AN ABSTRACT OF THE THESIS OF

Eva Andrea Wallner-Pendleton for the degree of Master of Science in

Veterinary Medicine presented on November 5, 1987 .

Title: The Early Pathogenesis of Pasteurella Multocida Infection

Studied by Immunohistochemical Techniques.

Abstract approved: Redacted for Privacy

masaxazu najumoto

Initial sites of localization and multiplication of Pasteurella

multocida were examined in chickens intravenously inoculated with

varying doses of bacteria. Sections of lung, liver, kidney, bone

marrow and spleen were prepared for light microscopic examination.

Tissue sections were stained with the hematoxylin and eosin stain,

Gram's stain or the avidin-biotin-peroxidase complex immunoenzymatic

technique. Of the three, immunohistochemistry proved to be far more

sensitive in localizing bacteria within the various organs. As

early as five hours postinoculation, bacteria were evident in low

numbers in splenic and hepatic mononuclear phagocytes. Bacteria

were not observed within the other organs or the blood. With

increasing time, bacteria were observed in large numbers within

these cells, causing necrosis of phagocytes, and liberating bacteria

into the sinusoids. In birds succumbing to the infection, bacteria

were seen in large numbers in all organs and in the blood, both

intracellularly and extracellularly. These results are consistent

with the hypothesis that P. multocida multiplies within hepatic and

splenic phagocytes in an early phase of infection. The possibility

that Pasteurella multocida may be able to resist intracellular

killing and may in fact multiply within the mononuclear phagocyte is

discussed.
 The Early Pathogenesis of

Pasteurella Multocida Infection

Studied by Immunohistochemical

Techniques

by

Eva Andrea Wallner-Pendleton

A THESIS

Submitted to

Oregon State University

in partial fulfillment of

the requirements for the

degree of

Master of Science

Completed November 5, 1987

Commencement June, 1988

 APPROVED:

Redacted for Privacy

U
Associate Professor, Veterinary Medicine and Microbiology

in charge of major

Redacted for Privacy

'--------D of the College of Veterinary Medicine

Redacted for Privacy

Dean of Graduate

Date thesis is presented November 5, 1987

Typed by for Eva Andrea Wallner-Pendleton

 ACKNOWLEDGEMENT

I thank Dr. Masakazu Matsumoto, my major professor, for his

assistance, encouragement, and constructive critique of this thesis.

I wish to thank my husband, Ken, for continual support, faith,

and love throughout these years, and baby George, for much happiness

despite trying times.

I wish to dedicate this thesis to Dr. George Wallner, my

father, who taught me the love of veterinary medicine.

 TABLE OF CONTENTS

Page

Chapter I

Literature review 1

Pasteurella multocida 2

Immunohistochemistry 11

Chapter II

In vivo multiplication of X-73 strain Pasteurella

multocida in chickens inoculated intravenously 15

Summary 16

Introduction 17

Materials and Methods 18

Results 18

Discussion 19

Chapter III

The early pathogenesis of Pasteurella multocida

infection studied by immunohistochemical techniques 21

Summary 22

Introduction 23

Materials and Methods 25

Results 30

Discussion 33

Bibliography 55
 LIST OF FIGURES

Page

Figure

1. Indirect immunoperoxidase method. 36

2. Avidin: biotinylated horseradish peroxidase complex

technique (ABC Technique). 37

3. Photomicrograph of chicken portal area with intracellular

bacteria. ABC stain 640X. 38

4. Photomicrograph of chicken sinusoid with bacteria.

ABC stain 800X. 39

5. Chicken liver with degenerating mononuclear cell within

sinusoid. ABC stain 640X. 40

6. Chicken liver with bacteria in inflammatory focus.

ABC stain 640X. 41

7. Chicken spleen showing intracellular bacteria within

mononuclear phagocyte. ABC stain 1600X. 42

8. Chicken spleen showing bacteria within macrophage.

ABC stain 800X. 43

9. Chicken hepatic sinusoid with bacteria.

ABC stain 1600X. 44

10. Chicken hepatic vein. ABC stain 1280X 45

11. Chicken inflammatory focus with bacteria in liver.

ABC stain 512X. 46

12. Chicken bone marrow with heterophilic granulocycte

depletion and cell necrosis. H&E stain 400X. 47

13. Normal chicken bone marrow. H&E stain 640X 48

 Page

14. Chicken spleen with fibrinoid necrosis. H&E stain 400X. 49

15. Chicken hepatic vein with heterophils. H&E stain 400X 50

16. Chicken glomerulus with thrombi. H&E stain 400X 51

17. Chicken renal blood vessel with bacteria.

Gram's stain 640X. 52

18. Chicken lung with interstitial thickening.

H&E stain 320X 53

19. Normal chicken lung. H&E stain 320X. 54

 The Early Pathogenesis of Pasteurella Multocida

Infections Studied by Immunohistochemical Techniques

Chapter I

Literature Review
 2

Pasteurella multocida

Pasteurella multocida is a bacterial pathogen responsible for

many diseases in cattle, swine, wild and domestic birds, and

rabbits. Infections in humans are occasionally observed secondary

to animal bites (9,11,28,31,49). Diseases caused by this agent

include acute septicemia, bronchopneumonia, rhinitis, and chronic

localized infections (49). In birds, the acute and chronic forms of

the disease have been collectively named "fowl cholera." The

disease has been recognized since the 1700's. In 1800, Louis

Pasteur was the first to isolate the organism and develope one of

the first vaccines against the disease (47). Despite its long

history, a satisfactory vaccine has yet to be developed, and more

information is needed concerning the pathogenesis and immune

mechanisms of this infection.

Pasteurella multocida has been classified as a Gram-negative,

facultatively anaerobic, nonmotile, nonsporeforming, rod-shaped

bacteria. Recently isolated organisms show bipolar staining

properties with the Giemsa, Gram's or Wright's stains. Pasteurella

multocida grows readily on sheep blood agar plates at 37 °C as

smooth, gray-white colonies with no hemolysis. The organism does

not grow on MacConkey's agar. Isolates usually produce oxidase,

catalase, and peroxidase and are indol-positive. No H2S, gas, or

urease are produced in appropriate media (47).

