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This is to certify that the

thesis entitled

APPLICATION OF ENZYME-LINKED IMMUNORSSAY AND
A MODIFIED GROWTH MEDIUM FOR AVIAN MYCOPLASMA

 

presented by

ASHRAF A. ANSARI

has been accepted towards fulfillment
of the requirements for

Ph.D. degreein POULTRY SCIENCE

 

 

Date Agril 22, 1980

0-7639

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.. “1 Place in book return to remove
:‘Ml’ . charge from circulation records

‘..4

 

 

 

 

© 1980

ASHRAF IBRAHIM ABOULATTA ANSARI

All Rights Reserved

APPLICATION OF ENZYME-LINKED IMMUNOASSAY AND A MODIFIED

GROWTH MEDIUM FOR AVIAN MYCOPLASMA

 

By

Ashraf A. Ansari

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Poultry Science

1980

ABSTRACT

APPLICATION OF ENZYME-LINKED IMMUNOASSAY AND A MODIFIED
GROWTH MEDIUM FOR AVIAN MYCOPLASMA

 

By
Ashraf A. Ansari

A medium was developed for growth and antigen production of avian

Mycoplasma. This modified Mycoplasma medium was named MSU-Mycoplasma

 

 

 

medium (MSU-medium). The medium contains Gibco Mycoplasma media, maltose

 

(or dextrose), L-histidine, cystine HCl, Waymouth medium, tris buffer,
penicillin, thallous acetate, phenol red and horse serum. Nicotinamide
adenine dinucleotide was added only to the media for the growth of M:

szpoviae (Ms). This medium was used for Hycoplasma gallisepticum (Mg)

 

antigen production for hemagglutination-inhibition (HI), tube agglutina-

tion test and Mycoplasma-Enzyme~Linked Immunosorbent Assay (MCYO—ELISA).

 

Comparative studies were conducted between MSU-medium and brain-heart
infusion rmxfiimn PPLO medium, tryptose phosphate broth, B-medium of
Fabricant and modified Frey medium. All media were inoculated with Mg_
organisms, and samples were taken every 4 hours for the first 56 hours

and at 3, 4, 5, 6, 7, 9, 30 and 60 days post-inoculation (P1) to determine
the pH and the total bacterial counts. A significant correlation between
bacterial counts and pH was demonstrated. The Mycoplasma organisms were
isolated from HSU-medium 30 and 60 days PI and the pH readings were 6.5

and 6.5 at 30 and 60 days PI. According to the results, the MSU-medium

Ashraf A. Ansari

is superior to other media which were compared for the growth of avian

Mycoplasma based on bacterial yield and the survival time of the

 

microorganisms in the media.

The identification of avian Mycoplasma species has been very

 

difficult because of the nature of the organisms. Cross reactions in
serological tests have.been reported by many authors. .Fluorescence
antibody technique was used for identification of avian Mycoplasma

in broth culture by direct and indirect methods. However, FA has some

limitations for expedient identifications of Mycoplasma organisms,

 

especially in mixed cultures. More research work is required to prepare
the specific labeled antisera to avoid non-specific reactions.

For this reason, protein A-Sepharose chromatography was employed
to isolate and purify IgG from normal and hyperimmune chicken and
rabbit sera. IgG molecules of subclasses l, 2 and 4 of mammals and
their fragments containing the Fc region have been‘reported to have the
affinity to bind to protein A. This activity to the IgG Fc fragments
has been shown to be correlated with IgG H-chain type. The two fractions
obtained by washing protein A-Sepharose 4 B column chromatography sen-
sitized by normal and hyperimmune chicken sera were identified by using
Ouchterlony immunodiffusion. The 0.1 M_sodium phosphate buffer, pH 7.0,
eluate contained all immunoglobulin fractions including IgG antibodies.
The l M_acetic acid, pH 7.0, eluate did not contain any chicken IgG.
It is believed that the non—binding properties of Fc region of chicken
IgG are because of the absence of some of the C-terminal amino acids
which are responsible for binding process in mammals. Another possible

reason for the non-binding properties was that other protein is present

Ashraf A. Ansari

in chicken serum which binds with protein A molecule and, therefore, the
binding process with IgG molecule is inhibited.

The enzyme immunoassay technique for the detection of avian Mycoplasma

 

antibodies of chicken was first adopted in the MSU Avian Microbiology Labora-

tory. The Mycoplasma-Enzyme-Linked Immunosorbent Assay (MYCO-ELISA) antigen

 

was prepared from Mg_whole bacterial cell or its disrupted cell suspension.
The affinity of antigen for binding or adsorption to the surface of poly-
styrene walls of microtiter plates was demonstrated by using 0.1 H_carbonate-
bicarbonate buffer, pH 9.6. Ninety micrograms of antigen protein per well
and 1:50 serum dilution were used to give high absorbance reading of 405 nm
(using spectrophotometer) as compared to the reading of control serum.

The conjugate consisted of IgG fraction of rabbit anti-serum and chicken
IgG labelled with horseradish peroxidase enzyme (HRP). 2,2' Azino di {3
ethyl-benzthiazolin-sulfonate (6)} was used as a substrate. A temperature
of 22 C (room temperature) for 2 hours was selected for routine incubation
of serum and conjugate. The results indicated that there was no cross

reaction between serum of Mycoplasma and serum of other bacterial or

 

viral disease infection. However, cross reactions on a large scale
between positive serum of flg_and Ms were observed.

A comparison study between HI and MYCO-ELISA test was conducted by
using sera obtained from naturally and experimentally flg_infected chickens.
The results indicated that the absorbance reading of positive sera gave
much higher ELISA titer than the HI titer. It is believed that enzyme
immunoassay can be the future method used to achieve a successful eradi-

cation program against Mycoplasma infections because of its simplicity,

 

reproducibility, sensitivity and the automation potential for a large

scale diagnosis in the laboratory or in the field.

Dedicated to the two men who have
contributed to my existence in

this life. My father, "Ibrahim”

the wise, the kind and the saint.

3Q; uncle, VAbdeZ-Daiem" the ambitious,

the disciplinary and the rich.

ii

ACKNOWLEDGMENTS

The author has often wondered why others used so much space and
time writing something rarely read: his acknowledgments. Now, after
having experienced the job of writing, publishing, meeting other
-scientists and industrial people and owing so much of his success to
the literally hundreds of people who gave of time, experience, support
and most important of all faith and hope, it is difficult to know
where to begin or stop.

First, I must thank my committee members for allowing so much
latitude and for their continued support and encouragement. I am
deeply thankfu1 to Dr. T.S. Chang for his guidance and personal support
during the course of this study and in the preparation of this dissertation,

Special thanks are due to Dr. R.F. Taylor, one of the very few
truly outstanding people I have come across during the several years of
my graduate and professional education, for his constructive guidance,
his never-ending words of encouragement and his special advice. His
inspiration, friendship and hospitality which he gave during the most
critical stage of this research will be always remembered. The critical
discussions and the learning experiences I gained without any doubt
contributed to my development as a researcher.

I deeply appreciate Dr. T.H. Coleman for his kindness, support and
the unlimited help in the preparation of this dissertation, his valuable

advice and the constructive conversation which was very rewarding at

iii

the end. Also, I am thankful to Dr. A. L. Rogers for his critical
evaluation of the research work and the dissertation. His encourage-
ment and friendship will be always remembered.

Special thanks to the late Dr. H. C. Zindel, Chairman of the
Department of Poultry Science, and his staff for providing the finan-
cial support and facilities which made this research possible and my
academic sojourn fruitful.

I wish to thank the people of the USDA-Regional Poultry Research
Laboratory, East Lansing, Michigan, for letting me borrow many chemical
and biological reagents which were indeed very valuable in speeding
up my research, especially Drs. E. Smith and A. Fadly. Also, I would
like to extend my thanks to Dr. W. L. Smith, Department of Biochemistry,
and Dr. H. C. Miller, Department of Microbiology and Public Health,
for their permission to use their research facilities.

The help which has been given by Dr. R. D. Angus, USDA-Veterinary
Service Laboratory, Ames, Iowa, in providing serum samples and M122;
plasma organisms and overall the valuable phone conversations are
deeply appreciated.

Special thanks to Mr. S. J. Hulkonen for preparing part of the
photographic work and Mr. P. Bowen and his crew, Messrs. V. Thurlby,

W. Blair, and W. Rhodes, in helping me keep and maintain my animals

at the Poultry Research Farm, MSU. The friendship which developed with
the people who shared the office and the laboratory with me, Mrs. B. J.
Scarbrough, I. R. Sutton and Messrs. M. A. McKinney and M. J. Gregoricka,
are unforgettable. Also, my sincere thanks to Mrs. V. E. Ross for her

support and cooperation. I would also like to express my thanks to

iv

Mrs. M. J. DeYoung, the librarian, for letting me occupy a table in
the library for three months without complaint.

My special thanks to my fellow members of the Poultry Science
Club of Michigan State University, especially G. A. DeVlaminck, G. H.
Carpenter, and W. H. Rhodes, for cheering me up and sharing a lot of
thoughts, feelings and memories and, most important of all, attending
the SOutheastern Poultry Convention at Atlanta, Georgia.

My most sincere thanks to my dear friend A. H. Golian for his
encouragement, moral support and his invaluable friendship. Also, my
thanks to Mrs. J. G. Gruber and Mrs. B. A. Fisher for typing this
dissertation under the usual pressure of a deadline.

My thanks extend to my family and friends in the USA and in the
old country, Egypt, without whose support the completion of my
Doctorate would have been impossible. They gave me hope and ambition
to pursue and to achieve a dream that has been with me for a long
time.

Finally, my acknowledgments will not be complete until I express
my most sincere thanks and admiration to the man who gave me the
precision of a scientist, the strong feeling toward life, and the
imaginations of an immigrant to America. I am forever indebted to a
very special man who had an invaluable influence on my development

and growing as a person. Thanks to you, Professor J. F. Williams.

TABLE OF CONTENTS

LIST OF TABLES. .
LIST OF FIGURES .

INTRODUCTION.

LITERATURE REVIEW .

051w?

Pleuropneumonia-like Organism (PPLO)

L—phase Organisms and Mycoplasma .
Classification of Mycoplasma .

Avian Mycoplasma . . .

l. Mycoplasma‘gallisepticum (Mg).

2. ‘Mycoplasma meleagridis (Mg).

3. Mycoplasma synoviae (Ms)

Protein A Chromatography .

1. Introduction .

2. Physical and Chemical Properties

3. Biological Properties.

4. Applications

Enzyme-Linked Immunosorbent Assay.

1. Introduction . .

2. Assays, Principles and Methods . .
3. Preparation and Characterization of ELISA
Reagents . . . .
Characterization of Enzyme- immunoassay .
Practical Application of ELISA .

Uses of ELISA in Veterinary Medicine .
Uses of ELISA in Avian Medicine.

 

 

 

 

 

\lO‘Mb

MATERIALS AND METHODS .

’ A.

B.

J

Mycpplasma Organisms . .

1. Source of the Organisms. . .

2. Growth and Maintenance Procedures.

3. Storage of the Organisms .

Culture Media. .

1. Media for Antigen Production . .

2. Media for Isolation, Growth and Maintenance.

3. A Modified Medium for Isolation and Antigen
Production (MSU-Mycoplasma Medium) .

4. Experimental Studies on the Effect of pH of
Differenti Mycoplasma Media on the Survival of
the Organisms.

 

 

 

vi

Page

ix

S6

56
56
56

7
58
58
58

58

61

J

RESULTS .

J

C.

/
/
/

D.

A.

Mycoplasma Antigen Production and Standardization.
1 Preparation of Mycoplasma Cells.

2 Antigen for Immunization . .
3 Antigen for Hemagglutination Inhibition Test .
4. Antigen for Enzyme- Linked Immunosorbent Assay.
P

l

2

3

 

reparation of Fluorescent Antibody Reagents .
Production of Rabbit Antisera.
Production of Chicken Antisera .
Conjugation of Rabbit IgG Fraction with
Fluorescein Isothiocyanate .
4. Fixation Procedures for Direct and Indirect
Methods. .
5. Direct Staining.
6 . Mi cros copy .
7. Indirect Staining.
Immunochemical Studies on Purification of Chicken
and Rabbit IgG Using Protein A- -Sepharose Chroma-
tography . . . . . . . . . . . .
Source of Protein A. .
Isolation and Purification of Rabbit IgG .
Immunodiffusion Tests for Rabbit Serum .
Isolation and Purification of Chicken IgG.
Immunodiffusion Tests for Chicken Serum.
Plasminogen and Their Affinity to Protein A.
Mycoplasma- Enzyme-Linked Immunosorbent Assay
(MYCO- ELISA). .
l. Antigen Production, Harvesting and Standardi-
zation . .
Source of Serum Samples. .
Preparation of Horseradish Peroxidase Conjugate.
Preparation of the Substrate .
MYCO-ELISA Protocol.

O‘U’IAMNi—I

 

(HAWK)

Cultural Media . .

1. Growth and Survival of Mg in Different Broth
Media. .

2. Changes of the pH of Different Mycoplasma Media.

3. Preservation of Mycoplasma Organisms . .

4. Standardization of Mg Antigen. . .

Immunofluorescence Identification of Mycoplasma

Isolates .

1. Antibody Titer of Rabbit and Chicken Antisera.

2. Direct Fluorescence Method . . . . .

3. Indirect Fluorescence Method . .

Purification and Assaying IgG by Protein A- Sepharose

Chromatography .

1. Purification of Rabbit IgG by Protein A.

2. Immunodiffusion Analysis for Rabbit Serum.

3. Purification of Chicken IgG by Protein A .

 

 

 

vii

Page

62
62
65
65
65
66
66
68

69

72
72
73
73

74
74
74
75
76
76
78

78

78
79
79
80
80

89
89

89
96
96
102

103
103
103
103

106
106
109
109

U14:-

Immunodiffusion Analysis for Chicken Serum .
The Effect of Chicken Plasminogen on Isolation
of Chicken IgG by Protein A-Sepharose
Chromatography . . . . . . .

D. Standardization of MYCO- ELISA.

l.
tration and Serum Dilution . .
2. Determination of the Optimum Conjugate
Concentration. .
3. Determination of the Optimum Temperature and
the Length of the Incubation Period. . .
4. Determination of the Specificity of MYCO- ELISA .
5 Determination of the Sensitivity of MYCO- ELISA .
DISCUSSION.
I A. Cultural Media . .
~/ B. Standardization of.Mg'Antigen. . .
C. Identification of Mycoplasma Isolate by Immuno-
fluorescence Methods .
l. Antisera Production. . .
2. Immunofluorescence Diagnosis for Mycoplasma.
0. Purification of IgG by Protein A .
E MYCO-ELISA . . . . .
APPENDIX.
LITERATURE CITED. . . . . . . . . . . . . . .,. . .

Determination of the Optimum Antigen Concen-

 

 

viii

Page
114
121
126
126
131
134
141
142
146

146
150

150
150
151
153
156

165

177

Table

LIST OF TABLES

Immunization schedule for rabbits.

Comparative study on the total bacterial count and
the pH of different_Mycop1asma media .

Comparative study on the total bacterial count and
the pH of differenthycoplasma media .

Comparative study on the total bacterial count and
the pH of different Mycoplasma media .

 

Comparison of antibody titer for Mg by HI and
MYCO-ELISA tests . . . . . . . . . . . .

Serotypes of avian Mycpplasma.

 

Avian Mycoplasma: serotypes, origin and pathogenicity .

 

Classification of avian Mycoplasma on biochemical and
serological bases.

 

ix

Page

67

90

91

97

143
171

172

174

Figure

10

11

12

13

14

15

16

LIST OF FIGURES

The competitive method of ELISA for assaying antigen .

The double antibody sandwich ELISA for measuring
antigen. . . . . . . . . . . . . . . . .
The indirect ELISA for measuring antibody.

Possible mechanisms of cross-linking reactions .

Spectral emission of HBO 200 W superpressure mercury
lamp (nm = mu)

a. Spectral transmission of exciter filters
b. Spectral transmission of barrier filters

Principle of MYCO-ELISA.
MYCO-ELISA procedures.

The total bacterial count (Log No CFU/ml) for Mg in
different media recorded in hours post-inoculation .

The total bacterial count (Log No CPU/ml) for Mg in
different media recorded in days post-inoculation.

The changes of the pH of different media for Mg by
hours post- inoculation . . . . . . . . . . .

The changes of the pH of different media for Mg by
days post- inoculation. . . . . . . .

Direct immunofluorescence staining of Mg organisms
showed a specific yellow-green fluorescence.

Control (negative) fluorescence fbr Mg organisms
stained with labeled heterologous antibody showed
no specific fluorescence .

Chromatography of hyperimmune rabbit serum on a
protein A-Sepharose column .

(38b) Immunodiffusion of rabbit immunoglobulin
fractions.

Page

43

44
45

49

175

176
176

82

83

93

99

100

105

105

108

111

Figure Page
17 Immunodiffusion of different rabbit IgG fractions. . . . 113

18 Chromatography of chicken hyperimmune serum on a
protein A-Sepharose column . . . . . . . . . . . . . . . 116

19 (aGb) Immunodiffusion of chicken immunoglobulin
fractions. . . . . . . . . . . . . . . . . . . . . . . . 118

20 (aab) Immunodiffusion of different chicken IgG

fractions. . . . . . . . . . . . . . . . . . . . . . . . 120

21 a. Immunodiffusion of rabbit IgG fraction . . . . . . . 123
b. Immunodiffusion of protein A, 1M acetic acid

eluate of chicken serum. . . . . . . . . . . . . . . 123

22 (aGb) Immunodiffusion of chicken immunoglobulin
against rabbit anti-chicken plasminogen. . . . . . . . . 125

23 Determination of Mg antigen concentration for MYCO-
ELISA using standard dilution of 1:200 HRP conjugate
and 1:50 reference control serum . . . . . . . . . . . . 128

24 Determination of the optimum serum dilution in
MYCO-ELISA using standard dilution of 1:200 HRP
conjugate and 90 ug antigen protein. . . . . . . . . . . 130

25 The effect of different concentrations of MSU-HRP
conjugate on the specific activity of MYCO-ELISA
using standard 1:50 reference control serum and 180
ug antigen protein per well. . . . . . . . . . . . . . . 133

26 The effect of different concentrations of Cappel-HRP
conjugate on the specific activity of MYCO-ELISA using
standard 1:50 reference control serum and 180 ug
antigen protein per well . . . . . . . . . . . . . . . . 136

27 The effect of different temperature of incubation of
serum and conjugate on the specific activity of MYCO-
ELISA using standard dilution of 1:50 reference control
serum dilution, HRP-conjugate dilution of 1:100 and
2 hours of incubation. . . . . . . . . . . . . . . . . . 138

28 The effect of various time of incubation at room
temperature (22 C) on the absorbance readings of
MYCO-ELISA using 1:50 and 1:500 positive serum control
dilution at MSU-HRP dilution of 1:100. . . . . . . . . . 140

29 The absorbance readings for MYCO—ELISA at 405 nm for
positive and negative serum for Mg at different
dilutions using standard 180 ug Ehtigen concentration
and 1:100 conjugate dilution . . . . . . . . . . . . . . 145

xi

INTRODUCTION

Chronic respiratory disease (CRD) had been an important flock
problem in chicken and turkey production in all areas of the United
States (Yoder, 1978 a). It appears to be world wide in distribution.
A drop in egg production of 10 to 20% over a period of two or four
months or longer, retarded growth and carcass condemnation constitute
the biggest loss to the poultry industry.

Isolation and identification of the causative agent from birds

infected with Mycoplasma is not always successfully achieved. Sero-

 

logical tests routinely used to detect subclinical and chronic myco-
plasmosis among susceptible flocks are not satisfactory. Opinions have
differed considerably about the specificity of the antigen used. Cross
reaction with other antibodies from related (either pathogenic or non-
pathogenic) microbes has been frequently reported. Furthermore,

Mycoplasma infections can develop slowly and may not be detected for

 

several weeks or months by currently and routinely used serological
tests. Therefore, a rapid and specific serologic test is needed for
diagnosis, since the National Poultry Improvement Plan for Eradiation
of mycoplasmosis in breeder flocks has been approved.

The first objective of this dissertation study was to develop a

culture medium for isolation and maintenance of avian Mycoplasma. This

 

medium was also used for Mg_antigen production for tube agglutination

test, hemagglutination-inhibition test and Mycoplasma-Enzyme-Linked

 

Immunosorbent Assay (MYCO-ELISA Test).

The second objective of this study was to establish a diagnostic
procedure for Mg_infections in chickens.
1. Develop immunofluorescence method to detect and differ-
entiate Mg from other species of Mycgplasma.
This method has the distinct advantage of rapid identification

.and determination of Mycoplasma. Pure culture of Mycoplasma

 

 

organism is not required.

2. Immunochemical studies to isolate and purify IgG fraction

from chicken serum using protein A-Sepharose column chromatography.
Protein A is a protein isolated from the cell wall of

Staphylococcus aureus. This protein A has the ability to interact

 

with a wide variety of IgG molecules to the Fc region in mammals.
No research has been recorded in the literature to isolate chicken
IgG by protein A chromatography. This purified IgG may be used in
preparation of the conjugate in immunofluorescence methods.

3. Apply the enzyme-linked immunosorbent assay method for

serological diagnosis of Mycoplasma infections in chickens.

 

Enzyme immunoassays are believed to be the simplest and a promising
serological technique that can be successfully applied in a wide range
of diagnosis and epidemiological investigations. This assay depends
on the assumption that either an antigen or antibody can be linked to
an enzyme and the complex will have both immunological and enzymatic

activities.

LITERATURE REVIEW

It is generally believed that Louis Pasteur first recognized that
a specific and very small microorganism caused contagious bovine pleuro-
pneumonia, although he was unable to isolate or to see the organism.
However, it was not until 1898 that Nocard and Roux accomplished the
first isolation of the microbe in a very interesting and ingenious way.
They filled collodion sacs with sterile broth media inoculated with lung
fluid from infected animals. The sacs were then placed in the peritoneal
cavity of a rabbit and left for several days. They soon were able to
grow the organisms in serum enriched broth ip_yi£393 but it was not until
1900 that Dujardin-Beaumet described colonial growth on a solid medium.
He showed that colonies were characterized by a dark center, as the
result of penetration of the organism into the medium, and a light
periphery. Later on, Bordet (1910) and Borrel e£_§l, (1910) described
in.detail the morphological appearance of thesepneomorphicorganisms
and later Elford (1931) showed the existence of viable forms 125 to
150 my in size, using gradacol filter. For this reason, as well as
many others, the organisms were thought by many to be a virus and,for

25 years, occupied this anomalous position.

A. Pleuropneumonia-like Organism (PPLO)
The accumulation of large amounts of serous fluid in the lungs and
pleural cavities as a result of the infection- by filterable organisms
gave rise to call those organisms Pleuropneumonia-like Organisms (PPLO),

which was primarly caused contagious bovine pleuropneumonia (Sabin, 1934,

Lu

\/

Foster, 1934).
Infectious sinusitis of turkeys was first recorded by Dodds (1905),
which appeared to be the first disease described in poultry caused by

what is known now as Mycoplasma. Graham Smith (1907) stated that the

 

sinus exudate from those birds was infectious, although the organisms
ordinarily isolated from it failed to produce sinusitis. These obser-
vations indicated the possibility that an ultramicroscopic agent was
the cause of the disease. Tyzzer (1926) was the first scientist to
report sinus infections in turkeys in the USA and he experimentally
used argyrocol for treatment.

During the following decade, the majority of researchers believed
that a virus (filterable agent) was the etiological agent responsible
for respiratory infections in turkeys and chickens. /The organism was
first described by Nelson (1935L who referred to the organisms seen in
embryonated eggs as coccobacilliform bodies. Also, Van Herick and Eaton
(1945) encountered filterable organisms in chicken embryos by cultivation
of the organisms on an enriched agar and demonstrated for the first time
that avian PPLOiarganisnm agglutinated chicken red blood cells and that the
immune system specifically inhibited the hemagglutination. Markam and
Wong (1952) demonstrated that PPLO organisms that did grow in all-free
media induced typical swelling and exudate from turkey sinus and air
sac infection and chronic respiratory disease (CRD) in chickens. Numerous
strains of coccobacilliform organisms (PPLO) isolated from chickens and

turkeys of different serotypes were subsequently described by Adler eg_

31: (1957). Adler §£_a13 (1958) isolated two species of PPLO from

chickens and turkeys. One of these PPLO organisms did not affect chickens

and has not been found in that species. However, PPLO in turkeys was not
taken seriously until Yamamoto (1965, 1966) demonstrated conclusively
that what was called at that time PPLO turkey sinusitis agent caused

air sacculitis in turkeys and he reported as Adler §£_§l, (1958) had,

the egg transmission cycle of the microbes. The researcher named this

organism M, meleagridis (MM) (Yamamoto g£_§l,, 1965).

 

Another PPLO infection was observed by Olson §£_al, (1954) and
Wills (1954) in broiler flocks in West Virginia and Texas. The symptoms
were described as swellings of the joints, caseous exudate accumulations
(arthritis) and, frequently, hepatosplenomegaly. The agent grew in
chicken embryos and produced lesions similar to those produced by CRD
agent. Later on, the organism was first cultivated by Lecce (1960) as

satellite colonies around a Staphylococcus. Chalquest and Fabricant

 

(1960) were able to grow PPLO in pure culture by replacing the

Staphylococcus with diphosphopyridine nucleotide or nicotinamide

 

adenine dinucleotide (NAD). They confirmed that the etiological agent
for the disease was PPLO organism after several ip_xi££g culture passages.
A typical joint lesion resulted after inoculation of the organisms into
chickens foot pads(Olsen §£_al,, 1964). The name M. synoviae (Mg)

was prOposed for the infectious synovitis agent.

B. L-phase Organisms and Mycpplasma

 

L-phase was defined as independent growth variants of bacteria that
lack a rigid cell wall and have a potential reversibility to the original
form (Klieneberger, 1931). Also, he reported the growth of an organism

resembling PPLO in a pure culture of Streptobacillus moniliformis.

