A study of orf virus isolates from sheep in New Zealand : a thesis presented in partial fulfilment of the requirements for the degree of Master of Philosophy in Virology and Immunology at Massey University

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(2) A STUDY OF ORF VIRUS ISOLATES FROM SHEEP IN NEW ZEALAND. A THESIS PRESENTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF PHILOSOPHY IN VIROLOGY AND IMMUNOLOGY AT MASSEY UNIVERSITY. TOMASA CRUZ BALASSU D,V,M, M.P.H.. ( UNIV,. - VET.. OF THE PHILIPPINES ). ( UNIV,. OF THE PHILIPPINES ). MARCHJ 1981.

(3) ii.. STATEf1EIH. The gradi ents d e p i c ted. in Figure 1 and the ge l d e p i c t e d. in F igure 3 were don e in c o l l abora t i on w i th Dr . Rob i ns on .. The rest of the work was my own .. !f301/D.A/J'. A---. T .C .. B a l assu. A.J..

(4) ABSTRACT. A pr e l imi nary s t udy New Z ea l and. is. of various o r f v i ru s i s o l a t e s i n. p r e s e nted .. pur i fy i ng the v i r u s. A me t hod o f i s o l a t i n g and. from s c ab mater i a l i� de s c r i b ed .. Th i r te e n o r f v i ru s i s o l ate s were compared by DNA r e s tr i c t ion endonuc l e a s e a n a l y si s .. T h e r e w a s ex t e n s iv e. h e t e r o g e n e i ty i n t h e E c o R. r e s t r i c t i on patter ns , 1 two o f the i s o l a te s w e r e s im i l a r .. P r opagat i on o f t h e v i r u s. .{. V!.. h oweve r ,. v-<.tJL o w a s a l so at temp t ed .. F i ve. o f the i s o lat e s w e r e s u c ce s s fu l ly adap ted to g r ow th in l am b te s t i s c e l l s a n d two o f the s e w e re p l aque -pur i f i ed tw i c e . T h e c h aracter i s t i c cytopath i c e f f e c t o f c e l l - adapted v iru s i s e a r ly round i n g and c l ump i n g tog e th e r o f i n f e c ted c e ll s . Th i s c e l l r ound i n g cou ld be s e e n a s e a r ly a s two h ou r s po s t - i n f e c tion a nd cou ld b e i n h i b i t e d by c y c l o h e x am ide . Ther e were two typ e s o f p laque� exh i b i t ed by the i s o l at e� i n l amb t e s t i s ce l l mono layer s , typ e s .. the. " open ". and " c l o s e d ". I n c l u s i o n bod i e s w e r e a l s o s e e n i n s t a i n ed i n f e c t e d. mono l a ye r s . 32. P- l a b e l l ing o f a plaque-pur i fied i s o l a t e w a s s u c c e s s f u l ly. pe r formed .. Low p a s s ag e. ( 2 3 p a s s ag es). in c e l l cu l tu r e o f. , 1 Barn H- I or H i nd I I I p a t t e r n s o f t h e DNA when c om p a r ed w i th p l aqu e-pur i f i e d i s o late No .. 2 d i d not a l ter the E coR. the o r i g in a l v i r a l DNA f r om s c ab m a t e r i a l ..

(5) 1V.. ACKNOWLEDGf'.lENTS. F i r s t o f a l l, I wou l d l ike to exp re s s my s 1ncere gr a t i tude t o the M in i s t ry o f F o re i gn A f f a i r s , New Zea l an d f o r g 1 v 1ng me th i s o p p o r tun i ty t o pursue h i gher s tu d ies in th i s c ou n t ry . I w i sh t o acknowledge the s up p o r t �nd a dv i ce o f Ms S arah C ra i g , Mr Don C a r s on and Mr Chr i s Wr i gh t .. Th e f inan c i a l. s u pp o r t ex ten ded b y the Dep t . o f Ve ter inary P a th o l o gy and P ub l i c Hea l th made th i s re search po s s ib le and o f wh i ch I am ver y g r a te fu l . My mo s t s 1ncere t h ank s a re due t o my su perv1 s o r , Dr. A . J . Rob in s on , for h i s enc ou r a gemen t and gu i dance throu gh o u t the c ou r se o f th i s wo r k .. I am a l s o gra te fu l t o the. techn i c a l a s s i s t ance o f Ms 1. F r a y and Ms G. E l l i s .. A s pec i a l th ank i s due t o Mr R . D i xon for h i s mod i f ied techn i que o f May-Grunw a l d - G iem s a s t a in wh i ch wa s very u s e fu l . The va l u ab le a s s i s t an c e o f Mr Pe ter W i l db o re and the pho t ograph i c wo rk o f Mr Tom Law a re s in cere l y a p p re c i a ted and a cknowled ged .. I am a l s o th ank f u l t o Mr s Jan S ch r ama. and Mr Rex Fau l d in g wh o se serv i c e s were o f mu c h he l p in the c omp l e t i o n o f th i s work . A g r a te fu l a cknmv l e d gmen t i s a l s o made t o the MAF Near D iv i s i o n ve ter i n ar i an s f o r p r ov i d in g the s amp l e s needed f o r th i s s tud y .. Fur ther a cknowle d gmen t i s due t o the Department. o f S c ien t i f i c and Indu s t r i a l Resear ch f o r the u se o f the e lec t r on mi c r o s c ope w i t h s pec i a l thank s t o Mr D ou g Hop c r o f t . F in a l l y , I w i sh t o a cknow l e d ge the f r iend s h i p and h o s p i t a l i ty o f the member s o f s t a f f and fe l l ow gradu a te s tu den t s o f the Dep t . o f Ve ter inary P a th o l o gy and Pub l i c Hea l th .. The f ine and c ompe ten t t yp ing o f Mr s V . Lobb i s. a l s o a c knowledged ..

(6) v.. TABLE OF CONTENTS. PAGE. Statem ent. ii. Ab s t r a c t. iii. A cknm..r l e d gmen t s. iv. Tab l e o f Con t e n t s. V. L i s t o f F igur e s. vii. In t rodu c t i on. ix. Ch ap t e r One. - Rev i ew o f L i t e r a t u r e C on t a g i o u s ec thyma and man. C h a p t e r T<;vo. 1 ln. s h e e p, goa t s ,. 1. Or f in o th e r h o s t / exp e r imen t a l an ima l s. 6. S c abby mou th i n New Z e a l an d. 9. D ia gn o s i s. 10. T r e a tmen t and Con t r o l. 11. Phy� i c o- c h eu i c a l p r o pe r t i e s o f th e V l ru s. 15. Mu l t i p l i c i t y o f s t r a in s. 17. G r o w t h o f o rf v i r u s in c e l l c u l ture. 19. - M a t e r i a l s and me th o d s. 27. I s o l a t i on and pur i f i c a t i on o f o r f V l ru s. 27. S p r ay d r op l e t c ou n t ing o f o r f v i ru s pa r t i c l e s. 29. M e a s u r emen t o f d en s i ty o f f r a c t i o n s f r om a r y l g r a d i e n t s .. 30. DNA e x t ra c t i o n. 30. Re s t r i c t i on endonu c l e a s e d i g e s t ion and g e l e l e c t r ophore s i s o f DNA. 31. P r o p aga t ion o f o r f v i r u s i n c e l l c u l tu r e. 31. P l a q u e - pu r i f i c a t i on o f v i ru s i s o la te s. 33. 3 2 P - l ab e l l ini o f o r f v i r u s DNA. 34. E f f e c t o f c y c l oh e xam ide o n o r f CPE. 36. C o v e r s l ip p r e p a r a t i on s. 36.

(7) vi. TABLE OF CONTENTS. PAGE 38. C h a p t e r Thr e e - Re s u l t s V i ru s pur i f i c at i o n. 38. Par t icle coun t i ng. 38. R e s t r i ctio n e ndonuc l e a s e ana lys i s. 38. Growth i n c e l l cu l tu r e. 40. Cytopa thic effe c t c e l l c u l tu r e. ( CP E ). in 42. P la q u e - purifi c a t ion and t i t r a t i o n of the v i r u s. 47. Exam i na t i on of May-Grunw a l dG i emsa s t a i n e d prepar a�ions. 47. Effe c t of c y c l ohexamide o n orf CPE. 48. Chap ter Four. - D i s cu s s i on. 48. Chapter F i ve. - Summ ary. 56. Appe n d i x I. - Li s t of Mate r i a l s. 58. Appendix I I. - L i s t of Equ i pment and App a r a t u s. 61. A pp e n d i x I I I. - Compo s i t i o n of Buffe r s and So l u t i o n s. 63. B ib l i og r a phy. 68.

(8) vii. LIST OF FIGURES AFTER P AGE. FIGURE l. 2. 3. 4. 5. S o d ium me t r i z o a t e ( l e f t ) and Na d i a t r i z o a t e ( r igh t ) grad i e n t s c on ta in ing v i r u s b and s . A m inor b and \vh i ch i s mo re d i s t ine t in Na m e t r i z o a t e i s s e en b e low the ma i n b and .. 38. A s p r ay drop l e t c o n ta i n i n g o r £ v 1 ru s p a r t i c l e s an d l a t e x b e a d s .. 38. Agar o s e g e l e l e c t r o p h ore s i s o f t h i r t e e n o r f v i ru s DNAs d i ge s t e d w i th E c oR r e s t r i c t i on 1 e n d onu c l e a s e . E l e c tro phor e s i s wa s in a 0 . 7 % a ga r o s e s l ab g el f o r 4 hou r s a t 8 0 V .. 38. Agar o s e ge l e l e ct r o p h o r es i s of i s o l a t e N o . 2. E l e c t r o ph o r e s i s wa s in a 0 . 7 % a ga r o s e s l ab g e l f o r 4 hours a t 8 0V . ( A ) Lane s c on t a in v i ru s DNA s d e r ived f r om o r i g ina l sc a b l e s i o n s d ig es t e d w i t h E c oR ( 1 ) , 1 B arn H-1(2) and H in d III(3). (B) L an e s show an au t o r a d iogra ph o f th e s ame g e l c o n t a in ing c o rre s p o n d i n g 3 2 p - l ab e l le d DNA d i ges t s d e r i v e d f r om a p l aquep ur i f i e d v i ru s p a s s ag e d 23 t im e s in LT c e l l s . Unino c u l a t e d c on t r o l l amb t e s t i s mono l ay e r . 134 .. X. 40. 46. 6. L amb t e s t i s mono l aye r , 2_lz h ou r s a f t e r i n fe c t ion w i th o r f n o . 1 0 P 1 0 s h owing e a r l y c e l l r ound ing . X 134 .. 46. 7. A mo r e genera l i s e d c e l l round i n g s e e n 5 h ou r s a f t e r in f e c t i on , l amb t e s t i s c e l l s . X 134 .. 46. 8a. LT , 48 hou r s p o s t - in fe c t i on . Mo s t c e l l s h ave d e t a c h e d f r om t h e f l a sk. X 134 .. 46. 8b. LT , 4 8 hou r s p . i . s h ow ing c l ump o f roun d e d up c e l l s . N o t e tha t s ome c e l l s h av e b e come en l a r g e d and e p i th e l i o i d . xsoo.. 46. 9. LT mon o l ayer i n fe c t e d w i th s c ab - d e r i v e d v i ru s i s o l a t e N o . 1 9 P 4 . No t e th a t s ome ce l l s have nume r o u s t iny c e l l u l ar p r o c e s s e s . xsoo.. 46.