Encapsulated organisms will show characteristic iridescence

under obliquely transmitted light. Nonencapsulated organisms show

blue or gray colonies under the same light. In addition, colonies

 3

may dissociate, that is, go from highly iridescent to blue or gray

on subculture in artificial media. This may result in a decrease in

virulence of that organism for its host species (22).

P. multocida isolates have also been grouped according to a

variety of serologic and immunologic tests. Little and Lyon

separated P. multocida isolates into three groups according to slide

agglutination and passive protection tests in mice (30). Carter,

using the indirect hemagglutination test, found four serotypes based

on capsular polysaccharides (8). Namioka and Murata (37) defined

somatic serotypes by the use of decapsulated organisms. Heddleston

devised the gel diffusion precipitin test and distinguished 16

serotypes based on heat stable antigens (17).

Fowl cholera is a disease of major economic importance to the

poultry industry. The disease can be particularly devastating in

turkey and waterfowl populations where peracute to acute septicemias

may result in the loss of much of the flock in a short period of

time (28). In natural infections, the chicken appears to be more

resistant with chronic infections more common (47). Transmission

occurs through bird to bird contact, possibly by direct exposure

through: chronic carrier birds, scratches or puncture wounds,

contaminated food or drinking water (4,27,39,42). Wild birds,

insects, and mammals are capable of carrying the organism and

introducing the infection into a flock (17).

In acute fowl cholera, few signs may be observed initially,

followed by anorexia, diarrhea, cyanosis, fever, and ultimately

death. The mortality and morbidity often escalate exponentially

 4

(47). In chronic fowl cholera, morbidity and mortality tend to be

lower. Depending on the site of bacterial localization, signs may

include swelling of the wattles, torticollis, lameness, sinusitis,

ophthalmia or respiratory signs from pneumonia (38). Gross necropsy

lesions in acute fowl cholera are typical of septicemia and include

pinpoint necrotic foci in the liver, petechial hemorrhages on

serosal surfaces of the heart, lung, spleen, as well as other

organs, and generalized venous congestion (28,46). Acute foci of

coagulation necrosis are seen in the liver and spleen. Heterophil

infiltrates are present in most organs, especially the liver and

spleen. Acute fibrinoid necrosis is present within arterioles in

the spleen with accompanying necrosis of RE and lymphoid cells.

Heterophil depletion has been described in the bone marrow with

necrosis of hemopoietic cells. Bacteria are observed in high

numbers in many organs, especially in blood vessels and hepatic

sinusoids. Disseminated intravascular coagulation resulting in

fibrin thrombi in the liver, lung, spleen and kidney has been

reported (41).

Pathogenesis of Pasteurella multocida infections: Bacteria are

present in the nasal and conjunctival secretions of infected birds

(27,42). The organism spreads via the aerosol route or by

consumption of contaminated feed or drinking water (39). By some

unknown mechanism, bacteria are thought to adhere to the mucosa of

the upper respiratory tract then enter the blood stream resulting in

septicemia (33). Virulent strains of the bacteria then multiply

freely within infected tissues and blood in susceptible animals.

 5

Large amounts of endotoxins are released in the process, resulting

in death. This theory of pathogenesis is supported by the

observations that birds are readily infected with live vaccine

strains administered via the drinking water and endotracheal route

(5,33). Birds appear to be refractory to infection when bacteria

are administered directly into the lower gastrointestinal tract

(27). The choanal slit swabbing method is also an effective means

of experimentally reproducing the disease. However, it is possible

that bacteria are inadvertently introduced directly into the blood

stream because of microtrauma produced on the choanal mucosa.

Animals can also become infected experimentally via intramuscular,

intraperitoneal, intravenous, and subcutaneous injection.

Septicemia and death often ensue when virulent organisms are

injected parentally into susceptible animals (11).

Virulence factors associated with Pasteurella multocida

infections in avians: The phenomenal rate in which P. multocida

multiplies in infected hosts, and it's ability to evade host

defenses is poorly understood. To date, significant exotoxins have

not been reported in avian pasteurella infections. It has been

postulated that the presence of a hyaluronic acid capsule might

prevent phagocytosis and uptake of bacteria into phagocytic cells

and allow the bacteria to multiply in the blood as if in liquid

media (11,34). However, research by Pabs-Garnon and Soltys (40),

and Tsuji and Matsumoto, (53) suggest that the organisms first

localize in the liver and spleen and re-enter the blood in later

stages of the disease. Recent experiments by Tsuji and Matsumoto

 6

further suggested that encapsulation had little effect on bacterial

uptake by phagocytic cells. However, they observed differences in

organism survivability within phagocytes when comparing encapsulated

and unencapsulated strains (53).

Besides the hyaluronic acid capsule and endotoxin, little is

known about other virulence factors of this organism. Resistance

plasmids have been studied by Hirsh et al. (24), which confer

antibiotic resistance to the organism to a variety of anti-

microbials. These R-plasmids were found to be nontransmissible and

not widely dispersed in nature. Borisenkova (6) examined the

production of hyaluronidase and neuraminidase in several high and

low virulent strains of Pasteurella multocida isolated from hens,

ducks, and turkeys, including an avirulent vaccine strain. In this

study, a direct correlation was found between levels of bacterial

hyaluronidase and virulence; however, the neuraminidase levels

remained the same in all strains. Hyaluronidase would allow for

rapid spread of bacteria in intercellular spaces, increasing

invasiveness of the organism and rapid bacteremia. The

neuraminidase might play a role in bacterial adherence at the site

of initial infection. It was concluded by the author that

hyaluronidase and neuraminidase may both play an important role in

pathogenesis of cholera infection.

Iron is an essential nutrient for many parasitic micro-

organisms. Some bacteria produce complex iron-binding molecules,

called siderophores, from which to obtain iron from the host.

Siderophore production has been observed by Hu et al., (26) in

 7

Pasteurella multocida organisms isolated from turkeys which died

from fowl cholera. Since iron is important in bacterial macro-

molecular synthesis, it has been hypothesized that reduction of

plasma iron may be an important part of the host's defense against

bacterial infections, including fowl cholera (26).

Resistance factors of the host: The roles of humoral and cell-

mediated immunity in Pasteurella multocida infection in poultry have

not been well studied. Major emphasis of research has been placed

on vaccine production and humoral immune response. Some, though not

always, linear correlations are observed between antibody titers and

protection. Antibody titers can be measured using several accepted

serological tests including agglutination tests or the enzyme-linked

immunosorbent assay (ELISA) (1,7,14,18,20).

