• TABLE OF CONTENTS
HIDE
 Title Page
 Acknowledgement
 Table of Contents
 List of Tables
 List of Figures
 Abstract
 Introduction
 Review of literature
 Cultural, morphological and physiological...
 Extracellular antigens of Moraxella...
 Lipopolysaccharide of Moraxella...
 Hemolysin of Moraxella bovis
 Piliation in Moraxella bovis
 General discussion
 Summary and conclusions
 References
 Biographical sketch














Title: Antigenic analysis of Moraxella bovis /
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Permanent Link: http://ufdc.ufl.edu/UF00097639/00001
 Material Information
Title: Antigenic analysis of Moraxella bovis /
Physical Description: 147 leaves : ill. ; 28 cm.
Language: English
Creator: Sandhu, Tirath Singh, 1937-
Publication Date: 1972
Copyright Date: 1972
 Subjects
Subject: Moraxella bovis   ( lcsh )
Cattle -- Diseases   ( lcsh )
Animal Science thesis Ph. D
Dissertations, Academic -- Animal Science -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Thesis: Thesis (Ph. D.)--University of Florida, 1972.
Bibliography: Bibliography: leaves 135-146.
Additional Physical Form: Also available on World Wide Web
Statement of Responsibility: by Tirath Singh Sandhu.
General Note: Typescript.
General Note: Vita.
 Record Information
Bibliographic ID: UF00097639
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000405708
oclc - 24663893
notis - ACF1949

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Table of Contents
    Title Page
        Page i
    Acknowledgement
        Page ii
    Table of Contents
        Page iii
        Page iv
    List of Tables
        Page v
    List of Figures
        Page vi
        Page vii
        Page viii
    Abstract
        Page ix
        Page x
        Page xi
    Introduction
        Page 1
        Page 2
    Review of literature
        Page 3
        Page 4
        Page 5
        Page 6
        Page 7
        Page 8
        Page 9
        Page 10
        Page 11
        Page 12
        Page 13
    Cultural, morphological and physiological characteristics of Moraxella bovis isolants and related organisms
        Page 14
        Page 15
        Page 16
        Page 17
        Page 18
        Page 19
        Page 20
        Page 21
        Page 22
        Page 23
        Page 24
    Extracellular antigens of Moraxella bovis
        Page 25
        Page 26
        Page 27
        Page 28
        Page 29
        Page 30
        Page 31
        Page 32
        Page 33
        Page 34
        Page 35
        Page 36
        Page 37
        Page 38
        Page 39
        Page 40
        Page 41
        Page 42
        Page 43
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        Page 45
        Page 46
        Page 47
        Page 48
        Page 49
        Page 50
        Page 51
        Page 52
        Page 53
        Page 54
        Page 55
        Page 56
        Page 57
    Lipopolysaccharide of Moraxella bovis
        Page 58
        Page 59
        Page 60
        Page 61
        Page 62
        Page 63
        Page 64
        Page 65
        Page 66
        Page 67
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        Page 72
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        Page 76
        Page 77
        Page 78
        Page 79
        Page 80
        Page 81
        Page 82
        Page 83
        Page 84
    Hemolysin of Moraxella bovis
        Page 85
        Page 86
        Page 87
        Page 88
        Page 89
        Page 90
        Page 91
        Page 92
        Page 93
        Page 94
        Page 95
        Page 96
        Page 97
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        Page 99
        Page 100
        Page 101
        Page 102
        Page 103
        Page 104
        Page 105
        Page 106
        Page 107
        Page 108
    Piliation in Moraxella bovis
        Page 109
        Page 110
        Page 111
        Page 112
        Page 113
        Page 114
        Page 115
        Page 116
        Page 117
        Page 118
        Page 119
        Page 120
        Page 121
        Page 122
        Page 123
        Page 124
        Page 125
        Page 126
    General discussion
        Page 127
        Page 128
        Page 129
        Page 130
        Page 131
    Summary and conclusions
        Page 132
        Page 133
        Page 134
    References
        Page 135
        Page 136
        Page 137
        Page 138
        Page 139
        Page 140
        Page 141
        Page 142
        Page 143
        Page 144
        Page 145
        Page 146
    Biographical sketch
        Page 147
        Page 148
        Page 149
Full Text





ANTIGENIC ANALYSIS OF MORAXELLA BOVIS


By





TIRATH SINGH SANDHU











A DISSERTATION PRESENTED TO THE GRADUATE
COUNCIL OF THE UNIVERSITY OF FLORIDA IN PARTIAL
FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA
1972













ACKNOWLEDGMENTS


The author expresses his sincere appreciation to the

Chairman of his Supervisory Committee, Dr. Franklin H.

White, for his encouragement, guidance and constructive

criticism during the course of this study and in the

preparation of this manuscript.

The author also acknowledges the interest and valuable

contributions of the other members of his Supervisory

Committee, Drs. Charles F. Simpson, Edward M. Hoffman,

George E. Gifford, George T. Edds and Alvin C. Warnick.

Sincere appreciation is expressed to Mr. Tom Carlisle

for the photomicrographs. The author further acknowledges

the help, cooperation and suggestions of other members of

the faculty, technical staff and his fellow graduate

students of the Department of Veterinary Science.













TABLE OF CONTENTS


ACKNOWLEDGMENTS . . . . . . . .

LIST OF TABLES . . . . . . . . .

LIST OF FIGURES. . . . . . . . . .

ABSTRACT . . . . . . . . .. . .

INTRODUCTION . . . . . . . . . .

REVIEW OF LITERATURE . . . . . . . .

SECTION 1. CULTURAL, MORPHOLOGICAL AND PHYSIOLOGICAL

CHARACTERISTICS OF MORAXELLA BOVIS

ISOLANTS AND RELATED ORGANISMS

Introduction and Review of Literature . . .
Materials and Methods . . . . . . .
Results . . . . . . . . . .
Discussion . . . . . . . . .
Summary . . . . . . . . . .

SECTION 2. EXTRACELLULAR ANTIGENS OF MORAXELLA

BOVIS

Introduction and Review of Literature . . .
Materials and Methods . . . . . . .
Results . . . . . . . . . .
Discussion . . . . . . . . .
Summary . . . . . . . . . .

SECTION 3. LIPOPOLYSACCHARIDE OF MORAXELLA BOVIS

Introduction and Review of Literature . . .
Materials and Methods . . . . . . .
Results . . . . . . . . . .
Discussion . . . . . . . . .
Summary . . . . . . . . . .


ii

v

vi

ix

1

3








14
15
18
22
24






25
27
35
55
57



58
65
72
80
84











Page


SECTION 4. HEMOLYSIN OF MORAXELLA BOVIS

Introduction and Review of Literature
Materials and Methods . . . .
Results . . . . . . . .
Discussion . . . . . . .
Summary . . . . . . . .

SECTION 5. PILIATION IN MORAXELLA BOVIS

Introduction and Review of Literature
Materials and Methods . . . .
Results . . . . . . . .
Discussion . . . . . . .
Summary . . . . . . . .

GENERAL DISCUSSION . . . . . .

SUMMARY AND CONCLUSIONS . . . . .

REFERENCES . . . . . . . .

BIOGRAPHICAL SKETCH . . . . . .


. . 85
. . 88
. . 95
. . 105
. . 108




. . 109
. . 113
. . 116
. . 123
. . 126

. . 127

. . 132

. . 135

. . 147












LIST OF TABLES


Table Page

1. Cultural and physiological characteristics
of MoraxeZla bovis isolants and other
related organisms . . . . . ... .21

2. Passive hemagglutination of Moraxella bovis
antisera (rough and smooth) with Moraxella
bovis lipopolysaccharides . . . ... .74

3. Hemagglutination-inhibition of Moraxella
bovis antisera with homologous and
heterologous lipopolysaccharides. . . .. 75

4. Optical density produced by lysis of sheep
erythrocytes in 4 ml of distilled water
for the standard curve. . . . . ... .90

5. Hemolytic activity of Moraxella bovis
hemolysin on erythrocytes of various
species of animals. . . . . . .. 99

6. Effects of calcium and magnesium ions on the
activity of Moraxella bovis hemolysin . .. 101

7. Effects of iodoacetic acid and cysteine on
the hemolytic activity of Moraxella bovis
hemolysin . . . . . . . . . 104












LIST OF FIGURES


Figure Page

1. Immunization scheme for the production of
antisera in rabbits against rough and
smooth type growth of various isolants
of Moraxella bovis on solid or in broth
culture media . . . . . . ... 28

2. Immunodiffusion analysis of Moraxella
bovis (IBH 68 712L) extracellular
antigens . . . . . . . . . 36

3. Immunodiffusion analysis of MoraxelZa
bovis (FLA-264 Ivan Park) extra-
cellular antigens . . . . . . .. 37

4. Immunodiffusion analysis of Moraxella
bovis (FLA-264 Ivan Park Rough type)
extracellular antigens against homologous
and heterologous antisera . . . ... 38

5. Immunodiffusion analysis of Moraxetla
bovis (FLA-264 Ivan Park Smooth type)
extracellular antigens against homologous
and heterologous antisera . . . ... 39

6. Immunoelectrophoretic pattern of extra-
cellular R antigens of FLA-264 (Ivan Park). 41

7. Fractionation of crude ammonium sulfate-
precipitated filtrate of Moraxella bovis
(FLA-264 Ivan Park Rough type) super-
natant fluid on Sephadex G-200. . . .. 42

8. Fractionation of crude ammonium sulfate-
precipitated filtrate of Moraxella bovis
(FLA-264 Ivan Park Smooth type) super-
natant fluid on Sephadex G-200. . . .. 43

9. Relationship of different R type antigens
of isolant FLA-264 (Ivan Park). . . .. 44

10. Immunoelectrophoretic pattern of extra-
cellular antigens (R-2 and R-3) of isolant
FLA-264 (Ivan Park) . . . . . ... 45





Figure Page

11. Immunoelectrophoretic pattern of extra-
cellular R antigens (R-1 and R-3) of
isolant FLA-264 (Ivan Park). . . . . 46

12. Immunodiffusion analysis of MoraxeZZa
bovis (IBH 68 712L Rough type) extra-
cellular antigens with homologous and
heterologous antisera. . . . ... 47

13. Immunodiffusion analysis of Moraxella
bovis (IBH 68 712L Smooth type)
extracellular antigens with homologous
and heterologous antisera. . . . ... 48

14. Fractionation of crude ammonium sulfate-
precipitated filtrate of Moraxella bovis
(IBH 68 712L Rough type) supernatant
fluid on Sephadex G-200. . . . ... 49

15. Fractionation of crude ammonium sulfate-
precipitated filtrate of MoraxeZla bovis
(IBH 68 712L Smooth type) supernatant
fluid on Sephadex G-200. . . . . ... 50

16. Immunoelectrophoretic pattern of R antigens
of isolant IBH 68 (712L) and their
relationship with R-antiserum to isolant
FLA-Vet.l (R). . . . . . . . 52

17. Effects of heat, formalin, and trypsin
treatments on the extracellular R-2
antigen of isolant FLA-264 (Ivan Park) . 53

18. Effects of heat, formalin and trypsin
treatment on the extracellular R-3
antigen of isolant FLA-264 (Ivan Park) . 54

19. UV absorption spectrum of Moraxella
bovis lipopolysaccharide (FLA-264, rough). 73

20. Immunodiffusion analysis of MoraxelZa
bovis lipopolysaccharide . . . ... 76

21. Local Shwartzman reaction produced by
MoraxeZZa bovis (Isolant FLA-264-R)
lipopolysaccharide (endotoxin) in a
rabbit . . . . . . . . . 79

22. Standard curve for determining units of
hemolytic activity of MoraxeZZa bovis
hemolysin. . . . . . . . ... 91









23. Hemolysin produced by Moraxella bovis
(Rough type of isolant FLA-264, Ivan
Park) in trypticase soy broth . . ... 96

24. Hemolysin production by Moraxella bovis
(Smooth type of isolant FLA-264, Ivan
Park) in trypticase soy broth . . ... 97

25. Hemolysin production by Moraxella bovis
(isolant IBH 68 712L) in trypticase
soy broth. . . . . . . . .. 98

26. Effect of trypsin (0.25%) on Moraxella
bovis hemolysin . . . . . . ... 102

27. Moraxella bovis isolant FLA-264 (Ivan
Park). Rough type cells with numerous
pili. . . . . . . . .... .117

28. Moraxella bovis isolant FLA-264 (Ivan
Park). Smooth type cells . . . ... 118

29. Purified pili from rough type cells of
Moraxella bovis (FLA-264, Ivan Park). ... .119

30. Immunodiffusion pattern of pill antigen
with homologous and heterologous type
antisera. . . . . . . . . ... 121

31. Immunodiffusion pattern of pili antigen
with homologous and heterologous rough
type antisera . . . . . . ... 122


viii


Figure


Page






Abstract of Dissertation Presented to the Graduate Council of
the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

ANTIGENIC ANALYSIS OF MORAXELLA BOVIS

By

Tirath Singh Sandhu

December, 1972

Chairman: Dr. Franklin H. White
Major Department: Animal Science

This study was undertaken to analyze lipopolysaccharide,

extracellular, and pili antigens of MoraxeZZa bovis, the

etiological agent of infectious bovine keratoconjunctivitis.

