The characterisation of strains of Mycoplasma ovipneumoniae by restriction endonuclease analysis : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science in Microbiology at Massey University, New Zealand

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T HE CHARACTERI SATION O F STRAI NS O F MYCOPLASMA OVI PNEUMONIAE BY RE STRICTION ENDONUC LEASE ANALYS I S

A t hes i s pres ent ed i n par t i a l fulfi l lment of t he requi rement s for the d egree of Mas t er of S c i ence i n Mi cro bi o l o gy a t Mas s ey Universi ty , New Zealan d .

A l i son Jane Mew 1 9 82

ABSTRACT

S tudi es of the pathogeni ci ty of Mycop l asma

ovipneumoni ae would be faci l i tated by an in vi tro met hod o f i dent i fying and c l as s i fying i so l a tes of thi s organi sm , and i t i s wi th the d evel opment o f such a me thod , and i ts po s s i b l e app lications , tha t thi s thesis i s mainly concerned .

T he me thod chosen was Res tri ction Endonucl ease Analys i s

(

REA

)

which has al ready been succes s ful ly appl i ed

to the i den t i f i cation of s ome vi rus es and bac t eri a .

I ni t i a l l y the REA pat t erns o f 2 s trains o f

M . ovipneumoni ae were obs erved . They di ffered markedly ln res tric tion p a t t ern , but the pa t tern exhi bi ted by any one o f

them was recogni s ab l e and reproduc i bl e . Furthermore ,

patterns di d no t change s i gni fi can t l y after l i mi t ed pass age i n vi tro . An ex t ension o f thi s s tudy to 8 i s o l a tes from wi dely di ff ering sources , showed that all gave markedly di fferent p a t t erns . I t was conc luded tha t , unl i ke the fi ndings for L ep to.spira and Rhi zobium , REA coul d no t be u s ed for the i denti fication of speci es , but i s an ex tremely powerful me t hod for i denti fying s trains of M . ovi pneumoni ae .

D es p i t e the marked het erog enei ty of i so l a t es from di fferen t s ources , the rel a tive s t abi l i ty of pat t ern of an i ndivi dua l i so l a t e , sugges t ed t ha t REA could be used t o examine t h e epi demi o logy o f indivi dual s trains o f

M . ovi pneumoni 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-mon t h p eri od , and from the lungs of t he s ame l ambs at s laugh ter . I t was shown t hat 54 i so l ates from the nas a l c avi ti es fi l l i n to 7 ma

j

or groups wi th respect to res tri c t i on pattern

(

a l t hough minor di fferences were detec t ed wi thin a group

)

. There was a t endency for t he s e groups to occur s equenti a l ly . None o f t he i s o l a t e s were s hown to p ers i s t for long perio ds , but a l ater s train could repl ace an earli er one . T he i so l at e s from the l ungs were more

homogeneous and t he predominant s train fell wi thin one of the ⁷ "nas a l" groups . Thi s sugges ts · that nas al i so l a tes

11

may vary ln their pathogeni ci ty for the lungs , a l though other explanat i ons are pos s i b l e

(

s ee General D i s cus s ion

)

.

No twi ths tanding the app arent s tabi li ty o f mas s cul tures of ^M. ovi pneumoni ae fo l l owing l imi ted pass age i n vi tro , the unexpectedly l arge number o f res tri c tion p a t t erns found wi th fi e l d s trains , l ed us to re-examine , i n more

detai l , the s t abi l i ty of cl oned i so l a tes . A mul tiply-c l oned i s o l ate was propagated i n vi tro and 8 sub-clones s el ec t ed before and , a fur ther 8 sub-c l ones a f t er 2 0 pas s ages . Some limi ted het ero g enei ty was detec t ed among the 8 sub-c l ones s el ected before pas s ag e , and a s omewhat grea ter degree of het erogenei ty was detected among sub-c lones s el ec ted a f t er pas s age . I t s houl d , 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 l ari ti es s een when i so l a tes from di fferent s ourc es were examined . L imi ted passage i n the pres enc e of sub-l ethal

concentra tions of an ti body did no t i ncreas e the hetero genei ty of patterns - i f any thi ng , t he revers e i s true .

Exp l anations ·for these findings and future experimen ts to confirm or d eny t he s e pos s i bi l i t i es are di s cus s ed .

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

TITL E PAGE ABSTRACT

ACKNOWLEDGMENTS L I ST O F CONTENT S LI ST O F FI GURES

LI ST O F TABLES

I NTRODUCTION . .