Multivalent, inactivated vaccines have been shown to signif-

icantly protect immunized hosts when challenged with homologous

strains of Pasteurella multocida, but field outbreaks continue to

occur despite immunization (21,36). Live, attenuated, and

naturally-occurring avirulent strains have been used parentally or

in the drinking water (4,5,20,35). Some cross-protection has been

observed with these vaccines against different serotypes.

Unfortunately, significant mortality directly attributed to the use

of live vaccines has been occasionally observed. Inactivated

bacterins produced in vivo in tissues or embryos have been shown to

induce some cross-protection in the same hosts. The mechanism for

this cross-protective immunity is unknown (19,44). Several antigens

have been isolated from Pasteurella multocida in avians and tested

 8

for their immunogenicity in the hopes of producing subunit vaccines.

Capsular polysaccharides have been isolated from turkeys by

Maheswaran (33) and identified as being composed of primarily

hyaluronic acid. A crude capsular antigen isolated through saline

extraction by Kodama et al. (29), proved to be immunogenic in

turkeys; however, the purified polysaccharide antigen was not.

Polysaccharide-protein complex antigens have some immunogenicity

which is lost following treatment with phenol. In addition, some

protective immunity is stimulated by potassium thiocyanide extracted

antigens. Many other antigens can be detected when pasteurella

organisms are sonicly disrupted, but their identification and

immunogenic potential have yet to be determined (1). It has been

assumed that both killed and live vaccines protect the host through

stimulation of the humoral immune system. Live vaccines may

stimulate both local and systemic antibody production. It is not

known whether these antibodies function as antitoxic, opsonic,

complement activating, or perhaps serve some other immune function.

Sensitivity to complement has been suggested by some to be an

important nonspecific factor in host resistance to infection with

fowl cholera. Snipes and Hirsh (51) demonstrated that P1059-1A, an

avirulent fowl cholera strain, was more sensitive to a heat-labile

substance in the serum than its virulent counterpart P1059-1. This

heat-labile substance was most likely complement according to the

authors. They concluded that the lipopolysaccharide configuration

may be different between the two strains, resulting in differences

in the activation potential of complement.

 9

Cellular Immunity: The primary cellular response in P.

multocida infections has been described as polymorphonuclear in

nature (11,46). Little, if any, mononuclear cell component has been

noted in the initial inflammatory cell response. The role of the

heterophil in the pathogenesis and natural immunity against

Pasteurella multocida infections in poultry is poorly understood.

Studies by Snipes et al. (50) indicated in vitro the ability of

mononuclear phagocytes to engulf and kill a virulent encapsulated

Pasteurella multocida strain in turkeys and its avirulent variant.

In those experiments, macrophages were able to phagocytose some

bacteria (one to three percent), though not very well. Most

phagocytized bacteria were destroyed. No differences were observed

between the virulent and avirulent strain regarding phagocytosis.

In another experiment by the same authors, an in vivo clearance was

performed with the same two strains. The virulent encapsulated

strain showed increased propensity for multiplication in the blood,

liver, and spleen when compared with the avirulent strain. They

postulated that the virulent strain may be able to multiply more

effectively in blood, resisting humoral bactericidal defenses of the

host (50).

Cell-mediated immunity was demonstrated in vitro against

Pasteurella multocida by Maheswaran et al. (35), via the lymphocyte

stimulation assay which is based on the principle that mitosis of

sensitized lymphocytes occurs three to five days after contact with

an antigen. This blastogenesis was shown to be antigen specific. A

similar experiment by Dua and Maheswaran (14) measured both humoral

 10

and cell-mediated immunity by the passive hemagglutination test and

the lymphocyte stimulation assay. Both humoral and cell-mediated

immunity was induced following vaccination with different strains of

P. multocida although the immunity did not persist for equal lengths

of time among animals vaccinated with different strains. Baba (2)

demonstrated that cell-mediated immunity was transferable from

immunized to normal chickens by the transfer of culture supernatent

from previously sensitized spleen cells. Peritoneal macrophages

from immunized hosts or macrophages sensitized with culture

supernatant fluid from immune spleen cells did not allow

intracellular proliferation of bacteria while normal macrophages

allowed considerable intracellular proliferation. Baba et al. (3),

in a previous experiment showed that surgical thymectomy had a more

deleterious effect on the survival rate of chickens challenged with

Pasteurella multocida than did hormonal or surgical bursectomy.

However, in a subsequent experiment with turkeys, Schlink and Olson

suggested that survival rate was negatively influenced by bursectomy

in young turkeys as well (48). This effect was less important with

increasing age of the birds, presumably because of the presence of

secondary centers of antibody-producing cells of bursal origin

scattered throughout the body. It appears likely that both cell-

mediated and humoral immunity may play important roles in protecting

poultry against Pasteurella multocida infection.

 11

Immunohistochemistry

Immunohistochemistry is a subclass of histochemistry, which is

the study of the chemical composition of tissues or cells without

the disruption of morphology. Immunohistochemistry combines the

principles of histochemistry and the immunologic specificity of the

antigen-antibody reaction. Antibody can be labeled (i.e.,

conjugated) with enzymes or fluorescent markers to specifically

locate antigens in histologically-prepared tissue sections. The

location of the antigen-antibody complex may then be demonstrated

with the addition of a substrate (enzyme histochemistry) or by the

presence of fluorescence (immunofluorescent histochemistry). Both

techniques are widely used in diagnostic pathology and research

(55).

Immunoperoxidase histochemistry: Horseradish peroxidase has

gained widespread use in enzyme histochemistry because of its

relatively low endogenous level in most tissue samples. The

indirect immunoperoxidase conjugated method is a classic example of

this technique and will be used to illustrate this method.

First, a tissue section, which contains the antigen in

question, is prepared histologically. Primary antibody prepared in

species B and directed against the antigen is added to the tissue

section. Secondary antibody, produced in species C and directed

against species B immunoglobulin, is next added to the slide. This

secondary antibody is also labeled with peroxidase enzyme. Finally,

the addition of the substrate chromogen chemical produces a colored

end product. In this example, the substrate is hydrogen peroxide

 12

and the chromogen is 3,3'-diaminobenzidine (usually referred to as

DAB). In this reaction, oxygen is liberated, oxidizing the DAB and

forming an insoluble brown end product at the site of antigen

localization (12). See Figure 1.