(9) viii. AFTER PAGE. F I GURE 10 ll. Un i no cu l a t e d c on t r o l f o e t a l bov ine l un g mono l ay e r . ( FBL ) . Xl34 . FBL, 5 h ou rs a f t e r i n fe c t i o n w i th s c a b ­ d e r iv e d i s o l a t e N o . l2 P 5 , show i n g rounded c e l l s w i th t iny p r o c e s s e s . X 5 00 .. 46 46. l 2 a FBL , 2 4 h ou r s a f t e r i nfe c t i on . Note tha t th e r e i s l e s s c l ump i n g o f roun d e d c e l l s and c e l l s tha t r ema i n e d a t t a c h e d t o t h e f l a sk b e c ome s p in d l e -sha p e d . X 1 3 4 .. 46. l2b FBL , 2 4 hou r s p o s t - i n f e c t ion s h owing c l ump i n g o f r oun d e d up c e l l s . X 500 .. 46. 1 3 a Open t y p e o f p l aqu e i n LT mono l aye r s ev e n d a y s p o s t - inocu l a t i o n w i th i s o l a t e No . 1 0 ( s e c on d p l a que-pur i f i c a t ion) . X 1 34 .. 47. 1 3 b C l o s e d t y p e o f p l aque in LT mon o l aye r s even day s a f t e r i n o c u l a t i o n w i th i s o l a t e N o . 1 2 P l l ( f i r s t p l aqu e-pu r i f i ca t i o n ) X 1 34 .. 47. 14. 55. Un ino c u l a t e d con t ro l LT mon o l ayer . Grunwa l d- G i ems a , X 1 2 5 .. May­. l 5 a LT , one ho ur p o s t- ino cu l a t i on w i th No . l OP l O . May-Grunwa l d- G i ems a , X 1 2 5 .. 55. 1 5 b LT , o n e ho ur po s t - i n o cu l a t i on a t h i gh e r magn i f i c a t i on . May-G runwa l d-G i em s a , X 3 1 0 .. 55. 16. 55. L T , t h r e e h ou r s a f t e r ino cu l a t i on , s h o w i n g t h e a ppe aranc e o f a f ew rounded up c e l l s . May-Grunwa l d-G i ems a , X 3 1 0 .. l 7 a LT , 6 h ou r s p o s t - ino c u l a t i on . N o t e th a t m o s t o f t he ce l l s h av e roun d e d up and s ta ined mo r e in t en s e l y than the c on t r o l . May-Gr unwa l d-G i em s a , X 1 25 .. 55. l 7 b H i gh e r magn i f i c a t i o n o f the ab ove . X 3 1 0 .. 55. l 8 a L T , 24 h ou r s a f t e r i n fe c t i on , c lump ing o f round e d u p c e l l s . G i em s a , X 1 2 5 .. 55. s h owing May-Grunwa l d -. l 8b H i ghe r magn i f i c a t i on o f the above . X 3 1 0 .. 55. LT,. 19. I t c an b e 2 4 h our s a f t e r i n fe c t i on . s e en t h a t s ome c e l l s c o n t a in p a ranuc l e a r i n c l u s i on b o d i e s s u r r ound e d b y a "ha l o" May-G runwa l d- G i em s a, X 3 1 0 .. 20. LT , 2 4 h ou r s p . i . s h ow ing an e n l a r g e d c e l l ( X ) c on t a in ing s ev e r a l nuc l e i ( a sync y t ium? ) May-Grunwa l d- G i em s a , X 3 1 0.. 55. 55.

(10) INTRODUCTION. Or f. i s common amon g s he ep i n New Z e a l and a n d the i nc i d e n c e. i n m a n a ppears to be incre a s i n g . ava i la b l e for sheep .. Th ere i s a v a c c i ne. A l though the v a c c i n e prote c t s l amb s. from t h e d i s e a s e i t perpe tu ate s the v iru s i n the· e nv i ronme n t .. There i s a n e ed for a l tern a t i v e vacci n a t ion. m e t hod s .. I t i s the a im of t h i s project to deve lop me thods o f i s o l a t i ng ,. prop a g a t i n g a n d pur i f y i n g orf v i r u s i n c e l l. cu l t ure an d i d e nt i f y i n g i s o l a t e s by re s tri c t i on e ndonu c l e a s e a na l y s i s i n prepara t i o n for v a c c i ne s tudi e s ..

(11) 1.. CHAPTER. ONE. REVIEW OF LITERATURE. CONTAG IOUS E C THYMA IN SHEEP , GOAT S AND HAN C on t a g i o u s e c thyma � s a p o xv i ru s d i s e a s e o f s h e e p , goat s and man.. It i s a l s o known by o th e r n ame s .. In s h eep , In En g l an d ,. i t 1 s c a l l e d c o n t a g i o u s p u s tu l a r d e rmat i t i s ( G l ov e r , 1n S c o t l an d , o r f ( Ma r t i n- S co t t , 1 9 5 5. 1 928) ;. a s c i t e d b y Tru eblo o d. and Chow , 1 9 6 3 ) f r om t h e Saxon f o r c a t t l e ( H o d g s on-Jone s , 1 9? 1 ) .. I t i s c omm on l y knmvn a s s c abb y mouth i n Au s t r a l i a. ( Ohman,. 1 94 1 ;. S u th e r l an d and Mou l e , undat e d ) wh i l e in the. Un i t e d S t at e s , i t is c a l l e d s o r emou th , i n f e c t i ou s d e rma t i t i s o r c ont a giou s ve s i cu lo-pu s t u l a r derma t i t i s ( S chm idt a nd Hardy ,. 1 9 3 2;. Trueb l o o d and Ch ow, 1 9 6 3 ) .. I n New Z e a l and ,. the. d i sea s e i s known as s c ab b y mou th to the fa rme r and e cth yma c on t a g i o s um or c o nt a g i o u s pu s tu l ar d e rmat i t i s t o v e t e r in a r i ans ( Pu r dy , 1 9 5 5 ) .. In human me d i c ine , i t i s mo r e p o pu l a r l y. r e f e r r e d t o a s o r f ( Pu r d y ,. 1955 ;. B e ck and T ay l o r , 1 9 7 4 ) .. Th e d i s e a s e i s p r eva l en t i n s h e e p-r a i s in g c oun t r i e s .. It. o c c u r s at any t ime o f th e y e a r but i s mo r e c on�on d u r i n g s p r i n g a n d s umm e r ma i n l y amon g l amb s a n d k i d s S chm i dt and Hardy , 1 9 3 2 ) .. ( G l ov e r , 1 9 2 8 ;. O l de r anima l s a r e mo r e r e s i s t a n t. d u e t o immun i ty f r om e i th e r v a c c ina t i on or p a s t i n f e ct i on ( B rune r and G i l l e s p i e ,. 1 973 ) .. Morb i d i t y c a n b e v e ry h i gh. a p p r o a ch in g 1 0 0 % ( S chmi d t and H a r d y ,. 1 9 3 2 ) b u t mo r t a l i ty. r a t e i n unc omp l i c a t e d c a s e s r a r e l y exc e e d s o n e p e r c en t ( N a t i on a l V e t . Me d . Pub l . , 195 1 ) .. 1 9 4 4 a s c i t e d b y H o d g s o n-Jone s ,. W i th s e c on d a r y c omp l i c at i on s , mor t a l i t y r a t e may. r a n g e f r om 2 0 - 5 0 % ( Aynaud , b y H owa r th ,. 1 92 9 ) .. 1 9 23 and J ac o to t , 1 9 2 4 as c i t e d. L o s s e s f r om s c ab b y mou th i n l amb s a r e. d u e t o t he l e s i on s o n t h e l i ps and mou t h wh i ch i n t e r f e r e w i th f e e d in g , o r r e fu s a l o f ewe s w i th i n f e c t e d t e a t s t o nu r s e th e i r y oun g , s ub s e q u en t ly a f f e c t ing g ro w th r a t e s ( Howa r th , 1 9 2 9;. Brun e r a n d G i l l e s p i e,. 1973) .. D e a th s a r e.

(12) 2.. u s u a l l y due t o c omp l i c a t i o n s c a u s e d b y , for i n s t an c e , i nv a s i on o f the l e s i o n s b y t h e s c r ewwo rm f l y a.mvL..<.c.a.na.. the. Coc.hR...<.omy..<.a.. ( Bou gh t o n and H a r d y , 1 9 3 5 ) o r t h e n e c r oba c i l l u s. SphvwphoJtM ne.c..�tophoJtM ( Ne'ivs om and Cro s s ,. 1 9 3 1 and 1 9 3 4 a ) .. A c a s e o f s c abby mou th a s s o c i a t e d wi t h Ve.Jtma-tophiltu., c.ongoR.e.M-i.-6 h a s b e en r e p o r t e d in New Z e a l and (Co o p e r e t a l . , 1 9 7 0 ) .. Th e. au thors a l s o c i t e d a c a s e o f s e c on d a r y c omp l i c a t i on s w i th S t r e p t o t r i ch o s i s among s h e e p in Keny a . Th e v 1 r u s e n t e r s the h o s t t hrough a b r a s ions o n the s k in o f t h e l i p s and fa c e .. S u c h ab r a s i o n s a r e c au s e d b y t h i s t l e s ,. h a r d s tubb l e s or p r i ck l e s o f r e d-bu r r o r s im i l a r p l an t s . Th e v i r u s c an a l s o b e t r ansmi t t e d t o t h e u d d e r o f ewe s b y s u c k l i n g l amb s .. The i n c ub a t i o n p e r i o d o f t h e d i s e a s e 1 s. a b o u t 7 2 h o u r s ( S u th e r l and and Mou l � und a t e d ) . Th e v 1 r u s 1 s h i gh l y e p i th e l i o t r o p i c ( B e c k and Tay l o r ,. 1974). and t h e l e s ion s p r o du c e d a r e ubu a l l y s e en o n t h e n o s t r i l s a n d c omm i s ure s o f the l i p s .. In mor.e s e r i o u s c a s e s i t may. e x t end t o o th e r p a r t s o f the b od y ,. f o r examp l e the th i gh ,. a x i l l a , c o r one t , vu lv a o r c or nea ( G l over , 1 9 2 8; 1 9 2 9;. S chm i d t and H a r d y ,. gum s ( G l ove r , 1 9 2 8;. 1932);. Howa r th ,. Howa r th ,. r o o f o f t h e mou th ,. 1 9 2 9 ; Va l d er e t a l . ,. v i s c e r a l o r gans ( News o n a n d Cro s s ,. 1931) .. t on gu e ,. 19 7 9 ) ;. Th e l a s t men t i on e d. c omp l i c a t i o n s w e r e du e t o Ac.tirto1nyc.e.J.> (Sphe.JtophoJtM) ne.c..�tophoJtM. A gene r a l i s e d c a s e o f t h e d i s ea s e w a s r e po r t e d among s h e e p i n Ken t b y Darb y s h i r e ( 1 96 1 ) .. The i n f e c t i on p e r s i s t e d in. t h e a c u t e f o rm f o r two mon th s invo l v in g t h e g a s t r o - in t e s t i n a l t ra c t a n d l e s i o n s e x t en d i n g t o t h e l u n g s a n d hea r t . a v e r a g e mo r ta li t y r a t e o f 1 8 % wa s r e c or d e d .. An. Bac t e r i a l. o r gan i sms w e r e i s o l a t e d b u t none w a s Sphe.JtophoJtUJ.J ne.c.JtophoJtU-6. A s e r 1 o u s ou tb r e ak o c cu r r e d in Ge rmany a f f e c t in g b o th l amb s and s h e e p .. H i gh mor t a l i t y wa s no t e d among t h e youn g .. In. a d d i t i on t o fo o t and g en i t a l l e s i o n s , d e e p u l c er a t ive s t oma t i t i s , ph a r yn g i t i s and o e s o phag i t i s w e r e ob s erved . Many o f t h e a f f e c t e d l amb s d e ve l o p e d l amen e s s and l o s t t h e i r.

