of coho salmon spawners showed a steady decline
in R. salmoninarum prevalence throughout the 4 years period. Data of this study
demonstrates the ability of both chinook and coho salmon females and males to
shed R. salmoninarum through the ovarian fluid and milt. Results also indicated
that over 90% of female and 80% of male chinook salmon tested in this study
from both watersheds were not shedding R. salmoninarum antigens along with
their gametes. The case was different in coho salmon as both male and females
shed R. salmoninarum antigens at different rates. The R. salmoninarum antigen
concentrations in Hinchenbrook gametes were significantly higher than in the MI-
adapted gametes.

The BKD testing of both feral spawners and their hatchery raised offspring
fingerlings demonstrates that the current testing and culling programs have been
successful in reducing R. salmoninarum transmission. This study also
demonstrates that some hatchery practices were more effective than others in
controlling the spread of R. salmoninarum and that a reliable improvement in the
reduction of R. salmoninarum prevalence within the hatcheries involved in this

study has been achieved.

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INTRODUCTION

Over the last 150 years, several attempts have been made to introduce
Pacific salmon species into the Great Lakes. The Michigan Department of
Natural Resources (MDNR) initiated the most recent and successful of these
introductions in 1966 for coho salmon (Oncorhynchus tshawytscha) and 1967 for
chinook salmon (Oncorhynchus kisutch). In 1966, MDNR stocked a total of
659,000 coho salmon yearlings in a tributary of the Manistee River and the Platte
River (Borgeson 1970). In 1967, similar number of chinook salmon smolts from
the west coast was introduced into Lake Michigan (Keller et al.1990). After
introduction, both chinook and coho salmon spread throughout the Great Lakes
basin where they have become the most popular sport and commercial fishery in

the State of Michigan (Dexter and O’Neal 2004).

Currently, the Maintenance of the salmon population in the Great Lakes is
primarily dependent upon the stocking of hatchery-propagated fish. This practice
requires the collection of gametes at the weirs from the fall returning runs, the
raising of the offspring at state hatcheries, and the subsequent release of
fingerlings at a weight 4-6 g/fish (Dexter and O’Neal 2004). The stocked
fingerlings move to the smolt stage at the various stocking sites in the rivers, and
then begin their migration to the Great Lakes (Seelbach 1985). Three to four

years later, spawning runs of salmon return with high fidelity to the Michigan

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streams in which they were either stocked or spawned. Once either natural or

man-assisted spawning has commenced, the fish perish (Dexter et al. 2004).

In 1967, multiple cases of coho salmon mortalities were reported by anglers
along the shores of Lake Michigan. MacLean and Yoder (1967) confirmed the
presence of clinical BKD in adult coho salmon that were dying along the shores
of Lake Michigan. The authors also reported that Lake Superior coho salmon
suffered higher BKD prevalence than those of Lake Michigan and the males had
higher prevalence than females. In 1986, clinical cases of were also observed in
spawning chinook salmon runs at the Little Manistee River Weir, Michigan. The
disease became more prevalent in subsequent years, and incidence ranged from
53-100% during 1986-1991 (Holey et al. 1998, Hnath and Faisal 2005).
Concomitant with the BKD surge occurring at the weirs, chinook salmon die-offs
were reported in 1986 and 1987 and reached a peak in 1989. Minimum
estimates of 20,000 adult fish were found dead (Johnson and Hnath 1991). The
majority of dead fish exhibited lesions indicative of BKD, hence BKD was
considered to be a major contributing factor to the chinook salmon kills. Starliper
et al. (1997) demonstrated that the R. salmoninarum isolates associated with
chinook and coho salmon mortalities in Michigan are more virulent than those
retrieved from other regions in North America.

Renibacterium salmoninarum is an obligate intracellular pathogen that is
transmitted both horizontally (Mitchum and Sherman 1981; Bell et al 1984) and

vertically (Evelyn et al. 1984, 1986). Renibacterium salmoninarum pathogenicity

relies upon a number of extracellular proteins (ECP) that possess

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immunosuppressive, proteolytic, hemolytic, and DNA degrading activities (Austin
and Rodgers 1980; Bruno and Munro 1986b; Turaga et al. 1987; Fredriksen et al.
1997). When ECP alone were injected into susceptible fish, 80-100% mortalities
may occur (Shieh 1988). The ECP contain a water-soluble, cell surface, 57- kDa
protein (p57) that has been demonstrated to be a major virulence factor of R.
salmoninarum (Getchell et al. 1985; reviewed in Wiens and Kaattari 1999).

The occurrence of vertical transmission of R. salmoninarum is well

documented (Evelyn et al. 1984; Lee and Evelyn 1989), but believed to be rare

(Hamel 2001). It seems that inclusion of R. salmoninarum-ECP into the eggs

increases the susceptibility of the offspring to R. salmoninarum infection (Brown
et al.1996). The studies of Brown et al. (1996) provided evidence that infra-ovum
inclusion of p57 can result in decreased immune functions when the resulting fry
are exposed to R. salmoninarum. The likelihood of vertical transmission of the
disease (from parent to offspring) or infra-ovum antigen inclusion
immunotolerance is related to the ovarian fluid infection levels (Hamel 2001).
Another complication results from the fact that the entry of R. salmoninarum-ECP
may cause life-long immunotolerance of the disease, greatly decreasing the
probability of survival to spawning (Hamel 2001).

Because R. salmoninarum can be transmitted vertically, MDNR adopted a
stringent visual inspection on each spawning female and the subsequent culling
of any gametes previously collected from spawning females showing overt signs
of clinical BKD. In addition, kidney swabs of individual fish were routinely

examined for the presence of R. salmoninarum soluble antigens using a

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monoclonal antibody based Field ELISA (FELISA) (Gary Whelan, MDNR
personal communication). This rapid field test allowed the detection of fish whose
kidneys are laden with R. salmoninarum antigen, and exclusion of their eggs
from further incubation (Beyerle and Hnath 2002; Hnath and Faisal 2005).
Neither health inspection, nor FELISA were performed on males because the
published studies of Klontz et al. (1983) and Evelyn et al. (1986) minimized the
role that male fish play in the vertical transmission of R. salmoninarum.

In the Great Lakes, chinook and coho salmon are not in their native range,
yet many of the stocked fish are able to survive and return to rivers to
successfully spawn. How returning salmon were able to co-exist with R.
salmoninarum in a non-native range is currently unknown and deserves further
investigation. This is particularly important since most of our knowledge on R.
salmoninarum-returning spawner salmon interactions originated from studies
performed in the Pacific Northwest, where the fish alternate between marine and
freshwater environments. Hence, the major thrust of this chapter is to identify
some of the basics of the R. salmoninarum- returning spawner salmon
relationship in Michigan waters such as the prevalence and intensity of R.
salmoninarum infection in returning spawning runs, the contribution of both males
and females to shedding of R. salmoninarum and its immunosuppressive soluble
antigens. Additional aim of this study is to determine the efficacy of the culling
program and hatchery practices on minimizing vertical and horizontal

transmission of R. salmoninarum.

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MATERIALS AND METHODS

Fish and sampling. In this study, samples were collected from three major
egg-take weirs, and three state fish hatcheries in Michigan (table 10). Chinook
salmon samples from returning spawners were collected from the Little Manistee
River Weir (LMRW, Manistee County, Lake Michigan watershed) and Swan River
Weir (SRW, at Rogers City, Presque Isle County, Lake Huron watershed).

LMRW is the major chinook salmon egg collection operating weir on the
Michigan-side of Lake Michigan, situated on the Little Manistee River, a major
trout tributary that annually receives millions of fingerlings via stocking. Swan
River Weir is the only egg-take facility on Lake Huron and is considered an ideal
tributary for the further development of pre-smolts to the smolt stage before

emigration to Lake Huron (Dexter and O’Neal, 2004).

Coho salmon samples were collected from two strains of coho salmon; the
Michigan-adapted and the Hinchenbrook strains. Adult coho salmon spawning
runs were sampled at the Platte River Weir (PRW), off the Platte Bay, at Beulah,
Ml (Lake Michigan watershed) during egg-takes in the falls of 2001 through 2004.
Platte River Weir is the only operating egg-take weir on the Michigan-side of

Lake Michigan that is used exclusively for collecting coho salmon gametes.

For the purpose of this study, kidney tissues were sampled from 303 and 653
chinook salmon spawners at the LMRW and SRW respectively (table 10). A total
of 383 and 273 kidney samples were sampled from Michigan adapted coho and

Hinchenbrook coho salmon spawners respectively at the PRW (table 10).

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Additionally, in 2004, ovarian fluid was collected from 280 and 60 chinook salmon
females at SRW and LMRW respectively. Moreover, ovarian fluid was also
collected from 60 Michigan adapted and 60 Hinchenbrook coho females at PRW.
Concomitantly, milt from 60 LMRW chinook, 60 PRW Michigan adapted coho,
and 60 PRW Hinchenbrook coho male spawners were collected in 2004.

Following an approximate 8 (for chinook salmon) and 18 (for coho salmon)
month period of egg incubation and fish rearing in three state fish hatcheries
(Wolf Lake state hatchery (WLH) in Mattawan city, Platte River state hatchery
(PRH) in Beulah city, and Thompson state hatchery (THH) in Manistique,
Michigan), kidney tissues of juvenile fingerlings were collected prior to their
release into the basins of lakes Michigan and Huron and subsequently analyzed
for the presence of R. salmoninarum. Within the period of 2002-2005, a total of
965 chinook and 480 coho salmon kidney samples were collected from juvenile
fingerlings at WLH (360 chinook salmon), PRH (425 chinook and 480 coho
salmon), and THH (180 chinook salmon) (table 11).

The sacrifice of feral spawners entailed exposing the fish to carbon dioxide-
Iaden water, followed by a blow to the head. Following gamete collection, the
abdominal cavity was cut open to examine individual internal organs for signs
associated with BKD, followed by the collection of approximately one gram of
tissue from anterior, posterior and middle kidney sections. Cross contamination
was avoided by using sterile dissecting tools for each fish. Coelomic fluid
samples were collected using sterile transfer pipettes from the egg/ovarian fluid

mixture, transferred to 5 ml sterile polypropylene tubes, and kept on ice until

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processed at the laboratory (Michigan State University, East Lansing, MI). Milt
samples were collected directly from the middle stream of milt into sterile 10-ml
polypropylene tubes. Fingerlings were euthenized by immersion in an overdose

of anesthesia using MS-222 (tricaine methan sulfonate, Finquel- Argent

Chemical Laboratories, Washington).

Sampling and sample processing: Samples from fish were analyzed
individually unless otherwise indicated. Kidney samples from the anterior,
posterior, and middle sections of the kidney were transferred in sterile 7.5 cm x
18.5 cm Whirl Pak® bags (Nasco, Forte Atkinson, WI), kept on ice, and were
softened as much as possible through multiple cycles of physical pressure. The
homogenized kidney tissues were diluted 1:4 (w/v) with Hank’s Balanced Salt
Solution (HBSS, Sigma Chemical Co, St. Louis, MO, USA) and then stomached
for 2 minutes at high-speed using the Biomaster Stomacher—80 (Wolf
Laboratories Limited, Pocklington, York, UK). In the case of either milt or ovarian

fluid, 1 ml of each sample was diluted 1:2 (v/v) in HBSS for Q-ELISA.

Measurements of Renibacterium salmoninarum antigen using the
Quantitative Enzyme-linked Immunosorbent Assay (Q-ELISA). Aliquots of
250 pl of each sample were transferred into 1.5 ml safe lock microfuge tubes, to
which an equal volume of 0.01 M Phosphate Buffered Saline Tween 20 (0.05 %
PBS-T20) (Sigma) with 5 % natural goat serum (Sigma) (Olea et al. 1993) and 50

pl CitriSoIv solution (Fisher Chemicals, Fairlawn, New Jersey, USA)

104

(Gudmunsdottir et al. 1993) were added. The solution was then thoroughly
vortexed, incubated at 100 °C on heat blocks with a rotary shaker for 15 minutes,
followed by 2 hours of incubation at 4 °C. After incubation, the mixture was
centrifuged at 60009 for 15 minutes at 4 °C. The aqueous supernatant of each
sample was carefully collected and then transferred to a 1.5 ml microfuge tube
for Q-ELISA. The Q-ELISA method used in this study was described by Pascho
and Mulcahy (1987); Alcorn and Pascho (2000). The positive-negative threshold
absorbances were calculated according to the method described by Meyers et al.
(1993) .The positive—negative cutoff absorbance for the kidney homogenate was
0.10. The Q-ELISA positive samples were assigned the following antigen level
categories: low (0.10 to 0.19), medium (0.20-0.99) and high (1.000 or more) as
developed by Pascho et al. (1998). Intensity of infection among certain group of

fish is determined by the prevalence of samples that possess high Q-ELISA

antigen concentrations.

Statistical Analysis:

Due to the nature of this study, descriptive statistics were heavily relied upon. For
year-to-year, salmon strains differences, gamete source, and hatchery
comparisons, the data was tested for normality, and then analyzed by an

analysis of variance (ANOVA), student t test (parametric) or Mann-Whitney Rank

Sum (nonparametric) Tests with an alpha = 0.05 (P=0.05).

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RESULTS

1. Renibacterium salmoninarum prevalence and intensity in kidney

tissues of returning salmon spawners. Renibacterium salmoninarum antigens
have been detected in the kidneys of chinook and coho salmon returning to both
weirs in every year sampling took place. Generally, there was a slight decline in
prevalence and intensity when the data of 2004 was compared with that of 2001.

a. Chinook salmon returning spawners. The prevalence of R.
salmoninarum in chinook salmon returning to the Little Manistee Weir (LMRW)
gradually decreased from 84% in 2001 to 20 % in 2003, before abruptly rising to
67% in 2004. Additionally, R. salmoninarum intensity from the same fish groups
declined from 5% in 2001 to 0% in 2002, and then increased slightly to 2.5% in
2003 and 2004. The prevalence of R. salmoninarum in the Swan River Weir
(SRW) constantly declined, but the intensity followed a different trend. The
prevalence of R. salmoninarum in the SRW chinook salmon declined from 46%
in 2002 to 12.3 % in 2004. However, R. salmoninarum intensity in the SRW fish
showed a slight decrease from 5% in 2002 to 3% in 2003, with a subsequent
increase to 4% in 2004 (Figure 7). Comparison of the prevalence and intensity of
R. salmoninarum in the feral spawner chinook from the two weirs demonstrated a
consistently higher prevalence in the LMRW chinook salmon, with the exception
of the samples taken in 2003. On the contrary, the intensity of the disease
seemed to be higher in the SRW salmon over the same period of analysis

(Figure 7). However, statistical analysis of the data revealed that difference

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between the prevalence of R. salmoninarum in chinook salmon collected from
the two different weirs and at the same hatchery in different years of collection
was insignificant.

To determine sex-related differences among chinook salmon from LMRW
and SRW, R. salmoninarum prevalence and intensity was determined in
separate groups of males and females returning to both weirs in 2002 and 2004.
Data showed no statistically significant differences between males and females
from both weirs, although there was a trend for males to have higher prevalences
and intensities of infection as compared to females (Figure 10).

b. Coho salmon returning spawners. The prevalence of R.
salmoninarum in Michigan-adapted coho strain decreased from 84% in 2001 to
64.8 % in 2002 then to 40 % in 2003 ending with 24.2 % in 2004. Additionally, R.
salmoninarum intensity from the same fish group showed no marked difference
through the period between 2001 and 2004 (F igure 8). The prevalence of R.
salmoninarum in the Hinchenbrook strain has decreased from 100 % in 2001 to
82 % in 2002 then to 29 % in 2003, before rising to 72.5% in 2004. The intensity
of infection in the same group of fish followed a similar trend in which the
intensity has decreased from 49 % in 2001 to 21.4 % in 2002 then to 10.4 %
before sharply increasing to 47.5 % in 2004 (Figure 8). Comparison of the
prevalence and intensity of R. salmoninarum in spawners of both coho salmon
strains demonstrates a consistently higher prevalence and intensity in the
Hinchenbrook coho with the exception of the samples taken in 2003. Additionally,

clinical examination of the Hinchenbrook, but not the Ml-adapted, strain revealed

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the presence of signs characteristic of BKD. Statistical analysis showed statistical
difference (P< 0.05) increases in both prevalence and intensity of R.
salmoninarum over the years in the Hinchenbrook strain as compared to the MI-

adapted strain. Moreover, data showed no statistically significant differences

between males and females from both weirs

2. Renibacterium salmoninarum prevalence and Intensity in ovarian
fluid and milt of returning salmon spawners. The shedding of R.
salmoninarum with the gametes was tested in 2004 returning spawners. As
displayed in tables 12, 13 and 14, R. salmoninarum and its soluble antigens were
passed along with the gametes of both sexes albeit not in all of the infected fish.

a. Chinook salmon returning spawners. While 28/60 (47%) LMRW female
salmon were infected; only 6 of these fish had R. salmoninarum antigens in their
ovarian fluids (table 13). Similarly, only half of the infected SRW females
exhibited R. salmoninarum antigens in their ovarian fluids. The same trend was
noticed in the LMRW males, with only 20% of infected fish had R. salmoninarum
antigens in their milt.

Table 13 summarizes the difference in prevalence between kidneys and
gametes of the same fish. More than 80% of the fish produced R.
salmoninarum-free gametes (sum of groups 1 and 2 in table 13), even though
some of these fish had R. salmoninarum in their kidneys. Indeed, a few of these
fish exhibited medium and high R. salmoninarum antigen titers in the kidneys. In

the case of LMRW males, the concentrations of R. salmoninarum soluble

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antigens in milt as determined by Q-ELISA coincided with that found in the

kidney tissues in all individual fish. Similar findings were observed in the LMRW,
with the exception of females# 5 and 29, which had very high R. salmoninarum
antigen concentrations in the ovarian fluid, while their kidney had low antigen
concentrations. Another LMRW female (# 43) shed the R. salmoninarum antigen
in a very low level in the ovarian fluid, while the kidney tissues tested negative. A
similar trend was noticed with the SRW chinook salmon, where 18 females shed
the R. salmoninarum antigen in the ovarian fluid, while the majority of females
exhibited a comparable titer of R. salmoninarum antigens in both ovarian fluids
and kidneys. Five fish (1.8%) shed the antigens in the ovarian fluids while their
R. salmoninarum antigen in their kidney was below the detection level (Group 4
in table 13).

b. Coho salmon returning spawners. Although 24.2% of the MI adapted
coho strain was infected, only 11.7 % of these fish had R. salmoninarum
antigens in their ovarian fluids. Similarly, while 72 % of the MI adapted coho
strain was infected, only 50 % of these fish had R. salmoninarum antigens in
their ovarian fluids (table 12). The same trend was noticed in the MI adapted
coho males, with only 5% of infected fish having R. salmoninarum antigens in
their milt samples. However, results demonstrated that the Hinchenbrook coho
males shed more R. salmoninarum and its antigens (55 %) in milt than that of the
MI adapted strains (5 %) (tables 12 and 14). The same trend was noticed when
comparing the prevalence and intensity of R. salmoninarum antigens shed along

with ovarian fluid of females of both strains (tables 12 and 14).