Along with antigenic studies, endotoxin and hemolysin

elaborated by this bacterium were also characterized.

Five isolants of M. bovis were used in this investi-

gation. These isolants had similar cultural, morphologic,

and physiologic characteristics except for the catalase

reaction. One isolant (FLA-264, Ivan Park) was catalase

positive while all other isolants were catalase negative.

On primary isolation most colonies were rough, dry, convex

or umbonate, adhesive, and autoagglutinable. These

colonies were called rough type (R), while the dissociated

smooth, nonadhesive and uniformly suspendable colonies

were called smooth type (S).

The extracellular antigens were isolated by ammonium

sulfate precipitation of cell-free culture filtrate,

purified on Sephadex G-200, and differentiated by imnuno-

diffusion and immunoelectrophoretic techniques. The R






type growth had two specific extracellular antigens in

addition to those present in culture filtrate of S type

cells of the same isolant. The extracellular antigens

produced by S type cells were identical in all the isolants.

The serotype specific extracellular antigens of two

isolants were identical with each other, but different

from those of a third isolant. The possibility of sero-

typing M. bovis isolants on the basis of their extra-

cellular R type antigens was suggested.

Lipopolysaccharides were isolated by phenol-water

method and were characterized by serologic and biologic

reactions. All the isolants, both R and S types, had an

identical O-antigenic determinant as indicated by immuno-

diffusion, passive hemagglutination and hemagglutination-

inhibition assays. Biologically, M. bovis lipopoly-

saccharides produced irreversible lethal shock, and

generalized and local Shwartzman reactions. The local

Shwartzman reaction was inhibited by neutralization of

endotoxin with homologous antiserum.

Moraxella bovis hemolysin was produced in an artificial

medium. The maximum hemolytic activity of the culture was

observed during the logarithmic phase of the growth. The

hemolysin was filtrable though .22 v Millipore filters, heat-

labile, and destroyed by treatment with formalin and

trypsin. There was no difference in the amount of hemo-

lysin production by R or S colony type of an isolant,

although differences were observed between two different

x







isolants. Partial requirements of sulfhydryl group and

divalent cations were indicative of the enzymatic nature

of M. bovis hemolysin.

A significant finding was the presence of pili on

R type cells, while the S type cells of the same isolant

did not have these structures. The colonial dissociation

was associated with loss of pili on repeated transfers

on artificial medium. The pili antigen was distinct from

extracellular antigens. The pili of isolant FLA-264

(Ivan Park) were serologically identical with the pili

of isolant IBH 68 (712L), but not with that of isolant

FLA-Vet.1.













INTRODUCTION

Infectious bovine keratoconjunctivitis, commonly

known as "pink eye", is an important ocular disease of

cattle associated with excessive lacrimation, con-

junctivitis and keratitis. This disease is prevalent

throughout the world and is more predominant in the

warmer months of the year. Although it is not a fatal

disease, it causes an economic loss to the cattle industry

due to conjunctival irritation and temporary blindness.

It has been found in cattle of all ages, but the most

susceptible are those under two years of age. The

affected animals lose weight quickly with resulting

economic losses.

Since 1889 when this disease was first recorded, it

has been a subject of sporadic studies, mostly concerning

its etiology, treatment and control. Although numerous

organisms have been implicated with this disease,

Moraxella bovis is considered the primary etiological

agent. In recent years most of the reported work has

been on isolation and characterization of Moraxella bovis,

and very little work has been done on its virulence,

antigenic aspects and toxins elaborated by this organism.

Experimental bacterins have been prepared and tested by

various investigators but with little success.

1




2

The antigenic analysis of Moraxella bovis and charac-

terization of toxins produced by this organism were thought

to be a step toward a better understanding of the patho-

genicity and antigenicity of this organism.

The purposes of this research were (1) to study antigens

of Moraxella bovis and their relationship in different

isolants, (2) to isolate and characterize endotoxin and

hemolysin produced by this bacterium and (3) to study

colony dissociation which occurs on artificial medium

after primary isolation.

For the sake of clear understanding, the main portion

of this manuscript has been divided into sections, with

an initial general introduction and review of literature

and a general discussion and summary at the end.













REVIEW OF LITERATURE

Bovine keratoconjunctivitis was first reported by

Billings in 1889, who detected the presence of thin, short

bacilli with rounded ends in sections of affected cornea, but

was unable to produce the disease in normal cattle and

rabbits by transfer of organisms from eyes of affected

animals. Mitter in 1915 isolated Micrococcus lanceolatus

(pneumococcus of Fraenkel) and a gram-negative, short,

thick, pleomorphic diplobacillus from an outbreak of keratitis

in cattle in Bengal, India. He identified the latter

organism as the bacillus of Morax-Axenfeld (Morax, 1896;

Axenfeld, 1897). Although he was unable to reproduce the

disease in normal cattle, he theorized that contact of

these organisms with an abraded surface was probably

necessary to reproduce the disease. In Scotland, Poels

(1911) isolated Bacillus pyogenes from eyes of infected

cattle as reported by Kappeyney and Ward (1917). He was

able to reproduce the disease by injection of the bacterial

culture into the cornea of normal animals. Two other

organisms, a gram-negative and a gram-positive cocci, were

also isolated along with Bacillus pyogenes, and these were

considered to be secondary invaders. A short, thick







gram-negative diplobacillus was isolated by Allen (1919)

in Canada from cultures of affected eye swabs on Loeffler's

blood serum medium. He associated this bacillus with that

of Morax-Axenfeld (isolated from infectious conjunctivitis

in man). Allen (1919) also referred to reports suggesting

worldwide distribution of bovine keratoconjunctivitis and

expected that it might be caused by the same organism in

other countries.

Jones and Little (1923) were able to isolate a gram-

negative, proteolytic and hemolytic diplobacillus from 24

cases of acute ophthalmia in cattle. They produced

characteristic inflammation by inoculation of pure broth

cultures beneath the eyelids in cattle, but laboratory

animals were refractory to freshly isolated culture or

exudate from the eyes of infected cases. During the same

investigation, Jones and Little (1923) studied cultural

and biochemical characteristics of this organism and re-

ported a characteristic three zone alkaline reaction in

litmus milk. These organisms produced hemolysis on horse

blood agar plates and liquified gelatin in ten days when

incubated at 22 C. The carbohydrates were not fermented.

These organisms were 1.5 2v in length and about 0.5p in

width, capsulated, nonmotile and nonsporing.

In attempts to infect flies, Jones and Little (1924)

reported that these organisms were rapidly destroyed in

the gut of flies, and did not live for three hours on

the external surface of flies, indicating that the disease

was probably spread through mechanical transmission.







This organism was later classified by Hauduroy,

Ehringer, Urbain, Guillot and MaGrou (1937) as Haemophilus

bovis and was placed in genus Moraxella in 1939 by Lwoff.

While comparing normal ocular bacterial flora with

that found in bovine keratoconjunctivitis, Baldwin (1945)

isolated MoraxelZa bovis (Haemophilus bovis of Hauduroy

et at., 1937) in 84% of 112 infected eyes. It was not

isolated from any of the normal eyes. Healthy cattle

were susceptible to ocular instillation of Moraxella bovis

(M. bovis) cultures, while sheep, rabbits, mice and guinea

pigs were resistant. Abrasion of the cornea or con-

junctiva was not thought to be necessary to produce an

infection. In summer, experimentally produced kerato-

conjunctivitis was more severe and had a shorter incuba-

tion period than that produced in winter. M. bovis was

persistent in the eyes of recovered animals, which were

thought to be carriers. Reid and Anigstein (1945) had no

difficulty in transmitting the disease by direct transfer

of ocular or nasal exudate from infected to normal cattle.

In addition to gram-negative diplobacilli with rounded

ends, conjunctival scrapings also revealed other organisms,

namely E. coli, Bacillus pyogenes, B. subtilis, Pasteurella

sp. and StaphyZococcus sp., but these organisms were in-

capable of producing keratoconjunctivitis in normal cattle.

From the experimental evidence they summarized that kerato-

conjunctivitis in cattle was caused specifically by M. bovis.







Farley, Kliewer, Pearson and Foote (1950) failed to

produce experimental infection in cattle with M. bovis iso-

lates obtained from Baldwin (1945), although they were

able to infect normal cattle by direct transfer of lachrymal

exudate. Organisms identical to M. bovis were isolated in

Scotland by Watt (1951) from four infected cases.

Earner (1952) isolated M. bovis from 92 of 95 acutely

affected cases, while 36 normal animals were not carrying

M. bovis in their eyes. Earner (19521 reported that experi-

mental infection was produced in four calves using a pure

culture of this organism. He suggested the possibility of

development of an immunizing agent since animals which re-

covered from an attack of bovine keratoconjunctivitis were

not susceptible to reinfection with M. bovis one year

after experimental infection. Sheep, rabbits and guinea

pigs were not susceptible to inoculation of heavy suspensions

of this organism. Dissociation of M. bovis to form rough,

intermediate and "dwarf" colonies on prolonged subculture

was observed by Earner (1952). He found that "pink eye"

caused by M. bovis in cattle in the state of Kansas was

infectious in nature and named it infectious bovine kera-

toconjunctivitis.

Jackson (1953) reported results similar to those of

Barner (1952). In addition to calves, he reported experi-

mental infection of four sheep and one goat with smooth

colonies of M. bovis. The dissociation of smooth virulent

form to rough avirulent phase was also observed by Jackson

(1953). He reported that serum agglutination titers







increased significantly 14 20 days after experimental

ocular instillation. In contrast to Jackson (1953),

Gallagher (1954) in Australia, could not infect sheep,

rabbits and guinea pigs, although six mice developed

lesions similar to keratoconjunctivitis after eight days,

and M. bovis was recovered from their eyes.

Moraxella bovis was isolated from eyes of infected

cattle in Scotland (Faull and Hawksley, 1954), England

(Formston, 1954) and in Argentina (Hoffman, 1956). In

Parbhani district of India, Ahmed and Rao (1956) reported

an outbreak of infectious bovine keratoconjunctivitis

(IBKC) among cattle and buffaloes, and M. bovis was re-

covered from the affected eyes. Morphological cultural

and biochemical characteristics of M. bovis were studied

by Seth and Chandrasekariah (1957) and were similar to

those reported by the previous authors, except that the

catalase reaction was negative, in contrast to the

catalase positive finding of Formston (1954). Virulent

cultures, injected intraocularly in guinea pigs and

rabbits, produced severe conjunctivitis and keratitis,

but when injected intravenously such cultures killed mice

in three to five days.

Henson and Grumbles (1960a) isolated M. bovis from

the lachyrmal fluid and nasal cavity, but not from aqueous

humor in acute cases of infectious bovine kerato-

conjunctivitis. The disease could be experimentally pro-

duced in calves and M. bovis was considered to be the

etiological agent. In the same year, Henson and Grumbles






(1960b) studied the susceptibility of laboratory animals

to M. bovis. Dermonecrosis or ophthalmitis was produced

in the rabbit when a viable culture was injected intra-

dermally or intraocularly, respectively. Chicken embryos

were highly susceptible and were killed by intravenous,

intraperitoneal and intracerebral inoculation of the

cultures. Henson and Grumbles (1961) reported that M.

bovis produced two toxins; a labile hemolytic toxin which

was associated with viable bacterial cells, and a dermo-

necrotic toxin which was demonstrated in the cell wall

of M. bovis and resembled those produced by other gram-

negative bacteria. Cooper (1960) reported colonial

variation in certain strains of M. bovis and concluded

that there might be strain differences due to dissociation,

in addition to possible virulence variation.

Although M. bovis was considered to be the etiological

agent of IBKC according to various reports of isolation

from typical lesions and production of the disease in

normal animals with pure cultures of this organism,

there had been reports in which bacteria other than M.

bovis, viruses, rickettsia and mycoplasma were associated

with this disease. Wilcox (1968) extensively reviewed

the literature concerning the involvement of bacteria

(including M. bovis), viruses and rickettsia in this

disease. Sykes, Dmochowski, Grey and Russell (1962)

isolated a herpesvirus from the eyes of cattle affected

with acute keratoconjunctivitis. Immunologically, this





9

virus was closely related to, or identical with infectious

bovine rhinotracheitis (IBR) virus. This herpesvirus

produced lesions similar to those seen in IBKC on con-

junctival inoculation in normal cattle (Sykes, Scanlon,

Russell and Dmochowski, 1964; and Bowen, Jardine, Scanlon,

Dmochowski and Russell, 1970). Bowen et al., (1970)

vaccinated animals with a commercial IBR vaccine and a

vaccine prepared of herpesvirus isolated by Sykes et aZ.,

1962. Both of these vaccines produced a high degree of

immunity against a challenge dose of herpesvirus. Wilcox

(1969) isolated a number of adenoviruses from infected

eyes of cattle. Experimentally produced infection with

six adenovirus isolates was mild and resulted in con-

junctivitis in 11 and a simultaneous keratitis in only

two out of 22 infected animals (Wilcox, 1970b). Pugh,

Hughes and Packer (1970) exposed cattle to IBR virus and

M. bovis infection. Cattle developed severe conjunctivitis

when IBR virus alone was instilled in the conjunctival

sac. Keratitis was not produced unless the cattle were

exposed to M. bovis after infection with IBR virus. They

concluded that IBR virus was not the primary etiological

agent of IBKC, but might enhance the pathogenic effects

of M. bovis by creating a more suitable environment for

these organisms.

