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This is to certify that the
thesis entitled

BACTERIAL PATHOGENS INFECTING LAKE WHITEFISH
(COREGONUS CLUPEAFORMIS) IN MICHIGAN

presented by

Thomas Peterson Loch

has been accepted towards fulﬁllment
of the requirements for the

MS. degree in Pathobiology and Diagnostic
Investigation

 

 

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6/07 p:lCIRC/Date0ue.indd-p.1

BACTERIAL PATHOGENS INFECTING LAKE WHITEFISH (C OREGONUS
CLUPEAFORMIS) IN MICHIGAN

By

Thomas Peterson Loch

A THESIS

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

MASTER OF SCIENCE

Department of Pathobiology and Diagnostic Investigation

2007

ABSTRACT
BACTERIAL PATHOGENS INFECTING LAKE WHITEFISH (COREGONUS
CL UPEAFORMIS) IN MICHIGAN
By

Thomas Peterson Loch

The lake Whiteﬁsh (Coregonus clupeaformis) is a commercially and ecologically
invaluable indigenous salmonid within the Great Lakes Basin (GLB). A recent decline in
its condition and abundance has alarmed managers, scientists, and the public at large,
necessitating research to determine what factors may be involved. Although diseases can
signiﬁcantly impact populations of ﬁshes worldwide, a meager amount of research has
focused on pathogens of lake Whiteﬁsh within the GLB. This study was undertaken to
determine what bacterial pathogens are present within four stocks of lake Whiteﬁsh
residing within Lakes Michigan and Huron and whether bacterial infections caused by
these microbes vary in prevalence spatially and temporally. Carnobacterium
maltaromaticum, the etiological agent of pseudokidney disease, Aeromonas salmonicida
subsp. salmonicida, the causative agent of furunculosis, and multiple motile, mesophilic
Aeromonas species, known to cause motile aeromonad septicemia, were among the
bacteria isolated from infected lake Whiteﬁsh within this study. Clinical signs of disease
associated with some of these infections were quite severe, and the prevalence of
infections caused by a portion of these microbes was at times, quite high. This is
considered the ﬁrst report of C. maltaromaticum and A. salmonicida salmonicida
infecting lake Whiteﬁsh within the GLB. Implications of these diseases on lake Whiteﬁsh

populations are discussed within.

ACKNOWLEDGEMENTS

First and foremost, I would like to thank my Major Advisor, Dr. Mohamed Faisal,
for the truly invaluable support that he has provided throughout my graduate career.
From the very beginning, he pushed me to excel in all aspects of my life, academic and
otherwise, and through his faith in my work, he bestowed into me a level of conﬁdence
that was not present before. Without his endless guidance and insight, that which I have
accomplished thus far, as well as the opportunities now on the horizon, would never have
been possible.

Utmost gratitude is also due to the members of my guidance committee, Dr.
Rocco Cipriano, Dr. Scott Fitzgerald, and Dr. Michael Jones, for their wealth of
knowledge that they made available to me, for their patience, and for their valuable
collaborations in this, as well as other studies.

I would also like to thank past and present members of the Michigan State
University Aquatic Animal Health Laboratory for their immense support and advice that
they provided to me. Special appreciation also goes to Wei Xu, Carolyn Schulz, Nathan
Nye, and Dr. P. Gary Egrie for their friendship, support, advice, and invaluable
assistance.

Sincere thanks to Natural Mortality Project members, particularly Mark Ebener,
Greg Wright, and Dr. Michael Arts, for their valuable discussion and support. I would
like to also thank the Great Lakes Fishery Trust for funding this project.

And last but most deﬁnitely not least, I would like to thank my mother and father,

Norma and Thomas, for their awesome parenting throughout life that enabled me to be

iii

who I am today, my sisters, Lisa and Christine, who have always been supportive of me,
regardless of the situation, Heather Papendick, for her friendship that keeps me aﬂoat
during rough times, and most of all, to my amazing children, Ethan Jacob and Hailee

Nicole, for the joy and endless love that they bring to me every minute of every day.

iv

TABLE OF CONTENTS

 

LIST OF TABLES .................................................................. vii
LIST OF FIGURES ................................................................. ix
GENERAL INTRODUCTION AND OVERVIEW 1
CHAPTER 1. REVIEW OF LITERATURE .................................. 4
1. Lake Whiteﬁsh background information ........................................ 4
11. Potential roles of disease in the decline of ﬁsheries ......................... 7
1) General Overview ....................................................... 7

2) Speciﬁc bacterial pathogens ........................................... 14

a) Carnobacterium spp ........................................... 14

b) Aeromonas spp .................................................. 16

CHAPTER 2. ISOLATION OF CARNOBA C TERI UM
[MAL TAROMA TIC UM FROM LAKE WHITEFISH (COREGONUS

CLUPEAFORMIS) IN LAKES MICHIGAN AND HURON ............... 25
1) Abstract ................................................................... 25
2) Introduction ............................................................... 27
3) Materials and Methods .................................................. 29

a) Fish and sampling ............................................... 29
b) Bacterial isolation ............................................... 29
0) Biochemical characterization .................................. 30
d) Conﬁrmation of isolate identiﬁcation with polymerase
chain reaction and gene sequencing ........................... 31
e) Analysis of the sequence data .................................. 32
t) Histopathological Analyses .................................... 33
g) Statistical Analyses .............................................. 33
4) Results ...................................................................... 34
5) Discussion .................................................................. 38

CHAPTER 3. ISOLATION OF AEROMONAS SALMONICIDA
SUBSPECIES SALMONICIDA FROM LAKE WHITEFISH (COREGONUS

CLUPEAFORMIS) IN LAKES MICHIGAN AND HURON ................. 6O
1) Abstract ..................................................................... 60

2) Introduction ................................................................. 62

3) Materials and Methods .................................................... 65

a) Fish and sampling ................................................. 65

b) Bacterial isolation ................................................. 65

c) Biochemical characterization ................................... 66
(1) Conﬁrmation of isolate identiﬁcation with polymerase

chain reaction (PCR) ............................................. 66
6) Detection of A.salm0nicida salmonicida with polymerase
chain reaction (PCR) ............................................. 67
f) Histopathological analyses ...................................... 68
4) Results ...................................................................... 69
5) Discussion .................................................................. 73

CHAPTER 4. ISOLATION OF MOTILE AEROMONAS SPP. FORM
LAKE WHITEFISH (COREGONUS CLUPEAFORMIS) IN LAKES

MICHIGAN AND HURON ......................................................... 85
1) Abstract ..................................................................... 85
2) Introduction ................................................................. 87
3) Materials and Methods ................................................... 89
a) Fish and sampling ................................................ 89
b) Bacterial isolation ................................................ 89
c) Biochemical characterization ................................... 90
d) Analysis of isolates by species, site, and season ............. 91
6) Statistical analysis ................................................ 91
4) Results ....................................................................... 92
5) Discussion .................................................................. 99
CHAPTER 5. CONCLUSIONS AND FUTURE RESEARCH ............... 118
REFERENCES ........................................................................ 124

vi

LIST OF TABLES

Table 1. Representative seasons during which lake Whiteﬁsh were collected and the
number of individuals sampled from each of the four sites throughout the course of
this study ........................................................................................... 43

Table 2. Dates, sites, organs of isolation, and identiﬁcation numbers of
Carnobacterium maltaromaticum isolates retrieved from lake Whiteﬁsh throughout
the course of this study ........................................................................... 44

Table 3. Percentage of Carnobacterium maltaromaticum isolates recovered from
infected lake Whiteﬁsh that were positive for characteristics of interest. All tests

were incubated at 22°C, with results being recorded at up to 7 days post-inoculation

with the following exceptions: methyl red, Voges-Proskauer, indole production,
Simmons citrate, and T81 reactions were read at 2 days. Production of acid from
carbohydrates was examined in phenol red broth base at a ﬁnal concentration of 1%.

All materials and reagents were purchased from Remel Inc. (Lenexa, KS) unless
speciﬁed otherwise. TSA=trypticase soy agar; CTSI=cresol red thallium acetate

sucrose innulin medium; TSI=triple sugar iron medium; ONPG= o-nitrophenyl-ﬂ-D-
galactopyranoside ............................................................................... 45

Table 4. Percent prevalence of Carnobacterium maltaromiticum infections in lake
Whiteﬁsh by site and season, as well as the total prevalence, throughout the course
of the study. (Nov. 2003-Aug. 2006) ........................................................ 46

Table 5. Sampling dates and sites from which A. salmonicida subspecies
salmonicida was recovered from lake Whiteﬁsh using culture techniques during
this three-year study (Nov. 2003-Aug. 2006) ............................................... 78

Table 6. Biochemical phenotypes of A. salmonicida salmonicida isolates

retrieved from lake Whiteﬁsh. +, positive reaction; -, negative reaction; (+) weak

and/or delayed positive reaction. All tests were incubated at 22°C, with results

being recorded at up to 7 days post-inoculation with the following exceptions:

methyl red, Voges—Proskauer, indole production, and T81 reactions were read at

2 days. Production of acid from carbohydrates was examined in phenol red broth

base at a ﬁnal concentration of 1%. All materials and reagents were purchased

from Remel Inc. (Lenexa, KS). All strains were Gram-negative, non-spore

forming, nonmotile, faculatative anaerobes that produced and alkaline over acid

TSI reaction without any gas or H28 production ............................................ 79

Table 7. Percent prevalence of Aeromonas salmonicida subspecies salmonicida

infections in lake Whiteﬁsh by site and season, as well as the total prevalence,
throughout the course of the study (Nov. 2003-Aug. 2006) ............................... 8O

vii

Table 8. Representative seasons with number of lake Whiteﬁsh sampled on each
occasion throughout the course of this study .............................................. 106

Table 9. Percentage of Aeromonas isolates (by complex) in this study that

were positive for the performed biochemical tests. Number in parenthesis =

number of isolates tested. All isolates were cytochrome oxidase positive,

facultative anaerobes that were resistant to the vibriostatic agent 0/129 and did

not produce H28 in Sulfur-Indole-Motility medium ....................................... 107

Table 10. Percentage of Aeromonas isolates (by species) in this study that were

positive for the performed biochemical tests. Number in parenthesis= number of

isolates tested. All isolates were cytochrome oxidase positive, facultative

anaerobes that were resistant to the vibriostatic agent 0/129 and did not produce

H28 in SulﬁIr-Indole-Motility medium ...................................................... 108

Table 11. Gross clinical observations in lake Whiteﬁsh collected from Lakes
Michigan and Huron in which Aeromonas spp. were the only detected bacterial
species .......................................................................................... 109

Table 12. Number of isolates belonging to each complex that were recovered
from lake Whiteﬁsh collected from Lakes Michigan and Huron between
October 2005-August 2006 .................................................................. 1 10

Table 13. Detected prevalence of the three Aeromonas complexes infecting lake
Whiteﬁsh collected from Lakes Michigan and Huron between October 2005-

August 2006 (BDN=Big Bay de Noc; NB=Naubinway; CHE=Cheboygan;

DV=Detour Village) .......................................................................... 111

Table 14. Detected prevalence of the four Aeromonas species infecting lake

Whiteﬁsh collected from Lakes Michigan and Huron between October 2005-

August 2006 (BDN=Big Bay de Noc; NB=Naubinway; CHE=Cheboygan;

DV=Detour Village) .......................................................................... 1 12

viii

LIST OF FIGURES

Figure 1. Four sites from which Lake Whiteﬁsh were collected for this study.

The Lake Michigan stocks were from management units WFM-Ol (Big Bay

de Noe) and WFM-03 (N aubinway); the Lake Huron stocks were from WFH-Ol
(Cheboygan) and WFH-02 (Detour). These sites were selected due to the

large spawning aggregations of Whiteﬁsh that inhabit these areas, as well

as their relevance in commercial ﬁsheries .................................................... 24

Figure 2. Appearance of Carnobacterium maltaromaticum colonies recovered

from lake Whiteﬁsh on Cresol Red Thallium Acetate Sucrose Innulin Medium

(CTSI) (Wasney et al. 2001 ). All isolates produced convex, entire colonies with a
reddish-purple center that were surrounded by a yellow halo ............................. 47

Figure 3. PCR patterns of Carnobacterium maltaromaticum isolates retrieved

from lake Whiteﬁsh. The approximate corresponding band size is denoted on the

left with the large arrows. The three small arrows in the upper left corner

correspond to the typical intergenic spacer region band pattern associated with C.
maltaromaticum (Pelle et al. 2005) ........................................................... 48

Figure 4. Portion of 16S and 23S rRNA sequence of Carnobacterium maltaromiticum
isolates recovered from lake Whiteﬁsh (denoted by WP) aligned with a C.
maltaromaticum sequence within Genbank (denoted by 2). The sequences were aligned
using the BLASTn software from the National Center for Biotechnology Information,
(NCBI) and analyzed using the nucleotide database from NCBI. Homology of the
generated sequences with that of the database was assessed using the nucleotide database
from NCBI. E-value=0.0; Nucleotide homology= 97%. Black=identical nucleotides;
grey=nucleotide with similar characteristic (i.e. both are purines, etc); white=nucleotides
differ ............................................................................................... 49

Figure 5. Putative piscicolin 126 precursor gene sequence of Carnobacterium
maltaromiticum isolates recovered from lake Whiteﬁsh (denoted by WP) aligned

with the sequence within Genbank (denoted by l). The sequences were aligned

using the BLASTn software from the National Center for Biotechnology Information,

(N CBI) and analyzed using the nucleotide database from NCBI. Homology of the
generated sequences with that of the database was assessed using the nucleotide
database from NCBI. E-value=lx10-138; Nucleotide homology=98%. Black=

identical nucleotides; grey=nucleotide with similar characteristic (i.e. both are

purines, etc); white=nucleotides differ ....................................................... 51

Figure 6. A phylogenetic tree of C. maltaromaticum isolates retrieved from lake
Whiteﬁsh based upon portions of the 16S rRNA and 23S rRNA genes. Generated
sequences were analyzed using the BLASTn software from the National Center for
Biotechnology Information, (N CBI, Bethesda, MD) to detect homologous sequences.
Homology of the generated sequences with that of the database was assessed

ix

using the nucleotide database from NCBI. Using the CLUSTAL W program

from Molecular Evolutionary Genetics Analysis (MEGA) 3.1., a phylogenetic

tree was constructed using the Neighbor-Joining method as the bootstrap test of
phylogeny. Accession numbers from NCBI precede scientiﬁc names ................... 52

Figure 7. Lumen of a lake Whiteﬁsh swim bladder ﬁlled with a turbid, mucoid

exudate from which C. maltaromaticum was recovered in pure culture. The

walls of the swim-bladder are severely thickened and opaque, with some areas

of extensive hemorrhaging also evident (denoted by arrow) .............................. 54

Figure 8. Stained section of a liver from a C. maltaromaticum infected lake
Whiteﬁsh exhibiting mild hepatocyte degeneration due to cytoplasmic
vacuolation (H&E stain, 20X magniﬁcation) ................................................ 55

Figure 9. Stained section of a liver from a C. maltaromaticum infected lake
Whiteﬁsh exhibiting mild bile stasis (H&E stain, 40X magniﬁcation) ................... 56

Figure 10. Stained section of a spleen from a C. maltaromaticum infected lake
Whiteﬁsh exhibiting congestion and swelling (H&E stain, 20X magniﬁcation) ........ 57

Figure 11. Stained section of the posterior kidney from a C. maltaromaticum
infected lake Whiteﬁsh exhibiting mild congestion
(H&E stain, 20X magniﬁcation) ............................................................. 58

Figure 12. Stained section of a lake Whiteﬁsh swim-bladder from which C.
maltaromaticum was isolated exhibiting severe epithelial hyperplasia

(right) compared with that of a normal swim-bladder (left; H&E stain, 20X
magniﬁcation) ................................................................................... 59

Figure 13. Extensive severe hemorrhaging within the musculature and vent
of a lake Whiteﬁsh infected with A. salmoncida salmonicida ........................... 81

Figure 14. F uruncule-like lesion on the dorsal musculature of a lake Whiteﬁsh
that was both culture and PCR negative for A. salmonicida salmonicida .............. 82

Figure 15. Musculature underlying a furuncule-like lesion, exhibiting severe
muscular degeneration and necrosis in a lake Whiteﬁsh .................................. 83

Figure 16. Skin and underlying musculature from a furuncle-like lesion found

in lake Whiteﬁsh exhibiting ulceration, necrotizing dermatitis & myositis, mixed
lymphocytes, macrophages, rare heterophils, ﬁbrin, and hemorrhage. No

bacterial colonies were observed (H&E stain, 10X magniﬁcation) ..................... 84

Figure 17. Prevalence of infection by motile Aeromonas spp. in lake Whiteﬁsh
from four sites within Lakes Michigan and Huron between
October 2005-August 2006 ................................................................... 1 13

Figure 18. Seasonal prevalence of motile Aeromonas spp. infections in lake
Whiteﬁsh collected from four sites within Lakes Michigan and Huron
between October 2005-August 2006 ......................................................... 114

Figure 19. Overall prevalence of lake Whiteﬁsh infected with motile Aeromonas
spp. by season and site between October 2005-August 2006 ............................ 115

Figure 20. Proportion of the total detected infections in lake Whiteﬁsh
caused by members of the three Aeromonas complexes by site ......................... 116

Figure 2]. Proportion of the total detected infections in lake Whiteﬁsh
caused by members of the three Aeromonas complexes by season ..................... 117

xi

GENERAL INTRODUCTION AND OVERVIEW

Historically, lake Whiteﬁsh (Coregonus clupeaformis) have been a staple
commercial ﬁshery within the Great Lakes Basin (GLB), beginning in the early 18405
and quickly expanding thereafter (Wells and McLain 1973). Although initial commercial
yields of lake Whiteﬁsh were not formally documented, anecdotal accounts suggest
substantially larger yields than those recorded in subsequent years. In the following
years, a variety of factors, such as overexploitation and interactions with invasive species,
brought about signiﬁcant declines in lake Whiteﬁsh abundance. Through the
implementation of a number of management strategies, lake Whiteﬁsh populations
rebounded (Ebener 1997) and above average harvests have been experienced (Mohr and
Nalepa 2005).

However, recent observations of declines in abundance, condition, and size at age
in harvested lake Whiteﬁsh have caused concern (Hoyle et al. 1999; Pothaven et al. 2001;
Madenjian et al. 2002; Mohr and Ebener 2005). Concurrently, there has been signiﬁcant
decline in the abundance of the preferred prey item (Diporeia spp.) of lake Whiteﬁsh
(N alepa et al. 1998), superceded by alterations in phytoplankton composition brought
about by the invasion of zebra (Dresseina polymorpha) and quagga mussels (D.
bugensis) into the GLB (Higgins et al. 2005; Bierman et al. 2005).

There is mounting evidence that infectious diseases exert signiﬁcant pressure on
the dynamics of animal populations and communities (Holmes 1996). For example,
infections with the fungus Icthyophonus hoferi in Atlantic (Clupea harengus) and Paciﬁc

Herring (Clupea pallasi) have caused population declines (Daniel 1933; Sindermann

1958; Mellergaard and Spanggaard 1997; Marty et al. 2003). Additionally, an epizootic
of bacterial kidney disease that occurred in Lake Michigan generated mass mortalities in
Chinook salmon that reduced the population by 50% or more (Holey et al. 1998).

There are many other examples of diseases affecting ﬁsh at the population level,
and often, outbreaks of these diseases can be linked to environmental conditions (Lafferty
and Holt 2003). Organisms residing in dysfunctional ecosystems do not resist infection
as well as their counterparts, which inhabit healthy ecosystems (Folke et al. 2004).
Diseases in wild ﬁsh populations also result from complex interactions among pathogens,
their hosts, and the environment (Snieszko 1973; Hedrick 1998; Reno 1998). To this
end, I postulate that signiﬁcant alterations in the Great Lakes’ ecosystem (e. g. food-web
shifts, interactions between exotic and native species, etc.) may have placed lake
Whiteﬁsh populations at an increased risk for disease epizootics.

One problem with the determination of a disease’s effect on a population of
organisms is the paucity of baseline data regarding the incidence and prevalence of
diseases in aquatic organisms (Lafferty et al. 2004). The lack of information on diseases
affecting coregonines, in general, and the GLB lake Whiteﬁsh speciﬁcally, makes efforts
to determine the effects that diseases are having and have had on their populations next to

impossible.

Study objectives

This study focused on the isolation and identiﬁcation of potential bacterial
pathogens infecting GLB lake Whiteﬁsh. In order to elucidate any temporal or spatial
associations, lake Whiteﬁsh were sampled from four stocks (two from Lake Michigan and
two from Lake Huron) at four different times per year (October-December; January-
March; April-June; July-September). Speciﬁc objectives were to: 1) isolate
Carnobacterium maltaromaticum, Aeromonas salmonicida subspecies salmonicida, and
motile Aeromonas spp. infecting lake Whiteﬁsh using standard bacteriological
methodologies and subsequently identify them using morphological, physiological, and
biochemical analyses; 2) determine if the detected bacterial infections of lake Whiteﬁsh
varied seasonally or by stock.

