Pathogenesis of ‘sheep-associated’ malignant catarrhal fever in rabbits

a t e d ' catarrhal fever athogemesis of ' s h e ~ ~ - ~ ~ ~ u c imalignant D. n u n l u n , H . w . REID, J. FINLAYSON. I. P O k , moreaun Kesearcn lnsnrure, 408 Gilmerton Road, Edinburgh, EH17 7JH

Pa~nugenesrs s~udiesur experilllrrcinlly pruduced sheep-associated malignant catarrhal fever (MCF) in laboratory rabbits are described. Animals were examined at intervals after inoculation. The principal change was a proliferation of lymphoid cells which began as soon as three days and became quite pronounced by 13 days after inoculation. The appendix, mesenteric lymph node and spleen were most obviously affected. The reason for this was a progressive increase in T-lymphocytes, which appeared to be a hyperplasia rather than neoplasia in T-dependent areas of these organs. Lymphoid cells also accumulated in interstitial spaces of nonlymphoid organs. The use of cyclosporin-A suppressed the lymphoid proliferation but rabbits still developed clinical MCF after a similar incubation period. It is suggested that the ragent of hICF might produce its effect by infecting and causi ng a dysfunction of lymphoregulatory cells, resiulting in bepion T-lymphot 'vto nrnlliferation. ..-.. nnlvrlnncll ,.". Te rminal net d be due Ito natural killer cell act ivity.

.

11 1s Ionly recently rnar cne cerrain rransmission of this for m of the ciisease t o I-abbits has been recorded (Buxtol2 and Re id 1980, Westbury and Denholm l concerns an analvsis of the .1982).. This r e ~ort lymphoprolifer~tiveresponse of this rabbit-passaged disease utilising histological and immunopathological procedures. The nature of the lymphoproliferation was further investigated by studying the effect of a potent 'T-lymphoc:yte suppr'essive age]It (cyclosplorinA ICS-/41) on the course of the disease.

I V 1 I I l C I I11

lIIUU3

Animal Conv reared New Zealand White ...yclung male a.na remale rabbits were ...,.- ~lsed. Following inocula tion rabbi ts were eltamined d.aily and I.ectal tempenItures recckrded.

1 "..,

in The sheepassociated agent had becu laboratory rabbits following isolation from a red deer (Buxton and Reid 1980). Washed mesenteric lymph node cells from clinically affected rabbits were stored at -80°C in aliquots containing 10 per cent fetal bovine serum and 10 per cent dimethvl sul~hoxide.

MALIGNANT catarrhal fever (MCF), an intectious lymphoproliferative disease of cattle and some other ungulates (Plowright 1968, Reid et al 1979), occurs in two distinct circumstances. One form is caused by alcelaphine herpesvirus 1 while the aetiology of the other is unknown. Circumstantial evidence implies that the causal agent in the latter is derived from les were fixed by i~~~~~~~~~~~~~ 111 1" ~ C LCIII I +ep; 3 11C thus it is referred to as the 'sheep-associated' for m. Experimental transmission of alcelaphone formol saline, processed to paraffin wax and 6 pm her.pesvirus 1 to rabbits, cattle and deer is readily thick se:ctions cut then stained with haematoxylin and act~ieved(Piercy 1955, Plowright et al 1960, Castro et eosin aIr Gordon and Sweet's method for reticulin. P-,--.a c ~ c c ~ ctissues d were either frozen in an isopentane/ ,I 1982) and has facilitated studies on the oatho:enesis of this condition (Plowright 1953a, ~ d j n ~ t o nsolid carbon dioxide freezing mixture and stored a t t al 1979, Rossiter 1980, Edington and P'atel 1981, -80°C or fixed for two hours at 4°C in Bouin's fixative (formalin adjusted to 5 per cent) before being 'atel and Edington 1981). In contrast, tra nsmission ... process ed to paraffin wax by the cold ethanol St 0 f the sheep associated form has not been so readlly ach~ieved. The reports of Pattison (1946) and Marie rnethod ( Pearse 1980 Da ubney (1959) fail to present convincing supportive evic3ence while that of Huck et al (1961) is C T I...--I. .-. infection implying Ithat the s ambiguous, loth were could have tkeen wildebeest or s Cryostat sections were cu t from froZen tissues;, airpresent in thc: park. dried for one hc)ur, fixed in 1 per cent hydl-ogen I1JJL