(13) 3.. h ooves ( Va l der e t a l . , 1 9 7 9 ) .. No secondary in fe c t i on wa s. men t i o ned. G ro s s l y , t he l e s i on s in s heep s t a r t a s d i s c r e t e , red dened swe l l in g s on t he l i p s f o l l owed by p a pu l e s , ve s i c l e s , pu s t u l e s and u l cer f o rma t i on in th ree t o f o u r d a ys .. In. unc omp l i c a ted c a ses , the d i se a s e i s s e l f- l im i t in g .. Scab. fo rma t i on o c c u r s wi t h i n one week and pee l s o f f t he s k in w i th in th ree t o four week s , Howa r th ,. 1 9 2 9;. leav i n g no s c a r ( G l over ,. Schm i d t and H a r d y ,. Mou l e , unda ted ) .. Some t ime s. 1 9 3 2;. 1 9 2 8;. Su t he r l an d and. c on t inued p ro l i fe r a t i on o f the. ep i thel ium o c c u r s and a den s e w a r t-l ike ou t gro w ths a p pea r , res emb l in g p ap i l l oma ( G l over ,. 1 928 ;. Se l b ie ,. ma l i gn an t f o rm rep o r ted b y V a l de r e t a l . ,. W i th secondary. les i o n s bec ome u l c e r a t i ve and ne c r o t i c w i thou t. s c ab f orma t i on ( New s on and Cro s s , 1 9 3 1 ;. D a r b y s h i re ,. The mi c r o s c o p i c a p pea r an c e o f s k in l e s ion s G l over ( 1 9 2 8 ). The. ( 1 9 7 9 ) p r o du ced. c au l i f l ower- l ike g rm..; t hs in the o r a l mu c o s a . c omp l i c a t i on s1. 1 944) .. 1 9 6 1) .. w a s des c r i bed b y. f o l l owi n g a r t i f i c i a l ino cu l a t i o n o f l amb s .. He d iv i ded the d i se a s e p r o ce s s i n t o three s t a ge s .. The f i r s t. s ta ge , o r p a pu l o-ve s i c l e s t a ge , wa s chara c te r i sed b y p r o l i fe r a t i o n o f the c e l l s o f the re te Ma l p i gh i .. The. ve s i c le s a r o s e be tween the more s uper f i c i a l c e l l s imme d i a te l y beneath the s t ra t um l u c i dum a n d were repre sen ted b y c o l lec t i o n s o f p o l ymo r ph o nu c lear leu c o c y te s . s t a ge , o r ve s i ca-pu s t u le ,. I n the s e c ond. there wa s fur the r de gene r a t ion o f. c e l l s f ormi n g an i r regu l ar ne two rk resemb l in g the " b a l l on i s an te " s ta ge o f va c c in i a .. The ve s i c le s bec ame. en l a r ge d an d deve l o ped i n t o p u s t u le s and f in a l l y rup t u re d t h r ou gh t h e s t r a tum l u c i dum .. C r u s t s then f o rmed f r om. c e l lu l ar deb r i s , f i b r i n and p o r tion s o f the s t r a t um l u c i dum l e a d i n g to the th i rd s t a ge , s c ab f o rma t i o n . c ru s t ,. Bene a th t he. the ep i thel ium regene r a ted and hea l in g o c c u r red .. A more de t a i led de s c r i p t i o n 1-v a s g iven b y Whee ler and C a\vley ( 1 9 5 6 ) a l s o ba sed on exper imen t a l inocu l a t i o n o f s heep . There wa s n o ev i den t ch ange on the four t h d a y p o s t - i n o c u l a t i o n ..

(14) 4.. The e a r l i e s t chan g e s w e r e " b a l l oon ing " d e gene r a t i on o f t h e u ppermo s t l ay e r o f pri ck l e c e l l s s e en on th e 4 th a n d 6 th days a n d wa s a lmo s t c omp l e t e b y the 7 th and 8 th d a y s .. Th e. r ema i n i n g c e l l wa l l s p r o du c e d a " b a ske t -weav e " a p p e a r an c e . This r e s u l t e d i n mu l t i l oc u l a t e d s uperf i c i a l l y p l a c e d v es i c l e s a nd p u s t u l e s .. B e t\¥ e e n the 1 1 th and 1 7 th d a y s ,. there. was ma r k e d p s eu d o - e p i th e l i oma t ou s hype r p l a s i a a n d granu l oma f orma t i on wh i ch l e d t o a p ap i l l oma pha s e .. Th i s f e a t u r e wa s. a lso d e s cr i b e d b y Se l b i e ( 1 944 ) i n na t u r a l ly o c cu r r in g d is e a s e i n l amb s , b u t \va s n o t d e s c r ib e d b y G l ov e r ( 1 9 2 8 ) . B etwe e n th e 2 2n d and 40 th d a y s, l e s i o n s gr adua l l y invo l u t e d . The p ap i l l oma a p p e a r an c e d i s a p p e a r e d , d e rma l i n f i l t r a t e s gradu a l ly r e s o l ve d , and r e ge n e r a t i on o c cur r e d .. The r e w a s. only s l i gh t c onn e c t iv e t i s s u e pr o l i fera t i on and thu s t h e h ea l e d l e s i o n s d i d no t l ea v e s c a r s .. In c l u s i o n b o d i e s w e r e. n ot d e s c ri b e d in t he s e exp e r imen t s b u t wer e d e s c r i b e d b y Abdu s s a l am ( 1 9 5 7 a ) .. He d e s c r ib e d i nc l u s i on b o d i e s th a t. m ea sur e d 4 t o 8 m i c r on s i n d i ame t e r and ap p e a r e d in t h e c yto p l a s m o f e p i d e rma l ce l l s .. Th e ma i n m i c r o s c o p i c f e a tu r e. h e s aw w a s p r o l i f e r a t io n and re t i cu l ar de gene r a t ion o f e p i d e rma l c e l l s l e ad i n g t o n e c r o s i s .. Th i s w a s a c c ompan i e d. b y t h e i n f i l t r a t i o n o f t h e cor ium w i t h po l ymo r ph onu c l e ar a n d mononu c l ear in f l amma t o r y c e l l s .. I n c on t r a s t t o o th e r. au t h o r s , he d i d n o t o b s e r v e "ba l l oon ing" d e g e n e r a t i on , bu t h e d id o b s erve t he mu l t i l o cu l ar f e a t u r e of v e s i c l e s . Th e gene r a l i s e d c a s e s r e p o r t e d b y Darbysh i r e ( 1 9 6 1 ). s h owe d. c a r d i a c , pu lmonary and g a s t r o - in t e s t in a l i nvo l v eme n t .. Th i s. i n c l u d ed myo c a r d i a l h a emo r rh a g e s , a c u t e myo c ar d i t i s , h aemo r rh a g i c pneumon i a w i t h ne c r o t i c f o c i i n t h e a p i c a l l ob e s o f the lungs , t r a c h e i t i s , gene r a l i s e d d e gene r a t i on o f t h e l iv er and in f l amma t o ry chan g e s in t h e ga s tr o ­ i n t e s t in a l t r a c t . c omp l i c a t i ons .. The s e l e s ions w e r e ma i n l y d u e t o s e cond a ry.

(15) 5.. As d i s c u s s e d b r iE· f l y above , o r f i s a l s o a z o o no s � s . Human o r f u su a l l y fo l l ow s c o n t a c t wi th in fe c t e d s h e e p (Ho d g s on-Jone s ,. 1 9 5 1 ) a n d i s t hu s a n o c cupa t i on a l di s e a s e. o f f a rme r s , s h e e p shear e r s , ve t er i n a r i an s ( Pu r dy ,. f r e ez i n g wo rk e r s and o c c a s io na l l y. 195 5 ) .. It h a s , howev e r , b e en s e en in. p e op l e no t d i r e c t l y invo lve d with h and l i ng s h e e p and goa t s ( K ew i s h ,. 1951 ) .. Th e s e in f e c t i o n s p r obab l y a r o s e from f omi t e s s u ch a s s h e epya r d r a i l s , w o o l h o oks and c l o th ing . No man t o man t r ansm i s s i on h a s b e en r e p o r t e d ( M o o r e , Lea v e l l ,. 1 9 7 3;. 1 9 6 8 ) bu t the r e i s n o th e o r e t i c a l r e a s on why i t. c ou l d n o t occ u r .. P r e s en c e o f woun d s and ab r a s i o n s on the. a rms a nd h an d s in c r e a s e s r i s k o f i n f e c t ion ( P a s k e t a1 . , 1 9 5 1 a s c i ted by P l a t t ,. 1958) .. The d i s e a s e i n man h a s b e en rep o r t e d and d e s c r ib e d in d e t a i l b y many wo rk ers (New s om and C r o ss , 1 9 34b; 1 9 5 1;. Pu r dy ,. Wh i t t l� ,. 1 95 5;. 1 96 1 ) .. 1 9 5 1;. N a g i n g t on and. Th e i n c ub a t i on p e r i o d ran g e s f r om th ree. d a y s ( C a rne e t a l . , 1 95 1 ). Hod g s on-Jon e s ,. Mu ir ,. 1 94 6;. Ho d g s on-Jone s ,. 1 95 1 ;. t o s ev en d a y s ( H o d g s o n-Jone s , 1 9 51;. Muir ,. Mu i r ,. 1 95 1 ) .. I t o c c u r s mo s t c ommon l y a s a s in g l e l e s ion o n t h e a rm o r h and .. Le s ion s app e a r a s d i s c r e t e , ra i s e d , c i r c u l a r ,. p u r p l i sh-r e d p a pu l e s ( C a r n e e t a l . , 1 9 4 6 ) ab ou t 1 - 3 cm . �n d i ame t e r � a rne e t a l . , 194 6; Ho d g s on- Jone s , 1 9 5 1 ). O f t en , the c e n t r e b e c ome s u l c e r a t e d and s u r r ound e d b y d e a d , whi t e " s o g g y " s k in wh i c h g iv e s a g r anu l oma- l i ke a p p e a ran c e ( Ho d g s on-Jone s , 1 9 5 1 ) and i s s ome t ime s d e s c r i b e d a s " c ig a r e t t e b u r n " l e s i on .. In th e e a r l y s t a g e s ,. the l e s i on. i s p a in l e s s b u t l a t e r b e c ome s s e n s i t ive t o t o u c h and i n un c omp l i c a t e d c a s e s h a s r e s o l v e d 5 - 8 w e e ks a f t e r i n f e c t i o n ( Ho d g s on-Jone s ,. 1 95 1 ) .. A mo r e s eve r e f o rm o f t h e d i s e a s e may s ome t ime s o c cu r . Kew i s h ( 1 9 5 1 ) r e p o r t e d me t a s t a t i c l e s i o n s on th e fa c e , a rm s and t runk a c c omp an i e d b y a c u t e f e b r i l e r e a c t i o n s ( S t e v e n s - John s t on Syn d r om e ? ) . A s im i l a r c a s e wa s r e po r t e d b y M o o r e ( 1 9 7 3 ) and E r i c k s on e t a l .. ( 1975) .. B l akemo r e. e t a l . , ( 1 9 48 a s c i t e d b y P l a t t , 1 9 5 8 ) d e s c r ib e d an e r y t h ema-mu l t i f o rme t y p e o f r e a c t i on . An unu s u a l c omp l i c a t i on w a s r ep o r t e d b y Ro y e r e t a l . ( 1 9 7 0 ) . E x t e n s i v e.

(16) 6.. ve s i cu l a- p u s tu l a r f o rearm s , h and s ,. eruption s were seen on the p a t ien t ' s. fee t and l e g s .. There wa s s we l l ing o f. reg i ona l l ymph g l and s , and t h r omb os i s o f the ven t r a l re t in a l ve i n o c c u r red wh i ch re s u l ted i n permanen t b l indne s s o f the r i gh t eye .. These c omp l i c a t i on s are , h owever , r a re .. There a re on l y a few rep o r t s o f h i s t o l o g i c a l exam ina t i on o f l e s i on s in man .. Whee l e r and Caw ley ( 1 9 5 6 ) des c r ibed. "ba l l o onin g " de gene r a t ion o f the p r i ck le cel l l ayer in the e a r l y s tages o f the d i se a s e wh i ch led t o ve s i cu l a t i on and s ome t imes changes a s s o c i a ted wi th a chron i c gr anu l oma . The m i cro s c o p i c p i c t u re wa s c on s i s ten t w i th the appearance o f t he g r o s s les i on s �¥h i ch are o f ten mu 1 t i l o cu l a ted ve s i c le s and a t o ther t ime s re semb le pyo gen i c g r anu l omu ( Whee ler and C a�¥ley , ( B l akemore e t a l . ,. 1956).. I nc l u s i o n s ma y be seen. 1 9 48 , a s c i ted b y Wheel e r and Cmv ley ,. 1956).. ORF IN OTHE R HOSTS E XPERIMENTAL ANIMALS. Al t h o u gh o r f i s reg a rded a s a d i s e a se p r ima r i l y o f d ome s t i c s heep and go a t s ,. i t h a s a l s o been rep o r ted a s o c cu r r i n g. na t u r a l l y i n o ther a n ima l s .. The d i sea se h a s been rep o r ted. in the mu s k ox ( Kummenej e and Kr o g s rud , 1 9 7 8 ) , reindeer s ( Kumn1ene j e and Kr o g s ru d ,. 1 9 7 9 ) , and a l s o i n b i gh o rn s heep ,. c h amo i s and th a r s ( Dav i s e t a l . , 1 9 7 0 a s c i ted b y Beck and T ay l o r ,. 1 9 74 ) .. The se i n fe c t i on s were bel ieved to be o f. o v i ne o r c a p r ine in o r ig i n .. There i s a l s o an unu s ua l c a se. in d o g s rep o r ted b y W i l k in s on e t a l . ,. ( 1 9 70 ) .. The d o g s. c ou l d h ave been in fec ted a f te r being fed w i th un s k i nned s heep c a r c a s se s . I n the mu sk ox , l e s i on s were chara c te r i sed b y c a u l i f l ower­ l ike p ap i l l omas on the l i p s , mu z z le , neck , eye l i d s , ches t and per i an a l reg i on .. Gro s s l y a · n d h i s t o p a t h o l o g i c a l l y ,. the se p ap i l l oma s resemb l e d c ommon \va r t s ( Kummene j e and Kro g s r u d ,. 1978).. The l e s i on s seen in the d o g s were. e s sen t i a l l y the s ame a s t h o s e found in s heep .. The.