This thesis consists of ﬁve chapters. The ﬁrst chapter will summarize the
background information and the available body of knowledge on the thesis’ topic. The
second chapter deals with the ﬁrst reported isolation of Carnobacterium maltaromaticum
from lake Whiteﬁsh. The third chapter reports on the isolation of A. salmonicida
subspecies salmonicida from lake Whiteﬁsh. The fourth chapter deals with the isolation
and identiﬁcation of four motile Aeromonas species, as well as a number of un-classiﬁed
presumptive Aeromonas spp. from lake Whiteﬁsh. Finally, I shall present my conclusions

and potential directions for future research.

CHAPTER ONE

REVIEW OF LITERATURE

1. Lake Whitefish background information

Lake Whiteﬁsh (Coregonus clupeaformis), a member of the family Salmonidae
and the subfamily Corgeninae, is among a decreasing number of salmonid species
considered indigenous to the Great Lakes Basin (GLB). Other endemic coregonines
collectively known as deepwater ciscoe, such as C. nigripinnis, C. johannae, C.
zenithicus, C. alpenae, C. reighardi, and C. kiyi) were once plentiful within the GLB, but
have dramatically declined in numbers or have been extirpated following the invasion of
non-native ﬁshes (Wells and McLain 1972). Despite these declines, C. clupeaformis is
the most plentiful coregonine inhabiting the GLB; the lake herring (C. artedi) and the
bloater (C. hoyi), are still present in some areas, albeit at signiﬁcantly lower numbers
than historically reported (Smith 1970). Coregonus clupeaformis is also native to Alaska
and most of Canada. Its range extends south into New England and central Minnesota.
Additionally, lake Whiteﬁsh have been introduced into a number of locales, such as areas
of the Northwestern United States, including Montana, Idaho, and Washington (Page and
Burr 1991).

The lake Whiteﬁsh is non-anadromous and is characterized by its coarse scales
numbering <110 on the lateral line, two dorsal ﬁns, including an adipose ﬁn, and does

not have teeth on the maxillary bone (Moyle and Cech 2000). Lake Whiteﬁsh can weigh

up to 9000 grams, measure up to 800 mm, and live up to 25 years, with sexual maturity
occurring by 6-7 years of age (Page and Burr 1991). Coregonus clupeaformis is
primarily benthivorous, feeding predominantly on the amphipod, Diporeia spp. (Hart
1931; Reckahn 1970), as well opossum shrimp (Mysis relicta), and various gastropod and
pelecypod molluscs (Ihssen et al. 1981; Brandt 1986; Christie et al. 1987). Diporeia spp.
are endemic amphipods that are extremely nutritious, having high lipid and caloric
contents (Johnson 1988, Cavaletto et al. 1996, Pothaven et al. 2001), and were once one
of the most abundant benthic invertebrates inhabiting the cold, offshore regions within
the Great Lakes (Cook and Johnson 1974).

Historically, lake Whiteﬁsh were an important ﬁshery within the Great Lakes,
until the 1940’s, when yields dramatically declined. The lake Whiteﬁsh ﬁshery continued
to struggle, and in the 19608-19708, populations reached all time lows (Mohr and Nalepa
2005). The reasons for these drastic declines varied among Lakes, but were largely
attributed to overexploitation, interactions with invasive species, such as the sea lamprey
(Petromyzon marinus), rainbow smelt (Osmerus mordax), and alewife (Alosa
pseudoharengus), and water quality/habitat degradation (Mohr and Nalepa 2005).
Through various lake management strategies, such as control of the sea lamprey,
improved commercial ﬁshery management, suppression of non-indigenous planktivores
via the introduction of exotic salmonids, and phosphorous abatement, the lake Whiteﬁsh
ﬁshery rebounded and produced above average harvest yields from the 19803 until
present (Mohr and Nalepa 2005).

Currently, lake Whiteﬁsh are the most commercially valuable ﬁsh species within

GLB (Bronte et al. 2003; Hoyle 2005; Schneeberger et al. 2005; Cook et al. 2005; Mohr

and Ebener 2005). A notable decline in lake Whiteﬁsh condition, grth rate, and size at
age has been reported in parts of Lake Michigan (Pothaven et al. 2001; Madenjian et al.
2002) and Lake Ontario (Hoyle et al. 1999). Furthermore, observations of depressed
growth rates and poor condition were reported in parts of Lake Huron (Mohr and Nalepa
2005). Concurrently, substantial changes within the Great Lakes’ food web were
reported. Exotic zebra and quagga mussels (Dresseina polymorpha and D. bugensis)
have driven signiﬁcant changes in both phytoplanktonic and ﬁlamentous algal
abundance, as well as alterations in water quality (Higgins et al. 2005; Bierman et al.
2005). Simultaneously, a number of studies have demonstrated declines in numbers of
Diporeia spp. within the Great Lakes, with the exception of Lake Superior, to the point
where certain areas once rich in Diporeia are now devoid of these organisms (Dermott
and Kerec 1997; Nalepa et al. 1998; Lozano et al. 2001). The high ﬁltering rates of
dreissenids have caused marked declines in diatoms within Lake Erie, Saginaw Bay, and
Lake Ontario, causing an abundance of cyanobacteria to be present in these areas (as
cited in Ebener and Arts 2007). When available, Diporeia spp. feed heavily upon
diatoms, which are a rich source of essential fatty acids (EFAs). These are subsequently
assimilated, thus providing an excellent source of EPA to both the Diporeia spp.
themselves, and those organisms that prey upon them (e. g. lake Whiteﬁsh). Essential
fatty acids are integral for maintenance of cell membrane integrity, improvement of cold
tolerance, mitigation of the stress response, and also contribute to a healthy immune
system function in ﬁsh and higher vertebrates (Snyder and Hennessey 2003).

Additional factors implicated in the decline in lake Whiteﬁsh condition include

density-dependent mechanisms (Madenjian et al. 2002), climate/temperature change, and

parasitism (Mohr and Nalepa, 2005). Furthermore, movement of lake Whiteﬁsh to areas
outside of their normal range in search of Diporeia spp. can change population
distributions, reduce weight, growth, and recruitment (State of Great Lakes 2005), which

may have broader implications on increased susceptibility to disease.

[1. Potential roles of disease in the decline of ﬁsheries

1) General overview

There is mounting evidence that infectious diseases play an important role in the
dynamics of animal populations and communities (Holmes 1996). In general, organisms
inhabiting healthy, functioning ecosystems may resist infections better than those
inhabiting dysfunctional ecosystems (F olke et al. 2004). Malnutrition (Beck and
Levander 2000), thermal stress from climate change (Harvell et al. 1999), and toxic
chemicals (Khan 1990) are among many stressors that can suppress immune functions, a
pertinent risk for animals lacking sufﬁcient energy (Rigby and Moret 2000), thereby
increasing susceptibility to infectious disease. Hence, stressed individuals should be,
generally, more susceptible to infection (Scott 1988; Holmes 1996).

However, an opposing prediction occurs when looking at things from the view
point that epizootics are more likely to occur and will have a greater impact on
populations with higher host densities (Anderson and May 1986). Thus, as stress
depresses population densities, the chance of an epizootic occurring also decreases

because contact rates between infected and uninfected individuals are reduced (Lafferty

and Holt 2003). For instance, a swim bladder nematode, Cystidicola stigmatura, which
parasitized lake trout (Salvenius namaycush) was purportedly extirpated from the Great
Lakes after numerous stressors (see previous section) had reduced trout populations
below transmission threshold levels (Black 1983). On the other hand, some stressors,
such as eutrophication, may increase host parasitism through elevated host densities
(Lafferty and Holt 2003). Eutrophication is the most commonly observed stressor found
in association with increased parasitism in both invertebrates and ﬁshes (Lafferty 1997).
Other possible effects of stressors include adverse consequences on the parasite itself, or
increased host susceptibility to toxins if already parasitized (Guth et al. 1977;
Stadnichenko et al. 1995). For example, some toxic metals and chemicals have been
reported to have negative effects on intestinal helminthes (Lafferty 1997) and free-living
stages of parasites (Evans 1982), while having negligible effects on their host

species.

Thus, diseases in wild ﬁsh populations result from complex interactions among
pathogens, the hosts they infect, and the environment within which they all reside.
(Snieszko 1973; Hedrick 1998; Reno 1998). The fact that different pathogens respond to
stressors or environmental changes in different ways, and that stressors can increase or
decrease disease, are both essential in understanding the effects diseases may have at the
population-level (Lafferty et al. 2004). For example, stressors tend to have mitigating
effects on infectious diseases when transmission is mainly between members of the same
population and host speciﬁcity is high, but tend to aggravate the effects of infectious
diseases when pathogens are generalists or persist in a resistant environmental reservoir

(Lafferty and Holt 2003). As such, the ecosystems within the GLB that are undergoing

signiﬁcant alterations (e. g. food-web shifts, interactions between exotic and native
species, etc.) may be at an increased risk for various epizootics caused by some
pathogens, but may be less susceptible to others.

Globally, a number of diseases caused by parasites, bacteria, fungi, and viruses
have generated large scale epizootics and/or mortalities in ﬁshes. For example, the
parasitic monogenean ﬂuke, Gyrodactylus salaris, decimated native Norwegian salmon
populations after being introduced with imported Atlantic salmon (Salmo salar) smolts
from Sweden (Johnsen and Jensen 1986; Sattuar 1988). Native populations of
Norwegian Atlantic salmon were in great peril, forcing Norwegian authorities to destroy
all resident ﬁsh in 30 rivers in an attempt to eradicate the parasite (Hindar et al. 1991).
Similarly, ichthyophoniasis, caused by the fungus Ichthyophonus hoferi, has been
associated with population declines in Atlantic herring (Clupea harengus; Daniel 1933;
Sindermann 1958; Mellergaard and Spanggaard 1997) and Paciﬁc herring (Clupea
pallasi; Marty et al. 2003). Marty et al. (2003) linked two disease epizootics caused by
Icthyophonus hoferi and viral hemorrhagic septicemia virus in Paciﬁc Herring inhabiting
Prince William Sound, Alaska, to increased natural mortality and poor subsequent
recruitment in the years following these outbreaks. Moreover, some aquatic disease
epizootics have caused enough devastation to spur mitigating legislation. Such was the
case in the issuance of the 1937 Diseases of Fish Act of Great Britain, which was enacted
in response to widespread furunculosis, a serious disease caused by Aeromonas
salmonicida, within wild ﬁsh populations in the United Kingdom (Hill 1996).

A decade ago, massive ﬁsh kills involving the Atlantic Menhaden (Brevoortia

tyrannus) occurred over a number of years in the Chesapeake Bay and its tributaries

(Hargis 1985; Blazer et al. 1999). Such acute mortalities were originally believed to be
caused by the toxin-producing heterotrophic dinoﬂagellate Pﬁsteria piscicida
(Burkholder et al. 1992, 1995, 2001), yet further investigations provided evidence that the
mortalities are caused by the oomycete, Aphanomyces invadans (Kiryu et al. 2002).

As is the case globally, many diseases present within the GLB can have
signiﬁcant effects on ﬁshes at the population level. Viral hemorrhagic septicemia virus
(V HSV), the etiological agent of viral hemorrhagic septicemia (VHS), is a rhabdovirus
that causes signiﬁcant pathology in infected hosts and is capable of generating large scale
mortality events. Viral hemorrhagic septicemia virus has invaded the Great Lakes,
spreading from the St. Lawrence River to the straights of Mackinac (Lake Huron),
causing massive ﬁsh kills in Great Lakes muskellunge (Esox masquinongy
masquinongy), lake Whiteﬁsh, walleye (Stizostedion vitreum), gizzard shad (Dorsoma
cepadianum), yellow perch (Percaﬂavescens), and freshwater drum (Aplodinotus
grunniens) (Elsayed et al. 2006; Faisal et al. in prep; Whelan 2007).

Infectious pancreatic necrosis virus (IPNV, Bimaviridae) is another disease that
has caused widespread infection and mortality in freshwater and marine ﬁshes worldwide
and has been isolated from many species of ﬁsh and invertebrates (Faisal and Hnath
2005). Although the presence of IPNV is necessary for disease to ensue, many other
factors are also important, such as viral strain, environment, and age of the host species
(Jarp et al. 1994; Hill 1982; Smail et al. 1992). This vertically transmitted virus causes
epizootics in a number of cultured ﬁshes, including members of the genera
Oncorhynchus, Salmo, Salvelinus, (Reno 1999) and has been isolated from numerous

sites within the GLB (Beyerle and Hnath 2002). Among the few GLB recovered isolates

10

that have been further analyzed, mortalities of up to 88% were generated in brook trout
(Salvelinusfontinalis) subjected to waterborne exposure (McAllister 2003).

Another viral disease of ﬁshes with serious implications is the large mouth bass
virus (LMBV). A member of the Family Iridoviridae, this ranavirus has been identiﬁed
as the causative agent of mortalities within numerous bodies of water within the southern
United States (Faisal and Hnath 2005). In 2000, substantial die-offs involving
largemouth bass (Micropterus salmoides) in a lake bordering Michigan and Indiana were
attributed to the LMBV (Grizzle and Brunner 2003), and subsequent evaluations indicate
that this virus was spreading northward, westward, and eastward in southern Michigan
(Faisal and Hnath 2005). This disease is primarily manifested within the swim-bladders
of infected individuals, where it causes varying degrees of hemorrhage (Faisal and Hnath
2005), with or without the presence of a brown/yellow exudate (Grizzle and Brunner
2003)

The microsporidians Glugea hertwigi and Glugea anomala have caused large
mortality events in rainbow smelt (Osmerus mordax) within Lakes Erie and Ontario and
in three-spine stickleback (Gasterostreus aculeatus) in Michigan’s upper-peninsula,
respectively (Nepszy et al. 1978; Faisal unpublished data). Myxobolus cerebralis, the
etiological agent of whirling disease, is a myxosporidian that can cause substantial
mortalities in both captive and wild ﬁsh populations (Gilbert and Granath 2003). For
example, Rognlie and Knapp (1998) implicated M. cerebralz's to cause a 90% decrease in
wild rainbow trout (Onchorhynchus mykiss) residing in a stretch of the Madison River,
Montana. This parasite has a two stage life cycle, during which it alternates between

oligochaete and teleostean hosts (Gilbert and Granath 2003), and is one of the most

11

pathogenic myxosporidians known to ﬁsh (Hedrick et al. 1998). Rainbow trout are most
susceptible to the disease, although many other salmonids, such as brook trout, brown
trout (Salmo trutta), chinook salmon, and Atlantic salmon are also infected (Gilbert and
Granath 2003). In 1966, whirling disease was detected in 3 commercial hatcheries within
Michigan (Yoder 1972). Despite great efforts to eradicate and contain this disease, M.
cerebralis was detected in the Manistee and Ausable rivers; however, the clinical form of
the disease has never been observed within the GLB (Faisal and Hnath 2005).

Bacterial diseases can also have major impacts on ﬁsh populations within the
GLB. For example, bacterial kidney disease (BKD), caused by a Gram positive
diplobacilli known as Renibacterium salmoninarum, has been reported from areas
wherever susceptible salmonid populations reside (Fryer and Sanders 1981; Klontz
1983), including the GLB. As early as 1955, BKD was reported in Michigan’s brook
trout (Allison 1958), and was later isolated from other salmonids inhabiting the GLB,
such as coho salmon (Onchorhynchus kisutch), as well as chinook (Oncorhuncus
tshawytcha) and kokanee salmon (Oncorhuncus nerka; Maclean and Yoder 1970). In the
late 1980’s, a substantial epizootic of BKD occurred in Chinook salmon residing within
Lake Michigan (Holey et al. 1998). Additionally, Eissa (2005) was able to isolate the
bacterium from hatchery raised brook trout. Bacterial kidney disease has also been
reported in salmonids of the genus Coregonus (Jonas et al. 2002). Renibacterium
salmoninarum has also been isolated from non-salmonid ﬁsh species, including Lake
Ontario sea lamprey (Petromyzon marinus; Eissa et al. 2004). The epizootic of BKD that
occurred in Lake Michigan generated mass mortalities in Chinook salmon that reduced

the population by 50% or more (Holey et al 1998). Factors that likely contributed to this

12

outbreak were heavy parasitic infection rates, elevated densities of Chinook salmon, and
depressed levels of prey species, namely alewives (Alosa pseudoharengus; Holey et al.
1998; Hansen and Holey 2001).

A recent example of disease effects at the population level is the massive ﬁsh kill
that involved common carp (Cyprinus carpio) in the St. Lawrence River, Quebec, Canada
(Monette et al. 2006). Over 25,000 dead or moribund carp were removed from a small
portion of the affected areas, while true numbers of affected individuals were most
probably higher. These deaths were ultimately attributed to opportunistic bacterial
infections by Aeromonas hydrophila and F Iavobacterium spp. that were secondary to
immuno-supression brought about by physiologic (i.e., spawning) and environmental (i.e.
high temperatures and low water levels) stressors.

Another group of bacterial diseases that generate more mortality than all other
pathogens combined within the hatcheries of the state of Michigan are caused by
members of the genus F lavobacterium (Records of Michigan DNR Fish Health
Laboratory; Faisal and Hnath 2005). F lavobacterium columnare, F. branchiophilum, and
F. psychrophilum, the etiological agents of columnaris disease, bacterial gill disease, and
cold-water disease, respectively, are yellow-pigmented, ﬁlamentous, Gram negative
bacilli that are capable of generating varying degrees of morbidity and mortality in both
captive and wild ﬁsh populations (Shotts and Starliper 1999). For example, F. columnare
reportedly caused 6% mortality in cultured tiger muskellunge (male Esox lucius x female
Esox masquinongy) within a 7 day period (Pecor 1978), severe ﬁn erosion in cultured
walleye (Clayton et al. 1998), and was responsible for signiﬁcant mortalities in a number

of wild ﬁshes (Becker and Fujihara 1978; Fijan 1968; Chen et al. 1982). Moreover, F.

13

columnare was isolated from spawning Chinook salmon at multiple weirs in Michigan,
where it was associated with varying degrees of pathological effects (Loch and Faisal,
unpublished data). F lavobacterium psychrophilum is responsible for high annual losses
of production coho salmon, brown trout and rainbow trout, particularly in facilities that
utilize open water sources reaching cold temperatures (near 0°C) in winter months. Cold
water disease has been reported to cause 85-90% mortalities in hatchery lake trout
(Schachte 1988). Flavobacterium branchiophilum is also capable of generating high
mortalities in cool and cold water ﬁsh species (Shotts and Starliper 1999), but is more
typically associated with high morbidity (up to 100%) via a chronic proliferative response

in gill epithelia (Noga 2000).

2) Speciﬁc bacterial pathogens

a) Carnobacterium spp.

Lactobacilli and other closely related species are Gram positive, non-spore-
forrning rods that constitute a normal component of the gastrointestinal and urogenital
ﬂora of vertebrates, including ﬁsh (Ringo et al. 1995; JoEborn 1998; Ringo and
Gatesoupe 1998; Gonzalez et al., 2000). In ﬁsh, lactobacilli have also been associated
with mortalities, septicemias, chronic inﬂammation, and necrotic changes within internal
organs (Rucker et al. 1953; Ross & Toth 1974; Cone, 1982). Based on extensive genetic
studies, biochemical fermentation patterns, and association with ﬁsh diseases, Hiu et al.

(1984) proposed a new Lactobacillus species; L. pisciciola. Studies demonstrated that L.

14

pisciciola was associated with post spawning morbidity and mortality, kidney
granulomas, massive chronic inﬂammation, and pseudomembrane formation (Herman et
al. 1985; Michel et al. 1986; Humphrey et al. 1987). Mature salmonids seem to be the
most susceptible to infection, though infections of fry and ﬁngerlings have also been
reported (Hiu et al. 1984). Additionally, Michel et al. (1986) isolated the bacterium from
the common carp.

Lactobacillus spp. went through a number of taxonomic changes when Collins et
al. (1987) grouped Lactobacillus divergens, L. piscicola, as well as some catalase-
negative, non-spore forming bacilli from poultry into a new genus; Carnobacterium. The
main characteristics separating Carnobacterium spp. from typical lactobacilli are their
inability to grow on acetate media and their production of oleic acid rather than cis-
vaccenic acid (Hiu et al. 1984). Carnobacterium pisciola has been isolated from
salmonid and non-salmonid species suffering from a range of infections, some of which
were associated with high mortalities (Baya et al. 1991; Starliper et al. 1992; Toranzo et
al. 1993). Due to kidney granulomas and pseudomembranes that are often associated
with C. pisciola infections, the disease in salmonids is often refered to as “pseudokidney
disease” (Austin and Austin 1987; Noga 2000). Based on recent phenotypic and
genotypic analyses by Mora et al. (2003), it was concluded that Lactobacillus
maltaromicus, originally isolated from milk, and Carnobacterium piscicola, were
synonyms. Therefore, Mora et al. (2003) reclassiﬁed these species into a combination
novum, Carnobacterium maltaromaticum.