Langford and Dorward (1969) isolated mycoplasma from

the eyes of calves in two outbreaks of IBKC along with

M. bovis.






Moraxella bovis was characterized by Pugh, Hughes

and McDonald (1966) in regard to morphological, cultural

and biochemical characteristics. The results were similar

to the findings of previous workers. MoraxelZa bovis

isolants were differentiated from related organisms,

namely M. liquefaciens, M. nonliquefaciens, Mima polymorpha

and Mima polymorpha var. oxidans.

Hughes, Pugh and McDonald (1968a) reported the enhancing

effect of ultraviolet radiation by sunlamp on M. bovis in-

fection in bovine eyes. The disease produced by UV irradia-

tion and M. bovis infection was more severe than that pro-

duced by M. bovis alone and was indistinguishable from

field cases of IBKC. Hughes, Pugh and McDonald (1968b)

exposed cattle to different strains of M. bovis and re-

exposed to homologous and heterologous strains. After first

exposure, 100% of the animals became infected and 66%

developed keratitis, while after a second and third ex-

posure 55% became infected, and only 3.4% (two cases)

developed keratitis. These two cases had been re-exposed

to heterologous strains of M. bovis. The reduction in

rate of infection was considered to be due to development

of immunity.

The virulence of different strains of M. bovis was

studied by Pugh and Hughes (1970a). It was reported that

rough forms of M. bovis became established in murine eyes

and produced disease similar to that produced by smooth

forms. Moreover, some of the eyes infected with non-

hemolytic strains developed keratoconjunctivitis. As







reported earlier (Pugh and Hughes, 1968) they concluded

that the disease might have been caused by hemolytic

organisms which dissociated from nonhemolytic ones. In

another study, Hughes and Pugh (1970) reported that solar

UV radiation might play a part in dissociation of non-

hemolytic M. bovis to hemolytic forms.

Laboratory animals have been reported to be refractory

to M. bovis infection, except mice, which developed con-

junctivitis and keratitis similar to that in IBKC

(Gallagher, 1954 and Pugh, Hughes and McDonald, 1968).

Contrasting views have been reported in the

literature about the development of antibodies to M. bovis

in experimental or natural infections (Reid and Anigstein,

1945; Baldwin, 1945; Barner, 1952; and Wilcox, 1970a).

This was probably due to the unavailability of a reliable

test to detect antibodies in serum. The plate and tube

agglutination tests were used mostly to detect serum

antibodies, but these tests were found to be unreliable

due to autoagglutination of smooth type organisms (Pugh

and Hughes, 1970b). An immunodiffusion test was used by

Mitchell and Burrell (1964) to detect serological relation-

ships among various species of Moraxella. They found that

M. bovis had common antigens with other Moraxella species.

Similar results were reported by Pugh, Hughes and McDonald

(1971) who also used the immunodiffusion test. Different

isolants of M. bovis were reported to have both common and

different or non-identical antigens, when bacterial cell






lysates were used as antigens in the test. The immuno-

diffusion test is used as a qualitative test and the

titer of antibodies cannot be determined by this test

when more than one antigen is involved. Immunization

studies with formalized bacteria were not successful

(Henson, Grumbles, and Dollahite, 1960). Partial immunity

to reinfection was reported when the animals were vaccinated

with viable M. bovis organisms (Hughes and Pugh, 1971).

From the literature, it seemed that there were anti-

genic differences among various isolants of M. bovis

(Pugh et al., 1971). A study of antigenic relationships

of various isolants could be a step toward development of

an effective bacterin. Moreover, from immunization studies

it appeared that some antigens were destroyed by formalin

treatments, or were released extracellularly. This fact

might be important in the development of immunity to

M. bovis.

Another area of importance was S/R dissociation of

M. bovis. According to Pugh and Hughes (1970a) IBKC was

caused by both smooth as well as rough forms, although

loss of virulence was reported when the smooth virulent

forms dissociated to rough variants (Cooper, 1960; Jackson,

1953). The mechanism involved in S/R variation was not

understood. Related to this area was the study and

importance of endotoxin (lipopolysaccharide) to find if

it was involved in S/R dissociation and virulence.







It was reported by various investigators (Jackson,

1953; Henson and Grumbles, 1960b, 1961; and Pugh, 1969)

that M. bovis produced potent toxins which might be

involved in the pathogenesis of this disease. Hemolytic

toxins and an endotoxin were demonstrated, but no work

on isolation and characterization of these toxins was

attempted.

The present investigation was undertaken to:

1. study antigenic relationships of different

isolants of M. bovis (both somatic and extracellular

antigens),

2. isolate and characterize hemolysins and endo-

toxins, and

3. study the basis of S/R dissociation in M. bovis.













SECTION 1

CULTURAL, MORPHOLOGICAL AND PHYSIOLOGICAL
CHARACTERISTICS OF MORAXELLA BOVIS ISOLANTS AND
RELATED ORGANISMS

Introduction and Review of Literature

Although cultural, morphological and physiological

characteristics of Moraxelta bovis have been studied by

various investigators (Barner, 1952; Pugh et al., 1966;

White, 1966, and Pugh, 1969), it was thought essential

to identify these isolants especially FLA-Vet.l and FLA-

264 (Ivan Park), and compare these with related species

of Moraxella, namely N. liquefaciens, M. nonliquefaciens

and Mima polymorpha var. oxidans. The literature was

reviewed in a previous section. In summary three isolants

used in this study (ATCC 10,900; 8613 [Md.] and IBH68

[712L]) were characterized by Pugh et al., (1966),

Baumann, Doudoroff and Stanier (1968) and Pugh (1969).

One Florida isolant, FLA-264 (Ivan Park), was characterized

and studied by White (1966). As these isolants were to

be used in studies of M. bovis hemolysins, endotoxin,

and extracellular antigens, they were characterized as

a group, with more emphasis being placed on their physio-

logical characteristics (production of enzymes and toxins)

and colonial dissociation.







Materials and Methods

Organisms

Five isolants of MoraxeZZa bovis and three Moraxella

bovis-like organisms were used in this study. These were:

MoraxeZla bovis isolants:

1. ATCC 10,900*

2. 8613 (Md.)*

3. IBH 68 (712L)*

4. FLA-264 (Ivan Park)**

5. FLA-Vet.l**

Moraxella bovis-like organisms:

1. M. liquefaciens (9833)*

2. M. nonliquefaciens (9893)*

3. Mima polymorpha var. oxidans (9714)*

Cultural characteristics

All the organisms were grown on tryptose blood agar

base with 5% sheep blood (BAP) and trypticase soy agar***

(TSA). Colony morphology was examined after 24 and 48

hours incubation at 37 C. Cellular morphology was studied

by gram stain and india ink-methyl violet capsule stain



All media were obtained from Difco Laboratories, Inc.,
Detroit, Michigan unless otherwise noted.

*Courtesy of Dr. G. W. Pugh, Jr., National Animal Disease
Laboratories, Ames, Iowa.

**Florida isolants.

***BBL laboratories, Baltimore, Maryland.




16

(Butt, Bonynge and Joyce, 1936). Motility was determined

in SIM medium, along with indol and H2S production. Growth

was also examined on EMB agar and Loeffler's blood serum

slants. Growth in broth culture was studied in trypticase

soy broth* (TSB) with and without 0.5% yeast extract.

Physiology

Utilization of sugars was determined in phenol red

broth with 1.0% carbohydrate, and triple sugar iron agar.

Cultures were examined for ability to ferment sucrose,

glucose, maltose, lactose, inositol, mannitol, dulcitol

and arabinose. The tubes were observed for acid and gas

production for 14 days after inoculation.

For the nitrate reduction test TSB with 0.2% (W/V)

potassium nitrate was used. The presence of nitrite was

detected by the addition of a-Napthylamine and sulfanilic

acid reagents after five days growth. The oxidase test

was made by flooding growth on TSA after 24 hours with 1%

solution of p-aminodimethylanaline oxalate. The catalase

reaction was determined by placing one drop of 3% hydrogen

peroxide on a colony on TSA and by adding 1.0 ml of same

strength hydrogen peroxide to a 48 hour old culture in 5

ml of TSB.

Hemolysis was determined on BAP after 24 and 48 hours

incubation.


*BBL Laboratories, Baltimore, Maryland.





17

Proteinase production was determined by liquefaction

and pitting of coagulated serum on Loeffler's blood serum

medium, casein hydrolysis in litmus milk and gelatin

liquefaction by the method described by Skerman (1967).

Extracellular lipase production was tested on

modified egg yolk medium (Graber, Latta, Fairchild and

Vogel, 1958) and by hydrolysis of Tween 80 (Baumann

et al., 1968). Observations were made for five days.

Production of DNase was tested on DNase test medium

by the method of Streitfeld, Hoffmann and Janklow, (1962).

Other media used for physiological tests were

Simmonscitrate agar, Urea agar, MR-VP medium, malonate

Broth, lysine and ornithine decarboxylase test media.

All the media were incubated at 37 C after inocula-


tion.







Results

There was no difference in the morphology of different

isolants of Moraxella bovis. The organisms were short,

plump rods, 1 3p in length and 0.5 lp in width with

rounded ends. There was an increase in pleomorphism

when the plates were incubated more than 48 hours. Un-

stained halos around the cells were seen when stained with

india ink-methyl violet capsule stain. Other organisms,

M. liquefaciens and M. nonliquefaciens were similar in

morphology to M. bovis. Mima polymorpha var. oxidans

cells were more pleomorphic.

After 24 hours incubation the colonies of M. bovis

on blood agar plates were 0.5 2 mm in diameter with

0.5 mm zone of beta hemolysis. The colonies increased to

2 4 mm after 48 hours incubation with a much larger

zone of hemolysis. Typical colonies of isolants IBH

68 (712L), FLA-264 (Ivan Park) and FLA-Vet.l, were convex

or flat, umbonate with entire edge and dry greyish-white.

A small depression on the surface of agar was noticed

when the colonies were moved. The colonies were firm,

friable and adherent. When suspended in physiological

saline solution (PSS) or distilled water the growth

autoagglutinated and formed clumps. The colonies of

isolants ATCC 10,900 and 8613 (Md.) were smaller in

diameter, convex, smooth and butyrous. There was a narrow

zone of beta hemolysis. These colonies formed uniform

suspensions in PSS or distilled water.






Colonial dissociation was noticed in the growth of

isolants IBH 68 (712L), FLA-264 (Ivan Park) and FLA-Vet.l.

The dissociated colonies were smooth, large,butyrous in

consistency and had irregular borders. They formed a

uniform suspension in PSS or distilled water like the

colonies of isolant ATCC 10,900 and 8613 (Md.). Some

small "dwarf" colonies were also observed. Dissociation

to nonhemolytic colonies was noticed with hemolytic

isolant IBH 68 (712L). The nonhemolytic variant had

both smooth and rough types. When incubated for a period

of 48 72 hours the nonhemolytic colonies showed a very

weak type of alpha hemolysis around the colonies.

The colonies of M. Ziquefaciens and M. nonliquefaciens

were nonhemolytic, 1 2 mm in diameter, convex, glistening,

butyrous and suspended uniformly in PSS. Mima polymorpha

var. oxidans produced even smaller (0.2 0.5 mm in diameter)

and nonhemolytic colonies.

The growth in trypticase soy broth was slight after

24 hours, but after 48 hours incubation M. bovis isolants

IBH 68 (712L), FLA-264 (Ivan Park) and FLA-Vet.l produced

granular growth with sediments which broke into coarse

particles on shaking. They also formed a surface ring

on the sides of the tube. The growth of the other two

isolants was uniform, without the sediment, like that

observed in the dissociated colony growth of isolants

IBH 68 (712L), FLA-264 (Ivan Park) and FLA-Vet.l.




20

The growth in TSB with 0.5% yeast extract was heavier

and the undissociated type formed a thin pellicle on the

surface. M. nonZiquefaciens and Mima polymorpha var.

oxidans produced diffused and slightly granular growth

in TSB, while M. Ziquefaciens had uniformly diffused

growth in the broth.

Moraxella bovis isolants and M. bovis-like organisms

did not ferment carbohydrates, but produced an alkaline

reaction. All the organisms were negative for MR, VP,

H2S, urea, malonate, ornithine and lysine decarboxylase.

MoraxellZa bovis isolants were negative for DNase.

Other reactions are summarized in Table 1. MoraxeZla

bovis isolants produced a proteolytic and a lipolytic

enzyme which hydrolyzed Tween 80. The isolants of M.

bovis differed from other organisms by being hemolytic

and by not reducing nitrate. With respect to cultural,

morphological and physiological characteristics, M. bovis

isolants constituted a homogenous group, except for the

catalase reaction (FLA-264 isolant was catalase positive)

and dissociation. Isolant ATCC 10,900 and 8613 (Md.) were

in dissociated form while other isolants were both in

smooth and rough forms (isolant IBH 68 [712L] had a non-

hemolytic variant).