(17) 7.. p r e d e l i c t i on s ite wa s a r ound th e h e a d .. Th e l e s i o n s took. th e f o rm o f c i r c u l a r a r e a s o f a c ute mo i s t d e rmat it i s w ith u l c e rat i o n and s c ab fo rmat i on .. H i st o l o g i c a l l y th e r e wa s. "b a l l o on i n g " d e g ene rat i on o f th e p r i ck l e c e l l l a y e r g iv in g r i s e to sma l l a r e a s o f a c anth o l y s i s a n d intra - e p ide rma l v e s i c l e f o rmat i on .. In s ome a r e a s , th e r e wa s r et i c u lar. d e gene r at i on o f the p r i c k l e c e l l s with sub - c ornea l ve s i c l e fo rmat i on .. I n s ome f o c i , th e s e d e generated c e l l s h a d b e en. i n f i ltr at e d wi th p o l ymo rphonu c l e a r l eu c o cyte g i v i n g r i s e to a s p on g i f o rm pus tu l e .. Th e ' b a s k et-\ve ave " a p p e a r a n c e du e to. v a c uo 1 i s at i on o f the c e l l s o f th e s t r atum c o rneum wa s ob s e rve d in s ome a r e a s . e p i d e rm i s .. Th e r e w a s u l c e r at i on and l o s s o f. H eavy neutr o ph il i c i n f i l tration i n th e u p p e r. d e rm i s a n d p u stu l e s with i n s ome o f the h a i r f o l l i c l e s wa s a l s o s een . Th e s ituat i on a s r e ga r d s c att l e i s l e s s un c e rta i n .. Or f i s. not s e e n i n c att l e that a r e run w ith i n f e ct e d s h e e p , and Howa rth ( 1 9 2 9 ). f a i l e d to r e p r o d u c e the d i s e a s e in a cow .. Howev e r , Bennet et a l . ,. ( 1 9 4 4 ) r e p o rte d s u s c e pt i b i l ity o f. c a lv e s t o exp e r imenta l i n f e c t i o n . Many attempt s have b e en ma d e to exp e r imenta l l y in f e ct oth e r an ima l s .. Th e s e exp e r iment s h av e p r o d u c e d var i ab l e r e s u lt s .. G l over ( 1 9 2 8 ) , Newsom and C r o s s ( 1 9 3 4 a ) , B ou ghton and H a r d y ( 1 9 3 5 ) , Gre i g ( 1 9 5 6 ) , P l owr i ght et a l .. ( 1 9 5 9 ) and. s eve r a l oth e r s c ited b y Ab du s s a l am ( 1 9 5 7 b ) f a i l e d to in f e ct rabb it s .. Ab du s s a l am , h oweve r ,. c l a imed s u c c e s s fu l. ino c u l at i on o f r ab b it s b y s c a r i f y i n g the s k in with ma s s �ve d o s e o f the v i ru s , and D a rb y s h i r e ( 1 9 6 1 ) p r o du c e d l e s i o n s i n th e rabb it b y mu l t i p l e i n j e c t i o n s o f 0 . 1 m l o f v i ru s i n o cu l um .. Se l b i e ( 1 9 4 4 ) and Hh e e ler and C aw l e y ( 1 9 5 6 ). a l s o d e s c r ib e d l e s i o n s in r ab b i t s a rt i f i c i a l l y i n f e ct e d w ith o r f v i ru s .. Se l b i e ( 1 9 4 4 ) a t th e t ime o f w r i t i n g. c l a imed the v i r u s h a d u nde r g o n e 1 9 p a s s a g e s i n r ab b its ..

(18) 8.. Th e i n f e c t i on i n rabb� t s w a s r e p o r t e d a s a m i l d d i s o r d e r . Wh e e l e r and Caw l ey ( 1 9 5 6 ) ob s e r v e d an in cub a tion p e r i o d o f s even t o n in e d ay s wh i l e S e lb i e ( 1 9 4 4 ) r epo r t e d 2 0 d a y s wh i ch vJa s r e d u c e d to s even d a y s o n t h e f i fth p a s s a g e .. Ab du s s a 1 am ( 1 95 7 b ) s aH f i r s t s ign of infec t i on on t h e 4 th and 5 th d a y s .. Th e f i r s t ind ica t i on o f· i n fe c t i o n wa s s e en. a s t iny ( 1 - 2 mm), e r y th ema tou s ma c u l o p apu l e s and th e d i s e a s e ra n a c ou r s e o f th r e e t o five d a y s (Hh e e 1 e r and Cawl ey , 1956) .. Ab du s s a l am ( 1 9 5 7 b ) o b s e r v e d e ry thema and p a pu l a t i on. o n the 4 th d a y .. Th e p a pu l a t i o n b e c am e mo r e mark e d and. s h owed a we l l de f i n e d m a r g i n o f c on g e s t ion on the b o r d e r o f th e l e s i on .. On the 5 th day ,. t h e papu l e s b e c ame c o v e r e d. w i th a d u l l , y e l l o�.;r i sh-wh i t e s c a l y s c ab .. In c on f l uen t. l e s i on s , c e n t r a l p a r t s c on t a i n e d a s e rous o r h a emo r rha gi c e xu d a t e \.vh i ch wa s ab s en t in d i s c r e t e l e s io ns . o f f on t h e 6 th day . days .. Scab s p e e l e d. Th e d i s e a s e ran a cou r s e o f ab out t en. S e l b i e ( 1 9 44 ) ob s e rv e d e s s en t i a l ly t h e s ame l e s i o n s. b u t gave emph a s i s t o th e a p p e ara n c e o f s c a l in e s s o f t h e l e s i on .. Th e ma in m i c r o s c o p i c f e a tu r e o f t h e in f e c t i on �n. r abb i t s \va s mi l d c hron i c in f l amma t o ry chan g e s and l a ck o f the "b a l l o on ing • • d e g e ne r a t i on a n d t h e granu l oma t o u s and p r o l i fe r a t iv e char a c t e r o f the d i s e a s e in s h e ep ( S e l b i e , 1 9 44 ;. Whee l e r and Caw l e y ,. 1 95 6 ;. Ab du s s a l am ,. 1 9 5 7a ) .. A. r e t i cu l a r t y p e o f d e g e n e r a t i o n o f t h e ep i d e rma l c e l l s , a s in sheep ,. ( Ab du s s a l am ,. 1 9 5 7 a ) �a s o b s e rved b u t no. c y t op l a sm i c or nu c l e a r i n c l u s i o n s w e r e s e en . Un l ik e t h e o r thopoxv i ru s e s , wh i ch r e ad i l y g r ow. on ch i ck. emb ryo s , o r f v i r u s d o e s n o t s e em t o b e e a s i l y p r o p a ga t e d in e g g s ( News om and C r o s s ,. 1 9 34a ;. 1958 ;. C on t r a r y t o th i s ,. D a r b y sh i r e ,. 1961) .. Greig ,. 1956 ;. Web s t e r ,. Ab d u s s a l am. ( 1 9 5 7 b ) r e p o r t e d a s u c c e s s fu l a t t emp t , bu t l e s i o n s r e gr es s e d a n d eve n tu a l l y d i s ap p e a r e d a f t e r t h e f o u r th p a s s ag e .. G rowth and mu l t i p l i c a t i on i n t h e c h o r i o a l l an t o i c · memb ra n e o f deve l o p in g emb r yo s wa s r e po r t e d ( S awhn e y , 1 9 6 6 a a s c i t e d b y B e c k and Tay l o� 1 974 ) and l a t e r d emon s t r a t e d b y e l e c t r on micro s c opy ( S awhn e y an d S p a s ova ,. 1973)..

(19) 9.. M i c e and �u �nea p � gs we r e f ound r e fra c t o ry t o in f e c tion . ( Howa r th ,. 1929 ;. Ab du s s a 1 am ,. News om and C r o s s , 1 9 3 4a ;. 1 9 5 7b ;. D a r b y sh i r e ,. 1 96 1 ) .. Greig , 1 9 5 6 ;. New s om and C r o s s. ( 1 9 3 4 a ) a l s o f ai l e d t o in fe c t pig s . In summa r y ,. i t i s e v i d e n t that s h e e p and goa t s r em a � n the. mo s t s u s c e p tib l e and the mo s t sui t ab l e anima l s f o r e x p e rimen t a l pu rp o s e s .. Howev e r , r abbi t s c ou l d b e u s e d t o. r a i s e n eu t r a l i s in g an tib o d i e s a gains t o r f v i ru s b y s t an d a r d me tho d s ( Ab du s s a l am , 1 9 5 7 b ) .. S CABBY HOUTH IN NEhT ZEALAND Al though the dis e a s e i s r e ga rde d a s c omm on �n thi s c oun t ry , the r e i s n o pub l i she d d a t a t o show the e x t en t o f the p r ob l em .. The inc i d e n c e in she e p and the e c onomi c l o s s e s. incu r r e d by the in fec tion have n o t b e en d e t e rmine d . The r e i s , howeve r , s ome d a t a. en. the p r eva l en c e of s c abby. mou th a f fe c t e d l amb s b e ing p r e s en t e d f o r s l au gh t e r a t New Z e a l an d f r e e z ing works .. B e l ow a r e the r e s u l t s o f a. pi l o t s u rv ey d one in two No r th I s l and s and one S ou th I s l and wo rk s .. The s e fi gure s h a v e b e en d e rive d f r om the. p r o du c t o f the av erage line s i z e and the ave r a ge numb e r o f a f fec t e d anima l s in a f f e c t e d l in e s ( Robin s on , Mon th. Au c k l and. L ongb u rn. 1 980 ) .. Oama r u. Oc t o b e r. 0. 49. 0. N o v emb e r. 0. 1 46. 293. D e c emb e r. 0. 65 9. 1 464. January. 0. 342. 1 464. 49. 122. 7 56. M a r-::h. 0. 122. 5 86. Ap r i l. 0. February. 122. Al tho u gh i t s eems t o o c cur t h r o u ghou t the y e a r ,. i t �s mor e. p r eva l e n t du ring the s umme r mon ths p r obab l y d u e t o the s e a s ona l na t u r e o f l amb in g .. I t i s no t no rma l l y a s e r � o u s. o r deva s ta t in g d i s e a s e a n d c a n b e con tr o l l e d b y v a c c ina t i on . The ava i l ab i l i ty and u s e o f che a p bu t e f fe c t iv e v a c c �n e s ma s ks the r i sk o f i n f e c t i on from na tura l l y o c cu r in g o r.