CHAPTER 1 Hi s tori cal Revi ew

1 ^• 1 1 ^• 2

D i scovery and charac t eri s a tion of res tri c ti on endonucl eases

App l i c a t i ons of res tri c tion endonuc l ea s e s

1 ^• 2 . 1 1 ^• 2 . 2 1 ^• 2 . 3

Mapping o f DNA Cloning o f genes

C l as s i fi ca t i on and i denti fi cation of

Page_

l i i lV V

X

x i i 1 6

6

9 9 1 ¹

mi croorgani s ms ¹⁴

a ) Gene t i c rel at ednes s between s p eci es b) Speci e s i denti fication of i so l a t es c ) I dent i f i c a t ion o f s t rains

1 ^• 2 . 4 Epi demi o lo g i c a l s tudi es . .

1 . 3 Effec t o f g enome s i z e on Res tri ction Endonuc l ease 1 8

Analys i s . . 2 1

CHAPTER 2 Development o f Res tri c tion Endonuc l ea s e Analys l s for Mycopl asma 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 ntroduc t i o n

Mat eri 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 tri c tion p a t t ern o f cul tures harves ted in death phase

2 . 2 . 1 2 . 2 . 2 2 . 2 . 3 2 . 2 . 4

Introduc tion

Materi al s and Methods Resul ts

Conclus ion

E ffect on res tri c tion pa t t ern o f freez i ng cel l suspensi ons pri or 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

Conclus ion

2 9

2 9 2 9 3 0 32

3 3

3 3 3 3 34 3 7

2 . 4 Res tri c tion Endonuc l ea s e Analys i s of M . ovi pneumoni ae :

Materi als and Me t hods 8

2 . 5

2 . 6

2 . 4 . 1

2 . 4 . 2 2 . 4 . 3 2 . 4 . 4 2 . 4 . 5

Propagation o f mycop l asmas for DNA extraction

Extrac t ion o f DNA from mycopl asmas Analys i s of Nuc l ei c Aci d Ext rac t . . Di ges tion o f DNA wi t h EcoRI

Gel el ec trophoresi s and pho to graphy

Reproducibi l i ty of Res tr i c tion Endonucl ea s e Analys i s of M . o vi pneumoni ae

2 . 5 . 1 2 . 5 . 2 2 . 5 . 3 2 . 5 . 4

I ntroduc tion

Materi a l s and Methods Resul ts

Conclusion

Res tri c tion Endonuc l ea s e Analys i s of 8 s trains of M . ovipneumoni a e

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 l a t i on and I dent i fi c a ti on of

Mycopl asma ovipneumon1 ae Prom a f l o c k

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 t i on 5 8·

3 . 1 . 2 Materi als and Methods 59

a) I so l a t i on o f M . ovi pneumoni a e from

nas al cavi ty 5 9

b ) I so l ation o f M . ovipneumoni ae from

lung t i s sue 6 0

c ) Cloning of i so l a t es 6 1

3 . 1 . 3 Resul ts 6 1

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

3 . 2 I dent i fi c a tion o f M . ovi pneumoni ae by g e l 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 tr1 ct1on Endonu c l eas e Analys i s 7 0 4 . 1 Compari s on of REA p a t t erns of four i so l a t e s

fol lowing s ingl e-cl oning and mul t ip l e-cl oning 7 0

4 . 1 . 1 I n t ro 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 lus i on 7 4

4 . 2 REA of nas al i s o l at e s obtai ned a t one t ime 7 5

4 . 2 . 1 I n t ro duc t i on 7 5

4 . 2 . 2 Mat eri a l s and Methods 7 5

4 . 2 . 3 Resu l t s 7 6

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

4 . 3

4 . 4

4 . 5

4.6

4 . 7

REA o f s eri al nas al i s o l a t e s

4 . 3 . 1 4 . 3 . 2 4 . 3 . 3 4 . 3 . 4

Introduc tion

Materi a l s and Methods Resul t s

D i s cus s i on

REA o f lung i s o l a tes

4 . 4 . 1 4 . 4 . 2 4 . 4 . 3 4 . 4 . 4

In troduc tion

Materi als and Me thods Resul ts

D i scus sion

Compari s on o f nas a l and l ung i s o l ates by REA

4 . 5 . 1 4 . 5 . 2 4 . 5 . 3 4 . 5 . 4

Introduc tion

Mat eri als and Me thods Resu l ts

Di s cus s i on

REA of i so l at es o f M . ovi pneumoniae obtai ned from a flock o f l ambs : cons ollda t ed resul ts

4 . 6 . 1 4 . 6 . 2 4 . 6 . 3 4 . 6 . 4

In troduc t i on

Mat eri als and Me tho ds Resul ts

D i s cus s ion

REA of mul tiple i so l ates of M . ovi pneumoni ae obtained from one pneumoni c lung

4 . 7 . 1 Introduc tion

4 . 7 . 2 Materi a l s and M e t hods 4 . 7 . 3 Resul ts

4 . 7 . 4 Conclusion

CHAPTER 5 ^An Inve s t iga t i on i n to the Source o f Heterogeneity o f Stralns of

