Pathogenic Mechanisms of Photobacterium Damselae Subspecies Piscicida in Hybrid Striped Bass Ahmad A

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2002 Pathogenic mechanisms of Photobacterium damselae subspecies piscicida in hybrid striped bass Ahmad A. Elkamel Louisiana State University and Agricultural and Mechanical College, [email protected]

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PATHOGENIC MECHANISMS OF PHOTOBACTERIUM DAMSELAE SUBSPECIES PISCICIDA IN HYBRID STRIPED BASS

A Dissertation

Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy

The Department of Pathobiological Sciences

Ahmad A. Elkamel B.V. Sc., Assiut University, 1993 May 2002 DEDICATION

This work is dedicated to the people in my life who encouraged each step of my academic career. My mother was anxious as I was for each exam or presentation. I have been always looking to my Dad as a model, and trying to follow his footsteps in academic career. My wife stood by me like no other one in the world, and her love and support helped me see one of my dreams come true.

ii ACKNOWLEDGMENTS

I thank God for answering prayers and helping me to achieve my dreams.

There are several extraordinary people who guided me through my doctoral

experience and without their help and support I could have not finished my project. I am and forever will be grateful to my advisor Dr. Ronald Thune for his advice, encouragement over years, insightful guidance, “tireless editing”, and most importantly for always being willing to listen. I feel extremely fortunate to be counted as one of his students. I would like to offer my deepest thanks to my minor advisor Dr. John Hawke for his assistance and help in my project. I would like also to offer my everlasting gratitude to all of my committee members, Dr. David Horohov, Dr. Philip Elzer, and Dr.

Richard Cooper for their aid, guidance, and advice throughout this long, and sometimes difficult, process.

To those of you in Dr. Thune’s lab, Denise Fernandez, Ron Miller, Heather

Harbottle, Pat Bohannon, Maria Smith, Ericka Judice, Karen Plant, Tara Landrum, Kim

Mayer, Jim Sellers, and Jennifer Benoit, I owe you a lot for all the help, encouragement, and friendship you gave to me, and for making the graduate school an experience I will never forget (it goes both ways).

I am uniquely and foremost grateful to my wife, Ghada, for her support, motivation, and understanding that sustained me to complete this work. Her contribution throughout my study is beyond words of recognition. I am eternally grateful for my

Mom and Dad, Mr. and Mrs. Elkamel, my sister Abeer, my brother in law Basem, and all of my family.

iii Finally, I thank the Egyptian Cultural and Educational Bureau for the financial support of my research. Also, I would like to thank Wally Landry for providing fish for my research. And thanks for all who I might forget to mention their name, but offered me any kind of support.

iv TABLE OF CONTENTS

DEDICATION...... ii

ACKNOWLEDGMENTS ...... iii

LIST OF TABLES...... viii

LIST OF FIGURES ...... ix

ABSTRACT...... xi

INTRODUCTION ...... 1

CHAPTER I. LITERATURE REVIEW...... 6 Striped Bass and Its Hybrids...... 6 Hybridization ...... 7 Production of Striped Bass and Its Hybrids...... 8 Broodstock and Hatchery Phase ...... 9 Phase I Culture...... 10 Phase II Culture...... 11 Phase III Culture ...... 12 Fish Photobacteriosis ...... 13 Geographic Distribution and Epizootics...... 13 Species Susceptibility ...... 15 Clinical Signs...... 15 Acute Disease...... 15 Chronic Disease ...... 16 Histopathology...... 17 Photobacterium damselae subspecies piscicida ...... 18 Taxonomy ...... 18 Morphology...... 19 Culture Characteristics...... 20 Biochemical Characteristics...... 20 Serological Characteristic ...... 21 Pathogenicity...... 21 Virulence Mechanisms of Photobacterium damselae ...... 23 Factors That Promote Colonization and Invasion of Host Surfaces...... 24 Flagella...... 24 Adhesion ...... 24 Invasion...... 30 Iron Acquisition ...... 37 Outer Membrane Proteins...... 39 Serum Resistance...... 40 Capsule...... 41 Lipopolysaccharide...... 43

v Plasmids ...... 43 Surviving Macrophage Killing ...... 46 Intracellular Replication...... 64 Apoptosis ...... 66 Factors That Cause Damage to the Host...... 76 Bacterial Toxins and ECP...... 76 Treatment and Prevention of Photobacteriosis ...... 78 Chemotherapy...... 78 Vaccination ...... 80 Study Objectives ...... 82

CHAPTER II. PHOTOBACTERIUM DAMSELAE SUBSPECIES PISCICIDA IS CAPABLE OF REPLICATING IN HYBRID STRIPED BASS MACROPHAGES . 84 Introduction...... 84 Materials and Methods...... 86 Fish...... 86 Macropahge Media ...... 86 Macrophage Isolation...... 87 Lysing Macrophages...... 88 Bacterial Strains...... 89 Determination of Optimum Gentamicin Concentrations...... 91 Killing Assay ...... 92 Microscopic Assays ...... 92 Light Microscopy...... 92 Electron Microscopy...... 93 Acid Phosphatase Cytochemistry ...... 93 Statistical Analysis...... 94 Results...... 94 Intracellular Survival/Replication of P. damselae...... 94 Light Microscopy and TEM...... 95 Inhibition of Phagolysosomal Fusion ...... 95 Discussion...... 103

CHAPTER III. INVASION AND REPLICATION OF PHOTOBACTERIUM DAMSELAE SUBSPECIES PISCICIDA IN FISH CELL LINES...... 110 Introduction...... 110 Materials and Methods...... 111 Tissue Culture Media and Cell Lines...... 111 Bacterial Strains...... 112 Invasion Assay...... 112 Microscopic Assays ...... 113 Light Microscopy...... 113 Electron Microscopy...... 113 Ruthenium Red Staining...... 114 Statistical Analysis...... 114 Results...... 114

vi Invasion and Replication of P. damselae in Fish Cell Lines ...... 114 Light Microscopy and TEM...... 116 Ruthenium Red Staining...... 125 Discussion...... 128

CHAPTER IV. INDUCTION OF APOPTOSIS IN HYBRID STRIPED BASS PHAGOCYTES BY PHOTOBACTERIUM DAMSELAE SUBSPECIES PISCICIDA INFECTION...... 132 Introduction...... 132 Materials and Methods...... 134 Fish...... 134 Phagocytes Media ...... 134 Head Kidney Cells ...... 135 Bacterial Strains...... 136 Apoptotis Assay...... 136 Flow Cytometry ...... 137 Statistical Analysis...... 138 Results...... 138 Induction of Apoptosis by P. damselae in Phagocytes...... 138 Discussion...... 139

SUMMARY...... 151

BIBLIOGRAPHY...... 154

VITA...... 181

vii LIST OF TABLES

Table 6.―Comparison of Photobacterium damselae colony forming units (CFU) recovered form epithelioma papillosum carpio (EPC), channel catfish ovary (CCO), and fathead minnow (FHM) cells, percent of infected cells, and number of CFU per infected cell at time 0 of incubation...... 119

viii LIST OF FIGURES

Figure 1.―Effect of distilled water on Photobacterium damselae. Numbers of bacteria after various exposure time periods were determined by standard plate count method and expressed as millions of CFU/ml. (CFU = colony forming units)...... 90

Figure 2.―Percent change in colony forming unit counts of Photobacterium damselae from time 0 of incubation for both free bacteria growing in media with gentamicin (B&G) and bacteria recovered from hybrid striped bass macrophages (RB). Multiplicity of infection was 1 bacterium to 100 macrophages ...... 96

Figure 3.―Percent change in colony forming unit counts of Escherichia coli from time 0 of incubation for both free bacteria growing in media with no gentamicin (B) and bacteria recovered from hybrid striped bass macrophages (RB). Multiplicity of infection was 1 bacterium to 10 macrophages...... 98

Figure 4.―(A) Light microscope photograph and (B) transmission electron micrograph of hybrid striped bass macrophages infected with Photobacterium damselae (P) at time 0 of incubation where macrophages show one or few bacteria in vacuoles, and (C) Light microscope photograph and (D) transmission electron micrograph at 6- hour incubation time where macrophages show several bacteria in spacious vacuoles...... 100

Figure 5.―Transmission electron micrographs of hybrid striped bass infected macrophages at 3-hour incubation time and stained for acid phosphatase. (A and B) Phagolysosomes of macrophages infected with Escherichia coli (E) show positive acid phosphatase activity (AP) as electron dense precipitation around the bacterium. (C and D) Phagolysosomes of macrophages infected with Photobacterium damselae (P) do not show any sign of acid phosphatase activity around the bacteria ...... 104

Figure 6.―Transmission electron micrograph of fathead minnow (FHM) cell with cytoplasmic extensions (CE) around Photobacterium damselae (P) at 30 minutes post infection...... 120

Figure 7.―Light microscope photographs of (A) fathead minnow (FHM) and (B) channel catfish ovary (CCO) cells infected with Photobacterium damselae (P) at 0 incubation time where cells show one or few bacteria in vacuoles, and (C) FHM and (D) CCO at 6-hour incubation time where cells show several bacteria in large vacuoles...... 122

Figure 8.―Transmission electron micrographs of epithelioma papillosum carpio (EPC) cells infected with Photobacterium damselae (P) at (A) 0 time of incubation, and (B) 12 hours of incubation that is stained with ruthenium red. The outer exposed surface of the cell is positive for ruthenium red staining that appears as electron dense precipitation, while the membrane of the vacuole containing P. damselae is negative indicating that this vacuole is not connected to the extracellular environment ...... 126

ix Figure 9.―Analysis of Alexa Flour® 488-annexin V/ propidium iodide (PI) double stained hybrid striped bass phagocytes after 12, 18, and 24 hours of incubation. The lower left area of the cytograms represents viable cells, which are negative for annexin V binding and exclude PI. The lower right area represents apoptotic cells that demonstrate annexin V binding, but are negative for PI indicating an intact cytoplasmic membrane. The upper area represents non-viable, dead cells that are positive for both annexin V and PI. Representative cytograms after 12, 18, and 24 hours of incubation are shown. (A) control phagocytes that are non-infected, non- treated with camptothecin (CAM), (B) phagocytes treated with 5 µl CAM, (C) phagocytes infected with formalin-killed Photobacterium damselae, and (D) phagocytes infected with virulent Photobacterium damselae ...... 140

Figure 10.―Percent change of numbers of early apoptotic and late apoptotic phagocytes infected with live (P.d.) and formalin-killed (FKB) Photobacterium damselae from the control at each incubation time point...... 148

x ABSTRACT

Photobacterium damselae subspecies piscicida, previously known as Pasteurella

piscicida, is an important pathogen of hybrid striped bass and many fish species cultured

in brackish water in the United States, Japan, Europe, and the Mediterranean. The

purpose of this study is to investigate virulence mechanisms that contribute to the

pathogenesis of this organism.

The ability of P. damselae to survive/replicate within hybrid striped bass

macrophages was evaluated with an in vitro killing assay. Results indicated that the

numbers of bacteria recovered from macrophages at 3, 6, 12, and 18 hours of incubation increased significantly over time. In contrast, the numbers of Escherichia coli control strain recovered from macrophages declined at the same designated incubation times.

Light and electron microscopy confirmed internalization, uptake, and multiplication of bacteria within spacious, clear vacuoles in the macrophages. Using acid phosphatase as a lysosomal marker, it was shown that P. damselae inhibits phagolysosomal fusion.

Invasion and replication of P. damselae within epithelioma papillosum carpio

(EPC), channel catfish ovary (CCO), and fathead minnow (FHM) cells lines was also evaluated using an in vitro invasion assay. All three cell lines were susceptible to invasion and supported replication of P. damselae. Fathead minnow cells were more susceptible to invasion than the other two cell lines as indicated by greater numbers of infected cells and recovered bacteria at time 0. Using light and electron microscopy, invasion of cells by bacteria was observed as early as 30 minutes after infection, and intracellular bacteria were observed in large, clear, membrane-bound vacuoles. The

xi intracellular location of P. damselae was confirmed using ruthenium red staining to

discriminate between the extra- and intra-cellular spaces.

Using flow cytometry, results indicated that P. damselae induces apoptosis in

phagocytes obtained from hybrid striped bass head kidney and after 12, 18, and 24 hours

of incubation, the relative numbers of cells infected with P. damselae showing signs of apoptosis increased over time and were significantly greater than the controls. The relative numbers of apoptotic cells that were infected with the formalin-killed strain increased, but not significantly, above the control after the same designated times of incubation.

xii INTRODUCTION

Hybrid striped bass (Morone saxatilis x Morone chrysops) culture began in the southeastern United States in the late 1980s and has rapidly developed into a major aquacultural industry, with production increasing from 500,000 lbs in 1987 to approximately 9.7 million lbs in 1999, valued at $ 25 million (Brent Blauch, Striped Bass

Growers Association, personal communication). Because hybrid striped bass can be cultured in freshwater, but excel in brackish water, there has been significant interest in mariculture production in coastal areas worldwide. Almost every state in the southern

United States has hybrid striped bass producers, but most of the production is in

Alabama, Louisiana, Maryland, Mississippi, North Carolina, South Carolina,

Pennsylvania, Texas, and Virginia (Kubota et al. 1970; Hawke et al. 1987; Noya et al.

1995b; Hawke 1996). The total southern regional production was an estimated 5 million lbs, about 50% of total United States production, with a farm-gate value of approximately

$12.5 million. In 1990, mariculture farms constructed in the coastal marshes of

Louisiana utilizing pond, net-pen, raceway, and cage culture techniques showed great promise for the culture of hybrid striped bass. That December, a newly emerged bacterial pathogen, Photobacterium damselae subspecies piscicida (formerly named Pasteurella piscicida), the causative agent of photobacteriosis, was isolated on one of the coastal farms (Louisiana Aquatic Diagnostic Laboratory, LADL, annual report 1990). Over the next several years, photobacteriosis, previously known as pasteurellosis, caused massive mortalities and was at least partially responsible for closing several hybrid striped bass farms on the Louisiana coast. In Louisiana alone, 50 cases of photobacteriosis with heavy mortalities have been reported in the last 12 years on coastal hybrid striped bass

1 farms (LADL annual report 2002). The loss of valuable food-size fish results in

significant monetary loss with losses due to photobacteriosis at one Louisiana fish farm

in 1991 and 1994 valued at $1 million (Hawke 1996).

Photobacteriosis epizootics in cultured hybrid striped bass generally result in

mortality rates ranging from 5 to 90% depending on the water quality and whether

medicated feed is used (Hawke 1996). Epizootics usually occur in the spring or fall.

Spring epizootics are characterized by sudden onset of mortality and a very steep

mortality curve that can reach 80% in as few as 10 days, while mortalities in fall

outbreaks are more prolonged, for unknown reasons. The rapid onset of mortality with little warning allows an infection to become established in cultured fishes before therapy can be initiated (Hawke 1996).

Due to the development of intensive aquaculture in coastal areas in the United

States, Japan, Europe, and the Mediterranean region, conditions favoring the establishment of disease by this highly pathogenic halophilic organism have been created.

In Japan, this pathogen has proven to be detrimental to wild and farmed fishes, and responsible for significant losses. Photobacteriosis causes severe losses in cultured

yellowtail juveniles (Kubota et al. 1970; Kusuda and Yamaoka 1972), and mortalities in

other cultured fishes, including ayu, red sea bream, black sea bream, oval file fish, and

red grouper. Prior to 1990 there were no reported cases of photobacteriosis in Europe.

Subsequently, epizootics were reported in many areas in Europe, beginning with a 1990

outbreak in northwestern Spain in a gilthead sea bream population (Toranzo et al. 1991).

Simultaneously, epizootics occurred in France, Turkey, and Greece, affecting mainly sea

bass, and in Italy, Malta, and Portugal, affecting populations of gilthead sea bream.

2 The causative organism of fish photobacteriosis was initially isolated from natural

populations of white perch and striped bass in 1963 during a massive epizootic in

Chesapeake Bay, USA (Snieszko et al. 1964). The pathogen was studied morphologically, physiologically, and serologically by Jansen and Surgalla (1968), who proposed the name Pasteurella piscicida. Based on rRNA sequence and DNA-DNA hybridization studies (Gauthier et al. 1995), the organism was renamed Photobacterium

damsela subsp. piscicida, and then corrected to Photobacterium damselae subsp. piscicida (Truper and DeClari 1997).

The only commercially available tools for controlling photobacteriosis are antibiotics applied after clinical signs appear. Because antibiotics are delivered in feed, and because sick fish often cease feeding, treatment prevents disease in the fish not yet infected. Unfortunately, in the United States, no antibiotics are cleared to treat bacterial infections in hybrid striped bass. Initially, in emergency situations, farmers were granted

“compassionate use” approval for Terramycin or Romet. Control of photobacteriosis in the United States is further complicated by the rapid emergence of resistant strains to these drugs (LADL annual report 1996). Similar observations of resistance have occurred in Japan in the 1980s during treatment of epizootics in yellowtail, when resistance to ampicillin, tetracycline, chloramphenicol, and quinolones was observed

(Robohm 1983).

Because of the complications associated with chemotherapy in disease control, immunization became the preferred method proposed for controlling the disease. Several vaccination approaches were conducted in yellowtail, including formalin-killed, heat- killed, live attenuated bacterins, (Kusuda and Hamaguchi 1987), and ribosomal antigens

3 (Kusuda and Hamaguchi 1988). Protection levels of all the vaccine preparations tested

were not satisfactory and none of the vaccines became commercially available.

Failure of current vaccine preparations is partly due to their inability to induce the

appropriate type of immune response required for protection against photobacteriosis.

The development of successful vaccines becomes easier as more is known about the

pathogenesis of the disease and the virulence mechanisms used by the causative bacteria

to establish and maintain an infection. For example, if a bacterium is able to survive

within host cells, it is protected from the humoral immune response, including the action

of antibodies and complement (Kaufmann 1999). At present, the pathogenic mechanisms

of P. damselae are not completely elucidated. Several studies, however, suggested

putative factors/mechanisms, including serum resistance (Magariños et al. 1994), an iron uptake system (Magariños et al. 1994), a capsule (Magariños et al. 1996a), adhesion and invasion of epithelial cells (Magariños et al. 1996b), and lipopolysaccharide (Nomura and

Aoki 1985). One of the major virulence mechanisms proposed in the pathogenesis of photobacteriosis is the potential intracellular life style of the bacterium, but data supporting this are contradictory. While morphologically intact bacteria within macrophages have been reported in vivo (Kubota et al. 1970; Hawke et al. 1987; Noya et al. 1995a; Noya et al. 1995b), suggesting an ability to survive in macrophages, in vitro studies have indicated that macrophages from three different fish species were able to kill

P. damselae (Skarmeta et al. 1995). Demonstrating the ability of P. damselae to survive intracellularly is a key point in designing a new vaccine or establishing a strategy for combating photobacteriosis. Studying the pathogenesis of P. damselae at the cellular and

4 molecular level may lead to new approaches to combating this disease and to the

development of efficacious vaccines.

Chapter I begins with a brief introduction to the current status of the culture of

striped bass and its hybrids. The current literature relating to Photobacterium damselae

subsp. piscicida is summarized and the chapter ends with an overview of previously

proposed virulence mechanisms of the bacterium. Chapter II evaluates the ability of the

bacterium to survive/replicate within hybrid striped bass macrophages, using an in vitro

killing assay and light and electron microscopy. Chapter III examines the invasion and

replication of the bacterium within three fish cell lines using an in vitro invasion assay,

with light and electron microscopic evaluation of the process. Chapter IV investigates

the ability of the bacterium to induce of apoptosis in hybrid striped bass phagocytes using flowcytometry and two-color analyses of annexin V/propidium iodide double stained cells.

5 CHAPTER I. LITERATURE REVIEW

Striped Bass and Its Hybrids

Striped bass (Morone saxatilis) has been a valuable fish since the colonials first landed on the North American shores. Because it was so popular, striped bass was one of the first fishes to be protected under a 1693 conservation law in the colonies. Taxes imposed on fishing for striped bass helped fund New England’s first public school system

(Harrell et al. 1990). While still a very popular food fish, wild caught striped bass in the fish marketplace are being supplemented by reliable, consistent supplies of their farm raised cousins, the hybrid striped bass (HSB).

Few fishes have as much to offer as the striped bass. It is an anadromous fish, adapting well to fresh, brackish, and salt water; it thrives in warm, cool, and cold water; it lives in oceans, estuaries, rivers, lakes, and ponds; and it eats a wide variety of fishes and shellfishes found in all of its habitats (Harrell et al. 1990). It was originally described along the Atlantic coast, from the St. Lawrence River in Canada to the St. John’s River in

Florida, and in the Gulf of Mexico, from western Florida to Louisiana (Harrell et al.

1990). In 1879 and 1881, federal authorities seined young-of-the-year striped bass from the Navesink River, New Jersey and released them into the San Francisco Bay, where a reproducing population soon became established. The first striped bass hatchery was established in 1906 on the Roanoke River, at Weldon, NC, from a prototype that had been operated since 1881. Early artificial spawning efforts were aimed toward release of larva to assist in maintaining the wild populations for continued harvests. A successful attempt to produce striped bass fry occurred as early as 1884 at Weldon, NC. Wild

6 females were caught and eggs were collected from ripe females and fertilized with sperm

of ripe, wild-caught males (Harrell et al. 1990).

Hybridization

The original objective of the Morone hybridization program, initiated in the

1960s, was to produce a fish that combined some of the more desirable characteristics of

the two parental species (Bayless 1972). Preferred characteristics included size,

longevity, and feeding habits of striped bass, and the adaptability of white bass (Morone chrysops) to adverse environmental conditions. As early as 1965, hybridization attempts

with striped bass female and white bass male (Bishop 1968) demonstrated that hybrids

(palmetto bass) showed heterosis (hybrid vigor) expressed as improved survival in the

larval, fingerling and sub-adult stages, superior early growth rates, greater disease

resistance, and increased general hardiness (Bishop 1968; Bayless 1972). As a result, this

hybrid has been artificially propagated and widely stocked as a food and sport fish

(Harrell et al. 1990). Additionally, the reciprocal of the original cross (sunshine bass),

male striped bass x female white bass has been used similarly, and its popularity with

commercial producers has increased over the last few years. Hybrid striped bass are

generally preferred by producers over striped bass because they appear to fare better in

warm waters (Harrell et al. 1990). In addition, acceptance of the hybrid, with its broken

stripe pattern that helps identify it as farm-raised, as an alternative to the striped bass in

the marketplace has been improving (Brent Blauch, Striped Bass Growers Association,

personal communication). It is of a particular interest that, unlike many fish hybrids, all

first-generation Morone crosses that have been reared to maturity, are fertile and can be

induced to spawn in a hatchery situation (Bayless 1972; Harrell et al. 1990). Several

7 additional crosses, and various backcrosses and outcrosses have also been made, but none have gained the acceptance of the first, original cross (Bishop 1968; Bayless 1972).

Palmetto bass continue to be the hybrid most widely-used in production and sport because of its rapid growth that can be exceptional under optimal conditions (Bishop

1968; Bayless 1972).

Production of Striped Bass and Its Hybrids

Since the mid 1980's, commercial production of HSB has been steadily improving

(Kohler 2000). Most farm-raised striped bass are a hybrid cross between white bass and

striped bass. Consumer acceptance of the hybrids, with their broken stripe pattern, as an

alternative to the striped bass has encouraged the expansion of the industry. Hybrid

striped bass represents a fish that is suitable for a variety of aquaculture techniques where

warm water resources are available. As a warm-water fish, hybrids grow best in waters

ranging from 75° – 85° F, but can survive in waters ranging from 40° – 90° F (Harrell et

al. 1990).

Hybrid striped bass fry and fingerlings grown in the United States are sold both

domestically and overseas. Food sized fish are sold both in the United States and

Canada, with some additional export to Europe and the Pacific Rim. Food sized fish are

predominately sold whole, on ice, from 1.25 to 3 lbs in size. Limited live fish markets

along the west coast and in the northeast United States and Canada demand fish in the

1.25 to 1.75 lbs size range. Fry and fingerling production in 1999 saw 185 million fry

hatched and about 17.5 million fingerlings sold (Phase I & Phase II) from about 12

fingerling producers. Less than a few dozen fingerling producers hatch nearly 200

million fry annually, with domestic usage of fingerlings exceeding 17 million fish (Brent

8 Blauch, Striped Bass Growers Association, personal communication). Fry and fingerling producers in the United States ship millions of fish annually to marine and brackish water facilities in Taiwan and the Mediterranean region. Countries such as Spain, Italy, France and Greece have significant aquaculture industries as well as the established mariculture operations in Japan and Taiwan (Harrell et al. 1990).

As an industry, United States striped bass growers number fewer than 100 farms nationwide that produced more than 9.7 million lbs of HSB in 1999, from farming operations that produce more than three million lbs per year to small operations that produce less than 20,000 lbs per year. The three largest farms (over 500,000 lbs/year), in the southeast and the west, produce nearly 60% of the total United States food fish production of HSB at 5.6 million lbs. Medium producers (100,000 – 500,000 lbs/year) number only 14, and contributed 25% of the total production with just less than 2.7 million lbs. Small growers (<20,000 – 100,000 lb/year) number over 50 farms and produce less than 1.5 million lbs all together, representing less than 15% of the total

United States production. Only 17 farms in the United States produce more than 100,000 lbs per year of these warm water fish (Brent Blauch, Striped Bass Growers Association, personal communication). The industry has two main components: fingerling production and grow-out. Some farmers are involved in both sectors, but most farms are specialized in either fingerling production or grow-out.

Broodstock and Hatchery Phase

Each spring, broodstock white bass and striped bass are collected from the wild and transported to fingerling farms where they are held for use in hybridization.

Attempts of broodstock domestication, especially white bass, are gaining success. The

9 striped bass female crossed to the white bass male (the original cross, palmetto bass), continues to have popularity among growers, while female white bass crossed with male striped bass (the reciprocal cross, sunshine bass) yields a consistently performing fish.

The original cross is the most common hybrid that is grown by farmers in the United

States due to its higher growth rate (Harrell et al. 1990).

Upon arrival at the farm, live adult fishes are segregated by species and sex, usually with females in areas where they can be readily captured and examined. When broodstock are first collected, they are often not ready to ovulate, and therefore not ready for spawning. Injecting gravid female fish with human chorionic gonadotropin can induce ovulation with greater control, leading to greater fertilization success. Reciprocal cross fry are smaller than original cross fry, and can barely be seen with the naked eye

(Harrell et al. 1990).