(20) ]0.. r e s i dua l v a c c 1ne v 1 ru s a s l on g a s v a c c ina t i on 1 s d one annu a l l y . Th e imp o r t an c e o f the d i s e a s e c an a l s o be me a suLe d b y i t s e f f e c t in man . .. Th e f i r s t human c a s e s in New Z e a l and w e r e. r e p o r t e d b y Mu i r and Pu r d y in 1 9 5 1 a n d 1 9 5 5 r e s p e c t ive l y . Mo r e r e c en t l y , ba s e d on a rep o r t b y th e Ac c i d e n t C ompens a t ion Commi s s i on , repor ted ,. t h e r e we r e i n c rea s i n g numb e r s o f human c a s e s. f r om two i n 1 9 7 5 t o 1 4 3 i n 1 9 7 9 .. C omp en s a t i on. c l a ims for o r f 1n man f r om 1 9 7 4 - 1 9 7 8 have amoun t e d t o $ 7 5 , 000 .. A l s o , f r om th i s d a t a , f r e ezing work e r s c omp r i s e d. 9 0 % o f the c ompen s a t i on c l a iman t s , t h e rema in ing p ro pcr t i on inc l u d e d f a rme r s , f a rm h and s , opera tor s ,. sheare r s , bale pre s s. f o o d p a cke r s and r e t a i l bu t che r s .. DIAGNOS I S Th e pr o l i f e r a t ive l e s i on s a r e c h a r a c t e r i s t i c f o r o r f b11t s h ou l d be d i f f e ren t i a t e d from o th e r i n f e c t i ons s u ch a s s h e e p p o x , go a t p o x and u l c e r a t ive d e rma t o s i s . Lab o r a t o ry c o n f i rma t ion c an b e a ch i eved by one o f s ev e r a l dia gno s t i c t e chnique s .. V e s i cu l a r f l u i d s f r om human. p a t i en t o r s c ab l e s i on fr om s h e e p c an b e ino c u l a t e d on t o s h e e p ( Na g in g t on , 1 9 5 7;. 1 9 6 8 ) , o r o n t o c e l l cul t u r e s ( Gr e i g ,. P l owr i gh t e t a l . , 1 9 5 8;. a n d Ch ow ,. 1 9 6 3;. N a g i n g t on ,. t e s t ( E r i ck s on e t a l . ,. S awhney , 1 9 6 6 c;. Trueb l o o d. 1 9 6 8 ) f o r f l u o r e s c en t an t ib o dy. 1975) .. Agar- g e l p r e c i p i t a t io n t e s t. c an a l s o b e e1np l o y e d when u s i n g o r f l e s i o n s t o de tec t a n t i gen bu t t h e t e chni qu e i s no t s en s i t ive 1n de t e c t ing an t ib o d i e s f r om c onva l e s c e n t s e r a ( S a whney e t a l . , 1 9 7 3 ) . S tud i e s f r om exp e r imen t a l and n a t u r a l c a s e s i n s h e e p h ave s h own t h a t t h e c omp l ement - f i xa t i on t e s t i s t h e mo s t s ens i t i v e fo r d e t e c t in g ant i gen ( Rome ro-Me r c ad o e t a l . , l9 7 3 a & b ) .. Ac c o r d in g t o t h e s ame au th o r s c omp l emen t­. f ix in g an t i g en p e r s i s t s in l e s i o n s or c l in i c a l ma t e r i a l f o r 6 t o 7 d a y s o r mo r e wh e r e a s i f b y e l e c t r on micro s c o p y , v i r i on s may b e masked b y h y p e rp l a s t i c ep i th e l i um and.

(21) 11 .. t i s s u e d eb r i s i n s u b acu t e and c h r on i c c a s e s . Harkn e s s e t a l . ,. However ,. ( 1 9 7 7 ) d e s c r ib e d a me th o d to ob t a in a. max ima l numb e r o f v i ru s p a r t i c l es f r om s c ab m a t e r i a l s t o o ve r c ome s ome d i f f i cu l t i e s in r e c ov er ing v i r u s p a r t ic l e s e s p e c i a l l y f r om o l d l e s i o n s wh e n e l e c t r on m i c r o s c opy i s emp l oy e d i n t h e r a p i d d i a gno s i s o f o r£ .. E l e c t r on. m i c r o s c o p y is p robab l y the mos t r a pid me ans o f d i a gn o s i s .. TREATMENT AND CONTROL T r e a tmen t o f o r £ l e s i o n s ln s h e e p i s u s ua l l y un s a t i s f a c t o r y and imp r a c t i c a l e x c e p t wh ere s e c on d ary inf e c t i on i s o c cu r ing .. Ho"l:.veve r , th e r e a r e a numb e r o f s u g ge s t e d. t r e a tmen t s and th e s e cou ld b e o f v a l u e i f on l y a f e w an ima l s a r e i nv o l ved .. A 5 % c o p p e r s u l ph a t e s o l u t i on can b e. app l i e d to t h e s k i n wi th a s wa b a f t e r r emova l o f t h e s c ab ( Su the r l an d an d Mou l e,unda t e d ) .. Oth e r med i c a t i o n s tha t. have been u s e d a r e 7 % i o d i ne , c r e o s o t e d i p , and 3 % ph eDo l in v a s e l i n e ( B e c k and Tay l o r ,. L i th i um an t imony R th i oma l a t e (6% ) a l s o known a s a n t h i oma l ine (May and B ake r ) c an b e u s e d ( S an d e r s on ,. 1 974) .. 1 9 76 ) .. Wh e r e p r ob l ems o f s c r ew­. worm f l i e s o c cu r , a p p r o p r i a t e r e p e l l an t s o r l a rv i c i d e s a r e r e c ommend e d (Me r ck Ve t . Manu a l ,. 1973) .. Con t r o l i s p r e fe rab l e t o tr e a tmen t and va c c lne s a r e ava i l ab l e wh i ch p r o d u c e a g o o d immun i ty ( G l ov e r , Howa r th , Har d y ,. 1 9 2 9; 1935) .. S chmi d t and H a r d y , 1 9 3 2 ;. 1 928 ;. B o u gh t on and. Immun i t y c ou l d l a s t f o r a s l on g a s two yea r s .. Al s o , i t h a s b e en no t e d tha t va c c ina t i on o f a l r e a dy a f f e c t e d l amb s r e du c e s t h e c o ur s e a n d s ev e r i ty o f t h e d i s e a s e ( B o u gh t on and Hardy ,. 1935) .. V a c c ine s are a lso s a f e t o u s e. i n · s u ck l i n g l amb s ( K e r r y and P owe l l ,. 1 9 71 ) .. R e c u r renc e o f. th e d i s e a s e i n v a c c ina t e d an ima l s wa s ob s e rv e d b y Trueb l o o d et al . ,. ( 1 9 63 ) a n d B e c k a n d T ay l o r ( 1 9 7 4 ) b u t wh e th e r th i s. wa s due t o a d i f fe r en t s t r a i n o f v i ru s , o r p o o r v a c c ina t io n t e chn i qu e wa s n o t d e t e rmine d ..

(22) 12 .. . V a c c ines c u r ren t ly u sed c ons i s t o f a su s pen s 1 on o f l ive v i ru s p rep ared fr om ve s i ca- p u s tu l ar les i on s p r o du ced an the s k i n o f hea l thy , 4 t o 6 mon t h s old l amb s .. The l ambs. a re k i l l e d and b led abou t the 5 th d ay p o s t-- i n o c u l a tion and t he ep i derma l pu l p is removed and gr ound u p w i th a mo r t a r and pes t le .. The g r oun d-up l e s i ons c an ei ther be d r ied. and kep t a t l ow temper a tu re or made i n t o a 1 0% s u s pen s i on i n a s u i t ab le bu f fer .. Bu f fe r s u sed are Mc l lva ine' s. c i t r i c a c i d and d i s od ium hyd r o gen ph o s pha t e s o lu t i o n ( pH 7 . 2 ) o r a s o l u t i on c o n s i s t i n g o f M / 5 0 p ota s s i um d ihydr o gen ph o s ph a te s o l u t i on , M / 5 0 d i s o d i um hyd r o gen ph o s ph a te ( pH 7 . 2 ) and 1 /10 v o l ume anae s the t i c e ther . W i th the l a t ter bu f fer the s u s pen s i on i s i n c u b a ted a t 2 ° t o 4 ° C f o r 1 2 t o 2 4 hou r s . The e ther i s then removed by bub b l ing wi th f i l tered air .. The s u s pen s i on , p repa red 1 n e i ther bu f fer , i s s to red a t - 2 0 ° to - 7 0 °C , t e s ted f o r. p o ten c y a n d d i l u ted t o 1 : 5 0 0 w i th n1 ix ture o f 7 . 5 p a r t s b u f fer and 2 . 5 par t s gly c e r i n and ch l o ro f orm a s a p re serv a t ive (B r i t i s h Ve t . C o dex , 1 9 53 ) .. I n l amb s ,. the. v a c c ine i s mo s t c ommon l y a dm i n i s tered by the a p p l i c a t i on o f the s u s pens i on t o t he s c a r i f ied s k in on the i n s i de o f the h in d leg . be u se d .. HoweveG. a lterna t ive va c c i na t io n s i te s c an. The ven t r o- l a te r a l s u r fa ce o f the t a i l o r non­. woo led area p o s ter i o r to the ax i l l a h ave been u sed in pregnan t or l a c t a t ing ewe s. ( Be c k and Tay l o r , 1 9 7 4 ) .. S uch. v a c c ine s p r o d u ce a s o l i d immun i ty bu t a t the s ame t ime the s c ab s p r o du ced by v a c c ina t i o n c on t a in l ive v i ru s p a r tic l e s . A t temp t s t o u s e t i s s ue cu l tu re- der ived v 1 r u s a s va c c 1ne a s an a l tern a t ive t o the v a c c i ne p re p a red a s des c r i bed ab ove , h ave been rep o r ted .. Ramy a r ( 1 9 7 3 ) s u g ge s ted u s in g v 1 rus. p a s s a ge d in p r ima ry l amb k i dney cel l s f o r va c c ine p r o du c t i on .. The p r o tec t i o n c o n fe r red by s u ch a v a c c ine. a g a in s t c h a l lenge w i th f ie l d v i ru s wa s l e s s than a yea r . E c thyma v i ru s p a s s a ged 2 8 t ime s in c a l f k i dney cel l s p r oved t o be o f l ow v i ru lence and immun i sed l amb s f o r s 1x mon th s. ( E r g i n and Kok l u ,. 1 9 7 7 ).. A s t ra i n o f the v i ru s. grown i n a v i an ce l l s wa s rep o r ted t o h ave l o s t i t s ab i l i ty.

(23) 13.. t o p r o d u c e sk in l e s i on s in lamb s and r abb i t s , h mvev e r , immuno genic p r o p e r t i e s \ve r e n o t t e sted .. its. Zach (19 7 9 ). r e p o r t e d p r o d u c i ng a s a f e and e f fe c t ive vac c ine f r om v i ru s c on t inuou s l y p a s s a ged ov e r a hund r e d t imes i n s he e p c e l l s . Howeve r , no cha l l en ge t e s t had b e en per formed in h i s exp e r iment s even tho u gh a r e t ro s p e c t i ve tr i a l �a s encou r a g ing .. A t i s s u e c u l t u r e - d er ive d vac c in e c on t a i n i n g. i n t e r f e r on indu c e r was a l s o r e p o r t e d b y Va l d e r e t a l . , ( 1 97 9 ) b ut the r e was n o t s u f f i c i e n t d a t a to a l l ow a c r i t i c a l eva l u a t i o n o f the e f f e c t ivene s s o f the va c c ine . I t ha s b e en sugge s t e d tha t p a s s a ge in human amn 1 o n c e l l s ( Na g in g t on and vJh i t t l e , T o s chkov ,. 1 96 1 ) and HeLa c e l l s ( S aHhn e y and. 1 9 7 1 ) ha s an a t tenu a t ing e f fe c t on the v i ru s. a s d e t e rm in e d b y l amb i n o cu l a t i o n b u t again the s e c l a ims h ave n o t b e en s u p p o r ted b y pub l i sh e d d a t a . L ive v 1 r u s v a c c ine ss p r e p a r e d f r om s c ab ma t e r i a l a r e v ery e f fe c t iv e b u t have s ome d i s a dvan t a ge s .. T i s s ue c u l t u r e ­. d e r ived v a c c in e s have g iven p r omi s in g r e su l t s b u t the i r c omme r c i a l a p p l i c a t i on h a s n o t b een a d e qua t e l y asse s s e d and , a s shown b y two \vorke r s , immun i ty may b e o f sh o r t d u ra t i on . K i l l e d v i ru s v a c c ine s d o no t s e em to be o f va l u e a s i mmun i s i n g a g en t s . Knowl e d g e o f the me chan i sm o f immun i ty to o r £ v 1 r u s in fe c t i o n may b e u s e fu l in the s e ar ch for a l t e rna t ive vac c 1ne s .. The r e a r e o n l y a f ew r e po r t s o f inv e s t i g a t i on. i n t o the me chan i sm o f immun i t y t o o r £ v i ru s i n f e c t i on . I t h a s b e en s t a t e d tha t o r £ v i ru s i s h i gh l y e p i th e l i o t r o p i c thu s indu c i n g • • s k i n immun i ty • • ( Aynau d , Trueb l o oJ e t a l , 1 9 3 5;. 1 9 6 3;. Darbyshir e ,. G l ov e r ,. 1 96 1 ) .. 1 9 2 8;. 1 9 2 3 a s c i ted by B o u gh ton a n d Ha r d y ,. I n the c o n t e x t o f c u r r en t. immuno l o g i c a l u nd e r s t an d i n g ,. s u ch s t a teme n t s a r e unhe l p fu l .. Ther e have , howeve r , b e en s ome ob s e rv a t i o n s ma d e whi ch a r e he l p fu l f r om the pra c t i c a l p o i nt o f v i ew and m a y g iv e s ome i n s i gh t i n t o the t y p e o f i��un i ty induc e d by o r f v 1 ru s . F o r examp l e ,. the s u s c ep t ib i l i ty t o i n f e c t i o n o f l amb s ,.