An interesting property of C. maltaromaticum is the ability of some strains to

produce a 126 kilo dalton protein, piscicolin, which has antimicrobial properties (Gibbs et

15

a1 2004). The effects of piscicolin in vivo in ﬁsh are currently unknown; however, in a
study by Ingham et a1 (2003), the antibacterial activity of systemically injected piscicolin
126 was studied in mice experimentally infected with Listeria monocytogenes. This
study showed a statistically signiﬁcant reduction in clinical signs associated with
listeriosis, as well as a decrease in the number of recovered colony-forming-units in
individuals that were injected with P126 15 minutes before and 30 minutes after being

experimentally infected with L. monocytogenes.

b) Aeromonas spp.

Most species belonging to the genus Aeromonas are oxidase-positive,
facultatively anaerobic, glucose-fermenting, Gram-negative bacilli that are ubiquitous to
the aquatic environment, including fresh, brackish, and marine waters (Hazen et a1 1978;
Alonso et al 1994). They are associated with disease in both poikilotherrnic and
homeothermic organisms, including an increasing number of human case reports (Austin
and Adams 1996; Janda and Abbott 1996). A number of the motile Aeromonas spp. can
cause infections in ﬁsh, typically termed motile aeromonad septicemia (Cipriano 2001;
Aoki 1999). Manifestation of disease is frequently associated with various environmental
or physiological stressors, such as warm water temperatures, poor water quality, parasitic
infections, or poor over-wintering conditions (Austin and Adams 1996; Aoki 1999; Noga
2000)

Disease manifests as a generalized septicemia typical of other gram-negative rods,

with acute, chronic, and sub-clinical forms (Cipriano 2001). Pathological effects induced

16

by infections with members of this complex include ﬁn/tail rot, dermal ulceration, and
hemorrhagic septicemias. These septicemias are characterized by small surface lesions,
which may lead to sloughing of scales, hemorrhaging in the gills and anus, ulcers,
abscesses, exophthalmia, and abdominal swelling (Austin and Adams 1996). The
presence of ascites within the peritoneum, anemia, and swelling of the kidney and liver
may also occur (Austin and Adams 1996). Motile aeromonads are also part of the natural
intestinal microﬂora present within healthy ﬁsh (Trust et a1. 1974); thus, their presence is
not necessarily indicative of disease.

The heterogeneous complex of Aeromonas species currently resides within the
family Aeromonadaceae (Colwell et al. 1986) and has undergone many reclassiﬁcations
within the last three decades (Carnahan 1993). In the mid to late 19703, the majority of
aeromonads belonged to one of two groups; those that grew at 35-3 7°C, were motile, and
were primarily associated with human infections, known as the mesophiles, and those
that grew better at lower temperatures and were responsible for infections in ﬁsh, termed
the pscychrophiles (J anda and Abbot 1998). Since then, the genus Aeromonas has
undergone much taxonomic ﬂux and the expansion of species belonging to this group has
been quite astounding (J anda and Abbott 1998).

Currently, the genus contains the following species according to the latest edition
of Bergey ’3 Manual of Systematic Bacteriology (Holt et al. 2000): Aeromonas
hydrophila, Aeromonas bestiarum, Aeromonas salmonicida, Aeromonas caviae,
Aeromonas media, Aeromonas eucrenophila, Aeromonas sobria, Aeromonas veronii
(biovars veronii and sobria), Aeromonasjandaei, Aeromonas schubertii, Aeromonas

trota, Aeromonas allosaccharophila, Aeromonas encheleia, Aeromonas popoﬂii, as well

17

as two homology groups without species names, Aeromonas sp. HGll and Aeromonas
sp. HG13. Additionally, three novel species have recently been described; Aeromonas
culicicola (Pidiyar et al. 2002), Aeromonas simiae (Harf-Monteil et al. 2004), and
Aeromonas molluscorum (Minana-Galbis et al. 2004).

In addition to the signiﬁcant taxonomic changes and expansion in the number of
species within the genus Aeromonas, the heterogeneity in biochemistry, genetics, and
serology among and between species are substantial (Cipriano 2001). For this reason, as
well as the lack of clear-cut phenotypic tables useful for the dichotomization of the
numerous groups of aeromonads, both newly and previously described (Abbott et al.
2003), identiﬁcation is never a simple process.

A number of Aeromonas spp. are associated with ﬁsh disease. For example, A.
sobria, and A. hydrophila have been recovered from ﬁsh exhibiting signs of Epizootic
Ulcerative Disease (EUI; McGarey et a1. 1991), and A. veronii biovar sobria has recently
been implicated as the possible etiological agent of EUI (Rahman et al. 2002).
Aeromonas sobria was recovered by Toranzo et al. (1989) from a large mortality event
involving adult gizzard shad (Dorosoma cepedianum). Aeromonas hydrophila has also
been recovered from a wide range of freshwater ﬁsh and marine ﬁsh (Austin and Adams
1996; Aoki 1999). Additionally, A. bestiarum, A. salmonicida, and A. veronii were
retrieved from common carp, albeit at varying degrees of virulence (Kozinska et al.
2002). The isolation of A. allosacchaophila from diseased elvers was reported by
Martinez-Murcia et al. (1992). Moreover, Aeromonas spp. cause disease in many warm

water ﬁshes, including minnows, baitﬁshes, channel catﬁsh (Ictalurus punctatus), striped

18

bass (Morone saxatalis), large mouth bass (Micropterus salmoides; Cipriano 2001), and
tilapia (Oreochromis niloticus; Faisal et al. 1984; 1989).

Although infections by motile aeromonads predominate in warmer temperatures,
infections can occur in cool and cold water species. For example, Aeromonas sobria was
reported to cause signiﬁcant mortalities in farmed perch (Percaﬂuviatilis) by Wahli et al.
(2005) and Michel (1981) isolated a motile aeromonad from diseased perch. Paniagua et
al. (1990) demonstrated that some motile aeromonads recovered from river water were
virulent to rainbow trout (Oncorhynchus mykiss) upon experimental infection.

A number of virulence factors associated with motile aeromonads have been
described. The presence of adhesins in strains of A. hydrophila, A. sobria, and A. caviae
were demonstrated to facilitate adherence to animal cell lines with mucous-cell receptors,
and the proportion of A. hydrophila strains able to bind was signiﬁcantly greater than that
of the A. sobria and A. caviae strains (Ascencio et al. 1998). The same authors were also
able to correlate speciﬁc binding of collagen, ﬁbronectin, and laminin to isolates from
diseased ﬁsh. Moreover, A. hydrophila can attach to selected ﬁsh cells, such as
erythrocytes, and tissue proteins via adhesins that are very speciﬁc for D-mannose and L-
fucose side chains on surface polymers (Trust et al. 1980; Ascensio et al. 1991).
Furthermore, a surface array protein (S-layer) was described from virulent isolates of A.
hydrophila (Dooley and Trust 1988) and from motile aeromonads recovered from
clinically diseased catﬁsh (Ford and Thune 1991). These S-layers are known to confer a
degree of protection from lytic action by complement and bacteriophages, and reduce

phagocytosis by leukocytes (Dooley et al 1988).

19

Extracellular products (ECPs) that play varying roles in virulence are also
produced by some motile aeromonads. For instance, ECPs with hemolytic and
proteolytic activity are capable of eliciting pathology when injected into ﬁsh (Allan and
Stevenson 1981). Extracellular metallo-proteases and serine-proteases have also been
identiﬁed (N ieto and Ellis 1986) and have been suggested to increase invasiveness,
protect the pathogen against the bacteriocidal effects of serum, and provide nutrients for
growth following host tissue destruction (Leung and Stevenson 1988). Many other ECPs
have also been identiﬁed, such as enterotoxins, hemolysins, hemagglutinins, and
endotoxins (Cahill 1990).

Aeromonas salmonicida is a non-motile, psychrophilic, obligate pathogen of ﬁsh
that causes furunculosis and other diseases that devastate populations of numerous ﬁshes,
including including salmon, trout, grayling, char (Cipriano and Bullock 2001), and
Whiteﬁsh (Coregonus spp.) from Finland (Rintamaki & Koski 1987). Additionally,
infections caused by A eromonas salmonicida have been reported in an increasing number
of non-salmonid species (Fijan 1972; Bootsma et al. 1977; Shotts et al. 1980; Wiklund
1995; Bemoth and Korting 1992; Wilson and Holliman 1994). Aeromonas salmonicida
also causes carp erythroderrnatitis (Fijan 1972), cutaneous ulcerative disease in goldﬁsh
(Shotts et al. 1980), and “head ulcer disease” in Japanese eels (Kitao et al. 1984).

In ﬁsh, diseases caused by Aeromonas salmonicida have been studied for quite
some time, beginning in 1890, when Emmerich and Weibel described a bacterium
associated with diseased trout within a hatchery. Initially known as Bacillus salmonicida
(syn. Bacterium salmonicida and Bacterium trutta) (Emmerich and Weibel 1890),

A eromonas salmonicida has become one of the most studied bacterial piscine pathogens.

20

F urunculosis, so-named for the presence of boil-like lesions that are known as
ﬁrruncles, which may be present in the musculature (Austin and Adams 1996).
Additional clinical signs include hemorrhages within the ﬁns, musculature, orbits of the
eye, and liver, exopthalmia, lethargy, bloody discharge from the nares and anus, swelling
of the spleen, and renal necrosis. Rates of mortality associated with these chronic
infections are typically low (McCarthy and Roberts 1980). Another form of disease
caused by A. salmonicida, termed acute furunculosis, manifests as a septicemia
accompanied by melanosis, inappetance, lethargy, petechiae in the ﬁns, and varying
degrees of splenomegaly. Mortalities typically occur within 2-3 days (McCarthy 1975).
Furthermore, a peracute form has also been found in association with high mortalities in
salmonid ﬁngerlings (McCarthy and Roberts 1980; Austin and Austin 1993). An
intestinal manifestation of furunculosis has also been described, in which intestinal
inﬂammation and anal inversion occurs (Amlacher 1961). Latent forms of A .
salmonicida salmonicida infection, recently termed covert infections (Hiney et al. 1997),
have also been identiﬁed. These individuals develop clinical disease only under certain
conditions of environmental and physical stress. Covertly infected individuals act as
reservoirs for the disease, capable of transmitting the disease to uninfected hosts (Hiney
et al 1997).

A number of virulence factors play major roles in the pathogencity of A eromonas
salmonicida. Virulence mechanisms ﬁt into two categories; cell-surface structures and
extracellular products (ECPs; Hiney and Oliver 1999). Aeromonas salmonicida can
produce an additional surface protein microcapsule (Kay and Trust 1997), traditionally

termed S-Layer, but known as A-layer in A. salmonicida for historical reasons (Udey and

21

Fryer 1978). This 50 kDa protein has been shown to be immunologically conserved
among many A. salmonicida strains (Kay et al. 1984) and strong evidence in favor of its
role as a primary virulence factor exists. The ability of the A-layer to bind
immunoglobulins and other extracellular proteins has been purported to “mask” the
bacterium’s own immunogenic receptors, thus allowing evasion of the host immune
system (Kay and Trust 1997). Other reported functions of the A-layer include promotion
of bacterial penetration and adhesion, inhibition of complement-mediated lysis, and
protection from dessication in low nutrient environments (Hiney and Oliver 1999).
Another major component of the cell surface of A. salmonicida is lipopolysaccharide
(LPS), a major constituent of all Gram-negative bacteria. The exact role that LPS plays
in the virulence of A. salmonicida is still not fully realized; however, Cipriano and
Blanch (1989) reported that only strains with an intact A-layer and LPS were virulent for
brook trout.

Extracellular products of A. salmonicida play a large role in the pathological
effects associated with infection, as evidenced by the killing of susceptible ﬁsh upon
injection with crude extracellular material (Munro et al. 1980; Ellis et al. 1981). Three
main types of ECPs have been identiﬁed by Ellis (1997), which are proteases, membrane
damaging toxins, and other toxins, such as H-lysin. Among these, glycerophospholipid-
cholesterol acyltransferase complexed with LPS has been suggested as the most
important factor in lethal toxicity and pathology associated with ECPs (Ellis 1997).

Currently, at least 5 subspecies of Aeromonas salmonicida are recognized; -
salmonicida, achromogenes, masoucida, smithia (Holt et al. 2000), and pectinolytica

(Pavan et al. 2000). Aeromonas salmonicida salmonicida is the etiological agent of

22

furunculosis, while subspp. achromogenes, masoucida, smithia are associated with
atypical forms of the disease that often involve external pathology (i.e. dermal
ulcerations) with or without septicemia (Cipriano and Bullock 2001). A. salmonicida
pectinolytica is a relatively new subspecies that was recently isolated from a heavily
polluted river near Buenos Aires city but has yet to be reported in association with

disease (Pavan et al. 2000).

23

93° 97° 96° 95° 94°

 

45° . 48°

 

 

 

 

47° _ 47°

   

     

  

Detour VI

 

Naubinway “38¢

   

460 Big Bay Den ‘80

 

 

45° 45°

 

9,0 9,0 960 9,0 9....
Figure 1. Four sites from which Lake Whitefish were collected for this study. The
Lake Michigan stocks were from management units WFM-0l (Big Bay de Noe) and
WFM-03 (Naubinway); the Lake Huron stocks were from WFH-Ol (Cheboygan)
and WFH-02 (Detour). These sites were selected due to the large spawning
aggregations of Whitefish that inhabit these areas, as well as their relevance in

commercial fisheries.

24

CHAPTER TWO

ISOLATION OF CARNOBAC T ERIUM IMALTAROIIM TIC UM FROM LAKE
WHITEFISH (COREGONUS CL UPEAFORMIS) IN

LAKES MICHIGAN AND HURON

ABSTRACT

Carnobacterium maltaromaticum, the causative agent of Pseudokidney Disease,
has been isolated from lake Whiteﬁsh (Coregonus clupeaformis) caught from Lakes
Michigan and Huron, USA, during a three-year study. Twenty-three C. maltaromaticum
isolates were recovered from kidneys and swimbladders of infected ﬁsh. The isolates
were Gram-positve, nonmotile, facultatively anaerobic, asporogenous rods arranged in
palisades. They did not produce catalase, cytochrome oxidase, or H28, and did not
reduce nitrate. Isolates grew on Cresol Red Thallium Acetate Sucrose Inulin Agar, a
selective and differential medium for Carnobacterium spp. Variability in mannitol and
innulin fermentation, as well as arginine dihydrolase production, considered a key in
Carnobacterium spp. dichotomization, was observed among our isolates. Ampliﬁcation
of selected 168 and 23S rRN A regions speciﬁc to C. maltaromaticum and subsequent
sequencing of generated amplicons, yielded a 97% nucleotide match with C.
maltaromaticum sequences deposited in GenBank. Additionally, sequencing of the
piscicolin 126 precursor gene yielded a 98% nucleotide match with GenBank sequences.

Phylogenetic analyses showed a high degree of relatedness of the lake Whiteﬁsh isolates

25

with C. maltaromaticum. Fish from which the bacteria was isolated showed
splenomegaly, pallor and mottling in the liver, congestion in the spleen and kidney, and
opacity/thickening of the swim bladder wall with accumulation of a mucoid exudate
within the lumen. Histopathological examination showed congestion within kidneys and
spleens, vacuolation and bile stasis within the liver, and varying degrees of hyperplasia
within the epithelium of the swim bladder. The prevalence of infection varied
signiﬁcantly between sites and seasons, with the highest infections occurring in winter

sampling periods.

26

INTRODUCTION

Lactobacilli and other closely related species are a normal component of the
gastrointestinal and urogenital ﬂora of vertebrates, including ﬁsh (Ringo et al. 1995;
JoEbom 1998; Ringo and Gatesoupe 1998; Gonzalez et al. 2000). However, in ﬁsh,
lactobacilli have also been associated with mortalities, septicemias, chronic
inﬂammation, and necrotic changes within internal organs (Rucker et al. 1953; Ross &
Toth 1974; Cone 1982). Based on extensive genetic studies, biochemical fermentation
patterns, and association with ﬁsh diseases, Hiu et al. (1984) proposed a new
Lactobacillus species; L. pisciciola. Studies demonstrated that L. pisciciola was
associated with post spawning morbidity and mortality, kidney granulomas, massive
chronic inﬂammation, and pseudomembrane formation (Herman et al. 1985; Michel et al.
1986; Humphrey et al. 1987). Salmonids seem most susceptible when they are sexually
mature, though infections in fry and ﬁngerlings have also been reported (Hiu et al. 1984).
Additionally, Michel and colleagues (1986) isolated the bacterium from the zebra danio
(Brachydanio rerio) and the common carp (C yprinus carpio).

Lactobacillus spp. have gone through a number of taxonomic changes. Collins et
al. (1987) grouped Lactobacillus divergens, L. piscicola, as well as some catalase-
negative, non-spore forming bacilli from poultry into a new genus; Carnobacterium.
Carnobacterium pisciola was isolated from salmonid and non-salmonid species suffering
from a range of infections, some of which were associated with high mortalities (Baya et
al. 1991; Starliper et al. 1992; Toranzo et al. 1993). Due to kidney granulomas and

pseudomembranes that are often associated with C. pisciola infections, the disease in

27

salmonids is often refered to as “Pseudokidney Disease” (Austin and Austin 1987; Noga
2000). Based on recent phenotypic and genotypic analyses, Mora et al. (2003),
concluded that Lactobacillus maltaromicus, originally isolated from milk, and
Carnobacterium piscicola, were synonyms. Therefore, Mora et al. (2003) reclassiﬁed
these species into a combination novum, Carnobacterium maltaromaticum.

Herein, I report on the retrieval of 23 bacterial isolates from kidneys and swim
bladders of lake Whiteﬁsh inhabiting Lakes Huron and Michigan, USA, whose
morphological, cultural, and biochemical characteristics resemble those of C.
maltaromaticum (Mora et al. 2003). This is considered a new host species and geographic

location record for C. maltaromaticum.

28

MATERIALS AND METHODS

Fish and sampling Between the fall of 2003 and the summer of 2006, lake
Whiteﬁsh were collected for bacteriological analyses from 4 representative stocks on four
sampling occasions per year (~30 ﬁsh/site/season, Table 1). The two Lake Michigan
sites, Big Bay de Noc (latitude 4527-4548.20; longitude 8535-8722) and Naubinway
(latitude 4553-4603; longitude 8513-8535), and the two Lake Huron sites, Detour Village
(latitude 4555-4556.97; longitude 8350.5-8419.12) and Cheboygan (latitude 4540.24-
4548; longitude 8424-843 7.87) were selected for this study due to their association with
large spawning aggregations and accessibility via commercial ﬁshing (Figure 1). A total
of 1286 lake Whiteﬁsh from the four sites were sampled throughout the course of this
study.

Fish were captured using commercial trap nets and, when necessary, commercial gill
nets, with only live ﬁsh being used for the study. The collected ﬁsh were transported
alive to the Chippewa Ottawa Research Authority (CORA) Fishery Enhancement Facility
near Hessel, Michigan. The ﬁsh were then chilled on ice and transported to the Aquatic
Animal Health Laboratory at Michigan State University, East Lansing, MI, where they

were immediately subjected to thorough internal and external examinations.

Bacterial isolation. Bacterial samples retrieved from lake Whiteﬁsh were
collected from kidneys and visible lesions. Tissue samples were streaked onto Trypticase
Soy Agar (TSA; Remel, Lenexa, KS) and Cresol Red Thallium Acetate Sucrose Inulin

(CTSI) agar (all ingredients are from Sigma Chemical Co., St. Louis, MO), a selective

29

and differential medium for Carnobacterium spp. (Wasney et al. 2001), and incubated at
22 °C for up to 72 hr. Periodic examination of bacterial grth was recorded, and
individual colonies were sub-cultured onto TSA and then incubated for 24 hours at 22°C.

A total of 23 isolates were recovered and examined (Table 2).

Biochemical characterization. Isolated bacteria were initially identiﬁed using a
battery of morphological and biochemical tests, including Gram reaction, cytochrome
oxidase, catalase reaction (3% H202), motility, indole production, hydrogen sulﬁde
production, oxidation/fermentation reaction (BD Scientiﬁc, Sparks, MD), methyl red,
acetoin production from glucose (V oges-Proskauer), nitrate reduction, citrate utilization,
TSI reaction, ONPG (o-nitrophenyl-B-D-galactopyranoside), lysine decarboxylase,
ornithine decarboxylase, arginine dihydrolase, esculin hydrolysis, phenylalanine
deaminase (BD Scientiﬁc), growth on acetate agar (pH 5.4), and growth on Cresol Red
Thallium Acetate Sucrose Inulin medium at pH 9.1, as described by Wasney et al. (2001).
Production of acid from the following carbohydrates was examined in phenol red broth
base at a ﬁnal concentration of 1%: adonitol, arabinose, cellobiose, dextrose, galactose,
glycerol, inositol, innulin, lactose, malonate, maltose, mannitol, mannose, melibiose,
rafﬁnose, rhamnose, salicin, sorbitol, sucrose, trehalose, and xylose. Results were
recorded up to 7 days post-inoculation with the following exceptions: methyl red, Voges-
Proskauer, indole production, Simmons citrate, and TSI reactions were read at 2 days.
All materials and reagents were purchased from Remel Inc. (Lenexa, KS) unless

speciﬁed otherwise.