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3. Renibacterium salmoninarum prevalence and intensity in kidney
tissues of hatchery-raised juvenile fingerlings. The prevalence of BKD in
juvenile spring salmon (originally hatched from the fall returning spawner
parents) raised in three hatcheries was followed for four consecutive years
(2002-2005; tables 15-16 and Figure 11). In general, R. salmoninarum
prevalence and intensity infection were lower in offspring when compared to
parent stocks.

a. Chinook salmon juvenile fingerlings. In case of chinook salmon, this
relationship was not present when the results of R. salmoninarum testing were
grouped by individual hatchery. However, offspring raised in certain hatcheries
tended to exhibit a consistent pattern. For example, chinook salmon fingerlings
raised in Thompson state fish hatchery (THSF H) exhibited the lowest prevalence
and intensity of R. salmoninarum in kidneys when compared to their cohorts
raised in other hatcheries. Year to year comparison, however, showed that over
the years, R. salmoninarum prevalence has declined in the other two hatcheries
as well. For example, R. salmoninarum prevalence in spring chinook salmon
fingerlings, originally spawned at LMRW and then raised in WLSFH, declined
from 100% in 2002 to 5% in 2005 (table 15). Similarly, the prevalence of spring
chinook salmon originally spawned at LMRW and raised at PRSFH declined from
100% in 2003 to 0% in 2004 and 5% in 2005. Likewise, spring chinook salmon
originally spawned from SRW parents and hatched in WLSFH showed a decline

in BKD prevalence from 35% in 2004 to 10% in 2005 (table 16). Sharp decreases

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in BKD prevalence was also noticed in the spring Chinook salmon from PRSFH
between 2003 and 2004 (92% vs. 0%) before slightly increasing to 8.3% in 2005.
However, statistical analysis revealed non-significant differences in prevalence
and intensity of infection when year-to-year comparison or hatchery-to-hatchery
comparisons were adopted.

b. Coho salmon juvenile fingerlings. The prevalence of BKD in spring
coho salmon juvenile fingerlings raised in the Platte River State Fish Hatchery
was followed up for three consecutive years (2003-2005). In general, R.
salmoninarum prevalence and intensity was lower in offspring when compared to
parent stocks (Figure 11). Year to year comparison, however, showed that over
the years, R. salmoninarum prevalence has steadily declined in the two coho
salmon strains. For example, R. salmoninarum prevalence in MI adapted coho
salmon fingerlings, originally spawned from MI adapted coho salmon spawners
at PRW and then raised in PRSFH, declined from 50 % in 2003 to 27 % in 2004
ending with 6.7 % in 2005. The same trend was noticed among the
Hinchenbrook coho fingerlings raised at the same hatchery in which the
prevalence was steadily decreased from 75 % in 2003 to 22 % in 2004 ending
with slight decline to 20 % in 2005. Comparing the R. salmoninarum prevalence
in the two strains of hatchery-raised coho salmon to that of their corresponding
parents demonstrated that fingerlings follow a statistically significant (P<0.05)
decline in the 2004 and 2005 samples of both strains. Data also demonstrated

that Hinchenbrook coho salmon fingerlings possessed statistically significant

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higher R. salmoninarum prevalence (P<0.05) than the Ml-adapted coho strain

through the 3 years testing period.

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DISCUSSION

Data generated in this study provide additional evidence that R.
salmoninarum infection is enzootic in Michigan and widespread in feral stocks of
feral spawner salmon in both lakes Michigan and Huron. As displayed in Figure
7, Lake Michigan’s Chinook salmon tends to have an overall higher prevalence
than that observed in Lake Huron’s Chinook salmon. In the case of SRW chinook
salmon, prevalence was much lower and intensity was relatively higher when
compared to the LMRW salmon. The stocking of chinook salmon in Lake Huron
is a relatively new event that started in the 19903, unlike in Lake Michigan, where
chinook salmon have been stocked since 1967 (Dexter and O’Neal, 2004). The
recent Chinook salmon introduction in Lake Huron has most likely contributed to
the relatively low R. salmoninarum infection prevalence observed in this study.
Renibacterium salmoninarum infection prevalence and intensity in returning feral
chinook salmon seem to fluctuate among years, although a reliable decrease
can be observed since the start of this study in 2001. However, the unexpected
surge in prevalence recorded in the 2004-LMRW salmon is alarming. A similar
increase in R. salmoninarum prevalence was observed in the Lake Michigan
stock of Hinchenbrook coho salmon, a strain of coho salmon that is especially
susceptible to R. salmoninarum. Further studies are needed in forthcoming

years to monitor R. salmoninarum infection levels in Lake Michigan salmon

stocks.

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Although Ml-adapted and Hinchenbrook strains belong to the same species
and residing in the same watershed, they exhibited marked differences in R.
salmoninarum prevalence and intensity. There are no published reports on R.
salmoninarum infection in the Hinchenbrook strain from New York, from which it
was imported, yet this strain is known in New York for its marked growth, high
survival and return rates (John Shachte, personal Communication). The
susceptibility of the Hinchenbrook strain to R. salmoninarum may be related to its
recent introduction into Lake Michigan where BKD is enzootic. The decrease in
R. salmoninarum prevalence over the years is a good indication of a beginning of
adaptation of the strain to its new environment. When the Ml-adapted strain was
first introduced from the Pacific Northwest, BKD-related epizootics were reported
along the shores of Lake Michigan (MacLean and Yoder, 1967). Adaptation in R.
salmoninarum-contaminated environments has been reported in the Arctic char
and the brown trout in Iceland (Jonsddttir et al. 1998). Genetic make up of the
two strains may have also played a role in the difference in R. salmoninarum
susceptibility. Winter et al. (1980) reported the presence of genetic basis for
differences in susceptibility to R. salmoninarum among of steelhead trout strains.
Further, Beacham and Evelyn (1992) also reported the presence of differential
susceptibility to R. salmoninarum in a number of chinook salmon strains. Other
important factors that may have contributed to differences between the two

strains is their diverse migration patterns in Lake Michigan (Dexter and O’Neal,

2004).

114

Based only on the data of this study, it is difficult to determine whether such
high levels of R. salmoninarum are associated with BKD-related mortalities. The
eruption of BKD in R. salmoninarum infected fish is influenced by a number of
factors including changes in habitat and forage fish availability. As reported from
other fish species and pathogens, changes in the spatial distribution of fish and
increased density of the fish population can affect the frequency of contact
between infected and non infected migrating fish which will be translated to rapid
spread and progression of infection (Reno, 1998). For example, the 1989 BKD
epizootic in chinook salmon was associated with a sharp decline in forage fish
availability (Holey et al.1998).

Combining the results of 2002 and 2004 kidney Q-ELISA data (Figure 9),
males tend to have an equal or slightly higher R. salmoninarum prevalence than
females. In the North Pacific USA, where much of Chinook salmon BKD data
has been generated, females seem to have a much higher R. salmoninarum
prevalence and intensity than males (Evelyn et al. 1986). Most of the studies,
however, were performed using experimental infection models.

The gradual decrease of prevalence and intensity among both chinook and
coho salmon coho salmon spawners could be due to the culling procedures
adopted in weirs which could minimized the possibility of vertical transmission
through infected females to their offspring. Also, the application of strict hygienic
measures in weirs by rinsing the spawner fish in iodophores (Ross and Smith
1972) before collecting eggs or milt, using sterile utensils for egg-milt mixing and

storage could have minimized the bacterial load transmitted to eggs and milt in

115

weirs. Moreover, efficient weir practices such as incubating fertilized eggs in
clean disinfected trays and incubators as well as hardening of eggs in 2-ppm
erythromycin solution (Amos 1977), could have reduced the prevalence and
intensity of BKD in the production fish before their release into the Platte River
(Maule et al.1996).

Results indicated that the 2003 Hinchenbrook coho salmon fingerlings
showed higher BKD prevalence (75%) than that of MI-adapted coho (50 %),
which appears consistent with the high R. salmoninarum prevalence results of
their 2001 parents. As demonstrated by Evelyn et al. (1986), the likelihood of
vertical transmission in heavily infected salmon is higher than in lightly infected
gamete donors. Moreover, data showed that female MI-adapted coho salmon
feral spawner showed slightly higher R. salmoninarum prevalence than males of
the same strain in the 2002 and 2004 samples. Female coho salmon are usually
stressed with the relatively large size of their egg-laden ovaries that occupy more
than 70 % of the fish body during the spawning season.

Interestingly, the data presented in this study demonstrates the ability of feral
spawner chinook and coho females to vertically transmit the bacterium through
the ovarian fluid (tables 12-13). This fluid surrounds the eggs once they have
been released into the body cavity following ovulation (Evelyn et al. 1986).

Most of the chinook spawner females that shed R. salmoninarum in their
ovarian fluid tested positive in the kidneys (table 13). Only in <2% of the chinook
females, R. salmoninarum was shed in the ovarian fluid, but not in the kidneys.

There are three explanations for the appearance of this phenotype. First,

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kidneys are infected in levels that are below the Q-ELISA detection limit.

Second, the fish were recovered from a prior R. salmoninarum infection, which
left remnants of R. salmoninarum-ECP that are known to be excreted in a slow
fashion (Sami et al. 1992). Indeed, R. salmoninarum antigen levels in these fish
were extremely low. Last, kidney samples may have given false negative results.
Most important, however, was the fact that over 90% of chinook salmon tested in
this study from both watersheds were not shedding R. salmoninarum antigens in
the ovarian fluid, which is much less than originally thought. As a result, high
levels of R. salmoninarum prevalence and intensity in hatchery fingerlings cannot
be attributed to vertical transmission alone. Moreover, the slow multiplication of
R. salmoninarum would not allow the infection to develop in < 6 month old
juvenile chinook salmon fingerlings to levels such as those observed In the
fingerlings of 2002 and 2003. Therefore, it is likely that horizontal transmission
plays a role in spreading infection among hatchery-propagated fish. There is no
doubt, however, that the exposure of offspring to the immunosuppressive R.
salmoninarum-ECP can predispose the fish to infection with R. salmoninarum or
other pathogens.

Data also demonstrated that over half of Hinchenbrook females and males
shed R. salmoninarum along with the gametes as opposed to 12% in females
and 5% males of the Ml-adapted strain. This extreme difference can account for
the higher prevalence and intensity of R. salmoninarum in the Hinchenbrook
strain as compared to the Ml-adapted strain. On the contrary, a higher percent of

infected adult Hinchenbrook coho salmon did not shed R. salmoninarum despite

117

the high levels of R. salmoninarum intensity in their kidneys (Group 2 in table 13).
By combining fish in Groups 1 and 2 of table 14, it became clear that a very high
percent of coho salmon shed R. salmoninarum-free gametes, a matter that may
lead to a reduction of R. salmoninarum infection incidence in the future. Group 4
on the contrary, are fish that shed R. salmoninarum antigen along with the
gametes despite their non-detectable R. salmoninarum levels in the kidneys.
Group 4 and group 3 are probably the cause of the continuous presence of R.
salmoninarum across generations. High portions of the Hinchenbrook females
shed R. salmoninarum in their coelomic fluid were tested positive in the kidneys.
Only in <2% of the Hinchenbrook and MI adapted coho females, R.
salmoninarum shed in the coelomic fluid, but not in the kidneys.

It has also been demonstrated that males shed R. salmoninarum in their milt,
sometimes in high concentrations, and thereby may contribute to the spread of
R. salmoninarum into eggs through the micropyle during fertilization. In addition,
the potential exposure of the embryo to immunosuppressive R. salmoninarum-
ECP from the milt cannot be ignored (Hamel 2001). It should be noted that only
10 out of 53 infected LMRW males shed R. salmoninamm antigen in their milt,
while the remaining males were able to keep their seminal plasma below the Q-
ELISA detection limits. There is a handful of papers that addressed the role of
males in the transmission of R. salmoninarum in chinook and coho salmon. The
studies of Evelyn et al. (1986) and Rocky et al. (1991) suggested that the role of
males in infecting eggs is minimal. Data of this study, however, contradicts other

reports and demonstrates that males are capable of shedding R. salmoninarum

118

more than females of the same stock. Further, we strongly recommend that the
testing and culling policy, currently applied to females only, be expanded to
include males.

The BKD testing of both feral spawners and their hatchery raised juvenile
fingerlings demonstrates that the current testing and culling programs have been
partially successful in reducing R. salmoninarum transmission. This study also
demonstrates that some hatchery practices were more effective than others in
controlling the spread of R. salmoninarum and that a reliable improvement in the
reduction of R. salmoninarum prevalence within the three hatcheries involved in
this study has been achieved. The aforementioned assumption coincided with
that of Maule et al. 1996 who reported variations in R. salmoninarum prevalence

among hatcheries in fingerlings derived from the same stock.

119

 

Salmon species and source Date collected Number of

 

 

fish tested
Chinook salmon spawners October, 2001 60
from LMRW October, 2002 63

 

October, 2003 60
October, 2004 120

 

 

 

 

 

 

 

 

 

 

Chinook salmon spawners October, 2002 59
from SRW October, 2003 34

October, 2004 560
Michigan adapted coho October, 2001 38
salmon spawners from October, 2002 165
PRW October, 2003 60

October, 2004 120
Hinchenbrook coho salmon October, 2001 49
spawners from PRW October, 2002 56

 

October, 2003 48
October, 2004 120

 

 

 

 

 

 

Table 10. Details of samples collected from spawning chinook and coho

salmon returning to egg-take weirs throughout the period from 2001-2005.

LMRW: Little Mansitee River Weir
SRW: Swan River Weir
PRW: Platte River Weir

120

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Rearing Date Egg-take Weir Number
hatchery Collected of fish
tested
Wolf Lake State March 2002 Little Manistee River 60
Fish Hatchery Weir
March 2003 Little Manistee River 60
Weir
March 2004 Little Manistee River 60
Weir
Swan River Weir 60
March 2005 Little Manistee River 60
Weir
Swan River Weir 60
Platte River March 2002 Little Manistee River 65
Weir State Fish Weir
Hatchery March 2003 Little Manistee River 60
Weir
Swan River Weir 60
March 2004 Little Manistee River 60
Weir
Swan River Weir 60
March 2005 Little Manistee River 60
Weir
Swan River Weir 60
Thompson March 2003 Swan River Weir 60
State FISh March 2004 Swan River Weir 60
Hatchery March 2005 Swan River Weir 60
Total 965

 

 

 

 

 

Table11. Chinook salmon juvenile fingerlings collected from Michigan

state fish hatcheries in the period from 2002-2005.

121

 

 

 

 

 

 

 

 

 

 

Fish species # of tested body Renibacterium % Q-ELISA positive titers
and source fluid samples salmoninarum High Medium Low
prevalence
Chinook 60 ovarian Fluid 6/60 1/60 1/60 4/60
salmon form (10 %) (1.5 %) (1.5 %) (7 %)
LMRW so Milt 10/60 8/60 2/60 0/60
(63.5 %) (13 %) (3 %) (0%)
Chinook 280 ovarian Fluid 18/280 11/280 2/280 5/280
salmon from (6.4 %) (4 %) (0.7 %) (1.78
SRW %)
Michigan 60 ovarian Fluid 7/60 5/60 1/60 1/60
adapted coho (11.7 %) (8.3 %) (1.7 %) (1.7
salmon from %)
PRW 60 Semen 3/60 0/60 0/60 3/60
(5 %) (0 %I (0 %l (5 %)
Hinchenbrook 60 ovarian Fluid 30/60 15/60 3/60 12/60
coho salmon (50 %) (25 %) (5 %) (20
from PRW %)
60 Semen 33/60 4/60 9/60 20/60
(55 %) (6.7 %) (15 %) (33.3
%)

 

 

 

 

 

 

 

 

Table 12. Renibacterium salmoninarum prevalence and intensity in ovarian

fluid and milt of the 2004 chinook and coho salmon spawners.

Data in this table was generated using a polyclonal antibody-based quantitative ELISA (Q-ELISA)
performed on kidney tissues. Prevalence was determined by % of Q-ELISA positive samples of
the total number of samples tested. Intensity was considered high if the antigen concentration 21;
medium if the antigen concentration = 0.20-0.99; and Low if the antigen concentration = 0.10 to

0.19.

SRW: Swan River Weir.
PRW: Platte River Weir.

122

 

 

 

 

 

 

 

 

 

 

 

Fish Group LMRW SRW
Females Males Females
1. Kidneys 8. Gametes negative 28/60 7/60 240/280
(46.7%) (11.7%) (85.7%)
2. Kidneys + & Gametes - 26/60 43/60 22/280
(43.3%) (71.7%) (7.9%)
3. Kidneys + & Gametes + with 5/60 10/60 13/280
same Renibacterium salmoninarum (8.3%) (16.6%) (4.6%)
anfigenlevel
4. Kidneys - & Gametes + with very 1/60 0/60 5/280
low Intensity (1.6%) (0 %) (1.8%)

 

Table 13. Renibacterium salmoninarum prevalence in kidney and gametes

of the 2004 chinook salmon spawners.

Data in this table was generated using a polyclonal antibody-based quantitative ELISA (Q-ELISA)
performed on kidney tissues. Prevalence was determined by % of Q-ELISA positive samples of
the total number of samples tested. Intensity was considered high if the antigen concentration 21;
medium if the antigen concentration = 0.20-0.99; and Low if the antigen concentration = 0.10 to

0.19.

123

 

 

 

 

 

 

 

 

Fish Group Hinchenbrook Michigan Adapted
Females Male Females Male
1. Kidneys 8| Gametes 10/60 15/60 40/59 45/60
"99mm (17%) (25%) (68%) (75%)
2. Kidneys + 8. 17/60 15/60 11/59 11/60
Gama” ' (28%) (25%) (19%) (18%)
3. Kidneys + 8. 32/60 25/60 6/59 1/60
$2,323,321? ’3'“ (53%) (42%) (10%) (2%)
salmoninarum antigen
level
4. Kidneys - 8. 1/60 5/60 1/59 2/60
3:133: :it“; "h my (2% ) (8%) (2%) (3%)

 

 

 

 

 

 

 

Table 14. Renibacterium salmoninarum prevalence in Kidney and gametes
of the 2004 coho salmon spawners (Shedding agreements possibilities

between kidney and gametes).

Data in this table was generated using a polyclonal antibody-based quantitative ELISA (Q-ELISA)
performed on kidney tissues. Prevalence was determined by % of O-ELISA positive samples of
the total number of samples tested. Intensity was considered high if the antigen concentration a;
medium if the antigen concentration = 0.20-0.99; and Low if the antigen concentration = 0.10 to
0.19.

124

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Gamete Donors Fingerlings tested 8 month following
Fish e -take
strain Year Renibacterium Year Hatchery Renibacterium
salmoninarum salmoninarum
prevalence prevalence
Little 2001 81.7 % Total 2002 PR 62.0 % Total
Manistee 43.3 % high 0.0 % high
Chinook 8.3 % medium 0.0 % medium
30.0 % low 62.0% low
WL 100.0 % Total
100.0 % high
0.0 % medium
0.0 % low
2002 63.5 % Total 2003 PR 100.0 % Total
0.0 % high 17.0 % high
6.3 % medium 8.0 % medium
57.1 % low) 75.0 % low)
WL 92.0 % Total
0.0 % high
50.0 % medium
42.0 % low
2003 20.0 % Total 2004 PR 0.0 % Total
1.7 % high 0.0 % high
1.7 % medium 0.0 % medium
16.7 % low 0.0 % low)
WL 35.0 % Total
2.0 % high
6.7 % medium
27.0 % low
2004 67.0 % Total 2005 PR 5.0 % Total
2.5 % high 0 % high
7.5 % medium 0 % medium
57.0 % low 5 % low)
WL 10.0 % Total
0.0 % high
0.0 % medium
10.0 % low

 

 

 

 

Table 15. Renibacterium salmoninarum infection prevalence and intensity
among the Little Manistee River chinook salmon feral spawners and their

corresponding hatchery raised fingerlings throughout the period from 2001

to 2005.

Data in this table was generated using a polyclonal antibody-based quantitative ELISA (Q-ELISA)
performed on kidney tissues. Prevalence was determined by % of Q-ELISA positive samples of
the total number of samples tested. Intensity was considered high if the antigen concentration 21;

125

 

medium if the antigen concentration = 0.20-0.99; and Low if the antigen concentration = 0.10 to
O. 1 9.

Note: PR (Platte River State Fish HatcherY). WL (Wolf Lake State Fish Hatchery). TH (Thompson
State Fish Hatchery).

126

 

Fish
strain

Gamete Donors

Fingerlings tested 8 month following
eggiake

 

Year Renibacterium
salmoninarum
prevalence

Year Hatchery Renibacterium
salmoninarum
prevalence

 

Swan
River
Chinook

2002 45.7 % Total

5.0 % high

1.7 % medium
39.0 % low

2003 PR 92.0 % Total

0.0 % high
50.0 % medium
42.0 % low

 

TH 0.0 % Total

0.0 % high
0.0 % medium
0.0 % low

 

2003 35.3 % Total
2.9 % high
0.0 % medium
32.3 % low

2004 PR 0 % Total

0.0 % high
0.0 % medium
0.0 % low

 

 

WL 35.0 % Total
3.3 % high
0.0 % medium
31.7 % low

 

TH 0.0 % Total

0.0 % high
0.0 % medium
0.0 % low

 

2004 13.7 % Total

4.1 % high
1.6 % medium
8.0 % low

2005 PR 8.3 % Total

0.0 % high
0.0 % medium
8.3 % low

 

WL 10.0 % Total

0 % high

1.7 % medium
8.3 % low

 

TH 2.0 % Total

0.0 % high
0.0 % medium
2.0 % low

 

 

 

 

 

 

 

 

Table 16. Renibacterium salmoninarum infection prevalence and intensity
among the Swan River Weir chinook salmon feral spawners and their
corresponding hatchery raised fingerlings throughout the period from 2001

to 2005.