Phase I Culture

After an initial 5 to 7 days post-hatch in the hatchery, fry are ready to consume food. Their initial diet is zooplankton and rotifers, which the farmer must produce. The standard practice for generating zooplankton is to fill a pond, assure that no other fishes are present, and apply sufficient organic and/or inorganic fertilizer until a phytoplankton bloom develops. As part of the pond’s natural cycle, zooplankton populations grow as they eat the phytoplankton, and eventually become populous enough to support the diet of the new fry. Fry are introduced to fertilized ponds about one to two weeks after the initial fertilization, at rates up to 500,000 fry per acre, depending upon the type and availability of zooplankton. Timing of ovulation, fry hatching, and pond fertilization must be scheduled very closely, because the fertilized pond develops in its own

10 ecosystem, larger aquatic organisms appear after about 3 weeks that might eat the 5 day

old fry. If the timing of these sequences of events alters in any significant way, the entire

process generally fails, with little to no survival, or survival of poor quality fingerlings.

After two to three weeks in the pond, fry are introduced to artificial feeds, augmenting the diminishing supplies of natural pond organisms. Around 45 days post-hatch, the small fish, already trained on feed, are at sizes ranging from 200 to 1000 per pound

(Harrell et al. 1990).

Phase II Culture

In pond culture, where growing seasons are controlled climatologically, the initial growing season, May to October, focuses on growing Phase I fingerlings to the next size

(Phase II). Phase I fish are stocked at rates up to 15,000 fish per acre and are fed high- protein, high-fat diets in various pellet sizes up to 1/8" diameter. Floating feeds are popular with farmers, as fish can be observed more readily in their feeding habits. Phase

II fish are recollected after about five months, regraded and sorted to size, and then restocked for final grow-out in less densely populated ponds, up to 5,000 fish per acre.

This second collection, grading and sorting helps minimize size variations and tends to maintain uniformity in the final harvest. Staging growth in this fashion also maximizes use of pond capacity, where several generations are present on the farm at any given time. Intensive culture facilities also stage grow-out from Phase I fingerlings by successively stocking and grading fish in various tank configurations. Intensive systems vary greatly in how stocks are managed, especially densities, feeding rates, growth rates, and time till harvest. Each farm has unique approaches to handling grow-out, customized to the resources and equipment available (Harrell et al. 1990).

11 Phase III Culture

Growers stock Phase II fingerlings at rates from 3,500 to over 5,000 fish per acre.

Feeding rates depend on fish size, water temperatures, dissolved oxygen concentrations, and pond conditions, up to a maximum of about 200 lbs per acre per day. After about 12 months, properly cared for Phase II fingerlings will reach food fish market size of about

1.5 lbs average. At that time they are seined, harvested, chill-killed, and packed whole on ice for market. Some growers have developed techniques to keep harvested fish alive, allowing sale for recreational fishing and live fish markets (Harrell et al. 1990).

Growers utilize ponds, tanks/raceways, and cages to produce hybrid striped bass, depending on the type and availability of land and water resources at their facilities. The first 10,000 lbs of HSB produced for sale came from tanks in 1986. The following year, pond growers contributed their first 10,000 lbs, while tank produced fish reached nearly

400,000 lbs. For the next ten years, tank cultured HSB maintained an edge over pond grown fish, reaching highs of over 5 million lbs per year in 1994 and 1995. In 1998 and

1999, pond harvests topped 5 million lbs and took over as the leading culture technique, representing 54% of all fish harvested. Cage culture harvests reached an annual high of

250,000 lbs in 1996, but fell to less than 40,000 lbs in 1999 (Brent Blauch, Striped Bass

Growers Association, personal communication).

Hybrid striped bass are presently grown in commercial food-fish pond operations in Alabama, Louisiana, Maryland, Mississippi, North Carolina, South Carolina,

Pennsylvania, Texas, and Virginia (Hawke 1996). Although most HSB production is conducted in freshwater systems (Smith et al. 1989), mariculture industries using pond, cage, net-pen, and raceway culture have developed in brackish water in coastal

12 Louisiana, Texas, and Florida (Hawke 1996). Throughout the remainder of this

dissertation, hybrid striped bass (HSB) will refer to the original (palmetto) cross of

female striped bass x male white bass.

Fish Photobacteriosis

Geographic Distribution and Epizootics

Due to strong demand, HSB production was expected to expand substantially and

become an important commercial aquaculture industry over the past ten years (Hodson et

al. 1987). Unfortunately, increased production of HSB in coastal areas was accompanied

by an increase in the incidence of disease. Photobacteriosis, previously named

pasteurellosis, is a devastating disease problem for HSB production in the United States

and several cultured fish species in Japan, Europe, and the Mediterranean.

Photobacteriosis is caused by Photobacterium damselae subspecies piscicida, formerly named Pasteurella piscicida.

In the United States, Snieszko et al. (1964) first documented photobacteriosis in

Chesapeake Bay during the summer of 1963, although at the time the disease was called pasteurellosis. This outbreak resulted in mass mortalities among wild white perch

(Morone americanus) and striped bass. The epizootic spread over a large area and it was estimated that almost half the population of white perch were killed (about 900,000 lbs) in the initial epizootic (Snieszko et al. 1964). A smaller outbreak was reported in Long

Island Sound in 1977 (Robohm 1979). Several outbreaks followed in coastal ponds in

Alabama (Hawke et al. 1987) and different coastal areas of the United States (Hawke

1996).

13 The recent evolution of coastal aquaculture in the United States and abroad has

created ideal conditions for this highly pathogenic, halophilic organism. The disease has

been reported to occur seasonally in coastal Louisiana HSB farms, with 50 cases reported

since 1990 and four-out-of-five farms closed as a result of photobacteriosis. Production losses due to photobacteriosis at one Louisiana fish farm in 1991 and 1994 were valued at over $1 million (Hawke 1996).

In Japan, photobacteriosis is one of the most economically important diseases of cultured fish, causing severe losses in cultured yellowtail juveniles (Seriola quinqueradiata) (Kubota et al. 1970; Kusuda and Yamaoka 1972). Pseudotuberculosis was the other name proposed for photobacteriosis in yellowtail (Kubota et al. 1970), as the affected fish usually show prominent white tubercles in several internal organs.

Subsequently, photobacteriosis spread to several farmed fish species including ayu

(Plecoglossus altivelis) (Kusuda and Miura 1972), black sea bream (Mylio macrocephalus) (Muroga et al. 1977; Ohnishi et al. 1982), red sea bream (Acanthopagrus schlegeli) (Yasunaga et al. 1983), oval file fish (Navodan modestus) (Yasunaga et al.

1984) and red grouper (Epinephelus okaara) (Ueki et al. 1990) in Japan, and snake-head fish (Channa maculata) in Taiwan (Tung et al. 1985).

In Europe and the Mediterranean, photobacteriosis was first reported in 1991 in juvenile gilthead sea bream (Sparus aurata) in the northwest of Spain (Toranzo et al.

1991). Simultaneously, epizootic outbreaks were reported from France (Baudin-

Laurencin et al. 1991) in a population of sea bass (Dicentrarchus labrax) and Italy

(Ceschia et al. 1991) in a population of gilthead sea bream. Other outbreaks were reported from sea bass in Greece (Bakopoulos et al. 1995), gilthead sea bream in Portugal

14 (Baptista et al. 1996), sea bass in Turkey (Candan et al. 1996), and gilthead sea bream in

Malta (Bakopoulos et al. 1997b). In 1994, photobacteriosis caused 50% mortality in a

population of market-size hybrid striped bass cultured in Israel (Hawke 1996).

Species Susceptibility

Although only fishes of the genus Morone have been found to be susceptible to

photobacteriosis in the United States, a variety of fish species have been diagnosed with

infection by P. damselae in Japan and Europe. In the United States, white perch

(Snieszko et al. 1964) and striped bass (Snieszko et al. 1964) and its hybrids (Hawke

1996) are susceptible to infection. In Japan, the most affected cultured species is yellowtail (Kubota et al. 1970), while other affected species of economic importance in

Japan include ayu (Kusuda and Miura 1972), black sea bream (Muroga et al. 1977), red sea bream (Yasunaga et al. 1983), oval file fish (Yasunaga et al. 1984), and redspotted grouper (Ueki et al. 1990). In Europe, gilthead sea bream (Toranzo et al. 1991) and sea bass (Baudin-Laurencin et al. 1991) were diagnosed with photobacteriosis.

Clinical Signs

Acute and chronic forms of fish photobacteriosis have been described, with some

variation depending upon the species as reviewed by Thune et al. (1993).

Acute Disease

Clinical signs of acute disease have been described in many reports (Kubota et al.

1970; Kusuda and Miura 1972; Robohm 1979; Hawke et al. 1987; Toranzo et al. 1991;

Hawke 1996). In the acute form, external pathological signs are usually inconspicuous

regardless of the species affected. Externally, striped bass appear normal with the

15 exception of pallor of the gills and petechiae in the opercular region, at the base of the

fins and inside the mouth. Striped bass also become lethargic and sink in the water

column, fail to regulate pigmentation, and show an increased rate of respiration. In HSB

and striped bass, redness of the operculum, pallor of the gills, and darkening of the skin

are the only consistent signs. Internally, an enlarged spleen and hemorrhagic kidney

provide the only clearly visible gross clinical signs. Other organs and physical features

appear normal with the exception of the liver which may be slightly mottled. Yellowtail

show evidence of edema and fail to regulate pigmentation. White perch show only slight

hemorrhage around the gill covers and the bases of the fins. Diseased gilthead sea bream

exhibit no apparent external clinical signs except rare individuals that display slight

hemorrhagic areas around the head and gills.

Chronic Disease

The chronic form of the disease may vary depending on the species affected and whether antibiotic feeds have been administered. In striped bass and white perch, small white miliary lesions may be seen in the swollen spleen and kidney (Wolke 1975). In yellowtail, chronic lesions are typified by 1 to 2 mm granuloma-like lesions that are composed of masses of the bacteria, epithelial cells, and fibroblasts. The lesions grossly

resemble true granulomas and have resulted in the disease being referred to as

"pseudotuberculosis" (Kubota et al. 1970). Chronic lesions in the spleen of gilthead sea

bream were found only in fish that died after the peak of mortality or when the

photobacteriosis outbreak was almost arrested with antibiotic therapy. The lesions seen

were very similar to those of the yellowtail, with typical whitish pseudotubercles being

present in the spleen (Toranzo et al. 1991).

16 Histopathology

Wolke (1975) described the histopathology of photobacteriosis in naturally

infected white perch and striped bass from Chesapeake Bay. In what was obviously a

chronic form of the disease, collections of necrotic lymphoid and peripheral blood cells

were present in the spleen, focal areas of hepatocytes undergoing coagulation necrosis

were apparent in the liver, and there was a conspicuous lack of an inflammatory cell

response (Wolke 1975). In cultured populations of striped bass fingerlings, acute

multifocal necrosis of the lymphoid tissue of the spleen, characterized by a loss of cells,

coagulation necrosis, karyorrhexis, and large colonies of bacteria were common. In the

liver, areas of acute multifocal necrosis with prominent karyorrhexis were common

(Hawke et al. 1987). Histopathological changes were noted in naturally and

experimentally infected gilthead sea bream. The spleen and kidney showed

circumscribed, acute necrotic changes and infiltration, with blood cells and masses of

bacteria plugging capillaries and interstitial spaces. In the liver, there was moderate

multifocal necrosis of the hepatocytes (Toranzo et al. 1991). In the spleen of infected

yellowtail, bacterial colonies were surrounded by inflammatory cells, and mature

granulomas containing eosinophilic substances and surrounded by epithelial cells were observed. Phagocytes containing bacteria and swollen into large globules were seen in the spleen and kidney, and it was theorized that swollen phagocytes blocked capillary blood flow in the organs resulting in ischemia (Kubota et al. 1970). The histopathological lesions of freshwater snakehead fish in Taiwan were very similar to those of the sea bream and striped bass, with no chronic epithelioid cell granulomas being observed (Tung et al. 1985).

17 Photobacterium damselae subspecies piscicida

Taxonomy

The Chesapeake Bay isolate was initially classified in the genus Pasteurella based on its morphological and biochemical features (Snieszko et al. 1964). Subsequent studies

(Janssen and Surgalla 1968) suggested the name Pasteurella piscicida, or the fish killer, which was never accepted by bacterial taxonomists. Although this species is clearly distinguishable from the other species within the genus Pasteurella by positive reactions for arginine dihydrolase and the methyl red test, and negative reactions for nitrate reduction and inability to grow at 37 and 42°C, the organism remained in this genus. The organism was also thought to belong to the genus Corynebacterium (Kimura and Kitao

1971) or Arthrobacter (Simidu and Egusa 1972). After studying 12 strains from various

locations, however, classification in the genus Pasteurella could not be supported (Koike

et al. 1975). A subsequent study, utilizing DNA hybridization techniques, indicated the

organism was 30% related to Pasteurella, Actinobacillus and Haemophilus (Pohl 1981).

The author also concluded that the fish pathogen described by Janssen and Surgalla

(Janssen and Surgalla 1968) deviates from Pasteurellaceae by its larger genome, its

cryophilia, its lack of nitrate reductase, and its unusual host range. Since then, this

organism has been extensively characterized, and strains have been shown to be

biochemically and serologically homogeneous (Magariños et al. 1992a; Magariños et al.

1996c). However, analysis of rRNA cistrons and 16S rRNA hybridization studies

provided the evidence that Pasteurella piscicida is more closely related to the family

Vibrionaceae and should not be classified in the genus Pasteurella (De Ley et al. 1990).

These results were subsequently confirmed by sequencing the 16S rRNA, thus providing

18 evidence that Pasteurella piscicida is closest to Photobacterium damsela, formerly

Vibrio damsela. Based on rRNA sequence and DNA-DNA hybridization studies,

Gauthier et al. (1995) renamed the bacterium Pasteurella piscicida as Photobacterium

damsela subsp. piscicida and the bacterium Photobacterium damsela as Photobacterium

damsela subsp. damsela. Finally, the names were corrected to Photobacterium damselae

subsp. piscicida (Truper and DeClari 1997), and Photobacterium damselae subsp.

damselae. Furthermore, analysis indicated that P. damselae subsp. piscicida is more

closely related to Photobacterium angustum and Photobacterium leiognathi than to P. damselae subsp. damselae (Thyssen et al. 1998). The authors concluded that there is not enough morphological and/or biochemical evidence to include P. damselae subsp. piscicida as a subspecies of P. damselae because the two subspecies differ in motility reaction, nitrate reduction, growth at 37 and 42°C, growth on TCBS and MacConkey media, β-hemolysis, production of urease and amylase, and the ability to ferment amidon, cellobiose, maltose and trehalose (Thyssen et al. 1998), as well as host specificity

(Magariños et al. 1996c). Throughout the remainder of this dissertation, Photobacterium damselae subspecies piscicida that causes disease in HSB will be referred to as P. damselae.

Morphology

Photobacterium damselae is a Gram negative, pleomorphic rod-shape bacterium.

It is usually 0.5 – 0.8 µm by 0.7 – 2.6 µm in size, exhibiting bipolar staining (Janssen and

Surgalla 1968). It is non-flagellated (Janssen and Surgalla 1968), non-motile in wet

mounts (Snieszko et al. 1964) and glucose motility stabs (Hawke et al. 1987). Bacteria in

log phase of growth can occur in chains and are longer and more rod-shaped than those in

19 later phases, which may become more coccobacilli (Hawke 1996). The classical

description of P. damselae includes coccobacilli that are non-capsulated (Koike et al.

1975). However, further studies demonstrated that the majority of P. damselae strains

constitutively produce capsular material that plays an important role in the pathogenesis

of this microorganism (Bonet et al. 1994). The authors found that the capsular material

was 99.6% carbohydrate and 0.4% protein, and completely different from those described

for Pasteurella species.

Culture Characteristics

Photobacterium damselae is an obligate halophilic bacterium that fails to grow at a NaCl concentration less than 0.5% or greater than 4.0%, or at a temperature below 15 or above 32.5°C (Snieszko et al. 1964). Optimum growth occurs at 1.0% – 2.5 % NaCl and temperature of 22.5 – 30°C (Hashimoto et al. 1985). Primary isolation is best achieved on brain heart infusion (BHI) agar supplemented with 2% NaCl or on blood agar (2% sheep blood) without added salt. Individual colonies on blood agar are grayish- white in color, opaque, 1 mm in diameter, smooth with an entire border, non-hemolytic, and clearly visible after 48 hours incubation at 27°C (Snieszko et al. 1964).

Biochemical Characteristics

Photobacterium damselae is a facultative anaerobe, producing acid, but not gas,

from glucose, mannose, maltose, fructose, and galactose (Hawke et al. 1987). It is

positive for oxidase, catalase, methyl red, and VP, but negative for indole production,

nitrate reduction, citrate utilization, and H2S production (Hawke 1996). Detailed

biochemical characteristics have been reviewed in several studies (Hawke et al. 1987;

20 Magariños et al. 1992a; Hawke 1996; Magariños et al. 1996c). Photobacterium

damselae has a unique code number (2005004) in the API 20E system (BioM'ereaux-

Vitek) (Kent 1982).

Serological Characteristic

Serologically, P. damselae constitutes a highly homogeneous group (Magariños et

al. 1992a). Serological homogeneity is supported by lipopolysaccharide (LPS) profiles,

in which different strains possess high molecular weight O-side chains in electrophoretic

analysis (Nomura and Aoki 1985; Magariños et al. 1992a). Both whole-cell and somatic

O antigens produced strong slide agglutination with antisera raised against various strains

of P. damselae. In addition, cross-agglutination tests failed to demonstrate the presence

of any serogroups (Romalde and Magariños 1997). Bakopoulos et al. (1997a), however,

demonstrated the existence of antigenic differences between Japanese and European

strains using six monoclonal antibodies produced against whole cells of P. damselae.

Pathogenicity

Many pathogens and microorganisms invade vertebrate bodies and attempt to

colonize them. Most of these attempts fail, either because the invaders are unable to

adapt evolutionarily to the new microenvrionment of the host or because the host’s

immune system prevents them from establishing a foothold in the body. When a

bacterium that is capable of causing disease becomes established in the body, this is

called an infection, and an infection that produces manifestations of damage, or

symptoms, is called a disease. By contrast, persistence of bacteria in a particular body

site without causing a disease (e.g., resident microflora) is often designated colonization

21 rather than infection. Infection does not necessarily lead to disease. If the host defenses are adequate, it may be infected by a disease-causing bacterium for a prolonged period without symptoms. This is called asymptomatic carriage, or asymptomatic infection.

Pathogenicity is defined as the ability of a pathogen to cause infection, while virulence is used to describe variations in the severity of disease. These two terms, however, are usually used synonymously. Virulence factor (mechanism of pathogenesis or virulence mechanism) denotes a bacterial product or strategy that contributes to virulence or pathogenicity.

Experimental infections demonstrate that P. damselae is highly pathogenic, not only for the host from which it was isolated, but also for other fish species, with 50%

3 6 lethal doses (LD50) ranging from 10 to 10 colony forming units (CFU) per fish

(Magariños et al. 1992b). Two strains, originally isolated from yellowtail and red grouper in Japan, were found to be non-virulent for gilthead sea bream or turbot

(Scophthalmus maximus), although the authors could not asses their virulence for the original hosts (Magariños et al. 1992b). In bath challenge experiments, P. damselae

5 proved to be virulent for yellowtail with a LD50 near 10 CFU (Kusuda and Hamaguchi

1988), and gilthead sea bream, but not for turbot (Magariños et al. 1995). Gilthead sea bream smaller than 5 grams are susceptible to disease, but fish greater than 20 grams are resistant (Noya et al. 1995a). Tung et al. (1985) conducted two infectivity trials by intraperitoneal injection and found the LD50 of P. damselae in snakehead fish to be 100

CFU per fish, making it the most susceptible fish on record. In HSB, however, LD50 of

P. damselae is one bacterium per gram of fish by intraperitoneal injection (Miller 2001).

Immersion LD50 of P. damselae in HSB is 687 CFU/ml of water, while fish challenged

22 with 125000 CFU/ml water died 72 hours post infection (Hawke 1996). On the other

hand, P. damselae cannot be considered as virulent for homoiothermic animals since

8 mouse pathogenicity assays showed LD50 values higher than 10 CFU (Magariños et al.

1992b).

Virulence Mechanisms of Photobacterium damselae

The discovery that certain bacterial traits, such as pili or capsules, could make a

significant contribution to the ability of bacteria to cause disease opened a new era of

disease research. Development of vaccines and therapeutic regimens occurred mainly by

trial and error, fortified with a lot of guesswork. The realization that certain traits were

responsible for virulence made it possible to develop vaccines and therapeutic agents by

rational design strategies for the first time. The realization that virulence factors were

bacterial traits designed specifically to subvert host defense mechanisms opened up

entirely new ways of understanding the infection process. Newer biochemical and

genetic analyses of the infective process indicate that bacteria produce many new proteins

and stop making many others as they adapt to the site of the body they are colonizing.

Bacteria that start at the mucosal surface, invade to underlying tissue, and end up in the

internal organs may go through several stages of adaptation as they encounter new

environments within the host’s body.

Deciding what to call a virulence factor has proven to be problematic. Some

bacterial traits and products, such as the ability to adhere to mucosal cells or to produce

toxic proteins, have a clear and direct connection to the infection process. Other bacterial

traits, such as the ability to obtain energy from sugar fermentation, are usually considered

to be housekeeping functions rather than virulence factors, even though the ability of a

23 bacterium to acquire carbon and energy from the host is clearly essential for infection.

Nonetheless, it is convenient to use the term "virulence factor" to denote features of

bacteria that are connected with their ability to cause disease, especially if the factor

indicated can be used as the target of a vaccine or a therapeutic strategy. Virulence

factors can be loosely classified into two categories: those that promote bacterial

colonization and invasion of the host and those that cause direct damage to the host

(Salyers and Whitt 1994).

Factors That Promote Colonization and Invasion of Host Surfaces

Flagella

Photobacterium damselae is non-motile and non-flagellated (Janssen and Surgalla

1968).

Adhesion

Host cell adhesion molecules, machinery, and associated signaling systems are predominantly designed for cell adhesion to other neighboring cells or to the extracellular matrix. However, in recent years, it was shown that host cell natural adhesion molecules like integrins and cadherins may also mediate adherence of other “nonconventional” surfaces, particularly bacteria.

A necessary step in the successful colonization and, ultimately, production of disease by microbial pathogens is the ability to adhere to host surfaces. This fundamental idea has led to an enormous amount of research over the last two decades that deals with

understanding how bacterial pathogens adhere to host cells. A class of bacterial surface

proteins called “adhesins” mediates the attachment of a bacterium to the host cell surface

24 (Salyers and Whitt 1994) or extracellular matrix (Finlay and Falkow 1997). Bacterial

adhesins are either located at the tip of a scaffold-like structure on the bacterial surface,

called pili or fimbria, or anchored in the bacterial membrane but surface exposed. Recent

investigations have revealed a great deal about biogenesis and the regulation of adhesin

factors, and, to a lesser extent, the identity of host receptors that are the targets of

microbial adhesin factors. The vertebrate cell surface contains many proteins,

glycoproteins, glycolipids, and other carbohydrates that could potentially serve as

receptors for bacterial adhesins (Finlay and Falkow 1997). Additionally, the extracellular

matrix provides a rich source of glycoproteins for adhesins to bind. Bacterial tropism to

colonize a restricted range of hosts, tissues, and cell types depends, among other factors, on the availability of specific host cell receptors for a given bacterial adhesin. Not all adhesins are essential virulence factors (Krogfelt 1991). The specific role of a particular adhesin in disease has been surprisingly difficult to define, since pathogens may express more than one type of adhesins, like Escherichia coli (Le Bouguenec and Bertin 1999;

Smith et al. 2001), or even many adherence factors, like Bordetella pertussis (Sandros and Tuomanen 1993).

Other pathogens are capable of binding soluble host molecules such as antibodies, complement, or extracellular matrix proteins. These molecules can then serve as a bridge between the bacterial adhesin and the natural host receptor for that molecule in a process called bridging mechanism (Cossart et al. 2000). Bridging mechanism adhesion is common among pathogens that have the ability to bind to the extracellular matrix, which serves as the underlying foundation for cells (Cossart et al. 2000). When tissue is damaged (possibly due to inflammation), the underlying extracellular matrix becomes

25 exposed. The YadA protein of Yersinia enterocolitica mediates binding to cellular fibronectin but not to plasma fibronectin, which may allow the organism to adhere to tissue rather than to bind circulating molecules (Tertti et al. 1992; Schulze-Koops et al.

1993). Mycobacteria also bind fibronectin, using three related bacterial molecules called the BCG85 complex (Westerlund and Korhonen 1993).

Fimbrial adhesion is mediated by rod-shaped protein structures, called fimbriae or pili, that project from the bacterial surface and bind to sugar residues of the host cell’s glycoproteins and glycolipids (Kubiet et al. 2000; La Ragione et al. 2000). Strictly speaking, "fimbriae" is the correct term for rod-like surface adhesins, because the term

"pili" was reserved for the longer, more flexible rod-like structures involved in conjugation.

Afimbrial adhesins are bacterial surface proteins that are clearly important for adherence but that do not assemble themselves into rod-shaped pili-like structures

(Garcia et al. 1996; Lalioui et al. 1999). Probably, in some cases, afimbrial adhesins mediate the tighter binding of bacteria to host cell following initial binding via pili. Little is known about the structure and attachment mechanism of the afimbrial adhesins. At least some of them may recognize proteins on host cell surfaces rather than carbohydrates.

Bordetella pertussis, the causative agent of whooping cough, is a respiratory mucosal pathogen that possesses several potential adherence factors that exemplify the complexity of bacterial adherence. Adherence factors of B. pertussis include at least four fimbrial genes and several afimbrial adhesins, including filamentous hemagglutinin

(FHA), pertactin, pertussis toxin, and BrkA (Sandros and Tuomanen 1993). Filamentous

26 hemagglutinin also contains an RGD tripeptide sequence that is a characteristic

eukaryotic recognition motif that binds to host cell surface integrins. Additionally, this

RGD sequence induces enhanced B. pertussis binding to monocytes by activating a host

signal transduction complex that normally upregulates the CR3 (complement receptor 3)

binding activity (Ishibashi et al. 1994). Both FHA and one of the binding subunits of

pertussis toxin (S2) have homologous sequences that mediate binding to host molecule,

lactosylceramides.

Yersinia species also contain several adhesins that mediate adherence to the host

cell surface or matrix. Perhaps the best-studied adhesin, which can also mediate invasion

under certain conditions, is invasin. This outer membrane protein adheres tightly to members of the β1-integrin family, including the fibronectin receptor (Isberg and Leong

1990). In addition to invasin, Y. enterocolitica expresses another adhesin that mediates a high level of adhesion but low levels of invasion called Ail (attachment invasion locus)

(Miller and Falkow 1988). The host receptor for this outer membrane protein has not been identified. Furthermore, Y. enterocolitica adhere through YadA to tissue rather than circulating molecules by binding to cellular fibronectin but not to plasma fibronectin

(Tertti et al. 1992; Schulze-Koops et al. 1993). Yersiniae also express a flexible fimbria whose assembly genes are homologous to those encoding Pap pili in E. coli and other related fimbriae (Salyers and Whitt 1994).

