Isolation and charaterisation of adenoviruses and reoviruses of domestic hens in New Zealand : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University, New Zealand

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ISOLATION

AND

CHAilAC'rERIS_4.TION OF ADEI'TOVIRUSES AND REOVIRUSES OF DOEESTIC HENS IN

NEvl ZEALAND

A

thesis presented in partial fulfilment of the requirements for the degree of

Master of Science in Microbiology at Massey University, New Zealand

Allan ^F

r

aser Green

1 974

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ii

ABSTRACT

This investigation uas undertaken to determine uhether or not adenoviruses or reoviruses could be recovered from domestic hens in Nevr Zealand. Using cul turcs derived from neonatal chicken kidney.

tissue

30

cytopathic agents vrere recovered from domestic hens.

Electron microscope examination follm.;ing staining by sodium phosphotungstate shoHed that 2 5 of these agents had the morphology of adenoviruses and the remaining

5

^agents ^vlere morphologically indistinguishable from reoviruses.

Some selected strains of adenoviruses and reoviruscs vrere subjected to physico-chemical tests and it was found that both groups of agents were u.11affected r .. y chloroform and the replication of adenovirus strains, but not the reovirus, vras inhibited by IDU.

These results provide confirmatory evidence for the identification of these agents as avian adenoviruses and reoviruses. The avian adenoviruses like the analogous mammalian agents l'rere found to have diminished thermostability in the presence of

1

^.^ON^Ca++.

Cross-neutralisation tests shov,red that four serologically distinct adenovirus strains 1vere recovered

nnd 1 6

of the remaining adenovirus isolates were assigned to one or other of the four sero

types. All

5

reoviruses were assigned to one serological type on the basis of neutralisation tests.

A preliminary scrUlil survey for neutralising antibody to the

4

adenovirus serotypes showed that antibody to one or more of these viruses was present in a high proportion of flocks of domestic fowl in the Manavmtu district of New Zealand and antibody to two of the serotypes vras particularly common.

This v1ork represents the first report of the recovery of avian adenovirus and reovirus from domestic hens in New Zealand. However their clinical and pathological significance remain to be investigated.

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iii

ACKN

0\VI,EDGEHENTS

I

vmuld like t o thank the Department o f �hcrobiology and Gene tics , IVJ:assey University, for providing t he oppo rt1mi ty, and facil itie s for this investigatio n .

I would also like to thank :

Dr J. Lohr for ass is tance in obtaining s pe c imens and sera , for the use of an egg incubator, and for helpful

sugges tions .

Mr R. Pat chell for gene rous ly supplying fertile eggs over the dura tion of this investigation.

Dr

^G.

Burgess and Mr P. Durham for us eful discussions .

The staff of t he Ele ctron �1icros cope Unit ( DSI R ) for the use o f their facilities , and for t echni cal advice .

'rhe

C entral Phot o graphic Unit , Massey Unive rsity.

Mrs C. Gradolf for the typing .

" The boys " ( Hereford Street e t al . ) for ke e ping my s pirits high .

In particuJ.ar , Dr

^J^.K.

Clarke , for his mos t appreciated

guidance and encouragement , and free beers at the

Majes tic .

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Abstrac t

A ckno1-1ledge men ts C ontents

List of Tables List of Plates List of Figures

Intro ducti o n General

C ONTENTS

I solation and characterization of avian adenovi ruses C larificati on of avian adenovi rus serol o gy

Re ovi ruses of domesti c hens Material s and ll1ethods

1

^•

Chick kidney ( CK ) cel l cul tures 2 . Virus isolatio n

( a ) Post mortem spe cimens

( b ) Svmbs from l ive birds

( c ) Inoculati on of spe c i mens into cell culture s

3 . Virus identification by electron microscopy

4 . Production and standardisation of reagents fo r

serological typing of virus isolates

( a ) Production of antisera to isolates

( b ) Virus standardisati on for neutral isation tests

( c ) Antiserum titration

5 . Serologi cal typing of virus isolate s and selection of prototype strains

6 . Cross-neutrali sation tests vvi th prototype strains 7 . Physi co-che mi c al tests

( a ) Chloroform sensitivity

( b ) IDU sensi tivity

( c ) Effect o f cations on heat stability 8. Screening tests for neutralising antibodies