Data in this table was generated using a polyclonal antibody-based quantitative ELISA (Ct-ELISA)
performed on kidney tissues. Prevalence was determined by % of Q-ELISA positive samples of
the total number of samples tested. Intensity was considered high if the antigen concentration a;

127

 

medium If the antigen concentration = 0.20-0.99; and Low if the antigen concentration = 0.10 to
0.19.

Note: PR (Platte River State Fish Hatchery), WL (Wolf Lake State Fish Hatchery), TH (Thompson
State Fish Hatchery).

128

 

Little Manistee River Weir

100%-

 

 

 

 

 

 

 

8 80%r
I:
% 60%-
5
E 40%-
20%
ml D Low
2001 2002 2003 2004 I Medium
I th
100%. Swan River Weir
80%-
at
O
5 50%-
E 40°/
0.
E
20%.
0%.
2001 2002 2003 2004

Figure 7. Prevalence of Renibacterium salmoninarum in feral chinook
salmon spawners collected from two of Michigan weirs between 2001 and

2005.

Data in this Figure was generated using a polyclonal antibody—based quantitative ELISA (Q-
ELISA) performed on kidney tissues. Prevalence was determined by % of Q-ELISA positive
samples of the total number of samples tested. Intensity was considered high if the antigen
concentration 21; medium if the antigen concentration = 0.20-0.99; and Low if the antigen
concentration = 0.10 to 0.19.

129

 

Michigan Adapted Coho salmon

60% [3 Low
5”“ I Medium l
I High

 

Prevalence
s
:1

 

2001 2002 2003 2004

Hinchenbrook Coho salmon

 

Prevalence

 

2001 2002 2003 2004

Figure 8. Renibacterium salmoninarum prevalences and intensities in feral
spawner, coho salmon strains returning to Platte River Weir throughout

2001-2004

Data in this Figure was generated using a polyclonal antibody-based quantitative ELISA (Q-
ELISA) performed on kidney tissues. Prevalence was determined by % of Q-ELISA positive
samples of the total number of samples tested. Intensity was considered high if the antigen
concentration 21; medium if the antigen concentration = 020-099; and Low if the antigen
concentration = 0.10 to 0.19.

130

 

Little Manistee River Weir

. , . 1
..
I ’7‘ ( a“ I

3’ A’s 436 '6’»

4”?

100

     

Prevalence

 

 

 

qr D Low
I Medium
I I-Igh

 

 

 

100,, Swan River Weir

Prevalence

20%1
1096a

 

 

Figure 9. Prevalence of Renibacterium salmoninarum in feral chinook
salmon spawner males and females collected from two of Michigan weirs
between 2001 and 2005.

Data in this Figure was generated using a polyclonal antibody-based quantitative ELISA (Q-
ELISA) performed on kidney tissues. Prevalence was determined by % of Q-ELISA positive
samples of the total number of samples tested. Intensity was considered high if the antigen
concentration 21; medium if the antigen concentration = 0.20—0.99; and Low if the antigen
concentration = 0.10 to 0.19.

131

Michigan Adapted Coho salmon

Prevalence

 

56
Female-02 Male«02 Female-04 Male-04

I High I Medium El Low

Hinchenbrook Coho salmon

too»
90%4
80%
70%
80%
50%
40%
30%-
20%
10921

A.

      
 

.
-

Prevalence

 

0]. 4
Female-02 Male-02 Female-04 Male-04

Figure 10. Renibacterium salmoninarum prevalence and intensity in
females and males of coho salmon spawners returning to Platte River Weir
throughout the period from 2002-2004.

Data in this Figure was generated using a polyclonal antibody-based quantitative ELISA (Q-
ELISA) performed on kidney tissues. Prevalence was determined by % of Q-ELISA positive
samples of the total number of samples tested. Intensity was considered high if the antigen
concentration 21; medium if the antigen concentration = 0.20-0.99; and Low If the antigen
concentration = 0.10 to 0.19.

132

Michigan Adapted Coho salmon

Prevalence

 

 

 

[- High a Medium 1:) Low ]

 

   

Hinchenbrook Coho salmon

Prevalence
s
a!

0 A 0 '° 0 ‘°
4) 4) 4)
“to.“ “2% “5% “a. “a “e
e, 0.4, g, ‘4 “v, $0.4,

Figure 1 1. Renibacterium salmoninarum prevalence and intensity of
infection in gamete donor feral spawner coho salmon strains versus their
corresponding 18-month juvenile fingerlings throughout 2001-2004.

Data in this Figure was generated using a polyclonal antibody-based quantitative ELISA (Q-
ELISA) performed on kidney tissues. Prevalence was determined by % of Q-ELISA positive
samples of the total number of samples tested. Intensity was considered high if the antigen
concentration 21; medium if the antigen concentration = 0.20-0.99; and Low if the antigen
concentration = 0.10 to 0.19.

133

 

 

CHAPTER FIVE

PREVALENCE, SHEDDING AND SPREAD OF RENIBACTERIUM
SALMONINARUM IN BROOK TROUT (SAL VELINUS FONTINALIS) WITH
SPECIAL EMPHASIS ON THE ASSOCIATED DISEASE EPIZOOTICS IN
MICHIGAN.

ABSTRACT

In order to determine the status of BKD in hatchery and wild populations of
brook trout in Michigan, (Renibacterium salmoninarum) R. salmoninarum
prevalence and intensity were detennined in representative samples from adult
hatchery raised and wild stocks as well as their offsprings from 2001 through
2005. The hatchery raised adult Iron River brook trout presented higher BKD
prevalence than the wild Cherry Creek strain. Generally, the BKD prevalence and
intensity in hatchery and wild brook trout strains gradually decreased throughout
the period from 2001 to 2004. The critical role played by hatchery practices to
control the spread and minimize the prevalence of BKD among Michigan brook
trout populations was discussed. Although most of the previous studies reported
an insignificant male role in transmission of Renibacterium salmoninarum (R.
salmoninarum), yet our results clearly demonstrated that males shed along with
their gametes more R. salmoninarum than females.

Number of BKD outbreaks among hatchery-reared brook trout populations

has increased dramatically throughout the period of late spring of 2003 to the

134

early fall of 2004. The affected brook trout populations have been assessed
using various diagnostic tools, such as clinical examination, Q-ELISA, culture,
histopathology, and immunohistochemistry. The possible causes that lead to the
initiation and progression of such outbreaks have been fully investigated.
Although Q-ELISA, nested PCR, and culture results for all of the examined cases
were positive, some of these cases were either barely within detection limits or
demonstrated negative results in histopathological and immunohistochemical
examination. This result is not surprising, as some of the specimens were
preserved in formalin for a relatively long period (i.e. enough to affect the IHC
power) even after performing improved antigen retrieval methods. Also,
representative samples of 3-year-old broodstock exhibiting chronic forms of the
disease had meager amounts of antigen remaining within their system, with trace
amounts being present in the antigen antibody complexes in granulomas that
occupied large portions of the kidney tissues. These were sequentially

presented as a very light antigen score using the IHC (1+).

135

INTRODUCTION

Brook trout (Salvelinus fontinalis) is an indigenous salmonid species in the
Great Lakes (Coon 1999) that has been artificially propagated and stocked in
Michigan’s public waters for years (Dexter and O’Neal, 2004). Two strains of
brook trout, the Assinica and Iron River strain, are being used by used by
Michigan Department of Natural Resources (MDNR) for supplementing resident
stream populations where there is a deficiency of natural recruitment (Dexter and
O’Neal, 2004). Assinica and Iron River brook trout are the two main strains of
brook trout that are being reared and stocked in Michigan. Assinica brook trout
are characterized by better survival and growth than domestic stocks (Gowing
1986). Iron River Brook trout are considered a pure native strain that is originally
from the Iron River in Michigan’s Upper Peninsula (Driver 1995). Unlike the
Assinica strain, Iron River brook trout are slow to reach maturity and are
characterized by a very slow growth rate because of their wild characteristics
(Dexter and O’Neal 2004).

Fish health poses major challenges to development and progress of the
brook trout restoration in the Great Lakes basin. Among these health challenges,
Bacterial Kidney Disease, caused by Renibacterium salmoninarum (R.
salmoninarum), is an eminent threat due to the enzootic nature of the pathogen
within Great Lakes waters (Eissa 2005-Chapter 3). Moreover, affinity of R.
salmoninarum for the kidneys, which possesses an essential lymphoid function

and its obligate intracellular nature, makes this pathogen and its soluble antigens

136

a major threat to the host by suppressing the fish immune system (Ellis 1999;
Fredriksen et al. 1997; Grayson et al. 2002).

A considerable number of studies have been performed on brook trout in the
USA which indicated that brook trout is the most susceptible salmonid to BKD
(Belding and Merrill 1935; Snieszko and Griffin 1955). However, most of these
studies involved the use of experimental infection, while a relatively few were
concerned with natural BKD infection (Allison et al. 1958; Mitchum et al. 1979;
Mitchum and Sherman 1981). For example, Bullock et al. (1971 ); Mitchum and
Sherman (1981) reported that brook trout mortalities from BKD are higher than
that of the brown trout.

Interestingly, the first report of BKD in the USA occurred in brook trout at a
Massachusetts State fish hatchery (Belding and Merrill, 1935). During the late
19403 and early 19503, R. salmoninarum infection caused mass mortalities in
brook trout at the federal hatcheries in Berlin, New Hampshire, Cortland, and
New York (Snieszko and Griffin, 1955). Mitchum et al. (1979) determined the
prevalence of BKD in dead and live brook trout collected from a small lake and
stream system in southeastern Wyoming, USA, where BKD epizootics had been
observed since 1972. They found that prevalence among dead fish and live fish
at upstream stations was 100% and 83%, respectively.

In Michigan, the first case of BKD was discovered in 1955 in brook trout
yearlings at the Oden and Marquette state hatcheries, where eggs were originally
imported from a hatchery in New England in which BKD had been endemic for

many years (Allison, 1958). Since the first report of the disease in 1955, none of

137

the published studies have reported any data about the recent occurrence of
BKD outbreaks, prevalence, or magnitude of the disease in brook trout in
Michigan or in the Great Lakes basin.

Thus, the aim of the current research was to investigate the status and
magnitude of BKD in brook trout in Michigan by assessing the R. salmoninarum
prevalence in the hatchery raised brook trout populations in Michigan. The role-
played by the male and the shedding of the bacteria along the gamete are also
investigated. Moreover, a number of recently erupted BKD outbreaks among

brook trout populations in Michigan were also investigated.

138

MATERIALS AND METHODS

Fish. To investigate the prevalence and intensity of R. salmoninarum
infection among brook trout (BKT) populations in Michigan waters, a total of 628
adult brook trout were collected from the hatchery raceways of Marquette State
fish hatchery (MSFH) and the Cherry Creek water stream outside the hatchery in
Marquette, Michigan, from 2001-2004. MSFH is the primary facility for brook trout
broodstock that are used for the production of fingerlings to be stocked in both
inland and Great Lakes waters.

Kidney tissue samples were collected from a total of 567 hatchery-raised
brook trout broodstocks (529 Iron River strain (IR-BKT) sample, 38 Assinica
strain (AS-BKT) sample) and 61 adult Cherry Creek wild brook trout (CC-BKT)
(Table17). Following approximately 18 months of egg incubation and fish rearing
in the Marquette State Fish Hatchery, MDNR releases fingerlings in the spring of
each year. Between 2002-2005, a total of 420 kidney tissues of pre-stocking
fingerlings were collected and analyzed for the presence of R. salmoninarum
prior to release into the basins of Lakes Michigan (table 17).

The sacrifice of broodstock was accomplished by exposing the fish to an
overdose of MS-222 (tricaine methane sulfonate, Finquel- Argent Chemical
Laboratories, Redmond, WA). Following gamete collection, the abdominal cavity
was cut open to examine individual intemal organs for signs of BKD, followed by

collecting about one gram of tissues from anterior, posterior and middle kidney

139

sections. Attention was given not to cross contaminate samples and dissecting
tools were replaced with sterile ones following dissection of each fish.
Fingerlings were sacrificed by immersion in an overdose of MS-222. In order to
ensure that the sample is representative, as much tissue as possible was
harvested from each kidney using sten'le dissection tools. A total of 200 coelomic
fluid samples were collected using sterile transfer pipette from the egg/ovarian
fluid mixture, transferred to 5 ml sterile polypropylene tubes, and kept on ice until
processed at the laboratory at Michigan State University, East Lansing, MI. Also,
a total of 200 semen samples were collected directly from the middle stream of
semen into sterile 10-ml polypropylene tubes. Fingerlings were sacrificed by
immersion in an overdose of anesthesia using MS-222 (tricaine methane
sulfonate, Finquel- Ardent Chemical Laboratories, Washington).

A number of BKD outbreaks of involving brook trout were recorded between
2003 and 2004. In late May of 2003, a total of 72 Iron River brook trout
fingerlings with a recent history of mortalities were submitted to the Aquatic
Animal Health Laboratory (AAHL) at Michigan State University from the
Marquette State Fish Hatchery (MSFH) in Michigan. A course of treatment was
prescribed and then followed by the collection of another 60 samples for post
treatment analysis. In Mid July 2004, a total of 9 adult three year-old Assinica
brook trout were submitted to the AAHL from the MSFH during an onset of
broodstock losses. In Mid September of 2004, a total of 30 brook trout yearlings
were submitted to the AAHL from Cedarbrook Fish Farm in Harn’sville, Michigan

that were experiencing increased mortalities.

140

Clinical examination. Fish were euthanized using an overdose of MS 222
(tricaine methane sulfonate) (Finquel- Argent Chemical Laboratories,
Washington). Fish were then externally examined for the presence of any
lesions, parasites, or abnormal growths on the skin or gills. Fish were dissected
and examined internally for any lesions, swelling, or color changes in the kidneys

other internal organs and viscera.

Sample processing. Kidney samples representing the anterior, posterior
and middle sections of the kidney were transferred in sterile 7.5 cm x 18.5 cm
Whirl Pak® bags (Nasco, Forte Atkinson, and WI), kept on ice, and were softened
as much as possible through multiple cycles of physical pressure. The
homogenized kidney tissues were diluted in 1:4 (w/v) Hank’s Balanced Salt
Solution (HBSS, Sigma Chemical Co, St. Louis, MO) then stomached for 2
minutes at high-speed in a Biomaster Stomacher-80 (Wolf Laboratories Limited,
Pocklington, York, UK). In the case of ovarian fluid, 1 ml from fluid sample was
diluted 1:2 (v/v) in HBSS for Q-ELISA testing. In the case of milt samples, 1ml

from each semen sample was diluted 1:2 (v/v) in HBSS for Q-ELISA testing.

Measurements of Renibacterium salmoninarum antigen using the
Quantitative Enzyme-linked Immunosorbent Assay (Q-ELISA). Aliquots of
250 pl volume of each samples were transferred into 1.5 ml safe-lock microfuge

tube to which an equal volume of 0.01 M phosphate buffered saline-Tween 20

141

(0.05 %) (PBS-T20) (Sigma) with 5 % natural goat serum (Sigma) (Olea et al.,
1993) and 50 pl CitriSolv solution (Fisher Chemicals, Fairlawn, New Jersey)
(Gudmunsdottir et al., 1993) were added. The mixture were thoroughly mixed by
vortexing then incubated at 100 °C on heat blocks with rotary shaker for 15
minutes and followed by 2 hours of incubation at 4 °C. After incubation, the
mixture was centrifuged at 60009 for 15 minutes at 4 °C. The aqueous
supernatant of each sample was carefully collected and then transferred to a 1.5
ml microfuge tube for Q-ELISA testing. The Q-ELISA method used in this study is
that described in details by Pascho and Mulcahy (1987) and Alcorn and Pascho
(2000). The positive negative threshold absorbances are calculated according to
the method described by Meyers et al. (1993) .The positive-negative cutoff
absorbance for the kidney homogenate was 0.10. The tested positive samples
were assigned the following antigen level categories: low (0.10 to 0.19), medium
(0.20-0.99) and high (1.000 or more) (Pascho et al., 1998). Intensity of infection
among certain group of fish is expressed by percent of fish with high titers of R.

salmoninarum soluble antigens using Q-ELISA.

Nested PCR. _A DNeasy tissue extraction kit (Qiagen-Valencia, CA, USA)
was used for the extraction of DNA from 100pl aliquots of kidney tissue
homogenates. The DNA was extracted according to the manufacturer's
instructions, with a few minor modifications from the method described by
Pascho et al. (1998). The tissue pellets were obtained by centrifugation at 6000 g

for 20 minutes at 4 °C and the pellets were incubated with lysozyme buffer

142

consisting of 180 pl of 20 mg lysozyme (Sigma), 20mM Tris-HCI, pH 8.0; 2 mM
EDTA (Sigma) and 1.2 % (v/v) Triton X 100 (Sigma) at 37 °C for 1 hour. The
nPCR method and primers recommended by Pascho et al. (1998) were
employed with slight modifications to the volume of DNA (5 pl for first round and
2 pl for second round PCR reaction), water, and master mixes (45 pl for first
round and 48 pl for second round nPCR reaction) .The controls were composed
of a PCR mixture containing no DNA template reagent (negative control), positive
R. salmoninarum and positive tissue control. A volume of 10 pl of the nPCR
product and controls were mixed with 2 pl of 6X loading dye (Sigma) and used on
a 2 % agarose gel (Invitrogen Life Technologies, Carlsbad, CA). Each
electrophoresis gel included a 1kbp DNA ladder with 100 bp increments
(Invitrogen). Gels were run in 1 X Tris Acetate Buffer (1 X TAE) gel buffer
(Sigma). Gels were visualized under the KODAK EDAS Camera System and UV
Trans-illuminator. Samples were considered positive when a 320 bp band was
detected. Molecular confirmation of the purified bacterial isolates was also
conducted using nPCR according to the method described by Chase and Pascho

(1998).

Histopathology. Kidney tissues fixed in 10 % neutral buffered formalin
solution were processed and embedded in paraffin. Five-micron sections were
routinely stained with Hematoxylin and Eosin (HE) using the method previously
described by Prophet et al. (1992). Slides were evaluated and given a score of 0,

+1 , +2, or +3 based on degree of pathological alterations.

143

lmmunohistochemistry (IHC). The methods used in the IHC were adopted
from Jansson et al. (1991) and Evensen et al. (1994). In brief, tissue sections of
kidney from BKT with clinical signs were used for immunohistochemical
evaluation of the expression R. salmoninarum soluble antigens. IHC staining was
performed on automated immuno-stainers. Paraffin embedded tissues that had
been fixed in formalin for less than 48 hours were deparaffinized in xylene,
rehydrated in graded ethanol, and rinsed in distilled water. Endogenous
peroxidases were neutralized with 3% hydrogen peroxide for 5 minutes. Antigen
retrieval was achieved by incubating slides in a citric buffer antigen retrieval
solution in a steamer (Black 8. Decker, Towson, MD) for 20 min, and non-specific
immunoglobulin binding was blocked by incubation of slides for 10 min with a
protein-blocking agent (Dako, Carpinteria, CA). Using the Dako autostainer
(Dako, Carpenteria, CA), slides were incubated for 30 minutes with a goat anti-
Renibacterium salmoninarum antibody (Kirkegaard & Perry Laboratories) at a
dilution of 1:100. A streptavidin-lmmunoperoxidase staining procedure (Dako,
Carpinteria, CA) was used for immunolabeling. The immunoreaction was
visualized with ABC (Dako, Carpinteria, CA). Sections were counterstained with
Mayer’s hematoxylin. Tissues that had been fixed in formalin for more than a
year were immunostained using the protocol described above as well as the
following method to enhance antigen retrieval. In this particular protocol
deparaffinization, antigen retrieval, and immunostaining of formalin-fixed paraffin

embedded tissues were performed on the Bench Mark Automated Staining

I44

System (Ventana Medical Systems, Inc.) using the Enhanced V-Red Detection
(Alk. Phos. Red) Detection System (Ventana Medical Systems, Inc.) and a goat
anti-Renibacterium salmoninarum antibody (Kirkegaard & Perry Laboratories) at
a dilution of 1:100. Antigen retrieval was achieved using the Ventana Medical
Systems Retrieval Solution CC1 (Ventana Medical Systems) for 60 min followed
by digestion with and Protease 3 for 4min (Ventana Medical Systems). Sections
were counterstained with haematoxylin. Positive IHC controls included a kidney
from a trout with strong Renibacterium salmoninarum soluble antigens
expression to which the appropriate antisera were added. For negative controls

the primary antibodies were replaced with homologous non-immune sera.

Statistical Analysis. Because of the nature of data collected in this chapter
required basic statistical description, data analysis was primarily relied on
descriptive statistics. For year to year and brook trout strains comparisons, the
data was tested for normality and then student t test (parametric) or Mann-

Whitney Rank Sum Test was (with an alpha level = 0.05).