. Buxton,

bid, J. Finr

. . dried and mmutes,

peroxlae In metnanol ror 10 rinsed in 0.01 M phosphate buffered saline (pss) p~ 7 - 5 containing 2 per cent egg albumen and exposed '-r 1 0 ; one hour to sheep anti-rabbit T-lymphocyte selu m , kindly donated by Dr David Catty (Bast et a1 - 19'79). They were then rinsed in Pss-egg albumen and tre:ated with pig IgG anti-sheep IgG conjugated with roxidase, for one hour, rinsed in 0.05 M trisdrochloric acid buffer, p H 7.6, treated with 3'3 iminobenzidine (4 mg in 10 ml) in the same tris ffer, plus 0.01 per cent hydrogen peroxide for 10 nutes, rin'sed in tris buffer alone, counlterstained th haemat1oxylin, delhydrated etnd mount

Sections, (5 pm thick:, of St M arie processed tissue i dewaxed . Endogerious peroxidase was an( we:re. cut . . blcx k e d wit..h 1 nor cent hydrogen peroxide in methanol for 30 minutes and then the sections treated with sheep Fab anti-rabbit Fab conjugated with peroxidase (Nakane and Kawaoi 1974) (kindly donated bv Dr H. R. P. Miller, Moredun Research In!stitute), r,insed antd develof be nzidine as described above.

TABLE 1: Onset of clinical signs i n rabbits infected w i t h MCF and treated with cyclosporin-A (experiment2)

Rablbit numlber

11%

wperrmenr .2: Effect c

Rectal tempera above4 (dpi

2 x 108 7~ ln8 -,. a"

17 1R

2 x 108 2 x 108 1x108 1 x 108 nil 1 x 105

I

E

5 1C 11 12 13 14 15 16

.

:perrment I: Sequentrat examrnalrons Twelve rabbits were each injec:ted into a n ear vein onI day 0 with 5 x 10' mesenter . . ic lymph node cells frr )m a rabbit reacting wlth MCF following 44 serial ralbbit passalges. One rabbit was killed on cjay 0, twoI on and theI onI each of d;ays 3 , 5 , 7 and 9 aftelr inoculati~ re]maining thlree were killed on d ay 13.

Cycl (20

Number of lymphocyte:i injected iv

iv lntra dpi Day Frornday + 1 t Frorndav-1

Results EXPER1

: ~ + ~ c i uI IAL.

EXAMINATIONS

CIinica Nonle of the rabbits killed on d ay 9 or t)efore develol3ed clinical signs of ill health. The three killed 40°C on day 13 developed rectal temperatiIres . ..above nn -..rlsv --,12 and displayed clinical signs slmllar to tnose alreadq describe d (Buxto,n and Rc:id 1980) with adipsia , anorexia and dullrless being most obvi OUS.

In the first. part of th

ent four rabbits (1 to I I I L I L ( v L n ~with ~ ~ I 2~ y 108 nph node cells from an affected rabbit following serial passages (Table 1) and ;a further t wo (5 and each received 1 x 108 of th e same cells intraveinously. Two rabbits (1 and 2) eacn received daily int.ramuscular injections of Cs-A (20 mg kg-]) (ki~ndlydonated by Sandoz, Basel) in Miglyol 812 (Dynamit, Nobel, West Germany) from day 1. In the second part of the experiment 11 other rabbits (8 to 18) were inoculated intravenously with lymph node cells from a reacting rabbit (46th serial passage). Rabbits 8 to 11 received 105 cells on day 0 d Cs-A (20 mg kg-') in Miglyol812, from day - 1 ti1 the end o f the experiment. Rabbit 7 was not 'ected but was similarly inoculated with Cs-A. lbbits 12 and 13 received 106 cells; rabbit 14, 105 Is; rabbit 15, lo4 cells; rabbit 16, 10-' ce:Ils; rabbit , 102.5 cells and rabbit 18, 102 cells (Table 1). Thus was estimated that 100 lymph node cells Iwere equal o r greater than one rabbit infectious do:se. .I. *' were earll