30

Conﬁrmation of isolate identiﬁcation with polymerase chain reaction and gene
sequencing. Representative colonies with the typical morphological and biochemical
characteristics of C. maltaromiticum, as listed in Bergey’s Manual of Detenninative
Bacteriology (Holt et al. 2000) were selected for this study. Single colonies were
resuspended in Trypticase Soy Broth (Remel), and incubated overnight at 22 °C. Their
DNA was extracted using a Qiagen DNeasy Tissue Kit (Qiagen Sciences, Valencia, CA)
according to the manufacturer’s protocol for Gram-positive bacteria. PCR ampliﬁcation
was performed using the following primers: 1) 163-4 (5 '-GCT GGA TCA CCT CCT
TTC T-3 ') and 23 S-7 (5'-GGT ACT TAG ATG TTT CAG TTC C-3'), which anneals to
positions 1526-1542 of the 16S rRNA gene and positions 207-189 of the 23S rRNA gene
in E. coli (Kabadjova et al. 2002; Pelle et al. 2005), and 2) PisA forward (5'-GTC ACA
GCA TTG ATG CGT ATC-3 ’) and PisA reverse (5 '-GAT GTG ATA CAG TCA GCA
TGT-3 '), which anneal to positions 1756-1777 and 2036-2057 on each side of the pisA
precursor gene for the piscicolin 126 protein produced by C. maltaromaticum (piscicola)-
JG126 strain (Pelle et al. 2005).

DNA template (4 ul) was combined with 2.5 ul of 10X PCR buffer containing 1.5
mMol MgCl2 (Invitrogen, Carlsbad, CA), 0.5 pl dNTP (Invitrogen), 1 ul of each primer,
17.5 ul distilled water, and 0.5 pl Taq Polymerase with loading buffer (Denville
Scientiﬁc, Metuchen, NJ). The PCR ampliﬁcation program was that of Pelle et al. (2005)
with slight modiﬁcations, which included an initial denaturation at 94°C for 10 min, 35
cycles of 1 min at 94°C, 1 min at 55°C, and 1 min at 72°C, and a ﬁnal step of 10 min at

72°C. The ampliﬁed products were subsequently run on 1.5% agarose gel for 30 min at

31

50 volts and then stained with ethidium bromide for viewing via UV exposure. A 1 Kb
plus DNA Ladder (Invitrogen) was used as a molecular marker.

Amplicons were puriﬁed using QIAquick PCR Puriﬁcation kit (Qiagen). Upon
puriﬁcation, a portion of the product was used for ligation with pGEM-T vectors
(Invitrogen) and incubated at 4°C overnight. The recombinants were then transferred
into DHS-a competent cells (Invitrogen). The transformed cells were then heat shocked
for 45 seconds in a 42°C water bath and subsequently placed on ice for 2 minutes.

S.O.C. medium (Invitrogen) was then added to the cells (0.9 ml at room temperature) and
the solution was incubated at 225 rpms at 37°C for 1 hour in an Incubating Shaker
(Labnet, Edison, NJ). Thirty ul of isopropyl-beta-D-thiogalactopyranoside (IPTG,
Denville Scientiﬁc) were then added and 500 pl of the bacterial suspension were
aliquoted to SOB + ampicillin agar plates, on which X-gal (Denville) had been previously
applied (BD Scientiﬁc). Plates were incubated at 37 °C overnight. Single, white,
colonies were then picked up, inoculated into 500 pl S.O.B. broth, and incubated at 220
rpms at 37 °C for 4 hours. Clones were then submitted to Michigan State University

Research Technology Support Facility for analysis.

Analysis of the sequence data. Generated sequences were analyzed using the
BLASTn software from the National Center for Biotechnology Information, (N CBI,
Bethesda, MD) to detect homologous sequences. Homology of the generated sequences
with that of the database was assessed using the nucleotide database from NCBI. Using

the CLUSTAL W program from Molecular Evolutionary Genetics Analysis (MEGA;

32

version 3.1), a phylogenetic tree was constructed using the Neighbor-Joining method as

the bootstrap test of phylogeny.

Histopathological Analyses. Samples of tissue with gross clinical abnormalities
were ﬁxed in 10% buffered formalin. The ﬁxed samples were then embedded within

parafﬁn, sectioned at 5 pm, and stained with hematoxylin and eosin (Prophet et al. 1992).

Statistical Analyses. Statistical analyses were performed using the Chi-Square

analysis of contingency tables through the Sigma Stat software (Jandel Corporation,

Carlsbad, CA).

33

RESULTS

Throughout the course of this study, a number of bacterial species were isolated
and characterized based on morphological, biochemical, and molecular attributes.
Among these, 23 isolates were presumptively identiﬁed as Carnobacterium
maltaromaticum. These isolates were subcultured onto both TSA and CTSI media.
Following 24-48 hours incubation on TSA, at 22°C, the isolates produced colonies that
were semi-translucent, whitish, entire, convex, and measured approximately 1-2 mm in
diameter. It is noteworthy that upon primary isolation, colonies were semi-translucent,
raised, striated, and with irregular margins. Growth on CTSI agar yielded complete,
convex colonies surrounded by a yellow halo with a reddish-purple center (Figure 2). In
21 isolates, the number of colonies present upon primary isolation varied from 1-50/10 pl
of kidney inoculum. This corresponds to a presence of approximately 1x102-5x103
colony forming units/ g kidney tissue in infected individuals. In the two cases that
involved the swim bladder, C. maltaromaticum growth was interconnected, profuse, and
covered the inoculated area.

Presumptive C. maltaromaticum isolates were then subjected to morphological,
cultural, and biochemical reactions. All 23 isolates were non-spore-forming, non-motile,
short, straight bacilli (1.0-1.5 pm by 0.5 pm) arranged in palisades that did not produce
catalase or cytochrome oxidase (Table 3). They were facultative anaerobes that did not
produce H2S, indole, phenylalanine deaminase, lysine decarboxylase, ornithine
decarboxylase, did not reduce nitrates, and gave a negative Simmons citrate reaction.

They all gave an acid slant over an acid butt Triple Sugar Iron reaction, with no H2S or

34

gas being produced, were able to hydrolyze esculin, and were positive for mixed acid
fermentation (MR+). Furthermore, all isolates produced acid from cellibiose, dextrose,
galactose, maltose, mannose, salicin, and sucrose. In addition, no acid was produced
from adonitol, arabinose, rhamnose, or xylose. No growth was achieved upon
inoculation of acetate or MacConkey agars.

There were, however, discrepancies among some isolates regarding a few of the
biochemical reactions. For example, the DV-l and DV-8 isolates did not produce
arginine dihydrolase, DV-l did not produce o-nitrophenyl-ﬂ-D-galactopyranoside, and
NB-6, 7, &13, DV-l, 2, 4, &7, and BD-l did not produce acetoin (VP negative).
Additionally, DV-8 produced acid from inositol, DV-3, DV-7, and CH-l produced acid
from rafﬁnose, and DV-7 and DV-8 produced acid from sorbitol. Furthermore, NB-lO,
DV-l , and DV-5 did not produce acid from melibiose and DV-l did not produce acid
from trehalose or lactose. Other detected variabilities occurred in glycerol, innulin, and
mannitol fermentation (Table 3).

Electrophoresis of the ampliﬁed rRNA from the 23 isolates generated 600 bp
amplicons, as well as the three typical intergenic spacer region bands associated with C.
maltaromaticum (Figure 3). Moreover, all isolates generated 300 bp bands when the
PisA primers were used for ampliﬁcation (Figure 3).

Sequencing of the 1526-1542 16S rRNA gene stretch, the 207-189 of the 23S
rRNA gene stretch, and the pisA precursor gene for the piscicolin 126 protein of our
Whiteﬁsh isolates showed high homology with the sequences contained within GenBank
(Figure 4 & Figure 5). For example, the 16S rRNA sequence of the C. maltaromaticum

isolates retrieved from lake Whiteﬁsh had highly signiﬁcant homology with the deposited

35

sequences in GenBank, as evidenced by the expectation value of 0.0 and the 97%
nucleotide match of our sequence with it’s homologue. The sequence corresponding to
pisA precursor gene from the lake Whiteﬁsh isolates had 98% nucleotide similarity with
GenBank sequences and an expectation value of e -139.

The sequences generated by representative C. maltaromiticum isolates retrieved
from lake Whiteﬁsh using portions of the 16S and 23S rRNA clustered most closely with
C. maltaromaticum isolates, followed by a cluster of C. gallinarum isolates (Figure 6).

The overall prevalence of C. maltaromiticum in the four lake Whiteﬁsh stocks
varied by season and by site (Table 4). The overall prevalence of Whiteﬁsh infected with
C. maltaromiticum varied among sites in a statistically signiﬁcant fashion (xz=18.587;
DF=3; P= < 0.001), with Naubinway having the highest prevalence, followed by Detour
Village. There were also statistically signiﬁcant differences in overall C.
maltaromaticum infections between seasons (x =27.740; DF =3; P=<0.001), with winter
prevalences being the highest, followed by spring. However, no statistically signiﬁcant
differences in the overall prevalence of C. maltaromaticum infections between Lakes
Michigan and Huron were observed (x2=0.611; df=1; P=O.435).

Lake Whiteﬁsh from Detour Village exhibited waves of low-level infections, with
peaks of infection occurring in the spring of 2005 and the winter of 2006, while high
prevalence was detected in the Naubinway winter 2005 samples, accounting for nearly
80% of the infections detected within this site throughout the three year study. The only
Carnobacterium infections that were detected in the other two sites came in the spring of

2006 and were found in only one individual from each sample.

36

In this study, we report on clinical signs and histopathology noticed in ﬁsh where
C. maltaromaticum was the only pathogen detected. Among the most common clinical
signs were mild to severe splenomegaly, varying degrees of hyperemia and friability
within the kidney, mottling and/or pallor in the liver, and varying degrees of congestion
within the testes. Additionally, of the two ﬁsh from which C. maltaromaticum was
isolated from the swim bladders, copius amounts of an opaque, mucoid, exudate was
present (Figure 7), along with hemorrhage and a general thickening/opacity of the swim
bladder wall.

In stained tissue sections of infected ﬁsh, there was mild to moderate hepatocyte
degeneration due to cytoplasmic vacuolation, as well as mild bile stasis, within the livers
(Figure 8 & Figure 9). Both spleen and kidney tissues had varying degrees of congestion
(Figure 10 & Figure 11). In the cases where C. maltaromiticum was isolated from ﬂuid
within the swim bladder, thickening of the swim bladder wall with ﬁbrous connective
tissue, some degree of neovascularization, as well as ﬁbrin and cellular debris within the
lumen, were evident. Additionally, the epithelial lining of the swim bladder exhibited

varying degrees of hyperplasia (Figure 12).

37

DISCUSSION

The morphological, biochemical, and molecular assays performed in this study
conﬁrm that the 23 isolates discussed were indeed C. maltaromiticum. This report is
considered the ﬁrst in which C. maltaromiticum was obtained from lake Whiteﬁsh within
the Great Lakes. C. maltaromiticum could potentially be the cause of the moderate
pathological effects observed in the infected lake Whiteﬁsh tissues. This is not totally
surprising, as C. maltaromiticum has been reported to cause diseased conditions and
mortalities (Cone 1982; Hiu et al. 1984; Herman et al. 1985; Michel et al. 1986; Baya et
al. 1991; Starliper et al. 1992; Toranzo et al. 1993). The isolation of C. maltaromiticum
from kidneys and swim bladders of lake Whiteﬁsh indicated that the infection may have
been systemic in nature.

When comparing the prevalence of C. maltaromiticum infections in the four
stocks of lake Whiteﬁsh, there was a statistically signiﬁcant difference among the four
sites. The highest prevalence was found within the Naubinway populations, but it is
interesting to note that most of the infections were detected in the winter of 2005
samples. We were unable to link other environmental factors to the high presence of C.
maltaromiticum at this particular site and time. However, in a parallel study conducted
by Sutton and Koops (ongoing study), lake Whiteﬁsh sampled from four stocks within
Lake Michigan, 2 of which were Naubinway and Big Bay de Noe, ﬁsh from the
Naubinway site had the lowest levels of docosahexaenoic acid (DHA) within dorsal
muscle tissues, which is known to play an important role in both neural development and

retinal function (Dr. Michael Arts, National Water Research Institute, Enivomment

38

Canada, pers. comm.) Additionally, levels of eicosapentaenoic acid (EPA) were
signiﬁcantly lower in Naubinway females, while EPA levels in males were signiﬁcantly
different from individuals collected from two of the four sites. EPA is a fatty acid
precursor of the anti-inﬂammatory prostaglandin G3, thus having pertinent effects on the
functionality of portions of the immune system (Dr. Michael Arts, pers. com.)

A high prevalence of C. maltaromaticum infections was observed in the winter
of 2005 in Naubinway Whiteﬁsh samples. It is of interest to note that no infections were
detected during the fall sampling periods, when spawning is occurring, as previous
research has proposed a link between post spawning stress and manifestation of disease
caused by C. maltaromaticum (Cone 1982; Herman et al. 1985; Starliper et al. 1992).
However, the fall sampling times correspondedto a period during which the ﬁsh were
still gravid/ready to spawn, which would mean that our winter sampling periods would
correlate more closely with a “post-spawning” time period. Thus, while a multitude of
factors likely play a role in the ability of C. maltaromaticum to generate disease,
spawning stress could potentially be a key predisposing and/or initiating factor.

Among the isolates that were retrieved in this study, some variations were
observed in their respective phenotypic characteristics, speciﬁcally, a number of the
carbohydrate fermentation reactions. Variability in biochemical proﬁles has also been
reported in previous studies that involved fermentation reactions of Carnobacterium spp.
(Hiu et al. 1984; Michel et a1. 1986; Starliper et al. 1992; Toranzo et al. 1993). Although
some of the phenotypes varied only by a single fermentation reaction, the substantial
variability that I observed may indicate that a number of strains are present in the Great

Lakes basin. Furthermore, two biochemical reactions that have been reported to be key

39

in the dichotomization of C. divergens and C. maltaromiticum, inulin and mannitol
fermentation (Montel et al. 1991), were variable among our isolates, despite the strong
homology between our sequences and that of GenBank. Some of the biochemical
variations seen in Carnobacterium spp. have been attributed to the use of different basal
mediums (Baya et al. 1991; Toranzo et a1. 1993); however, it is possible that these newly
isolated strains of C. maltaromiticum may truly have a multitude of biochemical
phenotypes that were not detectable genotypically due to the relatively small stretch of
DNA that was sequenced in this study. Thus, while current biochemical identiﬁcation
schemes are useful for presumptive identiﬁcation of Carnobacterium spp., molecular
techniques may, in some cases, be necessary for deﬁnitive identiﬁcation.

Concurrent with the biochemical variation discussed above was the variability in
pathological effects potentially associated with the C. maltaromiticum infections in lake
Whiteﬁsh. While a typical pseudokidney disease was not seen, the isolation of this
bacterium from the kidneys, along with the histopathological changes seen in the tissues
of infected individuals, suggests a systemic infection. For example, the vacuolation of
hepatocytes can be associated with the inhibition of protein synthesis, energy depletion,
disaggregation of microtubules, or shifts in substrate utilization (Hinton and Lauren,
1990). Furthermore, bile stasis can be an indication of inappetance (Andicoberry et al.
1999) that may have resulted from an ongoing C. maltaromitiucm infection
Additionally, the apparent congestion observed within some of the kidneys and spleens is
common in systemic bacterial infections due to an increase in angiogenic factors and
other substances that increase recruitment of cells that are integral in mounting an

immune response (Kumar et al. 2005) In the swim bladders infected with this bacterium,

40

signs of chronic irritation, such as hyperplasia, ﬁbrin deposition, and neovascularization,
and additional pathological effects, such as ﬁbrin and cellular debris within the lumen,
again illustrate the potential for disease generation.

The sequence generated from our isolates for the pisA precursor gene were nearly
identical to the sequences presently contained within GenBank, with the occurrence of
only a four base pair difference throughout the entire ~300 bp sequence. Additionally,
the 97% nucleotide match and extremely low E-value between the rRNA stretch
sequenced from our isolates and that of GenBank’s indicate that our isolates are closely
related to previously retrieved C. maltaromiticum isolates for which sequences were
deposited into GenBank. However, there were portions of our sequence that deviated
from the deposited sequence. These differences in genotype, as well as the variability in
biochemical phenotypes, may suggest that lake Whiteﬁsh isolates are distinct from
previously deposited C. maltaromiticum isolates, some of which were also retrieved from
ﬁsh.

In conclusion, C. maltaromaticum was isolated from lake Whiteﬁsh inhabiting
Lakes Michigan and Huron for the ﬁrst time, a new occurrence in this host species and
geographical location. Infected lake Whiteﬁsh exhibited a number of clinical and
histopathological alterations that could potentially have been caused by these infections,
although further investigation fulﬁlling Koch’s postulates is required. Although the
detected prevalence throughout the course of the study was not extremely high (1.8%),
infection prevalence did reach 20.8% in one seasonal sample. This ﬁnding may illustrate

that C. maltaromaticum can potentially infect large portions of endemic lake Whiteﬁsh

41

populations if conditions favorable to an epizootic occur. Implications of these outbreaks

of disease are currently unknown and warrant further investigation.

42

Collection Site

Sam Iin Bi Ba . Detour

Pergodg degNocy Naubinway Cheboygan Village
Fall 2003 35 30 30 34
Winter 2004 29 0 32 10
Spring 2004 30 30 2O 30
Summer 2004 22 20 30 20
Fall 2004 26 30 26 30
Winter 2005 16 30 15 30
Spring 2005 30 30 30 30
Summer 2005 30 30 28 30
Fall 2005 30 3O 0 30
Winter 2006 30 23 30 30
Spring 2006 30 3O 30 30
Summer 2006 30 30 30 30

Table 1. Representative seasons during which lake whiteﬁsh were collected and the
number of individuals sampled from each of the four sites throughout the course of

this study.

43

 

 

 

 

Sampling Date Collection Site Organ Isolate ID #
2/12/2005 Naubinway Kidney NB-l
“ Naubinway Kidney NB-2
“ Naubinway Kidney NB—3
“ Naubinway Kidney NB-4
“ Naubinway Kidney NB-5
“ Naubinway Kidney NB-6
“ Naubinway Kidney NB—7
“ Naubinway Kidney NB-8
“ Naubinway Kidney NB—9
“ Naubinway Kidney NB-lO
6/1/2005 Naubinway Kidney NB-l 1
“ Naubinway Kidney NB-l 2
6/7/2005 Detour Kidney DV-l
“ Detour Kidney DV-2
8/23/2005 Detour Swim Bladder DV-3
1/12/2006 Detour Kidney DV-4
“ Detour Kidney DV-5
“ Detour Kidney DV-6
3/28/2006 Naubinway Kidney NB- 1 3
5/25/2006 Bay de Noc Swim Bladder BD-l
5/3 1/2006 Detour Kidney DV-7
“ Detour Kidney DV-8
6/15/2006 Cheboyghan Kidney CH-l

 

Table 2. Dates, sites, organs of isolation, and identiﬁcation numbers of
Carnobacterium maltaromaticum isolates retrieved from lake Whitefish throughout

the course of this study.

44

 

 

 

 

 

 

 

 

 

Catalase 0% H28 0%
Cytochrome Oxidase 0% lndole 0%
Lysine Decarboxylase 0% Motility 0%
Omithine Decarboxylase 0% Methyl Red 100%
Arginine Dihydrolase 90% Voges Proskauer 63%
ONPG 95% Bile Esculin 100%

Phenyalanine Deaminase 0% Gas Production from Glucose 0%
Nitrate Reduction 0% Acid over Acid TSI Reaction 100%

Growth On:
TSA 100%] MacConkey Agar 0%
CTSI 100% Acetate Agar 0%
Acid Production from:

Adonitol 0% Mannitol 50%
Arabinose 0% Mannose 100%
Cellobiose 100% Melibiose 79%

Glucose 100% Rafﬁnose 10%
Galactose l 00% Rhamnose 0%

Glycerol 45% Salicin 100%

Inositol 5% Sorbitol 10%
Innulin 5 5% Sucrose 100%
Lactose 95% Trehalose 95%

Maltose 100% Xylose 0%

 

Table 3. Percentage of Carnobacterium maltaromaticum isolates recovered

from infected lake Whiteﬁsh that were positive for characteristics of interest. All

tests were incubated at 22°C, with results being recorded at up to 7 days post-

inoculation with the following exceptions: methyl red, Voges-Proskauer, indole

production, Simmons citrate, and TSI reactions were read at 2 days. Production of

acid from carbohydrates was examined in phenol red broth base at a ﬁnal

concentration of 1%. All materials and reagents were purchased from Remel Inc.
(Lenexa, KS) unless speciﬁed otherwise. TSA=trypticase soy agar; CTSI=cresol red

thallium acetate sucrose innulin medium; TSI=triple sugar iron medium; ONPG= o-

nitrophenyl-ﬂ-D-galactopyranoside.

45

 

 

 

 

 

 

 

 

 

 

 

Season
Collection Site Fall Winter Spring Summer Site Total
3’9 Bay °° N°° (cl/3i) (0.77/05) (11719:?) (03/302) 42.24%?)
Naumnway ((333) (212%)) (2229?) (ii/ifoy (1%./$.st
Chemyga" (0333) ((37/07) (11738?) (ti/Zoe) (2.3%?)
Dem” ”age ((332) (431%) (44/49?) (11%)) (gig/Z)
s°°°°"7°“' (align wigs) (8232/3) (21.333) (2337-3361

 

Table 4. Percent prevalence of Carnobacterium maltaromiticum infections in lake
whiteﬁsh by site and season, as well as the total prevalence, throughout the course of

the study. (Nov. 2003-Aug. 2006).

