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T HE CHARACTERI SATION O F STRAINS OF

MYCOPLASMA OVIPNEUMONIAE BY

RE STRICTION ENDONUCLEASE ANALYSI S

A t hesi s present ed i n partial fulfi l lment of

t he requi rement s for the degree of Mas ter of

S c i ence i n Micro bi ology a t Mas s ey Universi ty ,

New Zealand .

Alison Jane Mew

1 9 82

ABSTRACT

S tudi es of the pathogenici ty of Mycop lasma

ovipneumoni ae would be faci li tated by an in vi tro met hod o f

i dentifying and c l as s i fying isola tes of thi s organi sm , and i t

i s wi th the development o f such a me thod , and i ts possible

app lications , that this thesis i s mainly concerned .

T he me thod chosen was Res tri ction Endonucl ease

Analys i s ( REA ) which has already been succes s ful ly appl i ed

to the i dentification of some virus es and bac t eri a .

I ni t i ally the REA pat t erns o f 2 s trains o f

M . ovipneumoni ae were observed . They di ffered markedly ln

res tric tion pat tern , but the pat tern exhi bi ted by any one o f

them was recogni s able and reproducibl e . Furthermore ,

patterns did no t change s i gni fi cantly after limi ted passage

i n vi tro . An ext ension o f thi s s tudy to 8 i s o la tes from

wi dely di ffering sources , showed that all gave markedly

di fferent p at t erns . I t was conc luded tha t , unli ke the

findings for L ep to.spira and Rhi zobium , REA coul d no t be

used for the i denti fication of speci es , but i s an extremely

powerful me thod for i denti fying s trains of M . ovipneumoni ae .

D espi t e the marked heterogenei ty of i so l ates from

di fferent s ources , the rel a tive s tabi l i ty of pat tern of an

i ndivi dual i so late , sugges t ed tha t REA could be used t o

examine t h e epi demio logy o f indivi dual s trains o f

M . ovipneumoni ae wi thin a flock o f s heep . Hence , we

undertook a s tudy o f M . ovi pneumoni ae i so l a tes obtained by

s eri al swabbing of the nas al cavi ti es of a flock o f l ambs

over a 6-month p eriod , and from the lungs of the s ame l ambs

at s laugh ter . I t was shown that 54 i sol ates from the nasal

c avi ti es fi l l into 7 maj or groups wi th respect to res tri c tion

pattern ( al though minor di fferences were detect ed wi thin a

group ) . There was a tendency for these groups to occur

s equenti a l ly . None o f the i s o lates were s hown to pers i s t

for long perio ds , but a l ater s train could replace an

earli er one . T he i solates from the l ungs were more

homogeneous and t he predominant s train fell wi thin one of

the 7 "nasa l" groups . Thi s sugges ts · that nas al i so l a tes

11

may vary ln their pathogenici ty for the lungs , al though

other explanations are possible ( s ee General D i s cuss ion ) .

No twi ths tanding the apparent s tabi li ty o f mas s

cul tures of M. ovipneumoni ae fol l owing l imi ted passage i n

vi tro , the unexpectedly l arge number o f res tri c tion p a t t erns

found wi th fi eld s trains , l ed us to re-examine , i n more

detai l , the s t abi l i ty of cloned i so l a tes . A mul tiply-c l oned

i s o late was propagated in vi tro and 8 sub-clones s el ec t ed

before and , a further 8 sub-clones after 20 pas s ages . Some

limi ted het erogenei ty was detec t ed among the 8 sub-c lones

s el ected before pas sage , and a somewhat greater degree of

heterogenei ty was detected among sub-c lones s el ected a fter

pas s age . I t s hould , however , be emphasi s ed that the s e

di fferences were smal l compared to the to tal l ack o f

s imi lari ti es s een when i sola tes from di fferent sources were

examined . L imi ted passage i n the pres ence of sub-l ethal

concentrations of antibody did no t increas e the heterogenei ty

of patterns - i f any thing , t he revers e i s true .

Expl anations ·for these findings and future

experiments to confirm or deny the s e poss i bi l i t i es are

di s cussed .

Ill

ACKNOWLEDGMENTS

I am indebted to the Department of Microbiology and

Genetics, and to the Department of Veterinary Pathology and

Public Health, for providing the opportunity and facilities

for these studies.