 

Klieneberger (1931) first considered this to be a mixed culture of the

bacterium and a contaminant PPLO, but Diens and Edsall (1937) established
that these colonies which were called L-forms were derived from the
bacterium and not related to PPLO organisms. “McKay and Taylor (1954)

and Kelton and Gentry (1957) were the first people to postulate that PPLO
organisms isolated from chronic respiratory diseases in chicken were
actually L-form bacteria. During that period, the PPLO organisms were
included in order Mycoplasmatales (Freund, 1955) family Mycoplasmataceae
on the basis that all PPLO organisms require sterol for growth (Freund,
1955). Rogul g£_gl, (1965) stated that avian Mycoplasma (Avian PPLO)
were not derived from bacteria and the bases of DNA compositions (the
molecular percent of guanine plus cystine (G + C) in the DNA) and genetic
compatability or molecular hybridization indicated no relationship

between the two strains of avian Mycoplasma (M, gallisepticum and M:

 

 

gallinarum) and L-form of Haemophilus gallinarum. Also, the L-form could
be formed by many other species when wall synthesis was impaired by
penicillin and high salt concentration (Thomas, 1973). Hijmans e£_al:
(1969) published a list that contained 37 different bacterial species

which had been reported in the literature to grow as L-phase.'Hwn'indicated
that L-phase was generally not considered to be pathogenic, even when

it derives from a pathogenic bacterial phase. However, McKay 35.31.

(1966) and Hijmans §£_a13 (1969) reported that L-phase Haemophilus

 

parainfluenza, Salmonella urium andlfilndt>cholera were pathogenic.

 

 

C. Classification of Mycoplasma

Mycoplasmas appear to be ubiquitous in nature, occurring in both

 

animal and plant kingdoms. MycoplasM§_was recorded to be under class

Mollicutes (Edward and Freundt, 1967). The old name for this class

was Paramycetes,by Sabin (1941). Order Mycoplasmatales (Freundt, 1955)
was divided into two families, Mycoplasmataceae, which required sterol
for growth, and Acholeplasmataceae,which didn't require sterol for
growth. Family Mycoplasmataceae contains 37 known and documented species
(Freundt, 1973). This class was recorded to be prokaryotic organisms
bounded by a single 'triple-layered membrane; they lack cell wall precur-

sors such as muramic and diaminopimelic acids.

0. Avian Mycoplasma

 

The first clinical case describing an infraorbital sinusitis of

turkeys by Dodd (1905) was apparently caused by Mycoplasma E2: The

 

viral etiology remained unchallenged for years while research in this
disease continued. In 1952, the so-called "air-sac infection" and
chronic respiratory desease emerged as a serious problem, particularly
in broiler chickens and turkeys. The incredible amount of research
done by Nelson (1935) on coccobacilliform' coryza was completely over-
looked for many years. He succeeded in isolation of coccobacillary
bodies from chickens and showed upper respiratory disease signs of
unknown etiology. The organisms were propagated in tissue culture,
embryonating chicken eggs and on cell-free media. It was classified
by Smith g£_§1, (1948) on the basis of electron micrographic morphology
and cultural characteristics. The organism was placed in what was at
that time called PPLO organism, according to Sabhfs (1939) definition of

PPLO organisms. During the last 20 years, many aspects of Mycoplasma

 

infections in poultry have been investigated and published.

Avian Mycoplasma Serotyping

 

Many investigators attempted to classify various avian Myc0plasma

 

isolates by serologic techniques. Yamamoto and Adler (1958 a,b) char-
acterized 5 serotypes. Kleckner (1960) described 8 serotypes designated
A through H including the previous 5 serotypes described by Yamamoto and
Adler. Yoder and Hofstad (1964) characterized 12 serotypes (A through
L) and Dierks g£_§l, (1967) Eharacterized 19 serotypes (A through 5).

However, the three pathogenic serotypes A, H, and S remained separated

and distinct from the other serotypes,which represent M, gallisepticum

 

(Mg), M, meleagridis (MM) and M, sypoviae (Mg). Different serotypes of

 

avian Mycoplasma by different authors are summarized in Tables 5, 6, and 7

 

in the Appendix. The combination of serotypes I, J, K, N, Q and R into
one antigenically related group was in agreement among all authors. How-
ever, the fact that the metabolic inhibition test used by Barber and
Fabricant (1971a) was more reliable than complement fixation made their
classification more accurate than other tests, indicated by Frey

e£_gl, (1972). In addition to the species and serotypes in Table 5,
practically every investigator has added additional unclassified avian

Mycoplasma,which represented a problem in identification. Serotyping,

 

origin and pathogenicity of avian Mycoplasmaeumasummarized by Yoder

 

(1978),as shown in Table 6 in the Appendix.

Characterization of Avian Mycoplasma

 

 

Classification and characterization of avian Mycoplasma was an

 

obvious problem to many researchers for many reasons,as summarized by

Fabricant (1969).

(1)

(3)

Primary isolation of avian Mypoplasma often yields more than

 

one species from infected birds (Fabricant, 1960; Yoder and
Hofstad, 1964).

Definite identification of different Mycoplasma gp, depends on
the proper biochemical and serological tests applied and
standardized testing procedures (Fabricant and Freund, 1967).
Serologic tests performed using serum containing antibodies

against different species of Mycoplasma (pathogenic and

 

nonpathogenic) are of very limited value for identification

of Mycoplasma sp, isolates.

 

Characterization of avian Mycoplasma was mainly based on serotyping

I“ :

 

“ and biochemical reactions,as presented in Table 7 (Appendix). These

biochemical characterization studies were carried out by many authors

(Fabricant, 1969; Yoder, 1975, 1978a; Freundt, 1974).

These procedures of identification were built on:

(1)

(2)

(4)

Arginine decarboxylation: Indicated by alkaline change (deep
red color) in liquid basal medium with 1% arginine and phenol
red at a pH of 7.0.

Dextrose fermentation: The production of acid in liquid basal
medium supplemented with 0.5% dextrose (glucose) and phenol
red at pH of 7.6.

Tetrazolium reaction: Indicated by changes from colorless

to red in liquid basal medium containing 1 to 10,000 tetrazolium
red (2, 3, S-triphenyLZH-tetrazolium choloride). Some avian
Mycoplasma species will reduce the tetrazolium chloride which
acts as a growth indicator.

Hemagglutination: Using chicken or turkey red blood cells

19

depends on the source of the serum.

(5) Diphospho-pyridine nucleotide (DPN) or nicotinamide adenine
dinucleotide (NAD) requirements: M§_was the only avian M122;
plasma requiring NAD for growth (Chalquest and Fabricant,
1960).

(6) Serum requirements: The addition of 10-15% heat inactivated
serum (horse, swine, turkey or chickens) in the medium was

required by all avian Mycoplasma species. M, anatis (Roberts,

 

1964) and M, laidlawii var inocuum (Adler 95, 31,, 1961) did

 

grow on serum enriched media. But whether or not they required

serum needs more investigation.

Mycoplasmas

 

1. Mycoplasma gallisepticum (Mg)

 

Avian serotype A (Kleckner, 1960), avian serotype 5 A-5969, 801,

6’
X95 and others (Adler ggflgl., 1958) were described extensively in the
literature. This organism was the primary etiological agent of chronic
respiratory disease of chickens and turkeys. The disease in turkeys had
also been commonly called infectious sinusitis of turkeys. However, the
field condition popularly known as ”air sac” disease or complicated CRD
was not due to a Mg infection alone. Complicating infections which were
designated as airsacculitis and which produced fibrinous or fibrino-
purulent pericarditis and perihepatitis along with the massive airsacculi-
tis were responsible for the severe mortality. The economic impact of

the disease,represented in reduced feed conversion and decreased egg

production, weight loss, increased medication costs and downgrading of

carcasses and excessive condemnathn1rate at slaughter, made this disease

11

one of the major disease problems of the American broiler industry.
a. Chemical Compositions:

(1) Whole cell: Except for some variations, the Mycoplasmas re-

 

sembled other bacteria in their growth compositions. KThey lacked the
bacterial cell wall components such as muramic acid and diamino-pimelic
acid (Razin, 1969). Morowitz §£_gl, (1962) and Razin E: El: (1963)
studied the chemical composition and the microsopic morphology of Mg_
(Avian PPLO 5510:5969). A packed cell suspension contained approximately
80% water,which meant that the cell contains 20% dry weight. Protein
constituted about 80% of the dry weight. Total lipid determination
uniformly ran at between 10% and 12% of the dry weight. Carbohydrate
was usually less than 1%; RNA and DNA were 8.3% and 4% from the dry
weight of the whole cell, respectively (Morowitz g£_§l,, 1962). Gen-
erally, there was no great variation in the chemical composition of the
organisms of different strains except that most pathogenic strains had
a somewhat higher lipid content than the saprophytic strains. The Mg

contained a smaller amount of total carbohydrates than other Mycoplasmas

 

(Rauin g£_§l,, 1963).

(2) The Genome: Electron microscopy of the Mycoplasma gallisepticum

 

showed the absence of nuclear membrane (Morowitz g£_gl., 1967). The
genome size of two Mg was 1200 x 106 daltdns (Morowitz g£_g1,, 1967).

Other Mycoplasma species had a similar result. However, the Mycoplasma

 

 

genome was much smaller that that of the average bacteria; the genome

size of E, coli,for instance, was 2500 x 106 daltons. On the other hand,

the Mycoplasma genome was far larger than the genome of the virus, but

 

may be comparable to that of the bedsonia organisms (PLT group), or the

12

trachoma agent, the size of which was estimated at 600 x 106 daltons
(Razin, 1969).

(3) The Ribosomes: The ribosomes were similar to bacterial rib-
osomes, but showed some difference in base composition and sedimentation
behavior. Five percent of the total RNA in the cell was messenger
fractions (Morowitz SE.§l:: 1967). The RNA protein ratio was about 60:40
compared with about 40:60 in eukaryotic cell ribosomes. Electrophoresis
in acidic polyunnlamide gels separated 20 protein bands compared with 28
bands in E, gpli_ribosomes and the sedimentation of the ribosomal RNA was
approximately 166 (Kirk, 1966). The ribosomes were arranged in cylindrical
or helical corncob structures of over 50 ribosomes each (Maniloff §£_gl,,
1965,and Domermuth g£_§1,, 1966). 'The biophysical similarity between

the Mypoplasma and prokaryotic cell ribosomes was also apparent. Chloram-

 

phenicol and erythromycin, which specifically inhibited protein synthesis
on the 70 S microbial ribosomes, effectively inhibited protein synthesis

in many Mycoplasma including Mg (Tourtellotte §£_§l,, 1967). RNA was

 

synthesized by a DNA dependent RNA polymerase, and its synthesis can be
effectively inhibited by actinomycin D.

(4) Cell membrane: Mg, as well as other Mycoplasma, does not have

 

a cell wall or intracytoplasmic membrane; it possesses only plasma
membrane. In thin section, the plasma membrane seems to be composed of
two electron — dense layers with a less dense layer in between (Van Iterson
and Ruys, 1960). The three-layered structure corresponds to the unit
membrane which covers cells and organelle of various origins.

The plasma membrane can be separated from the cell, either by osmotic

lysis (Rottem 23 a1,, 1968) or by mechanical pressure using sonic or

13

ultrasonic oscillators (Pollack, §£_g1,, 1965). However, Mg_resisted the
osmotic lysis by the usual procedures (Rottem g: g£., 1968,and Robrish

and Marr, 1962). The washed Mycoplasma cells were suspended in a 2 M_'

 

glycerol solution, incubated for 10 minutes at 37 C and then rapidly
injected into deionized water. Glycerol freely penetrated through the
cell membrane and increased the internal osmotic pressure. The rapid
injection of glycerol-loaded cells into deinoized water caused rapid
penetration of water molecules, resulting in the sudden swelling of the
cell, rupture of the cell membrane, and releasing of the cell contents
into the medium.

The membrane consisted of 50-60% protein, 30-40% lipid and a very
little amount of carbohydrate (Razin §£_§l,, 1963). The major portion
of the lipids (60%) was phospholipids. Cholesterol and cholesterol
esters made up to about 18.8% of the total lipids. The controversial
issue of what is the structure and the organization of the protein and
lipids in the membrane still exists. The two main theories were that
the membrane is built of a bimolecular leaflet of lipid coated on both
sides with protein (Danielli and Davson, 1935) or lipoprotein subunits
(Green §£_§l,, 1967). Chu and Horne (1967) described a surface projec-

tion, resembling those of myxoviruses on negatively stained Mg, Mycoplasma

 

membranes were recently found to be solubilized by a variety of detergents,
anionic, cationic, nonionic and sodium dodecyl sulfate (SDS) with SDS
being the most effective (Rottem §£_gl,, 1968). SDS bound to different
molecular weight of protein to form protein 835 complex and to lipid to
form fat-SDS complex. However, a striking feature was discovered by

Razin g£_§1, (1965),in that the detergent solubilized membrane material

had the ability to reaggregate spontaneously to membrane—like structures

14

upon the removal of the detergent. The reformed membrane in this section
was shown to consist of typical triple-layered unit membrane about as
thick as the original one (Terry §£_§l,, 1967). Also, Magnesium (Mg++)
or some other divalent or polyvalent agents,but not the monovalent, were
essential in this aggregation.

b. Growth Requirements:

Mg and the rest of the Mycoplasma group were of special interest,

 

because they were the smallest organisms capable of growing in cell free
media. Mg required a rather complex enriched media with 10-15% heat in-
activated horse, swine or avian serum. The culture media available at
present are not entirely satisfactory. Most workers would agree that

more avian Mypoplasma gp, may be discovered if the quality of the present

 

media can be improved. Perhaps the most distinctive nutritional require-

ments of Mycoplasma are lipids and lipid precursors,especially cholesterol,

 

which incorporate in forming the plasma membrane. Serum, Tween 80 and
TEM-4T (diacetyl tartaric acid ester of tallow monoglycerides),which
contain fatty acids in various forms (saturated and unsaturated} are

found to be suitable for the growth of avian Mycoplasma (Lund and Shorb,

 

1966). Amino acid requirements have been found to be absolute for all
except glutamic and aspartic acids and cystine. Carbohydrate requirements
as a source of carbon and energy had been provided in monosaccharide form
as dextrose (Rodwell, 1969).

Vitamin requirement had been determined for a few strains of avian

Mycoplasma. Nicotinic acids, thiamine, nicotinamide adenine dinucleotide

 

(NAD or Coenzyme I, formerly DPN) and riboflavin are required for the

group of Mycoplasma (Tourtellotte §£_§l,, 1964). M§_ is unable to synth-

 

esiZe NAD from nicotinic acid or nicotinamide either because of the poor

15

penetration of the NAD into the cells or the absence of a nicotinamide
adenine dinucleotidase (Chalquest, 1962). The role of urea in metabolism
is still under investigation. Comparison of different media used for
isolation and antigen production has been recorded (Adler ggflgl., 1957;
Taylor gg_gl,, 1957; Fabricant, 1958; Ferey §£_§l,, 1968). Media for
isolation and maintaining have been developed by Adler g£_gl, (1954;
Yamamoto and Bigland (1964); Yoder and Hofstad (1964); Frey §£_gl, (1968);
and Yoder (1975).
c. General Morphology and Staining

The organisms in general were described by electron microscopy as
being spherical or elongated (tear drOp) in shape with a diameter of
0.15u - 0.5u (Morowitz §£_§l,, 1962). Maniloff and Morowitz (1967) studied
the ultrastructure and ribosomes of Mg,’flunrindicated that a Mycoplasma
cell is composed of 3 organelles; the cell membrane, the ribosomes and
the nucleoid.

(1) Membrane: The triple 3 layered plasma membrane had the

thickness of 110 A structure.

(2) Ribosomes and ribosomal structure: The cylindrical ribosomal

arrays,showing irregular packing, filled the intracytoplasmic space

between the nuclear area and the unit membrane. The RNA-protein

ratio of 0.68 was within the 0.6 to 1.7 range found for isolated

ribosomes and detached microsomal RNA particle.

(3) The nuclear material, DNA: It was in the center part of the

posterior section and had a fibrillar appearance.

(4) The bleb and infra-bleb regions: The bleb was described as

one of the very characteristic features of Mg_and so far this region

16

has never been found in any other Mycoplasma species. The bleb,
shaped approximately like an oblate ellipsoid, measured about

800 A by 1.300 A, excluding the bounding membrane. The bleb region
was reported to contain high amounts of lipids.

In summary, Mg differed from other Mycoplasma in several morpholo-

 

gical properties: a) the cell possessed a bleb which contained no nucleic
material, but protein and lipid and the function of the bleb are not yet
clear (infectious role), b) the ribosomes were arranged in helical
(polysome-like)arrangement, corncob gtructure; c) they were not
elongated or coccid, but they resembled a tear drop or a coca-cola
bottle, with the bleb in the top.‘ The Giemsa stained preparations of
isolates showed that the cells were coccoid forms and were approximately

0.4 p in diameter.

d. Modes of Replication: Mycoplasma, in general, have been reported

 

to have more than one mode of cell replication, depending on the various
cell and the environmental conditions. Kelton (1962) and Morowitz and
Maniloff (1966) showed that Mg multiplied by simple binary fission
(formation of two identical cells). Domermuth g£_§l, (1964) indicated

that Mg produced elementary bodies at one locus of the Mycoplasma fila-

 

ments. Several authors have suggested that Mycoplasma reproduced by

 

budding (Liebermeister, 1960; Dutta g£,g1., 1965).

e. Colonial Morphology: Mg_can grow either on serum—enriched agar or

broth medium. The initial isolates of Mycoplasma have been described

 

to grow better in broth than on agar media. Colonies of different sero-

types are different in sizes and heights of their centers. In general

17

the size of Mycoplasma is approximately 0.2-0.4 mm; however, it can be

 

as large as 0.8 mm such as serotype F (Yoder and Hofstad, 1964). The
colonies appeared to be tiny, smooth, circular, translucent masses of

dense raised central area with greater optical density (fried egg colonies).

f. Biochemical and Biological Properties: Mg_ferments glucose, maltose
and mannose with the production of acid, but not gas. It does not fer-
ment lactose, mannitol, dulcital, salicin, arabinose, xylose, trehalose,
rhamnose, sorbitol, abonitol or raffinose. Fermentation results are
varied with sucrose, galactose, fructose, and inulin (Dierks 33.31,,
1967; Yoder and Hofstad, 1964). The 86 isolate utilizes sucrose, whereas
strain A 5969 does not (Adler, 1970). Mg reduces 2, 3, 5, triphenyl-
tetrazolium chloride;therefore,it is incorporated into medium as a growth
indicator. All strains of Mg_hemagglutinate with red blood cells of
chicken and turkey as well as other mammalian and avian erythrocytes.

The hemagglutinating activity of Mg is attributed to neuraminidase and
neuramfifidase-like enzyme (Gesner and Thomas, 1966; Roberts, 1967). They
presented evidence that N-acetyl-neuraminic acid or a closely related
sialic acid provided binding sites for Mg at the erythrocyte surface.
The situation appeared analogous to hemagglutination by influenza A

virus and Myxoviridae group, where neuraminic acid provides sites for
virus attachment. Furthermore, the pathogenicity of Mg_may be attributed
to the function of a neuraminidase-like enzyme. Mg was shown to be
hemolytic, which meant that after attachment to the erythrocyte, the
organisms produced peroxide, changed the hemoglobin to methemoglobin and

led to lysis of the red blood cells (Thomas and Bitensky, 1966).

18

g. Physical Properties: The Mycoplasma are readily disrupleby mech-

 

anical means such as sonic and ultrasonic disintegrators and French
press. Razin 23.3}: (1964) found that pathogenic Mg_cultures are

resistant to osmotic effects while the nonpathogenic Mycoplasma are

 

susceptible. The osmotic lysis is heat-dependent. Mg can stand glycerol
osmotic shock, and has been stored for years in 50% buffered glycerol at
-40C to -60C without loss of viability. Most strains of Mg can be kept
for long periods of time in the frozen state at -60C in yolk, or broth
media, but not in albumen, serum, saline, feces, feather meal, chicken
muscle and muslin (Chandiramini gpflgl., 1976). Yodi g: 31' (1973)
indicated that adding sucrose was beneficial for lyophilization. Further-
more, no changes in antigenicity or pathogenicity took place during
storage for a month at -20C. Adler (1970) reported that lyophilization
in either milk, yolk, or serum broth was the most satisfactory method for
storage. Yoder and Hofstad (1964) indicated that the organisms remained
viable for fourteen years. They also found that stored broth cultures
remained viable for 2-4 years. Fabricant (1973) recovered the organism
from 60%, 35% and 13% of infected materials stored at 25 C for l, 2
and 3 years, respectively. Olesivk and Van Roekel (1952) reported that
infective allantoic fluid remained infective 4 days in the incubator, 6
days at room temperature and 32-60 days in the refrigerator.

No work has been done with the effectiveness of disinfectants or

other chemicals on Mycoplasma. However, it has been known for some

 

time that the Mycoplasma can be killed by the disinfectants. Mycoplasma

 

 

has been reported to be resistant to penicillin and thallium acetate

(Yoder, 1978).

19

h. Pathogenicity

The pathogenicity of Mg in different hosts varies according to the
nature of isolate, method of propagation, and number of passages before
it is inoculated into the host (Yoder, 1978). Also, the route of infec-
tions, the number of organisms, the presence of secondary infections, the
type of host, and other environmental factors, including stress and
vaccinations, affected the pathogenicity of the microbe. Roberts (1964)
indicated that the pathogenicity of Mg_could be attributed to the neur-
aminidase-like enzyme. However, this enzyme was reported to be in non-

pathogenic strains of Mycoplasma as well.

 

(1) Embryonated chicken eggs. Inoculation of broth cultures with
exudate containing Mg in seven—day-old embryonated chicken eggs via the
yolk sac route usually resulted in embryo deaths within five to 10 days
(Yoder and Hofstad, 1964). Stunting of the embryos, subcutaneous hemor-
rhages, and generalized edema were frequently noted. Hepatitis and
splenomegaly were the most obvious lesions (Van Roekel g£_§g,, 1952).
The periarthndar-abscesses were creamy-white to yellow nodules which
occurred in the subcutaneous areas adjacent to the mandible, wing, hip,
stifle, hock, and toe joints (Chute, 1960; Yoder and Hofstad, 1964).
However, Yoder and Hofstad (1964) stated that such lesions were produced
by serotypes of Mycoplasma other than Mg, Also, it is interesting that
isolates from trachea of chickens inoculated into chicken embryos pro-
duced "joint abscess" (Calnek and Levine, 1957; Yoder and Hofstad, 1964).
However, they failed to demonstrate that known pure isolates of Mg_
(serotype A) were capable of producing subcutaneous periarticular gran-

ulomas in chicken embryo.

20

(2) Chickens. Mg_produced a severe extensive airsacculitis and
caseated exudate in the air sac (Dierks §£_§l,, 1967). However, Yoder
and Hofstad (1964) indicated that moderate airsacculitis was produced
only occasionally, and that moderate to severe tendo-vaginitis was pro-
duced two to three weeks poSt inoculations. Also, they reported that
seven birds out of 19 developed airsacculitis lesions and 14 out of 22
developed tendo-vaginitis lesions. Yoder and Hofstad (1964) reported
high titer of antibody and formation of severe lesions after inoculation
of the tendo-vaginal cavity in the region of the hock and foot pad of
chickens while inoculated air sacs in the same chickens showed little or
no gross lesions and low antibody titer.

(3) Turkeys. The disease affects most of the turkeys in a flock,
although some of them may not exhibit sinusitis. Inoculated turkeys or
poults developed more severe airsacculitis. Only Mg (isolate serotype
A) produced sinusitis in turkeys. Yoder and Hofstad (1964) reported
that from 45 turkeys inoculated with serotype A, 31 birds showed sinusitis,
23 showed airsacculitis, 10 showedtendo—vaginitis and 39 birds showed
high antibody titer. Neurologic and arthritis syndromes of turkey poults
inoculated intravenously with Mg (strain 6)lrnm2been investigated by
Clyde and Thomas (1973).

(4) Game Birds. Yoder and Hofstad (1964) failed experimentally to

introduce Mg_infections into bobwhite quail (Colinus virginianus) by

 

inoculation of first yolk passage of isolate 894 of serotype A, which
was pathogenic for chicken embryos, chickens, and turkeys. However,
Madden §£_§l, (1967a) isolated pathogenic Mg_from bobWhite quail.

(5) Pigeons. The significance of Mycoplasma in pigeon nasal

 

21

cavities has not been evaluated. Yoder and Hofstad (1964) inoculated
three different isolates of Mg_into four groups of seven pigeons each
with no visible clinical signs or lesions. Winterfield (1953) isolated
the "turkey sinusitis agent” from pigeons via chicken embryo inoculation.
Gianforte §E_§1, (1955) and Mathey §£_al, (1956) isolated unclassified

strains of Mycoplasma from pigeons.

 

i. Epizootiology
(1) Natural and experimental hosts. Mg infection occurs naturally
in chickens and turkeys. It has also been isolated naturally from other

birds such as chukar partridges (Alectoris graeca) (Wichmann, 1957),

 

bobwhite quail (Colinus virginianus) (Madden, §£_§1,, 1967a), peacocks

 

(Pavo cristatus) (Willis, 1955) and pheasants (Osborn and Pomeroy, 1958a).

 

The organisms have been isolated from pigeons (Winterfield, 1953; Gianforte,
g£_§1, 1955). Van Roekel and Olesiuk (1953) reported that guinea fowls
and pheasants were readily infected.

(2) Spread and transmission. Mg_infections were reported as an
egg-borne disease in chickens (Van Roekel §£_§l,, 1952; Fahey and Crawley,
1954b) and in turkeys (Jerstad §£_§1,, 1949; Hofstad, 1957a). Yoder and
Hofstad (1964) isolated the organism from the oviduct of infected
chickens and semen of infected roosters, which indicated the congenital
or vertical transmission pattern. Under the modern pattern of the poultry
industry, where the breeder company provides the chicks to the grower in
flocks of 10,000 to 20,000 birds, under crowded conditions and confined
housing, a small number of newly hatched chicks will serve as a nucleus
for contact infections (horizontal transmission). The infections are

usually accelerated by vaccination programs or other stress factors.

Airborne infections by dust or droplets have been recorded (Fahey and
Crawley, 19553). The role of wild birds or other animal vector in
transmission did not appear to be significant in chickens, but since
turkeys were usually raised on range, airbbrne transmission had been
‘observed and animal vectors were potentially more important. Field
evidence suggested that the infection can be carried from one flock to
another by human visitors or employees (Yoder, 1978).

(3) Incubation period. Under natural conditions, it is very dif-
ficult to determine the exact data of exposure because many factors seem
to influence the onset and extent of the infections. Experimentally,
Delaplane and Stuart (1943) and Van Roekel 35 El: (1952) found the in-
cubation period to vary from four to 21 days. Sixty-five percent of 233
chicks had symptoms of nasal discharge between 11 to 18 days following
inoculation of infective turbinate material (Hofstad, 1952). Yoder
(1978) stated that numerous chicken and turkey flocks developed clinical
signs near the onset of egg production, suggesting a low level of inherent
infection (probably due to egg transmission) due to a series of stressfu1
events.