Table 1. Cultural and physiological characteristics of Moraxella bovis isolants and
other related organisms.
Mima
Moraxella bovis isolants M. M. polymorpha
ATCC 8613 IBH 68 FLA- FLA- lique- nonlique- var.
Reactions 10,900 (Md.) (712L) 264 Vet.l faciens faciens oxidans


+ +


Oxidase

Catalase

Nitrate

Hemolysis


+


+


+ +


Citrate


Growth on
EMB medium


Proteolytic
action on:
Gelatin

Loeffler's
serum

Litmus milk

Lipase action
on: Egg yolk
medium

Tween 80 med.


N.G.


N.G.


N.G.


N.G.


N.G. N.G. N.G. N.G.


N.G. N.G. N.G. N.G.


N.G.


N.G. Slight growth
dark violet
colonies


+ +


+ + + +


+ +


- Alkaline


+ +


N.G. No growth
- Negative reaction
+ Positive reaction






Discussion

All M. bovis isolants, including Florida isolants,

had similar cultural, morphological and physiological

characteristics except for the catalase reaction (FLA-264

was catalase positive, while all others were catalase

negative). Both types of catalase reactions have been

observed in isolants of M. bovis (Pugh et al., 1966).

According to Hughes et at., (1968a) production of catalase

did not play any role in the virulence or pathogenicity

of a particular isolant of M. bovis, as both catalase

negative and catalase positive isolants were able to

produce infectious bovine keratoconjunctivitis. Other

characteristics were similar to those reported by Pugh

et al., (1966), Wilcox (1970c), Pedersen (1970) and

Baumann et al. (1968). Most of the investigators have

reported smooth to rough dissociation in M. bovis

except Pedersen (1970) who noticed dissociation of rough

form to smooth type. On primary isolation, the colonies

of M. bovis are convex or flat, umbonate, firm, dry, and

adherent when touched with a loop. These colonies also

autoagglutinate on suspension in PSS or distilled water.

All these characteristics have been referred to as those

of rough type of growth (Burrows, 1968). On the other

hand, the dissociated type produced smooth, viscous,

butyrous colonies which formed a uniform stable suspension

in PSS. As both these types are virulent and able to

produce disease (Pugh and Hughes, 1970a), calling them

smooth colonies on primary isolation was probably a





23

misnomer. The results of the present investigation are in

agreement with that reported by Pedersen (1970). It is

recommended that convex of flat, umbonate, firm and adherent

colonies which autoagglutinate in saline solution (pre-

dominant type observed on primary isolation from acute

cases of disease) should be called rough type (R type)

colonies, while the dissociated type should be called

smooth type (S type) colonies. In the remainder of this

manuscript, the recommended terminology was used for

colony morphology and dissociation. Hemolytic to non-

hemolytic variation was observed in isolant IBH 68

(712L) which is similar to that reported by Pugh (1969).

The proteolytic and lipolytic enzymes produced by

M. bovis along with hemolysins and endotoxin may be

important factors involved in the pathogenicity of this

organism. Tween 80 hydrolytic enzymes have been reported

from Vibrio choZerae, Aeromonas sp., Pseudomonas

aeruginosa and other enteric gram-negative bacteria

(Chakrabarty, Adhya and Pramanik, 1970).







Summary

Five isolants of MoraxeZZa bovis, including two

Florida isolants, were characterized by their cultural,

morphological and physiological characteristics. Moraxella

bovis isolants were distinguished from related organisms

(M. Ziquefaciens, M. nonliquefaciens) by the nitrate

reduction test.

Moraxella bovis isolants produced proteolytic and

lipolytic enzymes which might be of significance in their

pathogenicity, along with hemolysins and endotoxin. On

primary isolation, M. bovis produced convex or flat,

umbonate, firm adherent colonies which autoagglutinated

in saline solution. These colonies were called rough

(R type), while the dissociated type formed smooth,

butyrous colonies which could be uniformly suspended in

saline solution and were called smooth (S type). The

dissociation of M. bovis after repeated transfer on

artificial medium was termed R to S type.













SECTION 2

EXTPACELLULAR ANTIGENS OF MORAXELLA BOVIS

Introduction and Review of Literature

In addition to their possible role in the production

of disease in susceptible animals, many extracellular

bacterial products are antigenic in nature. These extra-

cellular antigens are important in immunological charac-

terization of microorganisms as well as in the production

of immunity against disease.

Usually the immunizing agents or bacterins consist of

formalin or heat treated microbial cells which may be

devoid of or may have very little "essential immunizing

agents" if these antigens occur and are soluble in

extracellular fluid.

There are very few reports in the literature about

antigenic studies of Moraxella bovis. Mitchell and

Burrell (1964) reported that the cell fluids of various

species of Moraxella contained a few apparently different

antigens as indicated by 3 6 gel precipitin bands which

were formed in reaction with homologous antisera. At

least one antigen, the group specific antigen, was shared

by all MoraxeZZa species including M. bovis. Pugh (1969),

and Haug and Henriksen (1969a,b) demonstrated antigenic

relationships between M. bovis and other related organisms





26
namely M. liquefaciens, M. nonZiquefaciens, Mima polymorpha,

Mima polymorpha var. oxidans and HereZZea vaginicola.

Using bacterial lysates as antigens in immunodiffusion

tests, Pugh (1969) showed that different isolants of M.

bovis had common as well as nonidentical antigens. No

work was reported on the extracellular antigens of

M. bovis.

The present investigation was undertaken to isolate

and study the extracellular antigens of Moraxella bovis

with special emphasis on the relationships between rough

and smooth types of an isolant as well as among different

isolants.







Materials and Methods

Preparation of antisera

Twenty New Zealand white rabbits (4 6 pounds) were

used for the preparation of antisera against different

isolants of Moraxella bovis. These rabbits were divided

in two equal groups as shown in Figure 1. Those in group

1 were inoculated with cells grown on solid media (BAP),

while the ones in group 2 were inoculated with cultures

grown in broth (Typticase soy broth). Prior to inoculation,

serum was obtained from each rabbit and tested for pre-

cipitating antibodies by immunodiffusion test against

sonicated extract of M. bovis cells (isolant ATCC 10,900).

Fluids collected by swabs from both eyes of each rabbit

were cultured on BAP and TSB for presence of N. bovis.

The eye cultures were negative for N. bovis and no anti-

bodies were detected in the serum. All the animals were

housed in an air conditioned animal facility and received

standard commercial laboratory feed (Rabbit Chow) and

water.

Live cells or cultures were used as antigens for

production of antisera. For inoculation of rabbits in

group 1, the organisms were grown on blood agar plates.

After 24 hours incubation at 37 C the plates were checked

for contamination and purity of type. Rough (R) or

smooth (S) growth from uncontaminated plates was harvested

in physiological saline solution (PSS) and standardized

to contain approximately 1.0 x 109 cells per ml in a











Group 1
(10 rabbits)
Inoculated with growth from
solid medium


l.A. (5) .


1.B. (3) .


Group 2
(10 rabbits)
Inoculated with growth in
broth


Smooth type growth


Isolants: ATCC 10,900;
8613 (Md.); IBII 68 (712L);
FLA-264 (Ivan Park); and
FLA-Vet.l.

Rough type growth

Isolants: IBH 68 (712L);
FLA-264 (Ivan Park); and
FLA-Vet.l.


1.C. (2) .


Controls


2.A. (5).


2.B. (3) .


2.C. (2).


Figure 1. Immunization scheme for the production of anti-
sera in rabbits against rough and smooth type
growth of various isolants of Moraxella bovis
on solid or in broth culture media.







Petroff-Hausser counting chamber. Rough type growth

was suspended in 10% magnesium chloride solution to

facilitate cell counting (Pugh and Hughes, 1970b). The

quantities for each injection were frozen separately at

-60 C. When required for immunization, the proper tubes

were thawed at room temperature before injection.

To inoculate rabbits in group 2, R and S type cells

of different isolants were grown in trypticase soy broth.

After 48 hours incubation at 37 C the cultures were

standardized and frozen in the same manner as for group 1.

The inoculation schedule consisted of weekly in-

jections with 0.1, 0.2, 0.5, 1.0, and 2.0 ml of standardized

immunogens intravenously on day 1, 8, 15, 22, and 29 res-

pectively. Each rabbit was trial bled on day 35 and the

serum was tested for precipitins by the immunodiffusion

test. Sera from all rabbits except controls showed at

least one band of precipitation. All rabbits were bled

on day 37 and the sera were separated from clotted blood

by centrifugation. The sera were stored at -60 C until

used.

Specific absorption of antiserum

Specific R type antiserum was prepared by absorption

with homologous S type cells and/or heterologous R type

cells. Similarly, specific S type antisera were also

prepared. The cell type (to be used for absorption) was

grown on BAP and the growth was harvested in PSS. Growth

from two blood plates (100 x 15 mm) was used to absorb






0.5 ml of antiserum. The cells were centrifuged and

suspended in 0.5 ml of fresh PSS. An equal amount (0.5

ml) of appropriate antiserum was added. The mixture was

shaken and incubated at 37 C for 1 hour in a water bath

with occasional shaking. The absorbed serum was separated

from the cells by centrifugation and frozen at -60 C

until used.

Preparation and isolation of extracellular antigens

Moraxella bovis isolants FLA-264 (Ivan Park) and IBH

68 (712L) were used in this study. Extracellular antigens

of both R and S types of an isolant were studied. A pre-

liminary growth study of these isolants showed that the

end of the logarithmic growth phase was reached in about

9 10 hours when incubated at 37 C on a gyratory incubator

shaker (Model 425)* at 140 RPM.

Typticase soy broth was prepared, dispensed at 500

ml per flask and autoclaved at 15 Ib pressure for 15 minutes.

The organisms were grown on blood plates for 24 hours at

37 C. The plates were checked for contamination and

purity for R or S type. Growth from two blood plates

(100 x 15 mm) was suspended in 10 ml of sterilized TSB

which was used as an inoculum for each flask. After in-

oculation the flasks were incubated at 37 C for nine hours

on a gyratory incubator shaker at 140 RPM.



*New Brunswick Scientific Co., Inc., New Brunswick,

New Jersey.







Each flask was examined for contamination by gram

stain and by inoculating on a blood plate. The culture

from uncontaminated flasks was centrifuged in an IEC

International centrifuge at 1,290 G for three to four

hours at 4 C. The supernatant fluid was filtered

through a .22p Millipore* filter.

The antigens in cell-free culture filtrate were

concentrated and purified by precipitation with ammonium

sulfate. The filtrate was brought to 80 percent satura-

tion by adding 290 grams of ammonium sulfate per 500 ml

(Wannamaker, 1967) and the mixture stirred on a magnetic

mixer until ammonium sulfate was completely dissolved.

It was left at 4 C overnight to get complete precipitation.

The precipitate was removed by centrifugation at 13,820

G for 45 minutes at 4 C. The sediment from 500 ml of

filtrate was dissolved in 5 ml of cold PSS and dialyzed

against cold PSS to remove ammonium sulfate. A control

flask of uninoculated medium (TSB) was incubated and pre-

cipitated in the same manner as the inoculated culture

flasks. All the antigen preparations were kept at 4 C.

Purification and fractionation on Sephadex G-200**

Sephadex G-200 was suspended in distilled water and

allowed to swell for three days at room temperature.

Before packing, the gel was equilibrated with 0.1 M Tris-

HC1 0.2 M NaCl buffer, pH 8.0. A column (2.5 x 100 cm)


*Millipore Filter Corporation, Bedford, Massachusetts.

**Pharmacia Fine Chemical Inc., Picataway, New Jersey.






was packed according to the manufacturer's instructions.

The upward flow technique was used with a flow rate of 15

ml per hour. The sample was also equilibrated with

buffer before application. Four ml of the sample were

applied to the column and the fractions were eluted with

0.1 M Tris-HCl 0.2 M NaCl buffer, pH 8.0. The fractions

were collected in 5 ml volumes and protein was determined

by absorbance at 280 my in a Beckman DB spectrophotometer.

The fractions were also tested for the presence of anti-

genic material by immunodiffusion tests against homologous

antiserum. Selected fractions containing antigenic

peaks were collected and concentrated by ultrafiltration.

Immunodiffusion test

One percent suspension of purified Noble agar* was

prepared in PSS and one percent of merthiolate solution

(1:100) was added after the agar was melted and cooled

to 50 C. Both petri plates (100 x 15 mm) and glass slides

(25 x 75 mm) were used for the test. Ten ml of molten

agar were poured per plate or per frame of three slides.

Wells of appropriate sizes were cut and after the reactants

were placed in the wells, the plates or slides were left

in a moist chamber at room temperature. After 24 48

hours the precipitin bands were examined and the plates

or slides were washed, dried and stained with amido

black.



*Difco Laboratories, Inc., Detroit, Michigan.







Immunoelectrophoresis

Gelman equipment* was employed for immunoelectro-

phoresis. The chamber buffer was 0.05 M Tris-barbital,

pH 8.6. Half strength buffer containing 1% merthiolate

solution (1:100) was used for preparing 1.5 percent

purified Noble agar solution. Twenty-four ml of agar

solution were poured on a frame of six slides. After the

antigens were placed in the wells, electrophoresis was

carried out at room temperature at a constanL current of

6 ma per frame for three hours. Immediately after electro-

phoresis, antisera were added to the troughs and the slides

were incubated in a moist chamber at room temperature for

48 hours. After the precipitin lines were developed the

slides were washed, dried, and stained with amido black.