46

 

Figure 2. Appearance of Carnobacterium maltaromaticum colonies recovered from
lake Whitefish on Cresol Red Thallium Acetate Sucrose Innulin Medium (CTSI)
(Wasney et al. 2001). All isolates produced convex, entire colonies with a reddish-

purple center that were surrounded by a yellow halo.

47

  
 
 

300 bp—> '~ ~-

Figure 3. PCR patterns of Carnobacterium maltaromaticum isolates retrieved from
lake whiteﬁsh. The approximate corresponding band size is denoted on the left with
the large arrows. The three small arrows in the upper left corner correspond to the
typical intergenic spacer region band pattern associated with C. maltaromaticum

(Pelle et al. 2005).

48

Figure 4. Portion of 16S and 23S rRNA sequence of Carnobacterium
maltaromiticum isolates recovered from lake whiteﬁsh (denoted by WF) aligned with
a C. maltaromaticum sequence within Genbank (denoted by 2). The sequences were
aligned using the BLASTn software from the National Center for Biotechnology
Information, (NCBI) and analyzed using the nucleotide database from NCBI.
Homology of the generated sequences with that of the database was assessed using
the nucleotide database from NCBI. E-value=0.0; Nucleotide homology= 97%.
Black=identical nucleotides; grey=nucleotide with similar characteristic (i.e. both

are purines, etc); white=nucleotides differ.

49

 

 

WF

 

50

 

Figure 4

4O
39

80
79

120
119

160
159

200
199

240
239

280
279

320
319

360
358

400
398

440
438

480
478

520
518

560
558

600
598

619

616

 

 

 

 

l 40
Whitefish 4O
1 80
Whitefish 80
1 120
Whitefish 120
1 160
Whitefish 160
l 200
Whitefish 200
1 240
Whitefish 240
1 :‘aar TAR. . f: I :‘ - ‘- ' .f-gi ' 267
Whitefish 'TATAATTAA..GTCTCTTI‘ITTTT T T3717" ' 267

 

Figure 5. Putative piscicolin 126 precursor gene sequence of Carnobacterium
maltaromiticum isolates recovered from lake Whiteﬁsh (denoted by WF) aligned with
the sequence within Genbank (denoted by 1). The sequences were aligned using the
BLASTn software from the National Center for Biotechnology Information, (N CBI)
and analyzed using the nucleotide database from NCBI. Homology of the generated
sequences with that of the database was assessed using the nucleotide database from
NCBI. E-value=lx10-l38; Nucleotide homology=98%. Black=identical nucleotides;
grey=nucleotide with similar characteristic (i.e. both are purines, etc);

white=nucleotides differ.

51

Figure 6. A phylogenetic tree of C. maltaromaticum isolates retrieved from lake
Whiteﬁsh based upon portions of the 16S rRNA and 23S rRNA genes. Generated
sequences were analyzed using the BLASTn software from the National Center for
Biotechnology Information, (N CBI, Bethesda, MD) to detect homologous sequences.
Homology of the generated sequences with that of the database was assessed using
the nucleotide database from NCBI. Using the CLUSTAL W program from
Molecular Evolutionary Genetics Analysis (MEGA) 3.1., a phylogenetic tree was
constructed using the Neighbor-Joining method as the bootstrap test of phylogeny.

Accession numbers from NCBI precede scientiﬁc names.

52

aﬂ— AF405386.1| Pediococcus acidilactici
‘55 I———— AF405357.1| Pediococcus claussenii
26 AF405365.1| Pediococcus damnosus
l AJ635329. 1| Lactobacillus pentos us
97 IAJ616224. 1| Lactobacillus paraplantarum
AJ616222.1| Lactobacillus hilgardii

 

 

 

 

 

 

 

 
 
   

 

 

 

 

 

AY342344. 1| Weissella vin'descens
35 AY342335.1| Weissella minor
T‘ 33 AY342324.1| Weissella confusa
005383090 Bacillus subtilis
42 46 lZB4590.1| Bacillus cereus

 

 

284594.1l Bacillus thuringiensis
284592.1| Bacillus mycoides
— AJ295313. 1| Enterococcus rafﬁnosus
43 AJ295312.1| Enterococcus pseudoavium
4;l——A.1295299.1| Enterococcus avium
I DQO65846.1| Listeria welshimeri
39 I D0065845. 1| Listeria seeligeri
33 AJ295311.1| Enterococcus mundtii
AJ295309.1| Enterococcus hirae
AJ295304.1| Enterococcus durans
AJ295307. 1| Enterococcus ﬂamcens
AJ295300. 1| Enterococcus casseliﬂavus
34 AJ295298.1| Enterococcus asini
Whiteﬁsh isolate 82
a 61 Whiteﬁsh isolate 86
H 55 AF374295.1| Carnobacterium piscicola
AF374288. 1| Carnobacterium gallinarum
AF374294. 1| Carnobacterium divergens
99 J DQ472009.1| Staphylococcus xylosus

eel

 

 

 

 

 

 
 
  
  
  

 

 

 

72

 

 

 

 

 

' U1 1788.1| Staphylococcus aureus
30 AF139603.1| Streptococcus mutans
74 J———— AF255657.1| Streptococcus uberis

 

 

 

 

 

 

 

94 I——— 00839563.” Streptococcus thermophilus

0.01

Figure 6.

53

 

Figure 7. Lumen of a lake Whiteﬁsh swim bladder ﬁlled with a turbid, mucoid
exudate from which C. maltaromaticum was recovered in pure culture. The walls of
the swim-bladder are severely thickened and opaque, with some areas of extensive

hemorrhaging also evident (denoted by arrow).

54

 

Figure 8. Stained section of a liver from a C. maltaromaticum infected lake Whiteﬁsh
exhibiting mild hepatocyte degeneration due to cytoplasmic vacuolation (H&E stain,

20X magniﬁcation.

55

 

Figure 9. Stained section of a liver from a C. maltaromaticum infected lake Whitefish

exhibiting mild bile stasis (H&E stain, 40X magniﬁcation).

56

 

Figure 10. Stained section of a spleen from a C. maltaromaticum infected lake

whiteﬁsh exhibiting congestion and swelling (H&E stain, 20X magniﬁcation).

57

 

Figure 11. Stained section of the posterior kidney from a C. maltaromaticum

infected lake Whiteﬁsh exhibiting mild congestion (H&E stain, 20X magniﬁcation).

58

 

Figure 12. Stained section of a lake Whiteﬁsh swim-bladder from which C.
maltaromaticum was isolated exhibiting severe epithelial hyperplasia (right)

compared with that of a normal swim-bladder (left; H&E stain, 20X magniﬁcation).

59

CHAPTER THREE

ISOLATION OF AEROMONAS SALMONICIDA SUBSPECIES SALMONICIDA
FROM LAKE WHITEFISH (COREGON US CLUPEAFORMIS) IN LAKES

MICHIGAN AND HURON

ABSTRACT

Aeromonas salmonicida subspecies salmonicida, the etiological agent of
furunculosis, has been isolated from lake Whiteﬁsh (Coregonus clupeaformis) collected
from Lakes Michigan and Huron, USA, during a three-year study. Four A. salmonicida
subspecies salmonicida isolates were recovered from the kidneys of infected ﬁsh. The
isolates were Gram-negative, non-motile, coccobacilli that produced a brown diffusible
pigment on Trypticase Soy Agar. All isolates produced deep blue colonies on Coomassie
Brilliant Blue Agar. Ampliﬁcation of selected 16S rRNA regions speciﬁc to A.
salmonicida subspecies salmonicida via polymerase chain reactions and subsequent gel
electrophoresis analyses of the retrieved isolates yielded amplicons of the expected size
(512 bp). Kidney/spleen tissues from culture positive lake Whiteﬁsh, as well as culture
negative individuals with furuncle-like lesions, were assayed using the same primers.
Three of the four culture positive individuals were also PCR positive, while the
remaining culture positive lake Whiteﬁsh and the culture-negative, furuncle-like lesion
individuals were PCR negative. Clinical signs associated with infection included

extensive external hemorrhaging, exopthalmia, varying degrees of splenomegaly, hepatic

60

and renal pallor, spenic and renal congestion, friability of the kidney, ﬁbrinous adhesions
on the spleen and liver, as well as severe hemorrhagic enteritis. Histopathological
examination of culture positive lake Whiteﬁsh revealed multi-focal hemorrhage and mild
to moderate inﬁltration of lymphocytes and histiocytes in the fat under the skin and in the
muscultature, and massive diffuse congestion within the spleen. Histopathological
examination of furuncle-like lesions showed ulceration, necrotizing dermatitis and
myositis, along with an inﬁltration of mixed lymphocytes, macrophages, and infrequent
heterophils wihin the skin and underlying musculature, as well as hemorrhage and ﬁbrin
deposition; however, no bacterial colonies were observed. Detected A. salmonicida
subspecies salmonicida infections were limited to the Naubinway, Lake Michigan, and

Detour Village, Lake Huron sites.

61

INTRODUCTION

Bacteria belonging to the genus Aeromonas are oxidase-positive, facultatively
anaerobic, glucose-fermenting, Gram-negative bacilli that are ubiquitous to the aquatic
environment, including fresh, brackish, and marine waters (Hazen et al. 1978; Alonso et
al. 1994). They have been found in association with disease in both poikilotherrnic and
homeothermic organisms, including humans. In ﬁsh, diseases caused by these organisms
have been studied for quite sometime, beginning in 1890, when Emmerich and Weibel
described a bacterium associated with diseased trout within a hatchery. Initially known
as Bacillus salmonicida (syn. Bacterium salmonicida and Bacterium trutta) (Emmerich
and Weibel. 1890), Aeromonas salmonicida has become one of the most studied bacterial
piscine pathogens. This obligate pathogen is the etiological agent of furunculosis and is
known to infect a multitude of salmonids, including salmon, trout, grayling, char
(Cipriano and Bullock 2001) and Whiteﬁsh (Coregonus spp.) in Finland (Rintamaki &
Koski 1987). Additionally, infections caused by Aeromonas salmonicida have been
reported in an increasing number of non-salmonid species (Fijan 1972; Bootsma et a1.
1977; Shotts et al. 1980; Wiklund 1995; Bemoth and Korting 1992; Wilson and Holliman
1994). Aeromonas salmonicida has also been reported to cause carp erythroderrnatitis
(Fijan 1972), cutaneous ulcerative disease in goldﬁsh (Shotts et al. 1980), and “head ulcer
disease” in Japanese eels (Kitao et al. 1984).

Currently, at least 5 subspecies of Aeromonas salmonicida are recognized; -
salmonicida, achromogenes, masoucida, smithia (Holt et al. 2000), and pectinolytica

(Pavan et al. 2000). A. salmonicida salmonicida is the etiological agent of furunculosis,

62

while subspp. achromogenes, masoucida, and smithia are associated with atypical forms
of the disease that often involve external pathology (i.e. dermal ulcerations) with or
without septicemia (Cipriano & Bullock 2001). A. salmonicida pectinolytica is a
relatively new subspecies that was recently isolated from a heavily polluted river near
Buenos Aires city but has yet to be reported in association with disease (Pavan et al.
2000)

F urunculosis, so-named for the subacute or chronic form of the disease, is
sometimes characterized by the presence of boil-like lesions, known as furuncles, which
are present in the musculature (Austin and Adams, 1996). Additional clinical signs
associated with these forms of the disease include hemorrhages within the ﬁns,
musculature, and liver exopthalmia, lethargy, bloody discharge from the nares and anus,
swelling of the spleen, and renal necrosis. Rates of mortality associated with these
chronic infections are typically low (McCarthy and Roberts, 1980). Another form of
disease caused by A. salmonicida, termed acute furunculosis, manifests as a septicemia
accompanied by melanosis, inappetance, lethargy, petechiae in the ﬁns, and varying
degrees of splenomegaly. Mortalities typically occur within 2-3 days (McCarthy 1975).
Furthermore, a peracute form has also been found in association with high mortalities in
salmonid ﬁngerlings (McCarthy and Roberts 1980; Austin and Austin 1993). An
intestinal manifestation of furunculosis has also been described, in which intestinal
inﬂammation and anal inversion occurs (Amlacher 1961). Latent forms of A.
salmonicida salmonicida infection, recently termed covert infections (Hiney et al. 1997),
have also been identiﬁed. These individuals develop clinical disease only under certain

conditions of environmental and physical stress. Covertly infected individuals act as

63

reservoirs for the disease, capable of transmitting the disease to uninfected hosts, yet no
clinical signs are present (Hiney et al. 1997).

Herein, I report on the isolation of A. salmonicida salmonicida from the kidneys
of systemically infected lake Whiteﬁsh inhabiting Lakes Michigan and Huron, U.S.A. I
also report on the attempted detection of A. salmonicida salmonicida in culture negative

ﬁsh, with furuncle-like lesions, using a molecular assay.

64

MATERIALS AND METHODS

Fish and sampling. Between the fall of 2003 and the summer of 2006, lake
Whiteﬁsh were collected for bacteriological analyses from 4 representative stocks on four
sampling occasions per year (~30 ﬁsh/site/season, Table 1). The two Lake Michigan
sites, Big Bay de Noe (latitude 4527-4548.20; longitude 8535-8722) and Naubinway
(latitude 4553-4603; longitude 8513-8535), and the two Lake Huron sites, Detour
(latitude 4555-4556.97; longitude 83505-841912) and Cheboygan (latitude 4540.24-
4548; longitude 8424-8437.87) were selected for this study due to their association with
large spawning aggregations and their accessibility via commercial ﬁshing (Figure l). A
total of 1286 lake Whiteﬁsh from the four sites were sampled throughout the course of
this study.

Fish were captured using commercial trap nets and, when necessary, commercial
gill nets, with only live ﬁsh being used for the study. The collected ﬁsh were transported
alive to the Chippewa Ottawa Research Authority (CORA) Fishery Enhancement Facility
near Hessel, Michigan. The ﬁsh were then chilled on ice and transported to the Aquatic
Animal Health Laboratory at Michigan State University, East Lansing, MI, where they

were immediately subjected to thorough internal and external clinical examinations.

Bacterial isolation. Bacterial samples retrieved from the Whiteﬁsh were
collected from the kidneys, as well as visible lesions. Tissue samples were struck onto
Trypticase Soy Agar (TSA; Remel Inc., Lenexa, KS) and incubated at 22°C for up to 72

hr. Periodic examination of bacterial grth was recorded and individual colonies were

65

sub-cultured onto TSA and Coomassie Brilliant Blue Agar (CBBA) (Udey 1982), a
differential medium for distinguishing strains of A. salmoncida salmonicida that possess
an additional layer (poly-A layer) associated with the external cellular membrane.
Subcultures were then incubated for 24 hours at 22°C. A total of 4 isolates presumptively

identiﬁed as A. salmonicida salmonicida (Table 5) were recovered and examined.

Biochemical characterization. Isolated bacteria were initially identiﬁed using a
battery of morphological and biochemical tests, including Gram reaction, cytochrome

oxidase, catalase reaction (3% H202), motility, indole production, hydrogen sulﬁde

production, oxidation/fermentation reaction (BD Scientiﬁc, Sparks, MD), methyl red,
2,3-butanediol production from glucose (V oges-Proskauer), nitrate reduction, TSI
reaction, ONPG (o-nitrophenyl-,6-D-galactopyranoside), gelatinase, lysine decarboxylase,
ornithine decarboxylase, arginine dihydrolase, esculin hydrolysis, and production of gas
from glucose. Production of acid from the following carbohydrates was examined in
phenol red broth base at a ﬁnal concentration of 1%: arabinose, dextrose, galactose,
glycerol, maltose, sucrose, trehalose, and xylose. Tests were incubated for 10 days and
read periodically, with the following exceptions: methyl red, Voges-Proskauer, indole
production, and TSI reactions were read at 2 days. All materials and reagents were

purchased from Remel Inc. (Lenexa, KS) unless speciﬁed otherwise.
Conﬁrmation of isolate identiﬁcation with polymerase chain reaction (PCR).

Representative colonies with the typical morphological and biochemical characteristics of

A. salmonicida salmonicida, as listed in Bergey’s Manual of Determinative Bacteriology

66

(Holt et al. 2000) were selected for this study. Single colonies were resuspended in
Trypticase Soy Broth (Remel), and incubated overnight at 22 °C. DNA was extracted
using a Qiagen DNeasy tissue kit (Qiagen Sciences, Valencia, CA) according to the
manufacturer’s protocol for Gram-negative bacteria. PCR ampliﬁcation was performed
using the following Aeromonas salmonicida salmonicida primers: MIYl (5’-
AGCCTCCACGCGCTCACAGC-3’) and MIY2 (5’-AAGAGGCCCCATAGTGTGGG-
3’) (Miyata et al. 1996). Each 25 pl reaction contained 0.6 U Taq polymerase w/ loading
buffer (Denville Scientiﬁc Inc., Metuchen, NJ), 2.5 pl 10x PCR buffer containing 1.5
mmol MgC12(Invitrogen, Carlsbad, CA), 0.5 l dNTP (Invitrogen), and 2.5 pl of primers
MIYl and MIY2. The PCR ampliﬁcation program used was that of Byers et a1 (2002),
with modiﬁcations, which included an initial denaturation step of 94°C for 3 min, then
ampliﬁed for 30 cycles, with denaturation for 40 s at 94°C, annealing for 40 s at 60°C,
and elongation for 40 s at 72°C, and a ﬁnal extension was performed at 68°C for 3 min.
The expected size of the generated amplicon was 512 bp. The ampliﬁed products were
subsequently run on 1.5% agarose gel for 30 minutes at 50 volts and then stained with
ethidium bromide for viewing via UV exposure. A 1 Kb plus DNA Ladder (Invitrogen)

was used as a molecular marker.

Detection of A.salmonicida salmonicida with polymerase chain reaction
(PCR). Kidney tissues of culture negative lake Whiteﬁsh exhibiting furuncle-like lesions
were assayed with the same primers as above. DNA was extracted using a Qiagen

DNeasy tissue kit (Qiagen Sciences, MD) according to the manufacturer’s protocol for

67

the appropriate tissue. Assayed tissues included kidney/spleen homogenates. PCR

ampliﬁcation, as well as the PCR protocol, were performed as described above.

Histopathological analyses. Samples of tissue with gross clinical abnormalities

were ﬁxed in 10% buffered formalin. The ﬁxed samples were then embedded within

parafﬁn, sectioned, and stained with hematoxylin and eosin (Prophet et al. 1992).

68

RESULTS

Throughout the course of this study, a number of bacterial species were isolated
and characterized based on morphological, biochemical, and molecular attributes.
Among these, four isolates were presumptively identiﬁed as Aeromonas salmonicida
subspecies salmoncida. Following 24-48 hrs incubation at 22°C on TSA, all isolates
produced colonies that measured 1-2 mm in diameter and were opaque, smooth, entire,
and convex. The consistency of the colonies was typical of A. salmonicida salmonicida,
being friable and readily sliding across the culture medium upon attempted colony
removal. All isolates produced a brown diffusible pigment that was evident at 48 hrs of
incubation at 22°C on TSA. On CBBA, all isolates produced dark blue colonies. Colony
counts ranged from 9 to extremely profuse, interconnected, grth that covered the entire
inoculum streak per 10p] of kidney inoculum.

Morphologically, these isolates were Gram-negative coccobacilli, arranged in
bunches that measured 1.5 pm by 1 pm, although slight pleomorphism was often noticed.
Biochemical tests demonstrated that all isolates were non-motile, facultative anerobes

that did not produce indole, HZS, or 2,3-butanediol (Table 6). The lake Whiteﬁsh isolates

utilized the mixed-acid fermentation pathway (methyl red +), hydrolyzed esculin, yielded
an alkaline over acid TSI reaction without any gas production, and produced catalase,
cytochrome oxidase, and gelatinase. Acid production from fermentation reactions
occurred from the following sugars: arabinose, dextrose, galactose, glycerol, and maltose.
No acid was produced from sucrose, trehalose, or xylose. A eromonas salmonicida

salmonicida isolates recovered from lake Whiteﬁsh produced two atypical reactions; a

69

lack of gas production from glucose and no acid production from trehalose (Holt et al.
2000).

Gel electrophoresis of generated PCR amplicons derived from isolates of A.
salmonicida salmonicida retrieved from lake Whiteﬁsh were consistent with those
reported previously (Byers et al. 2002). Gel electrophoresis of the PCR amplicons
generated from culture positive kidney tissue yielded mixed results. Of the 4 tissues
assayed from culture positive individuals, three were positive, while one was negative.

The overall prevalence of lake Whiteﬁsh that were culture positive for A.
salmonicida salmonicida infections was 0.3% of the 1286 individuals that underwent
bacteriological examination (Table 7). Of the four populations that were studied, A.
salmoncida salmoncida infections were detected only in the Naubinway, Lake Michigan
site, and the Detour Village, Lake Huron site. The detected prevalence in these two sites
was 0.64% and 0.60%, respectively.