(4) Morbidity, mortality, and the role of secondary factors.
Chickens were susceptible to the infection at all ages, but they varied
‘in their susceptibility and the duration cf the disease. Young chickens
and turkeys were more susceptible to disease outbreak than older birds.
However, starting about 1950 to 1951, extensive outbreaks of a severe
respiratory disease were reported, especially in broiler-growing areas.

As early as 1954, Wasserman g£_§1, demonstrated that Escherichia coli

 

was the mOSt frequent complicating organism (especially O-group 2).

Other researchers reproduced a severe air sac infection experimentally
by infecting the chicken with a combination of E, 221;, Mg, and viruses
of infectious bronchitis or Newcastle disease (Gross, 1961a, 1962; Adler
g£_§l,, 1962). These relationships were further studied by Fabricant and
Levine (1962), confirming the observation of Gross (1961 a,b), who
determined that experimental lesions were indistinguishablefrom the
typical field cases. They concluded that E, gplg_did not readily invade
the lower respiratory tract unless it was previously infected with Mg_
or other respiratory viral or bacterial infections.

The role of viruses in establishing CRD such as reovirus and in-
fectious bursal disease needs more investigation. Mortality was very
low in adult chickens, but in.four-to eight-week-old broilers it was
higher than in laying hens. In complicated cases, mortality reached

about 30 percent.

j. Clinical Signs

(1) Chickens: Fabricant (1969) and Yoder (1979) summarized the
description of the disease in chickens and turkeys. The most character-
istic symptoms of the disease were tracheal rales, nasal discharge and
coughing. The duration of the clinical symptoms was recorded to be as
long as one to two months in experimentally infected chickens. However,
because of the long term duration of the disease and the long incubation
period, clinical signs may continue for a much longer time. In laying
hens, egg production declined but maintained at lowered levels. Food
efficiency decreased in broilers and significant levelSof lowered hatch-
ability were reported due to embryo mortality caused by Mg, However,

antibody titer against Mycoplasma reached high levels in some flocks

 

24

without any evidence of clinical signs, especially if the infection
occurred when they were young (Yoder, 1979).

(2) Turkeys: In turkeys, the disease has been described as prolonged,
progressive and severe, and manifested by swelling and distention of the
infraorbital sinuses and/or air sac infection. Subclinical infections
were rare (Fabricant, 1969). Tracheal rales, coughing and labored
breathing sometimes became evident if tracheitis or airsacculitis were

present. Lowered egg production in breeder flocks was reported.

K. Gross Lesions

The most consistent gross lesion associated with this disease was
the presence of mucoid to mucopurulent exudate in the trachea, bronchi,
air sacs, and nasal passages. Sinusitis was usually more prominent in
turkeys, but it has also been observed in chickens and other avian species.
The severe purulent, fibrinopurulent, or caseous exudates seen in the
pericardium, around the liver, and in the air sacs were due to secondary
E, ggli_infections (Gross, 19613, 1962). Domermuth and Gross (1962)
reported that salpingitis in chickens and turkeys occurred occasionally

due to the mycoplasmosis.

L. Histopathology

The microscopic lesions of CR0 have been studied by Jungherr 33.31:
(1953) and Van Roekel g£_§l, (1957). Thickening of the mucous membrane
of the affected tissues due to infiltration with mononuclear cells and
hyperplasia of the mucous gland were recorded as typical microscopic
descriptions of CRD. Focal areas of lymphoid hyperplasia, so-called

"lymphofollicular reaction% were commonly found in submucosa of the

25

respiratory tract. Barber (1962) recorded similar lesions in apparently
normal turkeys, and suggested that the presence of lymphofollicular lesions
might be of limited value. It is not clear whether the occasional gran-
ulomatous lesions seen in the lungs were due to Mg or to secondary bacterial

infections (Van Roekel §£_§E,, 1957).

m. Laboratory Diagnosis

(1) Specimen collection. Since Mycoplasma infections tended to be

 

respiratory and involved most birds in the flocks, five or ten tracheal
swabs were sufficient. Air sac swabs were cultured directly into the
media or from a suspension prepared in a mortar and pestle with a small
amount of nutrient broth. Sinus fluid was usually obtained from the in-
fraorbital sinus with a syringe and ZL-or 25-gauge needle. Specimens
of brain, heart, liver, kidney, spleen, oviduct, and cloaca were used
for cultures (Methods for Examining Poultry Biologics and for Identifying
and Quantifying Avian Pathogens, National Academy of Science, Subcommittee
on Avian Diseases, 1971).
(2) Identification of the organism

(a) Broth and agar media. Broth passage prior to plating on
agar increased the number of primary isolates. If there was no evidence
of growth after 96 hours, the cultures were incubated for another seven
days. Serial passages to a new broth tube every two to three days en-
hanced the multiplication of the organisms. Mg reduced phenol red when
it was present in the medium to yellow color as the medium became acid.
nge of the available media which have been published were suitable for

both isolation and antigen production of various avian Mycoplasmas (Olson

 

§£_§i,, 1965; Yoder and Hofstad, 1964; Yoder §£_§l,, 1972; Yoder, 1975).

26

The plates should be incubated in a moist atmosphere (sealed container
with a damp towel) at 37 C. Serotyping was required to achieve accurate
classification.

(b) Embryonated chicken eggs. Inoculation of seven-day-
embryonated chicken eggs via the yolk sac with the original exudate was
a very effective means to enhance the growth of Mg_(Yoder, 1978). Embryo
death has been recorded within five to ten days post inoculations. Lesions
such as hepatitis, splenomegaly, pericarditis, pneumonia and airsacculitis
were reported (Yoder and Hofstad, 1964).

(c) Staining. Cultures were further identified by preparing
Giemsa -stained smears that revealed small coccoid organisms often in
clumps when examined through the oil immersion lens of a microscope
(Yoder, 1979).

(d) Fluorescent technique. Fluorescent antibody (FA) was used
for differential diagnosis by applying FA directly to colonies on agar.
Corstvet and Sadler (1964) used high-titer FA conjugate prepared from
antiserum produced in rabbits.

(6) Biochemical reactions. Most of the laboratories working

with diagnosis of Mycoplasma did not require biochemical reaction studies

 

for differentiation of different Mycoplasma species. However, as mentioned

 

before and summarized in Table 7 (Appendix), different avian Mycoplasma

 

species had different biochemical reactions. Arginine decarboxylase,
dextrose fermentation, tetrazolium reaction, and DPN requirements were

the basis for diagnosis by biochemical reaction.

n. Serological Diagnosis

A positive serologic test, together with history and typical symptoms

27

of the disease, constitute a presumptive diagnosis of the disease.
Isolation and identification of the organism constitute the very import-

ant diagnostic steps for definite Mycoplasma diagnosis (Yoder, 1975).

 

Several serologic procedures are currently in use.

(1) Rapid serum plate test (RSP). The test was used primarily by
Adler (1954). Antigen was prepared according to the procedure of Adler
and Yamamoto (1956a). The test was performed by placing a drop of
serum on a rotating glass plate at room temperature and mixed with a
drop of stained antigen to make a spot about two cm. in diameter. The
results were read after two minutes. A positive reaction was characterized
by the formation of definite clumps, usually starting at the periphery
of the mixture. Positive and negative control sera were included when-
ever the test was run. Yoder (1979) reported that turkey serum reacted

to confirm the presence of Mycoplasma in the flock more slowly than

 

chicken serum when RSP test wusused. The main advantage to the test is
that it can be used in the field to give preliminary diagnosis and re-
latively easy, fast results. Many common human errors occurred, such as
improper temperatures of equipment, room, and antigen, improper light
for reading results, carelessness in placing the proper amount of antigen
and serum on the plate, or dirty plate and other equipment, causing in-
accurate readings. Other disadvantages includxlcross reactions; this
feature will be discussed in more detail later.

(2) Tube agglutination test (TAT). TAT was found to be more reliable
than the plate test using chicken serum by Jungherr g; g}, (1955). It
was also found more reliable than the plate test in testing turkey serum

(Hofstad, 1957b). The antigen prepared for the plate test could be used

28

in the tube test when diluted 1:20 in phenolized (0.25%) buffered saline
(pH 7.0). The tube test was conducted by mixing 0.02 to 0.08 ml of test
serum with 1.0 ml of diluted (1:20) antigen in a glass tube and allowing
the mixture to react for 18 to 24 hours at 37 C. A detailed review of
the recommended procedures of TAT is cited by The Methods for Examining
Poultry Biologics and for Identifying and Quantifying Avian Pathogens,
Subcommittee on Avian Diseases, National Academy of Science (1971). The
test usually was read after overnight incubation at 37 C. Clear super--
natant fluid with clumps of antigen covering the entire bottom of the
tube was indication of a positive test. The advantage of this test was
that it detected IgM and IgG antibody of Mg_ (Kleven and Pomeroy, 1971).
Yoder (1975) indicated that RSP and TAT are a sample screening procedure
for flocks, but some nonspecific reactions may result that could not be
confirmed by HI test.

(3) Hemagglutination inhibition test (H1). The HI test has been
used routinely to confirm various rapid serum plate or tube agglutination
tests and to differentiate Mg from M§, It has been recorded that the HI
test detected IgG antibody which appeared in the circulation several
weeks post infection (Kleven, 1975). The test was conducted by the
constant-antigen decreasing-serum method (Beta procedure) or vice versa
(Alpha method). This method required the use of a 4-unit antigen system.
Differences in the number of hemagglutination (HA) antigen units changed
the result markedly. The detailed procedure for H1 test was presented
in the Isolation and Identification of Avian Pathogens (Yoder, 1975).

Jungherr §£_gl, (1953) compared the HI test with slide and tube

agglutination tests and found that the HI test was the most reliable.

29

Many investigators arrived arbitrarily at the number of HA units to be
used in the test, which accounts for many unreliable results. Leach and
Blaxland (1966) reported wide variations in H1 results from laboratories
when a series of Mg_antisera were evaluated for H1. They attributed this
variation to the kind of HA antigen used. Kuniyasu and Ando (1966) found
that MngA antigen activity in broth developed to its peak on the fifth
day of incubation. HA activity of Mg antigen was not related to the
number of viable organisms, and various chemical treatments had no adverse
effect on the HA. In their studies, formalin destroyed the HA activity
of the antigen. Adler and DaMassa (1968) found that adding dex§39§e to
the culture medium for propagating Mg markedly reduced the sensitivity
of plate agglutination antigens, but they did npt observe any effect on
HI test. The sensitivity and the specificity of the antigen used varied
from one laboratory to another. The requirements of red blood cells,
buffers, and reagents made the test more expensive than the other two
serological methods.

(4) Complement fixation test (CFT). Frey and Hanson (1969) and
Marquardt and Newman (1971) described a direct complement fixation test
to detect antibody of Mg infections in chickens and turkeys. They used
guinea pig complement with normal unheated chicken or turkey serum dir-
ectly to quantitate antibodies against Mg, The technique showed as much
specificity and sensitivity as the HI test.

(5) Metabolic inhibition test (MIT). Barber and Fabricant (1971)
used this technique to classify Mg, They used microtiter equipment and
a constant antigen dilution in all wells (1000 units of metabolic activity

of the antigen/well). Hyperimmune rabbit antisera to serotype A (Mg) and

30

other serotypes were used. The test has been reported to be very
sensitive but modification is needed, especially for antigen production,
before it could be applied on a wide range.

(6) Growth inhibition test (GIT). For this test, small plates
(100 x 15 mm) containing agar medium and grid marks were used (Aycardi
SE §l°’ 1971). The plates were placed in an incubator at 37° C for one
hour to dry the surface. The Mycoplasma culture to be tested was diluted
serially in sterile broth medium, and 0.2 m1 of each dilution was spread
over the dry surface with a bent glass rod. When the inoculum had dried
(approximately 1/2 hour), filter paper discs of 5 mm diameter soaked in
hyperimmune sera were placed gently on the agar surface with sterile
forceps. The plates were incubated right-side up in a moist chamber at
37° C for 5 to 7 days, and then read. The zone of inhibition was measured
in mm. This test was used only in laboratory classification of different
serotypes of avian Mycoplasma, but it can be adapted for diagnostic
purposes.

(7) Agar-gel diffusion test (AGD). Aycardi ggugl., 1971) described
the procedures of preparing the diffusion antigen used in this test as
well as the methodology of the test. The diffusion medium consisted of
0.8 g agarose, 5 m1 borate buffer (pH 8.6), 95 m1 physiological saline,
one ml sodium azide (10% stock solution) to prevent contamination, and
one ml trypan blue (0.5% stock) to provide contrast. The test was per-
formed in 50 x 12 mm plastic plates with tight lids. Five m1 of diffusion
medium was deposited in each plate, and wells were made (5 mm in diameter)
and filled with approximately 0.05 ml of antiserum. Rabbits were used
only for serotyping of different Mycoplasma, but not for diagnostic

purposes.

31

(8) Fluorescent antibody technique (FA) (Immunofluorescent
technique (IF). The capacity of a substance to absorb light energy and
emit this energy at a longer wavelength is called fluorescence. Anti-
bodies complexed to a fluorescent dye (usually fluorescein isothiocyanate)
became highly fluorescent and retained their antigen specificity.

Several variations of the FA technique have been successfully
utilized, but those most commonly used for diagnostic purposes were the
following:

(a) Direct methods, in which fluorescein-labeled antibody

was applied to a preparation containing the corresponding
antigen. A smear made of the antigen (microorganisms,
tissue, etc.) and the labeled antibody was placed over
it. Complexing by homologous antigen and labeled anti-
body was revealed by fluorescent organisms when viewed
by fluorescence microscope.

(b) Indirect method consisted of a two-step procedure. The
first step allowed the reaction of antigen with specific
antibody. If the antigen and antibody were homologous,
the antibody was retained through complexing with the
antigen. The second step entailed the addition of
antiglobulin (an "antibody to the antibody”) labeled
with fluorescent dye. The antiglobulin complexing with
specific antibody resulted in fluorescing the organisms.

The manipulation of the FA made this test one of the most important
diagnostic techniques in modern medicine. For instance, tissue sections,

tissue culture, smears from various organs, exudates, or frozen tissue

32

can be used to diagnose the presence of such organisms(Goldman, 1968).
FA has been used in diagnosis of bacterial, viral, parasitic, and mycotic

infections, as well as different strains of Mycoplasma (Goldman, 1968).

 

Noel g3 21: (1964) demonstrated that indirect FA could be used to detect

Mg in infected tissues. Corstvet and Sadler (1964) used indirect FA to

 

detect flg_is tissue sections, tissue impressions and Mycoplasma colony

imprints. Identification of different avian Mycoplasma colonies was

 

accomplished by using FA (Kleven, personal communication, 1979).

0. Cross Reactions and Nonspecific Agglutination in Serological Tests

The cross reaction between different Mycoplasma positive serum and

 

Mycoplasma antigen was first recorded by Olson gg_al, (1965).When using the

 

serum plate test, they found definite cross reaction between Mg_positive
senmasamples and gg_antigen. Roberts and Olesiuk (1967) and Vardaman and
Yoder (1969) reported similar results when they employed serum plate tests.
Later on, Roberts (1970) reported non-specific agglutination reactions
with Mg_antigen. This cross reaction was sometimes due to partial cross
reaction of Mg_antigen with sera from chickens or turkeys during the

early stages of y§_infection, or after erysipelas vaccination (Boyer 93_
‘21., 1960; Olson g£_§l:, 1965). Various dead virus vaccines produced
rheumatoid factor-like antiglobulins and induced agglutinins to flg_antigen

(Roberts, 1970). Infections of chickens with Streptococcus faecalis or

 

Staphylococcus aureus had similar reactions (Thornton, 1973). Other

 

factors which have been suggested to cause mnrspecific agglutination
include contaminated serum (Roberts §£_gl,, 1967), storage of serum at
4° C (Windsor and Thornton, 1973), freezing and thawing of serum (Thornton,

1965) and inoculation of the birds with sterile media (Windsor and Thornton,

33

1973). Kleven (1975) summarized many different attempts that have been

used to eliminate the mnmspecific reaction.

p. Differential Diagnosis

HI test for identification of antibodies against M , ME; and flm_is
used routinely in the voluntary control program for chickens and turkeys
within various states and as a part of the National Poultry Improvement
Plan (1976).

Chicken: flg_was found to be the primary cause of CRD; other organisms
frequently caused complications (Biddle and Cover, 1957). Newcastle
disease and infectious bronchitis virus and/or antibodies may be present
as separate entities or as part of the complicated CRD problem. Infectious

coryza, fowl cholera and other Mycoplasma can be identified by cultural

 

and serological methods (Yoder, 1978).
Turkey: Repiratory disease and sinusitis in turkeys may be due to
fowl cholera, ornithosis, ߤ_infections, vitamin A deficiency, avian

influenza A, as well as Hg_(Yoder, 1978).

q. Host Immunological Response

After natural orartificialinfections with flg_the antibody response
was detected by RSP or TAT tests as early as a few days post infection
(Adler and Wiggins, 1973; Kuniyasu, 1969). Positive results from the
HI test can not be expected until approximately 2 weeks post infection
(Adler and Wiggins, 1973). Therefore, most of the diagnostic laboratories

employed RSP for screening and the HI for confirmation. Antibodies pro-

(duced in response to Mycoplasma infections belonged to the 198 or 78

 

class of immunoglobulins, depending upon the nature of the antigen eliciting

34

their formation, the route of infection, and the homogenicity or hetro-
genicity of the infections. It has been reported that immunoglobulin
M (IgM) (19S, agglutinating antibody) was the first antibody to appear
in chicken blood after infection with fig regardless of the route of infec-
tion (Kuniyasu, 1969; Adler and Wiggins, 1973). The persistence of IgM
in the blood varied according to the route of the infection. Antibody
can be detected up to 32 days after intravenous inoculation and 77 days
after infranasal inoculation (Kuniyasu, 1969). After the disappearance
or declining of the initial IgM antibodies, IgG 7S immunoglobulin pop-
ulation started to rise and stayed high for a much longer time (Kleven,
1975). This antibody was found to be less efficient than IgM in the
agglutination reactions. Roberts (1969) and Roberts and Olesiuk (1966)
found that RSP were mercaptoethanol sensitive and associated only with
IgM, not with IgG. The HI test was more sensitive in detecting IgG
antibodies and was not able to detect IgM, the initial antibody (Roberts,
1969 and Kuniyasu, 1969). This observation led Adler and Wiggins (1973)
to conclude the HI test was dependent on IgG activity exclusively. The
tube agglutination test was more sensitive to detect initial antibody
than the HI test. It was observed that TAT reacted with both IgG and
IgM. Therefore, the RPT was able to detect the early antibody response
(IgM) and the HI detected the late antibody response (IgG). Similarly,
turkeys infected with flg_had an early IgM response followed by IgG
(Kleven, 1975).

Roberts and Olesiuk (1966) studied, in detail, the immunologic
tolerance in chicken embryos inoculated with Hg, Their results showed

that chick embryos infected with flg_during early embryonic life start

3S

producing agglutinins (IgM) at 4 weeks of age. After subsequent challenge
with Hg, all chicks became serologically positive. Chicks infected at 1
day of age were all positive at 7 weeks. In summary, immunologic tolerance
was not detected under these conditions. Adler §E_§l: (1973) described
the function of the bursa of Fabricius in the development of resistance

and serologic response to Hg,

r. Treatment
Effective prophylactic and therapeutic drugs have been developed

against avian Mycoplasma: streptomycin, oxytetracycline, chlortetracyline

 

tylosin, erythromycin (Domermuth and Johnson, 1955; Yamamoto and Adler,
1956; Hamdy §£_al:, 1957; Kiser §£_§l,, 1961; Yoder §£_§l:, 1961). Some
strains of Hg_are resistant to streptomycin, spiromycin, erythromycin,

and tylosin. As deScribed by the above mentioned researchers Tylosin is
one of the most common antibodies used for treatment of mycoplasmosis in
.chickens and turkeys. However, constant results cannot be expected. It
can be injected subcutaneously at 3-5 mg per pound body weight or ad-
ministered at 2.3 g per gal. of drinking water for 3-5 days. Also,
antibiotic injection of infected breeding stock to control egg transmission

has been used.

5. Prevention and Control

There are several methods which have been used to establish an
effective control program:

(1) Medication of breeder flock

(2) Egg dipping

(3) Egg heating

(4) Vaccination
(5) General hygienic measures

A detailed review of those control measures was cited by Yoder (1978).

2. Mycoplasma meleagridis (gm)

 

This infection has only been found in turkeys. The organism was
designated "N" type (N strain PPLO, H serotype) and was recognized by
Adler g£_al, (1958) in several groups of newly hatched turkey poults
with air sac lesions which were free of Hg, In contrast to Mg_infected
poults, the lesions tended to decrease in severity and disappear as the
poults grew older. flm_was the causative agent of egg~transmitted disease
of turkeys in which the primary lesion was. airsacculitis in the progeny.
Other clinical signs were decreased hatchability, skeletal abnormalities
and poor growth performance. The organism is known to be a specific
pathogen of turkeys and has no public health significance. It has been
found generally that the organism is usually eliminated from the vagina
of naturally infected hens within 4 to 14 weeks following removal of the
source of infection (i.e., contaminated semen), while hens continuously
inseminated with contaminated semen maintain a high incidence of infection
(Kleven and Pomeroy. 1971b). The organism persisted for 55 to 344 days
in the male phallus (Yamamoto g£_§l., 1968). Passive antibodies (agglu-
tinins) were detected in a high percentage of poults from infected dams
and persisted for approximately 2 weeks after hatching. However, these
antibodies appeared not to protect against development of air sac lesions
in infected embryos (Yamamoto 23.22:, 1966b; Mohamed and Bohl, 1968).

Hamdy §£_gl. (1969) found that spectinomycin-lincomycin combination

(2 g per gal. for 10 days) was effective in reducing airsacculitis incidence

37

in turkeys experimentally infected with Em: High temperature and dipping
eggs using tylosin or gentamycin was reported as a successful procedure
to control the transmission cycle of flm_infections (Saif EE.§l:» 1970b
and 1971). The treatment of turkey semen with antibiotics to eliminate
flm_was not successful when tylosin (Kleven §£_§l:, 1971) or gentamycin

(Saif and Brown, 1972) was used.

3. Mycoplasma synoviae (gs)

 

This organism was first described by Olson g£_§l, (1954, 1956) and
Wills (1954) as the primary etiological agent of infectious synovitis
in chickens and turkeys. The infections occurred most frequently as a
subclinical upper respiratory infection. Airsacculitis developed after
secondary infections occurred when combined with Newcastle disease,
infectious bronchitis, or both. ME became systemic and resulted in
infectious synovitis, an acute to chronic infectious disease of chickens
and turkeys involving primarily the synovial membranes of joints and tendon
sheaths and producing an exudative synovitis, tenosynovitis, or bursitis
(Olson, 1978).

It has been recorded that the disease can be transmitted by eggs
(vertical) or by contact (horizontal) (Olson §£_§l:, 1965). No signif-
icant attempts have yet been made to control the disease. Extensive
studies on antibiotic treatment of clinically affected flocks indicated
that chlortetracycline (60 to 100 g per ton of feed) given continuously
will provide satisfactory control of infectious synovitis. A higher con-
centration of 200 g per ton of feed was needed to control synovitis
after infection had occurred in the flock.

The disease has been observed primarily in growing birds 4 to 12

38

weeks of age in the broiler-growing regions of the United States (Olson,
1956). It has been observed in turkeys at 10 to 20 weeks of age and has
been reported to be a greater problem in turkeys than in chickens (Olson,
1978). The synovial lesions were most frequent and severe in the hock
region, the foot pads, and the keel bursa. In the early stages of the
disease, a viscous, creamy-to-gray exudate, involving the synovial mem—

branes of the joints,tended to become caseous, or purulent exudate

developed as the disease progressed.

E. Protein A Chromotography

1. Introduction

Protein A was isolated from the cell wall of Staphylococcus aureus.

 

It was first described in 1940 by Verwey, named protein A by Grov g5.
El: (1964) Sjoquist §£_31: (1972) reported that protein A covalently
linked to the peptide part of the cell wall. Later on, Movitz (1974)
found that this linkage takes place within a very short time after syn-

thesis of protein A. Protein A from Staphylococcus aureus binds speci-

 

fically to the Fc region of human and other mammal immunoglobulin G
(IgG) of subclasses l, 2 and 4 (Forsejren and Sj5quist, 1966). This
information has been an invaluable tool for investigating cell surface
antigens and immunological reactions ig_!i££g, Early isolation methods
involved extraction of protein A from the bacteria by boiling, followed

by acid and ethanol precipitation (Jensen, 1959) or by mechanical dis-
integration, followed by precipitation, gel filtration and electrophoresis

(Lofkvist and Sjéquist, 1963, 1964). Following these procedures hetero-

geneous products resulted. Recently, protein A preparation method

39

has been improved by the introduction of lysotaphin digestion of the
bacteria followed by ion-exchange chromatography on DEAE-sephadex and
gel filtration on Sephadex G-100 (Sjoquist et al., 1972) and by the
use of affinity chromatography on immunoglobulin G (IgG) coupled to

Sepharose (Kronvall, 1973).

2. Physical and Chemical Properties

Protein A consists of a single polypeptide chain of molecular weight
42,000 containing several regions of internal homology (Sjoquist ggugl.,
1972; Bjork 35H§1,, 1972), but no significant amounts of carbohydrate
(Sjoquist 322$: 1972). Sjoquist 333;. (1972) indicated that protein
A contains no tryptophan or cystine residues but has 4 residues of
tyrosine per molecule. These tyrosines are responsible for the biologi-
cal activity of protein A.

The C-terminal amino acid of the protein chain was reported to be
lysine, but the amino terminal residue was "blocked" to Edman degradation
and no clear identification had been reported (Sjoquist §£H31., 1972).
The native structure of the molecule was recorded to be very stable and
was spontaneously reformed after removal of denaturing agents such as
6M guanidine hydrochloride (Hjelm 53.31., 1975) and about 50% of the

molecule was found to have an a-helical configuration.

3. Biological Properties

The most important biological property of protein A is its ability to
interact with many subclasses of IgG molecules from several species, mostly
mammals, such as humans, mice, guinea pigs, rabbits, and other domestic

and wild animals. Kronvall and Frommel (1970) studied protein A-IgG

40

reaction in serumobtained from different species representing seven classes
and 30 orders of living vertebrates. However, he obtained negative re-
actions with sera of most birds, such as penguins, ostriches, ducks, chick-
ens, and turkeys, except paleognathus species (the primitive flightless

bird, rhea) Rhea americana. Kronvall and Williams (1969) found that the

 

precipitation reaction was specific for human subclasses of IgG 1, 2, and
4, whereas IgG 3 was non-reactive. Sjoquist g£_§l, (1970) reported that
pure protein A was able to bind 2 molecules of IgG per molecule. No other
human serum proteins combined with protein A, including IgA, IgM, IgD,

and IgE (Kronvall and Frommel, 1970). The protein A content of the
swollen gel was reported to be 2 mg/ml and the binding capacity for human
IgG was approximately 25 mg IgG/ml gel. Later on, Forsgren and Sjoquist
(1966) indicated that protein A reacted with PC H—chain structure of IgG
globulin. In addition to that, since protein A reacts with Fc structures,
it is capable of blocking phagocytosis by competing with polymorphonuclear

leukocytes for Fe sites on the IgG molecule (Dosselt g£_al:, 1969).