:..:ante.

lllJrCrFU

Macroscopic pat hology The earliest ctlange obsc:wed was a thickening of the wa 11 of the a ppendix irI the rabbits killed on day -, >_ lyrrlpll 3 . --rn animals killea o_ -n uay 5 the mesenteric I----" -. node a,nd spleen were also slightly enlarged wh~ileon the seventh day the lungs showed al veolar emp)ly sema. On day 9 all these changes were more markecd. In rabbits killed on day 13 the append IX was grossly thickened, with clear mucoid contents while lymphc id nodules were clearly defined on the serosal surfacc:, some appearing blanched and necrot.ic. In ...- -L W U cases haemorrhagic foci of necrosis which 1were 5 to 6 mm in diameter and involved the whole thic:kness of the wall were present. The mesenteric lymph nodes were also very large (15 x 25 mm diameter), tl1e cut surface wet, the medulla pink and with focal areas of necrosis. The spleen was greatly enlarged while other lymph nodes were slightly bigger than normalI. The liver was pale and slightly swollen with whit e foci

.~ r

.

1 1 - 1

latarrhal fever 1 mm in ~ ~ ~ I I I C L111F LI W V uf the ~ I I I F CI ~ U U I Lnlilcu S ar this stage. Other tissuc:s appeared normal. i'

Lymphoid tissues. In the rabbit killed on day 0 the mesenteric lymph node was small with a simple meshwork of reticulin fibres. The medullary sinuses and thin surrounding cortex were populated with a mixture of mononuclear cells, a large proportion of which contained Ig (Fig la). A few primary nodules were present in the cortex. The appendices at this stage were also unstimulated, their walls thin and without obvious follicle formation. In the mesenteric lymph nodes of animals killed on day 3 the paracortical areas were full of moderate sized lymphoid

.:*L -.: cells vVILII ~rrcgurar uprrl ~ ~ u c l earlu i uabu cytoplasm while in the cortex early foll.icular development was present. At this stage apperidices were thickened and follicles clearly defined. 'rhese changes were more marked on day 5 and more .a,.,n nn -.. days 7 and 9. In mesenteric lymph node the paracortex and interfollicular zones were especially prominent by day 9 and densely populated by t.,-,h ?id cells in which the cytoplasmic membr a i r c 3 ryrlrplrc stained positive with sheep anti-rabbit T-lymph ocyte serum. Cells which stained positive for the pre sence of Ig vrere virtually absent. In appendices the walls were rnarkedly thickened due to very en1 follicle!; in the centres of which Ig-positive cells present as were a few sheep anti-rabbit T-lymph serum-]3ositive cells. The outer rim of each fc

G 1: (a) Day 0. (bl Day 13. Sections of mesenteric lymph node of rabbits experientally infect:ed with sheep associated MCF and stained with an immunohisto~emicalmethc~dfor rabbit lg. Positivecellsappear dark. x 90