9 . Staining CK monolayers

i i iii'

iv vi vii vi i

3 6 1 0 1 4 1 4 1 6 16 1 6 1 7 1 8

1 9 1 9 1 9 1 9

2 1

22

24

2 4

24

2 6

27

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Results

1 . Gr·ovrt h of

CK

ce l l cultures

2. Virus isolation and i dentification by electron microsco py

3. Serol ogical classif ication of viruses recovered from New Zealand domestic hens

(

_a

)

Adenovirus typing

(b)

l1.eovi rus typing 4-. Physico-chemical tests

V

28 28

29

38 38 38 41

(

a

)

Chloroform sensitivity 41

(b)

I DU

sensi tivit y : adenoviruses 41

(

c

) IDU sensi tivity: re oviruses 41

(

d

)

Heat stabilit y of avian adenoviruses and reoviruses in the presence of various cat i ons

5. Screening sera from domestic hens for neutralisj_ng antibody to avian adenoviruses

6. S taining of

C K

^cell^cultures

Di scussion

1 ^. Product ion of

CK

cell cultures 2. Isolation of cyt opathic agents

3. Identification of virus isolates by negative contrast

44

47 49

56 56 57

electron microscopy 58

4. Sero l o gical typing of iso lat es by neutralisation tests 60

5. Physico-chemical tests 62

6. The prevalence of adenovirus and reovirus infection of d omestic hens in Nel'r Zealand

(

a

)

Adenovirus .

(

b

)

Reovirus ^,

7. Clinical and pathological significance of avian

64 64 65

adenoviruses and reoviruses 66

(

a

)

Adenoviruses 66

(

b

)

Reoviruses 68

Appe

ndix 70

1;

Phosphate buffered saline and mineral salts - PBS

(

pH 7.5) 70

2. Trypsin solution 70

3. Earle's based solutions 70

4. Hank's solutions 5. Giemsa's stain References

72 73

74

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LIST

OF

^TABLES

1 .

Viruses of domest ic hens

2 .

C omparison of pro pe rties of avian adeno-like viruses and

3 . 4 .

5 . 6 . 7 . 8 .

9 . 1 0 .

mammal ian adenoviruses

Avian adenovirus serotype s : ove rseas designat ions

C omparison of pro perties of avian reoviruses and mammalian re ovi ruses

Vi ruses recovered from domestic hens using CK cell cul tures Results of typing adenovirus isolat es

C ross-neutralisation tests behmon prot otype strains of avian adenoviruses

S tabili t y of avian adenovi ruses and reoviruses in the presence of chlo roform

Replication of avian adenovi ruses in the pr esence of

IDU

Replica tion of the pro t otype strain of avian reoviruses in the presence of

IDU

1 1 .

Effect of cati ons on the heat stability o f avian adenoviruses

1 2 . 1 3 .

and reoviruses

Results of screening s e ra from domestic hens for the presence of neutral ising antibody t o avian adenovi ruses

C omparison of the proportion of sera posi tive for each of the

4

adenovi rus serot ypes •vi th the re la ti ve frequency of isolation of each serotype

vi

Page

2

7 9

1 1 37 3 9

40

42 43 4 5 46 48

5 1

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1 ' 2 and 3 4

5 6 6a

7 and 8 9

1 0

1 1 and 1 1 a

1 2 and 1 3 1 4 and 1 5

vii

I,IST OF PLATES

Page Chick kidney tissue dispersed by trypsj_n 30 Avian adenovirus CPE : cell rounding ( unstained ) 31

Avian adenovirus CPE: cell vacuolation ( unstaine d ) 31

Avian reovi rus CPE : syncytia formation ( unstained ) 32

Uninocula ted chi ck kidney cell monolayer ( unstained ) 32

Avian adenovi rus particles, negatively s tained 35 Avian reovirus parti cles, negatively stained 36

Mixture of adenovi rus and reovi rus, negatively staine d

Uninoculated chi ck kidney cell mono layer , Giemsa ' s stain

Adenovirus infe cted c e ll culture , Giemsa's stain Re ovirus infected c e ll cul ture, Giemsa's-stain

LIST OF

FIGURES

Distribution of titres of neutralising antibody to avian adenovirus type C in a smal l sample of randomly selected positive se ra

36

53 54 5 5

5 2