Avidin: Biotinylated Horseradish Peroxidase Complex Technique

(ABC technique): A modification of the immunoperoxidase procedure

has been developed employing avidin and biotin. The technique is

based on the fact that avidin has very high affinity for biotin.

The two substances form an essentially irreversible complex. Avidin

has four binding sites for biotin. Biotin can be easily conjugated

with enzymes, hence the term biotinylation. The resulting complex

of biotinylated enzyme and avidin has been termed "ABC."

The procedure involves incubating the antigen-containing tissue

section with primary antiserum, prepared the same way as in the

previously described immunoperoxidase technique. A secondary

antibody, directed against the species in which the primary antibody

was produced, is then added to the slide. This secondary antibody

is labeled with biotin. The avidin biotinylated horseradish

peroxidase is next added to the tissue section. Finally, the

hydrogen peroxide-chromogen complex is added, resulting in a colored

end product at the site of antigen localization in the tissue (25).

See Figure 2.

Advantages and Disadvantages to the ABC Technique: The ABC

technique is reported to be 40 times more sensitive than other

immunoperoxidase techniques (25). Highly diluted amounts of primary

antibody can be used, thus reducing the cost of production. There

 13

is less background staining reported with this method. Routinely

processed paraffin-embedded tissue sections, as well as tissue

smears and frozen sections, can all be stained with this technique.

These tissues may then be observed with an ordinary light

microscope, as opposed to the fluorescent technique in which only

frozen sections can be stained and fluorescent microscopy must be

used. Once stained, the DAB slides can be stored for years without

significant fading or deterioration. A wide variety of antigens may

be detected with this technique including tumors, hormones, toxins,

drugs, or infectious agents. The technique lends itself to

ultrastructural examination as well. A number of commercially

available kits have been developed containing standardized reagents

including secondary antiserum produced in a variety of hosts

(rabbit, goat, guinea pig, mouse, sheep, etc.). They also contain

quenching solution (to block endogenous tissue peroxides), diluted

normal serum (used as a blocking serum), and the ABC reagent. This

greatly simplifies the technique and standardizes the test.

Disadvantages to this technique include some background

staining occasionally observed. This may be due to high levels of

endogenous peroxides or nonimmunologic binding of antisera to the

tissue. This background staining is minimized by pretreating the

tissue with 0.3% H202 in methanol, then by the addition of normal

diluted serum to the tissue section. The normal serum is usually

produced in the same species as the secondary antibody. Incubation

times with the quenching solution or blocking serum can be varied

 14

depending on the amount of background staining present. Non-

specific staining can also be minimized by using the highest

dilution of primary serum that still stains well the antigen in

question. This dilution varies considerably and must be determined

for each antigen. The quality of the primary antiserum is also

important. Prompt fixation of the tissue is helpful in preserving

antigenic determinants and preventing diffusion of intracellular

antigen stores into extracellular spaces which may result in poor or

excessive staining (23).

The cost of the kits, and particularly the cost of producing

quality primary antiserum, can be high, especially if monoclonal

antibodies are used. Another disadvantage to the technique is the

suspected carcinogenicity of the chromogen substance. All necessary

precautions to prevent inhalation or skin contact should be used by

laboratory personnel (23).

As in all immunological tests, adequate controls are very

important in interpretation of results. A positive and a negative

control slide should be included. Ideally, they should both be

treated with positive antisera, and with negative preimmune sera to

test for specificity and the amount of background staining. The use

of absorption controls is advocated by some. The abolition of

positive staining after absorption of the antibody with the antigen

under test is considered by some to be the most sensitive test for

specificity. Other controls such as the use of DAB alone, or

omission of one or more reagents, or testing with primary antibody

produced against other antigens can also be tried (23).

 15

Chapter II

In Vivo Multiplication of X-73 Strain

Pasteurella multocida in Chickens

Inoculated Intravenously
 16

Summary

Chickens were inoculated intravenously with X-73 strain P.

multocida and portions of liver, spleen and blood were taken from

euthanatized animals at regular intervals. These samples were

examined for bacterial multiplication. The first four hours after

inoculation, the bacteria were present in roughly equal numbers in

the liver, spleen and blood. At eight hours, the liver and spleen

contained 100 times and 10 times the original inoculum per gram of

tissue respectively. No bacteria could be detected in the blood at

this time. The number of organisms steadily increased between 12

and 18 hours postinoculation, peaking at 109 number of organisms per

gram of tissue.
 17

Introduction

Fowl cholera is an acute or chronic septicemic disease

affecting poultry and wild birds. In the acute form, organisms can

be found widely disseminated in all organs and the blood at time of

death. It is widely believed that the bacteria can freely multiply

in the blood as if in artificial culture media, resulting in

septicemia. However, research by Paps-Garnon and Soltys (40) in

turkeys demonstrated that the bacteria first multiply in the spleen

and liver, and reenter the blood during the latter half of

infection. They concluded that Pasteurella multocida are probably

released into the blood at this time, rather than directly

multiplying intravascularly.

An in vivo clearance experiment by Snipes et al. (50) in

turkeys inoculated intravenously with 1059-1 strain P. multocida

demonstrated a similar, though much earlier, disappearance of

organisms from the blood stream, with a steady increase of bacteria

in the liver and spleen. They concluded that the disappearance and

subsequent reappearance of bacteria was due to dilution of the

organisms in the turkey vascular system rather than a specific

removal by liver and splenic phagocytes.

The purpose of this experiment was to determine if

multiplication of Pasteurella multocida in chickens with the X-73

strain occurs in a pattern similar to that observed in turkeys

inoculated with other Pasteurella multocida strains.