145

RESULTS

A. Prevalence of R. salmoninarum infection in captive and wild brook
trout stocks. Renibacterium salmoninarum antigens were detected in the
kidneys of Marquette State Fish Hatchery captive broodstock and fingerlings as
well as Cherry Creek brook trout. The prevalence of R. salmoninarum in the Iron
River broodstock exhibited a steady decline from 87% in 2001, to 80% in 2002,
to 60% in 2003, and to 43% in 2004. Similarly, the intensity of R. salmoninarum
in the Iron River brook trout (expressed by the percent of fish showing high titer
of R. salmoninarum antigen) exhibited a comparable decline. The percent of fish
with high R. salmoninarum antigen levels decreased from 17% in 2001 to 7.5% in
2004 (Figure 12).

Assinica brook trout Broodstock (BS AS-BKT) were tested only in 2003 and
2004. The results demonstrated a decrease in prevalence from 80 % in 2003 to
25% in 2004 (table 18). However, the intensity of the R. salmoninarum infection
in the BS AS-BKT did not exhibit a similar decline.

In the case of Cherry Creek brook trout (CC-BKT), samples were collected in
the falls of 2001-2004. Prevalence of R. salmoninarum in CC-BKT decreased
from 80% in 2001 to 67% in 2003. The intensity of R. salmoninarum showed no
consistent pattern (table 18).

The prevalence of R. salmoninarum in lR-BKT fingerlings between 2003 and

2005 were comparable to the parents and consistent with the high prevalence in

146

the gamete donor broodstock, although 2004 showed a significantly low
prevalence. However, the intensity of R. salmoninarum antigens in BKT
fingerlings was higher than those detected in their parents during 2003 (48% vs
17% in their parents) before sharply declining in subsequent years (table 18).

The prevalence of R. salmoninarum in 2005-AS-BKT fingerlings was lower
(28%) than those of their parent BS AS-BKT-2003. Similarly, the prevalence of R.
salmoninarum in 2005-AS-BKT fingerlings was much lower than that of the 2005-
IR-BKT fingerlings (28% vs 45% in lR-BKT fingerlings). The intensity of the R.
salmoninarum infection in the AS-BKT fingerlings showed a decline, which was
similar to the prevalence through the time of testing.

To compare the prevalence and intensity of R. salmoninarum in males and
females BKT, two hundred pairs of lR-BKT broodstock were tested in 2004.
Results indicated that the prevalence of R. salmoninarum infection was higher in
the kidney tissue of the females than males (48.5% in females vs 37.5% in
males). Similarly, the intensity of R. salmoninarum infection was clearly higher in
the females (11% in female vs. 4 % in male). The shedding of the bacterial
antigen along with the gametes was tested in broodstock IR-BKT in 2004. Data
shown in Figure (13) illustrated that R. salmoninarum antigen shed with the
gametes in both males and females IR—BKT. The prevalence of individuals that
shed the bacterial antigen along with the gametes is lower in males than in
females (10% in milt vs 15% in ovarian fluid). Also, the intensity of samples with
high R. salmoninarum antigen was much higher in females (2.5%) than in males

(0.5%). The majority of the females (20 out of 30) that shed the antigen in their

147

ovarian fluids exhibited similar levels of R. salmoninarum antigens in their
kidneys. However, 9 females shed the R. salmoninarum antigen along with the
ovarian fluid without detecting R. salmoninarum antigen in the kidneys and one
female shed the antigen at a low level in the ovarian fluid and tested negative for
the kidney tissue. Likewise, the majority of male shedders (13 out of 20) had
similar levels of R. salmoninarum in their kidneys with only 7 fish shed the

antigen without detected titer of R. salmoninarum antigen in the kidney.

8. BKD outbreaks among hatchery-raised brook trout. A number of BKD

outbreaks erupted among hatchery-raised brook trout during 2003 and 2004:

1. Iron River Brook trout. A major BKD outbreak has been observed in May,
2003. In this outbreak mortalities reached up to 50%. Externally, all fish
submitted for clinical examination had heavy infestations of skin monogenean
trematodes, sessile ciliates and Trichodina spp. lntemally, the majority of fish
exhibited typical signs of BKD, such as enlarged kidneys with whitish gray
discoloration and the presence of multiple creamy-whitish nodules scattered
throughout the kidney tissues (Figure. 14). Kidney samples were tested for R.
salmoninarum using the nPCR, Q-ELISA and culture. Results indicated that R.
salmoninarum was present in all samples using each of the three techniques. All
tested fish had high concentration of R. salmoninarum antigens in the kidney
tissue by Q-ELISA. Examination of the histopathological slides of affected

kidneys revealed the presence of typical granulomatous reactions with a necrotic

148

center that was surrounded by a fibrous capsule, with a mixture of epithelioid
macrophages, lymphocytes, and frequent giant cells. Gram-positive coccobacilli
were observed within the necrotic tissue as well as other layers of the granuloma.
When immunohistochemical (IHC) procedures were performed on the paraffin-
waxed kidney sections, high positive results (scored 3+) where bacterial cells
taking the dark red IHC staining were heavily distributed within the blue
background of the kidney tissues (Figure 15 & 16). The Iron River brook trout

fingerlings in the hatchery raceways were treated with 2.25% Aquamycin-100 in
food. The fish were fed Erythromycin between 75 and 150 mg/kg of fish weight to

comply with lnvestigational New Animal Drug (INAD) specifications and mortalities
gradually decreased until subsiding completely at the completion of treatment. In
early June of 2003, a total number of 60 erythromycin treated IR-BKT fingerlings
were further examined post treatment. Results showed 4 of 60 fish (6.7 %) still
had clinical BKD lesions. Kidneys of the examined fish were tested for the
presence of R. salmoninarum antigens using Q-ELISA. Results indicated that R.
salmoninarum antigens had sharply declined to 18.3 %, with only 5 %
demonstrating the high titer of the antigens. Culture from post treatment fish

showed that only 4 out of 60 fish (6.7 %) were positive.

2. Assinica Brook trout. In mid July of 2004, mortalities in the 3 year old
Assinica brook trout stocks in MSFH continued for a month before representative
samples were submitted for investigation. Random samples from fish in affected
raceways were externally examined, where skin scraping revealed heavy

infestations with monogenean (Gyrodacty/us sp.). A total of 9 clinically affected

149

fish were examined, where 7 out of 9 fish (77.8 %) exhibited heavy monogenetic
trematodes, sessile ciliates and fungal hyphae. lntemally, 5 out 9 fish (55.6 %)
exhibited grayish discoloration of the kidneys, with the presence of white
abscess-like nodules scattered throughout the kidneys. Kidney samples from the
9 fish were further tested using nPCR, Q-ELISA and culture. All samples were
positive when tested using nPCR, while a total number of 7 out of 9 (77.8 %) fish
were culture positive and 3 out of 9 fish (33.3 %) were Q- ELISA positive (1 high,
1 medium, 1 low). When submitted for histopathology, 75 % of the kidney
samples exhibited moderate muItI-focal granulomatous reactions (scored 1+) and
25 % exhibited severe multi-focal granulomatous reactions (Scored 3+) (Figure
18). The reactions were characterized by the presence of typical granulomas,
accompanied by a mixture of epithelioid macrophages, lymphocytes and
occasional giant cells. Interestingly, the granulomatous reaction replaced 50-60
% of the renal parenchyma (Figure 18). Centrally, the lesion was composed of
eosinophilic, caseous debris, surrounded by marked sheets, nests, and
laminated foci of epithelioid macrophages mixed with lymphocytes. The periphery
of the lesion was surrounded by mature fibrous connective tissue associated with
more destruction of the renal parenchyma (Figure 17). Despite the fact that most
of the samples showed the presence of typical granulomatous inflammation with
varying severity (1+ to 3+ scores), the results of the IHC technique performed on
the paraffin-waxed lesions were negative (Figure 18). The only sample with a

typical granulomatous reaction exhibited very few bacterial cells when the

150

antigen retrieval technique was performed on the paraffin embedded blocks of

the same samples (Figure 19).

3. Private Brook Trout Farm. In Mid September of 2004, mass mortalities
occurred in the brook trout yearlings of a private Brook Trout Farm. Mortalities
continued for two weeks. External parasitological examination of fish gills and
skin revealed the presence of large numbers of monogenean trematodes and
sessile ciliates. lntemally, 12 of 30 fish (40 %) showed pale gray, whitish
discolorations and swelling of the kidneys, with multiple abscess-like nodules
scattered all over the kidneys. Typical colonies of R. salmoninarum were
observed after 2 weeks on MKDM from a total of 18 out of 30 fish (60 %), which
was later confirmed with nPCR. Q-ELISA showed that 18 out of 30 fish (60%)
were positive for R. salmoninarum antigens, with an intensity of 67% (expressed
by percent of fish with high titers of R. salmoninarum soluble antigens using Q-

ELISA).

151

DISCUSSION

For decades, brook trout has been known for its high susceptibility to R.
salmoninarum infection (Snieszko and Griffin 1955; Mitchum and Sherman
1981). Brook trout infected with R. salmoninarum either naturally or
experimentally, suffer from high mortalities (Belding and Merrill 1935;

Snieszko and Griffin 1955; Mitchum et al. 1979).

Data obtained in this study demonstrated a high prevalence and intensity
of R. salmoninarum infection in both hatchery raised and wild stocks. This
concurs with previous reports. For example, Mitchum and Sherman, 1981,
recorded a relatively high prevalence and severity of R. salmoninarum
infection in wild and hatchery raised brook trout populations (58 %, 45 %
respectively).

A general trend of declining prevalence and intensity of R. salmoninarum
in IR-BKT broodstock was observed over the period of this study. This trend
might be explained by the improvement of hatchery hygienic practices.
Among these practices are the prophylactic erythromycin phosphate
administration and hardening of eggs in erythromycin containing water.
Evelyn et al. 1986 and Lee and Evelyn (1989) found that intramuscular
injection of broodstock with erythromycin phosphate dramatically minimized
the vertical transmission of R. salmoninarum. Also, Rinsing of broodstock in
iodophores solution before collecting gametes could minimize the R.

salmoninarum on the eggshell (Ross and Smith 1972). Maule et al. (1996)

152

described similar observations that lead to remarkable decrease of R.
salmoninarum prevalence among other salmonid species.

Although the Assinica strain demonstrated inconsistent R. salmoninarum
infection prevalence when compared to the Iron River, yet Iron River strain
showed higher intensity than Assinica strain. The Assinica strain is known for
its superior survival and growth (Gowing 1986), and these characteristics
could play a vital role in the general defense of the fish against severe
infections with R. salmoninarum. In addition, variable susceptibility of fish
strains to different diseases is not unusual. For example, some strains of
steelhead showed variable susceptibility to R. salmoninarum (Winter et al.,

1 980).

Wild populations of BKT (CC-BKT) showed a comparable prevalence to
that of the hatchery reared BKT. The fact that Cherry Creek supplies the
hatchery with water and that BKT exists in this water may explain the
similarity in infection levels.

Analysis of the data of R. salmoninarum prevalence and intensity of
infection among the Iron River brook trout pre-stocking fingerlings indicated
that fingerlings from 2003 exhibited the highest prevalence and infection
intensity (approaching 100 %). The 2003 pre-stocking fingerlings are the
offspring of the 2001 Iron River brook trout parent stocks that also exhibited
high BKD prevalence (83 %). Vertical transmission have probably played a
major role in this high incidence of infection, particularly that erythromycin

prophylactic administration was not implemented in 2001. On the contrary,

153

the 2004-2005 Iron River and Assinica brook trout offspring showed relatively
lower R. salmoninarum prevalence and intensity, although they originally
hatched from fertilized eggs collected from 2002-2003 parents with relatively
high R. salmoninarum prevalence and intensity. This could only be explained
by the strict hygienic measures adopted by the hatchery starting from 2002.

Data obtained from the Q-ELISA testing of gametes indicated that the R.
salmoninarum was shed with the gametes in both males and females (10% in
males versus 15% in females), with a higher intensity in females than males.
These results suggest a contribution of the male in the vertical transmission of
R. salmoninarum to the offspring, in addition to the role played by females.
Allison (1955) was the first to report vertical transmission in the brook trout,
albeit with circumstantial evidence that gametes from infected adults resulted
in infected offspring. Our data agreed with Wiens and Kaattari (1989), which
were able to detect the R. salmoninarum antigens in the milt of infected
males. However, the studies of Klontz (1983) and Evelyn, et al. (1986)
demonstrated that males play an insignificant role in the vertical transmission
of R. salmoninarum. However, the data may be complicated by
discrepancies between levels of R. salmoninarum in the ovarian fluid and
their levels in the kidney of corresponding individual fish.

BKD outbreaks were associated with severe clinical signs and high
mortalities in BKT in Michigan. The frequent occurrence of BKD epizootics in
hatchery raised brook trout populations during 2003 and 2004 without

affecting other salmonids, such as the lake trout residing in the same

154

hatchery, presumptively indicates that brook trout has higher susceptibility to
R. salmoninarum infection when compared to other char and trout species.
Although brook trout (Salvelinus fontinalis) are known for their high
susceptibility to R. salmoninarum infection (Snieszko and Griffin 1955;
Mitchum and Sherman, 1981), reports of the disease in this species were
scarce in Michigan. Data indicated that the clinical picture and the magnitude
of the mortalities described in the current study coincide with previous reports
about BKD-associated epizootics in brook trout in the United States (Belding
and Merrill 1935; Snieszko and Griffin 1955; Mitchum et al. 1979).

Outbreaks described in the current study were frequently coupled with
external parasitic infestations. It is not clear whether the external parasitic
infestation initiated or resulted from the BKD outbreaks. However, it seems
that many factors were involved in influencing mortalities associated with the
outbreaks. First, the rise in water temperature, along with the heavy density
in the hatchery raceways, favors the external parasites (Mo 1997; Rintamaki
and Voltonen 1994). Heavy external parasitic infestations open portals of
entry within the skin and gills of fish and facilitate the horizontal transmission
of the R. salmoninarum in water via the skin and gills. It is well documented
that the bacteria can survive in water and feces for up to 21 days (Austin and
Rodger 19803; Balfry et al. 1996). Also, a number of authors have
hypothesized that water and feces indirectly act as reservoir for the
transmission of R. salmoninarum from infected fish to other fish (Bullock

1975; Mitchum and Sherman 1981). Alternatively, the gradual increase of

155

water temperature during the time of the outbreaks was assumed by some
authors to initiate the progression of BKD infection (Belding and Merrill 1935;
Snieszko and Griffin 1955; Smith 1964; Sanders et al. 1978). The sequence
of BKD infections is usually associated with the production of R.
salmoninarum soluble antigens, which are known to suppress the immune
response of infected fish (Fredriksen et al. 1997; Turaga et al. 1987; Wiens
and Kaattari 1991, Ellis 1999), which drive the host to become vulnerable to
infection with other bacteria and parasites.

The development of the disease in the 2003-2004 outbreaks was
documented using both histopathology and IHC. Examination of kidney
sections stained with H&E indicated the presence of severe granulomatous
reactions, with typical granuloma components where the organism could be
detected both intra-cellularly and extra-cellularly using the Gram stain.
Previous records of histopathology support these pathologic pictures of the
kidney lesions during both experimental and natural BKD infections (Wood
and Yasutake 1956; Young and Chapman 1978; Bruno 1986; Sami et al.
1992). In case of the Iron River BKT mortalities, IHC stained slides indicated
the presence of an intense distribution of bacterial cells and their antigens
within the kidney tissues, as well as within the granulomas, which confirms
the assumption that the severe progression of the disease is associated with
high bacterial metabolic activities (Evenden et al.1993, Bruno 1986, Sami et
al.1992). Similar findings were reached by Hoffman et al. (1989); Jansson et

al. (1991); Evensen et al. (1994) and Lorenzen et al. (1996).

156

Some discrepancies were observed between histopathological findings
(positive with different scores) and IHC results (negative results of some of
positive histopathological section). The histopathological findings ranged from
mild multi-focal histiocytic inflammation (scored 1+), which appeared negative
using the IHC stains to severe granulomatous reaction replacing more than
60 % of the kidney parenchyma (scored 3+). These kidney sections were
negative when tested using regular IHC procedures and turned into very mild
positive after improved antigen retrieval procedures were performed.
Histopathological and IHC findings confirm the assumptions that the milder
irritation over longer period of time might resulted in typical granuloma in one
case, mild inflammation in others and negative results in the rest.

The Assinica BKT kidney samples were preserved in buffered formalin
solution for more than 1 year which is reported to be crucial factor in reducing
the sensitivity of IHC techniques for many antigens and epitopes (Sompuram
et al., 2004). This long time preservation in buffered formalin solution could
resulted in denaturation of the soluble antigens (p57 protein) and formation of
Methylene bridges, both inter- and intra-molecularly which will ultimately alter
the physical characteristics of the kidney tissues and also induces masking of
the antigens (p57) (Evensen et al., 1994). These deleterious effects of
formalin might be a good cause for the inability to detect the bacterial cells
within the affected kidney tissues using IHC, although they were positive with
other techniques like Q-ELISA, culture, and nPCR. An enzymatic digestion

and heat treatment methods (antigen retrieval methods) (Evensen et al.,

157

1994) were used intensively to retrieve the masked antigens from the IHC
processed tissues. Unfortunately the antigen retrieval methods were only able
to retrieve extremely few R. salmoninarum soluble antigens within a typical
granulomatous reaction of kidney tissue. One important disadvantage of the
antigen retrieval method using heat treatment is the unexpected dissociation
of the major soluble antigen (p57), which is known to be heat labile (Evensen
et al.1994).

In conclusion, this study supports the previous reports, which
emphasized that brook trout are highly susceptible to R. salmoninarum
infection. In addition, this study shed the light on the possible contribution of a
number of factors to development of BKD epizootics in Michigan hatcheries,
such as the seasonal changes and the presence of external parasites.
Further, the possible role of males and females in shedding the bacterium

and its soluble antigens was fully discussed.

158

 

Brook trout strain and life Date Collected Number of

 

stage fish tested
Iron River broodstocks October, 2001 54

 

October, 2002 45
October, 2003 30
October, 2004 400
Assinica broodstocks October, 2003 30
October, 2004 8
Iron River 18 month old pre- March, 2002 60
stocking fingerlings March, 2003 60
March, 2004 60
March, 2005 60
Assinica 18 month old pre- March, 2003 60
SIOCRIHQ fl“99'“"95 March, 2004 60
March, 2005 60

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 17. Details of samples collected from brook trout broodstocks
throughout the period from 2001-2004 and pro-stocking 18-month-old

fingerlings collected throughout the period from 2002-2005.

159

 

 

 

 

 

 

 

 

 

 

 

 

Parent stocks (Gamete 18 months old pre-stocking
Strain donorSI finge_rli_ngs
Year Renibacterium Year Renibacterium
salmoninarum salmoninarum
prevalence prevalence
Iron 2001 87.0 % Total 2003 98.0 % Total
River 16.7% high 48.0 % high
50.0 % medium 35.4 % medium
20.4% low 14.6 % low
Iron 2002 80.0 % Total 2004 20.0 % Total
River 15.55 % high 0.0 % high
13.3 % medium 10.0 % medium
51.1 % low 10.0 % low
Iron 2003 60.0 % Total 2005 45.0 % Total
River 10.0 % high 0.0 % high
0.0 % medium 12.5 % medium
50.0 % low 32.5 % low
Assinica 2003 80.0 % Total 2005 27.5 % Total
0.0 % high 0.0 % high
10.0 % medium 2.5 % medium
70.0 % low 25.0 % low

 

Table18. Renibacterium salmoninarum infection prevalence and intensity among
brook trout broodstocks and their corresponding 18 months pro-stocking
fingerlings throughout the period from 2001 - 2005.

Data In this table was generated using a polyclonal antibody-based quantitative ELISA
(Q-ELISA) performed on kidney tissues. Prevalence was determined by % of Q-ELISA

positive samples of the total number of samples tested. Intensity was considered high if
the antigen concentration 21; medium if the antigen concentration = 0.20-0.99; and Low

if the antigen concentration = 0.10 to 0.19.