4. Applications

The specific interaction with the Fc region of IgG made protein A
a powerful tool in cytochemical and immunochemical studies and in the
investigation of the surface structure of cells. Protein A labeled with
fluoresceinisothiocyanate(FITC) has been used for immunofluorescence
microscopy to demonstrate the presence of specific antigens in tissue
sections. FITC-labeled protein A was found to show less non-specific
reactivity with tissues and cells than FITC—labeled anti-IgG antibodies

(Pharmacia, 1975).

41

F. Enzyme-linked Immunosorbent Assay

1. Introduction

The enzyme-linked immunosorbant assay (ELISA) was developed indep-
endently by Engvall and Perlmann (1971) and van Weeman and Schurrs
(1971). Since then, many publications concerning the use of ELISA in
serodiagnosis of infectious diseases - pioneered mainly by Carlsson and
collaborators in Stockholm (1971) and by Voller and his associates

(1971) in London - have been reported.

2. Assays, Principles, and Methods
a. Reaction mechanism and methodology.

The basic ELISA test depends on two assumptions: (1) that antigens
or antibodies can be attached to a solid phase support yet retain immuno-
- logical activity, and (2) that either antigens or antibodies can be
linked to an enzyme and the complex retain both immunological and enzymatic
activities. Antibodies or antigens were shown to be readily attached to
paper discs or to plastic surfaces, such as polyvinyl or polystyrene,
either chemically or by passive absorption, and still retain their
activities. Voller §E_§l3 (1976) summarized the methodological aspects
of ELISA,which are described as follows:

(1) Competitive method (as seen in Figure 1).

This system was described for detection and measurement of

antigens and itvms carried out as follows:

(a) Specific antibody was attached to solid phase, which was
then washed by phosphate buffer.

(b) Test solution (fluid thought to contain antigenic

(2)

(3)

(C)

42

components) was then added. It was either mixed with
enzyme labeled antigen, or enzyme labeled antigen was
added after the starting time. Plates were then incubated.
Enzyme substrate was added. The reference wells
containing only enzyme-labeled antigen (step 2) showed
coloration. The inhibition of that color change in the
wells with test samples was proportional to the amount

of antigen in the test samples.

The sandwich method (Figure 2). This system was described for

the

out

(a)

(b)

(C)

(d)

The

detection and measurement of antigens and it was carried

as follows:

Specific antibody (to the antigen to be measured) was
attached to the solid phase,which was then washed.

The test solution was then incubated with the sensitized
solid phase for a given length of time, then was washed.
Enzyme labeled specific antibody was then incubated with
the solid phase, followed by washing.

Enzyme substrate was added. The color change was pro-
portional to the amount of antigen in the test solution

in step 2.

technique has been described as analogous to the immuno-

radio-metric assays of Miles and Hales (1968), and has the

the same sensitivity and specificity for high molecular

weight antigens.

Indirect method (Figure 3). This system has been used for

the detection and measurement of antibodies. It was carried

43

FlGURE 1
The compemwe method of ELISA for OSSJVIHQ antigen

1 ADSORB ANTIBODY 2'0 SURFACE

 

 

l l l
2aADD ENZWJE LABELLED :5 ADD Engvmg LABELLED
ANTEGEPJ + UNK‘JO‘J/N ANTIGEN Amncgn

‘4
Qt]

ACO
EHZ (“ME Suesmgrg

   

SUBSTRATE HYCRQL/Sil E '.-‘«E3£LLCC' Aru’cCEN

DIFFERENCE BETWEEN 3a 2.30 E UNKNO‘.‘/:‘J Armcguj

44

FIGURE 2
The Double Anthody Sandwuch ELISA for measurmg anugen

 

 

 

 

 

 

 

 

 

 

I ANTIBODY ADSORBED TO PLATE
WASH '
2 TEST SOLUTION CONTAII‘IH‘JG
ANTIGEN ADDED &
u use
3 ADDENZ‘/'.‘E LAE'ELLEC ‘
SPECIFIC ANTISQQ/ 5%:
!
WASH
a o
o o I .0. a
4 ADD ENZYME SUBSTRATE o ' 0' u .
. -"‘A‘- a
Z:
AMOLNT WYSI5=AMCAJTT ArrTCBI _'

 

 

 

PRESENT

45

FIGURE 3
The Indirect ELISA for measurIng antibody

 

 

 

 

 

 

1 ANTIGEN ABSORBEO TO PLATE
WASH
2 ADD SERUM ANY SPECIFIC
ANTIBODY AT TACHES TO ANTIGEN A
2
g
A
WASH
3 ADOENZY‘JE LABELLEO ANTIGLCBULN '
WHICH ATTAOiES TO ANTIBOCr’ ‘C—j‘
9
/
WASH
e' ‘ ‘ 5 °
4 A00 SUBSTRATE go ”.0
r «.o

AMOJNT HYDRCLYSEDE AMOINT
ANTHIJY PRESENT

 

“)8

 

 

 

 

 

46

out as follows:

(a) The relevant antigen was attached to the solid phase
which was then washed.

(b) The diluted test serum was added and incubated, followed
by washing.

(c) IAn enzyme labeled anti-species-globulin was added and
allowed to react for a given time before washing.

(d) A specific enzyme substrate was added. Degradation of
substrate resulted in a color change. The amount and
rate of color change wererelated to the amount of antibody

in the test serum.

b. Solid phase support

During the last few years a variety of solid phases have been used
in enzyme-immunoassay particulate materials, such as cellulose, agarose,
or polyacrylamide with covalently bound antigen or antibody (van Weemen,
1974). The use of a solid phase such as test tube, glass beads, or
discs simplifies handling procedures. Antigen can be passively absorbed
to such carriers, made of polyvinyl, polypropylene (Engvall and Perlmann,
1971), polycarbonate, glass, or silicone rubber (Hamaguchi §£_al:,
1976a, 1976b). For large-scale enzyme immunoassays, disposable micro-
titration plates were particularly convenient (Voller e£_al:, 1974).
Dipstick tests were also tried by Felgner (1977). Although various
carrier materials can be used, it was essential that each new type was
thoroughly tested to find the amount and reproducibility of uptake of
the antigen or antibody since these variables influence the results of

a test (Bidwell §£_§l:, 1977).

47

c. Equipment and mechanization (automation).

ELISA was carried out with only sensitized plates or tubes with
antigens or antibodies. The result was detected either visually or
using a spectrophotometer. Also, the technique consists of a series
of additions of reagents separated by washing steps. The reagent
additions were accomplished by using any of the commercially available
dispensers. The mechanization of practically all steps of the assay
was accomplished for macro-ELISA by Tuitenberg ggugl. (1977), and for
micro-ELISA (microplate assays) by Ruitenberg and Brosi (1978). The
automated line system included dispenser, a washer, and a spectrophoto-
meter. These authors indicated that by using line system for macro-
ELISA, as many as 4000 sennnsamples were processed daily with two persons.
Many companies offered equipment for macnr—and micro-ELISA, such as LKB
Ultralab System (Stockholm, Sweden), Finnpipette (Helsinki, Finland),
Olli (Kivenlahti, Finland), Organon Technika (055, The Netherlands) and
Dynatech Companies (Alexandria, Virginia, United States). In general,
reliable qualitative and quantitative determinations were achieved by

macro- and micro-ELISA.
3. Preparation and Characterization of ELISA Reagents

a. Choice of the enzyme.

Schurrs and van Weeman (1977) indicated that there was no single
enzyme ideal for use as a label in every conceivable ELISA. Instead,
the researchers had to determine which enzyme for a particular assay
system could be used. It has been recorded that factors such as turn-
over number of the pure enzyme (the number of substrate molecules con-

verted to product per enzyme site per unit of time), purity of the enzyme

48

preparation, and a substrate which produces stable, soluble, easily mea-
sured products would affect the sensitivity and specificity of ELISA
(Ishikawa and Kate, 1978; Rubenstein, 1978). The enzyme must be rea-
sonably cheap, available in a highly purified form, and suitable for
linkage with protein. The smaller the amount of enzyme label which

could be detected, the more sensitive the resulting assay would be.

b. Coupling procedures

Coupling enzymes (or protein in general) to other substances has
been carried out using several different reagents. The coupling has
been satisfactorily carried out using glutaraldehyde in either one
step (Avrameas, 1969) or two steps (Avrameas and Ternyck, 1971) or by
using periodate (Nakane and Kawaoi, 1974). The one-step method has
yielded satisfactory conjugates with alkaline phosphatase and horse-
radish peroxidase although the ratio of enzyme to antibody varies someé
.what (Avrameas, 1969). The two-step glutaraldehyde method gave conjugates
in which enzyme and antibody were present in equal ratio and this led
to more sensitive assays (van Weemen and Schuurs, 1974). The periodate
method (Nakane and Kawaoi, 1974) produced a good yield of high molecular
weight conjugates, which worked well in ELISA, as did the dimaleimide
linked conjugates of B-galactosidase, which had small complexes (Kato
93.31,, 1976b). Figure 4 shows possible mechanisms of cross-linking

reactions using sodium periodate and glutaraldehyde.

c. Purification of the conjugates
lEnzyme—labeled protein preparations obtained by any one of the above

procedures contained not only enzyme-protein conjugates but also other

49

@ Sodium periodate H

, cm-
cuow C. Pete“
cm .. c, cm-

§

® Glutataldehydo

H H H

@w’ , ch CH C’ § ~~_- ____. @N3CH_(CH2)3-CH3N_-

‘H 0” ‘4 2’3- *0 H’ .

H 9H0 CHO
H t I
’ ‘ CH-CH-CH _ _ HJcHI-CHo
@‘K + 3OHC-(CH2)3-CHO + H)“. ——> OHC-(CH2)3-| 2 c S 23
” .

Figure 4. Possible mechanisms of cross-linking reactions

protein derivatives and reagents. Isolated conjugates gave less back—
ground and had greater specific activity than unpurified preparations.
Sephadex G-200 (Avrameas and Ternyck, 1971) and Ultrogel (Polyacrylamide—
agarose gel) (Boorsma and Stearfkerk, 1976) were shown to be very
efficient materialsfor purification of the conjugate. Others, such as
Sepharose (Herrmann and Morse, 1974) and Bio-Gel (Kato §£_al,, 1976a,b)
columns,gave highly purified conjugate depending on the molecular
weight of both protein and enzyme. Purification of density gradient
centrifugation was also performed to purify horseradish peroxidase
conjugated to Fab fragments of IgG molecules (Mannick and Downey, 1973).
Gel filtration permits the separation of peroxidase antibody (or Fab)
conjugate from free antibody (of Fab) and free peroxidase. Ammonium
sulphate precipitation allows the elimination of free peroxidase, but

not of unconjugated antibody or Fab (Avrameas and Ternynck, 1971;

SO

Avrameas §£_al,, 1978).

d. Properties of the conjugates.

The properties of the conjugates have two parts: (1) immunological
properties, and (2) enzymatic properties. The immunological properties
of the immune reactant might be changed following conjugation to enzymes.
This reaction resulted in loss of antigenic determinants or binding
sites, or in a decreased affinity for the binding partner. In the end,
this might have affected the specificity, as well as sensitivity, of
the ELISA. Fab fragments of IgG molecule have been labeled with B-
galactosidase to yield more sensitive ELISA than a conjugate prepared
from whole IgG molecule (Kato §£_§13, 1976a). Like the immune reactant,
the enzyme might have suffered from the conjugation reaction. However,
little has been known about the degree of influence on the enzymatic
acitivity of the conjugate. Binding of 0.6 molecules of IgG (Kato‘§£_
al,, 19753) or 4 molecules of Fab fragment (Kato g£_§l:, 1975b) to one
molecule of B-galactosidase did not affect the enzyme activity after
measuring the Michaelis-Menton constant (Km) and the maximal reaction

veloc1ty (Vmax)'

e. Substrates

Substrates were usually chosen to give a colored product following
enzymic degradation. For visually red ELISA tests an insoluble or
partially insoluble product was acceptable, for example, S-amino-
salicylic acid (SAS) or diaminobenzidine (DAB) for peroxidase conjugates.
However, for all quantitative ELISA tests, the substrate products must

be soluble. For alkaline phosphatase conjugates, para-nitrophenyl

51

phosphate was proven to be an excellent substrate. The chromogen ABTS
2.2'-Azino-di-(3-ethyl benzothiazolin sulfonate-6) diammonium salt

has been used for peroxidase (Childs and Bardclay, 1975), but this
compound was expensive and its reaction products, being free radical,

were unstable.

4. Characteristics of Enzyme-immunoassay

Four factors are characteristic of ELISA, as well as other enzyme-
immunoassays. These factors are accuracy, specificity, sensitivity and
precision. Schuurs and van Weemen (1977) explained in detail the char-
acteristics of enzyme-immunoassays which cover ELISA. They indicated
that comparisons between sensitivity of different assays were difficult
because of the many variables involved. However, most authors compared
BIA with other tests, such as FA, HA, H1, or RIA, and found that EIA was
as sensitive (Farag g£_§l,, 1975; Ruitenberg and van Knapen, 1977; and
Huldt §£_§l:, 1975) or more sensitive (Engvall and Ljungstrom, 1975;

Ruitenberg g£_§l,, 1976; Ruitenberg §£_§13, 1977).

5. Practical Application of ELISA
a. Endocrinology.

Several attempts have been made to replace current radioimmunoassay
(RIA) with ELISA, but it has not yet become a generally acceptable test.
Van Weemen and Schuurs (1971, 1972, 1974, 1976) set up ELISA for measur-
ing human choriogonadotropin (HCG) and more recently Yorde g£_§l, (1976)
described a slightly different type of competitive enzyme immunoassay
for measuring HCG which gave results identical to those obtained by RIA.

Both insulin and thyrotropin have also been measured by enzyme-immunoassay

52

OMiedema §E_§l:, 1972, and Miyai g£_al,, 1976). Smaller molecular
weight hormones have also been measured, with a high sensitivity, includ-
ing progesterone (Dray 33.3}:, 1975, and Gross 93.31:: 1976),

oestriol (van Hell 33.31:, 1976) and cortisol (Comoglis and Celada,

1976).

b. Serum protein.

Immunoglobulin G was first measured using competitive ELISA methods
by Engvall and Perlmann (1971), Engvall 33 El: (1971) and Avrameas and
Guilbert (1971). Kato g£_al: (19753, 1976a) used the double antibody
sandwich method, which together with a fluorogenic substrate resulted in
an exceedingly sensitive assay. However, other techniques such as radial
gel diffusion or the more rapid nephelometric assays had adequate sen-
sitivity to detect IgG without applying FA. ELISA could be considered
a practical tool to measure IgE while other assays were not sensitive
enough to quantitate the antibody (Hoffmann, 1973). Other applications
of ELISA were measuring clotting factor VIII levels in the blood (Bartlett
§£_§l, 1976) and detection of snake venoms and antibodies to such venom
in persons suffering from snake bite (Theakston, 1977). Measuring of

Clq by ELISA was achieved by Ahlstedt §£_§l: (1976).

c. Detection and measurement of antigens of infectious agents.

As early as 1972, ELISA was used to measure the toxins on Vibrio
cholerae (Holmgren and Svennerholm, 1973), but it was not followed up
for bacterial antigens until recently;when Carlsson §£_§l, (1976) assayed

antigens from Brucella, Yersinia, and Salmonella and, more recently, by

 

 

Yolken g£_§l3 (1977b),who detected Escherichia coli toxins by ELISA.

 

53

More attention has been paid to viral antigen detection, and the first
practical microplate ELISA system was developed by Wolters §£.al, (1976,
1977) for the detection of hepatitis B surface antigen. Duermeyer 33-
a}: (1977) had developed an ELISA for hepatitis A, and it appeared to be
suitable for detection of the virus in fecal materials. Yoklen §E_§l:
(l977a) and Scherrer and Bernard (1971) used ELISA in detection of
infantile gastroenteritis caused by rotavirus. Fungal antigen detection

was achieved by Warren §£_gl: (1977) in detection of Candida albicans

 

antigen in infected patients.

d. Determination and assays of antibodies in infectious diseases.

The published literature indicated that the main impact of ELISA
has been the measurement of antibodies. Engvall g£_§l, (1971) first
showed that antibodies were measured by indirect ELISA using enzyme-
labeled antiglobulins as the indicator. Such enzyme-labeled anti-human
IgG, IgM, or IgA became available commercially to be used for ELISA, so
that it was not necessary for the investigator to carry out his own
enzyme-antibody conjugates. Initially, Carlsson §£_§l, (1972) set up

ELISA to measure antibodies to Salmonella antigens and they found that

 

ELISA was more sensitive than passive hemagglutination or the Widal

test. Other bacterial diseases, such as Brucella, Yersinia, Vibrio

 

 

cholera (Holmgren and Svennerholm, l973),rickettsia (Halle §£_§1: 1977)
and Treponema (Veldkamp and Visser, l975),were diagnosed by ELISA. The
first large-scale use of ELISA for antibody quantitation was in the area
of viral diagnosis and of epidemiological viral screening. Using ELISA
in viral diagnosis in humans started in 1974, when Vollner and Bidwell

described a simple microplate ELISA for detection of antibodies against

54

rubella infection and the results correlated with H1.
The high sensitivity of ELISA has been found to be advantageous in
the assay of antibody to herpesviruses adenoviruses, coxsackievirus,
cytomegalovirus, Epstein Barr viruses (Voller g£_§g:, 1976; Wallen 35_
31,, 1977) and rotaviruses (Yolken e£_§l:, 1977a), and other parasitic agents,

such as Plasmodium malaria, trypanosomes, amoebae, schistosomes, Toxoplasma

 

(Voller §:_al:, 1976) and Trichinella (Saunders §£-§13, 1977). Fungal

 

diseasescaused by Aspergillus, Candida (Hommel g£_gl,, 1976) and
Cryptococcus (Desgeorges §£_§13, 1979) were diagnosed by detection of

specific antibodies in the patients' sera.

6. Uses of ELISA in Veterinary Medicine
ELISA has been reported to be used in diagnosis of some animal dis-
eases using anti-animal species globulin conjugate. Saunders g; 31: (1977)

haveshown that the ELISA can be very successfully used for diagnosis of

 

Trichinella spiralis in pigs, Babesia divergens in cattle (Purnell EE.El:»
1976), and trypanosomiasis (Voller §£_gl,, 1976) can also be diagnosed by the
ELISA method. Other diseases such as brucellosis, hog cholera (Saunders

§£_§1,, 1977), calf rotavirus (Scherrer and Bernard, 1977) and Mycoplasma

 

superpneumoniae in pigs (Armstrong §£_§l:, 1979) were also diagnosed by

ELISA.

7. Uses of ELISA in Avian Medicine
ELISA has been reported to be used experimentally for detecting anti-

bodies in chickens infected with Mycobacterium avium serotype 2 (Thoen

 

§£_§l:, 1978) and reovirus (Slaght e£_§l,, 1978). Direct assays for

detection of avian leukosis-sarcoma virus wenedeveloped by Smith 93.31:

55

(1979). There are no published reports about diagnosis of Mycoplasma

 

infections in birds by using either direct or indirect ELISA methods.

MATERIALS AND METHODS

A. Mycoplasma Organisms

 

1. Source of the Organisms

. . _ . I. .
M, gallisepticum (Mg), Adler 56 F16 strain,and J. SXEOVlae (Mg),

WVU 853-T22 strain,were received in unknown passages in serum-enriched

 

brain-heart infusion medium from Dr. M.Y. Seif, Ohio Agriculture Research
and Development Center, Wooster, Ohio. M, gallinarum culture #2032 was
received in lyophilized form from Dr. E.E. Ose, Eli-Lilly Research Lab-
oratories, Greenfield, Indiana. Also, Mg was obtained in frozen form,
11th broth passage and 2nd embryo yolk culture (R#980),from Dr. H.W.
Yoder, USDA, Southeastern Poultry Regional Laboratory, Athens, Georgia.

All organisms were transferred and maintained in broth media, agar media

and chicken egg embryos.

2., Growth and Maintenance Procedures

a. Broth and agar media: Mycoplasma organisms were maintained by

 

serial subcultures in modified Frey and MSU-Mycoplasma media. They were

 

subcultured in broth media twice a week. Broth to broth transfers were
usually done by transferring 0.1 - 1 ml (after shaking the broth tube)
with a 1.0 ml sterile pipette. These media were incubated aerobically
and anaerobically with 10% CO at 370 C. Subculturing broth to agar was

2

done by streaking the agar with calibrated loops (vide supra) from the

 

sediment in the broth media and plates were incubated aerobically in

n

. o
moist atmosphere at 37 c. Agar to agar transfers were made by the

56

57

"push block" technique. A block of agar, 1 cm2, was cut out with a

sterilized scalpel. The block of colonies was placed face down on the
fresh medium and moved over the surface of the agar with the aid of a
sterile forceps. Subculturing agar to broth was done by transferring
a strip of agar containing colonies directly into broth media. Plates

were examined for Mycoplasma colonies over a 7-day period using a micro-

 

scope with oblique indirect lighting or a regular microscope at 20x: or
40x magnification lens.

b. Embryonating chicken eggs: Fertile chicken eggs were obtained
from the Department of Poultry Science, Michigan State University. Seven-
day-old embryos were inoculated with 0.1 m1 of broth culture containing
Mycoplasma suspensions by using a 27-gauge, 1/2 inch needle. In most cases
embryo-death occurred after 5-7 days and the yolk materials were harvested

by aspiration procedures with ZO-gauge, 1 1/2 inch needle.

3. Storage of the Organisms

Broth cultures containing Myggplasma organisms were centrifuged at

 

35,000 xg for 30 minutes under sterile conditions (Sorvall, RC-S, super-
speed refrigerated centrifuge). The sediments were diluted with 5 ml
of broth media and were frozen at -20 C in 100 x 13 mm screwcap tubes.
Some cultures were stored in the frozen egg yolk after the passage of

organisms was lyophilized and held at 5 C. The Mygoplasma culture

 

sediment to be lyophilized was mixed with 2 ml of broth media or
stablizing solutions (SPGA) as described for rickettsia (Bovarnick,
§£_al,, 1950), see Appendix. The lyophilized cultures were sealed under
vacuum. Titer and biochemical characteristics were determined for the
lyophilized cultures to ascertain viability and purity at certain time

intervals.

58

B. Culture Media
The medium which was selected for antigen production or for

isolation of Mypoplasma depended on two factors: the adaptability of

 

the Mycoplasma strain to the medium and the availability of the serum

 

and other enriched factors. All media used required the addition of
serum. Serum was inactivated for 30 minutes at 560 C before use. All
media (broth or agar base) were sterilized by autoclaving before adding
the serum. The other components of the medium were sterilized by fil-

tration (see the sterilization procedures below).

1. Media for Antigen Production

Three media were commonly used for large-volume antigen production:
brain-heart infusion medium (Vardmann, 1967), PPLO broth medium (Morton
and Lecce, 1954) and tryptose phosphate broth medium (Hall, 1962).
These media were used also in the primary isolations and for routine

serial passages of seed stock.

2. Media for Isolation, Growth and Maintenance

Many kinds of media for isolation of avian Mycoplasma were described

 

by several authors. Two media were used in our experiments for compara-
tive studies: B-media of Fabricant (Fabricant, 1959),which was prepared
with Difco beef-heart broth enriched with 10-15% horse or swine serum,
and a modified Frey medium (Yoder, 1975), which was used in broth and

agar forms with the addition of B-nicotinamide adenine dinucleotide (NAD).

3. A Modified Medium for Isolation and Antigen Production (MSU-Mycoplasma

 

Medium)

59

This modified medium was used in isolation of Mycoplasma organisms

 

and for antigen production as well. The Mycoplasma broth base (Gibco)

 

was used as a base medium. The MSU-Mycoplasma formula is as follows:

 

 

Ingredients Per Liter
Mypoplasma broth base (Gibco) 24.0 g
Maltose or Dextrose 2.0 g
L-histidine 2.48 g
Cystine HCl 0.1 g
Nicotinamide adenine dinucleotide (NAD) 0.1 g
Waymouth media 100.0 g
Tris (hydroxymethyl) aminomethane Hydrochloride 3.0 g
Penicillin 1000.0 IU/ml
Thallous acetate 0.1 g
Phenol red 0.025 g
Horse serum . 50-100 m1
Water to 1000 ml

Adjust pH to 7.8 with 20% NaOH

a. Procedures used for mixing MSU-Mycgplasma medium:

 

 

(l) Mycoplasma broth base (Gibco), maltose or dextrose (Difco),
and tris buffer base (Sigma) were dissolved in 700 ml deionized
distilled water (DDW) to make the base medium. Adjust the pH
to 7.9 - 8.0. Addition of 3 grams of yeast autolysate has

been recommended but it was not necessary because the Mycoplasma

 

broth base contained yeast extract.

(2) Thallous acetate (Fisher) was added to the base media before

60

or after autoclaving. One percent solution was prepared by
dissolving 0.5 gram in 5 m1 DDW and sterilized by filtration

(4) Phenol red solution was prepared by dissolving 0.3 g in

300 ml DDW (dissolving by heat) and sterilized by filtration;

25 ml was added to 1000 ml medium. Phenol red wasn't added to
the media for antigen production.

(5) L -histidine 1% solution was prepared by dissolving 2.48

gm in 10 m1 DDW and sterilized by filtration.

(6) Horse or swine or calf sera (Gibco) were used in the pre-
paration of different media for isolation and for antigen
production. All sera were heat inactivated at 56 C for 30
minutes and centrifuged for 30 minutes at 10,000 rpm to clarify.
They were then sterilized by filtration.

(7) Waymouth's medium (MD 705/1, Gibco) was added as a source
of amino acids and vitamins.

(8) Penicillin G (potassium salt) was reconstituted in sterile
distilled water and stored frozen; 100,000 IU/lOO m1 of

medium was added to the medium after mixing all other ingredients.
The final pH of the medium was adjusted to 7.7 -7.8 with sterile
10% NaOH.

Sterilization procedures:

(1) Autoclaving referred to heating for 15 minutes at 120 C

and 15 pounds of steam pressure.

(2) Filtration usually was achieved by successive passages through
20, 5.0, 3.0, 1.2, 0.45, 0.1 and 0.025 um Millipore filters.