M . oVlpneumonl a e

P age 7 9 7 9 7 9 80 8 2 8 3

8 3 8 3 84 8 6

8 7

8 7 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 p a t t erns o f M . ovipneumoni ae

o f pas s age in vitro 9 9

5 . 1 . 1 I ntroduc tion

5 . 1 . 2 Materi als and Me thods 5 . 1 ^.3 Resul t s

5 . 1 . 4 D i s cus sion

CHAPTER 6 General Discus s i on APPENDIX

BI BL I OGRAPHY

Page 9 9 1 00 . 1 01 1 0 6 1 0 7 1 1 5 1 1 7

L I ST OF F I GURE S

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 s on o f REA pat t erns o f DNA from 2

cul tures o f a s train o f M . ovi pneumoni ae ( M 1 4- 7 ) , harves t ed in the l o g phase and the dea th phas e 31 3 Compari s on of REA pat t erns o f D NA from 2

cul t ures of a s train of M . ovipneumoni ae

( s train 1 0 ) : the DNA immed1 a t ely ex trac t ed from the c e l l sus p ens i on , and fo l l owing thawing o f the c e l l suspens i on tha t had been s tored a t

-7 0°C ^. . ^{� · .} . . . . . 3 6

4 The Gel E l ectrophores i s E quipment 47

5 REA o f DNA from 2 s trains of M . ovipneumoni ae ( s trains 1 and 1 0 ) . E ach s tra1n was prop aga t ed in dup l i c a t e and t he DNA ex trac ted from each o f t he 4 c e l l suspens i ons. E a c h extrac t was

di ges t ed in dup l i c a te 51

6 REA of 8 s trai ns o f M . ovi pneumoni ae from wi dely

di ffering s ourc es 5 6

7 a ) The cumu l a t i ve propor t ion of lambs o f one f lock from which M. ovi pneumoni a e was r ec overed from the nas al cav1 ty by 3-weekly swabbi ngs b) T he proportion o f t hes e l ambs from whi c h

M . ovi pneumoni ae was recovered from the lungs

a t s l augh t er 6 3

8

9 1 0 1 1 1 2 1 3

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

REA of 4 i s o l a t es , s in g l y- and tri p ly-c l oned REA of 8 nasal i s o l a t e s from a flock o f l ambs a t

t he 7 th swabbing ( i . e . a g e o f l ambs ⁼ 2 0 7 days ) REA o f 8 s eri al nasal i s o l a tes obtained from

3

l ambs o f one flock

REA o f 8 l ung i s o l at e s from a flock o f l ambs a t slaughter

REA o f 8 nas a l and lung i so l a tes obt ai ne d from

3

l ambs o f one f lock

6 8 7 3 77 8 1 8 5 8 9

Figure 1 4 1 5 1 6 1 7

REA o f 1 0 i s o l a t es of ^M. ovipneumoni ae obtained from one pneumoni c lung

REA of 8 sub- c lones of ^M. ovipneumoni ae s train 5 pri or to the in vi tro p as s ag e exp erlment

REA of 8 sub- c l ones of ^M. ovi pneumoni ae s train 5 taken fo l l owing 20 pas s ages l n Vl tro

REA of 8 sub-c l ones of ^M. ovi pneumoni ae s train 5 t aken fo l l owing 3 pass ages l n Vl tro ln the pres enc e o f sub-inhibi tory conc entra tions of anti s erum

Page_

97 10 3 1 04

10 5

T abl e I II

L I ST OF TABLE S

F inal turbi di ty readings o f 4 cul tures o f

M. ovi pneumoni ae

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

I I I P ro tocol for preparation o f s tandard fluorome try curve

IV Ori gin of ^M. ovipneumoni ae s t rains as s hown in

26

30 4 3

F i gure 6 5 4

V I so l a t i on of ^M. ovi pneumoni ae from a ) the nas al cavi ti es o f a flock of l ambs swabbed at approx­

i mately 3-week i n t erva l s , and b ) from the lungs

at s l aughter ^· 6 2

VI Sourc e o f ^M. ovi pneumoni ae i so l a t es as shown 1n

Fi gure 9 7 1

VI I Ei ght nas a l i s o l a tes from the 7 th swabbing as

s hown in F i gure 1 0 7 6

VI I I Ei ght s eri a l nas al i s o l ates from 3 l ambs a s s hown

i n F i gure 1 1 7 9

I X E i ght iung i s o l ates a s s hown i n F i gure 1 2 8 3 X Ei ght nasal and lung i s o l ates a s shown 1n Figure 1 3 8 7 XI C l as s i fi c a tion o f i so l a tes of ^M. ovi pneumoni ae from

a £l ock of l ambs a) from the nas al caVl t l es s wabbed at approxima tely 3-week i n terval s , and

b ) from the lungs a t s�aughter 94