(24) 14 .. 7 2 h ou r s a f te r b i r th , b o rn t o in�une ewes , p r oves th a t no p r o tec t i ve immun i ty is d e r ived f r om the p l a cen t a o r c o l o s t rum (Bou gh ton and Hardy , 1 9 3 5 ; 19 6 8 ) .. R i ch ter and J an sen. Th i s imp l ie s tha t an t i b o dy , i f p r o du ced ,. in the. a f fec ted a n ima l is e i th e r o f a c l a s s which does no t p a s s th r o u gh the p l a c en t a o r in t o the c o l o s t rum , o r i t i s no t p r o tec t ive .. I t i s s t a ted ln Bux t on and F r a s e r ( 1 9 7 7 ). tha t. sera f r om immun i sed sheep c o n t a in p re c i p i t a t in g and c omp l imen t - f i x i n g a n t i b o d ie s to or£ v i ru s bu t the pro du c t i on o f neu t r a l i s in g an t ib o die s i s va r i ab le .. N a g i n g t o n and. Wh i t t le (19 6 1 ) rep o r t e d de t e c t in g s i gn i f i c an t leve l s o f neu t r a l i s in g an t i b o dies in c onva le s cen t s h eep and human p a t ient's sera .. On the o ther hand , T rueb l o o d e t a l ,. (1963). f a i led t o demon s tra te s i gn i f i c an t leve l o f neu t r a l i s ing an t i b o d ies in immune s heep sera .. N o inve s t i ga t i on s i n t o. ce l l u l a r immun i ty indu c e d b y o r f v i ru s i n fec t i on have been rep o r ted . More i s known ab o u t immun i ty t o or thop oxvi ruses .. I n v.(v o. and .(n v.(tk o s tu d ies w i th v a c c i n i a and r ab b i t pox s h owed tha t two f o rms o f v i ru s a re p r o du ced du r in g in fec t i on . Ab o u t 9 0 % o f the v i ru s p r o geny rema i n s in t r a ce l lu l a r ly and 1 0 % a re re l e a s ed f r om the cel l s , ( B ou l t e r and App leya r d ,. 1973) .. the ex t ra ce l l. u l a r f o rms The s e two f o rms o f Vlru s e s. a c t a s sep a r a te an t i gen s and ir1�un i ty t o one d o e s not c o n fe r p r o te c t ion t o the o ther , and th i s may be t r ue in o ther p ox v i ru se s , e . g . o r f .. Wher e the ex t r a cel l u l a r f o rm. i s i nva s ive a s i n sys t em i c p oxv i ru s i n fec t i on s , re s i s tance to i n fe c t i on wou l d be an i n terp l ay o f b o th humo r a l and c e l l u l a r types o f immun i ty .. The humo r a l immun i ty i s. d i re c ted t owards re l e a s e d v i ru s and the cel l u l a r immun i ty a t t a c k s the i n fec ted ce l l s .. An t ib o d ie s a g a i n s t the. re l e a s e d v i ru s p l ay a maJ o r r o le in the p reven t i on o f the s p re a d o f i n fec t i on ( Bo u l ter and App leya r d , 1 9 7 3 ) .. Th i s. a s pe c t has n o t been inve s t i ga ted f o r o r f v i ru s and i t may be o f v a l ue to l o ok i n t o t he s e two f o rms o f v i ru s in rel a t i on t o the imn1un i ty deve l o ped .. Howeve r , a s o r f i s. no t a s y s tem i c d i sea se , an t ibody d i rec ted a ga in s t v i ru s.

(25) 15 .. p ar t i c l e s may b e o f l i t t l e s i gn i f i c a n c e i n the p r o t e c t i on f r om o r p a thogene s i s o f th e d i s e a s e .. Ra ther , c e l l u l ar. iwnun i ty may b e i nvo l ve d . Th e r e a r e seve r a l. s p e cu l a t i o n s on the me chan i sm o f c e l lu l a r. immun i ty t o p oxv i ru s i n f e c t i on s .. C y t o t o x i c i ty o r d i re c t. a t t a c k o n the i n f e c t e d c e l l s i s a func t i on o f t h e s en s i ti s e d T-c e l l s tha t r e c o gni s e an t i gens expre s s e d e a r l y o n the s u r fa c e o f the c e l l s ( B l an d en , e t a l, , 1 9 7 7 ) .. D i r e ct. a t t a c k on the c e l l s by T- c e l l s i s s ome t im e s b l o c k e d b y th e p r e s en c e o f l ow c on c e n t r a t i on o f IgG an t ib od i e s on the s u r f a c e o f the c e l l s .. Howeve r , the s e a n t i b o d y- c o a t e d c e l l s. c an b e d e s t r o y e d by a me c h an i sm t e rme d ant i b o d y- d e p en d en t c e l l -m e d i ated c y t o t o x i c i t y ( ADCC ) .. Th i s l a t t e r me chan i sm. may b e exh i b i t e d by b o th pha go cy t i c and non- p h a g o cy t i c mye l o i d c e l l s and b y weak l y g l a s s - a dhe r e n t c e l l s w i th Fe r e c e pto r s kno\vn a s "K-c e l l s " .. Fur t h e rmo re ,. in t e r f e r on. p r o du c e d b y e i th e r th e s en s i t ised T- ce l ls o r t h e l ymph ok ine­ s t imu l a t e d ma c ro p hage s mi gh t p r even t th e i n t e r c e l l u l a r t r an s f e r o f v i ru s p a r t i c l e s ( Ro i t t ,. 1977) .. PHYS IC O-CHEMICAL PROPERT I E S O F THE V IRUS Con t a g i ou s e c thyma i s c a u s e d b y a v i ru s b e l on g i n g t o the fam i l y p oxv i r i d a e .. I t h a s b e en g r o u p e d w i th p a pu l ar. s t oma t i t i s v i ru s in the g e nu s P a r a p oxv i ru s ( In t e rn a t i on a l Cown i t t e e on Taxonomy o f V i ru s e s ,. 1979) .. Its s 1ze 1s. a p p r ox ima t e l y 2 6 0 x 1 6 0 nm w i t h an ax i s r a t i o o f 1 . 6 ( N a g i n g t on and Wh i t t l e ,. 1961 ) .. E l e c t r o n mi c r o s c o py o f the. v iru s p a r t i c l e s s t a ined w i th pho s ph o tun g s t a t e r ev e a l s two in t e r ch angeab l e f o rm s ( N a g in g t on and H o rne ,. t y p e 1 o r M ( Mu l b e r r y ) o f w oo l " app e a r an c e .. 1 962) .. Th e. f o rms show the cha r a c t e r i st i c "b a l l The t y p e 2 o r C ( c l e a r ). pho s pho tungs t at e p e rmeab l e .. f o rms a r e. Th e "b a l l o f wo o l " a p p e a r anc e. o f t h e t y p e 1 f o rms i s d u e t o t h e c r i s s - c r o s s i n g p a t t e r n s o f t h e tubul a r p r o t e i n t h r e a d s o n t h e s ur fa c e o f the. v i r i on s ( N a g i n g t on and H o rn e ,. 1 9 6 2;. N a g in g t o n e t a l . ,. 1964) ..

(26) 16 .. By ana l o gy w i th v a c c �n � a v � r u s ( We s t o o d e t a l . ,. 1 9 64 ). th e. type 2 f o rms a r e d ama g e d t y p e 1 v i r i ons wh i ch a l l ow p en e t r a t i o n o f s t a in .. Th e o r £ v i r i on c o n t a i ns a d o ub l e 6 s t rand e d l in ear DNA w i th a mo l e c u l a r we i gh t o f 8 9 x 1 0 (A . J . Rob in s on , p e r s . c omm . ) .. The v i r u s i s v e r y th e rm o s t ab l e and r e s i s t an t t o c h an g e s in pH . I t i s c omp l e t e l y i n a c t iv a t e d a t 6 0 ° C f o r 3 0 minu t e s bu t r e t a in s s om e in f e c t iv i ty wh e n h e l d a t 5 5 ° C f o r 3 0 minu t e s ( S mvhn ey ,. 1 972 ;. Bux t on a n d F r a s e r ,. 1977) .. In fe c t iv i ty i s r e du c e d by 1 . 5 - 2 l o g TCID wh e n the 10 50 ° ° v i r u s wa s k e p t at 3 6 C � o . 5 C fo r one week ( P l owr i gh t et al . ,. 1 9 5 9;. S awhney , 1 9 7 2 ) .. S c ab s d r i e d ove r s u l fu r i c. a c i d , p owd e r e d and s t o r e d i n w e l l - s t o p p e r e d s e a l e d g l a s s tub e s in i c e. b o x r e t a in e d i t s p o t ency for a t l e a s t 3 2. mon th s ( B o u gh ton and Hardy ,. 1 93 5 ) .. In ano th e r e xp e r imen t. by the s ame a u t h o r s , i n fe c t i o u s d r i e d s c ab ma t e r i a l wa s d iv i d e d into th r e e p o r tions and s t o r e d in s t o p p e r e d g l a s s b o t t l e s i n t h e d a rk a t 83 ° F . One o f the th r e e s amp l e s l o s t in f e c t iv i t y w i th in 5 4 d a y s , ano t h e r w i th in 64 d a y s , and t h e th i r d w i th in 1 2 0 d ays .. S c ab ma t e r i a l a l s o d r i e d o v e r. s u l fu r i c a c i d , s t o r e d in amb e r b o t t l e i n the r e f r i ge r a t o r a t 45 ° F r e t a in e d i n f e ct iv i t y f o r 2 2 y e a r s , 8 mon th s ( L iv in g s t on and Hardy ,. 1 9 6 0) .. F r e eze- thaw in g d o e s n o t. a f fe c t t h e t i t r e o f t h e v i ru s , i n s t e ad , a f t e r th e 4 th f r e e z i n g- thawing c y c l e ,. the t i t r e i s i n c r e a s e d .. Th i s may b e. d u e t o th e d i s ru p t i on o f v i r a l a g g r e g a t e s and r e l e a s e o f v i r u s f rom c e l l s ( P l owr i gh t e t a l. ,. 1 959 ;. S awhn e y 1 9 7 2 ) .. When e x p o s e d t o the s umme r h e a t in T e xas , s c ab s r ema in e d i n f e c tiou s on l y f o r f e'" mon th s b u t when l e f t o n t h e g round in s h ad e d a r e a s , r e t a i n e d i n f e c t i o u s p a r t i c l e s f o r y e a r s ( Bough t on and Hardy , 1 93 5 ) .. A t r o om t emp e r a t u r e i n t h e. l ab o r a t o r y , o r £ v i r u s c an b e i� f e c t ive f o r a s l o n g a s 1 5 y e a r s ( Bu x t on and F ra s e r , 1 9 7 7 ) .. I t i s on l y s l i gh t l y. s ens i t iv e t o e th e r and f l u o r o c a rb on ( Trueb l o o d a n d Chow , 1 9 63 ) b u t i n a c t iv a t e d by c h l o r o f o rm ( Bu x t on and F r a s e r ,.