Serum and phenol red were filtered first through No. l and 50

61

Whatman filter paper discs before passage through Millipore
filters.

All liquid broth media were incubated at 37 C overnight after
preparation to ascertain their sterility. They were then held
at 5 C until used. Agar plates were left at room temperature

overnight and then stored at 5 C.

 

4. Experimental Studies on the Effect of pH of Different Mycoplasma
Media on the Survival of the Organisms. . '
a. Culture media: The broth media used to evaluate growth were
brain-heart infusion medium, PPLO broth medium, tryptose phosphate broth
medium, B-media of Fabricant medium, modified Frey broth medium and MSU-

Mygoplasma medium. Exact formulation of each medium is detailed in the

 

Appendix. The broth media were used to support the growth of Mg, Agar
(Difco)was added at 1.5% to each broth medium to make the agar plate and
that was used to determine the viable organism counts.

b. pH: The initial pH of all media was measured before inoculating
the broth tube with Mg organisms. The pH of each medium was determined
at selected time intervals as follows: 4, 8, 12, 16, 20, 24, 28, 32, 36,
40, 44, 48, 52 and 56 hours and on the 3rd, 4th, 5th, 6th, 9th, 30th and
60th days post inoculation. The hydrogen ion concentration was measured
in an expanded scale pH meter (Model 7415, Leeds and Northrup).

c. Determination of numbers of viable organisms: Viable bacterial
count was determined by using dilution plate method (i.e.,counting the
total number of colony forming units in 1 m1 media (CFU/ml) from each
dilution). This was conducted by making a series of lO-fold dilutions

1 10

of the original from 10- to 10- in sterile distilled water. One-tenth ml

62

from each of these dilutions was placed on a Mycoplasma agar plate
(divided into two, 0.1 ml/half plate). The plates were incubated at

37 C for 7 days. The highest dilution showing any growth was recorded
as the plate titer. The number of colonies presented in a plate at a
given dilution was multiplied by the reciprocal of the volume of the
original sample presented in a plate. For example, a plate containing

a 1:103 diIUtion contained 10-3 ml of the original sample, so the number

3

of colonies was presented, multiplied by 1/10- or 103 to estimate the

number of colonies per ml of the original sample.
C. Mycoplasma Antigen Production and Standardization

1. Preparation of Mycoplasma Cells

 

a. Cell production: MSU-Mycoplasma medium (MSU) was used for the
growth of Mycoplasma organisms and for the preparation of antigen. The
medium was stored at 4 C in 3 different quantities as follows:

(1) Propagation medium tube: 16 x 150 mm screw cap tubes contain

9 ml of MSU. These tubes were used for serial transfers.

(2) Seeding medium flask: 300 ml Erlenmeyer flasks contain 90 ml

of broth medium.

(3) Antigen production medium flask: 2800 ml Erlenmeyer wide

bottom flasks contain 900 ml of broth medium.

The medium from the storage was warmed to incubator temperature
(37 C) before being inoculated. The original culture of Mg was serially
transferred in the broth medium (propagation medium tube) several times
before streaking on Mycoplasma agar plates. The inoculated agar plates

were incubated for 7-10 days at 37 C aerobically in a moisture-rich

Atmosphere to avoid dryness of the media. Examining the colonies under

a dissecting microscope with oblique indirect lighting or 20x or 40x

lens power ofa regular microscope showedtypical 0.1-1 mm diameter fried

egg colonies. Under aseptic conditions, a small block of agar containing
a colony was excised and transferred into propagation tube media. After
24 hours at 37 C, the contents of the tubes were transferred into the
seeding flasks,which contained 90 m1 of the medium. After 24 hours at

37 C, the contents of the seed flasks were poured into 900 ml of fresh
medium for antigen production. Antigen production flasks were agitated,
some for 2 hours at room temperature, then incubated for 4 hours at 37

C. Other flasks were incubated at 37 C for 15 hours and then agitated

for another 6 hours at room temperature. However, all flasks were agitated
on a variable speed shaker oscillating in a horizontal plane approximately
92 times per minute either in a room temperature or in a walk-in incu-
bator at 37 C. The incubation period was stopped when the pH of the
medium reached 6.8, which is the optimum pH to obtain the log phase of
growth. Sterility tests were routinely carried out with each passage of
the growing organisms to eliminate the possibility of bacterial contamina-
tion. Cultures in which any contamination was observed were immediately
discarded.

b. Cell harvesting: The antigen production media containing the
organisms were centrifuged at 35,000 xg for 30—45 minutes at 5 C using
Sorvall RC-S super speed refrigerated centrifuge. The sediments were
resuspended in small quantities in phosphate bufferedsaline (PBS) at pH
7.2 (see Appendix). The Myggplagga cell paste sediment was homogenized

in a sterile Ten Broeck tissue grinder with PBS or agitated with glass

64

beads and the final volume brought up to 10 ml per 1000 ml of the
original production medium, thus accomplishing a lOO—fold concentration

of Mycoplasma cells. This stock suspension was either kept frozen

 

at -20 C until needed or lyophilized using fresh broth media or sucrose
phosphate glutamate albumin (SPGA) as preservative stabilizing solution
(Bovarnic, Miller and Snyder, 1950) or proceeding to another step of stand-
ardization. The suspended antigen was diluted to a final concentration
equivalent to 2X of No. 10 McFarland tube (60 x 108nucroorganisms). This
was accomplished by using a dilution procedure to establish the concen-
tration that would provide a given percent transmission inzuiexciter and a
galvanometer lamp. The McFarland standard tube (2X a No. 10) was

diluted l:20,which gave a 54% transmission (O.D. = 0.25 :_0.01) reading

at wavelength of 540 millimicrons Uum)u5ing£1Coleman Junior II A
spectrophotometer, Model 6/20 A. The spectrophotometer was adjusted

to 100% transmission with a phosphate buffer as a blank. This was

matched by a series of antigen dilutions using PBS buffer to provide a
density of cells‘UDbe read directly in the spectrophotometer. The

antigen dilution that gave a reading of 54% :_2 transmission was then

used as equivalent to 2X No. 10 McFarland nephelometer tube. The stock
antigen solution was kept frozen at -20 C.

c. Sterility test: During the process of antigen production and
after thawing the antigen for further standardization, a sterility check-
up proceeded for bacterial and fungal contamination. A few milliliters
of the culture medium at different stages of the antigen production was
inoculated into blood agar plates with brain-heart.infusion and Sabouraud's

dextrose agar. It was noticed that the broth media in case of contamination

65

changes its color from rose red to a deep red.

2. Antigen for Immunization

Mycoplasma whole cell antigen has been described in the literature

 

as a particulate antigen. For the specific production of precipitating

antisera against Mg in rabbits, Mycoplasma broth medium was enriched

 

with rabbit serum instead of swine or horse serum. Organisms were har-
vested, washed twice with PBS,then homogenized as previously described.
The final volume of the antigen suspension was 1/100 fold concentration

of the growth medium volume (antigen production media). No preservative
was added to the concentrated antigen suspensibn. The antigen was diluted
to give 2X No 10 McFarland tube (60 x 108 microorganisms) using a spectro-

photometer before it was injected into the rabbits.

3. Antigen for Hemagglutination Inhibition Test

 

Mycoplasma whole cell paste was resuspended in pggpflgggliged phosphate
buffer at pH 7.0. Highly dense cell suspension with an absorbance reading
of 0.25 at a wavelength of 540 um at 1:10 dilution was referred to as
standard hemagglutination antigen (HA antigen). An equal volume of
glycerine was added and thoroughly mixed. This antigen was stored at

~50 to ~70 C.

4. Antigen for Enzyme-Linked Immunosorbent Assay
Preparation of soluble antigen in the form of disrupted cell sus-

pension was used for enzyme-linked immunosorbent assay. The preparation

of Mycoplasma cells was basically the same as described before. The

 

Mycoplasma cell paste was harvested and washed twice with PBS, pH 7.2.

 

The harvested cells were then resuspended in enough PBS to reach a 1/100 fold

66

concentration of the original volume. The total protein concentration
in the antigen suspension was determined by using Biuret method (Goznall
§£_§l,, 1949) and crystalline bovine albumin as a standard protein sol-
ution. Absorbance reading was recorded on a COleman Junior lIA spectro-
photometer Model 6/20 A at 560 um. The total protein content of the
antigen averaged 5.7 mg/ml. The antigen was solubilized by using sodium
dodecyl sulfate (SDS) (Sigma Chemical Co.). The antigen was diluted with
PBS, pH 7.2, to make a 3 mg/ml suspension and then incubated with SDS

(1 mg of detergent/mg of protein) for one hour at 37 C. The antigen

was then dialyzed against PBS,pH 7.2,containing 1 mM ethylene-
diaminetetraacetate (EDTA) (Fisher Chemical Co.) or other suitable
chelating agent (to prevent reaggregation of the solubilized antigen

on removal of SDA). The extract was centrifuged (20,000 xg for 30
minutes at 4 C). The supernatant fluid was removed and protein concen-
tration was determined using Biuret method. The antigen was diluted

and coated with 0.1 M_carbonate-bicarbonate buffer, pH 9.6.
D. Preparation of Fluorescent Antibody Reagents

1. Production of Rabbit Antisera

New Zealand rabbits were obtained from the Laboratory Animal Care
Service (LACS), Michigan State University, East Lansing, Michigan. The
weight of each rabbit was approximately 6 lbs. (2.7 kg) and the age was
3 months. They were fed Purina Rabbit Chow (Purina Company) and were
kept individually in a metal cage in the isolation room. The cage size
was as recommended by the LACS, 3.5 square feet per rabbit for floor and

14 inches high. Serum was collected from rabbits prior to immunization

67

and examined for the presence of agglutinating and precipitating anti-
bodies. Three rabbits were inoculated according to the immunization

schedule as shown in Table 1. One rabbit was kept uninoculated as a

 

 

 

 

control.
TABLE 1. Immunization schedule for rabbits
Days Route and Amount of Injection
Rabbit I Rabbit 11 Rabbit III
1 SC 1.0 ml IV 0.5 ml SC .25 ml + 0.25 IFA
7 IM 0.5 ml SC 1.0 m1 SC 0.5 ml + 0.5 IFA
14 FP 2.0 m1 IV 2.0 ml IV 2.0 ml
21 IV 3.0 m1 IV 3.0 ml IV 3.0 ml
28 and every
other week for
3 weeks 1P 5.0 m1 IP 5.0 ml 1P 5.0 ml
SC = subcutaneously
IM = intramuscularly (hindquarter)
FP = intrafoot pad
IV = intravenously
IP = intraperitoneally

IFA = incomplete Freund adjuvant

Rabbits I and II were injected with Mycoplasma bacterial suspension without
adjuvant. One ml of the bacterial inoculum contained 60 x 108 microorganisms.
Rabbit III was injected with combined Mycoplasma and incomplete Freund
adjuvant (Difco). Rabbits were bled (25-40 ml each bleeding) one week
after the third injection from the marginal ear vein and every other week
for 8 weeks after the first IP injection. The rabbits were terminally

bled by exsanguination. The antiserum was dispensed in 2 ml amounts into

68

screwcap vials and stored at -20 C. The antibody titer in serum was
determined in the three inoculated rabbits as well as the control one

by the HI and TA tests.

2. Production of Chicken Antisera

Two hundred male l-day-old SCWL from a Mycoplasma-free breeder flock,

 

unvaccinated for Marek's disease, were obtained from the Rainbow Trail
Hatchery, Inc., St. Louis, Michigan. The chickens were maintained in
metal cages and isolated in separate rooms at the Poultry Teaching
Research Center,Department of Poultry Science, Michigan State University,
East Lansing,Michigan. At 4 weeks of age, the birds were divided into

5 groups, 40 birds each group, according to the route of infections.
Birds were infected with live Mg_organisms prepared at the Avian Micro-

biology Laboratory, Michigan State University; each 1 ml of the Mycoplasma

 

suspensions contained 4 x 1010 organisms. The dose of the infections
was designated as follows: 0.2 ml intravenous, 0.2 ml subcutaneous,one
drop intranasal, 0.5 ml 'intraperitoneal inoculation. The last group

of birds was either sharing the room with the infected birds in separate
cages or sharing the same cages with the infected birds. Two to four
inoculations were administered to the chickemsat weekly intervals.

Serum samples were collected from some of the chickens before inoculation
and specific antibody titers for Mg and Mg_were determined by HI and TA
tests. Also, the mortality was recorded. The chickens were bled from
the wing vein 4 weeks postinoculation and every other week for another

4 weeks. They were terminally bled by exsanguination. Tissue samples

were taken for histopathology and isolation of Mycoplasma organisms was

 

achieved by using modified Frey Mycoplasma and MSU-Mycoplasma medium.

 

 

69

3. Conjugation of Rabbit IgG Fraction with Fluorescein Isothiocyanate

a. Fractionation of rabbit antisera with ammonium sulfate:
fractionation of rabbit antiserum with ammonium sulfate was conducted by
Herbert method (1971) with some modification. The method is summarized
as follows:

(1) Fivemlof 70% saturated ammonium sulfate (NH4)ZSO was slowly

4
added (dropwise) toza 5 ml serum sample with constant stirring.
Ajust the pH of the suspension to 8.0 with 1N NaOH.

(2) The suspension was agitated on an automatic shaker for 2 hours
in order to avoid mechanical trapping of the serum components other than
gamma globulin and the mixture was allowed to stand at 4 C for 24 hours.
The precipitated globulin was packed by centrifuging at 4 C for 30 minutes
at 1440 xg using a Sorval refrigerated superspeed centrifuge.

(3) The supernatant fluid was poured off and the precipitate was
redissolved in 5 ml of borate buffered saline (BBS), see Appendix.

(4) The globulin was reprecipitated as described in steps 1 and 2
and centrifuged immediately as before. The supernatant was discarded
and resuspended as in step 3.

(5) The globulin fraction was precipitated for the third time and
resuspended as in step 4.

(6) The globulin fraction was dialyzed against frequent changes of
0.85% NaCl solution, pH 8.0, at 4 C until sulfate was no longer detected
in the dialysate after overnight use. The presence of sulfate was de-
termined by adding a small volume of saturated barium chloride (BaClz)
solution to an equal volume of the well mixed saline dialysate. No

cloudiness resulted, which meant that the globulin solution was considered

70

to be substantially free of sulfate. This gamma globulin fraction was
considered to contain mostly IgG.

The isolation of immunoglobulin G (IgG) from the rest of gamma ,
globulin was experimentally approached by employing protein A (Pharmacia)
instead of ammonium sulfate.

b. Protein determination of gamma globulin fraction: The nitrogen
content of the crude gamma globulin and the total mg protein/ml of globulin
fraction were determined by applying the Biuret method (Goznall ggflgl.,
1949). Also, Bio Rad protein assay (Bio Rad Laboratories, Richmond,
California) was used. Standard solution of bovine gamma globulin was
used for preparation of the standard curve for both methods.

c. Conjugation of globulin with fluorescein isothiocyanate:

Rabbit gamma globulin fraction was conjugated with fluorescein isothio-
cyanate (FITC) (BBL, Baltimore, Maryland) according to the methods des-
cribed by Riggs §£_§1, (1958) and Herbert §£_§l, (1977).

The procedures used were as follows:

(1) Protein concentration of the globulin fraction was
determined. Rabbit I, 11 serum contained 38 and 44 mg protein/ml serum
simultaneously Rabbit II gamma globulin fraction was used in most of the
experiments. Ten ml of globulin was used and the total protein content
of the globulin was 10 x 44 = 440 mg.

(2) Tbs ml of globulin fraction was placed in a flask and 5
m1 of 0.2 M_Na HPO was added slowly with gentle mixing. The pH was

2 4

brought to 9.5 by drOpwise addition of 0.1 M_Na3PO4. The final volume

was brought to 20 ml by using 0.85% NaCl.

(3) The fluorescein protein ration used in the experiments was

71

17.6 ratio (FITC ug/mg protein). 50 the weight of FITC used was 440 x
17.6 = 7.74 mg. FITC solution was prepared by using 10 m1 dye solution
to 1 m1 of globulin (10 m1 x 10 m1 globulin = 100 ml FITC solution).

FITC (75%4mg)was dissolved in a volume of 0.1 M_Na NPO4 equal to 3/4 of

2
the final volume desired (75 ml). The pH was adjusted to 9.5 with 0.1
M_Na3PO4 and the final volume was brought up to 100 m1.

(4) The globulin fraction was placed in a dialysis tube
(Union Carbide) and the sac was tied so that there were no air bubbles.
The dialysis tube was placed in the FITC dye solution so that the globulin
was completely submerged. The reaction was allowed to proceed at 25 C
for 6 hours.

(5) The dialysis tube was removed and rinsed briefly under
cool tap water to remove all exterior dye.

(6) To remove the unreacted fluorescent material (UFM) the
labeled globulin was dialyzed against phosphate buffered saline (PBS),
pH 7.6, at 5 C for 24 hours. The low temperature of the buffer was to
reduce the labeling rate of the unreacting dye in the dialysis tube.
Dialysate PBS was used in a large volume and changed frequently. The
removal of UFM was completed when no fluorescence was visible in an
aliquot of the dialysate observed in the dark under a Wood's Lamp
(ultraviolet). The labeled globulin was centrifuged and the precipit-
ate was removed.

(7) Merthiolate was added as a preservative in a final con-
centration of 1110,000 to some of the prepared conjugate. The conjugate

was stored in 1 ml quantities in small screw cap tubes and kept in liquid

form at S C.

72

4. Fixation Procedures for Direct and Indirect Methods

-The avian Mycoplasmas used in this study were Mg, Mg, MM, These

 

organisms were grown on Mycoplasma broth and agar media as mentioned

 

before. The rabbit antiserum was prepared from Mg only. The Mycoplasma

 

antigens for the three different species mentioned above used in these
experiments were broth culture sediments and their agar colonies. A
thin smear of bacteria was prepared on microscopic slide (clean slide

by acetone) from broth culture sediments or bacterial colonies. The
smear was air dried at 37 C on slides prior to being subjected to

various fixing agents and conditions. Dry heat fixation for 1 minute

or exposure to 95% ethanol for 10 minutes were used in this study. Also,

agar blocks about cm2 with colonies of Mycoplasma were cut from the agar

 

plates with a sterile scalpel blade. The agar blocks were placed on a
microscope slide with colonies up. The colonies were fixed by using

the hot water fixation technique of Clark g£_gl, (1963). Hot distilled
water fixed the colonies on the slide as it dissolved away the surrounding
agar, thus leaving colonies relatively free of agar substances and there-

by the nonspecific fluorescence was reduced.

5. Direct Staining

The basics of the staining procedure used in this study were those
of Coons and Kaplan (1950) and Marshal 3E El: (1958). Rabbit antisera
conjugated to FITC was freshly diluted 1:10 and 1:20 with 0.85% NaCl,pH
7.0. The area of fixed antigen on the slide preparation was covered
with a few drops of the conjugate and the slide was placed on a moist
towel in a covered tray and kept at 37 C for 20 and 40 minutes. The

slide was then washed with PBS twice and was stored in fresh PBS for

73

2-3 minutes. Excess fluid was drained and the slides were mounted in

10% buffered glycerol, pH 7.2 (glycerol 9 parts, PBS 1 part), and a cover
slip was added. The specificity of the reactions was determined by

using 2 control systems labeled normal rabbit serum and homologous and
heterologous labeled anti-serum positive for Mg, Smears of Mg_and Mg_
were stained by the direct method to show the specificity of labeled

antibody against Mg_organisms.

6. Microsocopy

Fluorescence observations were made with Carl Zeiss photomicroscope
G 40 - 430 (W. Germany) by using a 40 X high dry Neofluor lens and an
epi-illuminator Fluorescein. Fluorescence was excited with a super
pressure mercury lamp HBO 200 w/4 which served as a radiation source.
The transmission curve for the light source is shown in Figure 5 (Appendix).
Permanent and exciter filters,combinations BG 12 and BF 004100 respec-
tively, were used as ideal filter sets in these experiments. The trans-
mission curve for the exciter and barrier filters is shown in Figure 6.
Photography was performed with Kodak (Ektachrome, 100 Tungsten filter

using the Leica Camera.

7. Indirect Staining
A thin smear of Mg_organism was prepared and fixed on a micro-
scopic slide as described before. A few drops of 1:10 rabbit unconjugated

antiserum for Mycoplasma was added to the slide and allowed to stand in

 

a moist atmosphere at 37 C for 20 minutes, then washed with PBS. The
slide was stained with FITC - labeled goat anti-rabbit IgG. The stained

slide was kept in a moist chamber at 37 C for 20 minutes, then was rinsed

with PBS, pH 7.2. The slide was mounted with a coverslip after the
addition of 10% buffered glycerol. Control slides were prepared as

in direct staining methods.

E. Immunochemical Studies on Purification of Chicken and Rabbit IgG

Using Protein A-Sepharose Chromatography

1. Source of Protein A
Protein-A-Sepharose CL-4B for chromatography was purchased from
Pharmacia Fine Chemicals, Piscataway, New Jersey. Protein A initially

was isolated from the cell wall of Staphylococcus aureus and covalently

 

coupled to Sepharose CL-4B by the cyanogen bromide method. Oweg of freeze-
dried powder was reported to be equivalent to approximately 3.5 ml of

swollen gel (Pharmacia Fine Chemical Publication, 1978).

2. Isolation and Purification of Rabbit IgG

Normal and immune serum were obtained from the same group of rabbits
which had been used in the immunization experiments. Protein concentra-
tion was determined by Biuret method. Immune serum from rabbit I
(immunized with Mg) containing 38 mg protein/ml and normal serum from
rabbit IV containing 26 mg protein/ml were used in these experiments.
Part of the serum (normal and immune) was used to recover IgG fractions
by successive precipitations with ammonium sulfate and recycling through
Sephadex G-200 with phosphate buffer saline pH 7.2. The IgG fraction
recovered from the column was dialyzed extensively for 3 days at 4 C with
0.85% NaCl pH 8.0.

Furthermore, rabbit IgG was isolated from both normal and immune

75

sera using column chromatography on Protein-A-Sepharose (Kessler, 1975).
Serum (10 ml) was applied to a column chromatography (1.1 x 4 cm) equili-
brated at 4 C with 0.1 M sodium phosphate, pH 7.0. The column was washed
with approximately 10 volumes of equilibration buffer. The IgG fraction
(35-70 mg) was then eluted with l M acetic acid and the pH was adjusted
with concentrated NH4OH to be 7.0. The neutralized IgG fraction was
dialyzed overnight at 4 C against 500 ml of 0.1 M sodium phosphate,pH
7.0. The buffer was changed twice during-the period of the dialysis.

The absorbance of the eluates of 0.1 M sodium phosphate and l M acetic
acid were measured at 280 nm using Hitachi Perkin Elmer l39-spectrophoto-

meter (Coleman Instrument Division).

3. Immunodiffusion Tests for Rabbit Serum

The method of immunodiffusion (Ouchterlony) was used in these experi-
ments as described basically by Ouchterlony (1968). Lines of precipita-
tions were formed as a result of antigen-antibody reactions. The numbers
of the precipitating band and the reaction of identity represented the
minimum numbers of homologous antigen-antibody systems. The two experi-
mental studies were conducted to compare the efficiency of protein A to
purify and isolate rabbit IgG to that of the ammonium sulfate precipita-
tion method.

First study: The center well contained 0.1 ml of goat anti-rabbit
serum and the surrounding wells contained eluates washed by 0.1 M sodium
phosphate, 1 M acetic acid and ammonium sulfate precipitates as shown
in Figure 16 (a,b).

Second study: 0.1 ml of goat anti-rabbit IgG was put in the center
well and 0.1 ml of different eluates washed by l M acetic acid,which has

higher absorbance reading than 2.0 at 280 nm was added to the other

76

surrounding wells. Also, 0.1 ml of ammonium sulfate precipitates was

added to several wells surrounding the center,as shown in Figure 17.

4. Isolation and Purification of Chicken IgG

Chicken normal and immune sera were obtained from the same group
of chickens used in the immunization experiments. The sera were delipi-
dated by centrifugation at high speed as 2700 Xg at 4 C for 20-30 minutes
and the lipid on the surface was removed by wooden stick or by Pasteur
type pipette. Also, precipitation of B-lipoprotein in the serum by
certain bivalent cations such as managanese chloride, cobalt chloride
and potassium bromide (Burstein and Praverman, 1957), was applied in
these experiments for the purpose of delipidation.

The IgG fraction was recovered by successive precipitation with
NaZSO4 and recycling through Sephadex G-200 with borate buffered saline
(BBS), pH 8.2. The eluate fraction was dialyzed extensively against PBS
for 2 days. Protein concentration was estimated by measuring the absorb-
ance at 280 nm and using bovine gamma globulin to draw the standard
curve. Protein A Sepharose(Pharmacia Fine Chemicals) was used to
isolate IgG fraction by the same method which has been described

previously with rabbit serum. The absorbance readings of 0.1 M sodium

phosphate and 1 M acetic acid elutes were measured at 280 nm.

5. Immunodiffusion Tests for Chicken Serum

Immunodiffusion experiments were conducted employing Ouchterlony
methods (Ouchterlony, 1968). Several studies were designed to determine
the specificity of the eluates.

First study (Figure 19 a,b): 0.1 ml of rabbit.anti£hicken globulin

77

(Rb-glb) was put in the center well, as shown in Figure 19a. One-tenth
m1 of different eluates washed by 0.1 M sodium phosphate (A-NP) which has
higher absorbance readings than 2.0 by spectrophotometer at 280 nm, as
shown in Figure 18. In the same study (Figure 19, b) 0.1 ml of Rb-glb
was put in the center well against 0.1 ml of the following: IgG fraction
purified by ammonium sulfate (AS), 1 M acetic acid washed eluate using
protein A-Sepharose (A) and control well contained chicken IgG fraction
(C-IgG).

Second study (Figure 20 a,b): One-tenth ml of rabbit anti-chicken
IgG (Rb-G) was placed in the center well. One-tenth m1 of 0.1 M sodium
phosphate washed eluate (A-ND), which has a higher absorbance reading
than 2.0, and control well contained 0.1 ml of chicken IgG fraction
(C-IgG), were placed in the surrounding wells, as shown in Figure 20a.
Furthermore, another Ouchterlony agar plate was designed in which the
center well contained 0.1 m1 of Rb-G and the surrounding wells contained
the following: .0.1 ml of l M acetic acid washed eluate of protein A-
Separose (A), 0.1 ml of IgG fraction purified by ammonium sulfate (AS)
and 0.1 ml of chicken IgG fraction (c—IgG) as a control well, as shown
in Figure 20b.