. . " " a -

-

9

Pow eid, J. Fifi . . -. was composed of a band of cells which did not s t a n day Y appeared normal. The majority ot these cells with either of these immunological methods. How- were shown to be T-lymphocytes with sheep antiever the interfollicular zones were grossly enlarged rabbit T-lymphocyte serum (66.9 per cent positive, and packed exclusively with cells which stained 30 per cent negative and 3.1 per cent could not be sitive with sheep anti-rabbit T-lymphocyte serum. allocated to either category) (Fig 2). Examination for By day 13 relatively few Ig-positive cells were the presence of Ig-positive cells revealed 1 a 8 per cent esent in the mesenteric lymph node (Fig lb), its t o be positive and 98.2 per cent negative. The focal eat increase in size being due to vast numbers of necrosis, seen macroscopically on day 13, consisted of eep anti-r abbit T-ly mphocyte serum-pa sitive cell:i clearly demarcated areas of degenerate hepatocytes the par: lcortex arid cortex. In addi tion foca I adjacent to sinusoids, lightly infiltrated by neutrobcrosis, of1:en centreci on the relatively ft:w corticalI phils and lined by increased numbers of Kupffer -- - -- In the appendix thrrt: .- -- .wab ..- - llicles, wab plcsrnl. a cells. Large multinucleate giant cells were scattered ss of architecture and reticulin fibres around intact infrequently in the livers of two rabbits (F:ig 3) llicles stained more densely while within follicles although whether they originated from lym phoid ticulin staining was granular. Furthermore within cells or hepatocytes was not clear. tollicles large cells with plentiful eosinophilic cyto-. Lunkg was normal on days 0 and 3 but rrom day 5 plasm, having the appearance of macrophages, hadI onwarcis interstitial thickening of alveolar walls replaced the smaller lymphoid cells normally present. caused by accumulations of reticulum cells became Peripheral lymph nodes showed similar but much less progressively more severe with nodules sometimes severe changes to me:senteric 1:ymph nocle and noI being formed. A1lveolar emphysema was also present and there was a mild arter:itis of a vt necrosis was present. .abbit Progressive enlargernent of. sp!.leens killed o n day 13. throi ~ g h o u the t . . . series was due t o an increase both In number and size Mild interstitial accumulations of lymphoid cells ~f periarteriolar lymphoid sheaths which were, were ot>served in the kidneys of four rabbits killed on populated by lymphoblast-like cells of uniform or after day 5 and in the bladder of one of these appearance. Follicle development was minimal. By animal!5 there were also focal infiltrations of jav 13 there was a much greater densitv of lvmohoid l.v m.~ hkid c cells deep to transitional epithelium. - . cel(1s than ncr m a l throug60ut th,e red pulp In the palpebral conjunctiva the lymphoid nodules in the subepithelial connective tissue were prominent Other timues. Liver was nornla1 on da)r 0 but by on day 3 and significantly enlarged by day 5. In . cells, wnlcn .. were snown t o be rahhitc killed later lymphoid proliferation was more rlny j ~ympnola -T-lymphocytes with sheep anti-rabbit T-lymphocyte widespiread and by day 13 had extended to the ser,urn, had accumulated around the periportal junctio n of the conjunctiva with the cornea ve:jsels of one rabbit. In animals killed thereafter contiguo u s with the lymphoid cells which had nilar, but more severe, inflammation was generally aLLuL,,dlatedin the corneal limbus. No abnormalities )ugh the 8vers of bc)th rabbits; killed on were fo ymus.

.

I.

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. . ..

.

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FIG 2: Lymphold accumulation in portal ,. sheepassoclated MCF. Immunoh~stocher.,~~,.

~~cally affected wlth

,,.