(27) 17 .. 1977) .. I t l S a l s o r e s i s t a n t t o g l y c e r o l ( S awhney ,. In s un@a ry ,. 1972) .. i t i s e v i d en t th a t o r f V l ru s l S t yp i c a l o f the. p oxv l ru s g r o u p , be i n g ve ry s t ab l e und e r norma l env i r onmen t a l c on d i t i o n s .. MULTIPLICITY OF S TRAINS I t has l on g b e en r e g a rd e d tha t th e r e i s on l y one s t r a in o f o r f v ir u s a n d tha t s t r a i n s f r om a l l o v e r the wo r l d a r e an t i gen i c a l l y s imi l ar .. C r o s s - p r o t e c t i on te s t s i n s h e e p and. s e r o l o g i c a l s tu d i e s by s ev e r a l au th o r s supp o r t th i s v i ew . S e d d on and McGr a th ( 1 9 3 1 a s c i t e d b y B o ugh t on and H a r d y , 1 935). found tha t t h e En g l i sh and Au s t ra l ian s t r a i n s sh owe d. c r o s s -p r o t e c t i on . Ha s s eb ,. G l ov e r ( 1 9 3 2 - 3 3 a s c i ted b y H o r gan and. 1 9 4 7 ) o b s e rv ed tha t v i r u s i s o l a t e s o f ovine o r i g in. f rom E n g l and , F ran c e and Ca l i f o r n i a c o n formed t o a s in g l e typ e .. A c a p r ine i s o l a t e f r om T an gan yika wa s i d en t i c a l w i th. t h e En g l i s h i s o l a t e bu t c omp a r i s on b e twe en the c a p r i n e i s o l a t e f r om Cyp r u s and t h e En g l i s h i s o l a t e w a s i n c o n c l u s ive . H o r gan and H a s s eb ( 1 9 4 7 ) a g a i n b y c r o s s -pro t e c t i on t e s t s ln s h e e p s h mved th a t s eve r a l o f th e i r i s o l a t e s w e r e immu no­ l o g i c a l l y i nd i s t in gu i s hab l e ·wh i l e o th e r s w·e r e c l o s e l y rela ted .. They d e s c r ib e d the d i f fe r en c e o f t h e r e a c t i on s. a s b e in g p a r t i a l t o c omp l e t e b u t exp l a ined th e s e d i f f e r e n t r e a c t i on s a s quan t i t a t iv e d i f fe r e n c e s in an t i gen o r an t i gens .. Howeve r , t h e y d i d no t ex c l u d e t h e p o s s ib i l i ty tha t. the s e mino r d i f fe r en c e s w ere du e t o an exp r e s s i on o f s t r a in var l an c e . Trueb l oo d e t a l . ,. ( 1 9 6 3 ) we r e o f th e op l n l on th a t s t r a in. v a r i an c e h a d n o t b e en f u l l y inve s t i ga t ed .. The o c cu r r en c e. o f the d i s e a s e i n v a c c i n a t e d an ima l s l ed t h em t o d ou b t t h e monova l en c e o f th e v i ru s .. S awhn e y ( 1 9 6 6b ) c i t e d S a rw ar. and B a ry a ( 1 9 6 1 ) as having i s o l a t e d a virus c au s i n g d e rma t i t i s i n s h e e p i n P ak i s tan \vh i ch d i f fe re d i n s ome r e s p e c t w i th the c l a s s i c a l e c thyma v i ru s .. A l s o c i t e d wa s.

(28) 18.. B r a ga z z i ( 1 9 5 9 ) who o b s e rv e d o u tb reak s w i th mo r ta l i ty r a t e o f a s h i gh a s 3 0 % and a s l ow a s 1 % wh i ch ind i c a t e d th e p o s s ib i l i ty o f d i f fe r ent s t ra ins .. Howeve r , a l though ,. th e s e. p a p e r s h ave n o t b e en c i t e d by the au th o r , th e p o s s i b l e c on fu s i on w i th o th e r d i s e a s e s s h ow i n g s imi l a r l e s i o n s s u ch a s s h e ep po x c ou l d a c c oun t f o r s u ch l ar g e d i f fe r e n c e i n v i r u l en c e .. V a l der e t a l .. ( 1 9 7 9 ) a l s o d e s c r i b e d an o u tb r e ak. o f o r £ wi th c l i n i c a l p i c tu r e th a t d i f f e r e d f rom u s u a l c a s e s . Th e y d e s c r ib e d th i s a s a ma l i gnan t f o rm , and c ou l d b e d u e t o a d i f fer en t s t r a i n o f the v i ru s . F r om the re s u l t s o f s e rum--ne u t r a l i s a t i ou and c ro s s p ro t c .: t i on t e s t s , S mvhney ( l 9 6 6 b ) conc l u d e d th a t there wa s a mu l t i -­ p l i c i ty o f s t r a in s o f c on t a g i ou s e c t h yma v i ru s .. He. t en t a t iv e l y c l a s s i f i ed Bu l ga r i an B2 i s o l a t e and t h e Eng l i s h i s o l a t e i n o n e an t i g e n i c g r o u p and the Ruman i an and S l ovak i an i s o l a t e s in ano the r .. He fur t h e r s h owe d th a t. an I r a n i an i s o l a t e r e s emb l e d the En g l i s h i s o l a t e . Bu l ga r i an i s o l a t e B l d i f f e re d f rom th e B 2 i s o l a t e .. Howeve r , He. s t a t e d fur th e r th a t a l tho u gh the s e s t r a ins d i d no t d i f f e r in t h e type o f Cy t o p a th i c e f fe c t t h e y p rodu c e d i n c e l l c u l t u r e , t i t r e s o f the Ruman i an and S l ovak i_ an i s o l a t e s w e r e a lways c om p a r a t iv e l y l ower than th o s e o f t h e Bu l ga r i an and Eng l i s h i s o l a t e s ( S awhney , 1 9 6 6 c ) . Ano th e r a t t emp t t o show s t r a in var i a t i on wa s r e p o r t e d b y P r e c au s t a and S t e l l man ( 1 9 7 3 ) .. T h e f ive i s o l a t e s s tu d i e d. we r e s im i l a r i n th e i r phys i c o-chem i c a l char a c t e r i s t i c s , h owever ,. they s h owed an t i ge n i c d i f fe r e n c e s .. n eu t r a l i s a t i o n t i t r e s and i nd i c e s ,. B a s e d o n the. they p l a c e d four o f t h e. i s o l a te s und e r o n e an t i g en i c s ub t y p e a n d t h e o th e r i n ano th e r sub t y p e a l though the l a t te r wa s v e r y c l o s e l y r e l a t e d t o t h e o th e r f ou r .. W i t t ek e t a l .. ( 1 9 8 0 ) in a c omp a r i s on o f. e i gh t p s eu d o c owp ox v i ru s i s o l a t e s and thr ee o r £ v i ru s i s o l a t e s d emon s t r a t e d d i f f e r enc e s i n the r e s t r i c t i on e n d o ­ nu c l e a s e p a t t e rn s o f the s e t h r e e o r £ v i ru s DNAs ..

(29) 19 .. I t app e a r s l ike l y tha t s t r a in v a r i a t i on d o e s e x i s t ln o r f v i r u s a s d e t e rm i n ed by s e rum-neu tra l i s a t i on and c r o s s ­ p r o t e c t i on t e s t s .. Howeve r , none o f th e s e s tu d i e s a r e. c on c l u s ive a n d t h e de f in i t ive exp e r imen t s have y e t t o b e d on e us ing p l a q u e - p u r i f i ed v i r u s .. GROWTH OF ORF V I RU S IN CELL CULTURE S S in c e Gre i g ( 1 9 5 7 ) r e p o r t e d the f i r s t s u c c e s s fu l a t t emp t t o g row o r f v i r u s i n c e l l c u l t u r e s , v i ru s have b e en o f t en r ep o r t e d .. in vi tro s tu d i e s o f th e. G r e i g ( 1 95 7 ) and Web s t e r. ( 1 9 5 8 ) u s e d p r ima ry emb ryon i c s k in c e l l s bu t th e l a t t e r d i d no t ob s e rve c y t o p a th i c e f fe c t ( CPE ) w i th a New Zea l and v a c c ine s t r a in of v i ru s . p l i c a t i on .. Th e r e wa s h owever , v i r a l mu l t i­. P l owr i gh t e t a l ,. ( 1 9 5 9 ) wa s ab l e t o p r o p a ga t e. a v i r u s s t r a in i n p r ima r y t e s t i s an d k i dney c e l l s o f o v i n e a n d bovine o r i g i n .. S imi l a r r e s u l t s we r e ob t a in e d b y. N a g iug t on a n d Hh i t t l e ( 1 9 6 1 ) , Ha c D ona l d and B e l l ( 1 9 6 1 ) and Trueb l o od and Chow ( 1 9 6 3 ) .. P r ima r y human amn i o n c e l l s. c ou l d a l s o b e u s e d t o p ro p a g a t e t h e v i ru s ( Na g in g ton and Wh i t t l e ,. 1961 ;. Ma cD ona l d and B e l l ,. 1961) .. N a g i n g t on and. \\Th i t t l e ( 1 9 6 1 ) a l s o u s e d monke y k i dney c e l l s 'vi th s u c c e s s b u t f a i l e d t o g r ow the v i r u s i n HeLa , HK2 and AM9 c e l l l ines . O th e r s u c c e s s fu l a t t emp t s t o p r o p a ga t e the v i r u s i n c e l l c u l tu :...· e s 've r e r e p o r t e d b y S awhn e y and Tos chkov ( 1 9 7 1 , i n HeLa an d l amb t e s t i s c e l l s; and S t e l lman ( 1 9 7 3 ). 1972). Ramy a r ( 1 9 7 3 ) and P r e c au s ta. in p r ima r y l amb k i dney c e l l s;. Ro s s i ( 1 9 7 3 ) i n c h i c k and d u c k emb ryo f ib r ob l a s t s .. and Th e s e. f ind ings s h ow th a t c e l l cu l t u r e - a d ap t e d or f v i ru s c a n b e p ro p a ga t e d i n a w i d e r ange o f c e l l t yp e s . Th e r e a r e d iv e r g e n t v i ews on th e c y t o p a thogen i c e f fe c t o f o r f v i r u s o n mono l ay e r c u l t u r e s .. A s men t i o n e d e a r l i e r. Web s t e r ( 1 9 5 8 ) d i d no t ob s e rve C P E w i th a N . Z. va c c l n e s t r a in o f t he v i r u s ino cu l a t e d o n t o emb ryon i c s h e e p s k in cel l s .. Howeve r , he c ou l d d emon s t r a t e v i ru s mu l t i p l i c a t i on ..

(30) 20 .. Th e a u th o r s tha t d i d ob s e rve a CPE. repor ted. a. w i d e range. 1 n the t ime i n t erva l b e twe en inoc u l a t i on and i t s a p p e a r anc e a s w e l l a s the c omp l e t i o n o f CPE . o f CPE \ve re s e en a t 1 8 h ou r s. S awhn ey and T o s chkov , Ro s s i ,. ( Na g i n g t on and Hh i t t l e ,. 1 9 7 2 ) a t 2 0 hou r s ( Ramya r ,. P r e c au s t a and S t e l lman , 1 95 7 ;. F o r e xamp l e , e a r l y s i gn s 1 96 1 ;. 1 9 73 ;. 1 9 73 ) o r a s l a t e as 3 6 h o u r s. 1 9 73 ) , and t·'\\7 0 d a y s ( S mvhney ,. ( G re i g ,. 1966c) .. Ob s e rva t i on o f c omp l e t e CPE a l s o var i e d from 2 4 hou r s t o 28 days .. Th i s w i d e v a r i a t i on c ou l d b e due t o the s i z e o f. ino c u l um , t y p e and b a t ch o f c e l l s a n d age o f cu l ture ( N a g ing t on ,. 1968) .. Youn g and r ap i d l y mu l t i p l y in g c e l l s a r e. mo r e s en s i t i ve t o v i ru s i n f e c t i on than o l d e r c u l tur e s . O th e r f a c t o r s th a t m i gh t a f fe c t p r o d u c t ion o f CPE a r e p a s s a g e a n d s t r a in o f v i ru s. ( S a� 1 n e y ,. l 9 6 6 c ) a n d t h e o r i g in. o f t h e s e rum u s e d in th e cu l t u r e me d i um ( N a g i n g t on ,. 1 9 68 ;. Z e b r ow s k i e t a l . , 1 9 7 4 ) . I n c lv s i on b o d i e s w e r e s e e n and d e s c r i b e d by s ome o f the au tho r s c i t e d ab ove .. P l owr i gh t et a l .. ( 1 9 5 9 ) ob s erved. i n t r a c y t o p l a sm i c e o s inoph i l i c ma s s e s a t twe l v e hour s p o s t ­ i n o cu l a t i on .. The s e ma s s e s we r e o f t en t ime s s u r r oun d e d b y. an i l l - d e f in e d h a l o .. S awhney and T o s chkov ( 1 9 7 1 ,. 1972). and Ro s s i ( 1 9 73 ) s aw s im i l a r inc l u s i o n s a t two d a y s p o s t ­ i n o c u l a t i on , t h e f o rm e r u s i n g H e L a a n d l amb t e s t i s c e l i s. and t�e l a t t e r , av i an c e l l s .. E o s i no ph i l i c i n c l u s i o n s. u s u a l l y p a r anu c l e a r we r e a l s o s e en b y Nagin g t o n ( 1 9 6 8 ) and Z e b rows k i e t a l .. ( 1 9 74 ) .. Th e behav i o u r o f o r f v i ru s 1 n. c e l l cu l t ur e s a s o b s e r v e d by t h e above au th o r s i s s umma r i s e d i n Tab l e I . Th e V JT u s s e ems t o s h mv a p r e fe renc e f o r p r 1ma ry c e l l s o f s h e e p o r human o r i g in , n o t mo r e than four d a y s o l d , and in the 1 s t to l O th s ub c u l t u r e p a s s a g e s ( N a g i n g t on , N a g i ng t o n. 1968) .. (1 9 6 8 ) ob s e rv ed th a t o r£ v i ru s f r om s h e e p \vou l d grrnv. i n i t i a l l y on s h e e p c e l l s o n l y . Howev e r , h e f o und tha t th i s s p e c i f i c i ty. wa s l o s t on s ub s e q u en t p a s s a g e s o r i f the.