Kidney tissues from 3 lake Whiteﬁsh exhibiting furuncle-like lesions, yet culture-
negative, were assayed with PCR using speciﬁc primers for A. salmonicida salmonicida.
Gel electrophoresis of kidney/spleen homogenate amplicons from these individuals
produced negative results.

Clinical signs associated with lake Whiteﬁsh from which A. salmonicida
salmonicida was retrieved included combinations of extensive petechial and echymmotic
hemorrhaging in the ﬁns, musculature and vent (Figure 13), head, and eyes, exopthalmia,
varying degrees of splenomegaly, pallor in the liver and kidney, congestion in the kidney
and spleen, friability of the kidney, and ﬁbrinous adhesions on the spleen and liver. In

one instance, there was also predominant pathology involving the gastrointestinal tract,

70

with a large amount of gas present within the stomach, along with severe hemorrhagic
enteritis. Additionally, a 5 cm by 5 cm shallow hemorrhagic ulceration was present on
the left lateral musculature of one infected individual.

Clinical signs associated with culture negative, furuncle-like-lesion positive lake
Whiteﬁsh included mild-moderate hemorrhaging within the ﬁns, varying degrees of
congestion in the liver, spleen, and kidneys, severe splenomegaly, and periodic
hemorrhagic enteritis. F uruncle-like lesions were raised, endurate, circular lesions
protruding from the dorsal and lateral musculature (Figure 14). The underlying
musculature was necrotically liqueﬁed and cavitated (Figure 15). In one instance,
previously healed muscular lesions were also evident.

Histopathological analyses of tissues retrieved from culture positive ﬁsh
demonstrated some microscopic pathological effects. For example, skin and the
underlying musculature taken from an individual with extensive severe external
hemorrhaging demonstrated multi-focal hemorrhaging in the fat under the skin and
seperating muscles, along with mild to moderate inﬁltration by lymphocytes and
histiocytes. The same individual also had massive diffuse congestion within the spleen.

Histopathological analyses of tissues retrieved from culture negative ﬁsh with
furuncle-like lesions produced a variety of microscopic lesions. One of the ”furuncular”
lesions involving the skin and underlying musculature exhibited ulceration, necrotizing
dermatitis and myositis, along with an inﬁltration of mixed lymphocytes, macrophages,
and infrequent heterophils (Figure 16). There was also predominant hemorrhage and
ﬁbrin deposition; however, no bacterial colonies were observed. The kidney and liver

from this individual did not exhibit histopathological alterations. Another “furuncular”

71

lesion exhibited superﬁcial & patchy muscle degeneration, with evident pallor,
vacuolation, and separation, as well as necrosis. No inﬂammation was present. Severe
and diffuse congestion, along with scattered macrophages containing brown-to-black
cytoplasmic hemosiderin pigment, was observed in the spleen of this individual.

Hemosiderosis can be indicative of extravascualr hemolysis (Kumar et al. 2005).

72

DISCUSSION

Morphological and biochemical analyses performed on four bacterial isolates
retrieved from lake Whiteﬁsh deﬁnitively validated their initial presumptive identiﬁcation
as Aeromonas salmonicida salmonicida. All four isolates were Gram-negative, non-
motile coccobacilli that were facultatively anaerobic, and produced cytochrome oxidase
and catalase. Additionally, the production of a brown, diffusible pigment, along with the
lack of indole and H2S production and sucrose fermentation, allows for the differentiation
of A. salmonicida salmonicida from other A. salmonicida subspecies (Popoff 1984).
Furthermore, the acid production from mannitol and maltose, as well as the hydrolysis of
esculin, further support an identiﬁcation of A. salmonicida salmonicida (Holt et al. 2000;
Bohm et al. 1986). The production of an A-layer, as evident by the deep blue appearance
of our colonies on CBBA, is a factor associated with virulence due to its role in different
binding activities, and is also critical for adherence and invasion of macrophages (Kay
and Trust 1997). Moreover, gel electrophoresis of generated PCR amplicons derived
from isolates of A. salmonicida salmonicida cultured from lake Whiteﬁsh were consistent
with those reported previously (Miyata et al. 1996). According to Byers et al. (2002),
MIY PCR is 100% speciﬁc to A. salmonicida salmonicida.

In all four culture-positive individuals, a signiﬁcant amount of pathology was
apparent, both grossly and microscopically. Clinically, lake Whiteﬁsh infected with A.
salmonicida salmonicida exhibited extensive hemorrhaging within the external
musculature, concurrent with the splenomegaly and congestion within the kidneys and

spleens, which are strongly suggestive of an acute septicemia. Experimental infection

73

studies performed by Klontz et al. (1966) and Ellis et a1. (1981) found splenomegaly and
congestion to be the most consistent gross clinical ﬁnding in salmonids infected A.
salmonicida salmonicida. Moreover, Klontz and co workers also found that the site most
prominently involved pathologically was the musculature. They found a marked increase
of macrophages and small lymphocytes, along with a moderate inﬁltration of neutrophils
and ﬁbroblasts and an increase in extravascular erythrocytes at 32 hours post-injection.
These ﬁndings correlate well with the histopathological results of this study.
Hemorrhagic enteritis and gastrointestinal involvement, similar to reports by Amlacher
(1961), were observed in one of the four culture-positive Whiteﬁsh. Klontz et al. (1966)
did not ﬁnd any pathological abnormalities in the stomach, pancreas, or large intestine of
intramuscularly infected ﬁsh. However, in ﬁsh injected by both intraperitoneal and
intramuscular injections, as was done by Ellis et al. (1981), inﬂammation of the vent and
rectum was only observed in LR injected individuals. This suggests that the route of A.
salmonicida salmonicida infection may play an integral role in the site-speciﬁc
generation of pathology and may help to explain the differences in pathological effects
from one individual to the next, at least in experimental infections. A possible difference
in mode of infection or infection target may exist for this particular isolate; however,
ﬁirther investigation is required. In addition, it is important to keep in mind that
experimental infections are vastly different from natural routes of infection, so caution
should be used when making comparisons of the two. When taken together, the clinical
and histopathological ﬁndings within these infected Whiteﬁsh are strongly suggestive of
the ability of endemic A. salmonicida salmonicida isolates to generate morbidity and

mortality, although under what conditions this can occur remains to be elucidated.

74

In acute A. salmonicida salmonicida infections, the onset is sudden, with death
typically occurring within 2-3 days (McCarthy 1975). Thus, a relatively small “window
of opportunity” is available for detecting acute A. salmonicida salmonicida infections, a
fact that most likely has an effect on studies attempting to quantify the prevalence of A.
salmonicida salmonicida within ﬁsh populations. As such, the overall 0.31% prevalence
that was observed in this study may not be representative of the true prevalence.

With regard to the 3 individual lake Whiteﬁsh that were culture and PCR negative,
yet displayed ﬁiruncle-like lesion that histologically were very similar to A. salmonicida-
induced furuncles, there are a number of possible explanations. First, the ﬁsh may have
recovered from the infection, yet the affected tissues were not fully healed. Second,
inhibitory substances may have inhibited the PCR reaction, a problem that has been
reported previously (Byers et al. 2002; Heie et al. 1997; Gustafson et al. 1992). These
inhibitive properties may also explain why the PCR was negative in one of the four cases
presented in this study, despite the successful isolation of A. salmonicida salmonicida
from the same tissue and conﬁrmation of the colony identity by PCR. Alternatively,
these lesions may have been caused other organisms that were not detected. Lastly, the
transient nature of A. salmonicida salmonicida infections (Scallan et al. 1993), as well as
its presence in varying sites depending on the stage of infection (Klontz et al. 1966) also
can be a source of “false negative” detection results via culture and non-culture based
‘proxy’ methods. Histopathological observations of the “ﬁiruncular” lesions were similar
to those seen by Klontz et al. (1966), although no bacterial colonies could be found. In

addition, splenomegaly was observed in these culture negative individuals. Therefore,

75

the possibility that the detection of A. salmonicida salmonicida was missed in these
individuals exists.

Although reported from other Coregonus spp. from Finland (Rintamaki & Koski,
1987), the isolation of A. salmonicida salmonicida from lake Whiteﬁsh (C. clupeaformis)
has never been reported. This occurrence is not considered surprising, as A. salmonicida
salmonicida is known to infect a plethora of salmonids (Cipriano and Bullock 2001) and
studies focusing on bacterial diseases of C. clupeaformis are few and far between.
Furthermore, A. salmonicida salmonicida has been isolated from a number of salmonids
and non-salmonid ﬁsh species in the Great Lakes basin (Faisal and Hnath, 2005; Faisal
and Loch, unpublished data; McCraw 1952).

In conclusion, A eromonas salmonicida salmonicida has been isolated from lake
Whiteﬁsh inhabiting Lakes Michigan and Huron. These infections were detected at a
relatively low overall prevalence of 0.3% throughout the course of this three-year study.
As such, the potential impact that A. salmonicida salmonicida derived infections may
currently have on lake Whiteﬁsh within the study sites would seem to be low.
Additionally, a number of lake Whiteﬁsh with furuncle-like lesions were observed, yet
this bacterium could not be detected histologically, by culture, or via PCR. These
individuals could potentially represent those on their way to recovery, thus suggesting
that clearing of the infection can occur, or alternatively, these lesions may have been
unrelated to A. salmonicida salmonicida. However, the pathological effects that were
observed in individuals from which A. salmonicida salmonicida was isolated, both
grossly and histologically, may indicate that severe disease can ensue upon infection.

Thus, while this disease was not extremely predominant in sampled lake Whiteﬁsh, its

76

presence could potentially affect lake Whiteﬁsh at the population level if conditions
become more favorable for disease causation through habitat alteration, nutritional stress,
depressed condition factors, and/or other environmental/physiological stressors.
Additionally, if lake Whiteﬁsh are somewhat resistant to this disease but are able harbor
the bacterium at certain times or under certain conditions, they may represent a possible

reservoir for A. salmonicida salmonicida derived infections in other endemic ﬁsh species.

77

Sagiftléng Collection Site Organ Isolate ID #

8/28/04 Detour Village Kidney DV-AS-l
9/1 1/04 Naubinway Kidney NB-AS-l
1/07/05 Detour Village Kidney DV-AS-2
6/28/06 Naubinway Kidney NB-AS-2

Table 5. Sampling dates and sites from which A. salmonicida subspecies salmonicida
was recovered from lake whiteﬁsh using culture techniques during this three-year

study (Nov. 2003-Aug. 2006).

78

 

 

 

 

 

Pigment Production on TSA + H2S -
Triple Sugar Iron K/A/-/- Indole -
Gas from Glucose - Motility -

ONPG + Methyl Red +
Arabinose (+) Voges Proskauer -
Galactose + Bile Esculin +

Glycerol + Catalase +
Maltose + Cytochrome Oxidase +
Sucrose - Lysine Decarboxylase (+)
Trehalose - Omithine Decarboxylase -
Xylose - Arginine Dihydrolase +
Nitrate Reduction + Gelatinase +

 

Table 6. Biochemical phenotypes of A. salmonicida salmonicida isolates retrieved
from lake whiteﬁsh. +, positive reaction; -, negative reaction; (+) weak and/or
delayed positive reaction. All tests were incubated at 22°C, with results being
recorded at up to 7 days post-inoculation with the following exceptions: methyl red,
Voges-Proskauer, indole production, and TSI reactions were read at 2 days.
Production of acid from carbohydrates was examined in phenol red broth base at a
ﬁnal concentration of 1%. All materials and reagents were purchased from Remel
Inc. (Lenexa, KS). All strains were Gram-negative, non-spore forming, nonmotile,
faculatative anaerobes that produced an alkaline over acid TSI reaction without any

gas or H2S production.

79

 

 

 

 

 

 

 

 

 

 

Season
Collection Site Fall Winter Spring Summer Site Total
. 0% 0% 0.0% 0% 0%
3'9 Bay °° N°° (0791) (0775) (0790) (0/82) (07338)
. 0% 0% 1.1% 1.3% 0.6%
N°“°'"‘”°V (0790) (0753) (1790) (1/80) (27313)
0% 0% 0.0% 0% 0%
Cheb°ygan (0756) (0777) (0/80) (0/88) (07301)
. 0% 1.4% 0% 1.3% 0.6%
Dem" V'"age (0794) (1770) (0790) (1 /80) (27334)
0% 0.4% 0.3% 0.6% 0.3%
s°°°°"T°t°' (07331) (17275) (17350) (27330) (4I1286

 

Table 7. Percent prevalence of Aeromonas salmonicida subspecies salmonicida
infections in lake Whiteﬁsh by site and season, as well as the total prevalence,

throughout the course of the study (Nov. 2003-Aug. 2006).

80

 

» \,...~’..

Figure 13. Extensive severe hemorrhaging within the musculature and vent of a

lake Whiteﬁsh infected with A. salmoncida salmonicida.

81

 

Figure 14. Furuncule-like lesion on the dorsal musculature of a lake Whiteﬁsh that

was both culture and PCR negative for A. salmonicida salmonicida.

82

 

muscular degeneration and necrosis in a lake Whiteﬁsh.

83

 

Figure 16. Skin and underlying musculature from a furuncle-Iike lesion found in
lake whiteﬁsh exhibiting ulceration, necrotizing dermatitis & myositis, mixed
lymphocytes, macrophages, rare heterophils, ﬁbrin, and hemorrhage. No bacterial

colonies were observed (H&E stain, 10X magniﬁcation).

84

CHAPTER FOUR

ISOLATION OF MOTILE AEROMONAS SPP. FORM LAKE WHITEFISH

(COREGONUS CLUPEAFORMIS) IN LAKES MICHIGAN AND HURON

ABSTRACT

A number of motile Aeromonas species have been isolated from lake Whiteﬁsh
(C oregonus clupeaformis) collected from four stocks within Lakes Michigan and Huron,
USA. Sixty-nine isolates from the A. hydrophila, A. sobria, or A. caviae complexes were
recovered from the kidneys of infected ﬁsh, some in pure, and some in mixed culture.
Representative isolates were cytochrome oxidase positive, faculatatively anaerobic
Gram-negative bacilli. Using biochemical identiﬁcation schemes to speciate
representative isolates, twenty-two were identiﬁed as A. hydrophila sensu stricto, nine as
A. jandaei, three as A. veronii bv sobria, and two as A. eucrenophila. The prevalence of
infection by motile aeromonads varied by site and season, with lake Whiteﬁsh sampled in
the summer having the highest prevalence. External clinical signs of disease in lake
Whiteﬁsh included congestion and or hemorrhaging in the ﬁns, general pallor, and
petechial/ecchymotic hemorrhagic foci within the musculature. Internal clinical signs
included varying combinations of congestion, mottling, hemorrhaging, pallor, and
multifocal necrotic foci within the liver; moderate to severe splenomegaly; congestion,

friability, mottling, and/or swelling in the kidneys; pronounced erythema within the

85

peritoneal cavity; and mild hemorrhagic enteritis. This study provided evidence on the

wide spread motile aeromonad infections present within the Great Lakes Basin.

86

INTRODUCTION

Lake Whiteﬁsh is an indigenous species within the Great Lakes basin that
comprises one of the most economically important ﬁsheries (Bronte et al. 2003; Hoyle
2005; Schneeberger et al. 2005; Cook et al. 2005; Mohr and Ebener 2005). The lake
Whiteﬁsh plays an integral role in the benthic food-web by efficiently cycling energy
harnessed from primary producers by benthic organisms into the upper trophic levels,
hence leading to a harvestable resource (Mohr and Nalepa 2005). Recent declines in
abundance, condition, and size at age in harvested lake Whitefish have been observed in a
number of the Great Lakes (Hoyle et al. 1999; Pothaven et al. 2001; Madenjian et al.
2002; Mohr and Ebener 2005), spurring much research on the factors that may be
contributing to these declinations.

Many diseases are well known to affect animal survival at the population and
community levels (Holmes 1996). One of the many important bacterial genera that are
known to cause disease in ﬁsh is the genus Aeromonas. These oxidase-positive,
facultatively anaerobic, glucose-fermenting, Gram-negative bacilli are ubiquitous to the
aquatic environment and have been found in both healthy (Trust et al. 1974) and diseased
poikilotherrnic and homeothermic organisms, including humans (Austin and Adams
1996; Janda and Abbott 1996). The motile, mesophilic members of the genus Aeromonas
often cause disease in association with various environmental or physiological stressors,
such as spawning, warm water temperatures, poor water quality, poor nutritional status,
parasitic infections, or poor over-wintering conditions (Aoki 1999; Noga 2000; Austin

and Adams 1996). Disease in ﬁsh, often termed motile aeromonad septicemia (MAS),

87

typically manifests in an acute, chronic, or sub-clinical form (Cipriano 2001), and can
cause a variety of clinical signs, such as ﬁn/tail rot, dermal ulceration, and hemorrhagic
septicemia (Aoki 1999). MAS is often characterized by small surface lesions that may
lead to sloughing of scales, hemorrhaging in the gills and anus, ulcers, abcesses,
exopthalmia, anemia, renal and hepatic swelling, and abdominal swelling due to presence
of ascites (Austin and Adams 1996).

The Aeromonas species that are most connected to diseases in ﬁsh are A. caviae
(Paniagua et al. 1990), A. allosaccharophila (Martinez-Murcia et al. 1992), A. bestiarum
(Ali et al. 1996), A. veronii bv veronii (Sugita et al. 1995), A. veronii biovar sobria
(Rahman et al. 2002), A. jandaei (Esteve et al. 1994), A. encheleia (Esteve et al. 1995), A.
eucrenophila (Huys et al. 1997), A. hydrophila (Santos et al. 1988; McGarey et al. 1991;
Monette et al. 2006), and phenospecies A. sobria (Toranzo et al. 1989; McGarey et al.
1991; Wahli et al 2005). Herein, we report on the isolation of multiple motile Aeromonas
species from systemically infected lake Whiteﬁsh stocks collected from lakes Michigan
and Huron, U.S.A. Characterization of the various isolates using morphologic and

biochemical criteria were conducted.

88

MATERIALS AND METHODS

Fish and Sampling. Between October of 2005 and August of 2006, lake
Whiteﬁsh were collected for bacteriological analyses from 4 representative stocks on four
sampling occasions (~30 ﬁsh/site/season, Table 1). The two Lake Michigan sites, Big
Bay de Noc (latitude 4527-4548.20; longitude 8535-8722) and Naubinway (latitude
4553-4603; longitude 8513-8535), and the two Lake Huron sites, Detour (latitude 4555-
455697; longitude 83505-841912) and Cheboygan (latitude 4540.24-4548; longitude
8424-843 7.87) were selected for this study due to their association with large spawning
aggregations and their accessibility via commercial ﬁshing (Figure 1). A total of 443 lake
Whiteﬁsh from the four sites were analyzed in this study (Table 8).

Fish were captured using commercial trap nets and, when necessary, commercial
gill nets, with only live ﬁsh being used for the study. The collected ﬁsh were transported
alive to the Chippewa Ottawa Research Authority (CORA) Fishery Enhancement Facility
near Hessel, Michigan. The ﬁsh were then chilled on ice and transported to the Aquatic
Animal Health Laboratory at Michigan State University, East Lansing, MI,
(approximately 4 hours) where they were immediately subjected to thorough internal and

external clinical examinations.

Bacterial Isolation. Bacterial samples retrieved from the Whiteﬁsh were

collected from the kidneys, as well as visible lesions. Tissue samples were inoculated

directly onto trypticase soy agar (TSA) (Remel Inc., Lenexa, KS) and incubated at 22°C

89

for up to 72 hr. Periodic examination of bacterial growth was recorded and individual

colonies were sub-cultured onto TSA and incubated for 24 hours at 22°C

Biochemical characterization. Isolated bacteria were initially identiﬁed as
presumptive Aeromonas spp. based on colony morphology, Gram reaction, cytochrome
oxidase, and oxidation/fermentation (BD Scientiﬁc, Sparks, MD) reactions, as well as
resistance to the vibriostatic agent 0/129 (2,4-diamino,6,7-di-isopropyl pteridine; Holt et
al. 2000; MacFaddin 2000). Following these criteria, representative isolates were then
subjected to additional biochemical analyses following the scheme and recommendations
of Abbott et al. (2003). Using tests for the hydrolysis of esculin, production of acetoin
(Voges-Proskauer), gas production from glucose, and acid production from arabinose,
isolates were initially dichotomized into one of the three complexes (A. hydrophila
complex, A. caviae complex, and A. sobria complex). Isolates that were esculin, Voges-
Proskauer, and gas from glucose positive (one exception), but arabinose variable were
placed into the A. hydrophila complex. Assignation to the A. sobria complex occurred
when isolates were esculin negative, Voges-Proskauer positive (one exception), gas from
glucose positive, and arabinose negative. Esculin variable, Voges-Proskauer negative,
gas from glucose variable, and arabinose negative isolates were assigned to the A. caviae
complex. In cases where results of these four tests were ambiguous for complex
assignation and/or speciation was possible, the Moeller Reactions (lysine decarboxylase,
ornithine decarboxylase, arginine dihydrolase), as well as other assays, including the

catalase test (3% H202), motility, indole production, hydrogen sulﬁde production, methyl

red, nitrate reduction, phenylalanine deaminase, gelatinase, and Simmon’s citrate were

90

used. Additionally, production of acid from the following carbohydrates was examined
in phenol red broth base at a ﬁnal concentration of 1%: arabinose, adonitol, cellobiose,
galactose, glycerol, inositol, lactose, maltose, mannitol, mannose, melibiose, rafﬁnose,
rharnnose, salicin, sorbitol, sucrose, trehalose, and xylose. Tests were incubated for 10
days and read periodically, with the following exceptions: tests for susceptibility to
O/129 were read at 1 day; methyl red, Voges-Proskauer, indole production, and Simmons
citrate reactions were read at 2 days. All materials and reagents were purchased from

Remel Inc. (Lenexa, KS) unless speciﬁed otherwise.