Department of Microbiology and Genetics

In particular, I would like to thank:

My supervisor, Dr J.K. Clarke, for his excellent guidance

and advice throughout the course of this study.

Mrs Alison Lee, Mr George Ionas, and Mr Andrew Fraser for

their friendship and help when thin�were hectic.

Professor D.F. Bacon for useful discussion, Mr R. Tucker,

and all other members of the academic and technical staff.

Department of Veterinary Pathology and Public Health

I would like to thank Dr A.J. Robinson for the supply

of EcoRI, and Lambda DNA, and who, together with

Dr R.B. Marshall, provided invaluable advice, discussion and

constant encouragement.

Special thanks must also go to Dr M.R. Alley for the collection

of specimens, Ms Gi�l Ellis for the preparation of lambda DNA

and EcoRI, and Dr K. Moriarty for use of laboratory space.

I would also like to thank:

Mrs Penny van Doorn for the excellent typing of this thesis.

The staff of the Central Photographic Unit.

And last, but definitely not least, warmest thanks go to

my husband Peter, for the construction and illustration of

the gel electrophoresis equipment and for his unfailing

support and forbearance throughout the preparation of this

thesis.

IV

CONTENTS

TITLE PAGE

ABSTRACT

ACKNOWLEDGMENTS

L I ST O F CONTENT S

LI ST O F FIGURES

LI ST O F TABLES

I NTRODUCTION . .

CHAPTER 1 Hi s torical Revi ew

1 • 1

1 • 2

D i scovery and charac t erisation of res tri c ti on endonucl eases

Applications of res tri c tion endonuc l eases

1 • 2 . 1

1 • 2 . 2

1 • 2 . 3

Mapping o f DNA

Cloning o f genes

Clas s i fi ca t i on and identi fication of

Page_

l

i i

lV

V

X

xii

1

6

6

9

9

1 1

mi croorgani s ms 14

a ) Genet i c rel atednes s between s p eci es

b) Specie s i denti fication of i so l ates

c ) I dent i fication o f strains

1 • 2 . 4 Epi demi o logical s tudi es . .

1 . 3 Effect o f genome s i z e on Res triction Endonucl ease

1 8

Analysi s . . 2 1

CHAPTER 2 Development o f Res tri c tion Endonuc l ease Analysl s for Mycoplasma ovlpneumonl ae

2 . 1 Growth s tudi es o f two s trains of M . ovi pneumoni ae in vi tro

2 . 1 • 1

2 . 1 • 2

2 . 1 • 3

2 . 1 . 4

I ntroduction

Materi a l s and Methods

Resul t s

Conclus ion

2-q

24

2 5

2 5

2 8

·-

2 . 2

2 . 3

E ffect on res tric tion pattern o f cul tures harves ted in death phase

2 . 2 . 1

2 . 2 . 2

2 . 2 . 3

2 . 2 . 4

Introduction

Materi als and Methods

Resul ts

Conclusion

E ffect on res tri c tion pa ttern of freezing cel l suspensi ons prior to DNA extrac tion

2 . 3 . 1

2 . 3 . 2

2 . 3 . 3

2 . 3 . 4

I n troduction

Materi als and Methods

Resul ts

Conclusion

2 9

2 9

2 9

3 0

32

3 3

3 3

3 3

34

3 7

2 . 4 Res tric tion Endonucl ease Analysi s of M . ovipneumoni ae : Materials and Met hods 8

2 . 5

2 . 6

2 . 4 . 1

2 . 4 . 2

2 . 4 . 3

2 . 4 . 4

2 . 4 . 5

Propagation of mycop l asmas for DNA extraction

Extrac tion of DNA from mycopl asmas

Analysi s of Nuc l ei c Aci d Ext rac t . .