(31) TAB LE. I:. CYTO PATH I C E F F E CT OF ORF V I RU S AS D E S CR I B ED BY D I F F ERENT AUTH O R S. Cell Type. Embr yon i c l amb s k i n. T ime o f. Virus Strain. CPE. Appe a r ance o f CPE. +. 3 - 4 d ays. S a s t r atchewan vacc i n e. N.Z.. s h e ep s k in. Strain. Sheep. We l come. t es t i s. Strain. Au thor. En l a r g em e n t r o u n d i n g o f c e l l s .. 2 isolates f r om C an a d a 1 i s o l a t e f r om. S u spended. Type of CPE. l. r r. c r e a s e d g r an u -. l a r i ty o f cytopl a sm w i t h even t u a l f r agme n t a t ion . Some c e l l s de tached f rom g l a s s , o t h e r c e l l s c l umped tog e th e r .. Comp l e t e C PE. Greig ( 19 57 ). in 7 days .. no C PE obs e r ved bu t w i t h pos s i b l e r ep l i ca t ion. -. !. Web s t e r ( 1958 ). +. nonspec i f i c r e a c t ion on f i r s t d a y . Med i um w a s. Plowr i gh t e t a l. change d . S h r i n k ag e and round i n g o f r ema i n i n g Comp l e t e CPE i n 4 days . cel l s . N e x t p a s s ag e , CPE comp l e te i n 7 days . CPE comp l e t e i n 2 - 3. ( 19 5 9 ). days on s•1bsequ e n t p a s s ag e s .. A l so m e n t ioned. d E t achme n t bu t d i d not m e n t i on c l ump i ng o f ce l l s . S he e p. P 3 f r om. embr yon i c. tes t i s. k i dney. cells. Calf. P 3 f r om. +. Wi thin. k i dney. tes t i s. +. 24 hrs. +. P4. s h eep. f rom. Rap i d C P E 8 0 - 9 0 % c e l l i n f e c t ion b y 4 th day n e x t p a s s ag e , s t i l l some normal cel l - i s l e t s on. +. c e n t r ipe tal s p r e a d of i n f e c t ion . c h a n g e s on s u bsequ e n t p a s s ag e s .. +. S im i l a r a s i n sheep k i dney. cells. k i dney. S u b s e qu e n t p a s s ag e s ,. Almo s t comp l e te CPE on 2nd and t h i r d d a y ,. P4. Goa t. 24 hrs. Compl e t e CPE i n 4 8 hou r s .. Comp l e te CPE be tween 2 - 4 days .. 7 th day . S u bsequ e n t p a s s a g e s s howed s i m i l a r e x t e n t t i l l 4 th day .. cells. f r om tes t i s. Within. k i dney -. --- - ---- -- --. S im i l a r. N I-'.

(32) ( cont inued ). TABLE I : Cell Type. I. Vir us. CPE. S tr a iP. Time. of. Appear ance. Author. Type o f CPE. o f CPE. Lamb. " S to c k S tr a i n ". embryonic. from Aberdeen. k idney. Un ive r s i ty. I. Early rounding. +. cells. ( t ime not spe c i f ie d ). f o l lowed by c l umping . 24. hour s .. S im i l a r CPE as. above .. CPE. seen in. and swe l l ing o f. Us ing heavy. inocul a. Mac Don a l d & Bell. ( 19 6 1 ). Almo s t compl ete CPE i n. 3 days . Human +. embryo n i c kidney amnion ! P 3. Human Human. f r om l amb. kidney. er.iliryo n i c l i ver 1. °. Human. amnion 2 0 Monkey kidney Cell. I. 2. Human. �. MK2. Mar g i n a l. Bovine. a r e rare .. f e tal kid n ey , s k i n , mu s c l e fetal. l. Fort Dodge. +. vacc ine ( US A ). rounded granular c e l l s 15. days. at the. 3 days. l. ,. rapidly ,. 5. day s .. of and. I solate Good. In monkey c e l l s ,. ho l e. after. later .. edge. c e l l s be come r e f r a c t i l e. cytop l a sm be c ome s granular . CPE more. f f e ct ". AM 9. #l -. f ur th e r change until. mono l ayer showed h o l e s. Tox i c. l i ve s :. kidney. no. +. H e La. Ovine. I so l ate. +. i so l a t e s. the in #. 18 hrs ,. Naging ton Wh i t tle. Then. &. ( 19 6 1 ). she e t . s ome the. 2 produced. i n c l u s ion bodies forma tion was. less. marked b u t more rounded c e l l s . Did no t d e s c r ibe CPE. comp l ete. in. type 6 days. o f CPE. Trueblood Chow. &. ( 19 6 3 ). + N N.

(33) TABLE 1 :. ( continued ). Cell. Virus. Type. S tr a i n. CPE. T ime o f Type of CPE. App e a rance. Author. o f CPE 1°. l amb. t e s ti s 1° l amb. Bulgarian B. 2. te s ti s 10 c a l f k i dney 10 pig k idney. omp lete. 2. -. 10. days. ! b omp l e t e � omp l e te. First. s i gn .. round ing of. Lo s s o f c e l l ul a r boundary the. cells. gaps o n ho l e s i n the mon o l ay e r ,. United. Eng l i sh and. Kingdom. re fa c tory be fore. cells ,. S awhney. f o l lowed by appearance o f. C z e c ho s lovakian Never. k idney 10 c a l f. !=. lysi s .. Bulg a r i an B. I n c l u s ions 2. ( 1966 ). c e l l s b e come more seen. in. isolates only .. Never. Rumanian. pomp l e te Not. German I r an i an. nention nter­. �e d i a·te Bovine testis Sheep tes tis Human. ORF Moneciwin. Rounding o f c e l l s w i th highly repa c t i l e. +. cytop l a sm and appearance o f ho l e s. ORAFLAM. Produced. " open typ e ". Naging ton. i n t he mono l ayer .. t l968 ). p l aque s. va c c i n e Field. s tr a in. amnion He la. Bulgarian strain. (PS. Lo s s o f c e l l u l a r boundary +. 48 hr s. Pyk no s i s. o f nuc l e i ,. from l amb. app e ar anc e. te s ti s ). 6 days .. of vacuo l e s .. Ob s e rved. in. 48-72. hour s .. S awhney and. g r anulation o f c y topla sm and Almo s t comp l ete CPE. in. Tos chkov ( 19 7 1 ). somewhat attenuating e f f e c t on. �irulence . Lamb testis. Cytoto x i c. Bulgar i a n strain. +. 18. hrs. cells. e ffect. lost. normal. mon o l aye r .. The r e. CPE. in. 4 - 5 day s .. in. 2 4 hour s .. At 4 2. shap e .. Ga L ' s. is. rounding .. cell. I n c l u s i on s. hours. and ho l e s. s e en .. 60-90%. appear. Comp l e te. in. S a whney and Tos chkov ( 197 2 ). N w.

(34) TABLE I :. 1. °. ( continued ). Cell. Virus. Type. Strain. l amb. k idney. Lo c a l. CPE. +. i s o l ate. I. I. Time o f Appearance. Comp l ete CPE. 2 4 hours. 4 vac c i n e. Author. Type o f CPE. o f CPE. in. 3 days .. Ramyar. Did not d e s cr ibe CPE. ( 197 3 ). +. s tr a i n. I. Duck. +. Not spe c i f ied. I. 3 6 hour s. First. s ign was. d i sp l a cement o f t h e nuc l i us ,. f ibrob l a s t s. c e l l u l a r body ,. I. Chick embryo. I. membrane . Ce l l s Virus. °. l a mb. Sheep. i so l a t e. +. w/ i n. 2 4 hr s. l I. I I. I. 1. °. bovine. cells Capr ine t e s ti s. I n c l u s i on bod i e s round e d and. F r e sh i so l ate from sheep S c ab ma t e ri al from goats. +. 3 - 4 days. ·. +. of. CPE i n 4 8 CPE. in. of. 4 8 th hour ,. Ly s i s o c c urred. in. 96. hour s .. 10. 7. c e l l ular in. P roduced. " open. type ". lys i s bordered by. 24 hou rs .. TC I D S 0 / 7 5 cm. 2. A mu l t ip l i c i ty. Precausta. &. St e l lman. ( 197 3 ). f l a s h produced. hou r s .. results. Mo r e. w i th 7 0. pronoun ced. in o t her. r e f r a c ti vene s s. pas s a ge CPE was '. seen. 3 - l l days .. I rregular. -. P la que s cells. i n fe c t ion. 3 rd day ,. cel ls ------ -----·- ---. pykno t i c. ( 19 7 3 ). of pathoge n i c a c t iv i ty .. Rounded c e l l s w i t h pykno s i s .. Inc r e a s e d. 3 days. s e e n at. swo l l en .. Comp l e t e CPE. showed l o s s. . pl aque s .. k i dney. Ro s s i. e n l a r g ement o f. vacuola tion a long cytop l a smi c. in some c e l l s .. f ibr obl a s t s. 1. are. lateral. c e l l ular pyknos i s ,. embryo. ceel. in. in. cells .. 2 4 ho • 1r s .. Zebrowski. e t al. ( 1974 ). and rounding o f c e l l s by. - 9 0 9o CPE by 5 th day . seen. te s t i s. typ e s .. By 5 th. Renshaw and Dodd. ( 19 7 8 ). l. I j '. !. ! '. I. N +'.

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FIGURE 1 :  Na me t r i zoa te ( l e f t )  and Na virus band s . d ia tr i z o a t e  ( r i gh t )  gradien t s  contain ing A minor band wh ich i s  more d i s t inc t in Na me trizoate is s e en b e l ow the main band

FIGURE 1 :

Na me t r i zoa te ( l e f t ) and Na virus band s . d ia tr i z o a t e ( r i gh t ) gradien t s contain ing A minor band wh ich i s more d i s t inc t in Na me trizoate is s e en b e l ow the main band p.49
FIGURE 3 :  Agarose gel electrophoresis of thirteen orf virus DNAs digested with EcoR1 restriction endonuclease

FIGURE 3 :

Agarose gel electrophoresis of thirteen orf virus DNAs digested with EcoR1 restriction endonuclease p.51
TABLE I I I  RECOVERY OF ORF VIRUS AND ORF DNA FROM SCAB HATERIAL

TABLE I

I I RECOVERY OF ORF VIRUS AND ORF DNA FROM SCAB HATERIAL p.52
FIGURE 5 :  X 134

FIGURE 5 :

X 134 p.61
FIGURE Grunwa l d-G iems a ,  LT , three h ours a f t e r  inocul a t ion ,  showing the appearance of a few rounde d  up ce l l s

FIGURE Grunwa

l d-G iems a , LT , three h ours a f t e r inocul a t ion , showing the appearance of a few rounde d up ce l l s p.77

References