Analysis of isolates by species, site, and season. Upon identiﬁcation to the
species level using morphological and biochemical proﬁles, an analysis of the

predominant species found in association with each season and/or site was conducted.

Statistical analysis. Comparisons between the ﬁsh stocks for differences in the
prevalence of motile A eromonas spp. by site or season were carried by the Pearson Chi
square ()6) analysis of contingency tables. Yates continuity correction was applied in
the calculation of values when the degrees of freedom (DF)=1. All calculations were
performed using the SigmaStat software package (Jandel Scientiﬁc Inc, San Rafael,

CA).

91

RESULTS

Throughout this study, a number of bacterial species were isolated and
characterized based on their morphological and biochemical characteristics. Among
these, 69 isolates were presumptively identiﬁed as motile Aeromonas spp. Twenty-two
Aeromonas spp. isolates were obtained in pure culture, while the remaining forty-seven
isolates were present in mixed cultures. Colony counts of primary cultures varied, with a
range of 1 colony forming unit (CF U) per 10 pl of kidney tissue inoculum to heavy
interconnected growth covering the inoculum streaks. Other bacterial species recovered
along with the motile aeromonads using standard culture techniques in this study
included nine isolates of Shewanella spp., one isolate of Carnobacterium
maltaromaticum, and rarely, members of the enteric bacteria (Family

Enterobacteriaceae). These bacteria will not be discussed further in this chapter.

Bacterial colonies suspected of being an Aeromonas spp. were subcultured onto
TSA and incubated for l8-24hrs at 22°C. Upon subculture, individual colonies ranged
from 0.75 mm to 1.25 mm in diameter, were cream to buff-colored, and were convex
with entire margins. Gram-stains performed on 18-24 hr old sub-cultures of suspect
Aeromonas isolates demonstrated Gram-negative bacilli (length of 0.75-2.5 pm; width of
0.3-0.5 pm) that were sometimes found in short chains. In some cases, evident
pleomorphism was present. F orty-eight Aeromonas isolates were selected for additional
analysis. The selected isolates, which were cytochrome oxidase positive, facultatively
anaerobic, and resistant to the vibriostatic agent 0/129, were further analyzed using

biochemical means, following the scheme of Abbott et al. (2003).

92

Results from the performed biochemical tests enabled the majority of isolates to
be grouped within the Aeromonas complexes (A. hydrophila, A. sobria, A. caviae),
however, six isolates could not be grouped in this manner (Table 9). Following the
Aeromonas identiﬁcation schemes of Abbott et al. (2003), of the 48 isolates, 25 belonged
to the A. hydrophila complex, 14 to the A. sobria complex, and 3 to the A. caviae
complex. When the 25 isolates within the A. hydrophila complex were speciated
according to Abbott et al. (2003), 22 were identiﬁed as A. hydrophila sensu stricto, while
three could not be speciated. Within the A. sobria complex, nine were identiﬁed as A.
jandaei, three as A. veronii bv sobria, and two could not be speciated. Of the three
isolates within the A. caviae complex, two were identiﬁed as A. eucrenophila, while one
could not be speciated.

As displayed in Table 10, the twenty-two A. hydrophila sensu stricto isolates were
positive for the Voges-Proskauer reaction, esculin hydrolysis, presence of lysine
decarboxylase and phenylalanine deaminase, and acid production from cellobiose,
galactose, glycerol, maltose, mannitol, mannose, and trehalose. All A. hydrophila
isolates were negative for H2S production, as well as acid production from adonitol,
inositol, lactose, sorbitol, rafﬁnose, rhamnose, and xylose. The remaining tests that were
examined yielded variable results.

Among the recovered isolates belonging to the A. sobria complex, nine were
identiﬁed as A. jandaei. Biochemical evaluation of these isolates produced unifome
positive results for catalase and indole production, motility, gas production from glucose,
and acid production from galactose, glycerol, mannitol, mannose, and trehalose (Table

10). Negative results were observed for H2S production, esculin hydrolysis, and acid

93

production from adonitol, arabinose, salicin, sorbitol, sucrose, rafﬁnose, rhamnose, and
xylose for all tested isolates. The remaining tests gave variable results

Biochemical examination results for the three isolates identiﬁed as A. veronii bv
sobria (Table 10) are as follows: All isolates were positive for indole production,
motility, Voges-Proskauer reaction, gas production from glucose, presence of lysine
decarboxylase and arginine dihydrolase, and acid production from sucrose. Moreover, all
3 isolates were negative for esculin hydrolysis, presence of ornithine decarboxylase,
production of H23, and acid production from arabinose. The only examined tests that
gave variable results for these isolates were Simmons citrate (33% positive) and mixed
acid production (33% methyl red positive).

The two isolates within the A. caviae complex that were identiﬁed as A.
eucrenophila yielded uniform positive results for mixed acid production (MR+), motility,
catalase and indole production, esculin hydrolysis, gas production from glucose, and
presence of lysine decarboxylase and arginine dihydrolase. Negative results for H2S
production, utilization of citrate, presence of ornithine decarboxylase, Voges-Proskauer
reaction, and acid production from arabinose were obtained for these isolates.

Among the 48 isolates selected for further analysis, 11 were retrieved both in pure
culture (in one instance, two distinct Aeromonas spp. isolated from one lake Whitefish)
and were all associated with varying signs of clinical disease (Table 11). Among the ﬁsh
from which these isolates were recovered, a portion were normal externally (5/10), while
a number of infected individuals presented with congestion and or hemorrhaging in the
ﬁns (3/10), pale coloration (2/10), and/or petechial and ecchymotic hemorrhagic foci

within the ventral musculature (1/10). Internally, some of the infected individuals

94

exhibited hepatic abnormalities such as congestion (1/10), mottling (2/ 10), hemorrhaging
(1/10), pallor (1/10), and multifocal necrotic foci (1/10). Additionally, moderate to
severe splenomegaly, with or without darkening, was apparent in 6/10 individuals.
Observed renal abnormalities included congestion (5/10), friability (5/10), mottling
(3/10), and/or swelling (1/10). In one case, pronounced erythema within the peritoneal
cavity was evident. Moreover, mild hemorrhagic enteritis was apparent in 3/10
individuals. Of the 11 motile aeromonad species that were retrieved in pure culture, six
belonged to the A. hydrophila complex, four belonged to the A. sobria complex, and one
belonged to the A. caviae complex.

The overall detected prevalence of motile aeromonads infecting lake Whiteﬁsh
during the period of this study was 14.45%. The prevalence of lake Whiteﬁsh infected
with motile Aeromonas spp. was higher in Lake Huron, at 15.71%, than in Lake
Michigan, 13.30%, but these differences were not statistically signiﬁcant. The
prevalence of lake Whiteﬁsh infected with motile aeromonads did vary by site (15.00%,
11.50%, 17.78%, and 14.17% for the Big Bay de Noe, Naubinway, Cheboygan, and
Detour sites, respectively), but differences were not statistically signiﬁcant (Figure 17).
However, differences in motile aeromonad prevalence among seasons were statistically
signiﬁcant (x°=22.098; DF=3; P=<0.001), with the highest prevalence occurring in
summer samples (Figure 18). The overall seasonal prevalence of motile aeromonad
infections in lake Whiteﬁsh by site is depicted in Figure 19.

The number of motile Aeromonas isolates identiﬁed to the complex level, and the
sites and seasons from which they were recovered can be found in Table 12, while the

prevalence of motile aeromonad derived infections can be found in Table 13.

95

Statistically signiﬁcant differences in the prevalence of infection caused by members of
the A. hydrophila, A. sobria, and A. caviae complexes were observed in this study
(96:17.85; DF =2; P=<0.001), with A. hydrophila complex members causing the highest
number of detected infections, followed by A. sobria complex. The proportion of isolates
belonging to each of the three complexes that were isolated from lake Whiteﬁsh at each of
the studied sites can be seen in Figure 20. Additionally, the proportion of isolates
recovered in the four sampled seasons belonging to each of the three complexes can be
found in Figure 21.

Of the 25 isolates belonging to the A. hydrophila complex, eight were recovered
from lake Whiteﬁsh caught from the Naubinway site, six from Cheboygan, seven from
Big Bay de Noc, and four from Detour. This corresponded to an overall prevalence of
5.64% in all sampled sites, with 7.08% prevalence in Naubinway lake Whiteﬁsh, 6.67%
in Cheboygan, 5.83% in Big Bay de Noe, and 3.33% in Detour. A. hydrophila complex
infections in lake Whiteﬁsh were most prevalent during the Summer sampling period
(8.33%), followed by the Spring (5.83%), Winter (5.31%), and Fall (2.22%) (Table 13).
Differences in the prevalence of infection by members of the A. hydrophila complex were
not statistically signiﬁcant among sites or seasons.

The 14 isolates identiﬁed as members of the A. sobria complex were recovered
from all four sites; six from Big Bay de Noe, four from Cheboygan, three from
Naubinway, and one from Detour Village. This complex was detected at an overall
prevalence of 3.16%, and were most prevalent in the Big Bay de Noc samples (5.00%),
followed by Cheboygan (4.44%), Naubinway (2.65%), and Detour (0.83%). Seasonally,

the prevalence of the A. sobria complex was most prevalent in Summer (6.67%),

96

followed by Winter (3.54%), Fall (1.11%) and Spring (0.83%) sampling periods.
Differences in infection prevalence by A. sobria complex were not statistically signiﬁcant
among sites and seasons. Infections with A. caviae complex members were detected at a
prevalence of 0.68% from all of the sampled sites. Big Bay de Noc (1.67%) and Detour
(0.83%) were the only sites where this complex was detected, with summer having the
highest prevalence (1.67%), followed by the spring (0.83%); however, these differences
were not statistically signiﬁcant.

When broken down by species, A. hydrophila sensu stricto was the highest in
prevalence among infected ﬁsh (22/443; 76:41.9; DF=4; P=<0.001;Table 14). The
prevalence of individuals infected with A. hydrophila was greatest in Naubinway samples
(6.19%), followed by Cheboygan (5.56%), Big Bay de Noc (5.00%), and Detour (3.33%).
The percent of infected individuals was greatest in the summer (6.67%), followed by
winter (5.31%), spring (5.00%), and fall (2.22%).

The next most common motile aeromonad species isolated in this study was A.
jandaei (A. sobria complex), with an overall prevalence of 2.03%. Prevalence of
infection was highest in Naubinway samples (2.65%), followed by Big Bay de Noe
(2.5%), Cheboygan (2.22%), and Detour (0.83%). The season of highest prevalence was
winter (3.54%), followed by summer (2.50%), fall (1.11%), and spring (0.83%). A.
veronii bv. sobria was the next most predominant motile aeromonad, with an overall
prevalence of 0.68%. This bacterium was prevalent only in Big Bay de Noc lake
Whiteﬁsh sampled within the summer (summer prevalence from BDN at 10.00%). Thus,
a prevalence of 2.50% was detected in Big Bay de Noc and summer samples, while all

other seasons and sites were negative. A. eucrenophila (overall prevalence of 0.45%)

97

was also isolated from individual lake Whiteﬁsh from both Big Bay de Noc and Detour
samples, with a prevalence of .83% in both sites. The seasonal prevalence was 1.67%

within summer samples, but negative in all other seasons.

98

DISCUSSION

Throughout the course of this study, numerous isolates identiﬁed as Aeromonas
spp. were recovered from systemically infected lake Whiteﬁsh. While a portion of these
isolates were found in mixed culture or in the presence of other pathogenic organisms,
many isolates were recovered in pure culture and were associated with obvious signs of
disease. For instance, ﬁsh infected with A. hydrophila are known to exhibit hemorrhage
in the trunk and ﬁns (Hoshina 1962). Similar observations were made in lake Whiteﬁsh
infected with motile aeromonads. Additionally, the degenerative changes observed in the
livers and kidneys of many lake Whiteﬁsh infected with motile aeromonads have also
been reported from other. ﬁsh species systemically infected with Aeromonas spp.
spontaneously (Huizinga et al. 1979, Miyazaki and Kaige 1985) and experimentally
(Bach et al. 1978). Furthermore, through rapid proliferation within the intestine, some
motile aeromonads can elicit a toxemia via adsorption of toxic metabolites through the
intestinal epithelium (Miyazaki and Jo 1985; Miyazaki and Kaige 1985), which among

other things, generates signiﬁcant pathology within the gastrointestinal tract.

A number of isolates were also recovered from individuals with marked external
ulcerations, along with a multitude of internal clinical signs, but were present along with
other pathogens. These include R. salmoninarum, the etiological agent of bacterial
kidney disease, C. maltaromaticum, the causative agent of pseudokidney disease, and
Shewanella spp., which cause shewanellosis, as well as a number of parasites, such as
Cystidicolafarionis, encysted metacercariae, and acanthocephalans. Interactions among

and between these pathogens in this host species are currently unknown.

99

Of the four species of motile aeromonads that were isolated from infected lake
Whiteﬁsh and identiﬁed according to Abbott et al. (2003), A. hydrophila sensu stricto was
the most prevalent, accounting for the highest proportion of infections in all sampled
seasons and sites. The prevalence of this bacterium was at least double that of the next
most predominant species in all sampled sites. The role of A. hydrophila in ﬁsh
infections is well known and, among the mesophilic aeromonads, is considered to be the
most important ﬁsh pathogen (N oga 2000). The next most prevalent motile aeromonad
species was A. jandaei, which was detected in over 2.00% of the lake Whiteﬁsh sampled
from 3 of the 4 sites. An interesting aspect of these infections was that they were most
prevalent in the winter sampling period. This in contrast to all other detected motile
aeromonad species, which were without exception, most prevalent in lake Whiteﬁsh
during the summer. Reasons for this seasonal difference in A. jandaei prevalence are
currently unknown. It is also interesting to note that this bacterium was always recovered
from organisms concurrently infected with another bacterial species, although the mixed
ﬂora was not consistent. In a previous study by Esteve and Garay (1991), A. jandaei ,
along with a number of other bacterial species (i.e. A. hydrophila, Pseudomonas
ﬂuorescens, Shewanella putrifaciens) were recovered from 3 disease epizootics in the
European eel (Anguilla anguilla), during which mortality rates of up to 80% were
observed. These isolates were later analyzed for their virulence via experimental
exposure, and only the A. hydrophila and A. jandaei isolates were able to reproduce the
clinical signs observed in the natural infections (Esteve et al. 1993).

Aeromonas veronii bv sobria was also isolated in this study, although its presence

was limited to Big Bay de Noc ﬁsh and only in the summer. Additionally, two of the

100

three A. veronii bv sobria isolates were recovered in pure culture, and the infected
individuals presented with clinical signs of disease, including renal mottling and pallor,
multi-focal necrotic foci within the liver, and mild hemorrhagic enteritis in the posterior
portion of the intestine. As such, the relatively high prevalence of this bacterium in lake
Whiteﬁsh collected from Big Bay de Noc in the summer, along with the observed gross
pathological effects, suggested that an A. veronii bv sobria-derived widespread infection
may have occurred. In a study by Rahman et al. (2002), virulence factors of A. veronii bv
sobria isolates recovered from deep hemorrhagic ulcers in a multitude of ﬁsh species in
Bangladesh, exhibited cytotoxic and hemolytic properties , in contrast to other
Aeromonas spp. recovered from environmental sources (Rahman et al. 2002).

The two isolates identiﬁed as A. eucrenophila were recovered from Big Bay de
Noc and Detour Whiteﬁsh in the summer. These isolates were recovered from individuals
with mixed infections exhibiting hemorrhagic and prolapsed vents, generalized erythema
within the peritoneum, including the internal lateral and ventral musculature, a small
amount of serosanguinous ﬂuid within the peritoneum, multi-focal hemorrhage within the
liver, moderate splenomegaly, a friable kidney, and gastroeneteritis/hemorrhagic enteritis.
In a study by Santos et al. (1999), the presence of a number of virulence factors
implicated in disease causation, such as aerolysin, siderophores, and other hemolytic
toxins, were detected in strains of A. eucrenophila recovered from a variety of freshwater
ﬁsh.

A number of atypical biochemical reactions were observed for the motile
aeromonad isolates recovered in this study. For example, 13.64% of the A. hydrophila

isolates recovered from lake Whiteﬁsh were ornithine decarboxylase positive, 9.10% were

101

arginine dihydrolase negative, and 4.55% were catalase negative, none of which was an
observed occurrence in A. hydrophila isolates studied by Abbott et al. (2003). Acid
production from cellobiose was a rare occurrence for A. hydrophila isolates analyzed by
Abbott, yet all of our A. hydrophila isolates were positive. Additionally, acid production
from melibiose has been rarely reported in Aeromonas spp., let alone in A. hydrophila,
but 66.67% of the isolates tested for this characteristic in this study were positive.
Additional atypical reactions recorded in this study include: lysine decarboxylase
negative (11.11%), ornithine decarboxylase positive (11.11%), arginine dihydrolase
negative (11.11%), and acid production from lactose positive (25.00%) strains of A.
jandaei, as well as lysine decarboxylase in A. eucrenophila (100%)

One possible explanation for these atypical reactions is the extremely
heterogenous nature of Aeromonas spp, which can account for large discrepancies in
phenotypic characteristics. Moreover, extrachromosomal elements can encode for
various metabolic activities, as well as antibiotic resistance, further perpetuating the
already diverse phenotypes associated with many Aeromonas strains (Abbott et al. 2003;
Walsh et al. 1995).

The diversity of recovered motile Aeromonas spp. was the highest in lake
Whiteﬁsh collected from Big Bay de Noc, with all four of the species isolated in this
study being represented. Conversely, motile aeromonad infections in Naubinway and
Cheboygan were only by A. hydrophila and A. jandaei. Detour Aeromonas isolates were
only slightly more diverse, with A. hydrophila, A. jandaei, and A. eucrenophila being
detected here. Reasons for the diversity in Big Bay de Noc, or lack thereof in the other

sites, are currently unknown, but may be explained by differences in migration rates,

102

increased reservoir abundance in particular sites, and/or increased susceptibility to motile
aeromonad infections in particular lake Whiteﬁsh populations, though further
investigation is required.

At the complex level, statistically signiﬁcant differences in motile aeromonad
prevalence were detected. Aeromonas hydrophila complex members were responsible
for the most infections caused by a motile aeromonad in this study, followed by A. sobria
and A. caviae complex members. In other studies, such as the one conducted by
Paniagua et al. (1990), A. hydrophila was the most common group of aeromonads
isolated from river water, and these isolates were found to be virulent more often than the
other less frequently isolated motile aeromonads.

Other studies have reported motile aeromonad infection prevalences of 7.7% in
wild ﬁsh populations in Croatia (Popovic et al. 2000), 2.5% in wild Nile tilapia
(Oreochromis niloticus), and 6.25% in wild Karmout catﬁsh (Eissa et al. 1994). Given
that motile aeromonads are extremely widespread in aquatic environments (Hazen et al.
1978; Alonso et al. 1994), and that the Great Lakes are currently in a state of ﬂux (see
literature review), the relatively high overall prevalence of motile Aeromonas infection
(14.45%) is not a complete surprise. By site, the detected prevalence of motile
aeromonads infection in lake Whiteﬁsh was highest in lake Whiteﬁsh from Cheboygan,
followed by Big Bay de Noe, Detour, and Naubinway; however these differences were
not statistically signiﬁcant. In preliminary results from other aspects of the Whiteﬁsh
Natural Mortality Project, lake Whiteﬁsh from Cheboygan have been demonstrated to
have the lowest lipid levels among all four sites, and thus may be under the most

nutritional stress, putting them at a higher risk for infection. However, many factors are

103

known to play a role in disease processes, and as such, further research will be necessary
to further elucidate differences among sites (Dr. Michael Jones and Dr. Michael Arts,
pers. comm).

Seasonally, overall motile aeromonad infections in lake Whiteﬁsh were most
prevalent in the summer. The observed seasonal pattern in this study is consistent with
what is considered typical in a number of other studies. High temperatures are frequently
observed in motile aeromonad infections (Aoki 1999). For example, Meyer (1970)
reported epizootics caused by motile aeromonads among warm water ﬁshes in the
southeastern US. to be most common in spring and early summer when water
temperatures are high. Moreover, mesophilic Aeromonas spp. present within the
environment have been detected in high densities when water temperatures are at their
highest point (Osborne et al. 1989). Additionally, pathogen surveys in wild and feral
populations of ﬁsh conducted at the Aquatic Animal Health Laboratory (AAHL)
(Michigan State University) have found a higher prevalence of motile aeromonad
infections in warmer months of the year (Loch and Faisal unpublished data).
Concurrently, clinical cases submitted to the AAHL involving motile aeromonad
infections in captive stocks are more frequent in the warmer months (Loch and Faisal
unpublished data).