Members of the E. coli adhesin family include the fimbrial adhesins F1845 and Dr

and two nonfimbrial adhesins, Afa-I and Afa-III (Nowicki et al. 1990; Guignot et al.

2000). All four of these adhesins use the Dra blood group antigen present on decay-

accelerating factor (DAF) on erythrocytes and other cell types as their receptor, although

27 they appear to recognize different epitopes of the Dr-antigen (Nowicki et al. 1990). It is

possible that the E. coli afimbrial adhesins have evolved from the related fimbrial

adhesins but have been altered such that the properties needed to polymerize a pilus are

missing yet the adhesin domain remains anchored on the bacterial surface. Recent work

indicates that the sequence of the adhesin molecule dictates whether it will be assembled

into a fimbria and that genetically switching the genes encoding these adhesins switches

the adhesin type (Cossart et al. 2000). Interestingly, enteropathogenic E. coli (EPEC) elicited a novel way to bypass classical activation via surface receptors and can potentially interfere directly with the cell machinery regulating the function of adhesion molecules (Salyers and Whitt 1994). Adherence of EPEC strains to cultured cells is characterized by effacing lesions, corresponding to the disruption of microvilli and followed by the formation of a pedestal-like structure at the site of bacterial interaction with the host cell. Initial adherence of EPEC to cells is mediated by BFP, a type IV pilus and is followed by an intimate adherence of the bacteria to the cell surface through specific proteins delivered with a type III secretory apparatus (Salyers and Whitt 1994).

One of these proteins, Hp90 (Tir), is inserted into the host cell membrane and activates host cell signaling pathways. Hp90 acts as a receptor for intimin, an outer membrane adhesin of EPEC that is responsible for intimate adhesion (Finlay and Falkow 1997). In other words, EPEC strains show how bacterial effectors that are translocated via a type

III secretion system contribute to the formation of a self-designed bacterial receptor that leads to tight attachment of bacteria and pedestal formation (Jarvis et al. 1995).

Shigella and Salmonella use a different method of adhesion to cells. These bacteria trigger the formation of numerous ruffles on the cell surface that usually leads to

28 engulfing of the bacterium in a macropinocytic process. Shigella IpaB and IpaC proteins can associate with the α5β1-integrins, but this association does not promote adhesion of the bacterium on the cell surface. This may be because the majority of the Shigella Ipa proteins are not found on the bacterial surface but are secreted into the media (Page et al.

2001).

Scanning electron microscopy studies did not reveal fimbriae on P. damselae adhered to CHSE-214 cells (Yoshida et al. 1997). Additionally, none of the strains tested

were able to hemagglutinate sheep, rat, rabbit, yellowtail, horse, rainbow trout

(Oncorhynchus mykiss), or chicken erythrocytes. Thus far, no fimbriae have been

reported from P. damselae.

Adherence assays, using different poikilothermic cell lines, demonstrated that P.

damselae is weakly to moderately adherent (Magariños et al. 1996b). The degree of

attachment displayed by P. piscicida was greater than that shown by a non-adherent

Vibrio anguillarum strain (Magariños et al. 1996b), and similar to that reported for other

known adherent bacteria such as Yersinia ruckeri (Romalde and Toranzo 1993).

However, when fish tissues were employed in the adherence experiments, P. damselae

showed a great binding capacity to fish intestines, with values ranging from 104 to 105

bacteria per gram of tissue depending on the fish species tested (Magariños et al. 1996b).

Furthermore, experiments to explore the nature of the substances implicated in adhesion

demonstrated that adherence was inhibited when heat-treated bacteria or sugar-treated

cell line monolayers were used, but was not affected by proteases or by treating the

bacteria with antisera raised against the LPS of P. damselae (Magariños et al. 1996b).

These results indicate that the molecules involved in adhesion could be glycoprotein

29 components of the bacterial cell surface. In addition, the authors could not correlate the

differences in the levels of adhesion detected among different P. damselae strains tested

with their pathogenic potential (virulent and non-virulent strains), since all the isolates

showed similar adhesive patterns. P. damselae strains are able to bind to various

poikilothermic cell lines and a mammalian cell line, but do not bind to glass, unlike

Aeromonas salmonicida A-l (A-layer positive) and Edwardsiella tarda 226 that showed a

high level of adhesion to the cultured cells as well as to the glass surface, suggesting some measure of specificity in the P. damselae-tissue cell interaction (Yoshida et al.

1997). Furthermore, persistence of the virulent strains of P. damselae could be an important factor in the pathogenesis of photobacteriosis in cultured gilthead sea bream

(Magariños et al. 1996a). The authors also suggested that persistence might be due in part to serum resistance and adherence.

Invasion

Some bacteria have evolved mechanisms for entering (invading) host cells that

are not naturally phagocytic (non-professional phagocytes) including epithelial cells

lining mucosal surfaces and endothelial cells lining blood vessels (Finlay 1990; Finlay

and Falkow 1990; Falkow et al. 1992). Bacteria that can invade these monolayers are

one step away from access to the blood stream and the rest of the body. Also, cells

provide the bacteria with an environment protected from host defense mechanisms and/or

exogenous antimicrobial agents like antibiotics, and thus contribute to the progression of

the disease (Plotkowski et al. 1994). Epithelial cells take up proteins and other factors from the outside using pinocytosis and receptor-mediated endocytosis, but they are generally not capable of engulfing large objects such as bacteria (Cossart et al. 2000). To

30 be taken up by epithelial cells, invading bacteria must induce their own internalization.

They do this by attaching to the host cell surface and causing changes in the host cell cytoskeleton that result in their engulfment by the host cell. Ultimately, host cell cytoskeletal components provide the machinery for bacterial uptake (Finlay and Falkow

1997). Rearrangements in actin filament structure often can be visualized in the vicinity of the invading organism. Microtubules also may be required for invasion of some pathogens (Salyers and Whitt 1994). In fact, the role of host actin and signal transduction mechanisms in bacterial uptake is indicated because the process can be inhibited by cytochalasins that inhibit actin polymerization (Finlay and Falkow 1988) or host tyrosine kinase inhibitors that block host cell signaling (Rosenshine et al. 1992). Bacterial surface proteins that provoke phagocytic ingestion of the bacterium by host cells are called

“invasins” or invasion factors (Isberg et al. 1987; Le Bouguenec and Bertin 1999), and can be involved in the adhesion process as well. Proteins that are essential for invasion of cultured cells by bacteria include invasin (inv) and Ail of Y. enterocolitica (Miller and

Falkow 1988), internalin (inlA) of Listeria monocytogenes (Gaillard and Finlay 1996),

IpaB to D of Shigella flexneri (Menard et al. 1996), and invA to H of Salmonella typhimurium (Galan 1996). There are three general mechanisms of how pathogenic bacteria invade host cells. These mechanisms are forcible entrance by disrupting the host cell membrane, zipper mechanism, where uptake is driven by specific receptor binding, and trigger mechanism, where tight receptor binding is not required (Finlay and Falkow

1997).

A small number of bacterial species appear to forcibly enter directly into host cells by a local enzymatic digestion of the host cell membrane following adherence to the

31 cell surface. One such pathogen, Rickettsia prowazeckii, produces phospholipases that appear to degrade the host wall localized beneath the adherent organisms, thereby enabling the pathogen to enter the cytoplasm directly (Walker et al. 1983). How the bacterium controls the enzymatic degradation to prevent host cell lysis and how the host cell reseals its membrane after invasion remain uncharacterized.

Zipper mechanism refers to the ability of the bacterium to induce local changes in actin organization of host cell. The bacterium appears to sink into the cell, or to pull the plasma membrane up around it (Cossart et al. 2000). The entry process appears to be driven by incremental interaction between bacterial surface ligands (adhesin/invasin) and cell adhesion molecules such as cadherins and integrins (Cossart et al. 2000).

There are several explanations for how zipper-mechanism invasion occurs. First,

Yersinia uses the zipper mechanism whereby invasin binds tightly to a family of β1 integrins, which are host molecules that bind to extracellular matrix proteins such as fibronectin (Isberg and Leong 1990; Leong et al. 1990). By binding tightly to integrins, invasin may mediate bacterial uptake via a “zipper-like” mechanism, zippering the host cell membrane around the bacterium as it enters by successive interaction between the adhesion molecules and the bacterial invasin (Isberg 1990; Isberg and Leong 1990).

Another explanation of the zipper mechanism is that, upon adherence to host cell, bacteria induce the host cell to use its own signal pathways and actin to behave similar to a phagocytic cell (Salyers and Whitt 1994). In actively phagocytic cells, cytoskeletal rearrangements involving polymerization and depolymerization of actin occur as an integral part of pseudopod formation. By causing similar actin rearrangements to occur in normally non-phagocytic cells, the bacteria are, in effect, forcing phagocytosis by

32 eliciting formation of pseudopod-like structures that mediate engulfment (Salyers and

Whitt 1994).

The most recent explanation is based on the reaction of the host cell toward

adhesion molecule signaling. Cadherins and integrins, as cell adhesion molecules,

mediate epithelial cell attachment to and spreading on their neighbors and the underlying extracellular matrix, respectively. Engagement of these receptors by their natural ligand results in receptor immobilization and clustering. These events initiate signaling cascades that can result in strengthening of the cell-cell and cell-matrix contacts. When a bacterial invasin engages the adhesion receptors on a host cell, it responds as it normally would, recruiting cytoskeletal elements to the location of the attachment and try to strengthen the attachment. However, since the bacterium is small compared to the responding cell, the attempt of the cell to spread against the bacterial surface quickly results in engulfment of the bacterium (Cossart et al. 2000).

Trigger mechanism is dramatically different from the zipper mechanism in morphological terms and requires a much more complicated type of bacterial machinery.

Unlike zipper mechanism, trigger mechanism does not require a successive specific receptor binding to achieve adhesion (Schaible et al. 1999). In these cases, brief contact between a bacterium and the surface of a host cell results in a rapid, large-scale cytoskeletal response, where explosive actin filament polymerization under the plasma membrane pushes out huge sheets or ruffles. The extending ruffles fold over and fuse back to the cell surface, trapping large membrane-bound pockets of extracellular medium within the cell, a process termed macropinocytosis (Cossart et al. 2000). The nearby bacteria are trapped by the infolding membrane ruffles, and find themselves within the

33 membrane-bound compartments. Where zippering invasion appears to be a modification

of cellular adhesion, triggering invasion bears much more resemblance to the response of

cells to growth factors (Cossart et al. 2000).

Salmonella typhimurium triggers a dramatic response from the epithelial cell

surface shortly after it contacts the host cell. The host cell surface extrudes outward,

from the point of bacterial adherence, with attendant localized membrane ruffling and

macropinocytosis (Finlay and Falkow 1990; Francis et al. 1993). Salmonella

typhimurium has multiple genetic loci involved in invasion that are clustered in one

region on the chromosome (58 to 60 min) called Salmonella pathogenicity island 1

(SPI1) (Finlay 1994; Galan 1996). Within this region, there are several different invasion

operons, including regulatory loci. Many of the proteins encoded in one gene cluster,

inv-spa, share considerable homology with those seen in other pathogenic bacteria.

During Shigella infection, the bacteria can invade a variety of cell types including

macrophages, M cells, and intestinal epithelia (Salyers and Whitt 1994). Shigella invades epithelial cells by a process that triggers global changes in the cellular actin cytoskeleton that result in membrane ruffle and macropinocytosis (Finlay and Falkow 1997). The trigger mechanism of Shigella invasion involves proteins whose genes are carried on a

220-kb virulence plasmid. Bacteria that have been cured of this plasmid lose invasive ability. The main system involved in this process is a type III secretion system that is activated when the bacteria come into contact with the host cells. This is not a process of transcriptional activation but, rather, mobilization of proteins already existing in the cytosol of the bacterial cell. The genes involved in this process are called the ipa

(invasion plasmid antigens) and the Mxi-spa (membrane expression of ipa-surface

34 presentation of antigens) genes. The Mxi-spa genes encode the secretory apparatus, while the ipa genes encode the secreted proteins. The protein products, IpaB, IpaC, and

IpaD, are all necessary for Shigella invasion (Page et al. 2001).

Despite the morphological and mechanistic differences between the trigger and zipper mechanisms for bacterial invasion of non-phagocytic cells, they have certain important traits in common. Both require that extracellular bacteria send signals across the host cell plasma membrane to induce local actin polymerization. Both involve bacterial activation of signal transduction cascades that are already present in the host cell, although used for other purposes. Both are mediated by bacterial expression of specific virulence factors, whose sole function is to induce these particular responses during host cell invasion. Importantly, both zipper and trigger uptake require energy derived from the host cell. The cytoskeletal and membrane rearrangements that are responsible for bacterial invasion require no energetic input from the bacterium; they

invade by persuasion rather than by force. This is in contrast to invasion of some

eukaryotic parasites, such as Toxoplasma gondii. In that case, the energy for invasion comes from the parasite, which actively pushes its way into the host cell, which is a passive victim (Schaible et al. 1999).

Photobacterium damselae is considered weakly to moderately invasive to several poikilothermic cell lines. Photobacterium damselae is able to invade those cell lines and to remain viable inside the infected cells at least for 2 days. Interestingly, there is no correlation between the adherence and invasion process, as a greater level of cell attachment of a particular strain does not necessarily correlate to greater invasiveness

(Magariños et al. 1996b). The invasive properties of Salmonella depend greatly on the

35 bacterial strain and the cell line used (Mills and Finlay 1994). Lopez et al. (2000)

showed that the uptake of P. damselae by EPC cells is time and bacterial-concentration dependant. The authors also suggested that internalization of P. damselae by EPC cells is receptor-ligand mediated (zipper mechanism), as more infected cells and greater numbers of bacteria per cell were found when more bacteria were used, up to a saturation where increased numbers did not increase invasion levels. Photobacterium damselae isolates also showed capacity to spread to adjacent cells from the initially infected cell, forming plaques of dead cells (Magariños et al. 1996b). Similar to what has been reported with

other Gram negative bacteria such as Pseudomonas aeruginosa (Plotkowski et al. 1994)

and Haemophilus ducreyi (Totten et al. 1994), invasion by P. damselae can be inhibited by cytochalasin D, indicating that actin and microfilment-dependent mechanisms are required for the internalization of P. damselae (Magariños et al. 1996b).

Moreover, invasiveness of heat-inactivated P. damselae was greatly reduced when compared to the non-treated viable cells. Ultra-violet-light inactivated bacteria, however, showed almost normal invasion rates, suggesting that engulfment by host cells

does not require P. damselae viability and that the integrity of the bacterial surface

components is necessary to permit interaction between bacteria and surface of the host

cell (Lopez-Doriga et al. 2000).

Incubation of P. damselae with fresh, normal sea bass serum reduces slightly, but

not significantly, the invasiveness of EPC cells, while incubation with heat-inactivated,

normal serum had no effect on bacterial invasiveness. Incubation of P. damselae,

however, with heat-inactivated antiserum against this strain significantly reduced the

invasiveness of the bacteria to EPC cells (Lopez-Doriga et al. 2000).

36 Iron Acquisition

Because iron is an essential factor for bacterial growth, the availability of iron is a critical factor in the pathogenicity of microorganisms invading hosts (Bullen 1981;

Griffiths 1987). The amount of free iron available to the invading pathogen within the host is quite low. Most of the iron in the body is found intracellularly as ferritin, hemosiderin, or heme, and that which is extracellular in serum or other body fluids is bound to high-affinity iron-binding proteins such as transferrin in blood and lymph, and lactoferrin in external secretions and milk. As a result, although there is an abundance of iron present in body fluids, the amount of free iron is too small to support bacterial growth (Griffiths 1987). In addition, the host can reduce the amount of iron bound to serum transferrin during an infection. This decrease is called the "hypoferraemia of infection". Consequently, pathogens must possess an effective iron acquisition system to remove the iron bound to the transferrin and lactoferrin. One of these transport systems involves the production of iron-sequestering compounds named siderophores and their corresponding membrane receptor proteins (Griffiths 1982). Siderophores are low molecular weight iron chelators that, along with their receptors, can effectively solubilize iron or remove it from other chelators and transport it into the cell (Neilands 1982).

There are two main classes of siderophores, phenolates and hydroxamates, which form tight, chelated complexes with iron. Siderophores are excreted into the medium, and the iron/siderophore complex is taken up by special siderophore receptors on the bacterial surface (Neilands 1982). The internalized iron/siderophore complex is cleaved to release the iron molecule within the bacteria. Some bacteria not only produce their

37 own siderophores, but also produce receptors capable of binding siderophores produced by other organisms (Salyers and Whitt 1994).

Magariños et al. (1994) first investigated the iron uptake mechanism in several strains of P. damselae, as well as the effect of iron overload on the virulence of these strains for fish. All P. damselae strains tested obtained iron by a siderophore-mediated mechanism, when grown in the presence of either the iron chelator ethylene-diamine-di

(O-hydroxyphenyl acetic acid) or human transferrin. Chemical tests and cross-feeding assays of siderophore-deficient, but receptor-positive mutant strains of V. anguillarum, E. coli, or S. typhimurium showed that P. damselae produced a siderophore which is neither a phenolate nor a hydroxamate. Cross-feeding assays and chromatographic analysis suggest that this siderophore may be chemically related to multocidin, a siderophore produced by Pasteurella multocida. In addition, data showed that all isolates utilized hemin and hemoglobin as alternative sources of iron in iron-restricted media, which is supported by the increase in the virulence of P. damselae strains when the fish were pretreated with these sources of iron. The authors also found a correlation between hemolysin production and utilization of heme compounds, as iron plays a regulatory role in the synthesis of proteolytic enzymes, since these enzymatic activities were expressed only when the strains were cultured in iron-restricted conditions. Therefore, although P. damselae has been classically considered a non-proteolytic bacterium in normal culture conditions (Magariños et al. 1992a), the low-iron environment within the fish could trigger the expression of other virulence factors. Similar results were reported in other fish pathogens for homoiothermic and poikilothermic animals (Woods et al. 1982;

Toranzo and Barja 1993).

38 Because of the strong indication that iron availability was involved in the

pathogenesis of P. damselae, Hawke (1996) constructed a stable, siderophore deficient P.

damselae mutant, LSU-P1, using transposon mutagenesis. Mutant strain LSU-P1 was

found to be non-virulent for hybrid striped bass juveniles by either immersion or

injection, but following immersion challenge, examination of gill tissue demonstrated

that bacteria penetrated the epithelium with no associated tissue damage. In contrast, the

wild type produced 100% mortality in 96 hours at the same immersion concentration

(Hawke 1996). The failure of LSU-P1 to establish an infection in hybrid striped bass by

immersion indicates that the siderophore is important in supplying iron for growth and

other invasive factors, and these results are supported by the fact that the mutant persisted

following intraperitoneal injection (Hawke 1996).

Outer Membrane Proteins

Some outer membrane proteins, as discussed above, may serve as virulence

factors by contributing to bacterial adhesion (adhesins) and invasion (invasins) of the

host. An essential part of the high affinity iron uptake systems (siderophres) is the

production of iron-regulated outer membrane proteins (IROMP) which are either

involved in the uptake or internalization of the siderophore/iron complex (Neilands

1982), or act as receptors for transferrin or heme compounds as alternative sources of

iron (Magariños et al. 1994). Iron regulated outer membrane proteins are not normally

synthesized in bacteria grown in iron-depleted media and are detected only following

growth under low iron conditions (Crosa and Hodges 1981). Induction of IROMP production under low iron conditions is documented for fish pathogens as V. anguillarum

(Crosa and Hodges 1981) and Y. ruckeri (Romalde et al. 1991). When various strains of

39 P. damselae are grown in iron-restricted media, three IROMP are expressed in all strains and a fourth one is expressed in some strains (Magariños et al. 1994).

Serum Resistance

The ultimate end product of the complement cascade is the membrane attack complex (MAC), which behaves as an integral membrane protein and is responsible for the formation a donut-like lesion in the bacterial cell membrane (Nahm et al. 1999). This lesion creates osmotic imbalance and results in death of the bacteria. Bacteria that are not killed by the MAC are called serum resistant. An indication of the importance of this trait is that many of the gram-negative bacteria that cause systemic infections are serum resistant (Nahm et al. 1999).

Virulent P. damselae strains are serum resistant and can grow in fresh rainbow trout serum, whereas non-virulent strains are sensitive to serum killing and their growth is totally inhibited in fresh serum. This inhibitory effect of the serum on the non-virulent strains, however, is totally lost if the complement is inactivated by heating at 56°C for 1 hour (Magariños et al. 1994). Serum resistance is associated with capsule production, with capsulated strains resistant to serum killing, while non-capsulated strains were serum sensitive. It is supported by the fact that when the serum was heated to destroy complement, the non-capsulated strains grew as well as the capsulated strains. On the other hand, when the strains were cultured in the medium YPGSA-2 to induce capsule expression, all the P. damselae isolates (originally capsulated and non-capsulated) were able to grow in normal serum (Magariños et al. 1996a). Photobacterium damselae was found to resist the bactericidal effects of normal HSB serum (Hawke 1996).

40 Capsule

Capsule was first described for P. damselae as a rigid integral capsule that excludes Indian ink stain (Bonet et al. 1994). Electron microscopy revealed the presence of a capsule on the external surface of the outer membrane of bacteria grown in glucose- rich YPGS-2 media, but it was absent in cells grown in BHIA-1 media (Bonet et al.

1994). Photobacterium damselae grown under iron-limited conditions had a significantly reduced amount of capsular material on their surfaces than those grown under iron- supplemented conditions (do Vale et al. 2001). The authors also reported that the amount of capsular material decreased with the age of the culture, irrespective to amount of iron in media.

Capsulation is a crucial virulence determinant for a number of bacterial species, providing protection from serum killing and phagocytosis (Amaro et al. 1994;

Yamaguchi et al. 1995). Some capsules prevent formation of C3 convertase (C3bBb) by failing to bind serum protein B (Salyers and Whitt 1994). Other capsules have a higher affinity for serum protein H than for B. If C3b complexes with H rather than B, it is degraded by serum protein I (Nahm et al. 1999). Capsules that are rich in sialic acid have a high affinity for H (Nahm et al. 1999). Preventing C3bBb formation means less C5b will be produced, and the MAC is less likely to form on the bacterial surface (Salyers and

Whitt 1994). The capsule itself is less likely to be opsonized by C3b, and bacteria will not be engulfed as efficiently by phagocytes (Salyers and Whitt 1994). The role of capsule in decreasing phagocytosis by macrophages has been reported in another fish pathogen, A. salmonicida (Garduno et al. 1993).

41 It was suggested that the property of serum resistance that is conferred by the

capsule is closely associated with the persistence of P. damselae in its host, since no

qualitative differences in protein or LPS composition of the outer membranes were

observed for the capsulated and non-capsulated strains (Magariños et al. 1996a).

Studies that describe the contribution of bacterial capsules to adhesion and

invasion of host cells are contradictory. It was suggested that a capsule might reduce the

adhesiveness of E. coli (Runnels and Moon 1984) and the adhesiveness and invasiveness

of Haemophilus influenzae (St Geme and Falkow 1991). On the other hand, Davis et al.

(1981) found that the E. coli capsule enhanced adhesiveness. Other studies postulated

that the level of encapsulation, rather than presence or absence of capsule, may affect adhesion of H. influenzae (St Geme and Cutter 1996).

Interestingly, no significant difference was found in adhesion to cell lines between virulent, capsulated P. damselae and non-virulent, non-capsulated strains (Magariños et

al. 1996a; Lopez-Doriga et al. 2000). Magariños et al. (1996a), however, demonstrated

that the relative adhesion of a virulent P. damselae strain when grown under low capsule

expression conditions on BHIA-2 was 10 times greater than when grown under high

capsule expression conditions on YPGSA-2. These data indicate that the thicker capsule

may interfere with adhesion of P. damselae to host cells. The authors postulated that

under iron-restricted conditions such as those encountered in vivo, P. damselae may not

be heavily capsulated, suggesting that the capsule is an important virulence factor to

avoid elimination by the host, although it may interfere with colonization.

Furthermore, the capsule plays an important role in lethality of P. damselae to fish based on the fact that non-virulent strains induced for capsule expression resulted in

42 a decrease of 4 logs in the LD50 values (by peritoneal injection) in sea bass and turbot

(Magariños et al. 1996a).

Lipopolysaccharide

The LPS of P. damselae has been isolated and subjected to morphological

analysis by polyacrylamide gel elecrophoresis (PAGE), gas chromatography,

spectrophotometry and immunoblotting techniques (Nomura and Aoki 1985). The LPS

of six P. damselae strains from diverse geographic regions in Japan and one isolate from

Chesapeake Bay, USA were all found to have identical LPS structure.

Lipopolysaccharide serves as a site for attachment of C3b and triggers activation

of the alternative pathway of complement cascade (Nahm et al. 1999). Two types of LPS

modifications affect the interaction between LPS and complement components. First,

attachment of sialic acid to LPS O-antigen prevents formation of C3 convertase, just as

capsules that contain sialic acid do (Salyers and Whitt 1994). Second, changes in length

of the LPS O-antigen side chains prevent effective MAC formation. It is not clear how

O-antigen side chain length prevents MAC killing, because C5b and some MAC

components still attach to LPS with longer O-antigen side chains. Possibly, the MAC

forms too far from the bacterial outer membrane to exert a bactericidal effect (Salyers and

Whitt 1994).

Plasmids

Plasmids have been found to confer a number of important attributes to bacterial

fish pathogens including antibiotic resistance (Aoki and Kitao 1985), iron sequestering

systems (Crosa et al. 1977), and resistance to phagocytosis (Stave et al. 1987). The

43 importance of plasmids in the virulence of P. damselae is currently unknown. The number of plasmid bands in Japanese strains of P. damselae, as visualized by agarose gel electrophoresis, ranged from three to seven, and most strains share three plasmid bands with molecular weights of 37, 15 and 5 MDa (Toranzo et al. 1983). American isolates from Chesapeake Bay and Long Island Sound possess two and five plasmid bands, respectively, that are slightly different in molecular weight from the Japanese strains.

Strain ATCC 17911 from Chesapeake Bay carries plasmids of 20 and 7 MDa molecular weight, while strain SB2-KK from Long Island Sound possesses plasmid bands of 65, 25,

18, 6.5, and 3 MDa (Toranzo et al. 1983). Examination of representative European,

Japanese and American (ATCC 17911) strains revealed similarities between the

European and American strains, with each containing a 20 and a 7 MDa band (Magariños et al. 1992a). When virulent and non-virulent strains from different geographic locations are compared, no individual plasmid or plasmid profile corresponds to a virulent phenotype (Magariños et al. 1992a).