Physical and chemical treatment of antigens

Two of the extracellular antigens, which were

specific to R type cells of isolant FLA-264 (Ivan Park),

were subjected to heat, formalin and trypsin treatment.

The treated antigens were tested by immunodiffusion.

The effect of heat was determined at 56 C for 30

minutes and at 100 C for 15 minutes. The antigens were

placed in capped glass vials and were kept in water baths

at 56 C and 100 C for 30 and 15 minutes, respectively.

After treatment antigens were centrifuged to remove pre-

cipitates. The supernatant fluid was tested by immuno-

diffusion.


*Gelman Instrument Company, Ann Arbor, Michigan.







The effect of formalin was determined by adding

formalin to make 1% of antigen solution. The mixture was

kept at room temperature for three hours and centrifuged.

The supernatant fluid was tested in the same manner as

heated antigens.

For trypsin treatment, the antigens were allowed to

react with an equal volume of trypsin solution (0.75 mg/ml)

in phosphate buffer saline, pH! 7.6 for three hours in a

39 C water bath. After centrifugation the supernatant

fluid was tested in the same manner as heated and formalin

treatment antigens.







Results

Figures 2 and 3 show the reactions of ammonium

sulfate precipitated (crude) extracellular antigens with

their homologous and hoterologous type antisera. The

sera, obtained from the rabbits in group 2 which were

immunized with organisms grown in broth, gave an additional

band of precipitin. This band was absent from the anti-

sera prepared by immunization with cells grown on solid

medium (group 1). This could not be due to medium pro-

teins since there was no reaction with trypticase soy

broth concentrated in the same manner as extracellular

antigens (Figure 2). Presumably this antigen was pro-

duced by the organisms and released in the broth as soon

as it was formed. This antigen was produced by both R

and S types of an isolant in broth. The R type crude

extracellular product gave 3 4 bands of precipitins

against its homologous antiserum, while the S type

produced 1 2 bands with homologous antiserum.

Two antigens were common in both R and S types of

an isolant (Figures 4 and 5). One of these showed com-

plete identity with all the smooth and rough antisera,

while the other also was common with partial identity

with FLA-264 (R), FLA-Vet.l (R and S) and 8613 (Md.).

Figure 4 shows a prominent band between R type crude

antigens from isolant FLA-264 and its homologous anti-

serum. This band was characteristic to only this isolant

and not any other. On electrophoresis of crude antigenic























T

3/1/


Immunodiffusion analysis of Moraxella bovis
(IBH 68 712L) extracellular antigens. Wells
1 and 2 contained crude extracellular antigens
from R and S type cells, respectively. Wells
3 and 5 contained antisera against R and S
cells grown in broth, while wells 4 and 6
contained antisera against R and S cells grown
on solid medium. Note a distinct band by
antisera 3 and 5 (broth cultures). Well 7
contained trypticase soy broth concentrated
in the same manner as extracellular antigens.


Figure 2.










































Figure 3. Immunodiffusion analysis of MoraxeZZa bovis
(FLA-264-Ivan Park) extracellular antigens.
Wells 1 and 2 contained crude extracellular
antigens from R and S type cells, respectively.
Wells 3 and 5 contained antisera against R
and S cells grown in broth while wells 4 and
6 contained antisera against R and S cells
grown on solid medium. Note a distinct band
produced by antisera 3 and 5.











































Figure 4. Immunodiffusion analysis of Moraxella bovis
(FLA-264 Ivan Park Rough type] extra-
cellular antigens against homologous and
heterologous antisera. Center well contained
crude extracellular antigens from FLA-264
(R). Peripheral wells contained antisera
against various isolants: 1. FLA-264 CR),
2. FLA-264 (S), 3. IBH 68 (R), 4. IBH
68 (S), 5. FLA-Vet.l (R), 6. FLA-Vet.l (S),
7. 8613 Md. (S), and 8. ATCC 10,900 (S).


U







































Figure 5. Immunodiffusion analysis of Moraxella bovis
(FLA-264 Ivan Park Smooth type) extra-
cellular antigens against homologous and hetero-
logous antisera. Center well contained crude
extracellular antigens from FLA-264 (S). Peri-
pheral wells contained antisera against various
isolants: 1. FLA-264 (R), 2. FLA-264 (S),
3. IBH 68 (R), 4. IBH 68 (S), 5. FLA-Vet.l (R),
6. FLA-Vet.l (S), 7. 8613 Md. (S), and
8. ATCC 10,900 (S).


o
@@^)
CU)~




40

material (FLA-264-R) and its reaction with R type specific

antiserum, two antigens were shown to be present in R

type extracellular material, while there was no reaction

with S type extracellular material (Figure 6).

On fractionation of R and S type crude extracellular

materials (FLA-264) on Sephadex G-200, four major anti-

gens were isolated from R and two from S type material

(Figures 7 and 8). S-1 and S-2 antigens were identical

to R-l and R-4 and these antigens could be removed from

R type antiserum by absorption with smooth cells. R-l

was overlapped with R-2 antigen as shown by an identical

line (Figure 9). From immunoelectrophoretic patterns, it

was evident that R-2, R-3 and a part of R-1 (contiguous

with R-2) are specific to only R type and not S type of

isolant FLA-264 (Figures 6, 10 and 11).

The serological reactions of crude extracellular

antigens from R and S types of isolant IBH 68 (712L)

with their homologous and heterologous antisera are shown

in Figures 12 and 13. Crude extracellular material from

rough type IBH 68 (712L) also gave two bands with the

homologous antiserum (Figures 12) which were not shown

by the antisera of any other isolant except R type FLA-

Vet.l. On Sephadex fractionation, three antigens were

recovered from R type (IBH 68) extracellular crude material

(Figure 14). Antigens R-l and R-3 from this isolant were

identical with S-1 and S-2 from smooth type crude material

of isolant IBH 68 (Figure 15) and also with that from S
















































Figure 6. Immunoelectrophoretic pattern of extracellular
R antigens of FLA-264 (Ivan Park). Wells 1
and 2 contained crude extracellular smooth
and rough antigens of FLA-264 (S) and FLA-264
(R), respectively. The trough contained R-
antiserum (FLA-264) absorbed with homologous
smooth cells.




i Positive precipitation reaction
.......... Fractions collected
--.. Fractionation of trypticase soy broth medium (Control)
0.4

R-.1.. a .2.... R ..... .4 .........

ISOLANT FLA-264 (IVAN PARK) ROUGH TYPE
0.3





S0.2-








0.1



20 30 40 50 60 70 80 90
FRACTION NUMBER
Figure 7. Fractionation of crude anrmonium sulfate-precipitated filtrate
of Moraxella bovis supernatant fluid on Sephadex G-200. Buffer,
0.1 M Tris-HCl 0.2 M NaC1 (pH 8.0); flow rate, 15 ml/hr;
Column size, 2.5 x 84 cm; fraction volume, 5 ml.







Is Positive precipitation reaction


.......... Fractions collected


S-I S-2
ISOLANT FLA-624 (IVAN PAR.....) SMOOT TYPE
ISOLANT FLA-624 (IVAN PARK) SMOOTH TYPE


S30 40 50 60 70 80
FRACTION NUMBER
Figure 8. Fractionation of crude anronium sulfate-precipitated
MoraxeZZa bovis supernatant fluid on Sephadex G-200.
Tris-HC1 0.2 M NaCI (pH 8.0); flow rate, 15 ml/hr;
2.5 x 84 cm; fraction volume, 5 ml.


filtrate of
Buffer, 0.1 M
Column size,
























A















Figure 9. Relationship of different R type antigens of
isolant FLA-264 (Ivan Park). Center well
contained R-antiserum (FLA-264) absorbed with
homologous smooth type cells. Wells 1, 2, 3,
and 4 contained extracellular antigens R-l,
R-2, R-3, and R-4 of FLA-264 (R), respectively.
Well 5 contained crude extracellular S anti-
gens of FLA-264 (S).

















































Immunoelectrophoretic pattern of extra-
cellular antigens (R-2 and R-3) of isolant
FLA-264 (Ivan Park). Wells 1 and 2 con-
tained extracellular antigens R-2 and R-3
of FLA-264 (R), and the trough contained R-
antiserum to FLA-264 (R).


Figure 10.

















































Immunoelectrophoretic pattern of extra-
cellular R antigens (R-1 and R-3) of isolant
FLA-264 (Ivan Park). Wells 1 and 2 contained
extracellular antigens R-l and R-3 of FLA-
264 (R), while the trough contained R-antiserum
to FLA-264 (R).


Figure 11.
















































Immunodiffusion analysis of Moraxella bovis
(IBH 68 712L Rough type) extracellular
antigens with homologous and heterologous
antisera. Center well contained crude extra-
cellular antigens from IBH 68 (R). Peripheral
wells contained antisera against various
isolants: 1. IBH 68 (R), 2. IBH 68 (S),
3. FLA-264 (R), 4. FLA-264 (S), 5. FLA-Vet.1
(R), 6. FLA-Vet.l (S), 7. 8613 Md. (S),
and 8. ATCC 10,900 (S).


Figure 12.










































Immunodiffusion analysis of Moraxella bovis
(IBH 68 712L Smooth type) extracellular
antigens with homologous and heterologous
antisera. Center well contained crude antigens
from IBH 68 (S). Peripheral wells contained
antisera against various isolants: 1. FLA-264
(R), 2. FLA-264 (S), 3. IBH 68 (R) 4. IBH 68
(S), 5. FLA-Vet.l (R), 6. FLA-Vet.l (S),
7. 8613 Md. (S), and 8. ATCC 10,900 (S).


0


O


Figure 13.






t'" Positive precipitation reaction


.......... Fractions collected


S......R ...R. ..


ISOLANT IBH 68 (712L) ROUGH TYPE


20 30 40 50 60 70 80 90
FRACTION NUMBER
Figure 14. Fractionation of crude ammonium sulfate-precipitated filtrate of
Moraxella bovis supernatant fluid on Sephadex G-200. Buffer,
0.1 M Tris-HCl 0.2 M NaC1 (pH 8.0); flow rate, 15 ml/hr;
Column size, 2.5 x 84 cm; fraction volume, 5 ml.


C
co
S0.2


H
E-i



0.1'
o

E-1
p4
0








E 1 Positive precipitation reaction

.......... Fractions collected


S-1
,..........


S-2
o........ o..o..... .... o.. .


ISOLANT IBH 68 (712L) SMOOTH TYPE


) 30 40 50 60 70 80 90 95
FRACTION NUMBER

Figure 15. Fractionation of crude ammonium sulfate-precipitated filtrate of
Moraxella bovis supernatant fluid on Sephadex G-200. Buffer,
0.1 M Tris-HCl 0.2 M NaC1 (pH 8.0); flow rate, 15 ml/hr;
Column size, 2.5 x 84 cm; fraction volume, 5 ml.


0.3-


0.2-





51

type of isolant FLA-264. Peak R-2 consisted of two anti-

gens which were not present in any of the other isolants

except R type of isolant FLA-Vet.l (Figure 16).

Antigens R-2 and R-3 of isolant FLA-264 were protein

in nature since they were destroyed by trypsin treatment

(Figures 17 and 18), although the optical densities of

their fractions from Sephadex G-200 at 280 mn were very

low. While antigen R-3 gave a wider band after heat

treatment at 100 C for 15 minutes, and it was partially

destroyed by formalin treatment (Figure 18), heat or

formalin treatment did not effect antigen R-2 of isolant

FLA-264 (Figure 17).




52











































Figure 16. Immunoelectrophoretic pattern of R antigens
of isolant IBH 68 (712L) and their relation-
ship with R-antiserum to isolant FLA-Vet.l
(R). Well 1 contained extracellular R-2
antigens of IBH 68 (R), while well 2 con-
tained crude extracellular antigens of IBH
(S). The trough contained R-antiserum (FLA-
Vet.l) absorbed with homologous smooth cells.
















































Effects of heat, formalin, and trypsin treat-
ments on the extracellular R-2 antigen of
isolant FLA-264 (Ivan Park). Center well
contained R-antiserum to FLA-264 (R). Peri-
pheral wells contained R-2 antigen with
different treatments; 1. native antigen,
2. heated at 56 C for 30 minutes,.3. heated
at 100 C for 15 minutes, 4. treated with
formalin, and 5. treated with trypsin.


Figure 17.

























2
Sil



a


Figure 18. Effects of heat, formalin and trypsin treat-
ment on the extracellular R-3 antigen of
isolant FLA-264 (Ivan Park). Center well
contained R-antiserum to FLA-264 (R). Peri-
pheral wells contained R-3 antigen with
different treatments; 1. native antigen,
2. heated at 56 C for 30 minutes, 3. heated
at 100 C for 15 minutes, 4. treated with
formalin, and 5. treated with trypsin.







Discussion

The results of the present study showed that rough

type MoraxeZla bovis produced 3 4 extracellular antigens

in the culture broth, while 1 2 antigens were produced

by smooth type cultures. The antibodies against one of

these antigens can only be produced if broth culture is

injected. The cells from solid medium did not induce

antibodies against this antigen.