160

 

Iron River Brook trout

 

 

  
  
 

IR-BKT-01 IR-BKT-02 IR-BKT-03 IR-BKT-IM

‘°°°"‘ Cherry Creek Brook trout
9070‘
8070‘
70%-
60%-
8 50%
,5; 40%-
.,>, 30%
OL- 20%
10%)

 

CC-BKT-M CC-BKT-02 CC-BKT-03

Figure 12. Prevalence and intensity of Renibacterium salmoninarum
among the adult brook trout collected through 2001-2003

Data in this Figure was generated using a polyclonal antibody-based
quantitative ELISA (Q-ELISA) performed on kidney tissues. Prevalence was
determined by % of Q-ELISA positive samples of the total number of samples
tested. Intensity was considered high if the antigen concentration a; medium
if the antigen concentration = 0.20-0.99; and Low if the antigen

concentration = 0.10 to 0.19.

lR-BKT: Iron River Brook trout

CC-BKT: Cherry Creek Brook trout

This figure Is In color

161

 

I High I Medium I Low

100% -
90% a
80%:
70%*
60% ‘
50% -
40% ‘
30% 1
20%:
1 0%‘

0% ‘

Prevalence

 

 

Figure 13. Renibacterium salmoninarum Prevalence and intensity in
kidneys and gametes of the Iron River brook trout broodstock in from
Marquette State Fish Hatchery. Samples were collected during the fall
spawning season of 2004.

Data in this Figure was generated using a polyclonal antibody-based quantitative ELISA
(Q-ELISA) performed on kidney tissues. Prevalence was determined by % of Q-ELISA
positive samples of the total number of samples tested. Intensity was considered high if

the antigen concentration 21; medium if the antigen concentration = 020-099; and Low
if the antigen concentration = 0.10 to 0.19.

This Figure is in color

162

 

Figure 14. An Iron River Brook trout fingerling with Bacterial
Kidney Disease. The kidney is swollen with multiple creamy-
whitish nodules (N).

The above case is from an outbreak of Bacterial Kidney Disease that
killed thousands of hatchery raised Iron River brook trout fingerlings

in May 2003.

This Flgure Is in Color

163

 

 

Figure15. Kidney tissue of Iron River brook trout fingerling exhibiting
heavy Renibacterium salmoninarum infection. Kidney section was
stained using an anti-Renibacterium salmoninarum antibody based
streptavidin -immunoperoxidase immmunolbeling (Magnification 400).
Sections were counterstained with Mayer’s hematoxylin (Blue
background)

Rs: Renibacterium salmoninarum soluble antigens with the red staining affinity.
Tu: Non-affected kidney tubules with blue counterstaining affinity.

This figure is in color

164

 

Figure 16. Kidney tissue of Iron River brook trout fingerling exhibiting
heavy Renibacterium salmoninarum infection after enhanced antigen
retrieval procedures using Alkaline Phosphatase Red and goat anti-
Renibacterium salmoninarum antibody. Sections were counterstained with
Mayer’s Hematoxylin (Blue background) (Magnification 400). This case is
from an outbreak of BKD that killed thousands of hatchery raised Iron River
brook trout fingerlings in May 2003.

Rs: Renibacterium salmoninarum soluble antigens with the red staining affinity.

Tu: Non-affected kidney tubules with blue counterstaining affinity. The marked increase in dark
red dots (retrieved bacteria) distribution within the kidney tissue after the antigen retrieval
compared to the picture in figure 16.

This figure is in color

165

 

 

Figure 17. Hematoxylin and Eosin stained slide of kidney showing a severe
granulomatous reaction that is replacing kidney tissues of a 3 years old
Assinica brook trout. Notice the fibrous capsule (FC) surrounding the
entire granuloma (Magnification 100). The case is from an outbreak of BKD

that killed captive 3 years old Assinica brook trout in mid September 2003.

This figure is In color

166

 

is

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with chronic multi-focal granulomatous reaction from a 3 years old
Assinica brook trout with absence of bacterial cells or antigen from
the affected kidney tissues. Kidney section was stained using an anti-
Renibacterium salmoninarum antibody based streptavidin -
Immunoperoxidase immmunolbeling (Magnification 400).

Sections were counterstained with Mayer’s hematoxylin (Blue
background). The case is from an outbreak of BKD that killed captive 3

years old Assinica brook trout in mid September 2003.

This figure Is in color

 

Figure 19. An ' h' ‘ h ' ' stained Kidney tissue section with

 

chronic multi-focal granulomatous reaction from a 3 years old Assinica
brook trout exhibiting a mild chronic presence of bacterial cells or antigens
after enhanced antigen retrieval procedures using Alkaline Phosphatase
Red and goat anti-Renibacterium salmoninarum antibody. Sections were
counterstained with Mayer’s Hematoxylin (Blue background) (Magnification
400).

Rs: Renibacterium salmoninarum soluble antigens with the red staining affinity.

This figure is in color

168

CHAPTER SIX

FIRST RECORD OF RENIBACTERIUM SALMONINARUM IN SEA
LAMPREY (PETROMYZON MARINUS) FROM THE GREAT LAKES
BASIN

ABSTRACT

Bacterial Kidney Disease (BKD), caused by Renibacterium
salmoninarum (R. salmoninarum), is a widespread problem with major
implications for salmonid fish. The mechanisms by which the bacteria have
reached high levels of infection previously unrecorded in the Laurentian
Great Lakes are presently unknown. Research involving reservoirs and
mechanisms of R. salmoninarum transmission in fish is lacking due to the
ecological complexity of heterogeneous habitats and the lack of adequate
funding. Surprisingly, we isolated R. salmoninarum from the kidneys of the
sea lamprey (Petromyzon mar/nus). The bacterium was cultured from
kidneys of 16% and 4% of Lake Ontario lampreys examined in 2003 and
2004 respectively, with bacterial colonies verified via nested polymerase
chain reaction (nPCR) and quantitative enzyme linked immunosorbent assay

(Q-ELISA).

169

INTRODUCTION

Bacterial Kidney Disease (BKD), caused by Renibacterium
salmoninarum, (R. salmoninarum) is a serious bacterial disease of
salmonines (Fryer and Sanders, 1981) that is widespread in the Great Lakes
basin (Hnath and Faisal 2005). Many facts pertaining to disease transmission
and reservoirs of infection are currently unknown. The majority of studies
conducted thus far suggest that R. salmoninarum exclusively infect
salmonids and that carrier fish are responsible for its distribution (Wood and
Yasutake 1956; Klontz 1983; Bullock and Herman 1988). However a few
studies have indicated that non-salmonid fish species such as Pacific hake
(Merluccius productus, Kent et al. 1998) and Pacific herring (Clupea
harengus pallasi, Paclibare et al. 1988) may harbor the pathogen. In
addition, certain fish, species such as Pacific herring (Traxler and Bell 1988),
Sablefish (Anoplopoma fumbria) (Bell et al. 1990), common shiners (Notropis
comutus) (Hicks et al. 1986) and fathead minnows (Pimephales promelas)
(Hicks et al. 1986) contracted the infection when exposed to R.
salmoninarum via intraperitoneal injection. The role that non-salmonid
species may play in the spread of BKD has not been investigated in the
Great Lakes basin.

In the Great Lakes basin, a number of non-indigenous species have

invaded the system and caused serious ecologic and economic losses (Lupi

170

and Hoehn 1998). Among these invasive species, is the sea lamprey
(Petromyzon marinus), which has been one of the most destructive of the
introduced species. The sea lamprey has been incriminated as a major factor
contributing to the collapse of the lake trout (Salvelinus namaycush) and the
lake whitefish (Coregonus c/upeaformis) fisheries in the Great Lakes during
the 19403 and 19503 from which these two fisheries have yet to fully recover
despite the advent of sea lamprey chemical control since 1958 (Smith and
Tibbles 1980).

To further reduce the number of sea lamprey and limit its spread, the
Great Lakes Fishery Commission began a large-scale experimental program
based on trapping male sea lamprey, sterilizing them, and subsequently
releasing the sterile males back into streams where they compete with fertile
males for spawning females. Field assessments indicated a decreased
hatch rate in streams where this strategy was practiced. Up to 40,000
sterilized sea lamprey are released annually, yielding major success in the
areas of implementation, such as the St. Marys River. Currently, males are
collected from different areas in the Great Lakes basin, transported into a
sterilizing facility in Hammond Bay, Michigan, and then released into selected
river systems basin-wide. These transfers of lamprey to different locations
may additionally transfer various pathogens concurrently, the probability of
which has raised major concerns regarding the possibility of resident fish

populations becoming infected.

171

To this end, this current study was initiated to determine if the sea
lamprey could be a new host range for R. salmoninarum infection and the
possible vector role that may contribute to the spread of Renibacterium

salmoninarum.

172

MATERIALS AND METHODS

Sea Lamprey (Petromyzon marinas). In the midsummer of 2003, 25
adult sea lamprey were moved from the Humber River and Duffins Creek,
Lake Ontario, and presented alive to the Aquatic Animal Health Laboratory
(AAHL) at Michigan State University to undergo fish health inspection and
thereby ascertain their suitability for transfer to the lamprey sterilizing facility
at Hammond Bay, then into the St. Mary’s River, both within the Lake Huron
watershed. In midsummer 2004, an additional 118 adult lamprey were
caught from Duffins Creek and Humber River, held separately, and brought
alive to the AAHL. Until examined, lampreys were maintained alive in well-

aerated chilled aquaria.

Dissection, sampling and sample processing. Lampreys were
euthanized with an overdose of MS 222 (tricaine methane sulfonate, Finquel-
Ardent Chemical Laboratories, Washington) and dissected under aseptic
conditions. Kidneys were removed aseptically and placed in sterile 7.5 cm x
18.5 cm Whirl-Pak® bags (Nasco, Fort Atkinson, WI) to which Hank’s
Balanced Salt Solution (HBSS) was then added at a ratio of 1:4 (weight/
volume) and stomached for 120 seconds using a high-speed Biomaster

Stomacher—80 (Wolf Laboratories Limited, Pocklington, York, UK).

173

Bacterial isolation and identification. A100 pl aliquots of stomached
kidney tissue were spread onto MKDM (Eissa 2005, Chapter 2). Inoculated
plates were incubated for up to 20 days at a sub-ambient temperature
incubator adjusted to 15 °C. Incubated plates were checked for colonial
growth on daily basis. Isolates were identified according to the standard
morphological and biochemical criteria for R. salmoninarum as described by
Sanders and Fryer (1980), Austin and Austin (1999) and Bruno and Munro
(1986). Biochemical tests included: motility, in motility test medium (BD and
Company Sparks, MD, USA), cytochrome oxidase determination on Pathotec
strips (Remel, Lenexa, Kansas, USA), catalase with 3 % hydrogen peroxide,
esculin hydrolysis using bile esculin agar (Remel), and a DNAse test using
DNAse test medium (Remel). Carbohydrate utilization was performed using
basal oxidation fermentation media (DIFCO-BD) that was prepared according
to manufacturer instructions prior to the addition of individual sugars.
Aseptically, 10 ml of filter sterilized (0.45 pm) 10 % sugar solution, was
added to autoclaved and cooled (48 °C) basal media to reach a final
concentration of 1 % with the exception of salicin, which was made as a 5 %
solution to reach a 0.5 % final concentration. Each of the following sugars
was added individually to the basal medium arabinose, glucose, lactose,

maltose, rhamnose, salicin, sucrose, sorbitol, xylose. All sugars were from

Sigma.

174

Nested PCR. A DNeasy tissue extraction kit (Qiagen-Valencia, CA,
USA) was used to extract DNA from 100pl aliquots of kidney tissue
homogenates. The DNA was extracted according to manufacturer's
instructions, with a few minor modifications from the method described by
Pascho et al. (1998). Tissue pellets were obtained by centrifugation at 6000
g for 20 minutes at 4 °C and the pellets were incubated with lysozyme buffer
consisting of 180 pl of 20 mg lysozyme (Sigma), 20mM Tris-HCI, pH 8.0; 2
mM EDTA (Sigma) and 1.2 % (v/v) Triton X 100 (Sigma) at 37 °C for 1 hour.
The nPCR method and primers recommended by Pascho et al. (1998) were
employed with slight modifications to the volume of DNA (5 pl for first round
and 2 pl for second round PCR reaction), water, and master mixes (45 pl for
first round and 48 pl for second round nPCR reaction) .The controls were
composed of a PCR mixture containing no DNA template reagent (negative
control), positive R. salmoninarum and positive tissue control. A volume of 10
pl of the nPCR product and controls were mixed with 2 pl of 6X loading dye
(Sigma) and used on a 2 % agarose gel (Invitrogen Life Technologies,
Carlsbad, CA). Each electrophoresis gel included a 1kbp DNA ladder with
100 bp increments (Invitrogen). Gels were run in 1 X Tris Acetate Buffer (1 X
TAE) gel buffer (Sigma). Gels were visualized under the KODAK EDAS
Camera System and UV Trans-illuminator. Samples were considered
positive when a 320 bp band was detected. Molecular confirmation of the
purified bacterial isolates was also conducted using nPCR according to the

method described by Chase and Pascho (1998).

175

Q-ELISA. The sample preparation and Q-ELISA protocol was adopted
from the methods detailed in Pascho and Mulcahy (1987) and Alcorn and
Pascho (2000). The positive negative threshold was determined according to
the calculations detailed in Meyers et al. (1993). The positive -negative cutoff
absorbance for the kidney homogenate was 0.10. The samples that tested-
positive were assigned the following antigen level categories: low (0.10 to

0.19), medium (0.20-0.99) and high (1.000 or more) (Pascho et al., 1998).

I76

RESULTS

Isolation, identification and confirmation of Renibacterium
salmoninarum. Renibacterium salmoninarum isolates were retrieved from
the kidneys of 4 out of 25 (in 2003) and 5 out of 118 (in 2004) adult sea
lamprey from the Duffins Creek/Humber River assemblage. The organism
was not isolated from blood samples in 2003 nor 2004 (table 19).
Morphologically, all isolates were Gram-positive diplobacilli or coccobacilli.
On MKDM agar plates, the isolates produced 1 mm diameter, white, shiny,
smooth, round colonies with raised surfaces. In MKDM broth, most of the
isolates produced white granular pellets with the exception of two isolates
(SL 14 and SLHR 15) that produced uniform turbidity with large white pellets.
Biochemically, all the retrieved isolates were non-motile, catalase positive,
cytochrome oxidase negative, esculin hydrolysis negative and DNAse
negative. The bacteria did not produce acid from any of the carbohydrates
that were tested (table 20). These bacterial isolates were identified as R.
salmoninarum and they were confirmed using nPCR (Figure 20) and Q-
ELISA.

Detection of R. salmoninarum in sea lamprey kidneys. Bands of 320
bp, characteristic of R. salmoninarum were visualized using the nPCR
technique in four (16 %) out of 25 lamprey kidney samples collected in the
mid summer of 2003 (table 19). Using the same technique the organism was
detected in 38 (66 %) out of 58 lamprey kidney samples collected in 2004

from the Duffins Creek site (table 19), while positive nPCR was recorded in 3

177

(5 %) out of 60 lamprey kidney samples collected from the Humber River site
that year. When the Q-ELISA technique was performed on the 2004
samples, R. salmoninarum antigens were detected in the kidneys of two (1.7

%) of the 118 lamprey and were low in titer (table 19).

178

DISCUSSION

Despite the fact that R. salmoninarum has been described as a
salmonid-specific pathogen, it has been isolated for the first time from the
sea lamprey (Petromyzon mar/nus) for two successive years (2003 and
2004). The prevalence in sea lamprey is relatively low when compared to the
prevalence found in salmonines. Despite the isolation of R. salmoninarum
from the kidneys of clinically affected sea lamprey, the bacterium was not
isolated from the blood nor any other intemal organs. It appears that sea
lamprey R. salmoninarum isolates possess the same affinity for kidney
tissues as that of salmonid isolates.

The morphological criteria and biochemical reactions of the retrieved sea
lamprey isolates coincided with those described for R. salmoninarum
(Sanders and Fryer 1980). The size of the detected amplicon band (320 bp)
in both kidney tissues and cultured isolates using nPCR was consistent with
that published for R. salmoninarum (Pascho et al., 1998; Chase and Pascho,
1998)

While all R. salmoninarum isolates obtained in this study were positive
with both nPCR and Q-ELISA, tissues from which the isolates were retrieved
were not always positive. This discrepancy could be attributed to the
presence of tissue inhibitors, present in the sea lamprey that may interfere
with PCR or ELISA reactions. As Makos and Youson (1988) reported, the

sea lamprey does not have a gall bladder, thus bile salts accumulate in the

179

muscles and kidneys. Biochemically, bile salts act as a detergent, which may
contribute, to the inhibition of diagnostic assays.
Inversely, in the case of Duffins Creek samples, other than SLDC6, nPCR
yielded positive results while no R. salmoninarum was isolated. This finding
could be explained by the presence of low numbers of bacteria that could be
detected with nPCR, but are less than the threshold that allows their
isolation. This threshold has been estimated by 100-colony forming
units/gram tissue in salmonids fish (Lee 1989). The nested PCR assay can
detect as little as 4-10 bacterial cells/gram tissue (Miriam, et al., 1997).

In summary, this study reports the sea lamprey as a host for R.
salmoninarum. The role played by Great Lakes sea lamprey in the

epizootiology of BKD in the Great Lakes requires further investigations.

180

 

 

 

 

 

 

 

 

Sea Site of Type of % Positive % % Positive

lamprey collection sample Cultured Positive nPCR Q-ELISA
Samples samples samples

2003 Humber/Duffins Kidney (4/25) (4/25) ND

SL1-25-03 Lake Ontario 16 % 16 %

2003 Hum ber/Duffins Blood (0/25) ND ND

SL1-25—03 Lake Ontario 0 %

2004 Hum ber River Kidney (4/60) (3/60) (2/60)

SLHR1-60 Lake Ontario 6.7 % 5 % 3.3 %

2004 Hum ber River Blood (0/60) ND ND

SLHR1-60 Lake Ontarion 0 %

2004 Duffin’s Creek Kidney (1/58) (38/58) 0 %

SLDC1-58 Lake Ontario 1.7 % 65.5 %

2004 Duffin’s Creek Blood (0/58) ND ND

SLDC1-58 Lake Ontario 0 %

 

 

 

 

 

 

 

Table 19. Prevalence of Renibacterium salmoninarum in kidneys and

blood of 2003-2004 Lake Ontario sea lampreys using nPCR, Q-ELISA

and culture.

181

Isolate ID Culture-based assays Q-ELISA

E

SL3-03
SL21-03
SL11-O3
SL14-03
SLDC 6-04
SLHR 9-04
SLHR14-O4
SLHR15-O4
SLHR16-O4

 

+
+
+
4.
+
+
+
+
4..

+++++++++
+++++++++

Table 20. Confirmation of Renibacterium salmoninarum isolates

using some culture based assays, serological and molecular assays.

Notice: SL3, SL21, SL11 and SL14 were the isolates retrieved from adult sea lamprey in
mid summer 2003. DC6 was an Isolate retrieved from adult sea lamprey number 6
collected from Duffins Creek, Lake Ontario. HR9, HR14, HR15 and HR16 were isolates
retrieved from adult sea lamprey collected from Humber River, Lake Ontario. Both DC
and HR isolate were retrieved from adult sea lamprey collected in the mid summer 2004.
Culture based assays: C (catalase), O (oxidase), E (esculin hydrolysis), D (DNAse), CHO
(carbohydrate utilization) and M (Motility). Serological assay: (Q-ELISA -Polyclonal
antibody based quantitative ELISA). Molecular assay: (nPCR-Nested polymerase chain
reaction)

182

 

Figure 20. Nested PCR assay performed on isolates showing the
characteristic 320 bp band of Renibacterium salmoninarum isolates.

Notice: SL3, SL21, SL11 and SL14 were the isolates retrieved from adult sea lamprey in
mid summer 2003. DC6 was an isolate retrieved from adult sea lamprey number 6
collected from Duffins Creek, Lake Ontario. HR9, HR14, HR15 and HR16 were isolates
retrieved from adult sea lamprey collected from Humber River, Lake Ontario. Both DC
and HR isolate were retrieved from adult sea lamprey collected in the mid summer 2004.
Samples were considered positive when a 320 bp band was detected.

L: 1kbp DNA ladder with 100 bp increments

183

CHAPTER SEVEN

CONCLUSIONS AND FUTURE RESEARCH

CONCLUSIONS

Although BKD exists in Michigan since 1955, only little is known about the
status and magnitude of the disease in salmonids and water basin of Michigan.
The only available information about the disease was from publications in
Europe, Canada and Pacific Northwest. Renibacterium salmoninarum, the
causative agent of BKD grows slowly in vitro and in vivo and incubation period
might extend to up to 12 weeks before obtaining bacterial growth. Further, most
of the published studies concerning these issues showed marked inconsistency.
Bacterial Kidney Disease is focal in its distribution within the affected kidneys,
thus targeting a part of the kidney other than the focus of infection (granulomas)
might lead to negative testing results. All the aforementioned factors were the
triggering factors that lead me to develop my research on BKD in Michigan
salmonines.