 18

Materials and Methods

Fourteen twelve-week-old specific pathogen-free male broiler

chickens were inoculated with 1.9 x 103 colony-forming units of

Pasteurella multocida R-73 strain intravenously as determined by the

standard plate dilution count method. Two birds from the group were

bled, euthanatized via electrocution and examined at necropsy at the

following intervals: one-half hour, four, eight, twelve, and

eighteen and a half hours. The blood was collected into sterile

heparinized vacutainer tubesl. Samples of liver and spleen were

collected separately into sterile plastic bags. The tissues were

homogenized and serially diluted 10-fold with brain-heart infusion

broth2. Likewise, serial dilutions were made with the heparinized

whole blood samples. Standard plate counts were performed to

determine the colony-forming units per gram of tissue or milliliter

of blood. Blood samples were not collected at 18% hours as the birds

were dead at this time.

Results

The results of the bacterial counts are summarized in Table 1.

 19

Table 1. Concentrations of P. multocida in the liver, spleen and

blood of chickens inoculated intravenously.

No. of Birds Hours PI Liver* Spleens Bloodb

2 0 0 0
4.5 X 102 4.7 X 102 2 X 103

2 4 1.8 X 103 4 X 102 2 X 103

1.0 X 103 0 0

2 8 6.6 X 105 0 0
3.6 X 106 2.3 X 105 0

2 12 1.0 X 105 3 X 107 1 X 107

1.1 X 106 1 X 106

2 18% 1.4 X 109 1.8 X 109 OP IMO

4.1 X 109 2.4 X 109

*Reported per gram of tissue

bReported per milliliter of blood

Discussion

In this experiment, a steady increase in the numbers of

bacteria per gram of tissue was observed with increasing time.

Between four and eight hours, the organisms could not be detected in

the blood, but were present in moderate numbers in the liver and

spleen. This would suggest that the bacteria may have been cleared

in these organs as has been hypothesized by others. In this study,

a few birds were able to clear bacteria from the spleen between four

and eight hours postinoculation while maintaining numbers in the

liver. This may be due to more effecient killing in the spleen.

Unfortunately, the small numbers of birds used in the experiment

make interpretation of this data difficult. At 12 and 18 hours,

 20

bacteria were present in high numbers in the liver, spleen and blood

in all birds, suggestive of very rapid multiplication of the

bacteria a few hours before death. This suggests that in vivo

multiplication of Pasteurella multocida in chickens with X-73 strain

occurs in a similar fashion as has been observed in turkeys

inoculated intravenously with pathogenic strains.

 21

Chapter III

The Early Pathogenesis of Pasteurella multocida

Infection Studied by Iamunohistochemical Techniques

 22

Summary

Initial sites of localization and multiplication of Pasteurella

multocida, X-73 strain, were examined in chickens inoculated

intravenously with varying doses of bacteria. Sections of lung,

liver, kidney, bone marrow, and spleen were prepared for light

microscopic examination. Tissue sections were stained with Gram's

stain as well as the avidin-biotin peroxidase complex (ABC)

technique. Immunohistochemical examination of tissues proved to be

far more sensitive in demonstrating P. multocida in low numbers

within tissues than the Gram's stain. As early as five hours post

inoculation with 1 x 103 colony-forming units (CFU) of the organism,

minute foci of inflammation were observed in the liver with

hyperemia observed in all organs. The organism was found within

isolated mononuclear phagocytes in the liver and spleen in low

numbers (approximately five to ten bacteria per cell). Bacterial

numbers increased tremendously with time within these cells in the

liver and spleen. The organism was not observed extracellularily in

the blood, lung, bone marrow, or kidney until later stages of

infection. Bacteria were not observed within the heterophils of

examined tissues. The results suggested possible intracellular

multiplication of these encapsulated bacteria within splenic and

hepatic mononuclear phagocytes.

 23

Introduction

Pasteurella multocida infections are of considerable importance

to the livestock and poultry industries. Infections have been noted

in man, cattle, chickens, turkeys, wild birds, and rabbits

(9,11,28,31,49). In poultry, the organism may produce an acute to

peracute fatal septicemia due to rapid multiplication of this Gram-

negative bacteria (11). Presumably, the cause of death is due to

endotoxic shock (11). The extraordinary rate at which the bacteria

multiply within infected hosts suggests potent virulence factors

exist in this bacteria, but the nature of such factors remains

largely unknown. Collins, using his mouse model, observed that the

host response to infection with P. multocida was primarily poly-

morphonuclear and that, in the absence of specific opsonins,

phagocytosis rates were very slow. His observations suggested that

the bacteria multiplied freely in an essentially extracellular

environment (11). Snipes et al. (50), when comparing a virulent

encapsulated P. multocida strain with its avirulent variant,

observed that virulence of the encapsulated organism was due to its

greater capacity to multiply extracellularity in the blood stream.

Their findings suggested that bactericidal and bacteriostatic

factors in the plasma such as complement or transferrin may have a

major role in determining survivability of infected fowl (50,51).

Investigations with turkeys by Paps-Garnon and Soltys (40), however,

indicated that the bacteria, following intravenous inoculation,

multiplied first in the liver and spleen and only later in the

blood. Experiments in turkeys conducted by Tsuji and Matsumoto

 24

suggested that P. multocida was readily engulfed by phagocytic cells

in the liver and spleen, regardless of the presence or absence of

hyaluronic acid capsule (53). In their experiments, differences

were observed in P. multocida survivability within the phagocytic

cells. The purpose of this experiment was to obtain information on

initial localization and multiplication sites of P. multocida within

infected tissues through the use of immunohistochemistry and clarify

questions concerning early pathogenesis of this infection.

 25

Materials and Methods

Experiment 1 Specific-pathogen-free eggs from Single Comb

White Leghorn chickens were obtained from a commercial source3 and

hatched. Sixteen, five-week-old chicks, raised in isolation

facilities within battery units, were used. Twelve birds were

inoculated intravenously in the brachial vein with 1.0 ml of P.

multocida in brain-heart infusion broth2 containing 1.0 x 103 CFU of

bacteria as determined by the standard plate dilution count

technique. Four birds served as uninoculated controls and were

housed separately. The inoculum was prepared by incubating

previously lyophilized X-73 strain° (type Al) of P. multocida

overnight on blood agar plates at 37°C. Ten colonies were randomly

selected and streaked for confluent growth on brain heart infusion

(BHI) plates2. Bacteria were then harvested from these plates after

five hours incubation at 37°C in BHI broth. Dilutions of the broth

suspension served as inoculum. Three inoculated and one control

chicken were euthanatized at the following intervals: 2.5, 5, 10,

and 20 hours postinoculation (PI). Sections of liver, spleen, lung,

kidney, and bone marrow were removed from each bird and fixed for 48

hours in 10% neutral buffered formalin. Isolation of P. multocida

from the livers of infected and control animals was attempted in

each bird according to standard microbiological methods.