Most strains of P. damselae, regardless of geographic source, have the same antibiotic resistance profile, possessing markers for kanamycin, erythromycin, and streptomycin (Magariños et al. 1996c). Early studies reported that P. damselae strains are sensitive to penicillin, chloramphenicol, tetracycline, and colistin, and are variably sensitive to kanamycin, neomycin, and erythromycin (Kimura and Kitao 1971; Robohm

1979; Toranzo et al. 1991). Photobacteriosis in Japanese fish farms is complicated by the emergence of drug resistant strains. Drug resistant strains carrying R-plasmids were first observed in 1980 following the heavy use of chemotherapeutics on fish farms (Aoki and

Kitao 1985). Strains resistant to tetracycline and ampicillin are now commonly

44 encountered on fish farms in Japan. Two-hundred eighty-one strains collected from diseased yellowtail were subjected to analysis of their resistance patterns. Plasmid DNA and transferable R-plasmids encoding various combinations of resistance to chloramphenicol, tetracycline, kanamycin, sulphamonomethoxine, and ampicillin were detected in 168 of the strains (Takashima et al. 1985). In mating experiments with E. coli strain RCS5, it was found that all the above drug resistance genes were contained on a single plasmid (Aoki and Kitao 1985). In a recent study, 12 chemotherapeutic agents were tested on strains of P. damselae from cultured yellowtail reared in Japanese fish farms from 1989 - 1991. Of 175 strains tested, 152 were found to be resistant to combinations of ampicillin, chloramphenicol, kanamycin, nalidixic acid, sulphamonomethoxine, and tetracycline (Kim and Aoki 1993). One hundred and forty nine of the 152 resistant strains were found to carry transferable R-plasmids (Kim and

Aoki 1993). In 1991 and 1994, strains of P. damselae isolated from Louisiana fish farms were resistant to Terramycin or Romet (sulphadimethoxine and ormetoprim) (LADL annual report 1998). Hawke (1996) compared the plasmid profile of five Louisiana strains, a representative strain from the US (Maryland), Greece, and Israel, and two strains from Japan. The author found that Louisiana strains had four identical plasmid bands, with the exception of one strain that contained only a single plasmid. Louisiana strains typically had two large plasmids that were not precisely sized, but appeared to be in the 30 - 40 kilobase (kb) range. The other two plasmids were estimated to be 8.0 and

5.0 kb in size. Examination of strains from other geographic locations indicated that they do not possess the same plasmid profile (Hawke 1996).

45 Surviving Macrophage Killing

Cells have evolved a variety of strategies to internalize particles and solutes,

including pinocytosis, receptor-mediated endocyotsis, and phagocytosis. Pinocytosis

usually refers to the uptake of fluids and solutes. Receptor-mediated endocytosis is the

specific process through which macromolecules, viruses, and small particles can enter

cells (Allen and Aderem 1996a). Pinoctyosis and receptor-mediated endocytosis share a

clathrin-based mechanism and usually occur independently of actin polymerization

(Allen and Aderem 1996a). By contrast, phagocytosis, the uptake of particles larger than

0.5 µm, occurs by an actin-dependant mechanism and is usually independent of clathrin

(Aderem and Underhill 1999).

Phagocytosis occurs primarily in specialized phagocytic cells such as

macrophages and neutrophils through an extraordinarily complex process.

Monocytes/macrophages and neutrophils have been referred to as professional phagocytes, or simply phagocytes, while other cells such as epithelial and endothelial are

considered non-professional phagocytes as they have limited phagocytic capacities

(Rabinovitch 1995). It was suggested that the major difference in phagocytic capacity

and efficiency of professional and non-professional phagocytes could be an array of

dedicated phagocytic receptors on professional phagocytes that increase phagocytic rate

and particle range, because transfecting fibroblasts and epithelial cells with cDNA

encoding Fc receptors (FcR) dramatically increased their phagocytic rate (Indik et al.

1995). However, it is clear that many other differences between professional and non-

professional phagocytes exist that affect both rate and efficiency of internalization

(Aderem and Underhill 1999).

46 Although phagocytosis by non-professional phagocytes occurs at a slower rate

than for professional phagocytes, it is morphologically similar (Jones et al. 1999). It is

important to know that the strategies used by various pathogens to induce phagocytosis

by host cells, either professional or non-professional phagocytes are quite diverse. Non-

professional phagocytes like fibroblasts and epithelial cells are clearly devoid of

receptors that enhance phagocytosis such as FcR and complement receptor (CR) that are

found on and used by professional phagocytes for standard recognition mechanisms

(Jones et al. 1999). Instead, pathogens that interact with the non-professional phagocytes

get internalized (invade) through a different set of receptors using either zipper or trigger

mechanisms (see invasion section above).

A group of microbes, termed intracellular pathogens, use phagocytosis to their

advantage as a mechanism of invasion. Their intracellular location allows them to avoid

the hostile extracellular environment, containing destructive host factors like specific

antibodies and complement. In principle, any type of tissue cell, including epithelial

cells, endothelial cells, hepatocytes, macrophages and dendritic cells, can potentially

serve as habitat (Schaible et al. 1999). After engulfment, some facultative intracellular

bacteria survive and replicate in professional phagocytes, but they can also proliferate outside of the host, and include the following bacteria (and diseases): Salmonella spp.

(typhoid fever and other salmonellosis), Francisella tularensis (tularemia), Brucella abortus (brucellosis), L. monocytogenes (listeriosis), Mycobacterium tuberculosis

(tuberculosis), Mycobacterium avium-intracellulare complex and other atypical mycobacteria causing opportunistic mycobacterial infections in the immunocompromised host, Legionella pneumophila (legionnaire's disease), and Mycobacterium leprae, the

47 agent for leprosy, although for this pathogen extracellular survival has not yet been proven. A second group of facultative intracellular bacteria enter non-professional phagocytes temporarily, exploiting this capability to invade the host through epithelial cells and include Shigella spp. (shigellosis). A third group of facultative intracellular bacteria are preferentially found in endothelial cells, but can also survive in macrophages, such as the cat scratch disease agent Bartonella henselae (Schaible et al. 1999). On the other hand, some intracellular bacteria depend fully on the host cell metabolism and cannot survive and replicate outside of their host cells and are hence called obligate intracellular pathogens. This group encompasses Rickettsia rickettsiae (Rocky mountain spotted fever), Ehrlichia canis (ehrlichiosis), and Chlamydia trachomatis (trachoma, lymphogranuloma venereum) (Schaible et al. 1999).

Professional phagocytes have evolved a variety of receptors that recognize conserved motifs on pathogens that are not found in eukaryotes referred to as pattern- recognition receptors, and their targets as pathogen-associated molecular pattern

(Janeway 1992). Binding to these receptors induces active phagocytosis and is called direct interaction (Cossart et al. 2000), non-opsonic recognition (Jones et al. 1999), or cellular recognition (Aderem and Underhill 1999), and only professional phagocytes perform such host-directed internalization process.

Several pattern-recognition receptors can be exploited by intracellular bacteria to induce phagocytosis (Schaible et al. 1999). These receptors possess lectin-like binding moieties in their extracellular domains that enable them to directly bind a broad array of sugar residues exposed on the surface of various bacteria. Mannose-type receptors, such as the macrophage mannose receptor (MMR), bind mannose, glucose, and fucose

48 residues, while galactose and fucose-type receptors bind galactose and fucose,

respectively (Stahl 1992; Stahl and Ezekowitz 1998). Another pattern recognition

receptor, CD 14, mediates phagocytosis through the binding of cell wall components like

the lipopolysaccharide (LPS) of gram-negative bacteria, or mycobacterial

lipoarabinomannan (LAM) (Pugin et al. 1994; Wright 1995). The macrophage scavenger

receptors, MSR-AI and MCR-AII, that usually recognize serum lipids and lipoproteins

can also bind cell wall components of gram-negative and gram-positive bacteria, and can

induce phagocytosis of various bacterial species including M. tuberculosis (Pearson

1996; Zimmerli et al. 1996). The MSR may play a more prominent role in bacterial

uptake than previously assumed, as mice with interrupted MSR-AI/II genes clear L.

monocytogenes infection less efficiently than wild-type mice (Suzuki et al. 1997).

Another mammalian surface protein that binds glycolipids, CD48, has been found to

serve as a receptor for the fimbriae of invasive FimH+ E. coli (Schaible et al. 1999).

Additionally, the host has evolved a series of soluble proteins that recognize

(opsonize) invaders and, in turn, are recognized by specific receptors on professional

phagocytes. This method of recognition is referred to as indirect or bridging interaction

(Cossart et al. 2000), opsonic recognition (Jones et al. 1999) or humoral recognition

(Aderem and Underhill 1999). Two important factors that opsonize bacteria are the

mannose-binding protein (MBP) that recognize mannose residues on the bacterial surface

and bind to the C1q receptor on macrophages (Nepomuceno et al. 1997), and the

pulmonary surfactant protein A (SP-A), which binds M. tuberculosis and mediates its uptake by alveolar macrophages via the SPR210 receptor (Gaynor et al. 1995). Major humoral components that opsonize infectious agents include fibronectin (Fn),

49 immunoglobulins (Ig), and complement components (C), where recognition occurs via the Fn receptor (FnR), Fc receptors (FcR), and complement receptors (CR), respectively

(Schaible et al. 1999). Fibronectin can induce phagocytosis via FnR of those bacteria expressing Fn-binding proteins, which include a family of secreted proteins of M. tuberculosis (Abou-Zeid et al. 1988). Immunoglobulin Fc receptors FcγR and FcαR expressed on professional phagocytes bind to the Fc portion of the heavy chain of IgG and IgA, respectively. Complement proteins, present in serum, opsonize bacteria by C3b or C3bi for phagocytosis via CRs on macrophages (Schaible et al. 1999). Several receptors that participate in phagocytosis of complement-opsonized particles, including

CR1, CR3, and CR4 are expressed on macrophages (Sengelov 1995). While FcRs are constitutively active for phagocytosis (Aderem and Underhill 1999), the CRs of resident peritoneal macrophages bind but do not internalize particles in the absence of additional stimuli (Wright et al. 1983). The signals required for particle ingestion and the arrangement of cytoskeletal proteins on the phagosome surface vary depending upon which phagocytic receptor is engaged (Aderem and Underhill 1999).

Although all types of phagocytosis require actin polymerization at the site of ingestion (Allen and Aderem 1996a), results of electron microscopy (EM) studies demonstrate that IgG- and complement-opsonized particles are internalized differently by macrophages (Kaplan 1977; Allen and Aderem 1996b). During FcγR-mediated phagocytosis, veils of membrane rise above the cell surface and tightly surround the particle before drawing it into the body of the macrophage (Kaplan 1977; Allen and

Aderem 1996b). Silverstein (Silverstein 1995) demonstrated that FcγR-mediated ingestion occurs by a zippering process, in which FcγRs in the macrophage plasma

50 membrane interact sequentially with IgG molecules distributed over the surface of the

ingested particle. On the other hand, EM data indicate that CR-mediated phagocytosis is

a relatively passive process that occurs by a variation of the classic zipper model;

complement-opsonized particles appear to sink into the cell with elaboration of small, if

any, pseudopodia (Kaplan 1977; Allen and Aderem 1996b). An additional difference

between FcR- and CR-mediated phagocytosis relates to their capacity to trigger the

release of inflammatory mediators. Immunoglobulin receptor-induced phagocytosis is

tightly coupled to the production and secretion of pro-inflammatory molecules such as

reactive oxygen intermediates and arachidonic acid metabolites (Wright and Silverstein

1983; Aderem et al. 1985). By contrast, CR-mediated phagocytosis does not elicit the

release of either of these classes of inflammatory mediators (Wright and Silverstein 1983;

Aderem et al. 1985).

Mechanism of phagocytosis used by professional phagocytes to internalize

bacteria depends greatly on the type of pathogen involved. Salmonella typhimurium is a

facultative intracellular pathogen that is capable of replicating in intracellular

compartments in macrophages (Buchmeier and Heffron 1989). After binding to the

surface of a macrophage, virulent S. typhimurium induces generalized membrane ruffling

which results in the internalization of the bacterium into a compartment resembling a

macropinosome (Alpuche-Aranda et al. 1994). This nascent vacuole is enormous relative

to the size of the bacterium and has been called a "spacious phagosome" (Alpuche-

Aranda et al. 1994). Non-virulent mutant strains of S. typhimurium bind to the surface of macrophages but do not induce membrane ruffling or macropinocytosis (Alpuche-Aranda et al. 1995), instead, these bacteria are phagocytosed into a vacuole with a tightly

51 opposed membrane (Alpuche-Aranda et al. 1995). Once internalized, the spacious

vacuole containing virulent bacteria persists, and the bacteria multiply (Alpuche-Aranda

et al. 1994). There are conflicting reports as to the maturation of the spacious vacuole;

while some suggest that the spacious phagosome acquires lysosomal markers such as

LAMP-1 and cathepsin L, others suggest that the spacious phagosome remains immature

and does not fuse with lysosomes (Rathman et al. 1997).

Initial Shigella invasion of the polarized epithelial barrier requires transmigration

through M cells of the mucosa associated lymphoid tissue (Salyers and Whitt 1994).

Resident macrophages beneath the M cells readily phagocytose the invading bacteria.

Within macrophages, the ingested bacteria rapidly escape from the phagosome into the

cytoplasm and begin a life in the host cell cytoplasm that involves direct cell-to-cell

infection (Salyers and Whitt 1994). In contrast, Salmonella remains entombed in the

phagosome where genes of a second pathogenicity island, SPI2, which is essential for

intracellular replication, are turned on in response to the acidic environment and a low

free Mg2+ concentration (Deiwick et al. 1998; Hensel et al. 1998).

The fundamental infection strategies of Yersinia initially appear to be markedly different than that of Salmonella or Shigella. Virulent Yersinia, unlike Salmonella and

Shigella, does not induce host cell ruffling and does not invade normally phagocytic

macrophages. Rather, virulent Yersinia rapidly inhibit the phagocytic machinery

(Silhavy 1997). Yet, like Salmonella and Shigella, the coordinated activity of type III

secretion machinery and the adherence factors allows the bacteria to translocate plasmid-

encoded proteins called Yops. Several Yops are translocated into host cells where they

52 interfere with normal cellular processes and essentially paralyze the cell, allowing

Yersinia to proliferate on their surface.

Legionella pneumophila is a facultative intracellular pathogen that invades and

replicates in macrophages (Shuman and Horwitz 1996). A bacterial surface protein,

major outer-membrane protein (MOMP), fixes complement component C3 to the surface

of the parasite, thereby facilitating binding to the macrophage surface through

complement receptors (Bellinger-Kawahara and Horwitz 1990). Removing complement

from the media, or blocking CR3, with a specific antibody prevents bacterial adhesion to

the macrophage surface (Bellinger-Kawahara and Horwitz 1990). After binding, the

parasite induces the formation of an extended host cell pseudopod that spirals around the

bacterium forming a structure termed a "coiling phagosome" (Horwitz 1984). Although

CR3 is enriched in coiling phagosomes, there is no evidence that these receptors signal coiling phagocytosis. Since the structure of this phagosome is very different than that induced by CR3, it is likely that an additional, yet uncharacterized, L. pneumophila signal must be required.

A plethora of macrophage receptors have been implicated in binding and internalization of M. tuberculosis (Ernst 1998). As with other bacteria, complement fixes

to the surface of the organism through the alternate pathway, allowing deposition of

complement proteins C3b and C3bi, which are recognized by CRI and CR3 (Schaible et

al. 1999). Blocking CR drastically reduces binding and invasion of M. tuberculosis but does not abolish it, suggesting that other receptors participate in their uptake. Consistent with this, M. tuberculosis has also been demonstrated to bind to the mannose receptor and

53 the scavenger receptor (Ernst 1998). In addition, SP-A enhances macrophage binding

and uptake of M. tuberculosis, probably by the SP-A receptors (Gaynor et al. 1995).

After pathogens attach to the phagocyte membrane, they are rapidly internalized

and become enclosed within a pathogenic vacuole, the phagosome. Phagocytes rapidly

hold an assault on pathogens within the phagosome that normally ends up with the killing

of invading pathogens. Although the actual killing of intracellular bacteria takes place

primarily by highly reactive toxic molecules, particularly reactive nitrogen intermediates

(RNIs) and reactive oxygen intermediates (ROIs), phagocytes use several strategies to make the chances of survival of the invading pathogens slim, like acidifying the phagosome, delivering digestive enzymes to the phagosome, limiting the available intracellular iron, and tryptophan degradation (Elsbach and Weiss 1983; Nathan 1983;

Nathan and Hibbs 1991).

The highly reactive antimicrobial nitric oxide radical (NO–) acts as an oxidizing

agent, and is derived from L-arginine and molecular oxygen in a reaction catalyzed by

nitric oxide synthase (NOS) (Kaufmann 1999). There are three distinct forms of NOS

called NOS1, NOS2, and NOS3. Nitric oxide synthase l and 3 are calcium dependent

and constitutively present in tissues to maintain house-keeping functions, while NOS2

(inducible NOS or iNOS) is a calcium-independent form that can be induced in a variety

of cell types in response to immunological stimuli such as IFNγ, TNF, and IL-l (Fang

1997; Schaible et al. 1999), as well as microbial products such as lipoteichoic acid of

gram positive bacteria and LPS (Raetz et al. 1991; English et al. 1996). The role of NO–

as a primary macrophage antimicrobial effector molecule has been addressed in vitro,

where macrophages activated with IFNγ can inhibit the intracellular replication of a

54 variety of intracellular bacteria, including L. pneumophila, Mycobacterium bovis BCG,

and M. tuberculosis in a NO-dependent manner (Flesch and Kaufmann 1991; Chan et al.

1992; Yamamoto et al. 1996; Rhoades and Orme 1997). Experimental infection of iNOS knockout mice with M. tuberculosis revealed that these mice are extremely susceptible to disease, resulting in a shortened survival time as well as increased bacterial loads within the lung, spleen, and liver (MacMicking et al. 1997). Also, inhibition of NO– in

experimental models of M. tuberculosis infection has resulted in prompt reactivation of

the growth of the bacilli suggesting a role for continuous NO– production in persistent

and latent infections (MacMicking et al. 1997). Yet, the contribution of RNIs to killing

of intracellular bacteria by human macrophages remains unclear (MacMicking et al.

1997).

Infectious organisms may encounter reactive oxygen species either from internal

metabolism of oxygen or respiratory burst activity of phagocytic cells. The toxicity of

oxygen reactive species is mediated by products resulting from the univalent reduction of

– molecular oxygen and includes the superoxide radical (O2 ), hydrogen peroxide (H2O2),

and the hydroxyl radical (OH–) (Keyer et al. 1995). Production of ROI is initiated by a membrane-bound NADPH oxidase, which is activated by IFNγ, LPS, or IgG/FcR binding

(Kaufmann 1999). Further metabolic steps catalyzed by superoxide dismutase and appropriate iron molecules result in the production of oxygen radicals, which are powerful oxidants and cause DNA damage as well as alterations in membrane lipids and proteins of intracellular bacteria (Fang 1997). Although it is generally thought that NO is more important in controlling intracellular bacterial replication, there is a certain amount of cooperation between the two systems. Simultaneous production of both NO and ROI

55 can lead to a variety of antimicrobial compounds that control intracellular bacteria (Fang

1997).

Phagosomes with internalized pathogens usually go through three stages, the early phagosome that is characterized by a slightly acidic pH 6, the late phagosome that is characterized by pH less than 5.5, and the phagolysosome, which results from the fusion of phagosome and lysosome, and characterized by more acidic pH and contains typical lysosomal enzymes (Kaufmann 1999). Immediately after phagocytosis, the phagosome becomes alkaline for a short time before acidification is initiated. The basic milieu is optimal for the activity of defensins and basic proteins, whereas the acidic pH is optimal for lysosomal enzymes (Kaufmann 1999). These enzymes reside in the lysosome and are delivered into the phagosome during maturation through several independent waves, and they reach their optimum activity during later stages in the phagolysosome (Kaufmann

1999). Lysosomal enzymes include defensins, bacterial permeability-inducing protein

(BPI), phospholipase A2, and cathelicidins (Ganz and Weiss 1997) which act by permeabilizing the bacterial membrane and have an inhibitory effect against a wide variety of pathogens in vitro (Porter et al. 1997). Purified defensins are microbicidal for certain intracellular bacteria, such as Salmonella enterica and L. monocytogenes

(Kaufmann 1999). A virulence factor of S. typhimurium for mice, phoP, has been implicated in resistance against defensins (Fields et al. 1989). Although the role of defensins and other lysosomal enzymes in control of intracellular bacteria is not clear, it is believed that their contribution to bacterial killing is small. Their major task is the degradation of already killed bacteria.

56 Intracellular bacteria have an obligate requirement for iron to maintain their growth within cells. In this way a competitive environment is established between host cell and pathogen for the intracellular iron pool. Host cells have an array of specific molecules available to enhance their iron supply, and limiting access of this to the intracellular pathogens to the iron supply is an effective measure for controlling their growth (Kaufmann 1999). However, because iron is required as a cofactor in other antimicrobial mechanisms, such as the generation of ROI, host cells must enact a difficult balance to generate sufficient intracellular iron to support these mechanisms but limit excess production so as not to favor growth of the bacteria. In light of this, as discussed above, successful intracellular pathogens have evolved ways to successfully scavenge intracellular iron from the host cell.

Increased degradation of the amino acid tryptophan has been associated with killing of Chlamydia psittaci (Byrne et al. 1986). Although it is possible that limiting the intracellular availability of essential amino acids provides a potent antimicrobial mechanism, little is known about its general role in resistance against intracellular bacteria.

Thus, phagocytosis is considered a major defense mechanism against invading pathogens. It is, therefore, not surprising that some pathogenic microbes have evolved mechanisms to counteract the antimicrobial capacities of macrophages, or subvert phagocytosis to maintain their intracellular habitat and evade killing.

Intracellular bacteria have necessarily evolved mechanisms to enhance their ability to survive in a hostile intracellular environment. One of the initial ways in which they do this involves the choice of receptor(s) for entry into the host phagocytic cells.

57 Entry into the cell via CR fails to induce the generation of ROI, making it a less

hazardous route of entry for the pathogen (Mosser 1994).

Although less is known about specific mechanisms by which intracellular bacteria

may interfere with killing by RNls, many intracellular bacteria produce superoxide

– dismutase and catalase, which detoxify O2 and H2O2, respectively (Franzon et al. 1990;

Barnes et al. 1996). Many of these defense mechanisms are not constitutively expressed

in bacteria but are rather induced once the microbes are resident inside the host cell

(Kaufmann 1999). Invading pathogens may face the bactericidal activity of ROI from the

respiratory burst of phagocytic cells. Consequently, enzymes involved in antioxidant defenses in pathogens have been associated with microbial virulence. Among these enzymes, superoxide dismutases (SODs), a family of three metallo-enzymes, are capable

– of inactivating O2 as the first reactive oxygen species formed in the metabolic reduction

of oxygen, into H2O2 (Barnes et al. 1999). Subsequently, the presence of catalase activity decomposes H2O2 into oxygen and water. Both SOD and catalase have been reported to

protect certain pathogenic bacteria during the respiratory burst following phagocytosis.

Thus, SODs of S. flexneri (Franzon et al. 1990), L. monocytogenes (Welch et al. 1979), S.

typhimurium (Tsolis et al. 1995), and the fish pathogen A. salmonicida (Barnes et al.

1996) have been reported to reduce the killing efficacy of the oxygen radicals, whereas catalase activity is thought to protect Staphylococcus aureus (Mandell 1975). However, the protective role of these enzyme activities is variable and catalase appears to make a limited contribution to virulence in S. flexneri (Franzon et al. 1990) and S. typhimurium

(Tsolis et al. 1995). Thus, the contribution of a particular detoxifying enzyme for reactive oxygen intermediates in protection against phagocytic killing mechanisms may

58 vary with the organism and the model system studied. Studies on survival of Nocardia spp. in polymorphonuclear leukocytes have demonstrated that bactericidal activity at early time points is due to oxidative metabolism, whereas killing after 3 hours was achieved by both oxidative and non-oxidative mechanisms (Beaman and Beaman 1984).

Resistance to early killing in phagocytes is, therefore, more likely to be mediated by enzymes which provide protection against oxygen-dependent bactericidal mechanisms.

The intracellular location of these enzymes has been reported to have an important role in the ability of bacteria to survive oxidative stress (Gregory et al. 1973).

Thus, a periplasmic location could contribute to the elimination of reactive oxygen species from external sources such as phagocytes, while a cytoplasmic location may be more relevant in dealing with radicals generated during cellular metabolism (Barnes et al.

1999). Also, cell wall components, including LAM and phenolic glycolipid I isolated from M. leprae, have been demonstrated to have a down regulatory effect on phagocytosis and the induction of the respiratory burst in murine macrophages (Schaible et al. 1999). Further more, low-molecular-weight fractions, particularly of mycobacteria, such as phenolic glycolipid I of M. leprae, scavenge ROIs (Neill and Klebanoff 1988;

Chan et al. 1989).

Intracellular pathogens have also developed several strategies to manipulate phagosome maturation to avoid exposure to the antibacterial activities of host macrophages, such as the toxic metabolites ROI, RNI, defensins, and other microbicidal peptides, low pH, and lysosomal enzymes (Nathan and Hibbs 1991; Haas and Goebel

1992). To achieve a successful intracellular life style, intracellular bacteria follow one or more of four general strategies: escape from the phagosome into cytoplasm, block

59 phagosome maturation, inhibit fusion between the phagosome and lysosome, or adapt to survival in a late phagolysosomal compartment (Schaible et al. 1999).

A few intracellular pathogens have chosen a simple but rigorous way to avoid the degradative pathway by escaping from the phagosome into the cytoplasm, as bacterial killing is focused on the phagolysosome in order to limit self-damage. In addition to avoiding the phagolysosomal conditions and associated defense mechanisms, this strategy provides the respective pathogens with a nutrient-rich environment in the cytoplasm (Moulder 1985). Listeria monocytogenes, a gram-positive rod, expresses several virulence factors, including a pore-forming sulfohydril-activated hemolysin

(listeriolysin, LLO) that promotes rupture of the phagosomal membrane and escape into the cytoplasm (Cossart 1997). A lecithinase and two different phospholipase-C molecules probably act in concert with LLO for both escape from the phagosome and spreading to other cells (Cossart 1997).

In general, Mycobacteria reside in tight vacuoles that resist maturation toward the late endosomal/lysosomal stage (Armstrong and Hart 1971; Frehel et al. 1986). These vacuoles represent early endosomal compartments that do not acidify (pH 6.5). It is not clear how Mycobacteria inhibit phagosome maturation. Ammonia, which is produced in vast amounts by Mycobacteria, has been suggested to buffer the phagosomal content

(Gordon et al. 1980), which is consistent with the fact that the ability of vesicles to fuse can be influenced by their pH (Clague et al. 1994). Also, M. tuberculosis as well as M. avium restricts phagosome acidification via the exclusion of the proton pump from the phagosome (Sturgill-Koszycki et al. 1994). Living in the environment of an early endosomal compartment seems important for mycobacterial survival. This is strongly

60 supported by data showing that the M. avium phagosome matures toward a late endosomal compartment in IFNγ-activated macrophages (Schaible et al. 1998). Similar to Mycobacteria, phagosomes containing L. pneumophila and S. enterica are arrested somewhere between the early and the late stages (Kaufmann 1999).