Initially, in order to overcome the slow growing nature and the longer
incubation period of the pathogen, an optimized tissue processing and culture
procedures together with a modified growth medium have been developed. The
protocol and modified medium have significantly shortened the incubation time
and allowed culturing of R. salmoninarum with relative ease throughout the
study. These achievements allowed the isolation of relatively large number of R.

salmoninarum isolates from both cultured and wild salmonines. These isolates

184

constitute a unique resource for future studies of R. salmoninarum and BKD in
the Great Lakes. Findings also demonstrated that R. salmoninarum is
widespread in Michigan.

Using the analysis of different diagnostic testing patterns produced from
different agreements and disagreements in results of diagnostic BKD testing
assays (Nested PCR, Quantitative ELISA, and Culture) I was able to track the
progress of natural R. salmoninarum infection in some of Michigan’s salmonid
stocks. The disagreement in results among the three assays was linked to
different phases of R. salmoninarum infection at the time of sampling. The testing
results demonstrated the presence of diagnostic testing patterns, with each of the
patterns representing a probable stage along the course of R. salmoninarum
infection.

Data generated in chapter four of this dissertation provided additional
evidence that R. salmoninarum infection is enzootic in Michigan watersheds.
Lake Michigan’s chinook salmon tend to have overall higher R. salmoninarum
prevalence than that in Lake Huron’s fish. Data also indicated that R.
salmoninarum infection and intensity in retuming feral salmon seem to fluctuate
among years, although a definitive decrease can be observed since the start of
this study in 2001. Data of this study demonstrated the ability of both Chinook
(Oncorhynchus tshawytscha) and coho (Oncorhynchus kisutch) females and
males to shed R. salmoninarum along with gametes. The BKD testing of both

feral spawners and their hatchery raised offspring fingerlings demonstrates that

185

the current testing and culling programs have been partially successful in
reducing R. salmoninarum transmission.

In chapter five, a gradual decrease in R. salmoninarum prevalence in the
hatchery raised and wild brook trout (salve/inus fontinalis) was noticed
throughout the period from 2001 to 2004. The role-played by hatchery practices
in minimizing the spread and prevalence of BKD among Michigan brook trout
populations was discussed. Despite the fact that most of the epidemiological
studies performed in the past diminished the role played by males in
transmission of the R. salmoninarum, yet data presented in this study indicated
that males can shed a fair amount of R. salmoninarum and its soluble antigens
into the milt which increase the possibilities played by male in spreading the
disease. Also, a number of BKD outbreaks involving the brook trout have been
investigated using a number of diagnostic techniques through the duration of the
study. Analysis of results presented in this chapter confirmed the previously
reported assumption that brook trout is highly susceptible to R. salmoninarum
infection.

Finally, data generated in chapter six indicated that the non salmonid adult
sea lamprey is a new host range for R. salmoninarum and might possibly play a
role in the dissemination of R. salmoninarum during the process of relocation of
adult stages between number of watersheds in great lakes including Lake

Michigan and Lake Huron watersheds.

186

FUTURE RESEARCH

The data obtained in this dissertation established a solid base for future
research on Renibacterium salmoninarum (R. salmoninarum) infection in
salmonids and non-salmonid species in Michigan. Further research is required to
study the effect of each ingredient of the modified media on growth of the
bacteria. Further, the chemical constituents of the R. salmoninarum metablites
released in culture media or into in the infected tissue are needed to be identified
in detail and wethere there is a dissimilarity or resemblance between In vivo and
In vitro production of metabolites. Molecular basis behind the antibiotic resistence
of some R. salmoninarum strains should be fully investigated.

Shedding of R. salmoninarum in male along with the milt has been proven in
the current study. Yet, whether the bacteria are shed in the seminal fluid or
passed on within the spennatozoal cells are needed to be elucidated.

Concurrent Infections with R. salmoninarum and external parsites or other
systemic bacteria as Aeromonas salmonicida and Fla vobacterium sp. were
noticed during the current study. The sequential mechanisms of such concurrent
infections needed to be fully investigated and addressed.

Renibacterium salmoninarum was isolated for the first time from the kidneys
of the adult parsitic seas lamprey. This work requires further research to
determine the mechanism of infection in lampreys. Also, the possible role palyed

by Sea lamprey in spreading the disease to salmonids needs to be illucidated.

I87

Kidneys are the primary targets of R. salmoninarum (Fryer and Sanders,
1981); however, other organs should al30 be assessed in future studies to better

understand BKD pathogenesis, particularly in natural infections.

188

REFERENCES

Alcorn, S.W., and Pascho, R.J. (2000). “Single-dilution enzyme-linked
immunosorbent assay for quantification of antigen-specific salmonid
antibody.” J. Vet. Diagn. Invest. 12: 245-252.

Alexander, G.R., Gowing, H., and Nuhfer, A.J. (1990) “Comparative catchability,
growth and survival of two wild stocks of brown trout.” Michigan

Department of Natural Resources, Fisheries Research Report 1966, Ann
Arbor.

Alexander, S.M., Grayson, T.H., Chambers, E.M., Coopers, L.F., Barker, GA,
and Gilpin, ML. (2001). “Variation in the spacer regions separating tRNA
genes in Renibacterium salmoninarum distinguishes recent clinical
isolates from the same location.” J. Clin. Microlgiol. 39: 119-128.

Allison, L.N. (1958). “Multiple sulfa therapy of kidney disease among brook trout.”
Prog. Fish. % 20 (2): 66-68.

Amend, D.F., and Fryer, J.L. (1968). “The administration of sulfonamide drugs to
adult salmon.” Pro; Fish-Ctfl 30:168-172.

Amos, K.H. (1977). “The control of Bacterial Kidney Disease in spring chinook
salmon.” M.S. thesis, University of Idaho, Moscow. 20pp.

Anonym, (1933). “Second intrim report of the Fumnculosis committee.”
Edinburgh: H.M.S.O., 81 pp.

Armstrong, R.D., Evelyn, T.P.T., Martin, S.W., Donlvand, W. and Ferguson, H.W.
(1989). “Erythromycin levels in eggs and alevins derived from spawning
broodstock Chinook salmon (Oncorhynchus tshawytscha) injected with the

drug.” Dis. Aguat. Org. 6: 33-36.

Austin B. (1985). “Evaluation of antimicrobial compounds for the control of
Bacterial Kidney Disease in rainbow trout, Sa/mo gairdneri Richardson.” 9L
Fish Dis. 8: 209-220

Austin, B. and Austin, DA. (1999). Bacterial Fish Pathogens, Diseases in
Farmed and Wild Fish. Third Edition. Spnnger-Praxis, Chichester, UK.

Austin, B., Bucke, D., Feist, S., and Rayment, J. N. (1985). “A false positive

reaction in the fluorescent antibody test for Renibacterium salmoninarum
with a "corynefonn" organism." Bull. Eur. Assoc. Fish Pathol. 5:89.

189

Austin, B., Embley, TM, and Goodfellow, M. (1983). “Selective isolation of
Renibacterium salmoninarum.” FEMS Microbiol. I._e_tL 17: 111-114.

Austin, B., and Rayment, J.N. (1985). “Epizootiology of Renibacterium
salmoninarum, the causal agent of Bacterial Kidney Disease in salmonid
fish.” J. Fish Dis. 8: 505-509.

Austin, B. and Rodgers, C.J. (1980).”Diversity among strains causing bacterial
kidney disease in salmonid fish.” Curr. Microbiol. 3: 231-235.

Awakura, T. (1978). “Bacterial Kidney Disease in mature salmonid.” Fish Pathol.
13.53-54.

Balfry, S.K., Albright, L.J. and Evelyn, T.P.T. (1996). “Horizontal transfer of
Renibacterium salmoninarum among farmed salmonids via the fecal-oral
route.” Dis. Aguat. Org. 25:63-69.

Bandin, l., Rivas, C., Santos, Y., Secombes, C. J., Barja, J. L., and Ellis, A. E.
(1995). “Effects of serum factors on the survival of Renibacterium
salmoninarum within rainbow trout macrophages.” Dis. Aguat. Org. 23:
221-227.

Bandin, l., Santos, Y., Bruno, D.W., Raynard, R.S., Toranzo, A.E., and Barja, J.L.
(1991). “Lack of biological activities in the extracellular products of
Renibacterium salmoninarum.” C_an. J. Fish. Aguat. Sci. 48: 421 -425.

Banner, C.R., Long, J.J., Fryer, J.L. and Rohovec, J.S. (1986). ”Occurrence of
salmonid fish infected with Renibacterium salmoninarum in the Pacific
Ocean.” J. Fish Dis. 9: 273-275.

Banner C.R., Rohovec J.S. and Fryer J.L. (1983). “Renibacterium salmoninarum
as a cause of mortality among Chinook salmon in salt water.” J. World
Maricult. Soci. 14:236-239.

Banner, C.R., Rohovec, J.S. and Fryer, J.L. (1991). “A new value for mol percent
guanine+cytosine of DNA for the salmonid fish pathogen Renibacterium
salmoninarum. ” FEMS Miami. Lett.79: 57-59.

Bauer, A.W., Kirby, W.M.M., Shen'is, JC. and Turck, M. (1966). “Antibiotic
susceptibility testing by a standardized single disk method.” Am. J. Clin.
Pathol. 45: 493-496.

Beacham, TD. and Evelyn, T.P.T. (1992). “ Population and genetic variation in

resistance of chinook salmon to Vibriosis, Furunculosis, and Bacterial
Kidney Disease.” J. flat. Anim. Hlth. 4(3): 153-167.

190

Belding, D.L. and Merrill, B. (1935). “A preliminary report upon a hatchery
disease of the salmonidae." Trans. Am. Fish. Soc. 65:76-84.

Bell, GR. (1961). “Two epidemics of apparent kidney disease in cultured pink
salmon (Oncorhynchus gorbuscha).” J. Fish. Res. Board. Can. 18: 559-
562.

Bell, G.R., Higgs, DA. and Traxler, GS. (1984). “The effect of dietary ascorbate,
zinc, and manganese on the development of experimentally induced
Bacterial Kidney Disease in sockeye salmon (Oncorhynchus nerka).”
Aguacult. 36: 293-31 1.

Bell, G.R., Hoffman, R.W. and Brown, LL. (1990). “Pathology of experimental
infections of the sablefish, Anop/oma fumbria (Pallas), with Renibacterium
salmoninarum, the agent of Bacterial Kidney Disease in salmonids.” ;
Fish Dis. 13: 355-367.

Bell, G.R., Traxler, GS, and Dworschak, C. (1988). “Development in vitro and
pathogenicity of an erythromycin- resistant strain of Renibacterium
salmoninarum, the causative agent of Bacterial Kidney Disease in
salmonids.” Dis. Aguat. Org. 4: 9-25.

Beyerle, J., and Hnath, JG. (2002). “History of Fish Health Inspections State of
Miclgggn 1970-1999.” Michigan Department of Natural Resources.
Technical Report 2002-2, May, 2002.

Borgeson, DP. (1970). “Coho salmon Status Report.” Michigan Department of
Natural Resources, Fisheries Management Report 3, Lansing.

Brown, L.L., Evelyn, T.P.T., Iwama, G.K., Nelson, W.S. and Levine, RP. (1995).
“Bacterial species other than Renibacterium salmoninarium cross react
with antisera against R. salmoninarum, but are negative for the p57 gene
of R. salmoninarium as detected by the polymerase chain reaction (PCR).”
Dis. Aguat. Org. 21: 227-231.

Brown, L.L., lwana, GK. and Evelyn, T.P.T (1996). “T he effect of early exposure
of coho salmon (Oncorhynchus kisutch) eggs to the p57 protein of
Renibacterium salmoninarum on the development of immunity to the
pathogen.” Fish ang Shellfish Immunol. 6: 149-165

Brown, L.L., Iwama, G.K, Evelyn, T.P.T, Nelson, W.S. and Levine, RP (1994).

“Use of PCR to detect DNA from Renibacterium salmoninarum within
individual salmonid eggs.” Dis. Aguat. Org. 18:165-171.

191

Bruno, D.W. (1986). “Histopathology of Bacterial Kidney Disease in laboratory
infected rainbow trout, Sa/mo gairdneri Richardson, and Atlantic salmon,
Salmo salar L., with reference to naturally infected fish.” J. Fish Dis. 9:
523-537.

Bruno, D.W. (1988). “The relationship between auto-agglutination, cell surface
hydrophobicity and virulence of fish pathogen Renibacterium
salmoninarum.” FEMS Micropiol. I993. 51 :1 35-1 40.

Bruno D.W. and Munro, A.L.S. (1986a). “Uniformity in the biochemical properties
of Renibacterium salmoninarum isolates obtained from several sources.”
FEMS Microbiol. I_._eit_. 33:247-250.

Bruno D.W. and Munro, A.L.S. (1986b).“Observation on Renibacterium
salmoninarum and the salmonid egg.” Dis. Aguat. Org. 1: 83-87

Bruno, D.W. and Munro, A.L.S. (1986c). “Haematological assessment of rainbow
trout, Sa/mo gairdneri Richardson, and Atlantic Salmon, Salmo salar L.,
infected with Renibacterium salmoninarum.” J. Fish Dis. 9: 195-204.

Bullock, G.L. and Conroy, DA. and Snieszko, SF. (1971). Diseases of Fishe_s_.
Pages 94-104 in Snieszko, SF. and Axelord, H.R. (Eds.). Bacterial
Diseases of Fishes, Book 2 A. T.F.H. Publications, Inc.

Bullock, G.L., Griffin, BR, and Stuckey, HM. (1980). Detection of
Corynebacterium salmoninus by direct fluorescent antibody tests. Can. J.
Fish. Aguat. Sci. 37: 719-721.

Bullock, G.L. and Herman, R.L. (1988).Bacterial Kidney Disease of salmonid
fishes caused by Renibacterium salmoninarum. Fish Disease Leaflet 78,
US Fish and Wildlife Service, Washington, DC.

Bullock, G.L. and Stuckey, HM. (1975). “Fluorescent antibody identification and
detection of the Corynebacterium causing the Kidney Disease of
salmonids.” J. Fish. Res. Board Can 32(11): 2224-2227.

Bullock, G.L. Stuckey, HM. and Chen. PK. (1974). “Corynebacterial Kidney
Disease of salmonids: Growth and serological studies on the causative
bacterium.” Aggl. Microbiol. 28(5): 81 1-814.

Bullock, G.L., Stuckey, HM, and Mulchay, D. (1978).”Corynebcaterial Kidney

Disease: Egg transmission following iodophore disinfection. Fish Health
News. 7:51-52.

192

Campos-Perez, J.J., Ellis, A.E. and Secombes, C.J. (1997). “Investigation of
factors influencing the ability of Renibacterium salmoninarum to stimulate
rainbow trout macrophage respiratory burst activity.“ Fish and Shellfish
Immunol. 7(8): 555-566.

Chase, D.M. and Pascho, R.J. (1998). “Development of a nested polymerase
chain reaction for amplification of a sequence of the p57 gene of
Renibacterium salmoninarum that provides a highly sensitive method for
detection of the bacterium in salmonid kidney.” Dis. Aguat. Org. 34: 223-
229.

Cipriano, R.C., Starliper, CE. and Schachte, J.H. (1985). “Comparative
sensitivities of diagnostic procedures used to detect Bacterial Kidney
Disease in salmonid fishes.” J. Wildl. Dis. 21 (2): 144-148.

Clark, RD. (1996). Status of chinook salmon in the Upgr Great Lakes. 1995.
Michigan Department of Natural Resources, Fisheries Division.
Administrative Report. Lansing, Michigan.

Clifton-Hadley, R.S., Bucke, D. and Richards, R.H. (1984).”Proliferative Kidney
Disease of salmonid fish; a review.” J. Fish Dis. 7: 363-377.

Coon, TC. (1999). Icthvophong of t_he Great Lakes Basin. In: Taylor, WW. and
Ferreri, C.P. (Eds), Great Lakes Fisheries and Management, A Binational
Perspective . Michigan State University Press, East Lansing, Michigan.

Crombach W. H. J. (1978). “Caseobacter po/ymorphus gen. nov., sp. nov., a
coryneform bacterium from cheese.” Int. J. Syst. Bacteriol. 28: 354—366

Daly, J.G. and Stevenson, RM. (1985). ”Charcoal agar, a new growth medium
for the fish disease bacterium Renibacterium salmoninarum.” A I.
Envirgnment. Microbiol. 50: 868-871.

Daly, J.G. and Stevenson, RM. (1988). “Inhibitory effects of salmonid tissue on
the growth of Renibacterium salmoninarum.” Dis. Aguat. Org. 42169-171.

Daly, J.G. and Stevenson, RM. (1990). “Characterization of the Renibacterium
salmoninarum haemagglutinin.” J. Gen. Microbiol. 136: 949-953.

Daly, J. G., S. G. Griffiths, A. K. Kew, A. R. Moore and G. Oliver. 2001.
“Characterization of attenuated Renibacterium salmoninarum strains and
their use as live vaccines.” Dis. Aguat. Org. 44,121-126.

Densmore, C.L., Smith, SA. and Holladay, SD. (1998). “In vitro effects of the
extracellular protein of Renibacterium salmoninarum on phagocyte

193

function in brook trout (Salvelinus fontinalis).” Vet. Immunol.
Immunopathol. 62: 349-357.

Dexter, JL and O’Neal, P. (2004). “Michigan Fish Stocking Guidelines II: with
Periodic Updates.” State of Michigan, Department of Natural Resources,
Fisheries Division Special Report. Number 32: 67 pp.

Driver, J. (1995). “Brook Trout in Draft Broodstock Management Proposal.”
Michigan Department of Natural Resources, Fisheries Division, Lansing.

Earp, B.J., Ellis, CH, and Ordal, E.J. (1953). “Kidney Disease in young salmon.
“Special Report Ser.No.1, Department of Fisheries, Washington State,
pp.73.

Eissa, A., Elsayed, E. and Faisal, M. (2004). “First record of Renibacterium
salmoninarum isolation from the sea lamprey (Petromyzon marinas)"
Abstract, Proceeding of the 29th Annual Eastern Fish Health Workshop,
North Carolina, USA. March, 2004.

Elliot, D.G., and Barila, T.Y. (1987). “Membrane filtration - fluorescent antibody
staining procedures for detecting and quantifying Renibacterium
salmoninarum in coelomic fluid of Chinook salmon (Oncorhynchus
tshawytscha).” C_an. J. Fish. Aggat. Sgi, 44: 206-210.

Eliott, DC. and McKibben, CL. (1997). “Comparison of two fluorescent antibody
techniques (FATS) for detection and quantification of Renibacterium
salmoninarum in coelomic fluid of spawning Chinook salmon
Oncorhynchus tshawytscha.” Dis. Aguatic Org. 30: 3743.

Elliott, D.G., and Pascho, R.J. (1991). Juvenile fish transportation: impact of
Bacterial Kidney Disease on survival of spring/summer chinook salmon
stocks. Annual Report, 1989. Prepared by the US. Fish and Wildlife

Service, Seattle, Wash., for the US. Army Corps of Engineers, Walla
Walla, Wash.

Elliott, D. G. and Pascho, R. J. (1995). “Juvenile Fish Transportation: Impact of
Bacterial Kidney Disease on survival of spring/summer chinook salmon
stocks.” Annual Report, 1993. US. Army Corps of Engineers.

Elliott, D. G. and Pascho, R. J., Palmisano, A.N. (1995). ”Broodstock segregation
for the control of Bacterial Kidney Disease can affect mortality of progeny

Chinook salmon (oncorhynchus tshawytscha) in seawater. “ Aguacult.
1 32: 1 33-1 44.

Ellis, A. E. (1999)."Immunity to bacteria in fish.” Fish and Shellfish Immgnol. 9:
291-308.

194

Ellis, R.W., Novotny, A.J and Harrell, L.W. (1978).”Case Report of Kidney
Disease in wild Chinook salmon (Oncorhynchus tshawytscha) in the sea.”
J Wildl. Dis. 14: 121-123

Emery, L. (1985). “Review of fish introduced into the Great Lakes, 1819-1974."
Great Lakes Fishery Commission Technical Report, volume 45: 31 pp.

Embley, T.M., Goodfellow, M., and Austin, B. (1982). “A semi-defined growth
medium for Renibacterium salmoninarum.” FEMS Microbiol. Lett. 14: 299-
301.