At least two "group specific antigens" were common in

R and S types of an isolant. The R type extracellular

product contained two additional antigens which did not

react with S type antiserum. These antigens were probably

serotype specific. By serological reactions of extra-

cellular antigens, isolants FLA-Vet.l and IBH 68 (712L)

were found to be identical, since absorption of antiserum

against either one by the cells of the other removed all

the antibodies. The serotype specific extracellular

antigens of a third isolant (FLA-264) were different from

those of all the other isolants. Moraxella bovis isolants,

therefore, can be serotyped on the basis of serotype

specific extracellular antigens. It was probably due to

different serotypes that animals immunized against one

isolant were more susceptible to challenge with a heterolo-

gous isolant than the same isolant (Hughes et al., 1968b;

and Hughes and Pugh, 1971). One of the serotype specific

antigens was partially destroyed by heat and formalin

treatment, which was probably the reason that formalinized




56

bacterin did not produce iriLunity against infectious bovine

keratoconjunctivitis (Henson et al., 1960). No study to

demonstrate protection by extracellular antigen was under-

taken in the present investigation. Hughes and Pugh

(1971) reported that some immunity was produced by intra-

muscular injections of viable M. bovis organisms, thus

suggesting that extracellular antigens might be involved

in immunity against infectious bovine keratoconjunctivitis.

It is evident from the present investigation that extra-

cellular products of 1M. bovis are prominent antigens.

The importance of extracellular antigens in immunity as

well as in pathogenesis of the disease needs to be

elucidated.

Another significant finding was that only the R type

of an isolant had serotype specific antigens. On dissocia-

tion of R type colonies to S type, there was loss of sero-

type specific antigens and only group specific antigens

were present in the culture fluid. The S type of various

isolants had similar extracellular antigens. For the

development of a bacterin, the importance of various

serotypes and extracellular antigens produced in liquid

medium should be taken into consideration.







Summary

Three to four extracellular antigens were present in

cell-free culture filtrate of the R type of Moraxella

bovis, while the S type of an isolant had only two anti-

gens. The S type antigens were common in both rough and

smooth types of an isolant as well as in different

isolants of M. bovis, so were called "group specific

antigens". In addition to the smooth antigens, R type

organisms of isolant FLA-264 (Ivan Park) produced at

least two more extracellular antigens which were different

from those of all the other isolants, and so were called

serotype specific antigens. The serotype specific anti-

gens of isolant (IBH 68) were different from those of

isolant FLA-264 (Ivan Park) but were identical with that

of FLA-Vet.l. Therefore, on the basis of serological

reactions of extracellular antigens three R type isolants

of M. bovis used in this study could be divided in two

groups, one isolant had different serotype specific

antigens than those of the other two while the latter two

isolants had common antigens but different from those of

the former.

Serotyping M. bovis isolants on the basis of extra-

cellular antigens was suggested in addition to their

importance in protection against the disease.













SECTION 3

LIPOPOLYSACCHARIDE OF MORAXELLA BOVIS

Introduction and Review of Literature

Gram-negative bacteria have a complex macromolecular

structure consisting of protein, lipopolysaccharide, and

phospholipid in addition to the mucopeptide layer in

their cell wall. This complex has been called Boivin

antigen, O-antigen, pyrogen, tumor-necrotizing substance

or Shwartzman toxin, but is commonly known as lipopoly-

saccharide (LPS). This structure is important antigeni-

cally as it is liberally exposed at the cell surface and

forms the somatic antigen which is the basis of identifi-

cation of various serotypes in different species of

Enterobacteriaceae. The differences between smooth (S)

and rough (R) types have been found due to certain

characteristic sugars present in smooth LPS in addition

to those in rough LPS. This complex is also capable of

eliciting a number of biological reactions in experimental

animals. Although the chemistry of LPS with respect to

its specificity and toxicity has been well worked out,

its involvement in the mechanism of disease induction

is still unknown.

The concept of endotoxin was developed by Pfeiffer

(1892) who defined it as a poison which was a part of

58





59

bacterial substance and was released only on lysis of the

cell. Endotoxin was reported as a constitutive part of

cell wall of gram-negative bacteria (Cumins, 1956; Milner,

Anacker, Fukushi, Haskins, Landy, Malmgren and Ribi, 1963;

and Shands, 1965). This complex formed somatic or O-anti-

gens which were used for immunological identification and

classification of gram-negative bacteria (Kauffmann,

1964). Considering physico-chemical and biological pro-

perties, endotoxins were tentatively defined by Milner,

Rudbach and Ribi (1971, p.55) as follows:

Endotoxic phospholiposaccharide (endotoxins)

are found principally at or near the cell surfaces of

gram-negative bacteria. As extracted by common pro-

cedures, they are macromolecular aggregates of sub-

units, united in various forms by hydrophobic bonds,

incorporating both the major somatic antigens of the

bacteria and a large number of toxic and other host-

reactive properties that are described collectively

as "endotoxic". All are stable to boiling in neutral

water. The probability of correctly identifying

a substance as an endotoxin increases rapidly with

the number of typical host responses that are demon-

strated. Of particular value in this regard are the

production, in suitable animals, of characteristic

biphasic fever, lethal shock after a latent period,

the Sanarelli-Shwartzman reactions, hemorrhagic

necrosis of transplantable tumors, leukopenia,





60

followed by leukocytosis, enhancement of antigenicity

of proteins, and nonspecific resistance to infection

or to damage by irradiation. Basic to many of the

pathophysiological effects of endotoxin is an injury

to the cardiovascular system by means not yet fully

understood, which alters or disrupts normal function.

The nonprotein nature of some bacterial antigens was

demonstrated by Boivin and Mesrobeanu (1933) and Mesrobeanu

(1936). Morgan (1936) was able to isolate a specific

polysaccharide from smooth types of B. dysenteriae (Shiga).

When extracted with diethylene glycol, this antigen was

found to be a complex mixture of phospholipin, poly-

saccharide and polypeptide (Morgan and Partridge, 1940),

and was reported as biologically toxic by Morgan (1937).

Lipopolysaccharide extracted by phenol water method was

free of proteins and was fully active immunologically as

well as biologically (Ribi, Anacker, Fukushi, Haskins,

Landy and Milner, 1964). Lipopolysaccharide from S type

organisms contained lipid and certain sugars in addition

to those present in the lipopolysaccharide of R type

organisms. These additional sugars were immunodominant

and represented O-antigenic group (Liideritz, Staub and

Westphal, 1966), while the lipid moiety (Lipid A) possessed

the endotoxic properties. It was reported by Ribi et al.,

(1964) and Nowotny (1969) that acid hydrolysis of LPS in-

activated toxic and biological activities. When Lipid A

was free from the rest of the complex, it was of low





61

biological activity (Kim and Watson, 1967) indicating that

lipid A alone was not responsible for all the toxic and

biological reactions. There had been two opinions about

this. Milner et al., (1971) believed that lipid A and

polysaccharide were not separable moieties, and removal

of fatty acids destroyed antigenicity just as it destroyed

toxicity. Luderitz, Westphal, Staub and Nikaido (1971)

hypothesized that polysaccharide in LPS and keto-deoxyoctul-

onate in glycolipid act as water solubilizing carriers

for lipid A, since it has been reported that free lipid A

of low toxicity became a potent toxin when put in a water-

soluble complex (Rietschel, Galanos, Tanaka, Ruschmann,

Luderitz and Westphal, 1971).

Irrespective of its source, the endotoxins elicit

similar biological reactions in experimental animals includ-

ing pyrogenicity, lethality, local and generalized

Shwartzman reactions, hemorrhagic necrosis of tumors,

nonspecific resistance to infection, leukocytic alteration,

abortion and other pathophysiological phenomena. These

reactions were reviewed by Thomas (1954) and Milner et al.,

(1971).

Although most experimental animals respond to the

lethal shock by endotoxin, wide variations occur in

different species. Rabbits were reported to be more

susceptible than mice (Ribi, Milner and Perrine, 1959).

In one experiment by Ribi et al., (1959), while the LD50

dose for rabbits was 23.4 mg/kg body weight, the dose for







a mouse was 22,090 mg/kg (about 1,000 fold difference).

The individual differences within a species were reported

as due to presentization by the presence of gram-negative

bacteria in the gut of an animal (Schaedler and Dubos,

1962).

The pyrogenic effects of endotoxin were described

by Bennett and Beeson (1950) and Bennett and Cluff (1957).

While in mice and rats the body temperature was depressed,

man and other animals showed an increase in the body

temperature (Milner et al., 1971).

The Shwartzman reaction was first noticed by

Sanarelli (1924). He reported that many rabbits died

when given sublethal doses of live cholera vibrio pre-

ceded by intravenous injection of culture filtrate from

E. coli or Proteus sp. Shwartzman, in 1928, observed

local tissue reactivity while he was trying to produce

natural resistance by multiple injections of cell-free

filtrate of a gram-negative bacterial culture. The

generalized reaction of Sanarelli (1924) was reinterpreted

as a phenomenon of tissue reactivity (Gratia and Linz, 1932).

Although the Shwartzman reaction has not been explained

in immunological terms, the cellular reactions were

reported due to an intravascular conversion of fibrinogen

to fibrin and blocking of function of the reticuloendothe-

lial system (Lee and Stetson, 1965).

Different methods of extraction of LPS were tried by

various workers. Boivin and Mesrobeanu (1935) used





63

trichloroacetic acid for extraction of O-antigenic complex

containing LPS, protein, and lipids. Morgan (1937) used

diethylene glycol, while phenol water extraction was first

tried by Palmer and Gerlough (1940) to extrace O-antigenic

preparation from Bact. typhosum. Morgan and Partridge

(1941) used the same method for extraction of LPS from

B. dysenteriae (Shiga). This method was modified by

Westphal and Jann (1965) to isolate lipopolysaccharide

from both rough and smooth types of bacteria. The problem

of contamination with ribonucleic acid was solved by

purification methods involving ultracentrifugation and

precipitation with quarternary ammonium compounds. It

was reported that ribonucleic acid was not extracted as

a contaminant along with LPS if the bacteria were pre-

treated with trichloroacetic acid (O'Neill and Todd, 1961)

or formaldehyde (Jeanloz, 1960).

The lipopolysaccharide of gram-negative bacteria were

able to coat erythrocytes from various species of animals

and produce hemagglutination in the presenceof reactive

antibodies (Neter, 1956). Passive hemagglutination and

hemagglutination inhibition tests were used for the study

of serological specificities of O-antigens of gram-negative

bacteria (Neter, 1956; Beckmann, Subbaiah and Stocker,

1964). Modification of LPS by either heat or alkali

treatment results in better absorption of antigen on the

surface of erythrocytes (Neter, Westphal, Luderitz,

Gorzynski and Eichenberger, 1956).




64
It was suggested that lesions produced in infectious

bovine keratoconjunctivitis might be due to the toxins

produced by Moraxella bovis (Jackson, 1953; Seth and

Chandrasekariah, 1956; Henson and Grumbles, 1961; and

Pugh, 1969). Henson and Grumbles (1961) demonstrated

two types of toxins produced by N. bovis in chick embryo

cultures. The hemolytic toxin was reported to be very

labile and cell-associated, while dermonecrotic toxin

was suggested to be a nonspecific endotoxin like that of

other gram-negative bacteria. Pugh (1969) exposed cattle,

rabbits, guinea pigs and embryonating chick eggs to whole

culture or growth products of M. bovis. The animals which

were given intravenous or intraperitoneal injections died

within 24 hours, while those given subcutaneous, intra-

dermal or intraocular injections produced severe local

reactions. A potent endotoxin was thought to produce

these effects.

In antigenic analysis of a gram-negative bacterium,

the study of its somatic antigen becomes necessary since

it forms one of the major portions of the cell and is

important antigenically. This study was undertaken to

isolate and characterize MoraxeZla bovis lipopolysaccharide

(endotoxin) in terms of its serological specificity and

its capability to produce biological reactions in

experimental animals.







Materials and Methods

Isolation and purification of lipopolysaccharide

MoraxeZZa bovis isolants, five in S and three in R

form, were examined for lipopolysaccharides or endotoxins.

The organisms were grown on blood agar plates (BAP) con-

taining 5% sheep blood. Fifty BAP (150 x 100 mm) were

inoculated with 24 hour old growth on BAP for each type

and incubated at 37 C for 24 hours. The plates were

examined for contamination, R-S variation and purity by

gram stain. The growth from uncontaminated plates having

pure R or S colonies was harvested in physiological saline

solution (PSS) containing 0.3% formaldehyde solution and

centrifuged at 1500 G for 2 4 hours. After pouring off

the supernatant fluid, the cells were washed twice with

PSS and three times with acetone. The pellet from the

final acetone wash was spread on the sides of the con-

tainer and dried at room temperature. The dried cells

were scraped off, ground, and sieved through double

thicknesses of gauze to remove pieces of agar.

The cell powder was weighed, suspended in 20 ml of

distilled water per gram of bacteria and maintained at 67

C in a water bath. Twenty ml of phenol (about 90%) were

brought to the same temperature and added to the cell

suspension. The mixture was vigorously stirred for 15

minutes at 67 C and was cooled to 10 C in an ice bath.