Experiment 2 - Sixteen, seven-week-old SPF Single Comb White

Leghorn chickens, raised in isolation, were used. Twelve were

inoculated with varying doses of X-73 strain via the brachial vein.

Four birds served as uninoculated controls. All animals were

 26

euthanatized at five hours PI. Of the twelve, three were inoculated

with 1 x 103, three with 1 x 105, three with 1 x 107 and lastly,

three with 1 x 109 CFU organisms. Tissue collection and fixation,

as well as inoculum preparation, were done in the same manner as in

Experiment 1.

Preparation of Tissues for Histopathology: Tissues fixed for

approximately 48 hours in neutral buffered formalin were dehydrated

in a graded series of ethanol, cleared with xylene, and processed in

paraffin. Sections were cut 5 pm thick and attached to glass slides.

Tissues were stained with routine hematoxylin and eosin stains.

Duplicate slides were also stained with Gram's stain.

Preparation of Sections for Immunohistochemistry: A commercial

kit (Vectastain9)5 was utilized for the immunoperoxidase

histochemical procedures. Reagents in the kit included normal goat

serum, biotinylated goat anti-rabbit IgG, avidin, and biotinylated

horseradish peroxidase. All reagents were prepared according to

manufacturer's instructions. Primary polyclonal antibody was

obtained from an adult New Zealand White rabbit hyperimmunized with

formalin-inactivated whole cell suspensions of X-73 strain given

subcutaneously at 0-, 28-, 55-, and 75-days. The rabbit was

exsanguinated on day 105 and its serum frozen at -70 C until use.

Two milliliters of inoculum emulsified in Freund's complete

adjuvant6 were used on all except day 55 at which time 0.5 ml

organism suspension was given. Normal rabbit serum was used as a

negative control serum. Both sera were diluted 1:1,500 as it was

predetermined that this dilution provided sections with minimal

 27

nonspecific background staining and maximum contrast. All steps in

the procedure were done in a moisture chamber kept at room

temperature. Wash steps were done with fresh Tris-saline buffer, pH

7.6.

ABC staining procedure: Tissue sections were deparaffinized

and rehydrated through a xylene and a graded alcohol series. The

slides were then rinsed five minutes in distilled water, and a 0.6%

H202 in methanol solution was applied to block endogenous tissue

peroxides for 60 minutes. Sections were washed for twenty minutes

in Tris-saline, then incubated with diluted 1.3% normal goat serum

for 20 minutes. The slides were subsequently incubated for 60

minutes with the primary antibody. After a wash step in Tris-saline

for ten minutes, sections were incubated for 30 minutes with .045%

biotinylated goat anti-rabbit IgG. After a ten minute wash, the

slides were incubated with the ABC reagent for 60 minutes. After a

ten minute Tris saline wash, the sections were covered with the

substrate-chromogen solution. The substrate consisted of a solution

of 0.1% 3,3'-diaminobenzidine (DAB) mixed with equal parts of 0.02%

H202. This solution was prepared a few minutes before each use.

Tris buffer, 0.1M, pH 7.2 was used to dilute the DAB solution.

Staining times varied from 2 to 5 minutes, depending on the rapidity

of the brown color change. Slides were washed in tap water for five

minutes and counterstained with Mayer's hematoxylin for one minute.

The slides were immersed in saturated lithium carbonate solution for

30 seconds and rinsed for 30 seconds in tap water. The slides were

dehydrated with 95% alcohol, absolute alcohol, and cleared in

 28

xylene, two changes each. Mounting of the slides was done with

PermountR7.

The following controls were used to assess the specificity of

the ABC technique and the primary antibody: a) a control slide

containing normal chicken tissues stained with the primary antibody;

and b) a control slide containing heavy numbers of P. multocida

bacteria stained with negative rabbit sera.

 29

'Becton Dickinson Vacutainer Systems, Rutherford, NJ 07070

2Difco, Detroit, MI.

3Spafas, Inc. Rural Route No. 1 Roanoke, Il 61561.

4A kind gift of Dr. K.R. Rhoades, National Disease Center, USDA,

Ames, Iowa.

'Vector Laboratories Inc., 1429 Rollins Rd., Burlingame, CA 94010.

6Pharmacia Fine Chemical Co., Piscataway, NJ.

'Fisher Scientific Company, Chemical Manufacturing Division, Fair

Lawn, NJ.
 30

Results

Microbiology: Pasteurella multocida organisms were readily

reisolated from inoculated birds in both experiments from swabs

taken from livers. Liver cultures of control birds remained

negative for P. multocida.

Gross and Microscopic Lesions: The gross lesions were

essentially identical in birds from both experiments. The severity

of the changes varied, however, with the bacterial dose given and

the time of euthanasia. A generalized cyanosis of skin and

musculature was observed. A light green fecal and urate pasting was

seen on the vent and tail feathers. Internally, the livers were

enlarged, pale, and friable with multiple pinpoint yellow or red

foci. Spleens were sometimes enlarged, though not consistently, and

mottled red and white in color. Petechial hemorrhages were

occasionally seen on serosal surfaces of internal organs. Watery,

green intestinal contents with reddening of the intestinal mucosa

was a common finding.

In Experiment 1, no visible changes were noted microscopically

at 2.5 hours PI in examined tissues. As early as five hours PI, a

generalized hyperemia was seen in all tissues. An influx of

heterophils was observed also in the liver with heterophils along

endothelial margins of hepatic blood vessels (Figures 10,15).

Minute inflammatory foci were occasionally observed in the liver,

consisting of equal numbers of heterophils and mononuclear

phagocytes (Figure 6). Gram's stained sections of liver, spleen,

kidney, lung, and bone marrow failed to demonstrate bacteria.