European Commission. (1999). “Bacterial Kidney Disease.” Report of the
Scientific Committee on Animal Health and Animal Welfare. European
Commission, Health and Consumer Protection, Brussels, p 1— 44 (also
available at: www.europa.eu.int/comm/food/fs/sc/scah/out36 en.pt_if

Evelyn, T.P.T. (1977). “An improved growth medium for the kidney disease
bacterium and some notes on using the medium.” Bulletin de L’ Office
lntemational des Epizooties 87: 511-513.

Evelyn, T.P.T. (1993). “Bacterial Kidney Disease - BKD.” In: Inglis, V., Roberts R.
J., and Bromage, N. R. (Eds.). Bacterial Diggses of Fish. Blackwell
Scientific Publications, Oxford, UK, pp. 177-195.

Evelyn, T.P.T. (1996). “Infection and disease.” In: Iwama, G. & Nakanishi, T.
(Eds), The fig immune system. Academic Press, San Diego, United
States. pp. 339-366.

Evelyn, T. P. T., Bell, G.R., Prosperi-Porta, L., and Ketcheson, J.E. (1989). “A
simple technique for accelerating the growth of the Kidney Disease
bacterium Renibacterium salmoninarum on a commonly used culture
medium (KDM2).” Dis. Aguat. Org. 7: 231-234.

Evelyn, T.P.T., Hoskins, G.E.,and Bell, GR. (1973). “First Record of Bacterial
Kidney Disease in an apparently wild salmonid in British Columbia.” LL
Fish. Res. Board. Can. 30: 1578-1580.

Evelyn, T.P.T., Ketcheson, J.E. and Prosperi-Porta, L. (1981). “The clinical
significance of Immunofluorescence-based diagnoses of Bacterial Kidney
Disease carrier.” Fish Pflol. 15: 293-300.

Evelyn, T.P.T., Ketcheson, J. E. and Prosperi-Porta, L. (1984). “Further evidence
for the presence of Renibacterium salmoninarum in salmonid eggs and for

195

the failure of providon-iodine to reduce the intra-ovum infection rate in the
water.” J. Fish. Dis. 7:173-182.

Evelyn, T.P.T., Ketcheson, J. E. and Prosperi-Porta, L. (1986 a). “Experimental
intraovum infection of salmonid eggs with Renibacterium salmoninarum
and vertical transmission of the pathogen with such eggs despite their
treatment with erythromycin.” Dis. Aggat. Org._1: 197-202.

Evelyn, T.P.T., Ketcheson, J.E., and Prosperi-Porta, L. (1986 b). “Use of
erythromycin as a means of preventing vertical transmission of
Renibacterium salmoninarum.” Dis. Aggat. Org. 2:7-11.

Evelyn, T.P.T., Prosperi-Porta, L., and Ketcheson, J.E. (1990). “Two new
techniques for obtaining consistent results when growing Renibacterium
salmoninarum on KDM2 culture medium.” Dis. Aguat. Org. 9: 209-212.

Evenden, A.J., Grayson, T. H., Gilpin, M. L. and Munn, C. B. (1993).
“Renibacterium salmoninarum and Bacterial Kidney Disease - the
unfinished jigsaw.” flin. Rev. Fish % 3287-104.

Evensen, 0., Dale, OB, and Nilsen, A. (1994). “lmmunohistochemical
identification of Renibacterium salmoninarum by monoclonal antibodies in
paraffin-embedded tissues of Atlantic salmon (Salmo salar L.), using
paired immunoenzyme and paired immunofluorescence techniques.” i

Vet. Diagn. Invest. 6: 48-55.

Faisal, M. and Hnath, J.G. (2005).”Fish health and dise_a_ses issues in t_hg
La_urenti_arn Great Lakes.” Pages 331- 350, In: Cipriano, R.C.,
Shchelkunov, I. S., and Faisal, M., editors. Health and Diseases of Aquatic
Organisms: Bilateral Perspectives. Proceedings of the Second Bilateral
Conference between Russia and the United States. 21 - 28 September
2003. Shepherdstown , West Virginia. Michigan State University. East
Lansing, Michigan.

Feltham, R.K.A., Power, A.K., Pell, RA. and Sneath, P.H.A. (1978). “A simple
method for storage of bacteria at -76 degree C.” J. Appl. Bact. 44:313-16.

Ferguson, H.W. 1989. “Systemic pathology of fish.” Iowa State University Press.
Ames, United States. 263 pp.

Flafio, E., Kaattari, S.L., Razquin, B. and Villena, A.J. (1996). “Histopathology of
the thyumus of coho salmon Onchorhynchus kisutch experimentally
infected with Renibacterium salmoninarum.” Dis. Aguat. Org. 26: 11-18

Frantsi, C. J., Flewelling, TC. and Tidswell, K.G. (1975).”Investlgations on
Corynebacterial Kidney Disease and Diplostomulum sp. (eye-fluke) at

196

Margaree Hatchery, 1972-1973,” Technical Seport Serial No. Mar/T-75-9.
Research Development Branch, Fish and Marine Services, Department of
Environment. Halifax, Nova Scotia, Canada. 30 p.

Fredriksen, A., Endresen, C. and Wergeland, H. I. (1997). “lmmunosuppressive
effect of a low molecular weight surface protein from Renibacterium
salmoninarum on lymphocytes from Atlantic salmon (Salmo salar L.)”. E_i_s_h,
999 Shellfish lmmgnol. 7: 273-282.

Freichs, G. and Roberts, R. (1989): “Fishfltologfi (Roberts, R., Second Edition)
Bailliere Tindal, London.

Fryer, J.L. and Sanders, J.E. (1981).”Bacterial Kidney Disease of Salmonid Fish.”
@nRgvMicrobiol. 35: 273-298.

Fryer, J.L. and Lannan, ON. (1993). “The history and current status of
Renibacterium salmoninarum, the causative agents of Bacterial Kidney
Disease in Pacific salmon.” Fish. Res. 17:15-33.

Getchell, RG. and Rohovec, J.S., and. Fryer, J.L. (1985). “Comparison of
Renibacterium salmoninarum isolates by antigenic analysis.” Fish Pathol.
20:149-159.

Goodfellow, M., Embley, TM. and Austin, B. (1985). Numerical taxonomy and
emended description of Renibacterium salmoninarum. J._§en. Microbfigl.
131: 2739-2752.

Gowing, H. (1986). “Survival and growth of matched plantings of Assinca strain
brook trout and hybrid brook trout (Assinca x domestic female) in six small
Michigan lakes.” North Am. J. Fish. Management. 6: 242-251

Grayson, T. H., Bruno, D. W., Evenden, A. J., Gilpin, M. L. and Munn, C. B.
(1995). “Iron acquisition by Renibacterium salmoninarum; contribution by
iron reductase.” Dis. Aguat. Om. 22:157-162.

Grayson, T.l-I., Coopers, L.F., Wrathmell, A.B., Roper, J., Evenden, A.J. and
Gilpin, M.L. (2002). “Host responses to Renibacterium salmoninarum and
specific components of the pathogen reveal the mechanisms of immune
suppression and activation”. J. Immnol. 106: 273-283.

Groman, BD. and Klontz, CW. (1983). “Chemotherapy and prophylaxis of
Bacterial Kidney Disease with erythromycin.” J. World Maricult. Soci. 14:
226-235.

Gudmondsdottir, S., Benediksdottir, E. and Helgason, S. (1993).”Detection of
Renibacterium salmoninarum in salmonid kidney samples: a comparison

197

of results using double sandwich ELISA and isolation on selective
medium.” J. Fish Dis. 16:185-195.

Gutenberger, S. K.,Giovannoni, S.J., Field, K.G., Fryer, J.L. and Rohovec, J.S.
(1991). “A phylogenetic comparison of the 168 rRNA sequence of the fish
pathogen, Renibacterium salmoninarum, to Gram-positive bacteria.”
FEMS Micropiol. L_e_rt_. 77: 151-156.

Gutenberger, S. K., Duimstra, J. R., Rohovec, J. S. and Fryer, J. L. (1997).
“Intracellular survival of Renibacterium salmoninarum in trout mononuclear
phagocytes.” Dis. Aguat. Org. 28:93-106.

Hamel, O. (2001)."The Dynamic and effects of Bacterial Kidney Disease in
Snake River spring chinook salmon (Oncorhynchus tshawytscha).” Ph. D.
Dissertation. University of Washington. Washington, DC.USA.

Harrell, L. W., C. K. Rathbone, M. E. Peterson, F. T. Poysky, M. J. Crewson and
M. S. Strom. 2000. Observations on the Efficacy of Azithromycin for
Chemotherapy of Bacterial Kidney Disease. Western Fish Disease
Workshop, Gig Harbor, WA, June 28-29, 2000.

Hardie, L. J., Ellis, A. E. and Secombes, C. J. (1996). “In vitro activation of
rainbow trout macrophages stimulates inhibition of Renibacterium
salmoninarum growth concomitant with augmented generation of
respiratory burst products.” Q3. Aggat. Org. 27:175-183.

Haukenes, AH and Moffitt, CM, (1999). “Concentrations of erythromycin in
maturing salmon following intraperitoneal injection of two drug
formulations.” J. Aguat. Anim. Hlth 11: 61-67.

Hayakawa,Y., Harada,T., Hatai,K., Kubota, S.S., Bunya, T. and Hoshiai, G.
(1989). “Histopathology of BKD (Bacterial Kidney Disease) occurred in

Sea-cultured Coho Salmon (Oncorhynchus kisutch).” Fish Pathol. 24 (1 )2
1 7-21 .

Hicks, B.D., Daly, J.G., Ostland, V.E. (1986). “Experimental infection of minnows
with Bacterial Kidney Disease bacterium Renibacterium salmoninarum.”

Abstract, Third Annual Meeting, Aquaculture Association of Canada, July
1986, Guelph, Ontario.

Hnath, J. and Faisal, M. (2005). Bacterial Kidney Disease: Historical

gersgectives. Technical Memorandum. Michigan Department of Natural
Resources, Lansing, Michigan.

Hoffmann, R.W., Bell, G.R., PfeiI-Futzien, C., and Ogawa, M. (1989). “Detection
of Renibacterium salmoninarium in Tissue Sections by Different Methods-

198

a Comparative Study with Special Regard to the indirect
Immunohistochemical Peroxidase Technique.“ Fish Pathol. 24(2): 101-
104.

Holey, M.E., Elliot, R.E., Marcqueneski, S.V., Hnath, J.G. and Smith, K.D. (1998).
Chinook salmon epizootics in Lake Michigan: Possible contributing factors
and management implications. J. Aguat. Anim. HILL); 10: 202-210.

Holt, J.G., Krieg, N.R. Sneath, P.H.A., Staley, J.T. and Williams, ST. (2001).
“Genus Renibacterium” in Bergey' 3 Manual of Detenninative
Bacteriology, 9th Ed., The Williams & Wilkins 00., Baltimore, pp. 567-568.

Hong, I; Longying, G; Xiujie, S and Yulin J. (2002). “Detection of Renibacterium
salmoninarum by nested-PCR.” J. Fish China / Shuichan Xuebao. 26(5):
453-458

Hongslo, E., T., Lindberg, R., Ljungberg, O. and Svensson, BM. (1991).
Detection of Reniacterium salmoninarum and Yersinia ruckeri by the
peroxidase-antiperoxidase Immunohistochemical technique in melanin-
containing cells of fish tissues. J. Fish Dis. 14:689-692.

Hopwood DA, and Ferguson HM. (1969). “A rapid method for lyophilizing
Streptomyces culture.“ J. Appl. Bact. 32: 434—6.

Hoskins, G. E., Bell, G. R., and Evelyn, T. P. T. (1976). “T he occurrence,
distribution and significance of infectious disease and neoplasms
observed in fish in the Pacific Region up to the end of 1974.” Fish. Mar.
§erv. Res_.Dev. Tgch. Reg 609: 37.

l-Isu, H., Bowser, PR. and Schachte, J.H. (1991). “Development and evaluation
of a monoclonal-antibody-based enzyme —linked immunosorbent assay for

the diagnosis of Renibacterium salmoninarum infection.” J. Aguat. Anim.
filt_h 3:168-175.

Hsu, H., Wooster, GA. and Bowser, PR. (1994). “Efficacy of Enrofloxacin for the
Treatment of Salmonids with Bacterial Kidney Disease, Caused by
Renibacterium salmoninarum.” J. Aguat. A_nim. Hlt_I_i, 6(3): 220-223.

Humphrey J.D., Lancaster C.E., Gudkovs, N. and Copland, J.W. (1987). “The

Disease status in Australia of Australian salmonids: bacteria and bacterial
diseases.” J. Fishfls. 10: 403—410.

Jansson, E. (2002). Bacterial Kidney Disease in salmonid fish: Development of
methods to assess immune functions in salmonid fish during infection by

199

Renibacterium salmoninarum. Doctoral Thesis, University of Upsala,
Sweden.

Jansson, E., Hongslo, T., Lindberg, R., Ljungberg, O., and Svensson, B. M.
(1991). “Detection of Renibacterium salmoninarum and Yersinia ruckeri by
the peroxidase-antiperoxidase immunohistochemical technique in
melanin-containing cells of fish tissues.” J. Fish Dis. 14: 689-692.

Jansson, E., Hongslo, T., Hoglund, J. and Ljungberg, O. (1996): “Comparative
evaluation of bacterial culture and two ELISA techniques for the detection
of Renibacterium salmoninarum antigens in salmonid kidney tissues.” 1%
Aguat. Org. 27:197-206.

Johnson, DC. and Hnath, JG. (1991). “Lake Michlggn Chinook Simon mortality-
1988”. Michigan Department of Natural resources, Fisheries Division
Technical Report No. 91 -4.1 1 pp.

Jonas, J.L., Schneeberger, P.J., Clapp, D.F., Wolgamood, M., Wright, G., and
Lasee, B. (2002). “Presence of the BKD-causing bacterium Renibacterium
salmoninarum in lake Whitefish and bloaters in the Laurentian Great
Lakes”. Arch. Hydrobiol. Spec. Issues Advanmnnol. 57: 447-452.

Jensdottir, H., Machuist, H.J., Snorrason, S.S., Gudbergssons, G. and
Gudmundsdottir, S. (1998). “Epidemiology of Renibacterium
salmoninarum in wild Arctic Charr and brown trout in Iceland.” J. Fish Biol.
53: 322-339.

Kebus, M.J., Collins, MT. and Brownfield, MS. (1992). “Effects of rearing
Density on the stress response and growth of rainbow trout
(Oncorhynchus mykiss).” J. Aguat. Anim. Hlth. 4:1-6.

Keller, M, Smith, K.D. and Rybicki, R.W. (1990).”Review of salmon and trout
management in l_.ake Michigan.” Fisheries Special Report No.14. Michigan
Department of Natural Resources. Charlevoix Fisheries Station. Box 205.
Charlevoix, Michigan 49720. April 1990.254.

Kent, M.L., Traxler, G.S., Kieser, D., Richard, J., Dawe, S.C., Shaw, R.W.,
Prosperi Porta, L. , Ketcheson, J. , and Evelyn, T.P.T (1998). “Survey of
salmonid pathogens in ocean-caught fishes in British Columbia, Canada.”
J. Aguat. Anim. Hlth 10(2): 2311-219.

Kettler, S., Pfeilputzlen, C. and Hoffman, R. (1986).”Infection of grayling

(Thymallus) with the agent of Bacterial Kidney Disease (BKD).” Bull. Eur.
Assoc. Fish Pathol. 6: 69-71.

200

Kimura, T. and Yoshimizu, M. (1981).”Rapid Methods for Detection of bacterial
Kidney Disease of salmonid (BKD) by coagglutination of antibody
sensitized protein A- containing Staphylococci.” Bull. Jap. Soc. Sci. Fish.
47 (9): 1173-1183. In Japanese with English summary. SFA 27 (1 ). (Lab.
Microbiol., Fac. Fish, Hokkaido Univ, Minato-3, Hakkodate 041, Japan).

Klontz, G.W. (1983). “Bacterial Kidney Disease irfialmonids: An Overview.” In:
Anderson, D.P., Dorson, M. and Dubourget, P.H. (Eds). Antigens of Fish
Pathogens: Development and Production for Vaccines and
Serodiagnostics. Collection Fondation Marcel Merieux, Lyons, France,
pp.1 77-200.

Kocik, J.E. and Jones, M.L. (1999).”Pacific salmonines and the Great Lakes
Basin." In “Great Lakes Fisheries Policy and Management: A Binational
Perspective", ed. W.W. Taylor and GP. Ferreri, pp. 455- 488. East
Lansing, MI: Michigan State University Press

Lee, E.G.H. (1989). “Technique for Enumeration of Renibacterium salmoninarum
in Fish Kidney Tissues.” J. Aguat. Anim. Hlth. 1:25-28.

Lee, E. and Evelyn, T.P.T. (1994). “Prevention of vertical transmission of the
Bacterial Kidney Disease agent Renibacterium salmoninarum by
broodstock injection with erythromycin.” Dis. Aguat. Org.18: 1- 4.

Lorenzen, E., Olesen, N.J, Korsholm, H., Heuer, OE, and Evensen, DE.
(1997). “First Demonstration of Renibacterium salmoninarum/ BKD in
Denmark.” Bull. Europ. Assoc. Fish Pa_thol.17 (3-4) 140-144

Lupi, F. and Hoehn, JP (1998). A partial benefit - cost analysis of sea lamprey
treatment options on the St. Marys River. A report submitted to the Great
Lakes Fishery Commission. Department of Agricultural Economics,
Michigan Sate University, East Lansing, Michigan, USA.

MacLean, D.G., and Yoder, W.G. (1970). “Kidney Disease among Michigan
salmon in 1967.” Progressive Fi§h. Cult. 32: 26-30.

lVIakos, B.K. and Youson, J.H. (1988).”Tissue levels of bilirubin and biliverdin in
the sea lamprey, Petromyzon marinus L., before and after biliary atresia."
Comp. Biochem. Physiol. A. 91 (4): 701-710.

Marcqueneski, S., Hnath, J., Homer, R., Eggold, P., Boronow, G., and Peeters,
P. (1997). “Effect of spring epizootics on the Lake Michigan chinook sport
fishery.” 59th Midwest Fish and Wildlife Conference, Milwaukee,
Wisconsin, December, 7-10, 1997.

201

Maule, A.G., Rondorf, D.W., Beeman, J., and Haner, P.(1996)." Incidence of
Renibacterium salmoninarum infections in juvenile hatchery spring

chinook salmon in the Columbia and Snake Rivers.” J. Aguat. Anim. Hlth.
8 (1): 37-46.

McIntosh, D., Austin, 3., Flal‘io, E., Villena, J.A., Tarazona, Martinez-Pereda and
J.V. (2000). Lack of uptake of Renibacterium salmoninarum by gill
epithelia of rainbow trout. J. Fish. Biol. 56: 1053-1061.

McIntosh D., P.G. Meaden and B. Austin 1996. A simplified PCR-based method
for the detection of Renibacterium salmoninarum utilizing preparations of
rainbow trout (Oncorhynchus mykiss, Walbaum) lymphocytes. Applied
Environmental Microbiology 62, 3929-3932.

Mesa, M.G., Maule, A.G., Poe, TR and Schreck, CB. (1999). “Influence of
Bacterial Kidney Disease on smoltification in salmonids: is it a case of
double jeopardy?” Aguacult. 174: 25-41.

Meyers.T.R., Korn, 0., Glass, K., Burton, T., Short, 8., Lipson, K. and Starkey,
N. (2003). “Retrospective Analysis of Antigen Prevalences of
Renibacterium salmoninarum (R. salmoninarum) Detected by Enzyme -
Linked Immunosorbent Assay in Alaskan Pacific Salmon and Trout from
1988-2000 and Management of R. salmoninarum in hatchery Chinook and
Coho salmon.” J. Aguat. Anim. Hlth. 15: 101-110.

Meyers. T.R., Short, 8., Farrington, C., Lipson, K., Geiger, H.J. and Gates, R.
(1993). “Establishment of negative —positive threshold optical density
value for the enzyme —Iinked immunosorbent assay (ELISA) to detect
soluble antigen of Renibacterium salmoninarum in Alaskan Pacific
salmon.” Dis Aguat. Org. 16: 191 -197.

Miriam A., Griffiths, S.G., Lovely, J.E. and Lynch, W.H. (1997). “PCR and Probe-
PCR assays to monitor broodstock Atlantic salmon (Salmo salar L.)
ovarian fluid and kidney tissues for the presence of DNA of the fish
pathogen Renibacterium salmoninarum.” J. Clin. Microbiol. 35:1322-1326.