The emulsion-like material was centrifuged at 3000 G for

one hour. The upper aqueous layer was taken off and the






remaining material was again extracted with 20 ml of

distilled water. The aqueous layers were pooled individually

for each type of organism and dialyzed against distilled

water at 4 C for five days to remove phenol. The dialyzed

material was freeze-dried for purification.

The lyophilized LPS was dissolved in distilled water

to give an approximately 3% solution. This solution was

centrifuged at 90,000 G for eight hours in a Beckman

"Spinco" preparative ultracentrifuge. The supernatant

fluid was taken off and the semitransparent pellet was

resuspended in distilled water. This was centrifuged

again at 105,000 G for four hours. This step was repeated

two more times and finally the pellet was dissolved in

10 ml of distilled water.

The lipopolysaccharide was further purified by pre-

cipitation with quartenary ammonium compound-hexadecyl-

trimethyl ammonium bromide. To 10 ml of LPS solution,

1.0 ml of 2% aqueous solution of hexadecyltrimethyl

ammonium bromide was added and the mixture was stirred

on a magnetic stirrer for 20 minutes at room temperature.

After centrifugation at 3,000 G to remove precipitated

ribonucleic acid, sufficient sodium chloride was added

to the supernatant to provide 0.5 M NaC1. This solution

was poured into a tenfold volume of cold ethanol and kept

overnight at 0 C. The resulting precipitates were

collected by centrifugation at 13,820 G for 30 minutes,

dissolved in 5 ml of distilled water and dialyzed against







deionized water for 3 4 days to remove sodium chloride.

The dialyzed solution was lyophilized for use in the tests.

The purity was determined by the UV absorption spectrum

(Graham, 1965) and immunodiffusion tests.

Passive hemagglutination and hemagglutination-inhibition

test

Preliminary trials with heated and alkali-treated

LPS indicated that heated LPS antigen gave higher titers

in passive hemagglutination tests than alkali-treated

LPS. In all the subsequent tests heated LPS was used as

an antigen to coat sheep erythrocytes (RBC). Sheep blood

was collected in sterilized Alsever's solution aseptically.

The cells were centrifuged at 3,000 G for 10 15 minutes

and washed three times with hemagglutination (HA) buffer,

Finally the RBC's were suspended in 1A buffer to 4%

suspension.

All the antisera for use in this test were obtained

by procedures mentioned previously in Section 2. These

were inactivated at 56 C for 30 minutes and were absorbed

with packed washed sheep red cells for two hours at 37 C.

The cells were removed by centrifugation at 3,000 G for

20 minutes and the absorbed antisera were stored at -60

C until further use. To prepare heated antigen for

coating RBC's, 5 mg of LPS was dissolved in 5 ml of PSS

and was heated for two hours in a boiling water bath.

The mixture was centrifuged to remove precipitates and

was dialyzed against HA buffer overnight. Two ml of heated







antigen were diluted to 10 ml with buffer and to this,

2.5 ml of 4% RBC suspension was added. The mixture was

incubated for one hour at 37 C with occasional shaking.

After incubation the cells were centrifuged, washed three

times with buffer and finally were suspended in 20 ml of

buffer (Neter et aZ., 1956; Lindberg and Holme, 1968).

For the passive hemagglutination test, serum was

diluted in twofold dilutions ranging from 1:5 1:1280.

To each tube containing 0.2 ml of diluted serum, 0.2 ml

of sensitized RBC suspension was added. The contents were

mixed by shaking and incubated at room temperature.

Hemagglutination in the tubes was read after four hours

and 18 hours. The hemagglutination titer was determined

as the last tube showing macroscopically visible hemag-

glutination. The controls consisted of sensitized RBC

suspension in HA buffer, normal rabbit serum with sensi-

tized cells and test serum with unsensitized RBC's. Each

test was run in duplicate. The replicates did not deviate

more than one dilution.

The hemagglutination-inhibition test was done using

heated LPS as inhibitor in varying amounts (Lindberg and

Holme, 1968). The serum used in the system was diluted

to contain four hemagglutination units (diluted four times

less than the highest dilution showing hemagglutination).

The inhibiting LPS was diluted in serial twofold dilutions

in concentrations ranging from 400 3.125 ig/ml. To 0.2

ml of inhibiting LPS dilution, an equal volume of appro-

priately diluted serum was added and the tubes were





69

incubated in water bath at 37 C for 30 minutes. Sheep RBC

sensitized with homologous LPS in 0.2 ml volume were added

to each tube and the mixture was incubated at room tempera-

ture. The tubes were read after four and 18 hours. The

minimum amount of LPS producing complete inhibition of

hemagglutination was recorded. Controls similar to those

used in the passive hemagglutination test were run along

with each test.

Immunodiffusion test

This test was done by the method of Ouchterlony

(1953). Glass slides (25 x 75 mm) were used. One percent

acetone-purified Noble agar was melted in PSS and 1.0 ml

of merthiolate solution (1:100) was added as a preserva-

tive. Ten ml of melted agar was poured on a frame of

three slides and was allowed to set for one hour. The

wells were cut using an immunodiffusion punch set (No.

71692)*, with a little modification to cut the center well

bigger (6 mm) than eight surrounding wells. After the

reactants were placed in the wells, the slides were kept

in a moist chamber for a period of 24 48 hours. When the

precipitin bands had developed (generally after 48 hours),

the slides were washed, dried and stained with amido black.

Generalized and local Shwartzman reactions

Lipopolysaccharides obtained from both R and S types

of isolant FLA-264 (Ivan Park) were used to study Shwartzman

reactions (Good and Thomas, 1953; Thomas, 1954).


*Gelman Instrument Company, Ann Arbor, Michigan.






Two rabbits (New Zealand white) were used for each

variant and one rabbit was kept as a control for the study

of generalized Shwartzman reaction. Initially 0.10 mg

of LPS/kg body weight was given intravenously as the first

injection, but subsequently the quantity was reduced to

0.05 mg/kg because 0.10 mg/kg killed rabbits within 24

hours of the first injection. The second injection was

also given intravenously 24 hours after the first injection

and in the same amounts as the first. The animals that

died after the first or second injection were necropsied

and tissues (lung, liver, spleen, kidneys and small

intestine) taken for bacteriologic and pathologic

examinations.

To study the local Shwartzman reaction, the backs

of rabbits were clipped and shaved. The first injection

was given intradermally at two sites. Three-tenths milli-

gram of LPS was dissolved in 0.6 ml of sterilized distilled

water and filter-sterilized through a 0.22 p Millipore

filter. At each site 0.1 0.2 ml was injected. The

second injection was given intravenously in amounts of

0.05 mg/kg body weight, 24 hours after the first injection.

The control rabbit was given 0.2 ml of sterilized distilled

water at each site intradermally while the second injection

was the same as in test rabbits. The skin lesions were

examined 4 to 12 hours after the second injection.

Specific inhibition of the local Shwartzman reaction

was shown when the first injection consisted of LPS with





71

and without homologous antiserum. Five-tenths milliliter

of LPS solution containing 0.5 mg of LPS was incubated

with 0.5 ml of homologous antiserum for 30 minutes at

37 C. The mixture was centrifuged to remove any precipitates

and was filter-sterilized. Intradermal injections were

made at two sites in an amount of 0.2 ml at each, while

LPS alone was injected at two other sites in the same

rabbit. The second injection was made in the same manner

as in the local Shwartzman reaction. The skin lesions

were examined 4 to 12 hours after the second injection.

Toxicity in mice

Due to small amounts of LPS available, the LD50 in

mice was noL attempted for each isolant. Lipopoly-

saccharides from rough and smooth forms were pooled

separately and injected to mice intraperitoneally.

Sixty-five mice (15 20 g) were used as 13 groups of

five mice in each group. Six groups were injected with

pooled LPS from R types, six groups with LPS from S

variants, while one group (control) was injected with

sterilized PSS. The amount of LPS injected was 500, 400,

300, 200, 100, and 50 pg/mice in each group. The animals

were observed for a period of five days.







Results

Purity

The purity of LPS preparations was tested by their

UV absorption spectra. The crude preparations (before

ultracentrifugation) showed a peak at 260 my (Figure 19-A)

indicating contamination with nucleic acid, although

the bacterial cells were treated with formaldehyde before

extraction (Jeanloz, 1960). This peak was eliminated

after ultracentrifugation and precipitation with hexa-

decyltrimethyl ammonium bromide (Figure 19-B). The puri-

fied LPS also gave a single band of precipitin against

homologous antiserum in the immunodiffusion test (Figure 20).

Passive hemagglutination and hemagglutination inhibition

tests

Heated LPS was more efficient in the passive hemag-

glutination test and gave higher titers than alkali-

treated LPS. The heterologous and homologous hemagglutina-

tion titers varied from 1:80 to 1:640 indicating little

serological differences in the LPS preparations of different

isolants, both R as well as S type (Table 2). Results of

hemagglutination-inhibition of homologous LPS-antiserum

system with homologous and heterologous LPS are shown in

Table 3. The amount of heterologous LPS (pg/ml) inhibiting

the system varied from one-half to two times the amount

of homologous LPS. Serologically there was no significant

difference in the LPS preparations of rough and smooth

types of the same isolant of M. bovis. At the same time








0.35


0.30


0.25


0.20


0.15


0.10


0.05


0.00





0.25


0.20


0.15


0.10


0.05


0.00


240 250 260 270
WAVE LENGTH (mi)
A.


280 290


280 290


UV absorption spectrum of MoraxeZZa bovis
lipopolysaccharide (FLA-264, rough).
A. Crude preparation. B. Purified lipo-
polysaccharide (0.1 mg/ml of distilled water).


240 250 260 270
WAVE LENGTH (mi)
B.


Figure 19.




Table 2. Passive hemagglutination of Moraxetla bovis antisera (rough and smooth)
with Moraxella bovis lipopolysaccharides

Lipopoly- Reciprocal of titers* of antisera to Moraxella bovis isolants
saccharides
from isolant FLA-264 IBH 68 (712L) FLA-Vet.l 8613 (Md.) ATCC
(Ivan Park) 10,900
R S R S R S S S


FLA-264
(Ivan Park) R 160 160 160 160 80 160 160 160

S 320 320 160 160 160 160 160 320


IBH 68
(712L) R 160 160 160 320 80 80 160 160

S 320 160 320 320 80 160 160 320


FLA-Vet.l R 320 320 320 640 640 320 320 320

S 320 320 320 640 160 320 320 320


8613 (Md.) S 160 160 160 320 160 160 160 320


ATCC 10,900 S 160 160 160 320 80 80 160 320


*The titer was determined as the highest dilution of serum, before adding lipopoly-
saccharide-sensitized erythrocyte suspension, giving visible hemagglutination.




Table 3. Hemagglutination-inhibition of MoraxeZZa bovis antisera with homologous
and heterologous lipopolysaccharides.
Hemagglutination-inhibition of the system* in Vg/ml of
Inhibiting inhibiting lipopolysaccharide
lipopoly-
saccharide FLA-264 IBH 68 (712L) FLA-Vet.l 8613 (Md.) ATCC
(Ivan Park) 10,900
R S R S R S S S


FLA-264
(Ivan Park) R 50 200 50 50 25 25 50 12.5

S 100 100 50 50 50 25 100 12.5


IBH 68
(712L) R 100 100 50 50 50 25 100 25

S 50 100 50 50 25 25 100 12.5


FLA-Vet.l R 50 50 25 50 25 12.5 50 6.25

S 50 50 25 25 25 12.5 50 6.25


8613 (Md.) S 50 50 50 25 25 12.5 50 6.25


ATCC 10,900 S 50 50 25 25 25 6.25 25 6.25


*Hemagglutinating system: homologous lipopolysaccharide (sensitized erythrocytes) and
antiserum (4 8 hemagglutinating units). Data recorded are minimal concentration
(pg/ml) of inhibiting lipopolysaccharide needed for complete inhibition of the homo-
logous system.















































Figure 20. Immunodiffusion analysis of Moraxella bovis
lipopolysaccharide. Center well contained
lipopolysaccharide from isolant FLA-Vet.l
(R). Peripheral wells contained antisera
against various isolants: 1. FLA-264 (R),
2. FLA-264 (S), 3. IBH 68 (R), 4. IBH 68
(S), 5. FLA-Vet.l (R), 6. FLA-Vet.l (S),
7. 8613 Md. (S), and 8. ATCC 10,900 (S).







no serological difference was detected in the lipopoly-

saccharides of different isolants. Similar results were

obtained in the immunodiffusion analysis when different

LPS preparations were tested with R and S type antisera.

A single band of precipitin developed which showed com-

plete identity with different antisera prepared against

R and S types of various isolants (Figure 20).

Shwartsman reaction

When the first injection was given in amounts of

0.10 mg LPS per kg body weight by the intravenous route,

both rabbits given R as well as S lipopolysaccharides

died within 24 hours after the first injection. These

animals showed symptoms and lesions similar to that pro-

duced by typical endotoxin lethal shock. The symptoms

which appeared after a latent period of about 30 minutes

consisted of listlessness, ruffled hair coat, redness of

conjunctiva, peripheral vascular dilatation, dyspnea,

diarrhoea and prostration before death. Histopathologi-

cally, the visceral organs (liver, lungs, spleen, small

intestine and kidney) showed edema, congestion and

hemorrhages. The liver showed centrilobular necrosis

and there was degeneration of some tubules in the renal

cortex.