,-,,,,,,,,dcytes. x 360

ver

:ell in the live?r of a rabbit clinically affcacted w ~ t hstjeep

FIG 3: A large m assc~ciatedMCF. I

)eriment 2,: Effect oJ'cyclosporin-A on MCF A,I1 rabbits injected with lymph node cells from . affecrea animals aeve~opedtypical clinical signs of MCF but, while this followed a mean incubation period of 15 days (range 11 to 18 days) in rabbits 1 to 6 and 8 to 15 (Table I), it was longer (21 to 34 days) in rabbits 16 to 18, which received between 102 and lo3 cells. In those given Cs-A as .well, the g:ross and histopathological changes were pr,ofoundly different from those described akhove. . L .. .--. ~ s - rrom In rabbits 1 and 2 whicn were glven n uay 1, changes seen post mortem were mild; one had a slightly enlarged mesenteric lymph node and appendix while the other had a few small white foci in the liver. In the slightly enlarged mesenteric lymph node the medulla appeared normal. There was n o follicle development in cortex but the paracortex and cortex were packed with lymphoid cells. The appendix was slightly thickened with rudimentary follicles containing clusters of large granular eosinophilic cells, possibly macrophages. In the liver there were interstitial accumulations of lymphoid cells around portal triads and in the lung there was alveolar emphysema and some interstitial thickening. The small white foci in the liver of the other rabbit were .. -- caused by focal necrosis of hepatocytes with surrounding lymphoid cells ancI , p;o~ifer;ition of Kul:~ffercells and bile dlJCtS. R abbits 8 to 11 given CS-A fro1m day, - 1- showed Fn.., abnormalities. The mesenterlc lympn noaes were nal in size with primary nodules present in a thin ex and no follicle formation (Fig 4a) in contrast nimals not given Cs-A (Fig 4b). The paracortex --.> uevrloped and the medullary cords were naa no1 sligtltly enlarged. The ar)pendix wzi s thin wal led in all cases with only rudimer ltary follic:les. the ct:ntres of

. .

. ' .

.

which contained large cells with eosinophilic cytoplasm. No abnormalities were found in spleen. 6 t h e liver there was centrilobular fatty degeneration with minimal periportal lymphoid infiltration while foci of necrosis infiltrated by neutrophil polymorphs and each surrounded by a zone of lymphoid inflamImation were present in one rabbit. Neutrophils \were present in lung capillaries of this animal. M a m n ~ a ~ y gland enlargement occurred in the two female rabbits and was due to a massive proliferation of both duct and alveolar tissue in which there was eosinophilic secretionI. No such changes v{ere seen i~I the two-rnale rabbits. Contrc31 rabbit 7 given Cs-A alcm e appeiwed normal.

Discussi The dlramatic patnologlcal cnanges seen nere are very sim~ilarto those associated with MCF cause1 infectiorI with alcelaphine herpesvirus 1. S virus, viral antigen nor histopatholol .neither . lesions suggestive of direct viral action are evide~ sites of tissue damage a number of hypotheses have been proposed to explain the pathogenesis of MCF. Mechanisms similar to graft rejection (Liggitt and DeMartini 1980b), transformation of lymphoid relic (Edington et al 1979, Hunt and Billups I 979, Denholm and Westbury 1982), direct virus-indlIced cytolysis (Liggitt and DeMartini 1980a) and cell I",. mediated responses t o virus infected vasc unat endothelium (Selman et al 1974, Liggitt and DeMartini 1980b) have been discussed. Plowr.ight (1968) proposed that virus may not be immedisltely .,.... -. responsible for the lesions and that hv~ersensiti lvlly to virus Ior virus-in duced antigens coulld be involved. This sug,gestion w as extendt:d by Reici and Bul(ton -7---

...

.

>.Buxfon,

FIG 4:

eid, J. Fit

Mesenreric lympn noae rrom cllnlcalcy arrecrea ramit wltn sneep assoclarea

MCF. H&E x 23. (a1Treated with Cs-A daily from day 1. (b) Not treated w ~ t h Cs-A

(1981) who postulate:d that the underly ing mech.anism could be profo und virus induced d ysfunctior1 of immunoregulator y mecharlisms, r esulting irI .." . - . -ilncontrolled lymphopro~~teratlon.Denholm and W'estbury (1982) suggested a T-lymphocyte prolifera.ticIn due to virus-induced destruction or inactivatiorI of ' T-suppressor cells with an accompanying: El-lymphocyte cytolysis. In contrast the studies. ported here indicate that the disease process starts on after inoculation and that cell destruction other an in the terminal stages of disease does not occur. The disease was characterised by the early appearance of interstitial acc:umulatio~7s of T-lymphocyte: ; in non-lymphoid orgal1s and a pr'ogressive T-lymphocyte proliferation accolmpanied t3y a relative decrease:

in Ig-.positive cells (B-lymphocytes) in certain lymph1aid tissues . These were mesenteric lymph node, appenciix. and .. . SF.)leen while peripheral lymph nodes were only sllghtly enlarged. This is in contrast to the patterr I seen in rabbits infected with alcelaphine herpes1virus 1 where the peripheral lymph nodes togethc:r with spleen are much enlarged, the thymus ..-.I--urlurrgoes degenerative changes (Plowright 1953a, Edington et al 1979) and the mesenteric lymph node is not so markedly affected (unpublished data). Similarly in this latter form of experimental MCF arteriti s is a feature (Plowright 1953a) whereas in ' the presenl : study it was not a prominent lesion. The proliferative response was hyperplastic Irather than ni:oplastic with retention of normal architc:cture

cver

h:lalignanf ( organs an.d tissue dlestruction occurring in trie Lerrrllrial SLi,IQeS. In MCF infected rabbits T-lymphocyte proliferation was prevented when Cs-A was administered from day - 1 and reduced when given from day + 1. As Cs-A is recognised as a potent T-lymphocyte suppressor which prevents initiation of T-lymphocyte mediated immune responses (Calne 1979), it seems likely that the proliferative process is initiated within first day after inoculation, a view supported by the histological observations on day 3 of experiment 1. ICs-A has also been shown to inhibit natural killer cel I activity in humans (Introna et al 1981) and in - .L a~nymic mice (Attallah et a1 1981), at high concentrations it also inhibits murine natural killer cells in vitro (Gui et al 1982). The massive proliferative responses found in the mammary glands of the two female rat')bits may be similar to the mammary dysplasia associated with alvec~ l a r and duct prolliferation replorted in human pati ents treated with Cs-A (Ralles anci Calne 1980). In the present study while Cs-A did J u p p l L J J T-lymphocyte proliferation it was not protective which suggests that the lymphoproliferative response is not directly involved in the tissue destruction. (:ell death in MCF h as been at tributed to natural killer cell activity (Re id et a1 1983) following the irnl .,,lation and culture:, from an experimentally infected rabbit, of large granular lymphocytes which had natural killer activity and t ransmittec1 disease. However large granular lymphoc:ytes have not been identified as yet within tissules by hi stological methods. A major l y m p h o r e g.*u--..l ~ t ur..lullction ~~ has bee n attributed t o large granular lymphocytes ( M Ioller 1979. Herberman and Ortaldo 1981) and Reid et al (1983) suggested that infection of these cell s by the MCF agent could cause a generalised imrnunological deregulation and result in extensive and tissue damage. The results I Y I~phoproliferation ~ rep orted here support the working hypothesis that the extensive T-lymphoproliferation seen is a noncific, benign, polyclonal response resulting from spe~ the malfunction of the large granular lymphocytes, rat1ier than a specific response to antigen in the tlssues, and tlhat it can be prevenlted by the administration of Cs-.A. i s in the re the ti ssue necr, ~ ~ seen Furthermo~ .. - C .L- 2: --...1 2 -2"re..-terminal stages U I trlr ulxabr c o u ~ ua l l x I I U I I I a11 --.. extt:nsion of natural killer ceI 1 cytotoxicity to nfected host tissues, a propert y which apparently uni~ develops late in disease., Thus it is; suggested that the a 1 1 1 1rial's death arises from.uncontrolled natural killer cell activity. (:on firmatiion of this concept should be SOU ght by attcrmpting tcI detect 'cy'totoxic cel Is in MCF affe:cted anim als. IY

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ices

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ATTALL.AH, A. M., URRITIA-SHAW, A., YEATMAN , T. J. & F01LKS, T. (1981) International Archiv and Applie,d Immunology 65, 465-469 BAST. E . J. E. G., CATTY, D., MANTEN-SL ID, R., JANSEIN. J. T. G., VELHUIS, R. H.. KUHULL. P. & . M A L L ~JS, E ~R. E. yean Journal 997-1003 BUXTON, D. & RE (1980) Veter 243-245 81 R.,.,;.,..,r . CALNE, R. Y. (1979) .......,..,.Y CASTRO, A. E., DALEY, G. < , M. A.. WHITENACK. D. L. & JENSEN, ; lerican Jour no1 of Velerinary Research 43, 5- 11 DAUBNEY, R. (1959) Veterinary IRecord 71,4! 33 nr-x,,,r,r . ULI~nuI -lVl, ..I J'. & WESTBU nr, n. A. IIWLI AUS rrarran