Di ges tion of DNA wi t h EcoRI

Gel el ectrophoresi s and photography

Reproducibi l i ty of Res tric tion Endonucl ease Analys i s of M . ovi pneumoni ae

2 . 5 . 1

2 . 5 . 2

2 . 5 . 3

2 . 5 . 4

I ntroduc tion

Materials and Methods

Resul ts

Conclusion

Res tri c tion Endonucl ease Analysi s of 8 s trains of M . ovipneumoniae

2 . 6 . 1

2 . 6 . 2

2 . 6 . 3

2 . 6 . 4

I ntroduc t i on

Materi als and Metho ds

Resul ts

Conc lus ion

3 8

3 9

4 1

4 3

46

49

49

49

5 0

5 2

5 4

5 4

5 4

5 5

5 7

CHAPTER 3 I so lation and I dent i ficati on of Mycoplasma ovipneumon1 ae Prom a flock of l ambs 5 8

3 . 1 I so l a tion o f M . ovipneumoni a e 5 8

3 . 1 • 1 I ntroduc tion 5 8 ·

3 . 1 . 2 Materi als and Methods 59

a) I solation o f M . ovipneumoni a e from nas al cavi ty 5 9

b ) I solation o f M . ovipneumoni ae from lung tis sue 6 0

c ) Cloning of i solates 6 1

3 . 1 . 3 Resul ts 6 1

3 . 1 . 4 D i s cussi on 6 4

3 . 2 I dent i fication o f M . ovipneumoni ae by gel precipi tin tes t s 6 5

3 . 2 . 1 I ntroduc tion 6 5

3 . 2 . 2 Materi a l s and Methods 6 6

3 . 2 . 3 Resul ts 67

3 . 2 . 4 Conclus ion 6 9

CHAPTER 4 Examination o f I so l ates from a F lock o f L ambs by Res tr1ct1on Endonucl eas e Analysi s 7 0

4 . 1 Compari son of REA patterns of four i so lates fol lowing s ingl e-cl oning and mul tip l e-cloning 7 0

4 . 1 . 1 Intro duc tion 70

4 . 1 . 2 Materi a l s and Methods 7 1

4 . 1 • 3 Resul ts 7 2

4 . 1 . 4 Conc lusi on 7 4

4 . 2 REA of nas al i s o l ates obtained a t one t ime 7 5

4 . 2 . 1 Intro duc tion 7 5

4 . 2 . 2 Materi als and Methods 7 5

4 . 2 . 3 Resu l t s 7 6

4 . 2 . 4 Di s cuss i on 7 8

4 . 3

4 . 4

4 . 5

4 . 6

4 . 7

REA o f s eri al nas al i solates

4 . 3 . 1

4 . 3 . 2

4 . 3 . 3

4 . 3 . 4

Introduc tion

Materi als and Methods

Resul t s

Discuss i on

REA o f lung i solates

4 . 4 . 1

4 . 4 . 2

4 . 4 . 3

4 . 4 . 4

Introduc tion

Materi als and Methods

Resul ts

D i scus sion

Compari son of nas al and l ung i s o lates by REA

4 . 5 . 1

4 . 5 . 2

4 . 5 . 3

4 . 5 . 4

Introduction

Materi als and Methods

Resu l ts

Di s cussion

REA of i so l ates of M . ovi pneumoniae obtai ned from a flock o f lambs : consolldated resul ts

4 . 6 . 1

4 . 6 . 2

4 . 6 . 3

4 . 6 . 4

Introduction

Materi als and Metho ds

Resul ts

Discus s ion

REA of mul tiple i so lates of M . ovi pneumoni ae obtained from one pneumonic lung

4 . 7 . 1 Introduc tion

4 . 7 . 2 Materi a l s and Methods

4 . 7 . 3 Resul ts

4 . 7 . 4 Conclusion

CHAPTER 5 An Investigation into the Source of Heterogeneity of Stralns of M . oVlpneumonl a e

Page 7 9

7 9

7 9

80

8 2

8 3

8 3

8 3

84

86

87

87

8 7

8 8

8 8

9 2

9 2

9 2

9 3

9 5

96

9 6

9 6

9 8

9 9

5 . 1 E ffec t on res tri c ti on patterns of M . ovipneumoni ae of pass age in vitro 9 9

5 . 1 . 1 I ntroduc tion

5 . 1 . 2 Materi als and Methods

5 . 1 .3 Resul t s

5 . 1 . 4 D i s cus sion

CHAPTER 6 General Discus s i on

APPENDIX

BI BLIOGRAPHY

Page 9 9

1 00 .