Third study (Figure 21 a,b): This study was designed to compare the
activity of the contents of the eluates of protein A-Sepharose column
chromatography washed by l M acetic acid for rabbit and chicken serum.

a. One-tenth ml of protein A-Sepharose, I‘M acetic acid washed eluate
of rabbit serum (Prt. A-Rb IgG-fret) was placed in the center well. One-
tenth m1 of normal rabbit serum (NRS), goat anti-rabbit serum (Gt-ant RS)
and goat anti-rabbit IgG (Gt-ant RIgG) were placed in the surrounding

wells, as shown in Figure Zla.

78

b. One-tenth ml of protein A-Sepharose, 1.M acetic acid washed
eluate of chicken serum (Pr A Ck IgG-frct) were placed in the center
well and 0.1 m1 of the following were in the surrounding wells: normal
chicken serum (NCS), rabbit anti-chicken globulin (Rb-ant Cglb) and

rabbit anti-chicken IgG (Rb-ant CIgG), as shown in Figure 21b.

6. Plasminogen and Their Affinity to Protein A

One-tenth ml of rabbit anti-chicken plasminogen (Rb-PL) was placed
in the center well. One-tenth m1 of different eluates washed by 0.1 M
sodium phosphate (A-NP), which has a higher absorbance reading than
2.0. The control well contained 0.1 ml of chicken plasminogen (PL),
as shown in Figure 21a. The Rb-PL and PL were obtained from Dr. E.
Smith, USDA Regional Poultry Research Laboratories, East Lansing,
Michigan. In another experiment, as shown in Figure 21b, 0.1 ml of
Rb-PL was added to the center well and 0.1 ml of the following was
added to the surrounding wells: different eluates of protein A-
Sepharose washed by l‘M acetic acid (A), chicken IgG fraction purified

by ammonium sulfate method (AS) and chicken plasminogen (PL) as a control.

F. Myggplasma—Enzyme-Linked Immunosorbent Assay (MYCO-ELISA)

 

l. Antigen Production, Harvesting and Standardization

'Mg antigen was prepared from the whole bacterial cell and from dis- .
rupted cell suspension, as described before in the chapter on antigen
production and standardization. The procedures of antigen preparation

are summarized as follows: .Mg organisms were grown in hBUemedium and the

79

cell paste was harvested, washed and protein concentration of the cell
suspension was determined. The antigen (cell suspension) was solu-
bilized by 808 to disrupt the plasma membrane, then the suspension

was dialyzed against PBS containing 1 mM_ethylenediaminetetraacetate
(EDTA). The protein concentration of the antigen was determined by
spectrophotometer. The antigen was diluted and coated by 0.1 M carbonate-

bicarbonate buffer, pH 9.6.

2. Source of Serum Samples

Blood from naturally and experimentally infected chickens was col-
lected and sera were separated. Some sera were inactivated by heat at
56 C for 30 minutes and stored at 5 C until used and other sera were
kept frozen at -20 C. Positive and negative serum references for avian

Mycgplasma were obtained from the National Veterinary Service Laboratory

 

(NVSL), Ames, Iowa. All serum samples were tested for antibody titer

against Mg and Mg_by the HI method before using for MYCO—ELISA test.

3. Preparation of Horseradish Peroxidase Conjugate

The conjugate used was IgG fraction of rabbit antiserum to chicken
IgG labeled with horseradish peroxidase enzyme (HRP) Type VI, Sigma
Chemical Co., St. Louis, Missouri. The conjugate was prepared by the
two-step glutaraldehyde method (Avrameas 35.31., 1978) with modification.
Ten ml of HRP was dissolved in 0.2 ml 1% glutaraldehyde solution in 0.1
filphosphate buffer, pH 6.8, and the preparation was incubated for 24 hours
at room temperature. Five mg of rabbit anti-chicken IgG (5 mg/ml) was
added to the peroxidase solution. Two-tenths ml of 0.5 M carbonate-

bicarbonate buffer, pH 9.5, was added and kept at 4 C for 24 hours.

80

0.1 ml of l M lysine solution, pH 7.0, was added and kept at room temp-
erature for 2 hours. The mixture was dialyzed overnight against PBS, pH
7.0, at 4 C. The conjugate was filtered through a sterile Millipore
membrane (0.22 um). An equal volume of glycerol and bovine serum albumin
(BSA) (5 mg BSA/ml) were added. The stock conjugate was stored in the
dark at 4 C until used. The conjugate remained viable for 10 weeks when
preserved in this manner. The use of optimum dilution of rabbit anti-
chicken IgG conjugated to HRP is essential for minimizing nonspecific
absorption to the control wells uncoated with antigen.

Initial experiments were conducted to standaradize the conjugate.
Conjugate dilutions of 1:50, 1:00, 1:200, 1:300, 1:400, 1:600, 1:800 and
1:1000 were used with serum dilution of 1:50 and 1:100 and antigen con-

centration of 90 mg/ml and 180 mg/ml.

4. Preparation of the Substrate
,_ . _ .- _ - . . _ _
2,2 A21no d1 3 ethyl benzthiazolin sulfonate (ABTS), (c18H16N406S4 NH4)

2, MG 5487, substrate was obtained from Boehringer Mannheim GMBH, Indi-
anapolis, Indiana. Preparation of the substrate was done as follows: 100
ul of ABTS Stock solution (22 mg/ml) was added to 20 ml of 0.1 citrate -
0.2 M phosphate buffer, pH 4.0, containing 20 ul of freshly prepared 0.3%
hydrogen peroxide (Saunders §E_§l,, 1977). The substrate was prepared

just before adding it to the wells. The ABTS stock solution should be

kept at 4 C in the dark at all times.

5. MYCO-ELISA Protocol
The indirect microtiter ELISA was performed as described by Engval

and Perlmann (1971) and Voller g£_§l, (1976). This procedure is

81

summarized in Figure 7. The MYCO-ELISA procedure is illustrated step

by step in Figure 8. Two hundred ul volumes of ELISAj g antigen diluted
in 0.1 M carbonate-bicarbonate buffer, pH 9.6, were added to each well
of the polystyrene substrate plates. The plates were incubated over-
night at 4 C. The plates were washed three times in PBS, pH 7.4, con-
taining 0.05% Tween 20. The buffer consisted of NaCl, 8.0 g; KH2P04,

2HPO'4°12H O, 2.9 g; KCl, 0.2 g; and Tween 20, 0.5 m1, as a

detergent. The buffer may be stored at 5 C for 4 weeks. The procedure

0.2 g; Na

for washing the plates consisted of shaking out the buffer in the wells,
refilling the wells with buffer, and then allowing this to stand for
1 minute.
The process was repeated two more times for 1 minute per washing.
Two hundred ul of serum sample, diluted with PBS, was placed in the
appropriate well. Each serum sample was tested in duplicate. The
plates were incubated for 2 hours at room temperature (22-24 C) in a
moist chamber or incubated at 37 C for 1 hour. The plates were then
washed 3 times with PBS-Tween 20 washing buffer, as described previously.
To each well, 200 pl of freshly prepared diluted conjugate HRP-
rabbit anti-chicken IgG was added and incubated at room temperature for
2 hours in a moist chamber. The plates were again washed three times
and 200 pl of ABTS substrate (freshly prepared) was added. After 30-45
minutes, or after the formation of green color in the positive serum
control, 50 ul of 0.45% hydrofluoric acid (HF) was added to each well
to stop the hydrolysis. The plates were then agitated well for 1-2 minutes.
The positive result is indicated by the formation of green color.

For quantitative measurement of the antibody level, the intensity

82

Fignm l PRINCIPLE 0F MYCO-ELISA

l. Mycoplasma antigen adsorbed to wells.

 

WASH

2. Serum sample is added, specific antibody,
if present, will bind to the antigen (Ag-Ab).

WASH

3. Enzyme-labeled antiglobulin is added, it
will attach to Ag-Ab. ‘

WASH

4. Substrate is added

Amount substrate hydrolyzed (color change) =
amount of antibody in serum sample.

 

 

 

 

 

 

 

 

83

 

 

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.44“: zo<m osz Away zmaupz< mm as _a com aa<

 

mxsomuoma <m~4muou>z .w 0.3m:

. —

84

 

 

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om zmmzh xmo.o + mmm IPHZ zm<3

.mmso:

 

N mom mm2h<mm¢2mh zoom h< mh<m=oz~ .mp<u~4a:o

 

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—: oom

85

 

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ON zmmze amo.o + mma zed: =m<3
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zmxudzu-ahz< »_mm<m do zoahu<ma wad op omp<a=azou
mmaodxoama zmdo<mmmao= ommaauma >s=mmma do _a com

86

 

 

 

 

 

 

 

 

 

 

 

 

pozcfiucoo w ohsmfim

.33“: =u<m op omaa< ma mxmpazoassm
mzHSON<HIHszm-s>IHm-mv-Ha-ozHN< .N.NH
whamhmmam mam< amz<amaa >s=mmaa me _a com

. a

87

 

 

 

 

 

 

 

 

 

 

 

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.m4¢z<m

22mm; 5: 2H >oom:.z< do #2322 “E. E. ._<zo:.mon_omn_
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P: mm .moz<:u monou ampudmzou m<z Aomhzou 2.5mm
muzmmmu—wm m>CHmoa mzh ~5th .mlo. 3::sz om an??? .m

88

of the reaction of the serum-control antigen reaction, measured by
absorbance reading, is subtracted from the absorbance-value of the
test serum sample. The blank is the antigen control wells, which do

not contain serum.

RESULTS

A. Cultural Media

1. Growth and Survival of Mg in Different Broth Media

Six different Mycoplasma media were used either for antigen pro-

 

duction or for isolation of the Mg organisms. Brain-heart infusion
medium (BHI), PPLO medium (PPLO), tryptose phosphate broth medium (TPB),
B-medium of Fabricant (BMF) and modified Frey media (MFM) were compared

to the new formula, MSU-Mypoplasma media (MSU). The growth curve of Mg

 

in four different medium formulas by hours or day post-inoculation (PI)
are illustrated in Figures 9 and 10. The total bacterial count was
calculated in the log10 number of colony forming units per ml (log No

CFU/ml) of the culture. MSU-Mycoplasma medium gave the highest CFU/ml
12

 

after 24 hours PI, which was 3.9 x 10 CFU/ml. The lowest bacterial

count was recorded for PPLO and TPB medium, which was 3 x 106 and 2 x

106, respectively, at 24 hours PI. The total CFU/ml for modified Frey

10 CFU/ml. The PPLO medium and TPB

medium at 24 hours P1 was 1.2 x 10
medium gave similar bacterial counts in the first 56 hours PI at each
reading. They gave the highest bacterial count at 32 hours PI, which was
0.3 x 107 and 0.4 x 107 CFU/ml for PPLO anad TPB media, respectively.

The total bacterial count for all media declined after 32 hours PI, as
shown in Table 3 and illustrated in Figures 9 and 10. The bacterial
count at 30 and 60 days was 0.0 for PPLO and TPB media. Modified

Frey medium gave total bacterial count of 0.1 x 101 CFU/ml at 30 days

89

 

esfleez

 

90

 

 

 

eofixm eeaxa. meaxe. ecsxw. modem. meaxm. - ado fleets
. . . . . . . . dddaiameed
e e m e e a N a m a e a m a :d emeedsde
x X. x. X. x. x. I
eea m eon a A ees m mes m H men 4 Ned m :do esid
e.e e.e e.a H.a m.a m.a e.a :d .
eax~.H eaxe.s eaxm.s esxa.e esxe.a eaxw.H I add ended:
ea OH a a e m seed
m.e e.e N.a m.a e.a w.a w.a :d eefldseez
x. x. x. x. x. x I .
Naea.e m Haea e m eHeH e N mes e m ecu a H mod ~ Dec :m:
m a e.a e.a a.a w.a w.a w.a :d
muse: muse: muse: muse: whoo: whoo: ouwfimoz an:
em em ea NA w a daemon <H .

 

 

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91

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some

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:e

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.mwnoa mammamoux: ucohommmw mo :9 exp use pcsoo Hewuouoma Hmuou ozu :o

waHm o>flpmhnasou

.m m4m<H

92

Figure 9. The total bacterial count (Log No CFU/ml) for Mg in
different media recorded in hours post-inoculation

-* MSU: MSU-Mycoplasma medium

 

Q MFM: Modified Frey medium
* PPLO: PPLO-medium

a TPB: Tryptose phosphate broth medium

 

203343002. PmOn. meDOI

av Vv av on an 0« Va 0.. up

 
 
 

       

  

    

 

    

 

 

 

   

vw

 

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or... .ll.
2.: 01.0
a: *II...

a ouswmu

ltu/n :10 ON 901

94

Figure 10. The total bacterial count (Log No CFU/ml) for Mg in
different media recorded in days post-inoculation

if MSU: MSU-Myc0plasma medium

 

OMFM: Modified Frey medium
a PPLO: PPLO-medium

D TPB: Tryptose phosphate broth medium

95

n.— aIIIIo
odd; .IIII.
2.2 orlllo
an: *IIII*

20....(1. 3002.

.meOn. w><0

 

 

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96

and 0.0 CFU/ml at 60 days,as shown in Table 4. MSU-Mycoplasma media

5 CFU/ml and 0.1 x 103 CFU/ml at 30 and 60

 

gave a reading of 0.3 x 10

days PI,respectively.

2. Changes of the pH of Different Mycoplasma Media

 

The changes of the pH of MSU-medium, MFM-medium PPLO-medium and
TPB medium were measured and recorded in Tables 2, 3 and 4 and illustrated
in Figures 11 and 12. The initial pH (before inoculation) of MSU-medium,
MFM medium, PPLO medium and TPB broth medium were 7.8, 7.8, 7.6 and 7.5,
respectively. The pH of all media dropped as a result of the growth of
Mg_in the media after 6 days P1 to reach 6.5, 6.1, 5.1 and 4.8, respectively.
The reduction in the pH value after 24-32 hours was accompanied by a rapid

loss of the viable Mycgplasma organisms. The pH readings of MSU-medium

 

stayed much higher (basic) than the pH of MFM at 24, 28, 32, and 36 hours
PI. Furthermore, MSU-media gave a higher pH reading (basic) as compared

to the other media at all times.

3. Preservation of Mycoplasma Organisms

 

Mg antigen was harvested by centrifugation procedures and stored
with or without the addition of preservatives. The main function of
preservative was to prevent the growth of any contaminating microorganisms

and to kill the Mycoplasma organisms. Merthiolate solution 1% was added

 

to the antigen suspension in a final concentration of 1:1000 and 125000.
The merthiolate in both concentrations did not inhibit the contaminating
microorganisms from growing in the antigen suspension. Phenol was added
to phosphate buffer in a concentration of 0.25%,which was used for washing

and as a diluent for Mycoplasma antigen. Phenol was proven to be very

 

97

 

Esflwoz

 

 

 

 

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m w v v v bonsai:
. . . . . . .. d _d
N e N e N e N a .m e a e a a : emeddsda
I I x . x . X . X . x . .
mod n e ecu e e sea e e ees m a sea o N ado 04d;
o.m e.m H.m H.m H.m H.m N.m :d
- x . x . x . x . x . x . . .
Hes A e mes e A eeH e e eea e a NeH a N Ned e N add eewmmu
A.e A.e N.e H.e H.e H.e H.e :d denuded:
. x O x o x I x o x O o o v
men A e mod n o Ned e e do. N e odes e e efieaxm o esedxe A Dad :2;
m.e m.e m.e m.e m.e m.e e.e :e ..
xma zen ken xmo xmo sea zen
eeoe seem nee see gem cue edm <H2m2

 

mwwoa mamQHmOUNz usopommmp mo :9 use pcm uczoo .eflpouoms .mHOH use :0

xwzum o>mumumgaeu

. .V m..m<...

98

Figure 11. The changes of the pH of different media for Mg

by hours post-inoculation

* MSU: MSU-Mycoplasma medium

 

O MFM: Modified Frey medium
* PPLO: PPLO-medium

D TPB: Tryptose phosphate broth

medium

99

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c a a q a n a - d u - q

d
-

 

or... ill.
I“: ollb
an: ill...

AH edewnd

 

100

Figure 12. The changes of the pH of different media for Mg
by days post—inoculation

i MSU: MSU-Mycoplasma medium

 

QMFM: Modified Frey medium
* PPLO: PPLO-medium

D'TPB: Tryptose phosphate broth medium

101

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o n v n

 

 

 

 

1.5.:
n.m.z

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06.

 

102

satisfactory as a preservative. Phenolized (0.25%) phosphate buffer
at pH 7.0 was used to standardize and to homogenize tube Mg_antigen but

not the hemagglutination (HA) antigen. Samples from Mycoplasma antigen

 

were taken for culturing on blood agar,thioglycollaterand MSU-Mycoplasma

 

media to isolate either Mypgplasma organisms or any other contaminants.

 

No growth was observed. The HA antigen was resuspended in phosphate buf-
fered saline: and then homogenized. Glycerol (Difco) was added as a
preservative to the antigen and also to prevent denaturation of the
antigen by frequent freezing and thawing. No preservative was added

to MYCO-ELISA antigen.

4. Standardization of Mg Antigen

The Mg tube antigen was standardized to a concentration equivalent
to the density of 2 x No. 10 McFarland nephelometer tube with phenolized
phosphate buffer,pH 7.0. This antigen concentration was found to give
comparable results to the USDA (Ames, Iowa) Mg_tube antigen. No dye
was used in the preparation of the tube antigen and the antigen was
allowed to age for several days before checking the sensitivity and
again before use.

The acitivity of HA antigen was checked out and the final product
was diluted using equal quantities of buffer and glycerin,which gave
HA titer of 1:320 or 1:640. High titer concentration of the antigen
was found to be unnecessary. No preservative other than glycerol was
added. Antigen in quantities of 5 ml was stored for future use at -40
- -70 C.

The Mycoplasma-Enzyme-Linked Immunosorbent Assay (MCYO-ELISA) 3Dti'

 

gen was prepared as described in the Materials and Methods. The harvested

Mycoplasma cell paste was homogenized with PBS at pH 7.2. An equal amount

 

of glycerin was added to make up suitable volume of the antigen. A
total protein content of the antigen had an average of 5-7 mg/ml with no
preservative added. Detergent solution, sodium dodecyl sulfate (SDS),
was used to dissolve the lipoprotein content of the plasma membrane of

Mycoplasma. A final concentration of 3-5 mg protein/ml was achieved.

 

_B. Immunofluorescence Identification of Mycoplasma Isolates

 

l. Antibody Titer of Rabbit and Chicken Antisera

Antibody titer in the serum was determined by HI method. Rabbit
red blood cell suspensions in 2 and 3% were used in the HI test and 0.5%
chicken red blood cell was used in the same test. The HI titer of rabbit
antisera ranged from 1:160 to 1:640 and the agglutination titer
ranged from 1:640 to 1:2560 for immunized rabbits. The antibody titer

for chicken antiserum ranged from 1:80 to 1:1280.

2. Direct Fluorescence Method

The Mycoplasma organisms stained with homologous conjugate displayed

 

typical yellow-green fluorescence,as shown in Figure 13. Control stained
slides showed no specific yellow-green fluorescence,as shown in Figure 14.
No significant differences in staining reaction were noticed with 1:10

or 1:20 diluted FITC conjugated rabbit antisera or in the time of antigen-

conjugate reaction at 37 C.

3. Indirect Fluorescence Method
Specific green fluorescence organisms were observed after adding

the labeled anti-globulin,as shown in Figure 13. No specific fluorescence

104

Figure 13. Direct immunofluorescence staining of Mg organisms
showed a specific yellow-green fluorescence

Figure 14. Control (negative) fluorescence for Mg organisms
stained with labeled heterologous antibody showed
no specific fluorescence

x

. .12-”..- -..-.....- .4 .1

. L.‘.._...-..a~:.u.i

105

 

Figure 14

106

was observed in the control slide labeled with heterologous antisera or

using other Mycoplasma antigen as Mg_or Mm,as shown in Figure 14.

 

Both methods (direct and indirect) were able to identify Mg organisms
in media culture and differentiate Mg from other species of avian

Mycoplasma. The dull yellow to yellowish green autofluorescence which

 

varied in intensity was common with the same old culture of Mycgplasma

 

when reacted with heterologous antisera. Care was exercised so that
autofluorescence would not be confused with minor cross-reactions or

poor staining from dilute antiserum. Autofluorescence tended to fade

much more quickly upon exposure to ultraviolet light and caused difficulty
in obtaining a good image. These characteristics were helpful in dis-

tinguishing positive fluorescence.

C. Purification and Assaying IgG by Protein

A-Sepharose Chromatography

1. Purification of Rabbit IgG by Protein A

The rabbit IgG was isolated from normal and immune serum by chroma—
tography fractions of the serum on a column of protein A coupled to
Sepharose 4B (2 x 10 cm),as illustrated in Figure 15. The flow rate of
the eluate was 60 ml/hour. The fraction numbers from 0-20 were the eluate
wash of the serum by 0.1 M sodium phosphate, pH 7.0. The high peak and
the large volume of the eluate,which has higher absorbance than 2.0,
indicated that the amount of immunoglobulin washed by sodium phosphate
buffer was greater than the amount of immunoglobulin washed by acetic
acid. The arrows in Figure 15 indicate where elution with 1M acetic

acid was started to release the bound IgG fraction. The initial protein

107

Figure 15. Chromotography of hyperimmune rabbit serum on a
protein A-Sepharose column

0.1M sodium phoSphate buffer was used as a washing
buffer. The arrow indicates where 1M acetic acid
was added to release the bound IgG.

108

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109

concentration of the immune serum was 44 mg/ml and the IgG fraction was

7 mg/ml. The two major peaks were obtained; one was the eluate of

sodium phosphate buffer wash,which apparently contained all immunoglobulin
except IgG,and the second was the eluate of acetic acid wash,which con-

tained only IgG fraction.

2. Immunodiffuison Analysis for Rabbit Serum

Agar immunodiffusion was used to study the reactions of different
protein A-Sepharose chromatography eluates against goat anti-rabbit serum
and goat anti-rabbit IgG. The results are illustrated in Figures 16
and 17.

First study (Figures l6a,b): The ammonium sulfate precipitating
fraction (AS) formed a multiple precipitating band against goat anti-
rabbit serum (Gt—s),as shown in Figure 163. The reaction of identity
(converging arc) was very clear among AS and A fractions. The identity
band between A8 and A fraction against Gtus was very clear,as illustrated
in Figure 16b. Protein A 0.1 M sodium phosphate eluate (A-NP) and AS
fractions had multiple precipitating bands and two identical bands.
Furthermore, A-NP did not form a reaction of identity with both A and AS
as one group.

Second study (Figure 17): The fractions of A and AS formed a
reaction of identity against goat anti-rabbit IgG fraction (Gt-G),as
illustrated in Figure 17. This reaction indicated that fractions A

as well as AS contain rabbit IgG.

3._ Purification of Chicken IgG by Protein A
Protein A coupled to Sepharose 4B in a column (2x10 cm) was used

to fractionate'chicken IgG from normal and hyperimmune chicken sera. A

Figure 16 (38b).

110

Immunodiffusion of rabbit immunoglobulin
fractions

Gt-s: Goat anti-rabbit serum

A: Protein A, 1M acetic acid eluate

AS: IgG fraction by ammonium sulfate

A-NP: Protein A, 0.1M sodium phosphate eluate

Figure 16

111

 

112

Figure 17. Immunodiffusion of different rabbit IgG fractions

Gt-G: Goat anti-rabbit IgG
A: Protein A, 1M acetic acid eluate
AS: IgG fraction by ammonium sulfate

113

Figure 17

 

114

typical elution pattern was illustrated in Figure 18. The flow rate was
60 ml/hour. The 0.1 M sodium phosphate buffer,pH 7.0, eluate gave the
highest peak,which was similar to the peak of phosphate buffer eluate

of rabbit serum. Furthermore, this fraction (from 0-15) was very rich
with immunoglobulin because of the high absorbance reading (higher than
2.0). The arrow in the same figure indicated the addition of 1 M acetic
acid, pH 7.0, to the column. A very small peak which had absorbance less
than 2.0 at 280 nm appeared after adding the acetic acid. These results
indicated that chicken IgG did not bind to the protein A as rabbit IgG

fraction did.

4. Immunodiffusion Analysis for Chicken Serum

First study (Figures 19 a,b): Several identity bands were formed
from all protein A, 0.1 M sodium phosphate eluate (A-NP) against rabbit
anti-chicken globulin (Rb-glb),as shown in Figure 193. Protein A, l M_
acetic acid eluate (A) did not contain any immunoglobulin and did not
form any precipitating band against(Rb-glb), as illustrated in Figure 15b.
However, IgG fraction precipitated by ammonium sulfate (AS) showed two
precipitating bands against Rb-glb and c-IgG showed very clear bands
against Rb—glb.

Second study (Figures 20 a,b): These figures illustrate that
all A-NP fraction contained IgG. The control well,which contained
chicken IgG (c-IgG),gave a band of identity with the two neighboring
wells A-NP. Protein A, l M acetic acid eluate did not contain IgG,as
illustrated in Figure 20b. There was no band of identity between A, A5
and c-IgG as fraction did form one precipitating bard against anti-

chicken IgG (Rb-G).

115

Figure 18. Chromotography of chicken hyperimmune serum on a
protein A-Sepharose column

0.1M sodium phosphate was used as a washing buffer.
The arrow indicates where 1M acetic acid was added
to release the bound IgG

dz 20...U< a...

116

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“Q
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117

Figure 19 (38b). Immunodiffusion of chicken immunoglobulin
fractions

Rb-glbz Rabbit anti-chicken globulin

A-NP: Protein A, 0.1M sodium phosphate eluate
A: Protein A, 1M acetic acid eluate

AS: IgG fraction by ammonium sulfate

C-IgG: Control, normal chicken IgG fraction

118

  
 

m~ oazuwu

119

Figure 20 (35b). Immunodiffusion of different chicken IgG
fractions

Rb-G: Rabbit anti-chicken IgG

A-NP: Protein A, 0.1M sodium phosphate eluate

c-IgG: Control normaT chicken IgG

A: Protein A, 0.1M fraction by ammonium
sulfate — -

  
 

Figure 20

121

Third study‘ (Figures 21 a,b): This immunodiffusion study was
designed to compare the content of the eluate of protein A-Sepharose
column chromotography washed by l M acetic acid for rabbit and chicken
serum. Band of identity was formed between protein A, l M acetic acid
eluate (Prt A-Rb IgG); goat anti-rabbit serum (Gt-ant RS) and goat
anti-rabbit IgG (Gt ant-RIgG frct),as shown in Figure 213. These results
indicated that 1 M acetic acid eluate of rabbit serum on protein A~
Sepharose chromatography contained IgG fraction. Normal rabbit serum
(NRS) formed several bands against Gt-ant RS which corresponds to the
numbers of homologous system between both wells. Chicken serum, 111
acetic acid eluate on protein A-Sepharose chromatography did not contain
IgG fraction,as shown in Figure 21b. The only precipitating band formed
was between normal chicken serum (NCS) and rabbit anti-chicken globulin

(Rb-ant Cglb).