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Velerinary Journal ! EDlNGTlON. N. & P,

(1981) Veler tiology 6, 107- .I12 . -. -. EDlNGTlUN, N., PAltL. J. K., RUSSELL, P. H. & P LOWKICiHT, W. (1979) European Journal of Cancer 15, 1515GUI, X., HO, M. & CAMP, P. E. (1982) Infec lion and Imr 36, 1123-1127 HERBERMAN, R. B. & ORTAL 1981) Scienc 24-30 HUCK, R. A., SHAND, A., ALLSOP, P. 11. & PATEFL ~ U I Y . A. B. (1x1) Veterinary Record 73,457-465 HUNT, R. D. & BILLUPS, L. H. (1979) Compalralive Immurrology and Microbiology of Infectious Direose 2, 27! i-283 INTRONA, M,, ALLAVENA, P., SPREAFICO, F. & MANTn... . VANI, A. (1981) Transplantolion 31. 113-111 LIGGITT , H. D. & DeMARTINI, C. (1980a) Vt 'rology 17, 58- 72 LIGGITT H. D. & DeMARTINI, C. (1980b) VtVerinary Pall iology ,7 9, 1 1 , (-1- 83 MOLLER., G. (1979) 1t1~~u"ology Reviews 44. NAKANE: , P. K. & KAWAOI, A. ( 1974) JournaI oj nrstocnemrsrry and Cylfochemisrry 22. 1084- 1091 I. R. & EDINGTON. N. (1981) Archives of Virology 68, PATEL.. - . .. 321-321 5 PATTISOIN, I. H. (ISM6) Journal of Compararlive Parhology and Theropc?ulics56. 254,-265 PEARSE, A. G. E. (1980) Histochelnistry, Theor etical and Ap r I.-"Aa" " k, "..-I. rl-. U I I I V L LLVIIUVII ~ ~ ~ . A.altu IYCW t u t n , C I I I Volume 1.4th edn. E>.,:"L..-"t. and Liv ingstone PIERCY, S. E. (1955) British Veter,;nary Journa3 111, 4 8 4 4Y I PLOWRIISHT, W. (IS63a) Proceeclings of the :YVth lnterna tional Veterinzkry Congress. Stockholm 1. 323-328 .or Lomoaratrve Palholopv,, 63. PLOWRII3HT. W. ( 1 9 ~ .5 Journal ~. 1 318-33: I PLOWRI(3HT, W. (I 968) Journa1 of the A I I rinary Medical Association 152, 795-8@ I PLOWRK3HT. W.. FEIRRIS. R. D. & SCOTT. c ialure 1W. ---,l.I6 .~7-1169 RALLES, K. & CALNE. R. Y. (1980) Lancet ii, 795 REID, H. W. & BUXTON, D. (1981) Proceedings of the Fifth International Congress of Virology. p 330 REID. H. W., BUXTON, D., CORRIGALL, W., HUNTER. n. n., McMARTIN, 1, D. A. & RUSHTON. B. (1979) Verer Record 104. 120-123 REID. H. W., BUXlTON, D.. P OW. I., FIIVLAYSON, BERRIE:, E. L. (I 983) Resean:h in Veterr'nary Scienn 103-109 ---KOSSITER. P. B. (1980) British Velerinary Jour SELMAN. J . E., WISEMAN, A., MURRAY, N. G. (1974) Velerinary Record 91,483-490 WESTBURY, H. A. & DENHOlLM. L. J. Velerinary Journal 58. 88-92 -

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Recei'vedfor pl rblication June r o,

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