Mitchum, D.L., Sherman, LE. and Baxter, GT. (1979). “Bacterial Kidney Disease
in Feral Populations of Brook Trout (Salvelinus fontinalis), Brown Trout

(Salmo trutta), and Rainbow Trout (Salmo gairdneri)” J. Fish Res. Board
Can. 36: 1370-1376.

Mitchum, D. L. and Sherman, L. E. (1981).”Transmisslon of Bacterial Kidney

Disease from wild to stocked hatchery trout.” C_an. J. Fish. Aguat. Sci.
36:547-551.

202

MO TA. (1997) Seasonal occurrence of Gyrodacly/us derjavini (Monogenea) on
brown trout, Sa/mo trutta, and Atlantic salmon, S. salar, in the
Sandvikselva River, Nonlvay. J. Parasitol. 83:1025—1029.

Moffitt CM. (1991). “Oral and injectable applications of erythromycin in salmonid
fish culture.” Vet. Hpman. Toxjcol. 3: 49-53.

Moffitt, CM. (1992). “Survival of juvenile chinook salmon challenged with
Renibacterium salmoninarum and administered oral doses of erythromycin
thiocyanate for different durations". J. Aguat. Anim. Hlth 4: 1 19-125.

Moffitt, CM. and Bjornn, TC. (1989). “Protection of chinook salmon smolts with
oral doses of erythromycin against acute challenges of Renibacterium
salmoninarum.” J. Aguat. A_nim. Hlt_l1 1: 227-232.

Murray, C.B., Evelyn, T.P.T., Beacham, T.D., Ketcheson, L.W. and Prosperi-
Porta, L. (1992). “Experimental induction of bacterial kidney disease in
chinook salmon by immersion and cohabitation challenges.” Dis. Aguat.
Org 12: 91-9.

Munro, A.L.S. and Bruno, D.W. (1988).“Vgccination against Bacterial Kidney
Disease.” In: Ellis, AE (Ed.) Fish Vaccination, Academic Press, London,
England. pp125-134.

O’Farrell, C. L. Elliott, D. G. and. Landolt, M. L. (2000). “Mortality and kidney
histopathology of chinook salmon Oncorhynchus tshawytscha exposed to
virulent and attenuated Renibacterium salmoninarum strains.” Dis. Aguat.
Org 43:199-209.

OIE, Office lntemational des Epizooties. (2003). “Bacterial kidney disease
(Renibacterium salmoninarum)” In: Manual of Diagnostic Tests for
Aquatic Animals, pp 94—104. 4th Edition. Paris.

Olea, l., Bruno, D.W. and Hastings, TS. (1993). “Detection of Renibacterium
salmoninarum in naturally infected Atlantic salmon, Salmo salar L., and rainbow
trout, Oncorhynchus mykiss (Walbaum) using an enzyme-linked immunosorbent
assay. Aguacult. 116: 99- 110.

Olsen, A.B., Hopp, P., Binde, M., Gronstol, H. (1992). Practical aspect of
bacterial culture for the diagnosis of Bacterial Kidney Disease (BKD).” Q13;
Aguat. Org.14: 207-212

Ordal, E.J. and Earp, B.J. (1956).”Cultivation and transmission of the etiological

agent of Bacterial Kidney Disease.” Proceeg. Soc. Experiment. gol. Med.
92: 85-88.

203

Paclibare, J.O., Albright, L.J. and Evelyn, T.P.T. (1988). “Investigations on the
occurrence of the Kidney Disease Bacterium Renibacterium
salmoninarum in non-salmonids on selected farms in British Columbia.”

Bull.AguacuIt. Assoc. Canada 88 (4): 113-115.

Pascho, R. J., Chase, D. and McKibben, C. L. (1998).”Comparison of the
membrane-filtration fluorescent antibody test, the enzyme-linked
immunosorbent assay and the polymerase chain reaction to detect
Renibacterium salmoninarum in salmonid ovarian fluid.” J. Vet. Diagn.
Invest. 10: 60-66.

Pascho, R.J. and Elliot, D.G. (2004).”Bacterial Kidney Disease” in AFS - FHS
(American Fisheries Society-Fish Health Section) FHS blue book:
“Suggested procedures for the detection and identification of certain finfish
and shellfish pathogens, 2004 edition.” AFS-FHS, Bethesda, Maryland.

Pascho, R. J., Elliot, D.G., Mallet, R.W., Mulcahy, D. (1987). “Comparison of five
techniques for the detection of Renibacterium salmoninarum in adult coho
salmon.” mans. gm. Fish Soc. 116: 882-890

Pascho, R.J., Elliot, D.G. and Streufert, J.M. (1991). “Broodstock segregation of
spring chinook salmon Oncorhynchus tshawytscha by use of enzyme-
Iinked immunosorbent assay (ELISA) and the fluorescent antibody
technique (FAT) affects the prevalence and levels of Renibacterium
salmoninarum infection in progeny.” Dis. Aguat. Org. 12: 25-40.

Pascho, R. J., Elliott, D. G. and Achord, S. (1993). “Monitoring of the in-river
migration of smolts from two groups of spring chinook salmon,
Oncorhynchus tshawytscha (Walbaum), with different profiles of

Renibacterium salmoninarum infection." Aguacult. Fish. Management
24: 1 63-1 69.

Pascho, R. J. and Mulcahy, D. (1987). “Enzyme-linked immunosorbent assay for
a soluble antigen of Renibacterium salmoninarum, the causative agent of

salmonid bacterial kidney disease.” an. J. Fish Aguat. Sci. 44(1): 183-
191.

Paterson, WD., Gallant, C., Desautels, D. and Marshall, L. (1979). ”Detection of
Bacterial Kidney Disease in wild salmonids in the Margaree River system
and adjacent waters using an indirect fluorescent antibody technique.” i
Fish. Res. Board Can. 36: 14644 468.

Paterson, W.D., Lall, SP. and Desantels, D. (1981). “Studies on Bacterial Kidney

Disease in Atlantic salmon (Salmo salar) in Canada.” Fish Pathol. 15: 283-
292.

204

Peddie, S. (2004).”Nephrocalcinosis.” Fish fsrrneg 27(3): 37.

Pickering, AD, and Pottinger, TC. (1989). “Stress responses and diseases
resistance in salmonid fishes: effects of chronic elevation of plasma
cortisol.” Fish Physiol. Biochem. 7:253-258.

Piganelli, JD., Wiens, GD., Xhang, J.A., Christensen, J.M. and Kaattari, S.L.
(1999). “Evaluation of a whole cell p57- vaccine against Renibacterium
salmoninarum.” Dis. Aguat. Org. 36:37-44.

Pippy, J.H.C. (1969). ”Kidney Disease in Juvenile Atlantic salmon (Salmo sa/ar)
in the Margaree River.” J. Fish. Res. Board. Can. 26:2535-2537.

Price CS, and Schreck CB. (2003). “Stress and saltwater-entry behavior of
juvenile chinook salmon (Oncorhynchus tshawytscha): conflicts in
physiological motivation.” C_an. J. Fish. Aguat. Sci. 60: 910-918.

Pridham TC. and Hesseltine CW. (1975). “Culture collections and patent
depositions." Adv.AppI. Microbiol. 19: 1-23.

Prophet, E., Mills, B., Arrington, J. (1992). “Laboratory Methods in
Hlstotechnology.” Armed Forces Institute of Pathology, pp 131-132.

Rathbone, C. K. Harrell, L. W, Peterson, M. E., Poysky, F. T. and Strom, M. 8..
(June 9-11, 1999). “Preliminary Observations on the Efficacy of
Azithromycin for Chemotherapy of Bacterial Kidney Disease.” Western
Fish Disease Workshop, Fish Health Section/American Fisheries Society
Annual Meeting, Twin Falls, Idaho,

Reno, PW. (1998). Factors Involved in the dissemination of disease in fish
populations.” J. Aguat. Anim. Hlth. 10: 160-171

Rhodes, LD., Rathbone, CK., Corbett, SC., Harrell, LW., Strom, MS. (2004).”
Efficacy of cellular vaccines and genetic adjuvants against Bacterial
Kidney Disease in chinook salmon (Oncorhynchus tshawytscha).” _Ei_s_p
Shellfish lmmgnol. 16(4): 461-474

Richards, R.H., Roberts, R.J. and Schlotfeldt, H-J (1985). “Bakterielle
Emrankungenper K_nochenfische.” In: Roberts, R.J. and Schlotfeldt, H-J
(Eds), Grundlagen der Fischpathologie, Verlag Paul Parey, Hamburg,
gerrnany, pp. 174—207

Rimaila—Pamanen (2002). “First case of Bacterial Kidney Disease (BKD) in

whitefish (Coregonus. Iavaretus) in Finland.” Bull. Eur. Assoc. Fish
Pathol., 22 (6): 403-404.

205

Rintamaki, P., and Valtonen, E.T. (1994).”Occurrence of Gyrodacty/us sa/aris at
four fish farms in northern Finland”. University of California, School of
Veterinary Medicine, Davis, CA, USA. pp. W-13.1

Rockey, D.D., Gilkey, L.L., Wiens, G.D. and Kaattari, S.L. (1991).”Monoclonal
antibody based analysis of the Renibacterium salmoninarum p57 protein
in spawning chinook and coho salmon.” J. Agpat. Anim. Hlt_h_. 3:23-30.

Ross, A.J., and Smith, GA. (1972). “Effect of two iodophores on bacterial and
fungal fish pathogens.” J. Fish. Res. Board. Canada. 29: 1359-1361

Ross, A. J. and Toth, R. J. (1974). “Lactobaci/Ius - a new fish pathogen? “Prog.
Fish Culturist. 36: 191.

Rucker, R.R., Berrner, A.F., Whipple, W.J., and Burrows, RE. (1951).
“Sulfadiazine for Kidney Disease.” ProLFish. Cult. 13 (3): 135-137.

Rucker, R.R., Earp, B.J. and Ordal, E.J. (1954). “Infectious diseases of pacific
salmon." T_rans. Pym. Fish. Soc. 83: 297-312.

Sakai, M., Atsuta, S. and Kobayashi, M. (1989). “Comparison of methods used to
detect Renibacterium salmoninarum, the causative agent of bacterial
Kidney Disease.” J. Aguat. Anim. Hlth. 1:21-24.

Sakai, M., Atsuta, S., and Kobayashi, M. (1993). ”The immune response of
Rainbow trout (Oncorhynchus mykiss) injected with five Renibacterium
salmoninarum bacterins.” Aguacult. 113: 11-18.

Sakai, MS. and Kobayashi, M. (1992). “Detection of Renibacterium
salmoninarum the causative agent of Bacterial Kidney Disease in
salmonid fish, from pen-cultured coho salmon.” Appl. Environment.
Microbiol. 58:1061-1063

Sakai, D.K., Nagata, M., Iwami, T., Koide, N., Tamiya, Y., Ito, Y. and Atoda, M.
(1986). “Attempt to control BKD by dietary modification and erythromycin
chemotherapy in hatchery-reared Masu salmon Oncorhynchus masou

Brevoort.” Bulletin of the Japanese Sociery of Scientific Fisheries. 52:
1 141 -1 147.

Sakai, M., Terutoyo, Y., and Masanori, K. (1995). ”Influence of the immuno-
stimulant, EF203, on the immune responses of rainbow trout
(Oncorhynchus mykiss) to Renibacterium salmoninarum.” Aguacult. 1 38:
61-67.

Sami, S., Fischter-Scherl, T., Hoffman, R.W. and Pfeil-Putzien, C. (1992).
”Immune complex mediated glomerulonephritis associated with bacterial

206

Kidney Disease in the rainbow trout (Oncorhynchus mykiss).” Vet. Pathol.
29: 1 69-1 74.

Sanders, J.E. and Barros, JR. (1986). “ Evidence by the fluorescent antibody
test for the occurrence of Renibacterium salmoninarum among salmonid
fish in Chile.” J. Wildl. Dis. 22: 255-257.

Sanders, J.E. and Fryer, J.L. (1980). “Renibacterium salmoninarum gen. nov.,
sp. nov., the causative agent of bacterial kidney disease in salmonid
fishes.” lntem. J. System. ficteriglr 30:496-502.

Sanders, J.E., Long, J.J., Arakawa, C.K., Bartholomew, J.L., and Rohovec,

J.S. (1992).”Prevalence of Renibacterium salmoninarum among downstream-
migrating salmonids in the Columbia River.” J. Aguat. Anim. Hlth. 4: 72-
75.

Sanders, J.E., Pilcher, KS. and Fryer, J.L. (1978). “Relation of water
temperature to Bacterial Kidney Disease in coho salmon (Onchorhynchus
kisutch), sockeye salmon (O. nerka), and steelhead trout (Salmo
gairdneri).” J. Fish. Res. Board can. 35:8-11

Secombes, C.J. (1985)."The in vitro formation of teleost multinucleate giant
cells.” J. Fish Dis. 82461-464.

Seelbach, P.W. (1985).”Smolt migration of wild and hatchery-raised coho and
chinook salmon in a tributary of Northern Lake Michigan.” Michigan
Department of Natural Resources, Fisheries Research Report 1935, Ann
Arbor.

Senson, P.R, Stevenson, R.M. (1999).”Production of the 57 kDa major surface
antigen by a non-agglutinating strain of the fish pathogen Renibacterium
salmoninarum.” Dis. Aguat. Org. 38 (1): 23-31

Shieh, HS. (1988). “An extracellular toxin produced by fish Kidney Disease
bacterium, Renibacterium salmoninarum.” Microbios. Leg. 38: 27-30.

Siegel, D. C. and Congleton, J. L. (1997). “Bactericidal activity of juvenile
chinook salmon macrophages against Aeromonas salmonicida after
exposure to live or heat-killed Renibacterium salmoninarum or to soluble
proteins produced by R. salmoninarum.” J. Aguat. Arrim. Hlt_h. 9:180—189.

Smith, I.W. (1964).” The occurrence and pathology of Dee Disease”. Freshwater
won Fish. Res. 3421-13.

207

Smith, BR, and Tibbles, J.J. (1980). “Sea lamprey (Petromyzon mar/nus) in
lakes Huron, Michigan and Superior: History of invasion and control, 1936-
1978.” Qgrn. J. Fish. Aguat. Sci. 37:1780-1801

Snieszko, SF. and Griffin, P.J. (1955). “Kidney Disease in brook trout and its
treatment.” Progressive Fish. Cult. 1723-13.

Sompuram, S.R., Vani, K., Messana, E., and Bogen, S.A. (2004).”A molecular
mechanism of formalin fixation and antigen retrieval.” Am. ngin. Pathol.
121: 190-199.

Speare, DJ. (1997). “Differences in patterns of meningoencephalitis due to
Bacterial Kidney Disease in farmed Atlantic and chinook salmon. Res. Vet.
Sci 62: 79-80.

Starliper, CE. (1996). “Genetic diversity of North American isolates of
Renibacterium salmoninarum.” Dis. Aguat. Org. 27: 207-213

Starliper, CE, Smith, DR. and Shatzer, T. (1997). “Virulence of Renibacterium
salmoninarum to salmonids.” J. Aggat. Am. Hlt_ty 9:1-7.

Starliper, C.E., William, BS, and Jay, M. (1998). “Performance of serum-free
broth media for growth of Renibacterium salmoninarum.” Dis. Aguat. Org.
34:21-26.

Stuart, SE, and Welshimer, H. J. (1974).”Taxonomic reexamination of Listeria
pirie and transfer of Listeria grayi and Listeria murrayi to a new genus
Murraya.” Int. J. Syst. Bacteriol. 24: p 177-185.

Suzumoto, B.K., Schreck, CB, and McIntyre, JD. (1977). Relative resistances
of three transferring genotypes of coho salmon (Oncorhynchus kisutch)
and their hematological responses to Bacterial Kidney Disease. J. Fish.
Res. Board of Canada. 34: 1-8.

Tanaka, K., Teraoka, H., Tamaki, M. Otaka, E., Osawa, S. (1968). Erythromycin-
resistant mutant of Escherichia coli with altered ribosomal protein
components. Science 162: 576-578.

Teska, JD. (1993). “In vitro enhancment of Renibacterium salmoninarum: the
aetiology of Bacterial Kidney Disease.” Biomed. Lett. 48: 27-33.

Teska, JD. (1994). “In vitro growth of the Bacterial Kidney Disease organism

Renibacterium salmoninarum on a nonserum, noncharcoal-based”
homospecies-metabolite” Medium.” J. Wildl. Dis. 30 (3): 383-388

208

Traxler, G. and Bell, G. (1988). “Pathogens associated with impounded Pacific
herring C/upea harengus pallasi, with emphasis on viral erythrocytic
necrosis (VEN) and atypical Aeromonas salmonicida." Dis. Aguat. Org. 5:
93-1 00.

Turaga, P. S., Weins, G. D. and Kaattari, S. L. (1987). “Analysis of
Renibacterium salmoninarum antigen production in situ.” Fish Pathol. 22:
209-214.

Weisblum, B., Siddhikol, C., Lai, OJ. and Demohn, V. (1971). Erythromycin-
inducible resistance in staphylococcus aureus, requirements for induction.
J. Bacteriol. 106: 835-847

Wedemeyer G. A., Ross, A. J. (1973). Nutritional factors in the biochemical
pathology of Corynebacterial Kidney Disease in the coho salmon
(Oncorhynchus kisutch). J. Fish. Res. Board Canada. 30:296—298.

Wellington, E.M.H., Williams, ST. (1979). “Preservation of actinomycete
inoculum in frozen glycerol.” Micropios. lg. 62151-157.

White, M.R., Wu, CC. and Albregts, S.R. (1995).”Comparison of diagnostic tests
for bacterial kidney disease in juvenile steelhead trout (Onchorhynchus
mykiss). “ . Vet. Diagn. Invest. 7(4): 494-499.

Wiens, GB. and Kaattari, S.L. (1989).”Monoclonal antibody analysis of common
surface protein (3) of Renibacterium salmoninarum.” Fish Pathol. 24:1-7.

Wiens, G.D. and Kaattari, S.L. (1991).”Monoclonal antibody characterization of a
leukoagglutinin produced by Renibacterium salmoninarum.” Infect.
Immunol. 59: 631-637.

Wiens, G.D. and Kaattari, S.L. (1999). “Bacterial Kidney Disease (Renib!acterii_l_m
salmoninarum)”. In: Woo, P.T.K. and Bruno, D.W. (eds.), Fish Diseases
and Disorders, CABI Publishing, New York, USA. pp. 269-302.

Wiens, G.D., Chein, M.S., Winton, JR. and Kaattari, S.L. (1999). Antigenic and
functional characterization of p57 produced by Renibacterium
salmoninarum. Dis. Aguat. Org. 37: 43-52

Winter, G.W., Schreck, CB. and McIntyre, JD. (1980). “ Resistance of different
stocks and transferrin genotypes of coho salmon, Oncorhynchus kisutch,
and steelhead trout, Salmo gairdneri, to Bacterial Kidney Disease and
vibriosis.” U.S. Natl. Mar. Fish. Serv. Fish. Bull. 772795-802

Wolf, K., and Dunbar, C.E. (1959).”Tests of 34 therapeutic agents for control of
Kidney Disease in trout.” Trans. Am. Fish. Soc. 88:117-124.

209

Wood, J.W. (1974). “Diseases of Pacific Salmon, Their Prevention and
Treatment.” Olympia, Wash: Dep. Fish. Hatchery Div., 82 pp.2nd Edition.

Wood EM, and Yasutake, W.T. (1956). “Histopathology of Kidney Disease in
fish.” Am. J. Pa_thol. 32: 845-857.

Wood, RA. and Kaattari, S.L.(1996). “Enhanced immunogenicity of
Renibacterium salmoninarum in chinook salmon after removal of the
bacterial cell surface-associated 57 kDa protein.” Dis. Awat. Org. 25: 71 —
79.

Woodall, A. N., and LaRoche, G. (1964). Nutrition of salmonid fishes. 11. Iodide
requirements of chinook salmon. J. Nutr. 82: 475-482.

Wright, G. and Faisal, M.(2005). Fish Health in Lake Michgan. In: The State of
Lake Michigan. Edited by Clapp, D. Great Lakes Fishery Commission.
Technical Report (In press).

Young, CL. and Chapman, GB. (1978). Ultrastructural aspects of the causative
agent and renal histopathology of Bacterial Kidney Disease in brook trout
(Salvelinus fontinalis). J. Fish. Res. Board. Can. 35:12341248.

Yoshimizu, M. (1996). “Disease problems of salmonid fish in Japan caused by

lntemational trade.” Bulletin de l’ Office lntematjonal c_les Epizooties 15(2):
533-549.

210

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