5. The Effect of Chicken Plasminogen on Isolation of Chicken IgG by

Protein A-Sepharose Chromatography

Immunodiffusion studies were conducted to study the effects of
chicken plasminogen on isolation and purification of chicken IgG by
protein A chromatography. Rabbit anti-chicken plasminogen (Rb-PL) was
put in the center well against A-NP, A and AS,as shown in Figures 223
and b. A precipitating band was formed between all A-NP eluates and
Rb-PL,as illustrated in Figure 223. Also, the band of identity was
formed between the central well which contained chicken plasminogen
(PL) and the two neighboring wells which contained A-NP. However, A
and AS fractions did not react against Rb-PL,as shown in Figure 22b.

These two figures (Figure 223 and b) indicated that plasminogen present

Figure

21.

a.

Immunodiffusion of rabbit IgG fractio

Prt A-Rb IgG-frct: Protein A,1M acetic acid
eluate _

NRS: Normal rabbit serum

Gt-ant RS: Goat anti-rabbit serum

Gt-ant R IgG: Goat anti-rabbit IgG

Immunodiffusion of protein A,1M acetic acid
eluate of chicken serum

Prt A-Ck IgG-frct: Protein A,1M acetic acid
eluate —

NCS: Normal chicken serum

Rb-ant Cglb: Rabbit anti-chicken globulin

Rb-ant CIgG: Rabbit anti—chicken IgG

123 -

 

MN 0.5m E

124

Figure 22 (36b). Immunodiffusion of chicken immunoglobulin
against rabbit anti-chicken plasminogen

Rb-PL: Rabbit anti-chicken plasminogen

A-NP: Protein A, 0.1M sodium phosphate eluate

PL: Chicken plasminogen

A: Protein A, 1M acetic acid eluate

AS: Chicken IgG—precipitate by ammonium
sulphate

125

  
 

NN ensued

126

in chicken serum did not bind to protein A-Sepharose molecule; instead

it got washed by 0.1 M sodium phosphate buffer.

D. Standardization of MYCO-ELISA

The use of MYCO-ELISA, as any immunologic assay, depends on the
strict standardization of all reagents and procedures used. Without
standardization, the performance of the test will be varied from one
laboratory to another. Thus, preliminary experiments were performed
to determine the conditions and reagents which should be used for the

detection of antibody to Mg,

1. Determination of the Optimum Antigen Concentration and Serum

Dilution

Sera positive and negative for Mg_(various dilutions) were titrated
against various concentrations of antigen using checkerboard design
experiments. Mg antigen was tested at 720, 360, 180, 90, 45, 21.5 and
10.75 pg protein concentration per well against 1:50, 1:100, 1:250,
1:500, 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000 and 1:64000 serum
dilutions. The test showed that 90 and 180 pg protein concentrations
gave similar absorbance reading at 405 nm after 30 minutes of conjugate
substrate reaction with 1:50 positive serum dilutions,as shown in Figure
23. The absorbance readings were 1.29 and 1.33 using 90 and 180 mg antigen
protein per well and 1:50 positive serum dilution. Furthermore, the re-
sult indicated that the optimum serum dilutions using 1:200 conjugate
dilution and 90 ug antigen protein per well were 1:50, 1:100 and 1:250,

as shown in Figure 24. The readings were 1.29, 1.15 and 0.99 at 1:50,

Figure 23. Determination of Mg antigen concentration for
. MYCO-ELISA using standard dilution of 1:200 HRP
conjugate and 1:50 reference control serum

128

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can ooh oeo eon oov can
Illrlllllllllllli

 

2mo.~z< 61 on!
MN etequ

ecu

 

m6

Oi

 

 

sorv 'a'o

129

Figure 24. Determination of the Optimum serum dilution in
MYCO-ELISA, using standard dilution of 1:200 HRP
conjugate and 90 ug antigen protein

130

 
 

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131

1:100 and 1:250 serum dilutions, respectively. It should be pointed
out that even with high serum dilution of l:32,000, the absorbance
reading at 405 nm (0.304) was higher than the negative serum at all
dilutions (lower than 0.3). However, serumcfijinions of 1:50 or 1:100

were considered thedilutions of choice for diagnostic purposes.

2. Determination of the Optimum Conjugate Concentration

The optimum concentration of horseradish peroxidase enzyme (HRP)
conjugated to rabbit IgG fraction anti-chicken IgG was determined.
Antiserum was obtained from two different sources, Miles Laboratories,
Inc., and Cappel Laboratories. The two antisera are essentially the
same except that the antiserum from Miles contains 0.1% sodium azide
as preservative. Furthermore, a labeled HRP with IgG fraction rabbit
anti-chicken IgG (heavy and light chain) was obtained from Cappel
Laboratories for comparison purposes (Cappel conjugate).

Both conjugates (MSU and Cappel conjugates) were titrated at
various dilutions (1:50, 1:100, 1:200, 1:300, 1:400, 1:600, 1:800 and
1:1000). Antigen concentrations of 90 and 180 ug protein were coated
to each well and 1:50 and 1:100di1utionscd’known positive serum were
titrated against each conjugate dilution.

MSU-conjugateIiilutions of 1:50 and 1:100 gave the highest absorbance
readings, as shown in Figure 25. The average absorbance readings was 2.49
and 2.25 for conjugate dilutionsof 1:50 and 1:100,respective1y. The
absorbance readings for the negative serum control using 1:50 and 1:100
conjugate dilutions were 0.623 and 0.595. MSU-conjugate dilutions of
1:200 and 1:300 gave absorbance readings higher than 1.0 Other conjugate

dilutions of 1:400 and 1:600 gave considerably higher absorbance readings

132

Figure 25. The effect of different concentrations of MSU-HRP
conjugate on the specific activity of MYCO-ELISA
using standard 1:50 reference control serum and
180 ug antigen protein per well

20:34.0 mama—.200

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134

than the controls. Cappel Laboratories conjugate, as shown in Figure

26, gave the optimum absorbance readings at 1:200, 1:300, and 1:400
conjugate dilutions which were 2.18, 2.06 and 2.01, respectively, at
antigen concentration of 180 ug per well and 1:50 serum dilution.
Furthermore, the absorbance readings of 1:600, 1:800 and 121000 conjugate
dilutions were 1.74, 1.44 and 1.44, using the same antigen concentra-
tion and serum dilution, which were much higher than the control absor-
bance reading. The negative serum control gave absorbance readings of
0.718, 0.723, 0.718, 0.701, 0.623, 0.595, 0.443 and 0.404 at 1:50, 1:100,
1:200, 1:300, 1:400, 1:600, 1:800 and 1:1000 Cappel conjugate dilutions,

as illustrated in Figure 26.

3. Determination of the Optimum Temperature and the Length of the
Incubation Period
Maximal binding of serum antibody and conjugate antibody to the
antigen or to the antigen-antibody complex is: essential to attain high
levels of sensitivity and to avoid non-specific reactions. Replicate
assays of differentdilutions of positive sera for Mg, 1:50, 1:500, 1:5000
and 1:50000, were incubated at various temperatures, 5 C, 22 C, 37 C,
50 C for differentlengths of time, 1, 2 and 3 hours, both the serum and
conjugate incubation periods. Two different antigen concentrations
were used, 90 and 180 pg protein per well. Also, two different conjugate
dilutions (1:100 and 1:200) were used in this experiment. The effect of
the temperature of incubation of serum and conjugate on the specific
activity of MYCO-ELISA using standard dilution of 1:100 and 2 hours of
incubation is illustrated in Figure 27. The effect of various time of

incubations at room temperature (22 C) on the absorbance reading of

Figure 26. The effect of different concentrations of Cappel
-HRP conjugate on the specific activity of MYCO-
ELISA using standard 1:50 reference control serum
and 180 ug antigen protein per well

136

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Figure 27.

137

The effect of different temperature of incubation

of serum and conjugate on the specific activity of
MYCO-ELISA using standard dilution of 1:50 refere-
nce control serum dilution, HRP-conjugate dilution
of 1:100 and 2 hours of incubation

8 pm

 

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Figure 28.

The effect of various time of incubation at

room temperature (22C) on the absorbance readings
of MYCO-ELISA using 1:50 and 1:500 positive serum
control dilution at MSU-HRP dilution of 1:100

140

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141

MYCO-ELISA using 1:50 and 1:500 positive serum dilution and 1:50 negative
serum control dilution with HRP-conjugate dilution of 1:100 is shown in
Figure 28. The highest absorbance reading for MYCO—ELISA was 1.54 at
22 C. The readings at 4 C, 37 C and 50 C were 0.73, 1.09 and 1.05,
respectively, as illustrated in Figure 28.

The specific activity increased with increasing time of the incubation.
The absorbance readings of positive Mg control serum at 1:50 dilution at
l, 2 and 3 hours of incubation were 0.498, 1.462 and 1.623. These readings
were much higher than the readings of the negative serum control,which
were .321, 0.462 and 0.523 at l, 2 and 3 hours of incubation,respectively.
The absorbance readings for positive serum control at 1:500 dilution were

a little lower than 1:50 serum dilution,as illustrated in Figure 28.

4. Determination of the Specificity of MYCO-ELISA

Positive known serum for 4 different subgroups of lymphoid leukosis,
2 different subgroups of Marek's disease and infectious bursal disease
were obtained from USDA Regional Poultry Research Laboratory, East Lansing,
Michigan. Positive and negative known chicken and turkey sera for M ,
Mg, Mg_and M, gallinarum were obtained from Veterinary Service Laboratories,
USDA, Ames, Iowa,and Southeastern Poultry Research Laboratory, USDA,

Athens, Georgia. Also, positive serum with high antibody titer for

 

Pasteurellanmltocida and Mycobacterium avium were obtained from other

 

sources to conduct the sensitivity test. The Mg antigen (MYCO-ELISA
antigen) reacted strongly with positive sera of Mg and ME, The HI
titers for both sera used in the test were 1:100 and the MYCO-ELISA was
1:8000. Positive sera for diseases other than Mg and Mg_showed no

reactions to Mg_antigen and very low absorbance readings similar to

142

that of control serum.

5. Determination of the Sensitivity of MYCO-ELISA

A comparison study between HI test and MYCO-ELISA test was made
by using sera obtained from naturally and experimentally Mg infected
chickens. The HI titersof 8 sera compared to those of MYCO-ELISA
titer'areshown in Table 5. The data indicated that MYCO-ELISA is far
more sensitive than the HI test. The absorbance readings at 405 nm
for all positive sera were higher than 2.5 times the absorbance reading
of the negative serum at the same dilution. The absorbance reading of
1:50 dilution of the serum withHI of 1:640 was 1.555, H1 of 1:320 was
1.329, H1 of 1:160 was 1:302 and HI of 1:80 was 0.991 in comparison to
the negative reference control serum,which was 0.295 at 1:50 dilution,
and it was much lower at higher dilutions,as shown in Figure 29. The
absorbance reading of the end point dilution for those positive serum
gave much higher ELISA titer than the H1. The ELISA titers for serum
number 8 (Table 5),which has HI of 1:640,was >32,000, HI of serum number
1 and number 5 was 1:320 and the ELISA titers were 1:32000 and 1:16000,
respectively. Again, the results recorded in Table 5, as well as results
illustrated in Figure 29, showed that correlation between the absorbance

readings of MYCO-ELISA and the HI antibody titer is obvious.

.coausawu Bowen onua we wcficmmu oucmnuomn<«

 

143

 

 

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<mHQmIou>E use H: xn.wm pom Hogan anonfiucm mo :omflpemsoo .m oflceh

144

Figure 29. The absorbance readings for MYCO-ELISA at 405nm

for positive and negative sera for Mg at different

dilutions using standard 180 pg antigen concentration
and 1:100 conjugate dilution

145

20:3...0 2.550

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DISCUSSION

A. Cultural Media

Six different culture media for Mycoplasma were used in these experi-

 

ments.. Brain-heart infusion medium, PPLO medium, tryptose phosphate
broth, B-medium of Fabricant and modified Frey medium were compared to

the MSU-Mycoplasma medium. Brain-heart infusion medium and B-medium of

 

Fabricant were not reported in the results because of the difficulty of
dealing with the small size colonies and lower numbers of CFU/ml of the

original media. The Mycoplasma broth base formula (Frey SE El" 1968),

 

manufactured and marketed by Gibco, contained the fbllowing: peptone

140 (pancreatic digest of casein) 7.5 gm, peptone 110 (papaic digest of
soy protein) 2.5 gm, yeast extract 5.0 gm, sodium phosphate dibasic 1.6
gm, potassium phosphate monobasic 0.1 gm. However, the contents of the
base medium were not enough to produce reliable numbers of the organisms.

The MSU-Mycgplasma medium (MSU-medium) contains the basic nutrient

 

requirements for_Mygoplasma organisms obtained from Mycoplasma broth

 

 

base (Gibco) plus other additives. Maltese (2 gm) or dextrose (3 gm)
were added to the medium to enhance the yield of viable organisms, which
is in agreement with Adler and DaMassa (1968). Many investigators stu-
died the production of Mg antigen by using basal medium containing high
levels of dextrose as a carbohydrate substrate. Modified Frey medium
and PPLO medium which contains 10 gm of dextrose are still commonly used

media for Mycoplasma isolation and antigen production. However, this

 

may have accounted for their failure also because the increase in

146

147

acidity of the medium will retard the growth of viable organisms.

The addition of 0.3% Tris buffer (Trihydroxy methyl aminomethane
hydrochloride) in MSU-medium was benificial to maintain the pH of the
medium around 7.3 for the first 48 hours of growth. The experimental
results showed that the pH of the culture medium fell to about 6.8 if
dextrose alone was added. These findings are in agreement with Vardamann
(1967) and Gill (1962). The addition of dextrose or maltose to the
basal medium would increase the total yield of the organisms. The
result of the sugar fermentation increased the media acidity,which de-
creased bacterial growth after 24 hours. The addition of Tris buffer
to the MSU-medium maintained the pH of the medium in the range of 6.5
1 0.5. The broth base (Gibco) contains 0.5% yeast extract,which acts
as a source of nitrogen and also provides maltase, the enzyme for maltose
fermentation. Dierks §£_§l, (1967) used autoclaved yeast autolysate and
had no problems with nonspecific enzyme activity except with avian
serotype F. However, Yoder and Hofstad (1964) earlier reported dif-
ficulty to grow Mg_in PPLO medium due to the presence of enzyme sucrose
of yeast origin. In our experiments, addition of 2-5 gm of yeast
autolysate presented no problems in growing the organisms.

L -histidine was added to the MSU-medium (0.25%) to prolong the
stationary phase and the survival of the microorganisms. It will also
delay the rise of the acidity of the media,which is in agreement with
Ajello and Romano (1975). It has been reported that L-histidine does

not interfere with urease activity of Mycoplasma (Ajello and Romano,

 

1975). Also, cystine hydrochloride was added to the MSU-medium as a

stabilizing agent. Coenzyme I (NAD) was added to the medium in 1%

148

solution for the growing of Mg and Mg, Waymouth medium MAB 87/3 (#78-027,
Gibco), 50 ml, was used in the MSU-medium as a source of amino acids and
vitamins,which was substituted for 70% of the serum requirements.

All avian Mycoplasma species required cholesterol and long chain
fatty acids for growth and morphological and osmotic stability. There-
fore, horse or swine serum, 5—10%, was added to the medium for Mg_
isolations and for antigen production. Thallium acetate was added in
0.01% quantity to the medium to inhibit Gram-negative bacteria. Pen-
icillin potassium G in a final concentration of 1000 units per ml was
added routinely to MSU-medium to inhibit Gram-positive bacteria. Phenol
red was added in quantity of 0.025 gm/liter as a pH indicator only to
the medium for isolation and not for antigen production. The closest
medium formula to MSU-medium is modified Frey medium. However, maltose,
L-histidine, yeast autolysate, tris buffer and Waymouth medium were not
included in the modified Frey medium formula. Both media used broth
medium (Gibco) as a basic ingredient.

The decline of the pH of the four media by hours and days P1 was
demonstrated in Figures 9 and 10. The pH of MSU-medium stayed in the
basic side much longer than the modified Frey medium, This probably was
due to the presence of Tris buffer and reducing the amount of dextrose
in the medium. There was not much difference in the pH reading of the
MSU-medium when either maltose or dextrose was used. The PPLO and
tryptose phosphate media gave the lower pH (acidic) reading than the
other media at all times. The lowest count of viable organisms was
obtained from PPLO and tryptose phosphate media throughout the experi-
ment as compared to MSU and modified Frey Media. The correlation between

the pH of different media and viable bacterial counts was observed.

149

When the pH declined (acidic), the viable bacterial count decreased.

However, DaMassa and Adler (1969) reported that Mycoplasma organisms

 

grew over a wide pH range,which is in agreement with our research
results. It was also shown that Mg grew at low pH (4.8) but not for

a long time. After 9 days PI, Mg organisms were isolated from all
media. The pH range of all media was 4.8 - 6.5. After 30 days PI,
the organisms were only isolated from MSU- and modified Frey media and
the pH of these media was 6.5 and 6.1, respectively. At 60 days PI,
the organisms were isolated from MSU-medium only (0.1 x 103 CFU/ml)and
not from the other media. and the pH was 6.6. The reason for this
probably was due to the presence of Waymouth's media,which provided
enough nutrient support for the growth of the organism for a much longer
period of time than did media containing serum only. The pH of MSU-
medium stayed close to the neutral pH reading for a long time. The

MSU-medium seems to be superior to other media that are currently used

to isolate avian Mycoplasma regarding bacterial yield and the survival

 

time of the microganisms in the media.

The reaquirements of growth for avian Mycoplasma were studied in

 

detail for the three most common strains Mg, Mg and Mg_(Yoder and
Hofstad, 1964; Kerr and Olson, 1967 and Anderson, 1968). However,no
data have been published to characterize the requirements of the growth

for non-pathogenic:strains such as M, gallinarum, M, anatis, and M,

 

laidlawii var inocuum. More growth and maintenance media should be
formulated which would be able to differeniate between the pathogenic

and non-pathogenic Mycoplasma isolates. This can be accomplished by

 

finding the requirements for growth for the nonpathogenic strain and

/

150

developing a medium which allows the growth of the pathogenic strains.

’1 B. Standardization of Mg_Antigen
In this study, Mg serotype A (S6) was the only antigen prepared.
However, there is no reason to doubt that the same procedures cannot

be used to prepare different Mygoplasma antigen by the same methods

 

(Sabry, 1968; Fabricant, 1969). In order to obtain a high yield of

antigen, Mycoplasma organisms were added to the culture medium in the

 

log phase of growth and also when passed to another culture medium
(Sabry, 1968). The volume of culture was increased through these
passages at 24-hour intervals until a one liter volume had been inocu-
lated. The agitation was very helpful to prevent clumping the organisms.
Also, using glass Ten Broeck tissue grinder or glass beads was very
beneficial to obtain homogenized antigen suspension. Glycerol proved
to be a good preservative for HA antigen. The antigen was thawed and
frozen 3 number of times with very little loss in hemagglutinating
activity. Without glycerol, the freezing and thawing process was very
detrimental to the HA antigen. These results seem to agree with the
research results of Dierks (1964). No preservative was added to MYCO-
ELISA antigen to avoid any interference with the enzymatic activity of

the conjugate.

C. Identification of Mycoplasma Isolate by

 

Immunofluorescence Methods
The fluorescence antibody technique (FA) has been used in diagnosis
of a wide variety of diseases.

1. Antisera Production

151

The purpose of these experiments was not to determine the effective-
ness of such vaccination program or different route of vaccination. All
vaccinated rabbits and chickens produced a high titer of antibody against
Mg antigen. This hyperimmune serum was used in both immunofluorescence
and MYCO-ELISA experiments. The antibody titer in both hyperimmune sera
was higher than 1:80 in chickens and 1:60 in rabbits using HI test at
all times of the immunization program. The response of the rabbits in
developing specific antibodies for Mg was influenced by the route of
vaccinations. The production of agglutinin antibodies was very high
following the injection of small amountsof antigen with incomplete
Freund adjuvant subcutanemniy (SC) (Rabbit No. 3) one week after the
second injection. The antibody titer was 1:1280 using HI test for
Rabbit No. 3. The SC and intramuscular (1M) injections (Rabbit No. 1)
gave higher antibody titer one week after the second injection in
comparison to intravenous (IV) and SC injection programs (Rabbit
No. 2). The antibody titer of Rabbit No. l was 1:640 and for Rabbit
No. 2 was 1:320 using HI method.

Also, the HI titers in RabbitsNo. l and 3 were very similar in
the long run for 8 weeks. Thus, SC injections alone or SC with adjuvant
followed by several IV injections or the combination of IM, intrafoot
pad PP and IV injections for several weeks were very effective to
produce high antibody titer. These results are in agreement with

Morton and Roberts (1967).

2. Immunofluorescence Diagnosis for Mycoplasma

 

The use of direct FA for the identification of avian Mycoplasma

 

in broth culture has the distinct advantage of rapid identification

152

of Mycoplasma growth without having to plate and incubate for 5-10 days

 

until typical Mycoplasma colonies develop; The observation of fluores-

 

cence indicated the presence of specific Mycoplasma growth without having

 

to depend on colony growth and morphology criteria. The identification

of pathogenic from nonpathogenic Mycoplasma species has been very dif-

 

ficult because of the unavailability of specific serological tests for

each Mycoplasma species. Furthermore, the identification of Mg, Mg_and

 

Mg_dependsprimarly on serological tests such as rapid plate test (RSP),
tube agglutination (TAT) and hemagglutination inhibition test (HI).
Cross reactions with serological tests had been reported by Olson gg_
31, (1965), Roberts and Olesiuk (1967) and Vardeman and Yoder (1969).
Vaccines such as erysipelas bacterin (Boyer gg_§l,, 1969; Olson gg_gl,,
1965,and Roberts, 1970), contaminated serum (Roberts gg_gl,, 1967) and
frequent freezing and thawing of the serum (Thornton, 1969) have been
reported to cause cross reactions in serological diagnosis of avian

Mycoplasma. Also, Streptococcus faecalis and Staphylococcus aureus

 

 

 

infections in chickens had been demonstrated to cause non-specific agglu-

tinations. Direct FA can identify different species of Mycoplasma

 

organisms obtained from broth media if the primary identification of
the organism from the host failed. Noel gg_gl, (1964) used direct and

indirect fluorescent methodsto diagnose avian Mycoplasma organisms from

 

air sac lesions and turkey sinus exudate. However, Noel gg_gl, (1964)
and Barile gngl, (1962) showed thattissue exhibited a high degree of
green fluorescence because of non—specific uptake of the labeled globulin.

The fluorescent Mycoplasma organisms which took up the labeled antibodies

 

were not readily differentiated from the backgrounds,which were having

similar color. Clark gg_gl, (163), Costvet and Sadler (1964) and Stewart

(1967) methods showed nonspecific reactions in identifying mixed cultures

of Mycoplasma growth on an agar median. However, each method has some

 

limitations for expedient identifications of Mycoplasma organisms and

 

mixed Mycoplasma cultures. The identification of specific avian Mycoplasma

 

organisms by direct FA method by fixing the organisms on a slide from
both media was satisfactory and not as time consuming as the agar block
method for colony identification (Figures 13 and 14).

It has been postulated that different PPLO contamination in tissue
culture was L-forms of different bacteria. This transformation to L-form
occurred in the presence of specific antibodies (Rothblat and Morton,

1958). Whether the nonpathogenic avian Mycoplasma are L-forms of the

 

pathogenic strains or are entirely different strain of Mycoplasma is
not clear and needs more investigation. The cross reaction in serological
tests made the identifications more difficult. Also, the preparation of

different non-pathogenic Mycgplasma antigen is needed to confirm such a

 

positive serological test for Mg, Mg_and MM, Until this can be accomp-
lished, FA has the potential advantage for characterization of different

Mycoplasma organisms isolated.

 

Another advantage is that small numbers of organisms can be identified
when large numbersof contaminants are present. The disadvantage of the
test is the cost of initial fluorescent microscopy equipment and-necessity
of adequately trained personnelto avoid non-specific reactions, auto-

fluorescence and over—labeling of the conjugate.

D. Purification of IgG by Protein A
The IgG binding properties of protein A made affinity chromatography

with protein A—Sepharose CL-4B molecules the method of choice for

154

isolation of IgG antibodies in mammals. Recent studies have indicated
that the protein A reactivity to the IgG Fc fragments was correlated

with the IgG H-chain type. Also, the activity shbwn by Fc fragment is
not found in Fc' subfragment nor in pepsin components 11 or III (Kronvall
gg_gl,, 1970). The IgG molecules of subclasses l, 2 and 4 and their
fragments containing the Fc region hanathe affinity to bind to protein

A but not IgG-3 (Hjelm gg_gl,, 1975). Rabbit IgG was isolated by
chromatography fractionation of the serum on a column protein A, coupled
to Sepharose. Two major peaks were obtained (Figure 15). The first peak
was the eluate of 0.1 M sodium phosphate,pH 7.0 which apparently contained
all immunoglobulin and IgG3 molecules. The second peak was the eluate

of l M acetic acid, pH 7.0, which contained only IgG 1, 2 and 4 fraction,
as shown in the Ouchterlony agar immuno-diffusion test (Figures 16 and
17). The same protein A-Sepharose column was used to purify IgG fraction
from normal and hyperimmune chicken sera. The acetic acid eluate wash
gave a very small peak which had a spectrOphotometer absorbance reading
at 280 nm less than 2.0,as shown in Figure 18. Immunodiffusion studies
for the eluates indicated that chicken IgG does not bind to protein A
molecules,as illustrated in Figures 19, 20 and 21. No reactions were.
noticed with any of the acetic acid eluatesagainst rabbit anti-chicken
IgG (Rb-ani CIgG). The significant difference between rabbit and chicken
IgG is that acetic acid eluate did not form any precipitating band
against either Rb-ant Cglb or Rb-ant CIgG,as illustrated in Figures 213
and b. The reasons for the non-binding properties of chicken IgG to
protein A-Sepharose molecule may be due to: l) the presence of other

protein which binds to the protein A molecule and inhibits the binding

155

of protein A to the IgG molecule; 2) the amino acid configuration of
the Fc region of chicken IgG is different from mammal's IgG and subsequ-
ently possesses different binding properties and antigenic determinants.
Recent studies on IgG molecules of chickens and other birds such as
pheasants and quail indicated that they are closely related in respect
to a number of properties that are not usually associated with mammalian
IgG (Leslie andEmnedict,1970). The differences between avian and mammal-
ian IgG were summarized as follows: the IgG of these birds dissociated
when reduced in the absence of a dispersing agent (Leslie and Benedict,
1969), it aggregates especially Fc but not Fab fragments in high salt
concentration (1.5 M Nacl) solution where rabbit IgG does not (Kuba and
Benedict, 1969), and has a molecular weight of about 1.7 x 105 daltons
(Hersh e_t__a_l_., 1969). Also, Leslie and Benedict (1970) indicated that
the IgG molecules of these birds share certain antigenic determinants.
Leslie and Benedict (1970) digested rabbit and chicken IgG by papain and
other reducing agents to produce products that had a sedimentation co-
efficient of 3.4 5. Two antigenic components were identified by immuno-
electrophoresis from chicken IgG, Fab-fragment (antigen binding) desig-
nated S' and S" and PC fragment (crystallizable) which was designated
as F. According to their preliminary analysis, chicken IgG contained
greater amounts of serine, glycine and alanine and lesser amounts of
glutamine and tyrosine amino acids than were found in rabbit, human or
horse IgG. We suggest that the nonbinding prOperties of the Fc region
of chicken Inginebecause of the absence of some of the C terminal amino
acids whichzxnaresponsible for binding process in mammals. Investigations

to date have not clarified why the Fc region of rabbit and other mammalian

156

IgG bindsto protein A molecules; conversely, the lack of binding of
chicken IgG to protein A is equally vague.