 31

Examination of immunohistochemically-stained tissue sections showed

a very small number of macrophages containing P. multocida in the

liver and spleen (Figures 3,4,7,8). The organisms were usually

within cells associated with an inflammatory focus and numbered

about five to ten organisms per cell. (Figure 4). No bacteria were

seen in ABC-stained sections of lung, bone marrow, or kidney at five

hours PI. In birds inoculated with 1 x 103 CFU bacteria both 10 and

20 hours PI, mononuclear phagocytes in the liver and spleen appeared

to contain larger numbers of organisms (often too numerous to count)

(Figure 9). Necrosis of these cells was common, releasing bacteria

into the sinusoids (Figure 5). Fibrinoid necrosis was observed in

the spleen (Figure 14). Infiltration of these organs with

heterophils and mononuclear cells became more pronounced (Figure

15). Low numbers of bacteria were evident in the blood at this

time. In dead or moribund birds, bacterial dissemination was wide-

spread in all organs (Figures 11,15). They were readily observed

with H &E, Gram's and immunohistochemical stains both intra and

extracellularly. Few bacteria were seen in the kidney within blood

vessels (Figure 17). The lung and bone marrow showed large numbers

of bacteria in the blood. Necrosis of blood cell precursors, as

well as a depletion of heterophilic granulocytes was observed in the

bone marrow (Figures 12,13). The severity of the congestion

increased in all organs at death. Fibrin thrombi were observed

within glomerular blood vessels in the kidney (Figure 16). An

interstitial pneumonia and nephritis consisting of infiltrating

mononuclear cells and hetrophils were observed in tissues taken from

 32

dead or moribund birds (Figure 18,19). Occasional thrombi were

present within blood vessels of the lung and spleen.

In Experiment 2, the microscopic lesions in birds dosed with

1 x 103 and 1 x 105 CFU bacteria and killed at five hours PI were

essentially the same as Experiment 1, when similar bacterial doses

were given and the birds killed at five hours PI. Birds inoculated

with 1 x 107 and 1 x 105 CFU organisms and killed at five hours

demonstrated changes similar to birds killed at 10 and 20 hours PI

in Experiment 1. However, necrosis of cells was not as prominent in

this group microscopically, perhaps because not as much time had

elapsed between inoculation of bacteria and euthanasia.

 33

Discussion

Immunohistochemistry using the avidin-biotin-peroxidase

technique has shown to be a highly specific and sensitive test for

the demonstration of antigens in tissue, particularly for the

demonstration of microorganisms (12,23,25,54). In the present

experiment with this technique, it was possible to demonstrate P.

multocida within phagocytic cells during very early stages of

infection when no bacteria were observed with conventional H&E and

Gram-stained tissues. Cell types were recognizable in

immunohistochemistry-prepared sections; whereas, they were often

difficult or impossible to recognize in gram-stained tissues. Some

background staining did occur, however, especially in sections

containing large amounts of blood such as the bone marrow and lung

tissue sections. This was probably due to large amounts of

endogenous peroxides found in red blood cells. Control slides were

very helpful in interpretation of some sections where this was a

problem. Several techniques were employed to minimize background

staining and artifacts (12,23).

The histopathologic changes noted in birds that died from the

infection were similar to those previously described with some

notable differences (46). The early lesions suggest that these

organisms are readily phagocytized and may in fact have multiplied

within splenic and hepatic mononuclear phagocytes. Little, if any,

phagocytosis by heterophils was seen by the authors. It may be that

the heterophil is less effective at phagocytizing Pasteurella

multocida in chickens. It should be noted, however, that even under

 34

high magnification, cell borders between inflammatory cells were not

always distinct so that absence of phagocytosis by heterophils

cannot be stated with absolute certainty. The numbers of bacteria

within mononuclear cells appeared to increase with time, suggesting

intracellular multiplication, because it is unlikely that large

numbers of bacteria could gain entrance into a cell via phagocytosis

alone. This data supports that the reticuloendothelial cells of the

liver and spleen appear to be the main sites of initial localization

and multiplication of P. multocida infection in the chicken and does

not support that the organism freely multiplies in the blood as was

previously presumed. Multiplication of the bacteria in the blood

would result in bacteria being present in all organs equally. This

was definitly not the case in this experiment. Although phagocytic

cells are present in many organs, the organism appears to be picked

up primarily in the liver and spleen. This type of localization and

multiplication pattern is not uncommon with facultatively

intracellular bacteria (10,15).

An interesting microscopic finding in this experiment was the

significant mononuclear component of the inflammatory response seen

in the lung, kidney, and spleen in birds which had succumbed to the

disease or were necropsied late in the infection. Previous reports

on the microscopic lesions of acute fowl cholera describe a mostly

heterophilic response. A mixed inflammatory cell response may be

characteristic of some avian bacterial infections including fowl

cholera. The information obtained from this experiment suggests

that the mononuclear phagocyte may play a key role in natural

 35

immunity against this infection. Baba (2,3) demonstrated that cell-

mediated immune protection exists in chickens against P. multocida.

The possibility exists that chronic carriers of fowl cholera

may harbor organisms intracellularly. This might protect the

bacteria from harmful serum factors such as antibodies or

complement. To date, most research has focused on increasing

humoral immunity in birds. However, the most effective vaccines

that provide some cross protection are the live-organism vaccines

and killed vaccines derived from organisms grown in vivo. Perhaps,

these vaccines stimulate cell-mediated as well as humoral immunity.

According to Baba (2), the activation of macrophages by previously

primed T-cells may be an important resistance mechanism which

increases the effectiveness of intracellular killing by these cells.

More research is needed to explore the mechanisms by which

virulent P. multocida organisms avoid intracellular killing in

susceptible host.
 36

E>
Antigen 1° antibody

2°antibody peroxidase

H202 DAB
Colored end product

Figure 1. The indirect immunoperoxidase method.