Organisms like Salmonella (Buchmeier and Heffron 1991) and Mycobacteria

(Armstrong and Hart 1971) use mechanisms that avoid or inhibit targeting of the internalized bacteria to the phagosome-lysosome fusion pathway. The inhibition of phagolysosomal fusion is reported to be an active process, and generally does not occur when the pathogen is killed or inactivated before cellular uptake (Buchmeier and Heffron

1991). Neither heat-killed nor glutaraldehyde-fixed S. typhimurium inhibited phagolysosomal fusion in murine macrophages (Buchmeier and Heffron 1991).

Similarly, heat-killed or inactivated M. leprae (Sibley and Krahenbuhl 1987) and M. tuberculosis (Clemens et al. 1995) failed to prevent phagolysosomal fusion. The mechanism by which invading pathogens inhibit phagolysosomal fusion depends on the type of pathogen and the pathway of its entry (Sibley and Krahenbuhl 1987). The binding of some invading pathogens to cell surface receptors or their opsonization may dictate whether or not phagolysosomal fusion takes place. Mycobacterium tuberculosis failed to inhibit phagolysosomal fusion after entry via FcR, but not after entry via CR3

(Armstrong and Hart 1975). On the other hand, coating with antibodies had no effect on the inhibition of phagolysosomal fusion in cells infected with M. leprae (Sibley and

Krahenbuhl 1987) or S. typhimurium (Buchmeier and Heffron 1991). A mechanism of evading phagolysosomal fusion successfully used by C. psittaci is the evasion of receptor-mediated entry by altering membrane signals or recognition sites during

61 phagocytosis and by attaching to unusual sites on host cells. When the surface integrity of C. psittaci is altered by heat or antibody treatment, the pathogen loses its ability to prevent phagolysosomal fusion. In these later instances, it may be that the host cell, rather than the pathogen, directs entry, as in case of opsonized bacteria, through FcR

(Eissenberg and Wyrick 1981).

Another group of intracellular pathogens survive and even thrive under acidic and hydrolytic harsh conditions in late phagosomal/lysosomal compartment. Even more importantly, the phagolysosomal milieu may provide a vital source of nutrients for these organisms (Schaible et al. 1999). Unlike its relatives of the Chlamydia genus, Coxiella burnetii resides in a spacious macrophage phagosome showing late phagosomal/lysosomal characters as acidification (pH 5.2), cathepsin D, acid phosphatase, and vacuolar proton ATPase (Heinzen et al. 1996; Mege et al. 1997).

Early histopathological studies of yellowtail affected with photobacteriosis suggest that P. damselae can survive within fish macrophages (Kubota et al. 1970).

Histological evidences were based on observing apparently intact bacteria free and within phagocytes in internal organs. In contrast, a later study using experimentally infected gilthead sea bream with a highly virulent P. damselae strain concluded, based on electron microscopy, that both macrophages and granulocytes obtained from the peritoneal cavity of 20-30 gram fish may be involved in phagocytosis and killing of P. damselae (Noya et al. 1995a). Engulfed P. damselae were observed in the phagosomes of cells that suffered severe degranulation, and during the first 5 hours following infection, almost all internalized P. damselae showed severe morphological alterations. At the same time, the granulocytes themselves showed signs of degeneration, and the authors suggested that

62 this might also be a result of intense degranulation (Noya et al. 1995a). In another study,

the authors suggested that the degenerative changes of gilthead sea bream granulocytes

infected with P. damselae might be a result of interaction with the bacteria or

accumulation of toxic substances produced by the bacteria in the peritoneal cavity (Noya

et al. 1995b). In contrast, macrophages of smaller gilthead sea bream (0.5, or 5 to 10

grams), studied by electron microscopy, showed unaffected P. damselae in the peritoneal cavity (Noya et al. 1995a), kidney, and spleen (Noya et al. 1995b). Macrophages, on the other hand, contained a large number of unaltered and apparently dividing bacteria, suggesting a role for macrophages in disseminating bacteria throughout the body (Noya et al. 1995b). In advanced stages of infection, aggregates of degenerated macrophages full of apparently intact bacteria were observed in several organs (Noya et al. 1995b). It was suggested that the inability of macrophages to kill internalized bacteria might be due to macrophage deficiency or to insufficient macrophage activation (Noya et al. 1995a;

Noya et al. 1995b). In contrast, Skarmta et al. (1995) evaluated the ability of a virulent and a non-virulent strain of P. damselae to survive in vitro contact with macrophages obtained from sea bass, gilthead sea bream, and rainbow trout (300-500 gram) using a colorimetric assay and concluded that the phagocytic cells of three tested species were able to actively kill P. damselae. The virulent strain, however, was killed to a greater extent than the non-virulent strain. To explain the unusual finding, the authors suggested that the difference in killing of two strains might be due to differential stimulation of the respiratory burst, phagocytosis, or resistance to oxygen free radicals.

Barnes et al. (1999) concluded that all the strains of P. damselae studied have iron-SOD activity. Several types of SODs have been found in different bacterial species

63 such as E. coli (Gregory et al. 1973), L. pneumophila (St John and Steinman 1996) and A. salmonicida (Barnes et al. 1996). In many cases, one type of SOD is located in the

– periplasmic space of the cell, possibly contributing to the elimination of O2 generated extracellularly or at the membrane, while the other types may be found in the cytoplasm.

Only one type of SOD enzymes, the ferric SOD located in the periplasm, was identified in P. damselae (Barnes et al. 1999). Interestingly, P. damselae is susceptible to reactive oxygen species generated in a cell-free system. Exposure of bacteria to photo-reduced riboflavin in the presence of methionine resulted in a significant reduction in bacterial survival, however, in the presence of high number of P. damselae (107 CFU) the

bactericidal activity of cell-free generated superoxide anion was substantially reduced

regardless of concentration (Skarmeta et al. 1995). The periplasmic location of SOD

suggests that P. damselae should be able to protect against exogenous superoxide anions and this may, in fact, be the case. However, catalase was absent from the periplasm of P.

damselae, and there was no induction of catalase in response to sublethal concentrations

of peroxide (Barnes et al. 1999). Furthermore, the addition of both SOD and catalase

– simultaneously did not make a significant difference in protection against O2 compared

with the addition of catalase alone (Barnes et al. 1999). The authors suggested that P.

– damselae contains sufficient SOD activity to inactivate exogenous O2 but that the

susceptibility to killing results from accumulation of H2O2.

Intracellular Replication

Many pathogenic bacteria and intracellular parasites grow within infected host

cells, including S. typhimurium, S. flexneri, Yersinia pestis, C. trachomatis, Coxiella burnetii, and L. pneumophila (Schaible et al. 1999). Salmonella gain access to their host

64 by penetrating through intestinal epithelial cells (Darwin and Miller 1999) and surviving

within phagocytic cells (Alpuche-Aranda et al. 1994). They also grow inside vacuoles of

cultured epithelial cells (Finlay and Falkow 1990). There are at least three non-

auxotrophic loci in S. typhimurium required for growth inside epithelial cells, but not required for extracellular growth. Salmonella typhimurium with a mutation in any of these loci are defective for intracellular replication, yet are able to invade various epithelial cell lines and survive in the phagocytic cells at rates similar to virulent strains.

Most importantly, replication defective mutants are totally avirulent in mice (Leung and

Finlay 1991), suggesting that S. typhimurium must replicate intracellularly during a stage of its pathogenesis. In contrast, there are many Salmonella mutants that do not survive within phagocytic cells, but are able to invade and replicate in epithelial cell lines (Fields et al. 1986; Gahring et al. 1990), suggesting that invasion, intracellular survival, and replication are genetically separate processes.

The pathogenic process of S. flexneri encompasses several steps including

bacterial entry into epithelial cells (Menard et al. 1996), intracellular replication (Cersini

et al. 1998), and intra- and inter-cellular spreading (Goldberg et al. 1994), which allows

bacteria to infect neighboring cells. Main virulence-associated phenotypes such as

invasion and spreading are related to the bacterial cell division process. Shigella

auxotrophic mutants having high generation times move slowly, as shown by the

unchanged number of infected HeLa cells after three hours of infection, and the inability

to form observable plaques of lysis (Cersini et al. 1998).

In addition, Brucella proliferate within host macrophages, and virulence is

associated with the ability to multiply intracellularly (Ugalde et al. 2000). It has been

65 proposed that Brucella abortus rough mutants are less virulent for the two reasons; high

sensitivity to lysis by complement and inability to replicate intracellularly (Riley and

Robertson 1984). In addition, pigeon macrophages infected with Streptococcus bovis died after seven hours of incubation, and the authors suggested that the intracellular replication of the pathogen resulted in the release of substances toxic for macrophages, indicating that intracellular replication is an essential virulence factor in the pathogenesis of S. bovis in macrophages (De Herdt et al. 1995).

The ability of P. damselae to adhere to and invade several fish cell lines has been previously demonstrated (Magariños et al. 1996b; Yoshida et al. 1997; Lopez-Doriga et al. 2000), while its ability to replicate intracellularly within epithelial cells has not been reported. On the other hand, Lopez et al. (2000) concluded that P. damselae is able to invade but not replicate in EPC cells. Although the number of intracellular bacteria was time-dependant, the authors attributed the significant increase to further uptake of bacteria from the media, rather than intracellular replication of the bacteria. The authors, however, reached their conclusion based only on microscopic observations of numbers of infected cells and intercellular bacteria per infected cell.

Apoptosis

Multicellular animals often need to get rid of cells that are in excess, devoid of

function due to age or use, or potentially dangerous. To this end, they use an active,

dedicated, molecular program. As important as cell division and cell migration, regulated

(programmed) cell death, or apoptosis, allows the organism to tightly control cell

numbers and tissue size, and to protect itself from rogue cells that threaten homeostasis.

Optimum body maintenance means that billions of cells will die on a daily basis just to

66 counter the numbers of new cells that arise by mitosis. It is now clear that, during development, apoptosis is important for establishment and maintenance of tissue architecture to achieve proper organ function (Jacobson et al. 1997). Both the immune system and the nervous system arise through overproduction of cells followed by the death of those that fail to establish productive antigen specificities or functional synaptic connection (Krammer 2000; Yuan and Yankner 2000). Apoptosis can be induced by many physiological and pathophysiological stimuli, including specific receptor molecules, such as CD95 (Fas), or the tumor necrosis factor (TNF) receptor, and many stress factors such as growth factor withdrawal, irradiation, ultraviolet light, heat shock,

cytotoxic drugs, H2O2, ceramide treatment, bacteria, bacterial toxins, or viruses (Itoh et al. 1991; Baffy et al. 1993; Hockenbery et al. 1993; Beutler and van Huffel 1994;

Stevenson et al. 1994; Bose et al. 1995; Collins 1995). Apoptosis is distinctive from necrosis in many aspects. The term ‘apoptosis’ is sometimes used to describe only the changes in morphology, or sometimes to describe the underlying mechanism, which is a cell-autonomous, active process in which a specialized signaling pathway is active in killing the cell and organizing its disposal. In contrast, the term necrosis is reserved for the accidental cell death which results in relatively slow disintegration of the cell, or the large scale death of cells. Apoptotic cell death is an active process that requires participation of the dying cell and changes in cellular biochemistry, whereas, necrotic cell death is a passive process due to an acute cellular injury (Gulbins et al. 2000). Because apoptosis does not result in the release of intracellular material into the extracellular space, it usually does not result in an inflammatory response, whereas necrosis leads to

67 cell disintegration and the induction of an unspecific and/or specific immune response

(Gulbins et al. 2000).

Apoptosis is a highly conserved biological process requiring the regulated activation of several signaling cascades, which finally result in typical biochemical and

morphological alterations of the cell. The dying cell starts to sever attachments to other

cells and extracellular matrix and to round up, and then starts to show protrusions from

the plasma membrane, commonly referred to as ‘blebs’. Vacuoles have been observed to

appear in the cytoplasm of a dying cell. Staining of DNA allows the direct observation of

the condensation of the cell nucleus, which often starts as a condensed ring/sphere along

the nuclear envelope that will extend to encompass the entire nucleus and, at a later stage,

the fragmentation of the nucleus into several particles occurs. The entire cell condenses

and is reorganized into so-called ‘apoptotic bodies’, which are taken up by phagocytes

and neighboring cells (Kerr et al. 1972). Apoptotic bodies are membrane-bound vesicles

that vary in size and composition; they can contain the entire contents of the cell in

various combinations, such as cytosolic elements.

Cells dying by apoptosis in most situations display a very similar pattern of

morphological changes. This uniformity has contributed much to the emergence of the

understanding that apoptotic cell death, in probably all physiological situations, is

implemented by the same intracellular pathway. However, exceptions have been

reported and this uniform view may be an oversimplification. Undoubtedly,

morphological and biochemical differences can be demonstrated in different situations of

cell death in vivo. Most of the morphological changes that were observed by Kerr et al.

(1972) are caused by a set of cysteine proteases that are activated specifically in apoptotic

68 cells (Hengartner 2000). These “death proteases” are homologous to each other, and are

part of a large protein family known as the caspases (Alnemri et al. 1996). Caspases are

highly conserved through evolution, and can be found from humans all the way down to

insects, nematodes, and hydra (Hengartner 2000). Because they bring about most of the

visible changes that characterize apoptotic cell death, caspases can be thought of as the central executioners of the apoptotic pathway. Indeed, eliminating caspases activity, either through mutation or the use of small pharmacological inhibitors, will slow down or even prevent apoptosis (Earnshaw et al. 1999).

Pathogenic organisms can intervene in the host apoptotic pathway to their own favor. At least three pathogenic strategies are deployed by bacteria that involve

programmed cell death, including activation of apoptosis to destroy cells, utilization of apoptosis to initiate inflammation, and inhibition of host cell apoptosis to maintain the intracellular niche. On the other hand, infected host cells can undergo apoptosis as a defense against invading pathogens.

The most obvious scenario where killing host cells would be beneficial to pathogens is the induction of apoptosis in professional phagocytes, like

polymorphonuclear cells and macrophages, since these are the most dangerous cells that

are programmed to prevent the establishment of an infection. Apoptosis is triggered in

host cells in response to in vitro infections by a variety of extra- and intra-cellular

bacterial pathogens (Chen and Zychlinsky 1994; Chen et al. 1996a; Guzman et al. 1996;

Monack et al. 1996). This is not simply a manifestation seen in cell culture; bacterial-

induced apoptosis has been shown to occur in the tissues of infected animals as well

(Zychlinsky et al. 1996; Monack et al. 1998). Bacteria that produce exotoxins, like

69 Corynebacterium diphtheriae (Kochi and Collier 1993), P. aeruginosa (Kochi and

Collier 1993), Actinobacillus actinomycetemcomitans (Kato et al. 1995), and Bacillus anthracis (Hanna et al. 1993), may benefit from killing macrophages before they ingest and destroy the bacteria. Another situation where host cell killing would benefit the bacteria is the induction of apoptosis of epithelial cells, thus allowing sloughing off of the infected epithelium. This would serve as a vehicle for clearance and bacterial dissemination as suggested in P. aeruginosa infections (Pier et al. 1996).

While cell necrosis is a powerful stimulus for inflammation, in most cases of apoptosis, the release of proinflammatory cellular contents and cytokines is suppressed.

The data with for Salmonella and Yersinia suggest that this suppression plays an important role in some stage of the pathogenesis of infection (Brubaker 1991; Nakajima and Brubaker 1993). As noted, this is not what appears to be the case with respect to the utility of apoptosis in shigellosis. In Shigella infections, induction of apoptosis in macrophages allows the efficient release of mediators that trigger the acute inflammation typical of shigellosis (Zychlinsky and Sansonetti 1997). Surprisingly, in contrast to other situations where immune modulators are suppressed, Shigella uses apoptosis to deliberately incite the host’s inflammatory machinery. The subsequent infiltration and diapedisis by leukocytes disrupts the tight-junction of the bowel epithelium, thereby permitting a massive invasion by bacteria in the colon, followed by a massive invasion and disruption of the bowel mucosa (Zychlinsky et al. 1994; Zychlinsky and Sansonetti

1997).

The inhibition of apoptosis could be beneficial for intracellular pathogens, this mechanism has yet to be clearly described. Although S. typhimurium can induce

70 apoptosis in macrophages, Lundberg et al. (1999) reported that three epithelial cell lines,

HeLa (human), Caco-2 (human), and MDCK (canine), did not undergo apoptosis following infection with S. typhimurium. In addition, epithelial cells are resistant to

Shigella- (Mantis et al. 1996) and Yersinia-induced (Mills et al. 1997) apoptosis, although

they are able to undergo caspase-mediated apoptosis when treated with anti-CD95 antibodies or TNF-α. Lundberg et al. (1999) suggested that epithelial cells, in contrast to macrophages, might lack a component of the signal transduction pathway targeted by the

putative Salmonella factor(s) triggering apoptosis (e.g., the surface receptors), or

alternatively, they might contain an inhibitor(s) of this specific apoptotic pathway.

Apoptosis can be also used as a defense mechanism against pathogens such as

Mycobacteria. Molloy et al. (1994) showed that macrophages infected with Bacillus

Calmette-Guérin (BCG) are stimulated to undergo apoptosis, resulting in a great

reduction in the number of surviving bacteria compared with other mechanisms that do

not activate apoptosis. Interestingly, activation of the CD95 receptor, a different

apoptotic stimulus, in M. avium-M. intracellulare infected macrophages did not result in

bacterial killing (Laochumroonvorapong et al. 1996). Another study showed that

apoptosis of Shigella-infected macrophages causes the release of mature interleukin-1,

which induces an acute inflammatory response that eventually clears the infection

(Zychlinsky and Sansonetti 1997).

Pathogens can induce apoptosis by a variety of mechanisms, including activation

of cell surface receptors, induction of a second messenger, inhibition of host cell protein

synthesis by bacterial A-B toxins, disruption of membrane integrity by pore-forming

hemolysins, and regulation of caspase functions by a bacterial protein translocated via

71 type III secretion directly into the cytoplasm of infected cells (Chen and Zychlinsky

1994; Monack et al. 1996; Monack et al. 1997; Cossart et al. 2000). Staphylococcal enterotoxins induce apoptosis by engaging T-cell receptor (TCR) on T-cells (Jenkinson et al. 1989). The exact mechanism by which non-specific activation of TCR by superantigens as S. aureus enterotoxin B induce apoptosis is still unclear. This phenomenon of activation-induced cell death (AICD) is largely CD95-mediated and reflects interaction of cell-surface CD95 with its ligand, which is upregulated on activated T cells (Nagata and Golstein 1995). Bordetella pertussis induces apoptosis in the macrophage-like cell line J774, and in murine alveolar macrophages (Khelef et al.

1993). One of the virulence factors produced by B. pertussis is adenylate cyclase hemolysin (AC-Hly), which is directly involved in induction of apoptosis by producing a large amount of intracellular cAMP (Khelef et al. 1993). It is not clear how the cAMP is involved in programmed cell death. Corynebacterium diphtheriae produces diphtheria toxin (DTx) that induces apoptosis in target cells by interfering with RNA translation, and thus protein synthesis (Morimoto and Bonavida 1992). The enzymatic activity of

DTx catalyzes ADP-ribosylation of eukaryotic Elongation Factor 2 molecule that terminates all protein synthesis (Moynihan and Pappenheimer 1981). There are various bacterial pore-forming toxins that can destroy eukaryotic cells by disrupting the membrane, which leads to necrosis by osmotic lysis. These toxins can also, at lower concentration, induce apoptosis in host cells by forming very small pores in the host cell membrane that are not big enough to cause necrosis (Jonas et al. 1994). It is possible that small membrane permeability defects may induce other cellular physiological changes that indirectly trigger apoptosis, like increase in intracellular Ca2+ levels with leukotoxin

72 of A. actinomycetemcomitans (Iwase et al. 1992), influx of Na+ with α-toxin of S. aureus

(Jonas et al. 1994), and efflux of K+ and influx of Ca2+ with hemolysin of E. coli (Bhakdi and Tranum-Jensen 1988).

Direct induction of apoptosis by invasive bacteria was first demonstrated in S. flexneri, an etiologic agent of bacterial dysentery, which caused macrophage killing both in vitro and in vivo (Zychlinsky et al. 1992; Zychlinsky et al. 1996). Subsequent studies established that a single Shigella gene product, IpaB, was necessary and sufficient to induce apoptosis (Chen et al. 1996b) through interaction with caspase-1 (Hilbi et al.

1998). The ipaB gene is one of several essential, plasmid-encoded genes in an operon whose gene products are secreted by a type III secretion system and introduced into the cytoplasm of host macrophages (Mounier et al. 1997). In Salmonella there is a similar set of virulence genes that are part of the sip operon found on the bacterial chromosome in pathogenicity island 1, SPI1. The sip operon contains five genes: sipA, sipB, sipC, sipD, and sipE (Darwin and Miller 1999). These genes are homologous to the corresponding genes of the ipa operon of Shigella (Hermant et al. 1995). Recently,

Hersh et al. (1999) showed that SipB is necessary for Salmonella-induced macrophage apoptosis and that SipB functions by directly interacting with caspase-1. This finding is underscored by the observation that peritoneal macrophages lacking caspase-1 are insensitive to Salmonella-induced cytotoxicity (Hersh et al. 1999). Salmonella gene product SipB, as IpaB, induces apoptosis in macrophages, but not in epithelial cells, through a similar mechanism, using type III secretion system (Chen et al. 1996a). In addition, Salmonella that lose the capacity to induce apoptosis are measurably less

73 virulent after oral challenge (Jones and Falkow 1994), therefore, the capacity to induce

apoptosis is an important virulence determinant.

Yersinia is not a facultative intracellular bacteria. Yet, like Salmonella and

Shigella, Yersinia uses adherence factors and a type III secretion system to translocate

plasmid-encoded Yops. Yersinia YopJ, or YopP in Y. enterocolitica, a secreted protein,

is necessary for inducing apoptosis of macrophages in vitro (Monack et al. 1998).

Furthermore, a Y. pseudotuberculosis strain that no longer makes YopJ is significantly

attenuated in the mouse model of systemic Yersinia infection, with almost a 100-fold

increase in the oral LD50 over wild-type strain in Balb/c mice (Monack et al. 1998). The

exact mechanism by which YopJ induces apoptosis is not known, but it was recently

shown that it does not interact with caspase-1 (Monack and Falkow 2000), as do

Salmonella and Shigella. Clearly, the ability of wild-type Yersinia to kill one of the host’s first lines of defense, macrophages, gives the bacteria an advantage early in the infection.

Viable cells maintain an asymmetric distribution of different phospholipids between the inner and outer leaflets of the plasma membrane (Bretscher 1972).

Apoptotic cell death is accompanied by a change in plasma membrane structure and characterized by surface exposure of phosphatidylserine (PS), while the membrane integrity remains unchallenged (Martin et al. 1995). A novel technique was designed to detect apoptosis by targeting the loss of phospholipid asymmetry of the plasma membrane (Vermes et al. 1995).

Phosphatidylserine exposure during apoptosis seems to be a universal phenomenon (Martin et al. 1995) and not limited to mammalian cells, but also occurs in

74 insect and plant cells (O'Brien et al. 1997). Martin et al. (1995) showed that PS exposure

during apoptosis occurs in most, if not all, cells under the action of most, if not all,

triggers of apoptosis. The authors measured PS exposure in several murine and human

cell lines exposed to a wide variety of initiating stimuli and all resulted in PS

externalization. Furthermore, evidence was provided that loss of membrane asymmetry

is a rather early phenomenon in the apoptotic process, since PS exposure preceded both

the characteristic morphological changes as well as nuclear condensation. Direct comparison of the terminal dUTP nick end-labeling (TUNEL) assay and the annexin V affinity assay indicates that PS externalizations can be measured prior to the detection of

DNA strand breaks (O'Brien et al. 1997). All experimental evidence obtained thus far indicates that loss of membrane asymmetry is a very early phenomenon during apoptosis

(Castedo et al. 1996; Martin et al. 1996), initiated at a time following the caspases proteolytic cascade but possibly preceding nuclear condensation and breakdown of intracellular cytoskeletal and nuclear matrix constituents. This cell surface exposed PS may function as a tag for a specific recognition by macrophages and for phagocytosis of the dying cell (van Engeland et al. 1998). To date, the molecular machinery responsible for cell surface exposure of PS remains unidentified.

The availability of labeled annexin V facilitated studying of PS exposure at the outer membrane leaflet in cells undergoing programmed cell death, since annexin V, in the presence of calcium, preferentially binds to phospholipid species such as PS (Tait et al. 1989) which is normally absent in the outer leaflet of the plasma membrane of viable cell. Annexin V is not able to bind to normal, vital cells since it cannot penetrate the phospholipid bilayer. In dead cells, however, the integrity of the plasma membrane is

75 lost, and PS in the inner leaflet of the membrane is available to bind applied annexin V.

As apoptotic cells expose PS at the outer leaflet of the plasma membrane while the integrity of which has not yet been compromised, a membrane impermeable DNA stain, such as propidium-iodide (PI), cannot penetrate and stain the nucleus (Vermes et al.

1995). In this way vital, apoptotic, and dead cells can be discriminated and analyzed either by flow cytometry or fluorescence microscopy. Using flow cytometry, bivariate analysis shows that vital cells are negative for both annexin V and PI, apoptotic cells are annexin V positive, but PI negative, while dead cells are positive for both annexin V and

PI.

Factors That Cause Damage to the Host

Bacterial Toxins and ECP

The extracellular products (ECP) secreted by a variety of bacterial fish pathogens are important virulence factors because they can contribute to the development of the disease or enabling the bacteria to counteract the host's defense mechanisms. Fish pathogens of the genus Aeromonas hydrophila are known to produce proteases and hemolysins which are virulence factors (Thune et al. 1982) and the catfish pathogen

Edwardsiella ictaluri has been shown to produce chondroitinase (Shotts et al. 1986).

Some fish pathogens, such as E. tarda (Ullah and Arai 1983) and Y. ruckeri (Romalde et al. 1991), are known to produce potent dermatotoxins. The fish pathogen A. salmonicida produces a phospholipase, glycerophospholipid cholesterol acyltransferase, which serves as a primary extracellular toxin (Lee and Ellis 1990). The halophilic pathogen

Photobacterium damselae subsp. damselae (Vibrio damsela) produces a cytolysin

(damselysin) with phospholipase D activity (Kreger et al. 1987).