1 0 1

1 06

1 0 7

1 1 5

1 1 7

L I ST OF F IGURES

Figure

1 Moni toring pH o f s haking and s ta tionary cul tures of two s trains of M . ovi pneumoni ae ( s trains 2 and 1 o) 2 7

2 Compari son of REA pat t erns of DNA from 2 cul tures of a s train of M . ovi pneumoni ae ( M 1 4-7 ) , harves t ed in the log phase and the dea th phas e 3 1

3 Compari son of REA pat t erns of D NA from 2 cul tures of a s train of M . ovipneumoni ae ( s train 1 0 ) : the DNA immed1 a tely extract ed from the cel l suspens i on , and fol lowing thawing of the cell suspensi on that had been s tored a t -70°C . . � · . . . . . . 3 6

4 The Gel E l ectrophoresi s E quipment 47

5 REA of DNA from 2 s trains of M . ovipneumoni ae ( s trains 1 and 1 0 ) . Each s tra1n was propagat ed in duplicate and the DNA extrac ted from each of t he 4 cell suspens i ons . Each extrac t was di ges t ed in dup l i c a te 51

6 REA of 8 s trai ns of M . ovi pneumoni ae from wi dely di ffering s ources 5 6

7 a ) The cumulative proport ion of lambs o f one f lock from which M. ovipneumoniae was recovered from the nas al cav1 ty by 3-weekly swabbings

b) T he proportion of thes e l ambs from which M . ovipneumoni ae was recovered from the lungs a t s l aughter 6 3

8

9

1 0

1 1

1 2

1 3

The Gel Precipi tin Tes t for the identi fi cation of i s o l a tes

REA of 4 i so l at es , s ingly- and tri p ly-c l oned

REA of 8 nasal i s olates from a flock of l ambs a t the 7 th swabbing ( i . e . a g e of l ambs = 2 0 7 days )

REA of 8 s eri al nasal i s o la tes obtained from 3 l ambs of one flock

REA of 8 l ung i s o l ates from a flock of l ambs a t slaughter

REA o f 8 nasa l and lung i so la tes obt ai ned from 3 l ambs of one f lock

6 8

7 3

77

8 1

8 5

8 9

Figure

1 4

1 5

1 6

1 7

REA of 1 0 i solates of M. ovipneumoniae obtained from one pneumoni c lung

REA of 8 sub-clones of M. ovipneumoni ae s train 5 prior to the in vi tro p as s ag e exp erlment

REA of 8 sub-clones of M. ovi pneumoni ae s train 5 taken fol lowing 20 pas s ages l n Vl tro

REA of 8 sub-clones of M. ovipneumoni ae s train 5 t aken fol l owing 3 pass ages l n Vl tro ln the pres ence of sub-inhibi tory conc entrations of anti s erum

Page_

97

10 3

1 04

10 5

T abl e

I

II

L I ST OF TABLES

F inal turbidi ty readings of 4 cul tures of M. ovi pneumoni ae

S pec tropho tometry profi l es of Nuc l ei c Acid Extrac ts from exponenti al and death phas e cul tures

I I I Pro tocol for preparation o f s tandard fluorometry curve

IV Origin of M. ovipneumoniae s t rains as s hown in

26

30

4 3

F igure 6 5 4

V I so lation of M. ovipneumoniae from a ) the nas al cavi ti es of a flock of l ambs swabbed at approx­i mately 3-week interva l s , and b ) from the lungs at s l aughter

·

6 2

VI Source of M. ovi pneumoni ae i so l a t es as shown 1n Fi gure 9 7 1

VI I Ei ght nas al i s o la tes from the 7 th swabbing as s hown in F i gure 1 0 7 6

VI I I Eight s eri al nasal i s o lates from 3 l ambs a s s hown i n Fi gure 1 1 7 9

I X E i ght iung i s o l ates a s s hown i n Fi gure 1 2 8 3

X Ei ght nasal and lung i solates as shown 1n Figure 1 3 8 7

XI C l as s i fi c ation of i so l a tes of M. ovipneumoni ae from a £lock of l ambs a) from the nasal caVl t l es swabbed at approxima tely 3-week interval s , and b ) from the lungs a t s�aughter 94