In conclusion, the clinical ﬁndings associated with those Aeromonas species that
were recovered in pure culture, along with the relatively high prevalence of infections
detected in a portion of the sampled sites or seasons (i.e Cheboygan Summer samples
greater than 40% prevalence) suggest that this complex of bacteria may be capable of

causing signiﬁcant amounts of disease in lake Whiteﬁsh populations, especially during

104

periods of high water temperatures. Furthermore, the high proportion of Aeromonas
infections detected in the presence of additional pathogens (>80%) suggests that even if
many of the A eromonas infections are occurring secondarily to other infections, the
possible augmentation of disease processes through the synergistic effects of multiple
pathogens may have serious implications on infected individuals. For instance, numerous
reports of infestations with parasites and fungi have been shown to predispose ﬁsh to
infections with motile aeromonads (Noga 1986; Esch and Hazen 1980; Egusa 1978). In
addition, immuno-supression brought about by physiologic (i.e., spawning) and
environmental (i.e. high temperatures and low water levels) stressors can predispose
individuals to signiﬁcant infections with motile aeromonads, as illustrated by the recent
massive ﬁsh kill in common carp (Cyprinus carpio) residing in the St. Lawrence River

(Monette et al. 2006), which was partially attributed to infection by A. hydrophila.

105

Sampling Season

Period 80 NB C DV Total
Fall 05 30 30 0 30 90
Winter 06 30 23 30 30 113
Spring 06 30 30 30 30 120
Summer 06 30 30 30 30 120
Site Total 120 113 90 120 443

Table 8. Representative seasons with number of lake whiteﬁsh sampled on each

occasion throughout the course of this study.

106

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

A. h dro hlla A. sobria A. caviae
Test “1:04.398 cgmplgx complex complex
(n=25) (n=14) (n=3)
Catalase 97.92% (48) 96.00% (25) 100.00% (14) 100.00% (3)
Simmon's Citrate 20.83% (48) 24.00% (25) 21.43% (14) 0.00% (3)
Indole 91.67% (48) 96.00% (25) 100.00% (14) 66.67% (3)
Motility 95.83% (48) 96.00% (25) 100.00% (14) 100.00% (3)
Methyl Red 54.17% (48) 52.00% (25) 50.00% (14) 100.00% (3)
Voges-Proskauer 83.33% (48) 100.00% (25) 93.33% (14) 0.00% (3)
Esculin 59.57% (47) 100.00% (25) 0.00% (14) 66.67% (3)
213:3: 91.67% (48) 96.00% (25) 100.00% (14) 66.67% (3)
Decimggase 94.74% (38) 100.00% (22) 91 .67% (12) 66.67% (3)
03:31:33; 9 13.64% (44) 12.00% (25) 14.29% (14) 0.00% (3)
5'9“" 86.36% (44) 92.00% (25) 85.71% (14) 66.67% (3)
Dihydrolase
Pgi'm‘i‘ﬂrg" 88.89% (18) 100.00% (8) 80.00% (5) 0.00% (1)
Arabinose 4.65% (43) 8.00% (25) 0.00% (13) 0.00% (3)
Adonitol 0% (8) 0% (5) 0.00% (3) ND
Cellobiose 84.62% (13) 100.00% (6) 66.67% (6) 0.00% (1)
Galactose 100% (8) 100.00% (5) 100.00% (3) ND
Glycerol 87.50% (8) 100.00% (3) 100.00% (4) 0.00% (1)
Inositol 20.00% (5) 0.00% (3) 50.00% (2) ND
Lactose 10.00% (10) 0.00% (6) 25.00% (4) ND
Maltose 100% (5) 100.00% (3) 100.00% (2) ND
Mannitol 90.90% (11) 100.00% (5) 100.00% (5) 0.00% (1)
Mannose 90.00% (10) 100.00% (5) 100.00% (4) 0.00% ( 1)
Melibiose 42.86% (7) 66.66% (3) 25.00% (4) ND
Salicin 20.00% (10) 40.00% (5) 0.00% (5) ND
Sorbitol 9.09% (11) 12.50% (8) 0.00% (3) ND
Sucrose 23.53% (17) 20.00% (5) 27.27% (11) 0.00% (1)
Raffinose 0% (8) 0% (5) 0% (3) ND
Rhamnose 0% (12) 0% (8) 0% (4) ND
Trehalose 100% (8) 100.00% (5) 100.00% (3) ND
Xylose 0% (8) 0% (5) 0% (3) ND

 

 

Table 9. Percentage of Aeromonas isolates (by complex) in this study that were
positive for the performed biochemical tests. Number in parenthesis= number of
isolates tested. All isolates were cytochrome oxidase positive, facultative anaerobes
that were resistant to the vibriostatic agent 0/129 and did not produce H28 in

Sulfur-Indole-Motility medium.

107

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

. A. veronii bv A.
Test A. hydrophila % {#323396 sobria eucrenophila
Positive (n=22) (n=9) % Positive % Positive
(n=3) (n=2)
Catalase 95.45% (22) 100.00% (9) 100.00% (3) 100.00% (2)
Simmon's Citrate 27.27% (22) 22.22% (9) 33.33% (3) 0.00% (2)
Indole 95.45% (22) 100.00% (9) 100.00% (3) 100.00% (2)
Motility 95.45% (22) 100.00% (9) 100.00% (3) 100.00% (2)
Methyl Red 54.54% (22) 66.67% (9) 33.33% (3) 100.00% (2)
Voges-Proskauer 100.00% (22) 88.89% (9) 100.00% (3) 0.00% (2)
Esculin 100.00% (22) 0.00% (9) 0.00% (3) 100.00% (2)
gﬁjggg': 95.45% (22) 100.00% (9) 100.00% (3) 100.00% (2)
Decgfgdgliase 100.00% (20) 88.89% (9) 100.00% (3) 100.00% (2)
Deggg‘ggse 13.64% (22) 11.11% (9) 0.00% (3) 0.00% (2)
Daﬂgﬁse 90.90% (22) 88.89% (9) 100.00% (3) 100.00% (2)
szgﬁggge 100.00% (8) 80.00% (5) ND ND
Arabinose 4.55% (22) 0.00% (8) 0.00% (3) 0.00% (2)
Adonitol 0.00% (5) 0.00% (3) ND ND
Cellobiose 100.00% (6) 66.67% (6) ND ND
Galactose 100.00% (5) 100.00% (3) ND ND
Glycerol 100.00% (3) 100.00% (4) ND ND
Inositol 0.00% (3) 50.00% (2) ND ND
Lactose 0.00% (6) 25.00% (4) ND ND
Maltose 100.00% (3) 100.00% (2) ND ND
Mannitol 100.00% (5) 100.00% (5) ND ND
Mannose 100.00% (5) 100.00% (4) ND ND
Melibiose 66.67% (3) 25.00% (4) ND ND
Salicin 40.00% (5) 0.00% (5) ND ND
Sorbitol 0.00% (7) 0.00% (3) ND ND
Sucrose 20.00% (5) 0.00% (8) 100.00% (3) ND
Rafﬁnose 0.00% (5) 0.00% (3) ND ND
Rhamnose 0.00% (7) 0.00% (4) ND ND
Trehalose 100.00% (5) 100.00% (3) ND ND
Xylose 0.00% (5) 0.00% (3) ND ND

 

Table 10. Percentage of Aeromonas isolates (by species) in this study that were
positive for the performed biochemical tests. Number in parenthesis= number of
isolates tested. All isolates were cytochrome oxidase positive, facultative anaerobes
that were resistant to the vibriostatic agent O/129 and did not produce H28 in

Sulfur-Indole-Motility medium.

108

 

 

 

 

 

 

 

 

 

 

 

_ 0)
E E E 0 2 2 3 i 3
" .9 w 9 3 E .2 E 3’ _ E 2
Bacterial 5 g 9 E .g a 1; E E g E ‘GE,
Identiﬁcation - org 0 o E 8 O 0 u
“ t: E a O = 0! o I:
E o g o :l g I 5 g 5 m
g o a? I E l.l.l < m <
A. h ydrophila sensu + _ _ _ + + + _
stn‘cto
A. hydrophila complex - + + - - + + +
A. h ydrophila sensu _ + _ _ + + + _
stncto
A. h ydrophl'la sensu _ _ _ + + + + _
stn'cto
A. hydrophila complex + - - - + - + .
A. hydrophila complex - - - + + + + -
A. janadei at. A. _ + _ _ + + + +
eucrenophila
A. veronii bv sobria + - - - - - +
A. veronii bv sobn'a + - - - + - - +
A. sobria complex + - - - - - + -

 

 

Table 11. Gross clinical observations in lake Whitefish collected from Lakes
Michigan and Huron in which Aeromonas spp. were the only detected bacterial

species.

109

A. hydrophila A. sobria A. caviae
complex complex complex

7 6 2

3
4
1

Detour Vil
Season
Fall 2 1
Winter 6 4
7 1
8

Summer 0

 

Table 12. Number of isolates belonging to each complex that were recovered from

lake whitefish collected from Lakes Michigan and Huron between October 2005-

August 2006.

110

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Complex Site Season
Identity
BDN NB CHE DV Fall Winter Spring Summer
A. hydrophila 5.83% 7.08% 6.67% 3.33% 2.22% 5.31% 5.83% 8.33%
complex (7/120) (8/113) (6/90) (4/120) (2/90) (6/113) (7/120) (10/120)
A. sobria 5.00% 2.65% 4.44% 0.83% 1.11% 3.54% 0.83% 6.67%
complex (6/120) (3/1 13) (4/90) (1/120) (1 /90) (4/1 13) (1/120) (8/120)
A. caviae 1.67% 0.00% 0.00% 0.83% 0.00% 0.00% 0.83% 1.67%
complex (2/120) (0/1 1 3) (0/90) (1 /1 20) (0/90) (0/1 1 3) (1/120) (2/120)

 

Table 13. Detected prevalence of the three Aeromonas complexes infecting lake

whiteﬁsh collected from Lakes Michigan and Huron between October 2005-August

2006 (BDN=Big Bay de Noc; NB=Naubinway; CHE=Cheboygan; DV=Detour

Village).

111

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Identification
BDN NB CHE DV Fall Winter Spring Summer
A h dro bile 5.00% 5.19% 5.56% 3.33% 2.22% 5.31% 5.83% 3.33%
' y p (5/120) (7/113) (5/90) (4/120) (2/90) (6/113) (7/120) (10/120)
A. veroniibv 2.50% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 2.50%
sobria (3/120) (0/113) (0/90) (0/120) (0/90) (0/113) 0/120) (3/120)
A .andael. 2.50% 2.65% 2.22% 0.83% 1.11% 3.54% 0.83% 2.50%
" (3/120) (3/113) (2/90) (1/120) (1/90) (4/113) (1/120) (3/120)
. 0.83% 0.00% 0.00% 0.83% 0.00% 0.00% 0.00% 1.57%%
A'eucre"°ph”a (1/120) (0/113) (0/90) (1/120) (0/90) (0/113) (0/120) (2/120)

 

Table 14. Detected prevalence of the four Aeromonas species infecting lake

whitefish collected from Lakes Michigan and Huron between October 2005-August

2006 (BDN=Big Bay de Noe; NB=Naubinway; CHE=Cheboygan; DV=Detour

Village).

112

 

 

20%
18%
16%
14%
12%
10%
8%
6%
4%
2%
0% iiii . 2255222 .

Big Bay de Noc N aubinway Cheboygan Detour Village

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Prevalence

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 17. Prevalence of infection by motile Aeromonas spp. in lake whitefish from

four sites within Lakes Michigan and Huron between October 2005-August 2006.

113

 

30%

 

25%

 

20%

 

15%

Prevalence

 

10%

5%

 

 

0%

 

Figure 18. Seasonal prevalence of motile Aeromonas spp. infections in lake whitefish
collected from four sites within Lakes Michigan and Huron between October 2005-

August 2006.

114

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

50%
45% f
40% /
X
35% . .
8 300/ '
5 ° 1. /' WWW BDN
g 25% I" —I— NB
0 t + CHE
9': 20% Mi". - ->< - Dv
X A??? - ._ I~ www
15% S .1“ (‘55.. I '
k\ f / V
10% F” 2 I \
15W ‘ S \ I \ I
5% “gig; \ ‘ A
00A! 4 r ‘ x ‘ — X 1
Fall Winter Spring Summer

Figure 19. Overall prevalence of lake whiteﬁsh infected with motile Aeromonas spp.

by season and site between October 2005-August 2006.

115

100% -
90% -
80% -
70% ‘

 

60% -
50% ~
40% ‘
30% ‘
20% 1

[[I A. caviae complex

'5 A. sobria complex

B A. hydrophila complex

 

 

 

10% .
0% 1

 

 

 

Figure 20. Proportion of the total detected infections in lake whitefish caused by

members of the three Aeromonas complexes by site.

116

100% -
90% r

80%

70% -
60% ~
50% ~
40% -
30% -
20% ~
10% -

0% -

 

 

//

\ S Ill

1

 

[11] A. caviae complex
A. sobria complex
E A. hydrophila complex

 

 

 

IIIIIIIII
WWWWW%%%%

§
\

IIIIIIIIII
WWWWWW%%%W

lll||lllIll||||||||||||||Illlllll|||||||llllllllllllllllllIIIW

 

Fall Winter Spring Summer

Figure 21. Proportion of the total detected infections in lake whitefish caused by

members of the three Aeromonas complexes by season.

117

CHAPTER 5

CONCLUSIONS AND FUTURE RESEARCH

CONCLUSIONS

Despite the ecological and economic importance of indigenous lake whiteﬁsh
inhabiting the GLB, extremely little is known of the infectious diseases that afﬂict them.
It is well established that diseases can play a signiﬁcant role in the dynamics of ﬁsh
populations, and that there may be an increased risk of diseases in disturbed
environments. Disturbances in the Great Lakes, such as habitat alterations, invasions by
exotic organisms, and climate/temperature changes, may increase the susceptibility of
lake whiteﬁsh to disease. To ascertain whether this is truly a problem, one must have
some idea as to what pathogens are present within lake whiteﬁsh populations and
whether these organisms are capable of generating disease in this speciﬁc host. The
potential of these pathogens to generate mortality is also important. Although the base of
knowledge regarding diseases of ﬁsh within the GLB is ever expanding, a lack of
research on lake whiteﬁsh diseases is still a major impediment to understanding what
may or may not be playing a role in their population dynamics.

Through this study, I was able to isolate a number of bacterial species infecting
lake whiteﬁsh inhabiting the GLB, and thus gain some understanding as to what effects
these infections may have on this particular host species. In Chapter two, I found that C.
maltaromaticum, the causative agent of PseudoKidney Disease, which in itself is not
extremely well-studied, is present within populations of lake whiteﬁsh, and that

infections caused by this bacterium can potentially be associated with varying degrees of

118

pathology. Some of the lake whiteﬁsh from which C. maltaromaticum was recovered
exhibited signiﬁcant degenerative changes within their livers, spleens, and most
dramatically, swim-bladders, thus providing evidence that this bacterium may indeed be
capable of causing disease in lake whiteﬁsh. Moreover, through the use of biochemical
analyses, molecular assays, and genetic analyses, I was able to deﬁnitively identify these
isolates as C. maltaromaticum, yet demonstrate unique phenotypic characteristics and
some degree of phylogenetic variability was present among lake whiteﬁsh strains and
among previously isolated strains. The prevalence of this pathogen was not uniform
across space and time, with the majority of infected individuals coming from a single site
and sampling occasion (Naubinway, winter). The 20.8% prevalence that was detected in
the winter Naubinway samples is considered quite high for a wild ﬁsh population and
illustrates the potential of this bacterium to generate large-scale epizootics under
conditions favorable to disease causation. This was also considered the ﬁrst report of C.
maltaromaticum from this host species and within this geographic locale.

Within Chapter 3, I describe the isolation of Aeromonas salmonicida subspecies
salmonicida, the etiological agent of furunculosis, ﬁ‘om lake whiteﬁsh within the GLB,
an occurrence that has never been previously reported. Thorough clinical examination
and histopathological assessment, I observed the destructive capability that this bacterium
has on lake whiteﬁsh that may succumb to infection, thus validating the fact that a serious
bacterial pathogen is endemic within populations of GLB coregonines. However, the low
overall prevalence of infection that was detected throughout this study may indicate that
this bacterium may not currently be a signiﬁcant threat to lake whiteﬁsh populations

within Lakes Michigan and Huron. However, if conditions more conducive to disease

119

generation by A. salmonicida salmonicida should arise, the impact on lake whiteﬁsh
populations could be potentially devastating. Through the use of biochemical assays and
PCR, I was able to deﬁnitively identify this bacterium as A. salmonicida salmonicida. I
also attempted to detect A. salmonicida salmonicida in lake whiteﬁsh that presented with
furuncle-like lesions, yet were culture negative. While unable to detect the presence of A.
salmonicida salmonicida molecularly in these individuals, histopathological examination
of a portion of the furuncle-like lesions demonstrated nearly identical tissue alterations to
what has been previously reported in ﬁsh experimentally infected with this bacterium.
Moreover, infections in lake whiteﬁsh caused by A. salmonicida salmonicida were
detected in only two of the four sampled sites.

Finally, in Chapter 4, I describe the isolation of motile Aeromonas spp. from lake
whiteﬁsh within the GLB. This heterogenous group of bacteria was present in all four
sampled sites and seasons, with a tendency towards higher prevalence in summer, as
might be expected given the higher water temperatures at this time. When assigned to
their respective complexes, members of the A. hydrophila complex were the most
prevalent group, followed by members of the A. sobria complex. A total of four distinct
Aeromonas spp. were detected, including A. hydrophila sensu stricto, A. jandaei, A.
veronii bv sobria, and A. eucrenophila. A. hydrophila sensu stricto was the predominant
species, a common occurrence among studies looking at motile aeromonad infections in
ﬁsh, followed by A. jandaei. Some of the sampled sites had a more “diverse” population
of Aeromonas spp. that caused infections than others. Moreover, a large percentage of
the isolated motile aeromonads were found in the presence of an array of other

pathogens, thus suggesting that often, these bacteria may infect the host secondarily.

120

Clinical ﬁndings in lake whiteﬁsh infected only with Aeromonas spp. were quite varied,
but in general, demonstrated the ability of some of the isolated strains to cause

pathological changes in infected lake whiteﬁsh.

121

FUTURE RESEARCH

The data presented within this Thesis established a solid base for future research
that may involve bacterial pathogens of lake whiteﬁsh within the GLB. However, much
more work is needed if one wants to determine what bacteria are capable of affecting lake
whiteﬁsh at the population level. This Thesis focused on non-fastidious bacterial
pathogens of lake whiteﬁsh, but many other bacterial species, such as the facultatively
intracellular Mycobacterium spp., the obligately intracellular Rickettsia—like organisms,
obligate anaerobic Clostridium spp., and many others cannot be detected via the
methodologies used in this study. Studies speciﬁcally targeting these organisms are
necessary to determine their presence and/or rule out any impact, regardless how small,
they may have on lake whiteﬁsh.

Moreover, studies focusing on the interactions between multiple pathogens that
were found to infect a single host concurrently could aid in the elucidation of disease
processes in these organisms. It is likely that these pathogens may impact one another
negatively in some situations, while in others they may act synergistically in the
generation of disease.

Further research on C. maltaromaticum isolates recovered in this study could be
fruitful, as the presence of a piscicolin precursor gene was detected in our isolates. This
bacteriocin is known to have antibacterial properties; thus by further investigating its role
in viva, one may be able to elucidate a novel role this protein may play in many processes

involving bacterial infections. In addition, an assessment of possible virulence factors

122

that this bacterium may have could shed light onto the pathogenesis of C.
maltaromaticum.

Additional studies attempting to quantify the prevalence of A. salmonicida
salmonicida infections in lake whiteﬁsh through the use of serological assays, such as the
Quantitative Enzyme Linked Immunosorbent Assay (Q-ELISA), could help in the
determination of whether whiteﬁsh are able to recover ﬁ'om these infections or if they are
always fatal. As was seen in this study, this bacterium is capable of causing severe
disease in an infected host, and as such, knowledge of how lake whiteﬁsh, as a now
known susceptible host species, are able to manage these infections would be useful.
Moreover, further molecular characterization of the isolates retrieved in this study might
allow one to determine how closely related these strains are to strains recovered in other
areas and host species within the GLB.

Further characterization of the motile Aeromonas spp. isolated within this study
via molecular techniques would allow for a deﬁnitive species identiﬁcation of all isolates.
Through the sequencing of housekeeping genes within the Aeromonas genome,
relatedness of isolates to one another can be determined and sources of these infections
can sometimes be traced. In addition, comparison of strain variation among the four
sampled stocks could tell us much about migration of lake whiteﬁsh, sources of infection,
etc. Analysis of the virulence factors associated with the Aeromonas isolates recovered
in this study could also aid in the determination of how likely these bacteria are to cause
disease in this host species and where they may tend to localize in the body upon

infection.

123

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