76 The ECP of P. damselae strains were shown to be lethal for different fish species

including gilthead sea bream, sea bass, turbot and rainbow trout, with LD50 values

ranging from 1 to 4.6 µg of protein per gram of fish, and to fish cell lines, and

homoiothermic cell lines. In addition, these ECPs contained toxins with lethal effect for

mice, and therefore, with strong activity at 37°C. Although slight variations in the ECP

activities among strains were described, specifically for the caseinase, gelatinase and

dermonecrotic activities, all the P. damselae isolates possessed strong phospholipase, cytotoxic, and hemolytic activities in their ECP. The fact that the ECP activities were lost after heat treatment indicated that toxicity for fish and mammals is not associated with the LPS, and is probably proteinaceous (Magariños et al. 1992b).

It has been demonstrated that iron plays an important regulatory role in the

synthesis of proteolytic enzymes by P. damselae, since these enzymatic activities were

expressed only when the strains were cultured in iron-restricted conditions (Magariños et

al. 1994). Therefore, although the pathogen is classically considered a non-proteolytic

bacterium in normal culture conditions (Magariños et al. 1992a), the low iron

environment within the fish could trigger the expression of other virulence factors, as

occurs in other pathogens for poikilothermic and homoiothermic animals (Woods et al.

1982; Toranzo and Barja 1993). On the other hand, there were mild or undetectable

lesions adjacent to the injection site following intramascular injection, and in the spleen

following intraperitoneal injection of purified ECP into gilthead sea bream (Noya et al.

1995b). The authors felt the lack of tissue destructive ability was the result of the low

proteolytic activity of the P. damselae ECP compared to ECP of other fish pathogens,

such as A. salmonicida (Kawahara et al. 1990) and V. anguillarum (Delacruz and Muroga

77 1989). Furthermore, the histopathological changes surrounding the colonies or

aggregates of bacteria were usually mild or undetectable, suggesting either that toxins

released by bacteria are not very destructive (Noya et al. 1995b) or toxins are not released in vivo. In addition, Zorrilla et al. (1999) found that the P. damselae strain tested lacks proteolytic activities such gelatinase, caseinase, and elastase. The authors have suggested that P. damselae must possess a different mechanism of tissue damage, probably of

multifactorial origin. These findings are in agreement with the fifth hallmark of idealized

intracellular bacteria set by Kaufmann (1999), who suggested that intracellular bacteria

would express little or no toxicity for host cells by themselves, and that pathology is

primarily a result of the immune reaction.

Treatment and Prevention of Photobacteriosis

Chemotherapy

The only tools for controlling the disease after clinical signs appear are

antibiotics. Because antibiotics are delivered in feed, and because sick fish often cease

feeding, the treatment actually prevents the disease in the fish not yet infected. Studies

on the in vitro sensitivity of P. damselae to chemotherapeutic agents showed that this

microorganism is sensitive to penicillin, chloramphenicol, tetracycline, novobiocin,

ampicillin, oxolinic acid, nitrofurantoin, trimethoprimsulphametoxazole, and colistin, and

has a variable sensitivity to erythromycin, kanamycin, streptomycin, and neomycin

(Kimura and Kitao 1971; Simidu and Egusa 1972; Robohm 1979; Toranzo et al. 1991).

From a theoretical point of view, most epizootics can be controlled by treatment with

chloramphenicol (Tung et al. 1985), ampicillin (Robohm 1983), or oxytetracycline

(Toranzo et al. 1991). New drugs such as florfenicol, bicozamycin, and phosphomycin

78 are also useful for controlling photobacteriosis outbreaks (Kitao et al. 1992; Kim and

Aoki 1993; Sano et al. 1994).

Besides the development of resistance, other factors are suggested for the failure of chemotherapy, including the cessation of feeding in infected fish, when the antibiotics are usually administered as a feed supplement. It is well known that the indiscriminate use of some chemotherapeutics in the treatment of bacterial infections of cultured fish has resulted in the appearance of strains resistant to such compounds (Aoki and Kitao 1985;

Brown 1989).

The use of chemotherapy in the treatment of cultured yellowtail affected by photobacteriosis in Japan was effective until 1980. After 1980, plasmid or chromosomal coded resistance to ampicillin, tetracycline, chloramphenicol, and quinolones was observed (Robohm 1983). In addition, P. damselae is a suspected intracellular pathogen dwelling within macrophages during an infection (Kubota et al. 1970; Noya et al. 1995b;

Lopez-Doriga et al. 2000), which can protect them from exposure to antibiotics and render the antibiotic ineffective in the treatment of photobacteriosis.

Control of photobacteriosis in the United States is hindered by the fact that no antibiotics are cleared to treat bacterial infections in hybrid striped bass. Initially, in emergency situations, farmers were granted compassionate use approval for

Terramycin or Romet. Resistant strains, however, emerged rapidly (LADL annual report 1996). Currently, these drugs are available for use with HSB under an investigational new animal drug application (INADA) through the Food and Drug

Administration (FDA).

79 Vaccination

Due to complications associated with chemotherapy in disease control,

immunoprophylaxis is the preferred method for controlling photobacteriosis. The first

significant interest in vaccination against P. damselae began in Japan in the early 1980’s

following the demonstration of protective antibodies in yellowtail after an outbreak of

pseudotuberculosis (Kusuda and Fukuda 1980), as well as the emergence of drug-

resistant strains of P. damselae carrying transferable R-plasmids in farm-raised yellowtail populations (Aoki and Kitao 1985). A number of studies to analyse the effectiveness of

vaccination as a preventive measure against photobacteriosis have been performed.

Fukuda and Kusuda (1981) found that passive immunization of yellowtail with rabbit

anti- P. damselae sera protected fish from subsequent challenge for 24 hours, which

corresponded with the time at which the rabbit antibodies were no longer detectable in

the serum. Yellowtail were found to be protected from P. damselae injection challenge

following immunization by intraperitoneal injection of formalin-killed cells in Freunds

complete adjuvant (FCA), high pressure spray, mixing antigen with the feed,

hyperosmotic infiltration, and immersion. Survival rates following challenge ranged

from 60 to 88% in oral and immersion treatments to 100% in injection and spray

treatments. The response of yellowtail to formalin killed (FKB), heat killed (HKB), and

live attenuated bacterins (LAB) was examined by Kusuda and Hamaguchi (1987). The

FKB and HKB reduced mortality from 81.3% in controls to 57.3% and 78.7%

respectively, while the LAB reduced mortality to 25.3%. No difference in antibody titer

among the treatments was seen (Kusuda and Hamaguchi 1987). The LAB increases

phagocytic activity over controls from 4.0% to 19.0%, compared to the FKB and HKB,

80 which increased 4.8% and 8.0% respectively (Kusuda and Hamaguchi 1988). Kusuda

and Hamaguchi (1987) noted that the results indicated that protection from P. damselae

may be based on the activation of phagocytes. It is important to note that the LAB was a

spontaneously attenuated mutant, with potential for reversion to the virulent form.

Purified LPS from P. damselae, delivered either by immersion or spray, was more protective (survival 87% compared to 40% in controls) than whole cell (40% survival) or lysed cell preparations (73% survival) after challenge by gastric intubation with 25 mg of cells per fish (Fukuda and Kusuda 1985). A vaccine prepared from ribosomal antigens of

P. damselae and administered by intraperitoneal injection was compared with vaccines produced from outer membrane fraction (OMF), LPS, precipitated antigen (PCA), and

ECP, while formalin killed cells (FKC) were used as a control. Of all the vaccine preparations tested, ribosomal antigen produced the greatest phagocytic activity, but produced the lowest levels of specific antibody against P. damselae (Kusuda et al. 1988).

Ninomiya et al. (1993) obtained similar results with a P. damselae ribosomal vaccine by

injection, which provided more protection than injected FKC. In the ribosomal

vaccinated group, agglutinating antibody titers against FKC did not increase throughout

the experiment, however, in the FKC vaccinated group a slight increase was noted at 2

and 4 weeks. The increase in antibody titers in the FKC group did not provide protection,

indicating that another immune mechanism must be involved. Although vaccination

would seem to be the best approach to control photobacteriosis, conventional vaccination

trials with killed bacterins are not satisfactory. No commercial vaccines for preventing

photobacteriosis are currently available, and the current trend in vaccine formulations

against P. damselae involved the development of live attenuated bacteria.

81 Study Objectives

At present, little is known about the mechanism(s) by which P. damselae avoids the host immune response and establishes an infection. For the ultimate control and treatment of photobacteriosis, it is necessary to understand its pathogenesis and the factors that make P. damselae a successful pathogen. Faced with ineffective treatment and preventive measures, unlocking the mechanisms required by the pathogen for invasion, spread, and survival in its fish host may ultimately provide the knowledge necessary for designing more effective means of control.

Histological observations of P. damselae in macrophages of gill capillaries, spleen, kidney, and to a lesser extent liver (Kubota et al. 1970; Hawke et al. 1987; Noya et al. 1995b; Hawke 1996), and its adherence and invasion capability to poikilothermic cell lines (Magariños et al. 1996b; Yoshida et al. 1997; Lopez-Doriga et al. 2000), led to the hypothesis that P. damselae is a facultative intracellular pathogen and its intracellular life-style is the key factor in the pathogenesis of photobacteriosis in hybrid striped bass.

Consequently, the purpose of this study was to evaluate the interaction of a virulent P. damselae strain with macrophages obtained from hybrid striped bass and

different poikilothermic cell lines using in vitro assays. The identification of P. damselae as an intracellular pathogen will indicate that stimulation of cell mediated immunity will be required to protect against photobacteriosis, and further indicate the need for live, attenuated vaccine to control the disease.

Specific study objectives are: 1) to evaluate the ability of P. damselae strain to survive and multiply within macrophages obtained from hybrid striped bass, 2) to investigate the mechanism(s) that enable P. damselae to survive/multiply within HSB

82 macrophages, 3) to investigate the ability of P. damselae to invade/multiply with

different poikilothermic cell lines, 4) to use light and electron microscopy to visually evaluate the interaction of P. damselae with HSB macrophages and cell lines, and 5) to use flow cytometry to evaluate the ability of P. damselae to induce programmed cell death (apoptosis) of infected HSB phagocytes.

83 CHAPTER II. PHOTOBACTERIUM DAMSELAE SUBSPECIES PISCICIDA IS CAPABLE OF REPLICATING IN HYBRID STRIPED BASS MACROPHAGES

Introduction

Commercial farming of hybrid striped bass (Morone saxatilis x Morone chrysops) is a rapidly growing aquaculture industry in the United States and abroad. Production of hybrid striped bass (HSB) in the United States has steadily increased since 1989 and reached 9.7 million lbs in 1999, valued at $ 25 million (Brent Blauch, Striped Bass

Growers Association, personal communication). Presently, HSB are grown in commercial operations in Alabama, Louisiana, Maryland, Mississippi, North Carolina,

South Carolina, Pennsylvania, Texas, and Virginia (Hawke 1996). Although most HSB production is conducted in fresh water systems (Smith et al. 1989), mariculture industries using pond, cage, net-pen, and raceway culture have developed in brackish water in coastal Louisiana, Texas and Florida (Hawke 1996). In addition, U.S. fry and fingerling producers ship millions of fish annually to marine and brackish water facilities in Taiwan and the Mediterranean region (Brent Blauch, Striped Bass Growers Association, personal communication).

Photobacteriosis is a devastating disease problem for HSB production in

Louisiana and several cultured fish species in Japan, Europe, and the Mediterranean.

Photobacteriosis is caused by Photobacterium damselae subspecies piscicida, formerly named Pasteurella piscicida. The disease seriously threatens the expansion of commercial hybrid striped bass production in warm water coastal areas (Hawke et al.

1987; Hawke 1996). Photobacteriosis is also a serious disease of cultured fish in Japan, causing severe economic losses in yellowtail (Seriola quinqueradiata) (Kubota et al.

84 1970; Kusuda and Yamaoka 1972), and mortalities in red sea bream (Acanthopagrus schlegeli) (Yasunaga et al. 1983), black sea bream (Mylio macrocephalus) (Muroga et al.

1977; Ohnishi et al. 1982), oval file fish (Navodan modestus) (Yasunaga et al. 1984), and red grouper (Epinephelus okaara) (Ueki et al. 1990). In addition, photobacteriosis has been reported in cultured gilthead sea bream (Sparus aurata) in Spain (Toranzo et al.

1991) and Italy (Ceschia et al. 1991), and in sea bass (Dicentrarchus labrax) in France

(Baudin-Laurencin et al. 1991) and Greece (Bakopoulos et al. 1995).

Although detailed pathological features of infected fish are known (Kubota et al.

1970; Hawke et al. 1987; Toranzo et al. 1991; Hawke 1996), the pathogenesis of the disease remains largely unexplored and the literature is somewhat contradictory. Several investigators have proposed that P. damselae is a facultative intracellular pathogen.

Unfortunately, the evidence presented in such studies is histological, with observation of bacteria in macrophages of gill capillaries, spleen, kidney, and to a lesser extent liver

(Kubota et al. 1970; Hawke et al. 1987; Noya et al. 1995b; Hawke 1996). Noya et al.

(1995a), on the other hand, concluded that both macrophages and granulocytes of gilthead sea bream are involved in phagocytosis and killing of virulent P. damselae in vivo. Further, Skarmeta et al. (1995) reported that sea bass, gilthead sea bream, and rainbow trout (Oncorhynchus mykiss) macrophages could effectively kill P. damselae in an in vitro killing assay. The demonstration, however, that live attenuated vaccines provide protection, while killed vaccines do not (Kusuda and Hamaguchi 1987), supports the hypothesis that P. damselae is an intracellular pathogen (Kaufmann 1999). Because of the contradictory nature of the evidence, an in vitro killing assay was developed for

85 HSB macrophages isolated from the head kidney, and the ability of P. damselae to

survive and replicate within them was evaluated.

Materials and Methods

Fish

All fish were obtained from a brackishwater HSB farm with no history of photobacteriosis. Fish were acclimated for a minimum of two weeks in a recirculating system with 3 ppt salinity, 5 ppm dissolved oxygen, and the temperature at 27°C.

Macropahge Media

Optimal media formulation for HSB macrophages was designed based on catfish leukocyte media (Miller and McKinney 1994) with normal HSB serum instead of catfish serum and an osmolality of 345 mOsm to match HSB serum. Normal HSB serum was collected by caudal-vein puncture, filter sterilized, and stored at -20ºC. All components of the media were obtained from Sigma (St. Louis, MO) except L-15, AIM-V, and L- glutamine (Gibco BRL, Grand Island, NY). Macrophage isolation and washing media

(MIWM) contained 243 ml of AIM-V media, 243 ml of L-15 media, 5.5 ml stock solution of non-essential amino acids (NEAA), 5.5 ml stock solution of sodium pyruvate

(SP), 0.6 ml stock solution of 2-Mercaptoethanol (2-ME), and 2.4 ml of 1 M HEPES buffer. The NEAA stock solution is a 1:100 dilution in media of 100X NEAA (Sigma, catalog number M-1745), SP stock solution is a 1:100 dilution in media of 100 mM SP

(Sigma, catalog number S-8636), and 2-ME stock solution is a 50 mM solution in media.

Macrophage incubation media (MIM) contains 45 ml of L-15, 45 ml of AIM-V, 1.2 ml of

NEAA stock solution, 1.2 ml of SP stock solution, 0.1 ml of 2-ME stock solution, 1.5 ml

86 of 7.5% sodium bicarbonate solution, 1.00 ml of 200 mM L-glutamine, and 5 ml of

normal hybrid striped bass serum.

Macrophage Isolation

Skarmta et al. (1995) isolated kidney macrophages from sea bass using a discontinuous percoll density gradient. The authors described a band of cells at the interface between 34% and 51% percoll that they suspected was enriched for macrophages, but no confirmatory stains were done. Using HSB head kidney cell suspensions and a non-specific esterase stain to identify macrophages (Ellsaesser et al.

1984), the band between 34% and 51% percoll yielded only 35% macrophages, and evaluation of other density gradient procedures resulted in a maximum of 45% macrophage enrichment. Furthermore, the use of a continuous percoll density gradient resulted in formation of four bands, with macrophages distributed among all four bands and a maximum enrichment of only 50% macrophages. Consequently, we decided to evaluate attachment to enrich for macrophages.

Briefly, macrophages were isolated from fish euthanized in 1 g/L of tricane methanesulfonate (Argent Chemical lab, Redmond, WA). A cell suspension was obtained by aseptically dissecting the head kidney and dispersing the cells through a cell dissociation sieve (Sigma) with double stainless-steel mesh (140 µm and 280 µm) with

HSB MIWM. Viable cell counts were determined using Trypan blue dye exclusion

(Sigma), and the cell suspension was seeded directly at a rate of 1 X 107 cells/well in flat-

bottomed 24-well plates (Sarstedt Inc., Newton, NC) with 1 ml/well HSB MIM. Cells

were allowed to adhere overnight (18 to 24 hours) at 28ºC in a 5% CO2 atmosphere, and

then washed 3 times with HSB MIWM to remove non-adherent cells and enrich for

87 attached macrophages. Macrophages were again identified using the non-specific

esterase stain (Ellsaesser et al. 1984). Esterase staining is regarded as the most reliable

cytochemical maker for mammalian macrophages (Kaplow 1981) and was successfully

adapted for several species of fish, including catfish (Miller et al. 1985), Atlantic salmon

(Jørensen et al. 1993) and goldfish (Wang et al. 1995). The number of total cells seeded

and time of incubation are the main factors determining final macrophage yield. When 1

X 107 cells were seeded and incubated for 18 to 24 hours, macrophages composed 80% of the adherent cell population, with a total yield of about 5 X 105 macrophages per well.

Another modification to the methods of Skarmeta et al. (1995) is the use of HSB

serum as a media supplement factor rather than fetal bovine serum (FBS). In preliminary

studies, HSB macrophages grown in media with FBS tended to clump and darken in

color compared to cells grown with HSB serum. In addition, adjusting the osmolality of the media to match HSB serum provided more stable growth conditions for macrophages.

Lysing Macrophages

Preliminary studies using cold sterile distilled water as per Skarmeta et al. (1995)

to lyse infected macrophages and release intracellular bacteria resulted in the recovery of

no bacteria. Because of the halophilic nature of P. damselae, however, and their

demonstrated inability to survive in distilled water while being prepared for

electroporation (Cutrin et al. 1995), the effect of distilled water on P. damselae was

evaluated. This study demonstrated that direct exposure of P. damselae to distilled water

resulted in an 80% decline in bacterial numbers after only 15 seconds and almost 100%

decline after only 60 seconds of exposure (Figure 1). Consequently, the ability of a

variety of commonly known chemical/mechanical lysing agents to lyse HSB

88 macrophages, including Tween 20, EDTA, SDS, saponin, and sonication was evaluated.

Results indicated that 5% of a saponin lysing solution (SLS) containing 40 mg/ml

saponin (Sigma) and 2.5% (w/v) polyethylenglycol (Amresco, Solon, OH) in normal

saline lysed HSB macrophages without any detrimental effect on P. damselae. In fact,

increasing the concentration of SLS up to 20%, increasing the time of exposure of

bacteria to SLS up to 15 minutes or lowering the number of bacteria exposed had no

effect on P. damselae survival. Saponin lysing solution is added to wells, thoroughly

mixed, and left for 3 minutes to lyse cells and release intracellular bacteria. As a final

evaluation, in a direct comparison of distilled water and SLS, no colonies were recovered

after 0 or 6 hours of incubation of macrophages and P. damselae when distilled water

was used, while 1.25 X 104 and 7.17 X 104 colony forming units (CFU)/ml were

recovered after 0 and 6 hours of incubation, respectively, when SLS was used.

Bacterial Strains

Photobacterium damselae subsp. piscicida strain LA91-197 (Hawke 1996) was grown to mid-log phase (optical density of 1 to 1.2 at 600 nm) in brain heart infusion

(BHI) broth (Difco, Detroit, MI) with 2% NaCl (BHI-salt) at 28ºC in a Cel-Gro Tissue

Culture Rotator (Lab Line, Melrose, IL) at 200 revolutions per minute. Escherichia coli strain QC779 (Carlioz and Touati 1986), a superoxide dismutase (SOD) double negative mutant, was used as a killing control and was grown in Luria-Bertani (LB) broth (Gibco

BRL) at 37ºC in the same model rotator.

300

250

200

150

100

Million CFU/ml

0 Control 15 30 60 120 240 Exposure Time in Seconds

90 Determination of Optimum Gentamicin Concentrations

Bactericidal and bacteristatic concentrations of gentamicin (Sigma) for P.

damselae and the bactericidal concentration for E. coli were determined using a standard dilution method. Briefly, bacteria were incubated in HSB MIM with varying concentrations of gentamicin at 28°C or 37ºC in a 5% CO2 atmosphere. At determined

time intervals, samples were removed, and CFU/ml were determined by standard plate

count methods using BHI-salt agar for P. damselae at 28ºC, or LB agar at 37ºC for E.

coli. The minimal bactericidal concentration of gentamicin, following a one-hour

exposure for both P. damselae and E. coli strains, was determined to be 10 µg/ml.

Although the bacteristatic concentration for P. damselae was determined to be 0.9 µg/ml

for up to 18 hours, a gentamicin concentration of 1 µg/ml was added during incubation to

ensure control of the extracellular growth of intracellular bacteria released from lysed

macrophages.

To evaluate possible intracellular accumulation of gentamicin, HSB macrophages

were incubated for 1 hour in HSB MIM containing 0, 10, 20, 50, 100, or 200 µg/ml

gentamicin. Cells were washed three times with MIWM and lysed with a 5% final

concentration of SLS. Lysates were used to saturate blank sensitivity discs that were

placed on Mueller-Hinton agar (Difco) freshly inoculated with P. damselae. Intracellular

levels of gentamicin were determined by evaluating zones of growth inhibition around

lysate-saturated discs. Results indicated no detectable concentration of gentamicin from

lysed macrophages, even at the 200 µg/ml maximum concentration.

91 Killing Assay

For P. damselae the multiplicity of infection (MOI) was 1 bacterium to 100 macrophages. After infection, plates were centrifuged at 50 X g for 5 minutes to bring bacteria in contact with macrophages. Cells were incubated with MIM at 28ºC in a 5%

CO2 atmosphere for 30 min, after which 10 µg/ml gentamicin was applied for 1 hour to

kill extracellular bacteria. Cells were washed 3 times with MIWM and then MIM

containing a 1 µg/ml gentamicin was applied to control replication of bacteria released

from lysed, infected macrophages. Plates were incubated at 28ºC in a 5% CO2

atmosphere and cells were lysed at 0, 3, 6, 12, and 18 hours with 5% final concentration

of SLS. Numbers of CFU of bacteria recovered were determined by standard plate count

method using BHI-salt agar. Photobacterium damselae with MIM and the 1 µg/ml dose

of gentamicin without macrophages acted as a media control. Escherichia coli was

assayed the same way except that a MOI of 1 bacterium to 10 macrophages was used, the

assay was done without gentamicin, and recovered E. coli were grown on LB plates.

Escherichia coli grown in wells with MIM, but without macrophages or gentamicin acted

as a media control.

Microscopic Assays

Light Microscopy

The same assay procedures as above were used to conduct light microscopic studies for P. damselae, except that cells were seeded in wells with 13-mm tissue-culture- treated therminox cover slips, and infected at a MOI of 1 bacterium to 1 macrophage.

92 Instead of lysing cells, however, cover slips were mounted on labeled slides and stained

(LeukoStat™, Fisher Scientific, Pittsburgh, PA).

Electron Microscopy

For transmission electron microscopy (TEM), the MOI was 10 bacteria to 1

macrophage, and the cover slips were processed for TEM. Briefly, primary fixation was

for 6 hours in 1.25% glutaraldehyde and 2% formaldehyde in 0.1 M sodium cacodylate

buffer, with pH adjusted to 7.4. Cells were post-fixed for 1 hour in 1% osmium

tetraoxide (OsO4) in distilled water. Cells were stained for 2 hours with 2% uranyl acetate in 0.2 M sodium acetate buffer with pH adjusted to 3.5. Ethanol-dried cells were infiltrated and then embedded in epoxy resin. Ultra thin sections were cut on a Sorvall®

model MT600 ultra microtome, mounted on 300-mesh copper, and stained for 10 minutes

with 5% uranyl acetate in distilled water. Sections were washed 3 times with DD water

and then stained for 2 minutes with lead citrate. Stained sections were examined using a

Zeiss® model EM 10C electron microscope under various magnifications.

Acid Phosphatase Cytochemistry

The same microscopic assay procedures as above were used to conduct an acid

phosphatase staining assay for P. damselae and E. coli with a MOI of 20 bacteria to 1 macrophage. After 3 hours of incubation, the cover slips were processed for acid

phosphatase staining as discribed by Robinson and Karnovsky (1983) and then processed

for TEM as above. Briefly, cerium (Sigma), an electron-dense element, was used as a

capture agent, and β-glycerophosphate (Sigma) was used as a phosphate-containing

substrate for assaying acid phosphatase enzyme activities within the phagosome after

93 fusion with the enzyme-carrying lsyosome. Positive reactions appeared as a fine,

electron-dense precipitate of cerium phosphate accumulation within active

phagolysosomes. One hundred bacteria-containing vacuoles were counted for both

bacteria, and the percent of vacuoles that show active reaction was determined.

Statistical Analysis

The experimental design was completely randomized with a factorial arrangement of treatments and data were analyzed by analysis of variance following a natural log transformation of the percent change data that were adjusted to a positive integer scale

(SAS 1999). The response variable was log percent change of treatment over time 0.

When the overall model indicated significance at p ≤ 0.05, Scheffe’s test was used for pairwise comparison of main effects, and a least square means procedure was used for pairwise comparison of interaction effects.

Results

Intracellular Survival/Replication of P. damselae

Photobacterium damselae was internalized within macrophages and increased significantly in numbers by 105, 510, 1091, and 1385% over time 0, after 3, 6, 12, and 18 hours of incubation, respectively, (Figure 2; Table 1) despite the presence of gentamicin in the media. In contrast, the number of P. damselae in media without macrophages, but

also with gentamicin, increased 253% at 3 hours, 188.2% at 6 hours over time 0, but then declined 21 and 71% below time 0 at 12 and 18 hours, respectively. Escherichia coli was actively killed by macrophages, with bacterial numbers decreasing by 47, 80, 91, and

96% from time 0 after 3, 6, 12, and 18 hours of incubation, respectively (Figure 3; Table

94 2). At the same time, the number of E. coli in media without macrophages or gentamicin

increased by 130, 277, 625, and 867% after the same designated incubation times.