 37

1° antibody Biotinylated
2°antibody

+ Avidin
H202 DAB
Peroxidase

Figure 2. Avidin: biotinylated horseradish peroxidase

complex technique (ABC technique).
 38

Figure 3. Chicken portal area and vein (V) in the liver from a bird
injected with 1 x 105 CFU bacteria and killed five hours PI. Note a
macrophage laden with bacterial antigen (arrow).. ABC stain 640X.
Figure 4. Liver from a chicken injected with 1 x 105 CFU bacteria
and killed five hours PI. Note P. multocida within vacuole (arrow)
of mononuclear phagocyte. Bacteria-laden cell is within hepatic
sinusoid (S). ABC stain 800X.
 40

Figure 5. Liver of chicken showing degenerating mononuclear

phagocyte within sinusoid (arrow). ABC stain approximately 640X.
 41

Figure 6. Inflammatory foci in liver of a chicken injected with

1 x 105 CFU organisms and killed five hours PI. Note a macrophage
containing P. multocida (arrow). Surrounding heterophils do not
contain organisms (H). ABC stain 640X.
 42

Figure 7. Splenic mononuclear phagocyte (arrow) containing

bacteria. The bird was inoculated with 1 x 103 bacteria and
necropsied five hours PI. ABC stain 1600X.
 43

49 it'l,i.V4t ic,4167
ektk w .04,
j 14 v tr,T., -iTIR a.kl'i-T

4i
iftir
4 . 41
.1.: , oft k, oll
it' ;'Obis,u,.. 42.
re,_,., 4-4 cjil'

,,.,..."::
J.01.7., 17,11.1:45:::.S1.1:44t%ct,14)::4,....,- ifrilei,
zic *41007 is ; s -, ,..... r $:
'44i.
,...i.,.4ilk. klikivie Ner:w ' ,
7....
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.... ,i, -
ION ..

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- ----;/"'-'
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;elv t:',.-- * 4
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,

4.,r$ ) `a Psi 'we .-4-:

,
\
444- 't4

::(ti: -",.$0 'tf 0, ,,',`=.)%- 11`1A 3 ',t4kt ei 4 i 440 k' ir ...::

4
:47t04,'
14 fr,,47-- atil vii
,-),,i.-
mss
*ii141e. m. t.t-r):4-t -.t el i--7,-.
,..

V4,4 ,..41':'' i411)4! fetj444

..:....zitt. 1:.:LIA\ Li_ It ,: a% aikv ''. At 10' fee kr i&c.S.
eb:11
Sili-k
-et
'4.t

Figure 8. Splenic macrophage containing Pasteurella multocida

bacteria (arrow). Chicken injected with 1 x 103 CFU organisms and
killed five hours PI. ABC stain 800X.
 44

Figure 9. Liver from chicken inoculated with 1 x 103 CFU bacteria

and killed 10 hours PI. Note bacteria within sinusoid (arrow) both
extracellular and within necrotic phagocyte. ABC stain 1600X.
 45

Figure 10. Hepatic vein (V) from a chicken inoculated with

1 x 105 CFU bacteria and killed five hours PI. Bacteria absent from
blood or heterophils (H). ABC stain 1280X.
 46

--.
''''-',...."'""` "r"...ii7. F P "",,."' r .;fe Sot.; ilr, orLar-gsrogra;,.._-44^,:= vr:. , -a
*,, ` iielb - , .44 i t" , I'l oie -- r ,1 ' k ,4- , .1' 4 4. 1 ; )=- 0 ,-''. ..-- I t '
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i
. *.- S -Ntitte ikii e
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I

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ar
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-.4 7,, 4 1/P "
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iie ,
tvt- A. s.0.
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4., e
.4 il ,
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' *i. 1 , A.;., ao 43.
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.".r tit *' 't.i :. if( 411th 14 le lik e
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4
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. ,
r ,:.-46.1,..4 -10`
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.,
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4.,,, i I
1- t'... ;50 7-4. 0! 40 N'.;,
I*
a tow or 0';".4
: IteL ii,. ,,,,1,...: ..4 f ,,
k ,- ,,'. -
,;',fiE. Wait :::2.6Ciict ....:.:Lkiri:.-- A.,544.,,,:-::::;d-i.......;

Figure 11. Inflammatory focus in a chicken liver (AB) containing

large numbers of organisms. The bird had been injected with
1 x 103 CFU bacteria and killed 20 hours PI. ABC stain 512X.
 47

Figure 12. Bone marrow from a chicken inoculated with 1 x 103 CFU
bacteria and dead at 20 hours PI. Note heterophilic granulocyte
depletion and necrosis of blood cell precursors. H&E stain 400X.
 48

Figure 13. Normal chicken bone marrow with abundant heterophilic

granulocytes. H&E stain 640X.
 49

Figure 14. Chicken spleen demonstrating fibrinoid necrosis (N).

The bird was inoculated with 1 x 103 CFU bacteria and killed ten
hours PI. H&E stain 400X.
 50

Figure 15. Chicken hepatic vein (V) demonstrating accumulation of

heterophils along endothelial margins (H). H&E stain 400X.
 51

Figure 16. Chicken kidney demonstrating thrombi (TH) within

glomerular tufts in uppermost glomerulus (G). H&E stain 400X.
Figure 17. Chicken kidney in bird injected with 1 x 109 CFU
bacteria and killed five hours PI. Note large numbers of bacteria
(B) within blood vessel (V). Gram's stain 640X.
 53

.-,retopirr T1,7047/.7..1
.-ter er- 4,f, "pg,
. 1P7 ; e. ;lir "4, 416.4 40:;.;414115:1 P.1 'et,--11!;74
' "Phs. ner-r, -....
4 ate ("3 . 1.4.4
4 ctk i .:4 1 Iry to44:.
4.
t
.. 3. **-- ' '
.... 4 4.1..... -vit.,- ,.._ 11.--e
...1 40,
im:,7?-ve
I .. _
0..." kg-0-s 4 7:414 e; VIA, /:
Ipto,o, 0.14:-. 0 ',loc. :
, ...O.' .4 0 .
'4.4.$" le *!Ir :
A,

te 1.

*:,;:tii, * a Vii: f :4 c-4"e V? .":

4 >4,s4 .4^41..
aAs.P. -.... 0
'v. ,..
II ZAN) 6,.. 1r%
tlell *"
%. 'V
. 4, :for *t.
itlfr44 i
4
?, ...Atte '
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, *24
., t:di
;
..

t"---4 ,
4 ,......4 VI/di!
, 0a
il f 'Iftlekl.
84-........ 7 'JO '
bi,

Figure 18. Chicken lung demonstrating interstitial thickening and

hyperemia. The bird was inoculated with 1 x 103 CFU and necropsied
20 hours PI. H&E stain 320X.
 54

Figure 19. Normal chicken lung. H&E stain 320X.

 55

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