A possible reason is that the chicken Fc-H chain of IgG did not
bind covalently to protein A-Sepharose molecule because other protein
molecules present in the chicken serum did bind to protein A molecules
and did inhibit the binding process with IgG molecule. Quigley gg_gl,
(1974) described an assay system to purify chicken and mammalian
plasminogen on lysine-sepharose column. For this reason, immunodiffu-
sion study was conducted to study the effects of chicken. plasminogen
on purification of chicken IgG by protein A-Sepharose chromatography.
The chicken plasminogen did not bind covalently to protein A molecules.
Different sodium phosphate eluates gave an identical band with the
control chicken plasminogen well against Rb-Pl,as illustrated in Figure
22. Also, this figure showed that plasminogen did not have the affinity
to bind to protein A-Sepharose molecules. The acetic acid eluate of
the column as well as the ammonium sulfate did not give any precipit-

ating band against Rb-Pl.

E. MYCO-ELISA

The enzyme immunoassay technique for the detection of Mycoplasma

 

antibodies of chickens was first adopted in the Michigan State University

Avian Microbiology Laboratory. The standaradization to determine the

optimum concentration of the test reagents and procedure was jperformed.
The MYCO—ELISA antigen was prepared from Mg_whole bacterial cell

or its disrupted cell suspension. This antigen showed strong affinity

for binding or adsorbing to the surface of polystyrene walls of micro—

titer plate (U-form, Immunolon substrate plates, Cook Laboratory,

157

Alexandria, VA). These disposable plates were more convenient for
large scale use and very economical since only small volumns of
reagents were needed. It is known that polystyrene plates (Dynatech
Laboratories) can be coated and sensitized with many protein and

lipoprotein antigens (Voller and Badwell, 1976). The Mycoplasma

 

antigens retain their immunologic activity after coating the surface

of the wells. The process of coating the plates with Mycoplasma

 

protein antigen was done by passive adsorption to the polystyrene
plates using alkaline solution such as 0.1 M_carbonate-bicarbonate
buffer,pH 9.6. This method was also used by other researchers (Engvall
and Perlmann, l97l,and Hamaguchi gg_gl,, 1976a,b). The optimum con-
centrations of antigen used in MYCO-ELISA were 90 and 180 ug antigen
protein per well. Both concentrations gave the same absorbance
reading at 405 nm with 1:50 serum dilution (Figure 23). Based on the
results of experiments conducted at MSU, it is concluded that serum
dilutions of 1:50 or 1:100 are the dilutions of choice for diagnostic
purposes (Figure 24).

Since this technique was first applied in this laboratory for

diagnosis of avian Myggplasma, materials for comparative discussion

 

 

were not available. Thoen gg_gl, (1978) used 0.5 ug of Mycobacterium
gzigg_antigen protein per well and serum dilution of 1:10 in their
experiments for detection of antibodies from M, ggggg_infections in
chickens. Bruggmann gg_gl. (1977) indicated that optimum sensitization
for ELISA microtiter plates was obtained with 2.5 - 5.pg solubilized
antigen protein with SDS or 20 pg when the antigen was solubilized

ultrasonically for Mycoplasma suippeumoniae.

 

158

The results of HRP conjugate experiments indicated that MSU
conjugate is more reliable than Cappel conjugate when it is used in
high concentration. This is because of the high reading of the negative
serum control with Cappel-HRP conjugate. The readings were 0.723 and
0.404 at 1:100 and 1:1000 conjugate dilution, respectively (Figure 26).
MSU-HRP conjugate gave a much lower absorbance reading with the
negative control serum (0.285 and 0.134 at 1:100 and 1:1000 conjugate
dilution, respectively) (Figure 25). Using positive serum 1:50 dilution
and 90 ug antigen protein per well with both conjugates the results showed
that 1:50 dilution MSU-conjugate gave similar absorbance readings as
Cappel-»conjugate at the same dilution. The absorbance readingswere
2.39 and 2.41,respectively. However, MSU-conjugate dilutions of 1:200
or 1:300 were the dilutions of choice to be used in MYCO-ELISA since it
will give high absorbance reading with the positive serum in comparison'
to the control serum. Cappel-HRP conjugate can be used in 1:600,
1:800 or 1:1000 dilutions with expectation that negative serum control
will give high absorbance readings in comparison to MSU-HRP conjugate.
The reason for this probably is due to non-specific binding of rabbit
anti-chicken IgG conjugated to HRP made by Cappel Laboratories. Smith
gg_gl, (1979) reported that 1:200 dilution of rabbit antiserum (prepared
against avian Myeloblastosis virus group antigen) conjugated to HRP
gave high absorbance ELISA readings. Also, Thoen gg_gl, (1978) indicated
that 1:100 rabbit antichicken gamma globulin labeled with HRP is sensitive

enough to be used in ELISA to detect antibody for Mycobacterium avium

 

in chickens.

The room temperature of 22 C was the best incubation temperature

159

for high absorbance reading and subsequently for maximum binding of
the antibody and the labelled conjugate to the sensitized well with
the antigen. The absorbance reading at 22 C was high, 1.462 at 1:50
serum dilution as compared to 0.734, 1.092 and 0.981 at 5, 37 and 50 C,
respectively (Figure 27). The results indicated that the absorbance
readings, after 2 or 3 hours incubation period at 22 C,were relatively
close using 1:50 or 1:500 serum dilution, 1.542, 1.290 and 1.623, 1.464,
respectively (Figure 28). However, the reading, after one hour of
incubation at 22 C, was very low, 0.498 and 0.401 at 1:50 and 1:500
serum dilution, respectively. Low levels of activity at low incuba-
tion temperatures or at one hour of incubation can be attributed to
incomplete binding of available antibody. The decrease in activity
at higher temperature (37 C or 50 C) is presumably due to a combination
of temperature denaturation effects, dissociation of the enzyme-anti-
globulin complex, and release of the absorbed antigen to the surface of
the wells at these temperatures. A temperature of 22 C (room temperature)
for 2 hours was therefore selected for routine incubation of serum and
conjugate. These results are in agreement with other research results
using the same conjugate but on different disease systems (Wallen gg_
31,, 1977,3nd Castellano g£_gl,, 1977). However, different incubation
temperature and length of incubation were suggested by other researchers
(Smith g£_gl,, 1979,and Bruggmann gg_gl,, 1977).

The time which was needed for complete enzyme-substrate reaction
depended on both the quantity of enzyme present and the length of the
reaction period. The time for reaction of labeled enzyme with the

substrate must be long enough so that one can recognize various amounts

160

of bound enzyme, yet short enough to avoid depletion of the substrate.
The difference is the amount of enzyme bound to the polystyrene well,
and those of the control groups became more apparent as the time of
substrate reaction increased. A substrate reaction time of 30 min was
arbitrarily chosen for high dilutions of serum because it gave a high
level of sensitivity while avoiding depletion of substrate. However,
the change in the color of the positive control serum is the main
indicator, at that time 0.45%, HF should be added to stop the hydrolysis
of the substrate.

All buffer solutions and washing buffer used in these experiments
did not contain any sodium azide as a preservative bacause of its
interfering effect in the enzyme-substrate reaction. Persijn and Jonder
(1978) reported that addition of a lower concentration of sodium azide
completely stops color development in a peroxidase—labelled enzyme
immunoassay using 2.2'-azino-di(3-ethy1benzthiazoline sulphonic acid
(6)) as a substrate.

To determine the specificity of MYCO—ELISA, positive chicken serum
for viral and bacterial diseases as well as other species of avian

Mycoplasma such as Mg and Mg_were used in these experiments. There was

 

no cross reaction between Mycoplasma and other bacterial or viral

 

diseases. However, the result of specificity tests showed that cross
reaction occurs in a large scale between positive serum of Mg and Mg,

The common Mycoplasma antigen, phospholipids and glycolipids of the cell

 

membrane are the major antigenic determinants which cross react with

other positve serum for other avian Mycoplasma (Kahane and Razin, 1969).

 

Other experiments ig_vivo showed that whole cell or membrane antigen of

161

Mg stimulated antibody formation (Williams and Taylor-Robinson, 1967).

Mg as well as many other Mygoplasma are bounded by a single lipoprotein

 

membrane (Rettem gg_gl,, 1968) and this lipoprotein can be readily
isolated by osmotic lysis of the organisms (Razin, 1968, 1969). Dis—

aggregation of Mycoplasma membrane by strong detergent such as sodium

 

dodecyl sulfate (SDS) resulted in soluble antigen clear solution. How-
ever, use of this antigen did not increase the specificity of the test

among avian Myc0plasma species. There aresufficient data on the homogeneity

 

of the antigen solution. A striking feature was discovered by Razin gg_
El: (1975) in that the detergent solubilized membrane material had the
ability to reaggregate spontaneously to membrane like structure on the
removal of the detergent. They reported that reassembly of solubilized
membrane (lipid and protein) after removal of SDS by dialysis or by
Sephade usually occurs if the divalent cation Magnesium (Mg++) is
present. The addition of 1 mM disodium ethylene diaminatetra acetate
(EDTA) to MYCO-ELISA antigen to prevent the aggregation of the antigen,
by binding to the calcium ions, did increase the titer but not the

specificity. Myggplasma organisms do not have a cell wall or intra-

 

cytoplasmic membrane; it possesses only plasma membrane. Therefore when
this plasma membrane was lysed by carbonate—bicarbonate buffer at pH 10,
it was used as an antigen in serological diagnosis 0f.ME and Mg (Goel,
1973 and Villegas gg_gl,, 1976). They discovered that membrane antigens
were somewhat more sensitive in the RSP and TAT tests whereas the HI
test showed no major difference between the whole and membrane antigens.
Separation of the membranes is therefore a prerequisite for the

s pecificity of these antigens.

162

Several methods, including lysis by osmotic schock (Razin, 1963),
detergent (Rottem gg EL" 1968) and mechanical pressure using sonic or
ultrasonic oscillators (Pollack gg.g},, 1965),were used to separate the
plasma membrane of the organism. However, the Mycoplasma organisms were
insensitive to osmotic shock whentransferred from 0.25 M sodium chloride

to water (Razin E£H21°’p1964)' The Mycoplasma membranes for immunological

 

studies were obtained from Mg by injecting the bacterial suspensions in
2 M glycerol into water (Kahane and Razin, 1969, Rottem‘gghgl,, 1968).
The majority of the organisms remained unlysed by this method. Ultra-
sonic treatment leaves variable amounts of organisms unlysed and dis-
aggregates the membrane into minute fragments. The future of ELISA as

a specific test for diagnosis of different avian Mycoplasma species

 

relies on extraction of different components of the organisms and testing
the antigenic determinants for each component.

A comparison study between HI and MYCO-ELISA test was conducted
using sera obtained from chickens naturally and experimentally infected
with Mg, The results, in Table 4, showed the sensitivity of the ELISA

method in detecting Mycoplasma antibodies. The absorbance readings at

 

405 nm for all positive sera were higher than 2.5 times the absorbance
reading of the negative sera at the same dilution. LISA titer for 5
serum samples out of 8 gave 100 times higher titer than the titer of H1
test for the same serum (Table 4). HI titers of the sera were 8:320,
1:160, 1:80, 1:640, 1:160 and the MYCO-ELISA antibody titers for the
same serum were l:32,000, l:l6,000, 1:8000, l:32,000 and l:l6,000,
respectively. The absorbance reading of the end point dilution for

positive sera in another study (Figure 29) gave much higher ELISA titer

163

than the H1. The ELISAtiters of sera whichhave HI of 1:640, 1:320,
1:160 and 1:80 were >l:32000, 1:16000, 1:6000 and 1:8000. These results
strongly suggest that the MYCO-ELISA is much more sensitive than the

current HI test for detecting Mycoplasma antibodies. The HI test has

 

been used as the most efficient and accurate method to differentiate Mg
from Mg, Furthermore, our data would suggest that H1 is a more specific
means of differentiation than MYCO-ELISA.

The official blood tests for Mg and Mg_are RSP test, TAT test and
HI test, or a combination of two or more of these tests as recommended
by the National Poultry Improvement Plan (USDA, November, 1976). The
plan stated that the HI test should be used to confirm the positive
results of Mg for other serological tests, namely RSP and TAT tests.
Villegas gg_gl, (1976) stated that the RSP as a screening test followed
by the HI test for confirmation are the most commonly used procedures.
The RSP has the advantage of speed, simplicity and sensitivity but for
different causes produces occasional non-specific reactions with sera
from birds (Roberts, 1967 and 1969). .Mg antigen used in the RSP test has
been reported to cross react with sera from chickens or turkeys that
have been vaccinated within 2 weeks for erysipelas (Boyer 33.31,, 1960,
and Olson gghgl., 1965). The HI test has been reported to be more highly
specific than the other two tests. It may not attain significant
titer until several weeks later even though the RSP test could be
positive, because HI measures only the IgG and not the IgM (Villegas
gg-gi,, 1976). The cross reaction and non-specific reaction which had
been reported with HI test indicate that HI test is not completely

specific for detection of Mg antibodies (Yoder, 1978b) either. Outbreaks

164

of typical Mg infections in chickens were characterized by a low
percentage of reactors to the RSP test,which often failed to give a
meaningful result. Based on this discussion, there is a definite need

for a simple method to diagnose Mycgplasma infections in chickens and

 

turkeys. A successful national eradication program for Mycoplasma
requires a dependable method for disease detection with accurate
specificity and sensitivity test results.

ELISA has been reported to be more sensitive than radioimmunoassay
(RIA), virus neutralization test, passive hemagglutination test, tube
agglutination test and hemagglutination-inhibition test in the detection
of antibodies for several viral, bacterial and parasitic agents (Gilman
and Docherty, 1977; Castellano gg_gl,, 1977; Voller and Bidwell, 1975;
and 1976; Saunders and Clinard, 1976).

It is believed that enzyme immunoassays can be the future method

used to achieve a successful eradication program for Myc0plasma infections

 

because of their simplicity, reproducibility, sensitivity and the auto-
mation potential for a large scale diagnosis in the laboratory or in
the field. The obvious existing disadvantage is the lack of specificity;
however, current sophisticated biochemical, microbiological and immuno-
logical techniques will undoubtedly lend themselves to address the

problem of molecular differences present in Mycoplasma plasma membrane.

 

Once the variants are delineated, specific MYCO-ELISA techniques
applicable to all the pathogenic and non-pathogenic strains will soon

be feasible.

APPENDIX

165

ADJUVANTS
Incomplete Freund Adjuvant (Difco)
Arlacel (Mannide monoaleate) 1.5 m1
Bayol F (paraffin oil) 8.5 ml
Sterilized by autoclaving at 121 C and 15 lb pressure for

thirty minutes.

166

 

 

ANTICOAGULANTS
1. Citrate Solution
Sodium Citrate 2.0 gr
Distilled water 100.0 ml
2. Modified AlSever's Solution
Glucose 2.050 gr
Sodium Citrate 0.800 gr
Sodium Chloride 0.420 gr
Citric Acid 0.055 gr
Distilled water 100.000 ml

STABILIZING SOLUTION

1. Sucrose Phosphate Glutamate Albumin Solution (SPCA)

 

Sucrose 74.60 gr
MonOpotassium phosphate 0.45 gr
Dipotassium phosphate (3H20) 1.64 gr
Sodium glutamate 0.82 gr
Bovine albumin powder (fraction V) 10.00 gr

Distilled water sufficient to make 1000 m1
Sterilize by filtration
Store at SCC

2;?

BUFFERS

Phosphate buffer saline (PBS), pH 7.2

Stock solution:

Sodim chloride (NaCl) 170 gm
Potassium dihydrogen phosphate (KH3P04) 13.6 gm
Sodium hydroxide (NaOH) 3.0 gm
Distilled water 95. — 1000 ml

One milliliter of stock solution is diluted 1:20 with
distilled water to give a buffered saline having a pH
of 7.1 to 7.2.

Borate buffimr saline (BBS), pH 8.4

Stock solution:

Boric acid 6.184 gm
Borax (Na B40 .10H20) 9.536 gm
Sodium Chioride 4.384 gm
Distilled H20 to 1 liter

Phosphate buffered glycerol:

 

For immunofluorescence

Prepare phosphate buffer, pH 8.0

0.1 M_NaZHPO 94.5 ml
0.1 M_NaZH P 4 5.5 ml
Combine and mix thoroughly nine volumes glycerol and
one volume phosphate buffer, pH 8.0

0.1 M phosphate buffer, pH 6.8

 

For Preparation 0f glutaraldehyde solution

Solution A:
Monobasic sodium phosphate 1.38 gr
(NaH? PO . H20) '
Distilled water 100.00 ml

Solution B:
Dibasic sodium phosphate
(Na HPO4 . 7H 0 4.68 gr
DistiIled water 100.00 ml
Mix 77 ml solution A to 33 ml solution B

8.

168

0.05 M carbonate - bicarbonate buffer pH 9.5

 

As a coating buffer

Na2 CO 1.59 gr
Na 1iC03 2.93 gr
Dis illed water 1000.00 m1

Store at 4 C for not more than 2 weeks

Citrate - phosphate buffer, pH 4.0

 

Buffer for the preparation of the substrate for MYCO-ELISA

0.1 M citric acid 307 ml
0.2 M_dibasic sodium phosphate 193 ml
Deionized water 500 ml

Phosphate buffer'saline, pH 7.4

 

MCYO-ELISA washing buffer

Sodium chloride 8.0 gr

Monabasic potassium phosphate 0.2 gr
(KH PO )

Dibasic sodium phosphate 2.9 gr
(Na HPO . 12 H 0)

Potassium chloride . 0.2 gr

Tween 80 0.5 ml

Deionized distilled water to 1 liter
Store at 4 C for not more than 30 days

Phosphate buffer saline, pH 7.2 - 7.4

 

For serum and for conjugate dilution

Sodium chloride 37.0 gr
Potassium chloride 0.2 gr
Dibasic sodium phosphate 1.2 gr
Monobasic potassium phosphate 0.2 gr

Deionized distilled water to 1 liter
PBS for serum dilution add 0.1% bovine serum albumin (BSA)
PBS for conjugate dilution add 0.05% Tween 20.

169

MEDIA

Brain heart infusion medium (Vardaman 1967):

 

per liter
Brain-heart infusion broth (Difco) 37.0 gm
Dextrose 3.0 gm
Yeast autolysate (Albimi) 5.0 gm
Thiamin HCl 0.005 gm
Tris buffer 3.0 gm
Trypticase 0.5 gm
Thallium acetate 0.1 gm
Swine serum 100.0 ml
Penicillin 100,000 units

B-medium of Fabricant (Fabricant and Freundt, 1976):

Brain-heart infusion broth (Difco) 37.0 gm
Cystine HCL 0.1 gm
Swine serum 100.0 m1
Thallium acetate 0.1 gm
Penicillin- ‘ 100,00 units

Modified Frey Mycoplasma medium (Yoder, 1975):

 

 

 

per liter
Mycoplasma broth base 22.5 gm
Dextrose 10.0 gm
Swine or horse serum 100-200 ml
PEenol red 25n¥_
Penicillin G potassium 100,000 units
Thallous acetate (1:4000—l:2000 2.5-5.0 ml 10% Sal
PPLO broth medium (Morton and Leece, 1954):
per liter

PPLO w/o CV 21.0 gm
Yeast autolysate 10,0 gm
Dextrose 5.0 gm
Thallium acetate 0.1 gr
Horse serum 100.0 ml

Penicillin 100,000 units

Tryptose_phosphate broth

 

Tryptose phosphate broth

Maltose

Horse serum
Yeast autolysate
Thallium acetate
Penicillin

Waymouth gynthetic media

 

Amino acids: mg/l
L-Alanine
L-Arginine HCl
L-Asparagine
L-Aspartic acid
Cysteine (free base)
L-Cystine
L-Glutamic acid
L-Glutamine
Glycine
L-Histidine (free base)
L-Isoleucine
L-Leucine

L-Lysine HCl
L-Methionine
L-Phenylalanine
L-Proline

L-Serine
L-Threonine
L-Tryptophane
L-Tyrosine
L-Valine

Special agar, 1% in PBS

 

170

(Hall, 1962):

(Difco)

(Gibco, MAB 87/3 #78-027)

Vitamins: mg/l

11.20 Ascorbic acid
75.00 Biotin

24.00 Calcium pantothenate
60.00 Choline Cl
61.00 Folic acid
15.00 i-Inositol
150.00 Nicotinamide
350.00 Pyridoxine HCl
50.00 Riboflavin
121.00 Thiamine HCl
25.00 Vitamin B12
50.00

240.00

50.00

50.00

50.00

12.80

75.00

40.00

40.00

65.00

Ionagar No. 2 (Oxoid) or Noble agar (Difco)

PBS, pH 7.2

per liter
29.5 gm
2.5 gm
125.0 m1
10.0 gm

0.1 gm
100,000 units

17.00

250.00
.50
.00
.00
.00
.00
.00
.20

OOHI—II—II—a

1 gr
100 ml

171

Table 6. Serotypes of avian Mycoplasma

 

Aycardi et al. (1971) Barber and Fabricant (19713)

Frey et al. (1972)

 

 

 

Agar gel Cfowth
Diffu- inhi- Micro complement
sion bition Metabolic inhibition fixation
A A A A
BM BM B BM
CDOP C CO DCOP

D DP

OP
EG EG EG EG
IJKNQR IJKNQR IJKNQR IJKNQR
F F F F
H H H, H
L L L L
S S S S

 

172

 

 

 

 

 

 

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173

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174

 

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LITERATURE CITED

Adler,
Adler,
Adler,
Adler,

Adler,

Adler,

Adler,

Adler,

Adler,

Adler,

Adler,

LITERATURE CITED

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H. E., 1958. A PPLO slide agglutination test for the detec-
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H. E., 1970. Avian mycoplasmal diseases, In: The Role of
Mycoplasma and L-Forms of Bacteria in Disease. Editor, J. T.
Sharp, p. 240, Charles C. Thomas, Publisher, Springfield, IL.

 

 

H. E., and A. J. DaMassa, 1968. Effect of dextrose in medium
for the preparation of flycoplasma gallisepticum plate antigens.
Appl. Microbiol. 16:558.

 

H. E., J. Fabricant, R. Yamamoto, and J. Berg, 1958. Symposium
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identification of pleuropneumonia-like organisms of avian
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H. E., D. A. McMartin, and H. Ortmayer, 1962. The effect of
infectious bronchitis virus on chickens infected with Myco-
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H. E., M. Shifrine, and H. Ortmayer, 1961. Mycoplasma inocuum
sp. a saprophyte from chickens. J. Bact. 82:239.

 

H. E., and A. D. Wiggins, 1973. Interpretation of serologic
tests for Mycoplasma gallisepticum. World Poult. Sci. J. 29:
345.

 

H. E., and R. Yamamoto, 1956a. Preparation of a new pleuro-
pneumonia-like organism antigen for the diagnosis of chronic
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H. E., and R. Yamamoto, 1956b. Studies on chronic coryza
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H. E., R. Yamamoto, and R. A. Bankowski, 1954. A preliminary
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177

A -

178

Adler, H. E., R. Yamamoto, and J. Berg, 1957. Strain differences of
pleuropneumonia—like organisms of avian origin. Avian Dis.
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Ahlstedt, 5., L. A. Hanson, and C. Wadsworth, 1976. A Ciq. immuno-
sorbent assay compared with thin layer gel filtration for
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Ajello, F., and N. Romano, 1975. Effect of L-histidine on the sur-
vival of a T-strain ongycoplasma. Appl. Microbiol. 29:293.

 

Armstrong, C. H., M. J. Freeman, L. L. Sands, and D. 0. Farrington,
1978. The enzyme linked immunosorbent assay (ELISA) for diag-
nosing Mycoplasma pneumoniae of swine. Proc. 21th Ann. Meeting
Amer.Assoc.Vbt. Lab. Diag., Buffalo, NY.

 

Arnason, B. G., and B. D. Jankovic, 1967. Immunoglobulins after
surgical bursectomy in chickens. J. Immunol. 99:917.

Avrameas, 8., 1969. Coupling of enzymes to proteins with gluta-
raldehyde. Use of conjugate for detection of antigens and
antibodies. Inununochemistry 6:43.

Avrameas, S., and B. Guilbert, 1971. Dosage enzymeimmunologique de
proteines é laide d'immunoadsabents et d'antigene marques aux
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Avrameas, 5., and'I. Ternyck, 1971. Peroxidase labeled antibody and
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Avrameas, S., T. Ternyck, and J. L. Guesdon, 1978. Coupling of enzymes
to antibodies and antigens. Scand. J. Immunol. 8:87.

Aycardi, E. R., D. P. Anderson, and R. P. Hanson, 1971. Classifica-
tion of avian Myc0plasmas by gel-diffusion and growth-
inhibition tests.i Avian Dis. 15:434.

 

Barber, T. L., and J. Fabricant, 1971. Identification of Mycoplasmatales:
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Barile, M. F., W. F. Malizia, and D. B. Riggs, 1962. Incidence and
detection of pleuropneumonia-like organisms in cell culture by
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Bartlett, A., K. M. Dormandy, C. M. Hawkey, P. Stableforth, and A.
Voller, 1976. Factor VIII related antigen: measurement by
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Biddle, E. S., and M. 8. Cover, 1957. The bacterial flora of the
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