Light Microscopy and TEM

Light microscopic examination of HSB anterior kidney macrophages infected

with P. damselae (Figures 4 A and C) revealed numerous bacilli, some of which appeared

to be actively dividing. Both number of infected cells and number of bacteria per

infected cell increased over the time of incubation (data not shown). Bacteria always

appeared to be within spacious, clear vacuoles, with no sign of bacteria free in the

cytoplasm. Transmission electron micrographs (Figures 4 B and D) confirmed bacterial

uptake and internalization, with bacteria visible within well-defined, membrane-bound,

spacious phagocytic vacuoles. At early incubation times, P. damselae were found in

relatively tighter vacuoles that enlarged at later incubation times. Also, the number of

bacteria per vacuole increased at each incubation time up to 12 hours, which was the last

incubation time tested. At later incubation times, vacuoles containing numerous bacteria

compressed other cell components of the macrophage to the edge of the cell, and many of

the infected cells were degenerative, with the bacteria-containing vacuole separated form

the outside by only a thin cytoplasmic layer (Figure 4 D). In addition, many lysed cells

and ghost cells were observed.

Inhibition of Phagolysosomal Fusion

Transmission electron micrographs of acid phosphatase stained cells showed active reaction within vacuoles containing E. coli, indicating successful phagolysosomal fusion and formation of a phagolysosome (Figures 5 A and B). Actually, 40% of E. coli

1600% 1400% 1200% 1000% 800% B&G 600% RB

Percent change 400% 200% 0% -200% 0 hrs 3 hrs 6 hrs 12 hrs 18 hrs Incubation Time

Table 1.―Intracellular survival of Photobacterium damselae in hybrid striped bass macrophages. Values represent the percent change of colony forming unit (CFU) counts over time 0 of incubation, and are the mean of 3 experiments (3 wells for each treatment per experiment) ± standard deviation. Mean in the same row with the same letter designation are not significantly different (p<0.05) in the Least Square Means procedures. Means in the same column with the same number designation are not significantly different (p<0.05) in the Least Square Means procedures.

Percent change in CFU count

0 hour 3 hour 6 hour 12 hour 18 hour

0 ± 0 253 ± 54.9 188.2 ± 182.5 -20.7 ± 60.8 -71.1 ± 23.2 Free

P. damselae A, 1 B,1 B,1 A,1 C,1

0 ± 0 105.3 ± 6.7 510.3 ± 121.8 1091 ± 591.5 1384.8 ± 471.1 Recovered

P. damselae A, 1 A,1 B,2 B,C,2 C,2

1200%

1000%

800% ge n a 600% B ch t 400% RB rcen e

P 200%

-200% 0 hrs 3 hrs 6 hrs 12 hrs 18 hrs Incubation Time

Table 2.―Killing of Escherichia coli by hybrid striped bass macrophages. Values represent the percent change of colony forming units colony forming unit (CFU) counts over time 0 of incubation, and are the mean of 3 experiments (3 wells for each treatment per experiment) ± standard deviation. Mean in the same row with the same letter designation are not significantly different (p<0.05) in the Least Square Means procedures. Means in the same column with the same number designation are not significantly different (p<0.05) in the Least Square Means procedures.

Percent change in CFU count

0 hour 3 hour 6 hour 12 hour 18 hour

0 ± 0 130.4 ± 74.3 277 ± 65.6 709.2 ± 224.8 1070.4 ± 221.1 Free

E. coli A, 1 B,1 C,1 D,1 E,1

0 ± 0 -47.1 ± 10.4 -80.4 ± 2.2 -91.5 ± 4.1 -95.7 ± 1.2 Recovered

E. coli A, 1 B,2 C,2 D,2 E,2

Figure 4.―(A) Light microscope photograph and (B) transmission electron micrograph of hybrid striped bass macrophages infected with Photobacterium damselae (P) at time 0 of incubation where macrophages show one or few bacteria in vacuoles, and (C) Light microscope photograph and (D) transmission electron micrograph at 6-hour incubation time where macrophages show several bacteria in spacious vacuoles.

100 101 102 containing vacuoles demonstrated phagolysosomal fusion after 3 hours of incubation.

This percent matches the average killing percent determined by the direct plate count

method after the same incubation time. By contrast, none of the vacuoles containing P.

damselae had evidence of phagosome-lysosome fusion (Figures 5 C and D).

Discussion

Results have demonstrated that P. damselae is capable of intracellular survival and replication in HSB head kidney macrophages. In the killing assay, HSB macrophages were unable to kill P. damselae, even at a low MOI of 1 bacterium to 100 macrophages, conditions that strongly favored killing of bacteria by macrophages. In contrast, the double SOD negative E. coli mutant was easily killed by HSB macrophages,

even at an MOI of 1 bacterium to 10 macrophages, conditions that favored bacterial

survival. The ability of P. damselae to replicate intracellularly is further supported by the

increased number of bacteria in macrophage cultures containing gentamicin, while the

number of bacteria grown in media containing the same dose but with no macrophages

increased slightly, then declined from 6 to 18 hours of incubation due to the extreme

sensitivity of P. damselae to gentamicin.

Light microscope and TEM results show that P. damselae enter the macrophage

and is enveloped in a relatively small vacuole that quickly enlarges into a spacious

phagosome (SP). In Salmonella typhimurium a similar SP develops, and mutants with

reduced ability to induce formation of SP are defective in survival in macrophages and

are attenuated in mouse challenge studies (Alpuche-Aranda et al. 1995). Interestingly, it

was further demonstrated that strains of mice that were resistant to S. typhimurium

infection are deficient in the ability to form and/or maintain SP, indicating that unknown

103

104 105 106 host factors are involved in SP formation and maintenance (Alpuche-Aranda et al. 1995).

Furthermore, TEM results indicated that P. damselae has the ability to inhibit

phagolysosomal fusion, similar to Mycobacterium tuberculosis (Armstrong and Hart

1971), Mycobacterium avium (Frehel et al. 1986), and S. typhimurium (Buchmeier and

Heffron 1991). Comparison of P. damselae and E. coli intracellular performance

indicated a strong correlation between the ability to inhibit phagosome-lysosome fusion and survival of these two organisms within HSB macrophages.

Results presented here are in contrast to those of previous studies. Noya et al.

(1995a) examined peritoneal exudate cells from two different sizes of gilthead sea bream using light and electron microscopy after intraperitoneal injection of a virulent strain of

P. damselae. They reported that bacteria within granulocytes and macrophages of the larger fish were morphologically altered and looked damaged, while bacteria within macrophages of smaller fish remained unaffected (Noya et al. 1995a). They concluded

that P. damselae could actively multiply within peritoneal macrophages of 0.5-gram fish, with 100% mortality of all fish within 4 – 5 days post injection. In contrast, there were no mortalities in larger fish (20 – 30 gram) and no clinical signs of disease developed within 3 weeks post injection. They suggested that the difference in resistance between the two groups of fish could have resulted from a macrophage deficiency, or a reduction in their activation due to the absence of some peritoneal fluid components in younger fish. In another study, Noya et al. (1995b) suggested that P. damselae is capable of replicating in macrophages of 5 – 10 gram gilthead sea bream based only on electron microscopic evidence. The difference in these results, in which macrophages from larger

HSB were unable to kill P. damselae, could be due to species differences, where

107 macrophages of gilthead sea bream are capable of killing P. damselae, while HSB macrophages are not. Alternatively, the difference could be due to the fact that peritoneal macrophages used in the previous study are activated, while head kidney macrophages are not.

Skarmeta et al. (1995) evaluated the ability of a virulent and a non-virulent strain

of P. damselae to survive in vitro contact with macrophages obtained from sea bass,

gilthead sea bream and rainbow trout, using a colorimetric assay, where cold distilled

water was used to lyse macrophages and recover intracellular bacteria. The authors

concluded that phagocytic cells from the three fish species tested were able to kill both

strains of pathogen, with the virulent strain being killed to a greater extent than the non-

virulent strain (Skarmeta et al. 1995). Results presented here demonstrating that P.

damselae is extremely sensitive to exposure to distilled water, however, indicate that their

results were an artifact of the methods.

In conclusion, intracellular pathogens have evolved a variety of strategies to

circumvent host cell mechanisms designed to take up and kill these invading

microorganisms, and have devised successful strategies that enable them to survive and

replicate within potentially lethal phagocytic host cells. To achieve a successful

intracellular life style, intracellular bacteria follow one or more of four general strategies:

escaping from the phagosome into cytoplasm, blocking phagosome maturation, inhibiting

fusion between the phagosome and lysosome, or adaptating to survival in a late

phagolysosomal compartment. Results presented here indicate that P. damselae is

capable of intracellular survival and multiplication in HSB macrophages and that this is

108 at least partially accomplished by preventing fusion of the phagosome and lysosome to produce a phagolysosome with bactericidal activities.

109 CHAPTER III. INVASION AND REPLICATION OF PHOTOBACTERIUM DAMSELAE SUBSPECIES PISCICIDA IN FISH CELL LINES

Introduction

Photobacteriosis, previously known as pasteurellosis, is a serious bacterial disease affecting different economically important marine fish species, including striped bass

(Morone saxatilis) (Snieszko et al. 1964) and its hybrids in the United States (Hawke

1996), juvenile yellowtail (Seriola quinqueradiata) in Japan (Kubota et al. 1970; Kusuda and Yamaoka 1972), gilthead sea bream (Sparus aurata) and sea bass (Dicentrarchus labrax) (Baudin-Laurencin et al. 1991; Ceschia et al. 1991; Toranzo et al. 1991; Candan et al. 1996) in Europe and the Mediterranean. Photobacteriosis is caused by the bacterium Photobacterium damselae subspecies piscicida, formerly known as

Pasteurella piscicida.

Photobacteriosis is a devastating problem in the hybrid striped bass (Morone saxatilis x Morone chrysops) mariculture industry in Louisiana. From the fall of 1990 through fall of 2000, photobacteriosis was diagnosed from 50 submissions of diseased hybrid striped bass to the Louisiana Aquatic Diagnostic Laboratory (Louisiana Aquatic

Diagnostic Laboratory annual report 2002). The outbreaks of photobacteriosis occurred on mariculture farms located in the coastal parishes of Louisiana and resulted in losses of over one million dollars in 1991 and 1994 (Hawke 1996).

The ability to invade non-phagocytic cells is a key determinant of virulence for several pathogenic bacteria, including Escherichia coli, Salmonella, Shigella, and

Yersinia (Finlay and Falkow 1988; Finlay 1990; Jouve et al. 1997; Schaible et al. 1999).

Presumably, this invasion tactic ensures a protected cellular niche for bacteria to replicate

110 or persist, because they are protected from the host defense mechanisms and/or

exogenous antimicrobial agents, including antibiotics (Plotkowski et al. 1994).

The pathogenesis of photobacteriosis is poorly understood. Different studies have

reported putative factors/mechanisms important in the virulence of P. damselae,

including a capsule (Magariños et al. 1996a) and an iron uptake system (Magariños et al.

1994). Based on the observation that P. damselae is capable of invading different fish

cell lines, it was suggested that P. damselae avoids host defense mechanisms by

persisting intracellularly in non-phagocytic cells (Magariños et al. 1996b).

Photobacterium damselae invaded various poikilothermic cell lines and remained viable

inside the infected cells for up to 2 days without replicating (Magariños et al. 1996b). In

addition, Lopez-Doriga et al. (2000) concluded that P. damselae is able to invade, but not

replicate in, EPC cells. Thus, the ability of P. damselae to invade non-phagocytic cells is

well demonstrated, but its ability to replicate intracellularly has not been reported. Based

on the previous demonstration that P. damselae is able to invade and replicate in hybrid

striped bass macrophages (Chapter I), this study was conducted to re-evaluate the ability

of P. damselae to invade and replicate in various fish cell lines.

Materials and Methods

Tissue Culture Media and Cell Lines

Three different fish cell lines were used in this study, including epithelioma papillosum carpio (EPC), channel catfish ovary (CCO), and fathead minnow (FHM) cells. All cell lines were grown in 25 cm2 flasks (Corning, Corning, NY) containing

minimal essential medium (MEM, HyClone, Logan, UT) with 25 mM HEPES buffer

(Sigma, St. Louis, MO) and 10 % fetal bovine serum (Sigma) (supplemented MEM) at

111 28°C. Cells from confluent flasks were seeded in flat-bottomed 24-well plates (Sarstedt

Inc., Newton, NC) and grown under the same conditions until they reach 90-95 %

confluency.

Bacterial Strains

Photobacterium damselae subsp. piscicida strain LA91-197 (Hawke 1996) was grown to mid-log phase (optical density of 1 to 1.2 at 600 nm) in BHI broth (Difco,

Detroit, MI) with 2% NaCl (BHI-salt) at 28ºC in a Cel-Gro Tissue Culture Rotator (Lab

Line, Melrose, IL) at 200 rpm.

Invasion Assay

The invasion assay was conducted under the same conditions that were

experimently determined in chapter I. Briefly, the multiplicity of infection (MOI) was 1

bacterium to 1 cell, and plates were centrifuged at 50 X g for 5 minutes to bring bacteria

in contact with cells. Cells were incubated with supplemented MEM at 28ºC for 30 min,

after which 10 µg/ml of gentamicin was applied for 1 hour to kill extracellular bacteria.

Cells were washed 3 times with MEM-HEPES and then supplemented MEM containing a

1 µg/ml of gentamicin was applied to control replication of bacteria released from

ruptured, infected cells. Plates were incubated at 28ºC and cells were lysed at 0, 3, 6, 12,

and 18 hours with a 5% of saponin lysing solution containing 40 mg/ml saponin (Sigma)

and 2.5% (w/v) polyethylenglycol (Amresco, Solon, OH) in normal saline. Numbers of

colony forming units (CFU) of bacteria recovered were determined by standard plate

count methods using BHI-salt agar. Photobacterium damselae with supplemented MEM and the 1 µg/ml dose of gentamicin without cells acted as a media control.

112 Microscopic Assays

Light Microscopy

The same assay procedures described above were used to conduct light microscopic studies, except that cells were seeded in wells with 13-mm tissue-culture- treated therminox cover slips at a MOI of 10 bacteria to 1 cell. Instead of lysing cells, however, cover slips were mounted on labeled slides and stained (LeukoStat™, Fisher

Scientific, Pittsburgh, PA).

Electron Microscopy

For transmission electron microscopy (TEM), the MOI was 10 bacteria to 1 cell,

and the cells were kept in suspension prior to processing for TEM. Briefly, primary

fixation was for 6 hours in 1.25% glutaraldehyde and 2% formaldehyde in 0.1 M sodium

cacodylate buffer, with pH adjusted to 7.4. Cells were post-fixed for 1 hour in 1%

osmium tetraoxide (OsO4) in distilled water. Cells were stained for 2 hours with 2%

uranyl acetate in 0.2 M sodium acetate buffer with pH adjusted to 3.5. Ethanol-dried

cells were infiltrated and then embedded in epoxy resin. Ultra thin sections were cut on a

Sorvall® model MT600 ultra microtome, mounted on 300-mesh copper, and stained for

10 minutes with 5% uranyl acetate in distilled water. Sections were washed 3 times with

DD water and then stained for 2 minutes with lead citrate. Stained sections were examined using a Zeiss® model EM 10C electron microscope under various magnifications.

113 Ruthenium Red Staining

The same microscopic assay procedures as above were used, and cells were

stained with ruthenium red and processed for TEM to discriminate between intracellular

compartments and the extracellular environment (Luft 1971b). Ruthenium red is an

inorganic stain that binds polysaccharides on the cell surface and appears as an electron-

dense deposition on the exposed outer surface of cells (Luft 1971a).

Statistical Analysis

(SAS 1999). The response variable was log percent change of treatment over time 0.

Results

Invasion and Replication of P. damselae in Fish Cell Lines

Photobacterium damselae was capable of invading and replicating in all three cell

lines. In EPC, P. damselae increased in numbers by 76, 421, 689, and 985 % after 3, 6,

12, and 18 hours of incubation, compared to time 0, despite the presence of gentamicin in

the media (Table 3). On the other hand, the numbers of P. damselae controls in media

without cells, but with gentamicin, declined 52, 82, 98.5, and 99% below time 0 after 3,

6, 12, and 18 hours , respectively. Photobacterium damselae also invaded CCO cells,

114

Table 3.―Intracellular replication of Photobacterium damselae in epithelioma papillosum carpio (EPC) cells. Values represent the percent change of colony forming unit (CFU) counts over time 0 of incubation, and are the mean of 3 experiments (3 wells for each treatment per experiment) ± standard deviation. Mean in the same row with the same letter designation are not significantly different (p<0.05) in the Least Square Means procedures. Means in the same column with the same number designation are not significantly different (p<0.05) in the Least Square Means procedures.

Percent change in CFU count

0 hour 3 hour 6 hour 12 hour 18 hour

0 ± 0 -52.2 ± 35.9 -81.7 ± 11.2 -98.5 ± 1.1 -99.3 ± 0.4 Free

P. damselae A, 1 B,1 C,1 C,D,1 D,1

0 ± 0 76.4 ± 31.2 421.2 ± 141.2 688.7 ± 316.4 984.6 ± 112.6 Recovered

P. damselae A, 1 B,2 C,2 C,D,2 D,2

115 and numbers of intracellular bacteria steadily increased by 124, 461, 620, and 851% after

3, 6, 12, and 18 hours of incubation, compared to time 0 (Table 4). On the other hand, P.

damselae recovered from FHM cells increased only 32% after 3 hours and 10% at 6

hours, but then declined 75 and 84% below time 0 at 12 and 18 hours, respectively (Table

5). Interestingly, the actual numbers of P. damselae CFU recovered from infected FHM

cells were one log greater than those recovered from EPC and CCO cells at 0 time of

incubation (Table 6). Although numbers of CFU recovered from FHM appeared not to

increase over time, and even declined after 6 hours of incubation, they were still

significantly more than the free P. damselae control at each time point. In contrast, the numbers of P. damselae in media-only controls, declined 69, 88, 96, and 99% below time

0, at 3, 6, 12, and 18 hours, respectively.

Light Microscopy and TEM

Photobactrium damselae proved to be invasive to cells and engulfment was seen

30 minutes post infection in FHM cells, which developed cytoplasmic extensions around bacteria (Figure 6). Photobacterium damselae showed the highest rate of invasion in

FHM cells where 30% of the cells were infected at time-0 incubation, compared to only

3% in EPC and CCO cells at the same time point (Table 6). Furthermore, the number of bacteria per infected cell averaged 3.0 in FHM cells, compared to 1.5 in EPC and CCO cells at time 0 (Table 6). Microscopic examination of cells of all three cell lines infected with P. damselae revealed numerous bacilli, which appeared to be actively dividing

(Figures 7 A, B, C, and D). Both numbers of infected cells and numbers of bacteria per infected cell increased over time (data not shown). Bacteria always appeared to be within

116

Table 4.―Intracellular replication of Photobacterium damselae in channel catfish ovary (CCO) cells. Values represent the percent change of colony forming unit (CFU) counts over time 0 of incubation, and are the mean of 3 experiments (3 wells for each treatment per experiment) ± standard deviation. Mean in the same row with the same letter designation are not significantly different (p<0.05) in the Least Square Means procedures. Means in the same column with the same number designation are not significantly different (p<0.05) in the Least Square Means procedures.

Percent change in CFU count

0 hour 3 hour 6 hour 12 hour 18 hour

0 ± 0 -68.5 ± 4.9 -87.9 ± 16.9 -95.6 ± 4.4 -99.2 ± 0.4 Free

P. damselae A, 1 B,1 C,1 C,1 D,1

0 ± 0 123.9 ± 130.4 460.8 ± 95.1 620.3 ± 256.7 851.3 ± 393.9 Recovered

P. damselae A, 1 A,2 B,2 B,2 B,2

117

Table 5.―Intracellular replication of Photobacterium damselae in fathead minnow (FHM) cells. Values represent the percent change of colony forming unit (CFU) counts over time 0 of incubation, and are the mean of 3 experiments (3 wells for each treatment per experiment) ± standard deviation. Mean in the same row with the same letter designation are not significantly different (p<0.05) in the Least Square Means procedures. Means in the same column with the same number designation are not significantly different (p<0.05) in the Least Square Means procedures.

Percent change in CFU count

0 hour 3 hour 6 hour 12 hour 18 hour

0 ± 0 -68.5 ± 4.9 -87.9 ± 16.9 -95.6 ± 4.4 -99.2 ± 0.4 Free

P. damselae A, 1 A,1 B,1 B,C,1 C,1

0 ± 0 32.1 ± 32.8 9.9 ± 34.5 -74.7 ± 28.4 -84.7 ± 21.6 Recovered

P. damselae A, 1 A,2 A,2 B,2 B,2

118

Percent of infected Recovered CFU/ml CFU/cell cells

EPC cells 147 3% 1.5

CCO cells 278 3% 1.5

FHM cells 2827 30% 3.0

119

Figure 6.―Transmission electron micrograph of fathead minnow (FHM) cell with cytoplasmic extensions (CE) around Photobacterium damselae (P) at 30 minutes post infection.

120 121

Figure 7.―Light microscope photographs of (A) fathead minnow (FHM) and (B) channel catfish ovary (CCO) cells infected with Photobacterium damselae (P) at 0 incubation time where cells show one or few bacteria in vacuoles, and (C) FHM and (D) CCO at 6- hour incubation time where cells show several bacteria in large vacuoles.

122 123 124 large, clear, membrane-bound vacuoles with no sign of free bacteria in the cytoplasm.

Intracellular replication of bacteria in all cell lines tested was observed at advanced

incubation times as indicated by the presence of vacuoles with numerous bacteria

(Figures 7 C and D). Due to the increased invasion of P. damselae in FHM cells, bacteria

accumulate more rapidly in FHM cells and are released from lysed cells as early as 3

hours of incubation. Transmission electron micrographs (Figures 8 A and B) confirmed

bacterial uptake and internalization, with bacteria visible within large well-defined,

membrane-bound phagocytic vacuoles. Also, the number of bacteria per vacuole

increased at each incubation time up to 12 hours, which was the last time examined. At

later incubation times, in EPC and CCO cells, vacuoles containing numerous bacteria

compressed other cell components to the edge of the cell and were separated from the

outside by only a thin cytoplasmic layer. In addition, many of the infected cells were

degenerative with many lysed, ghost cells present. These changes were, however,

observed as early as 3 to 6 hours of incubation in FHM cells due to the difference in

invasiveness.

Ruthenium Red Staining

Transmission electron microscopy of ruthenium red stained cells confirmed the intracellular location of P. damselae, with active staining on the outer membrane, but none on the P. damselae vacuolar membrane, indicating that these vacuoles are closed intracellular compartments with no connection to the extracellular environment (Figure 8

B).

125

126

127 Discussion

The ability of P. damselae to adhere to and invade several fish cell lines has been previously demonstrated (Magariños et al. 1996b; Yoshida et al. 1997; Lopez-Doriga et al. 2000). This is, however, the first study to report the ability of P. damselae to replicate within fish cell lines. In this assay, the ability of P. damselae to invade all three cell lines was confirmed following the application of gentamicin to kill extracellular bacteria. The intracellular location of P. damselae was further confirmed by ruthenium red staining of the external cell surface, but not of the P. damselae vacuolar membrane.

In EPC and CCO cells replication of P. damselae within infected cells was demonstrated by the significant increase in bacterial numbers despite the presence of a low dose of gentamicin in the media, a dose which resulted in a decline in numbers of bacteria when in media without cells. On the other hand, a comparison of the numbers of

CFU recovered from infected cells over time suggested that P. damselae replicated poorly in FHM cells. However, microscopic observations indicated that 30% of FHM cells were infected at 0-time of incubation, compared to 3% for CCO and EPC. In addition, an average of 3.0 bacteria was observed per infected FHM cell, compared to 1.5 for EPC and CCO. These numbers combined with numbers of CFU recovered at those times, clearly indicate that FHM cells are more susceptible to infection than the other two cell lines, despite the decline in CFU over time. There are two reasons for the discrepancy. First, the high initial infection levels tend to mask the percent increase in numbers of bacteria recovered. In addition, having more bacteria per cell at time 0 resulted in a more rapid accumulation over time and the death of infected cells earlier than the EPC and CCO cells, which started with lower numbers per cell at time 0. This is

128 supported by the observation of lysed FHM cells as early as 3 hours of incubation, which exposed released bacteria to the detrimental gentamicin dose, resulting in a decrease in numbers of bacteria recovered at 12 and 18 hours of incubation. Replication of P. damselae within FHM cells is further supported by the microscopic observation of cells packed with bacteria from 3 to 6 hours of incubation, compared to cells with one or two bacteria at time 0.

Several factors, including the use of a low MOI, optimum doses of gentamicin, and the determination of direct counts of CFU recovered from infected cells were important factors in optimizing the assay, and demonstrating that P. damselae is capable

of invading and, also, replicating within infected cells. Suboptimal combinations of these

factors, however, may have resulted in the failure of previous studies to demonstrate

replication of P. damselae in EPC cells (Magariños et al. 1996b; Lopez-Doriga et al.

2000).

In an in vitro assay, using gentamicin to control extracellular bacteria, Magariños

et al. (1996b) found that numbers of P. damselae recovered form infected cells did not

increase over time of incubation. The authors, however, used a MOI of 100 bacteria to 1

cell, and infected the cells over a 5 hour period, compared to a MOI of 1 to 1 and 30

minutes of infection in the present study. This would result in larger numbers of bacteria

per cell at 0 time of incubation and a rapid accumulation of intracellular bacteria by

replication, resulting in cell lysis and release of intracellular bacteria to media. In

addition, the authors first measured the numbers of CFU recovered from infected cells

after 24 hours of incubation. With such a high MOI and the prolonged infection time,

any detectable increase in bacterial numbers would occur within the first 6 to 9 hours of

129 incubation, followed by release of bacteria and exposure to 10 µg/ml of gentamicin for 15 to 18 hours. In the present study, exposure to 1 µg/ml of gentamicin resulted in a 96% decline in bacterial numbers after only 12 hours exposure.

In a previous study, microscopic observation of fluorescein-stained P. damselae in the EPC cells indicated an increasing number of intracellular bacteria per cell at 3 to 6 hours of incubation (Lopez-Doriga et al. 2000). However, when extracellular bacteria were removed by washing at 3 hours, the numbers of intracellular bacteria did not increase from 3 to 6 hours. The authors concluded that P. damselae does not replicate in

EPC cells and that the increase from 3 to 6 hours was due to the uptake of additional extracellular bacteria. The difference in results compared to those reported here could be due to the lower incubation temperature (22 versus 28°C), or to variation in the bacterial strains used.

Many pathogenic bacteria and intracellular parasites grow within infected host cells, including Salmonella typhimurium, Shigella flexneri, Yersinia, Chlamydia trachomatis Coxiella burnetii and Legionella pneumophila (Schaible et al. 1999). An intracellular life-style confers several advantages to pathogens; they become inaccessible to humoral and complement-mediated attack, they no longer require a specific adherence mechanism to maintain their site of infection, and they have access to a nutrient-rich environment. Salmonella typhimurium mutants that are defective for intracellular replication, yet are able to invade various epithelial cell lines and survive in the phagocytic cells are totally avirulent in mice (Leung and Finlay 1991), suggesting that S. typhimurium must replicate intracellularly during that stage of its pathogenesis in order to cause disease.