In a second trial to produce a generalized Shwartzman

reaction, the amount of LPS was reduced to 0.05 mg/kg body

weight. One animal which was injected with LPS from

FLA-264 (Ivan Park) survived after a second injection.




78

This animal was killed 36 hours after second injection and

necropsied. Both the animals showed degeneration and

necrosis of tubules in the renal cortex in addition to

edematous and hemorrhagic lesions in liver, lung and

spleen.

A typical local Shwartzman reaction was produced in

rabbits by M. bovis lipopolysaccharides. After 4 to 12

hours of intravenous injection, the area at the intra-

dermal injection site showed hemorrhagic necrosis (Figure

21). Specific inhibition of the local Shwartzman reaction

was shown when first injection consisted of LPS incubated

with homologous antiserum (Figure 21).

Lethality

Although LPS of MoraxclZa bovis produced irreversi-

ble lethal shock in rabbits during attempts to produce

the generalized Shwartzman reaction, it was not lethal

to mice even in amounts of 500 pg per mouse when injected

intraperitoneally. The animals showed listlessness, red-

ness of conjunctiva, rapid breathing and peripheral

vascular dilatation, but recovered in one to two hours

after the injection.












































Local Shwartzman reaction produced by
Moraxella bovis (isolant FLA-264-R) lipopoly-
saccharide (endotoxin) in a rabbit. The
reaction was inhibited when lipopolysaccharide
was incubated with homologous antiserum prior
to intradermal injection.


Figure 21.


Xln/






Discussion

Various isolants of a species can be differentiated

by biochemical characteristics, antigenic relationships

or phage susceptibility. Gram-negative bacteria, parti-

cularly enteric, have been classified into different

serotypes on the basis of serological specificity of their

somatic or O-antigen (and H-antigen if motile).

According to cultural, morphological and biochemical

characteristics, different isolants used in this study

constitute a homogeneous group except for catalase reaction

(isolant FLA-264 is catalase positive while all others are

catalase negative). Although Pugh (1969) reported the

presence of common as well as nonidentical antigens in

bacterial lysates of different isolants on the basis of

the immunodiffusion tests, the differences in somatic

antigen have not been studied in detail in different

isolants of Moraxella bovis. The study of somatic antigen

was also related to endotoxin and colonial variation from

rough to smooth types on an artificial medium. The

possibility of an endotoxin was suggested by various

workers (Henson and Grumbles, 1961; and Pugh, 1969), but

it was not isolated or characterized. Colony dissociation

was observed by Barner (1952) after four months of multiple

transfer on a solid culture medium. The basis of this

variation was not known and it could have been due to the

differences in the LPS structure as already known in the

enteric gram-negative bacteria. Endotoxin was isolated





81

and purified in order to study its serological specificity

in an effort to show antigenic relationships among

different isolants of M. bovis, and to explain R/S varia-

tion on the basis of differences in lipopolysaccharide.

Some serological studies of Moraxella species were reported

by Haug and Henriksen (1969a,b) using agglutination and

agglutination absorption tests, but these tests were

found unreliable because of interference due to auto-

agglutination of M. bovis antigen (Pugh, 1969). The

passive hemagglutination test was used for antigenic

studies of LPS from many gram-negative bacteria, including

Neisseria and Hemophilus species (Neter, 1956).

During this study LPS was extracted by the phenol-

water method (Westphal and Jann, 1965) both from rough

as well as smooth types of M. bovis. The UV absorption

spectra of crude and purified LPS preparations showed that

nucleic acid was still extracted even if the cells were

treated with formaldehyde before extraction. This is in

contrast to Jeanloz (1960) who reported that contamination

of LPS with nucleic acid could be avoided if bacterial

cells were pretreated with formaldehyde. Ultracentri-

fugation and precipitation with hexadecyltrimethyl

ammonium bromide was still required to obtain a pure

preparation of LPS. In passive hemagglutination and

hemagglutination inhibition tests, heated LPS was used as

an antigen to coat sheep erythrocytes, as it was found

more efficient and sensitive than alkali-treated LPS

(Neter et al., 1956).







The serological tests show that LPS from different

isolants do not have different serological specificities,

indicating that there are common somatic antigens in the

isolants used in this study. Unlike the colony dissociation

in enteric organisms, there does not seem to be any

difference in lipopolysaccharides of rough and smooth

types of 1. bovis. This dissociation may be due to pili

which are present on the surface of R type cells but

are absent on the smooth variant of some isolants as will

be discussed in a later section.

It appears from this study that rabbits are much more

susceptible to the lethal action of LPS than mice. An

amount of 0.1 mg of LPS per kg body weight was lethal to

rabbits when given by intravenous route, while mice were

resistant to even 0.5 mg per mouse (20 25 grams body

weight). Henson and Grumbles (1960b) reported similar

results. Mice were resistant to intraperitoneal injections

of inactivated culture fluid of M. bovis. This is also

in agreement with Ribi et al., (1959) who reported that

rabbits were about 1000-fold more susceptible to

the lethal effect of endotoxin than mice. The symptoms

and lesions shown by rabbits were indistinguishable from

those produced by endotoxins of other gram-negative bacteria

(Thomas, 1954).

MoraxeZZa bovis LPS produced Shwartzman reactions

similar to the other endotoxins. The dermal Shwartzman

reaction was inhibited when LPS was treated with homologous





83

antisera, indicating that toxicity of LPS can be neutralized

by homologous antibodies as shown by Radvancy, Neale and

Nowotny (1966).

It has been suggested that the lesions produced in

infectious keratoconjunctivitis may be due to toxins

produced by Ioraxella bovis, and lesions similar to

keratitis and corneal opacity were produced by intraocular

injection of viable and nonviable cultures of N. bovis

(IIenson and Grumbles, 1960b; and Pugh, 1969). The lesions

could be due to LPS if injected intraocularly, but it is

difficult to imagine that these lesions are produced by

LPS in natural infection, particularly when this organism

does not appear to invade tissue. Possibly, endotoxin

may be one of the factors which make the tissues more

susceptible to the action of a virulent or toxic factor

as hemolysin or a proteinase produced by M. bovis. It

has been reported that mice become more susceptible to

experimental infection with even avirulent strains of

gram-negative bacteria when given concomitant inoculation

of endotoxin (Rowley, 1956).







Summary

Lipopolysaccharides were isolated from rough and

smooth variants of different isolants of Moraxella bovis

by phenol-water method. They were purified and character-

ized by serological and biological reactions. Serologically,

no difference was found in the lipopolysaccharide pre-

parations from rough and smooth types of different

isolants as indicated by immunodiffusion, passive hema-

gglutination and hemagglutination-inhibition tests. Colony

dissociation (R-S) in M. bovis is probably not due to

lipopolysaccharide. The isolants used in this study

constitute a homogeneous group with regard to lipopoly-

saccharide or somatic antigen. Local and generalized

Shwartzman reactions were elected by LPS of M. bovis

in a manner similar to the endotoxin of other gran-

negative bacteria. Inhibition of local Shwartzman

reaction was shown by homologous antisera. Rabbits were

found to be much more susceptible to the lethal action

of this lipopolysaccharide, while mice were resistant

to as much as 500 pg of LPS per mouse.













SECTION 4

HEMOLYSIN OF MORAXELLA BOVIS

Introduction and Review of Literature

Although MoraxeZZa bovis has been considered as the

etiological agent of infectious bovine keratoconjunctivitis,

the mechanism by which this organism causes disease is

still unknown. It is well known that the endogenous and

exogenous metabolic products of different microorganisms

play an important role in their ability to produce disease

in a susceptible host. The production of cytolytic toxins

by bacteria has been associated with virulence, although

it is true that bacterial products such as hemolysins

and enzymes are produced by both pathogenic as well as

nonpathogenic bacteria. As products of pathogenic

bacteria, the extent to which these may be associated with

the mechanism of disease is not understood, but the

possibility that these do contribute to the disease cannot

be overlooked.

Velde reported in 1894 that a filtrable toxin of

staphylococci produced hemolysis. The culture filtrate

of these bacteria could lyse rabbit but not human erythro-

cytes (Kraus and Clairmont, 1900). The hemolysin was

thermolabile and was called alpha-hemolysin (Wilson and

Miles, 1964). Beta-hemolysin, another toxin produced by

85





86

staphylococci was demonstrated by Bigger (1933). It lysed

sheep, ox and human erythrocytes but not rabbit. The

hemolysis was produced when the tubes were kept at 4 C

after incubation at 37 C ("hot-cold lysis"). Morgan and

Graydon (1936) reported a third heoolysin fronn staphylococci

which caused lysis of erythrocytes of varion: species of

animals but not the horse. A fourth hcaolysin, delta--

toxin, was demonstrated by William and IIar *-. (1947).

Two kinds of hemolysins produced by sti-ptococci

were demonstrated by Todd (1934). O-streptolysin was

oxygen labile but could be reactivated by reduction with

0.1% sodium hydrosulfite, while S-lysin was oxygrn stable

and could be easily extracted from streptococci by

shaking with serum. Bernheimer (1948) reported that S-

lysin was a protein and antigenic when present in the

bacterial cell, while O-lysin produced hemolysis rapidly

and was antigenic in the free state.

The hemolytic activity of CZostridium perfringens

was found to be due to lecithinase (Macfarlane and Knight,

1941). Alpha-toxin was thermolabile and hemolytic for

the erythrocytes of most animal species, but not of the

horse and goat.

Widholm (1953) demonstrated that E. coli hemolysins

could not be separated from bacterial cells by filtration.

This nonfiltrable hemolysin was heat-labile. Snyder and

Koch (1966) demonstrated the production of both filtrable

and nonfiltrable hemolysins by E. coZi. These hemolysins







were heat-labile and could be inactivated by formalin

treatment.

Hemolysins have been reported from a variety of

bacteria, including leptospira (Russell, 1956), Vibrio

cholerae (Watanabe and Seaman, 1962) and Listeria

monocytogenes (Njoku-Obi, Jenkins, Njoku-Obi, Adams and

Covington, 1963).

Early investigators observed that MoraxclZa bovis

produced beta-hemolysis on blood agar plates (Jones and

Little, 1923; and Barner, 1952). Henson and Grumbles in

1961 demonstrated the presence of hemolysins in chick

embryo cultures of M. bovis. The hemolytic toxin was

heat labile and was inseparable from the viable bacterial

cells. There was complete loss of activity by formalin

treatment or by filtration through a Seitz EK pad.

The purpose of the present investigation was to

characterize hemolytic toxins of MoraxeZla bovis produced

in whole broth medium and in cell-free culture filtrate.







Materials and Methods

Hemolysin assay procedure

The activity of hemolysin was determined by its

lytic action on sheep erythrocytes. Sheep blood was

collected aseptically in sterile Alsever's solution.

The blood was centrifuged at 3,000 G for 10 15 minutes

to collect the cells. The red blood cells were washed

three times with PSS containing 0.01 M calcium chloride

which was also used for the dilution of hemolysin and

suspension of erythrocytes. A 1% final suspension of

washed erythrocytes was used.

The assay was made by preparing twofold dilutions of

hemolysin ranging from 1:2 1:1024. An equal amount of

1% sheep erythrocyte suspension was added to 1.5 ml of

each dilution of hemolysin. The tubes were shaken and

incubated at 37 C for four hours followed by 12 18

hours at 4 C. The tubes were agitated and centrifuged

in a clinical centrifuge at full speed for 10 minutes.

The hemoglobin content in the supernatant fluid was

determined by optical density at 540 mu on a Spectronic

20*. A tube containing 1.5 ml of the medium (when hemo-

lysins were determined in the culture medium) or 1.5 ml

of PSS 0.01 M CaC12 plus an equal quantity of 1%

erythrocyte suspension was included in each test to

serve as a control.

The hemolytic activity was measured in units. A

series of dilutions of 1% sheep erythrocytes were made in


*Bausch and Lomb, Rochester, New York.





89

distilled water, keeping the final volume as 4.0 ml in each

tube as given in Table 4. These tubes were incubated and

their optical densities recorded in the same manner as

in the experimental trials. A standard curve (Figure 22)

was constructed from an average of 10 different runs by

plotting percent hemolysis against optical density. The

straight line to 75% hemolysis showed a direct relation-

ship between optical density and percent hemolysis. One

hemolytic unit (HU) was arbitrarily defined as that amount

of hemolysin which would produce 1% hemolysis (lyse 1% of

erythrocytes) in a standard suspension. The units of

hemolysin in a particular sample were calculated by

multiplying the reciprocal of hemolysin dilution by the

hemolytic units indicated in that dilution. The dilution

having an optical density of approximately 0.575, i.e.

50% hemolysis, was used to calculate hemolytic units.

Production of hemolysin

Two isolants of N. bovis (IBH 68 [712L] and FLA-264

[Ivan Park]) were used for hemolysin production. Both R

and S types of each isolant were studied. Hemolysin was

produced in 100 ml of trypticase soy broth which was

dispensed in nephloflasks. The inoculum was prepared by

growing R or S types of organisms in 10 ml of trypticase

soy broth. These cultures were incubated at 37 C for

48 hours after which they were kept at 4 C. Before inocula-

tion the cultures were shaken and 2 ml was used to

inoculate 100 ml of medium. Duplicate flasks for each type




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