130 In conclusion, this study indicates that P. damselae is able to invade and replicate intracellularly in various fish cell lines, in contrast to pervious studies suggesting that P. damselae is able to invade, but not replicate in epithelial cells. The ability of P. damselae to invade and replicate within epithelial cells could be an important factor in its pathogenesis, similar to other fish pathogens (Lawson et al. 1985).

131 CHAPTER IV. INDUCTION OF APOPTOSIS IN HYBRID STRIPED BASS PHAGOCYTES BY PHOTOBACTERIUM DAMSELAE SUBSPECIES PISCICIDA INFECTION

Introduction

The culture of hybrid striped bass (Morone saxatilis x Morone chrysops) is an

important and growing aquaculture industry in the United States and abroad. Hybrid striped bass (HSB) production in the United States has increased from 500,000 lbs in

1987 to approximately 9.7 million lbs in 1999 (Brent Blauch, Striped Bass Growers

Association, personal communication). Increased production of HSB in the coastal marshes of Louisiana, utilizing pond, net-pen, raceway, and cage culture techniques, has resulted in the emergence of the aquatic pathogen, Photobacterium damselae subspecies piscicida, the causative agent of photobacteriosis, an important pathogen in coastal aquaculture worldwide. Intensive aquaculture in coastal areas in the United States

(Hawke et al. 1987; Hawke 1996), Japan (Kubota et al. 1970; Kusuda and Miura 1972;

Yasunaga et al. 1983; Yasunaga et al. 1984), and the Mediterranean region (Baudin-

Laurencin et al. 1991; Ceschia et al. 1991; Toranzo et al. 1991; Bakopoulos et al. 1995;

Baptista et al. 1996), ideal environmental conditions have been unintentionally established for this highly pathogenic, halophilic organism.

Although detailed pathological features of fish infected with P. damselae are known (Kubota et al. 1970; Toranzo et al. 1991; Hawke 1996; Magariños et al. 1996b), pathogenesis of the disease remains largely unexplored. At present, little is known about the mechanism(s) by which P. damselae avoids the host immune response and establishes an infection. A key point in the ultimate control and treatment of photobacteriosis is to understand its pathogenesis and the factors that make P. damselae a successful pathogen.

132 Bacterial pathogens possess a variety of strategies to take control of host cells,

including induction or suppression of apoptosis. The induction of bacteria-associated

apoptosis might not reflect a host response to infection, but rather a bacterial strategy to

promote disease. Species of Shigella (Zychlinsky et al. 1992), Salmonella (Chen et al.

1996a), Yersinia (Mills et al. 1997), Listeria (Guzman et al. 1996), and Mycobacterium

(Placido et al. 1997) are capable of directly modulating components of the apoptotic

pathways.

Phagocytosis and killing of microorganisms are two major tasks that professional

phagocytes have to fulfill. For this purpose, monocytes/macrophages and granulocytes

possess a variety of receptors and enzymatic mechanisms enabling them to engulf and

kill microbes (Elsbach and Weiss 1983; Nathan 1983; Morel et al. 1991). Pathogens

induce apoptosis, as a virulence factor, in the professional phagocytes because they are a

physical barrier to bacterial colonization.

It has been reported that peritoneal granulocytes of gilthead sea bream (Sparus

aurata) infected with intracellular P. damselae showed signs of degeneration, and the authors suggested that this is due to intense degranulation of the infected cells (Noya et al. 1995a). In another study, the authors suggested that the degenerative changes of

gilthead sea bream granulocytes infected with P. damselae are a result of either direct

interaction with the bacteria or the accumulation of toxic substances produced by the

bacteria in the peritoneal cavity (Noya et al. 1995b). Furthermore, using electron

microscopy, macrophages of smaller gilthead sea bream (0.5, 5 and 10 gram) showed

unaffected and apparently dividing P. damselae in the peritoneal cavity (Noya et al.

1995a), kidney, and spleen (Noya et al. 1995b). In addition, P. damselae is able to

133 invade various poikilothermic cell lines and remain viable inside the infected cells at least

for 2 days, producing noticeable damage to the infected cells (Magariños et al. 1996a).

Furthermore, Yoshida et al. (1997) reported that Chinook Salmon embryo cells (CHSE-

214), an epithelial cell line, rounded up and detached after they were infected with P. damselae. All these efforts describe typical morphological changes associated with apoptotic cells (Kerr et al. 1972), and suggest that P. damselae, as an intracellular pathogen, may be capable of inducing apoptosis in fish professional and non-professional phagocytes. Consequently, the ability of P. damselae to induce apoptosis in HSB phagocytes obtained from a head kidney cell suspension was evaluated.

Materials and Methods

Fish

All fish were obtained from a brackishwater HSB farm with no history of photobacteriosis. Fish were acclimated for a minimum of 2 weeks in a recirculating system with 2 ppt salinity, 5 ppm dissolved oxygen, and the temperature at 27° C.

Phagocytes Media

Optimal media formulation for HSB phagocytes was designed based on catfish

leukocyte media (Miller and McKinney 1994) with normal HSB serum instead of catfish

serum and an osmolality of 345 mOsm to match HSB serum. Normal HSB serum was

collected by caudal-vein puncture, filter sterilized, and stored at –20°C. All components

of the media were obtained from Sigma (St. Louis, MO) except L-15, AIM-V, and L-

glutamine (Gibco BRL, Grand Island, NY). Leukocyte isolation and washing media

(LIWM) contained 243 ml of AIM-V media, 243 ml of L-15 media, 5.5 ml stock solution

134 of non-essential amino acids (NEAA), 5.5 ml stock solution of sodium pyruvate (SP), 0.6

ml stock solution of 2-Mercaptoethanol (2-ME), and 2.4 ml of 1 M HEPES buffer. The

NEAA stock solution is a 1:100 dilution in media of 100X NEAA (Sigma, catalog

number M-1745), SP stock solution is a 1:100 dilution in media of 100 mM SP (Sigma,

catalog number S-8636), and 2-ME stock solution is a 50 mM solution in media.

Leukocyte incubation media (LIM) contains 45 ml of L-15, 45 ml of AIM-V, 1.2 ml of

NEAA stock solution, 1.2 ml of SP stock solution, 0.1 ml of 2-ME stock solution, 1.5 ml of 7.5% sodium bicarbonate solution, 1.00 ml of 200 mM L-glutamine, and 5 ml of normal hybrid striped bass serum.

Head Kidney Cells

Fish head kidney has proven to be a rich source of macrophages and neutrophils for in vitro culture in several fish species (Finco-Kent and Thune 1987; Jørensen et al.

1993; Sørensen et al. 1997). Briefly, phagocytes were isolated from fish euthanized in 1 g/L of tricane methanesulfonate (Argent Chemical lab, Redmond, WA). The cell suspension was obtained by aseptically dissecting the head kidney and dispersing the cells through a cell dissociation sieve with double stainless-steel meshes (140 µm and

280 µm) with HSB LIWM. Viable cell counts were determined using Trypan blue dye exclusion (Sigma), and the cell suspension was directly seeded at a rate of 5 X 105

cells/100 µl of LIM in 1.5 ml polypropylene eppendorf tubes (Eppendorf Scientific Inc,

Westbury, NY). Cells were incubated at 28°C in a 5% CO2 atmosphere.

135 Bacterial Strains

Photobacterium damselae subsp. piscicida strain LA91-197 (Hawke 1996) was grown to mid-log phase (optical density of 1 to 1.2 at 600 nm) in brain heart infusion

(BHI) broth (Difco, Detroit, MI) with 1% NaCl (BHI-salt) at 28°C in a Cel-Gro Tissue

Culture Rotator (Lab Line, Melrose, IL) at 200 revolutions per minute. Formalin-killed

P. damselae were prepared by adding an equal volume of 0.6% formalin in normal saline to an aliquot of P. damselae culture that was grown to mid-log phase, and incubating for several days at room temperature. Bacteria were harvested by pelletting at 5°C, and then resuspended in 0.3 % formalin in normal saline (Garvey et al. 1977).

Apoptotis Assay

The multiplicity of infection (MOI) was 10 bacteria to HSB head kidney cell for

both live and formalin-killed P. damselae strain. After infection, tubes were centrifuged

at 50 X g for 5 minutes to bring bacteria in contact with macrophages, and then gently

resuspended by inverting the tube several times. Cells were incubated with LIM at 28° C

in a 5% CO2 atmosphere for 30 min, after which 10 µg/ml of gentamicin was applied for

1 hour to kill extracellular bacteria (Chapter I). Cells were washed 2 times with LIWM

by pelleting and resuspending, and then 100 µl of fresh LIM was added to each tube.

Cells were incubated at 28°C in a 5% CO2 atmosphere and were processed for flow

cytometry after 12, 18, and 24 hours of incubation. As a positive control, apoptosis was

induced in uninfected HSB macrophages with camptothecin (CAM, Sigma), DNA

topoisomerase I inhibitor. A stock solution of CAM was prepared at a concentration of 3

mg in 1 ml DMSO (Sigma). Positive control uninfected HSB macrophages were assayed

the same way except that instead of infecting them with P. damselae, 5 µl of CAM was

136 added. As a negative control, macrophages that were non-infected and non-treated with

CAM were assayed in the same way as the infected group.

Flow Cytometry

Vybrant™ Apoptosis Assay Kit #2 (Molecular probes Inc., Eugene, OR) was used to differentiate between viable, early apoptotic, and late apoptotic/dead cells using recombinant annexin V conjugated to Alexa Flour® 488 (AF 488) green dye, and red- fluorescent propidium iodide (PI) as the nucleic acid binding dye. Manufacturer recommendations were followed throughout the assay. Briefly, at the end of each incubation time, cells were pelleted by centrifugation and then gently resuspended in 100

µl of the binding buffer (kit provided) in polystyrene tubes (Becton Dickinson Labware,

Lincoln Park, NJ). To each tube, 5 µl of AF 488-labeled annexin V and 1 µl of PI were added, after which tubes were incubated at room temperature for 15 min.

Stained cells were analyzed by two-color fluorescence using a FACScan flow cytometer (Becton Dickinson, San Jose, CA) equipped with a 15 mW air-cooled 488 nm argon-ion laser. Green fluorescence emission (annexin V-AF 488) was detected with a

530/30 nm band pass filter, while red fluorescence (PI) was detected with a 585/42 nm band pass filter. A total of 10,000 cells were analyzed on each sample using Cellquest analytical software (Becton Dickinson). Percentages of viable, apoptotic, and dead cells for each sample were measured using a bivariate histogram display of annexin V versus

PI fluorescence.

Based on the fact that apoptotic cells expose phosphatidylserine (PS) on the plasma membrane (Martin et al. 1995), investigations into the exposure of PS is facilitated by the finding that annexin V, in the presence of calcium, preferentially binds

137 to PS (Tait et al. 1989), which is normally absent in the outer leaflet of the plasma

membrane of viable cells. Annexin V is not able to bind PS on normal, vital cells

because it cannot penetrate the phospholipid bilayer. In dead cells, however, the integrity

of the plasma membrane is lost, and PS in the inner leaflet of the membrane is available

to bind applied annexin V. Because apoptotic cells expose PS on the outer leaflet of the

plasma membrane before its integrity is compromised, a membrane impermeable DNA

stain, such as PI, cannot penetrate and stain the nucleus (Vermes et al. 1995). Both apoptotic and dead cells bind labeled annexin V resulting in green fluorescence, but apoptotic cells exclude PI, while dead cells do not. As a result, apoptotic cells show only green fluorescence, while dead cells show both red and green fluorescence.

Statistical Analysis

The experimental design was completely randomized with a factorial arrangement of treatments, and data were analyzed by analysis of variance following a natural log transformation of the raw percent change data (SAS 1999). When the overall model indicated significance at p ≤ 0.05, Scheffe’s test was used for pairwise comparison of main effects, and a least square means procedure was used for pairwise comparison of interaction effects.

Results

Induction of Apoptosis by P. damselae in Phagocytes

The analysis of different groups of AF 488-labeled annexin V/PI double stained

HSB phagocytes after 12, 18, and 24 hours of incubation is shown in figure 9.

Phagocytes infected with P. damselae that were at an early stage of apoptosis

138 significantly increased by 49, 81, and 126% over the control base line, after 12, 18, and

24 hours of incubation, respectively (Table 7). Phagocytes at a late stage of apoptosis, that were marked as dead, also increased by 51, 72, and 146% after the same designated incubation times (Table 8). In contrast, phagocytes infected with formalin-killed P. damselae that were at an early stage of apoptosis increased slightly, but not significantly, by 8, 10, and 15% over the control base line after 12, 18, and 24 hours of incubation, respectively (Table 7). Phagocytes at a late stage of apoptosis increased slightly, but not significantly, by 9, 10, and 13% after the same designated incubation times (Table 8).

Control CAM-treated phagocytes at an early stage of apoptosis increased significantly by

182, 255, and 313 % over the control base line at 12, 18, and 24 hours of incubation, respectively (Table 7), while cells at a late stage of apoptosis increased by 108, 136, and

175 %, respectively, at the same designated incubation times (Table 8).

Discussion

This is the first report indicating that P. damselae is capable of inducing apoptosis in phagocytes. Pathogen-induced cell death in phagocytes generally occurs by programmed cell death, or apoptosis, a process whereby a cell actively participates in its own demise. The obvious benefit to the pathogens is the removal of dangerous cells that are programmed to prevent the establishment of an infection. Several studies have reported that the intracellular P. damselae is cytotoxic for fish phagocytes (Noya et al.

1995b), and fish cell lines (Magariños et al. 1996a; Yoshida et al. 1997). The description of the way that P. damselae kills the host cell in (Yoshida et al. 1997) is close to reported typical signs of apoptosis (Kerr et al. 1972).

139

Figure 9.―Analysis of Alexa Flour® 488-annexin V/ propidium iodide (PI) double stained hybrid striped bass phagocytes after 12, 18, and 24 hours of incubation. The lower left area of the cytograms represents viable cells, which are negative for annexin V binding and exclude PI. The lower right area represents apoptotic cells that demonstrate annexin V binding, but are negative for PI indicating an intact cytoplasmic membrane. The upper area represents non-viable, dead cells that are positive for both annexin V and PI. Representative cytograms after 12, 18, and 24 hours of incubation are shown. (A) control phagocytes that are non-infected, non-treated with camptothecin (CAM), (B) phagocytes treated with 5 µl CAM, (C) phagocytes infected with formalin-killed Photobacterium damselae, and (D) phagocytes infected with virulent Photobacterium damselae.

140 12h

18h

24h

141 12h

18h

24h

142 12h

18h

24h

143 12h

18h

24h

144

Table 7.―Comparison of apoptosis induction in hybrid striped bass phagocytes exposed to virulent or formalin-killed Photobacterium damselae, or camptothecin (CAM). Values represent the percent change from non-infected non-treated with CAM controls at each time point, and are the mean of 3 experiments (3 tubes for each treatment per experiment) ± standard deviation. Means with “*” are significantly different (p<0.01) from the control at that time point in the Least Square Means procedure. Means in the same row with the same letter designation are not significantly different (p<0.01) in the Least Square Means procedures. Means in the same column with the same number designation are not significantly different (p<0.01) in the Least Square Means procedures.

Percent Change

12 hours 18 hours 24 hours

49.12* ± 15.73 81.23* ± 29.19 126.08* ± 30.79 Virulent A, 1 B, 1 C, 1

7.85 ± 2.61 10.11 ± 2.40 14.65 ± 3.62 Formalin-killed A, 2 A, 2 A, 2

182.2* ± 67.4 254.97* ± 85.45 313.57* ± 94.74 CAM A,3 B,3 C,3

145

Table 8.―Comparison of late apoptosis in hybrid striped bass phagocytes exposed to virulent or formalin-killed Photobacterium damselae, or camptothecin (CAM). Values represent the percent change from non-infected non-treated with CAM controls at each time point, and are the mean of 3 experiments (3 tubes for each treatment per experiment) ± standard deviation. Means with “*” are significantly different (p<0.01) from the control at that time point in the Least Square Means procedure. Means in the same row with the same letter designation are not significantly different (p<0.01) in the Least Square Means procedures. Means in the same column with the same number designation are not significantly different (p<0.01) in the Least Square Means procedures.

Percent Change

12 hours 18 hours 24 hours

50.55* ± 10.38 71.87* ± 23.52 145.80* ± 35.19 Virulent A, 1 B, 1 C, 1

8.83 ± 4.33 9.50 ± 4.48 12.94 ± 3.45 Formalin-killed A, 2 A, 2 A, 2

108.34* ± 27.3 135.86* ± 35.59 175.00* ± 38.14 CAM A,3 B,3 C,1

146 Results reported here confirm that P. damselae is cytotoxic for phagocytes

through a mechanism that involves the induction of programmed cell death, or apoptosis.

Apoptosis is initially indicated by the binding of PS, exposed on the outer membrane

leaflet, to AF 488-labeled annexin V, while the cytoplasmic membrane remains intact as

indicated by the exclusion of PI. Cells in late stages of apoptosis cannot exclude PI

because the integrity of the cytoplasmic membrane is compromised due to the cumulative

effect of the apoptotic changes. Thus, most cells that stained with both annexin V and PI,

in this assay, are presumably at later stages of apoptosis or had already died by apoptosis.

This is supported by the finding that formalin-killed bacteria failed to induce apoptosis in

HSB head kidney phagocytes, which consequently led to lower levels of dead cells

(Figure 10).

Although cells incubated with the formalin-killed strain of P. damselae showed a slight increase in the relative number of early and late apoptotic cells, this increase was not significantly different from the base line of the control. These results clearly show that a live P. damselae is required to induce apoptosis in infected host cells and are in accordance with other studies where infection with live bacteria is necessary for the induction of apoptosis, while killed bacteria failed to have the same effect (Placido et al.

1997; Menzies and Kourteva 1998; Rodrigues et al. 1999; Jendro et al. 2000). The necessity for a live-bacterial infection in other pathogen-induced apoptosis results because the protein(s) responsible for induction of apoptosis are actively synthesized and exported by bacteria.

Pathogens can induce apoptosis by a variety of mechanisms, including activation of cell surface receptors, induction of a second messenger, inhibition of host cell protein

147

300%

250%

200%

150%

100%

50%

12hr FKB 12hr P.d. 18hr FKB 18hr P.d. 24hr FKB 24hr P.d.

Early apoptotic Late apoptotic

148 synthesis by bacterial A-B toxins, disruption of membrane integrity by pore-forming

hemolysins, and regulation of caspase functions by a bacterial protein translocated via

type III secretion directly into the cytoplasm of infected cells (Chen and Zychlinsky

1994; Monack et al. 1996; Monack et al. 1997; Cossart et al. 2000). Staphylococcus

enterotoxins induce apoptosis by engaging T-cell receptor on T-cells (Jenkinson et al.

1989) in a process that involves interaction of cell-surface CD95 with its ligand, which is

upregulated on activated T cells (Nagata and Golstein 1995). One of the virulence

factors produced by Bordetella pertussis is adenylate cyclase hemolysin that is directly involved in induction of apoptosis by producing a large amount of intracellular cAMP

(Khelef et al. 1993). Corynebacterium diphtheriae produces diphtheria toxin that induces apoptosis in target cells by interfering with RNA translation, and thus protein synthesis

(Morimoto and Bonavida 1992). There are various bacterial pore-forming toxins that, at lower concentration, can induce apotosis in host cells by forming very small pores in the cell membrane that are not big enough to cause necrosis (Jonas et al. 1994).

The ability of facultative intracellular bacteria to induced apoptosis in host macrophages was first demonstrated by showing that Shigella flexneri, an etiologic agent of bacterial dysentery, killed macrophages both in vitro (Zychlinsky et al. 1992) and in vivo (Zychlinsky et al. 1996). The Shigella gene product, IpaB, was necessary and sufficient to induce apoptosis (Chen et al. 1996b) through an interaction with caspase-1

(Hilbi et al. 1998). Recently, Hersh et al. (1999) showed that SipB, a part of the sip operon, is necessary for Salmonella-induced macrophage apoptosis and that SipB also functions by directly interacting with caspase-1. Both IpaB in S. flexneri (Mounier et al.

1997) and SipB in Salmonella typhimurium (Chen et al. 1996a) require type III secretion

149 systems to trigger apoptosis in macrophages. In addition, Salmonella that lose the

capacity to induce apoptosis are measurably less virulent after oral challenge (Jones and

Falkow 1994). Like Salmonella and Shigella, Yersinia induces apoptosis in

macrophages, but not in epithelial cells using a type III secretion system and the

adherence factors to translocate plasmid-encoded Yops (Silhavy 1997). A single

bacterial protein, YopP of Yersinia enterocolitica, or YopJ in Yersinia

pseudotuberculosis, is required for the induction of apoptosis in macrophages (Monack et

al. 1998). Further, a Yersinia pseudotuberculosis strain that no longer makes YopJ is

significantly attenuated in the mouse model of systemic Yersinia infection (Monack et al.

1998).

Phagocytes serve as sentinels that guard the potential points of pathogenic

invasion and deeper tissues against infection. Included in their antimicrobial arsenal is

the ability to produce potent cytokines that induce inflammation and recruit other

immune cells, including neutrophils (Secombes and Fletcher 1992). Potential pathogens

confront phagocytes soon after gaining access to host tissues, and the outcome of this

encounter is important in determining whether the host will mount an effective immune

response or whether the pathogen will successfully colonize the host. Clearly, the ability

of P. damselae to kill one of the host’s first lines of defense, phagocytes, provides an early advantage following first contact with the host immune cells.

150 SUMMARY

The ability of a virulent Photobacterium damselae subspecies piscicida strain to survive/replicate within macrophages obtained from hybrid striped bass was evaluated using an in vitro assay. Results indicated that the number of bacteria recovered from macrophages at 3, 6, 12, and 18 hours of incubation increased significantly by 105, 510,

1091 and 1385%, respectively, from time 0, despite the presence of gentamicin in the media. Growth and multiplication of the intracellular bacteria were observed at a very low multiplicity of infection (1 bacterium to 100 macrophages). In contrast, the number of P. damselae in media without macrophages, but also with gentamicin, increased 253% at 3 hours, 188.2% at 6 hours over time 0, but then declined 21 and 71% below time 0 at

12 and 18 hours, respectively. The numbers of the Escherichia coli control strain recovered from macrophages declined below time 0 by 47, 80, 91, and 96%, respectively, at 3, 6, 12, and 18 hours. Light and electron microscopy confirmed internalization, uptake, and replication of bacteria within spacious, clear vacuoles in the macrophages.

At later incubation times, numerous bacteria per vacuole and many lysed cells and ghost cells were observed. Using acid phosphatase as a lysosomal marker, it was shown that none of the P. damselae vacuoles showed evidence of phagolysosomal fusion, while, 40

% of E. coli containing vacuoles demonstrated phagolysosomal fusion after 3 hours of

incubation.

In an in vitro invasion assay in which intracellular growth was controlled by

gentamicin, channel catfish ovary (CCO), fathead minnow (FHM) and epithelioma

papillosum carpio (EPC) cells, were all shown to be susceptible to invasion and to

support replication of P. damselae. Numbers of bacteria recovered from EPC and CCO

151 cells increased significantly after 3, 6, 12, and 18 hours when compared to time 0.

Numbers of bacteria recovered from FHM cells increased significantly after 3 and 6

hours, and then declined at 12 and 18 hours, while still being significantly different from

time 0. Fathead minnow cells were actually more susceptible to invasion than the other

two cell lines as indicated by the greater number of infected cells and recovered bacteria

at time 0 and 3 hours of incubation. The decline in bacterial numbers in FHM cells at 12

and 18 hours of incubation was a result of lysing of the infected cells, the subsequent release of intracellular bacteria, and exposure of bacteria to gentamicin in the media. The

numbers of P. damselae controls in media without cells, but also with gentamicin,

declined at 3, 6, 12, and 18 hours below time 0. Using light and electron microscopy,

invasion of bacteria was observed as early as 30 minutes after infection, and intracellular

bacteria were observed in large, clear, membrane-bound vacuoles with no sign of bacteria

free in cytoplasm. Photobacterium damselae showed the highest rate of invasion in FHM

cells where 38% of the cells were infected at time 0 of incubation, compared to only 3%

in EPC, and CCO cell, at the same time point. Furthermore, there was an average of 3.0 bacteria per infected FHM cell, compared to 1.5 in EPC and CCO cells at time 0. The intracellular location of P. damselae was confirmed using ruthenium red staining, with active staining on the outer membrane, but none on the P. damselae vacuolar membrane, indicating that these vacuoles are closed intracellular compartments with no connection to the extracellular environment.

Two-color analysis of annexinV/propidium iodide double staining was used to investigate induction of apoptosis by P. damselae in a population of phagocytes

(macrophages/neutrophils) obtained from hybrid striped bass head kidney. Results

152 indicated that after 12, 18, and 24 hours of incubation, the relative numbers of cells

infected with virulent P. damselae that show signs of apoptosis are significantly greater

than the control by 49, 81, and 126% respectively, while relative numbers of infected

cells that show signs of late apoptosis are also significantly greater than the control by 51,

72, and 146% after the same designated incubation times. The relative numbers of

apoptotic cells that are infected with the formalin-killed strain increased, but not

significantly, by 8, 10, and 15% above the control after 12, 18, and 24 hours of

incubation, respectively, while the relative numbers of late apoptotic cells increased, but

again not significantly, by 9, 10, and 13% after the same designated incubation times.

These results indicate that viable P. damselae can induce programmed cell death in

hybrid striped bass phagocytes. Control CAM-treated phagocytes positive for apoptosis

increased significantly by 182, 255, and 313 % over the control base line at 12, 18, and

24 hours of incubation, respectively, while late-apoptotic cells increased by 108, 136, and

175 %, respectively, at the same designated incubation times.

Future research should focus on several avenues opened by this study. Factors that enable P. damselae to evade killing by macrophages should be further studied and

exact mechanisms should be investigated. Using regular transposon mutagenesis or

signature tagged mutagenesis will generate a variety of mutants that can be evaluated for

their ability to survive and replicate in macrophages, invade and replicate in fish cell lines, and induce of apoptosis. Such live attenuated mutants could represent promising vaccine candidates for combating photobacteriosis.

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180 VITA

Ahmad Elkamel was born on October 12, 1970, to Mr. and Mrs. Abd Elhady

Elkamel of Assiut, Egypt. After graduation from high school, he applied to the Faculty

of Veterinary Medicine, Assiut University, and studied there for the next 5 years. He

received his B.V. Sc. in June 1993 and applied for a job at the aquatic animal disease

division of the Veterinary Medicine Department, Faculty of Veterinary Medicine, to

teach the lab part of the fish disease class. He started his graduate study program in the

Department of Veterinary Microbiology and Parasitology, School of Veterinary

Medicine, Louisiana State University in spring 1997. He plans to continue with his

academic career in the fish disease field backed by the skills and lab experience gained through his studies at LSU.

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