Lymphocyte-Mediated Immune Cytotoxicity in Dogs Infected

Transcription

1 INFECTION AND IMMUNITY, Aug. 1982, p /82/8592-9$2./ Vol. 37, No. 2 Lymphoyte-Medited Immune Cytotoxiity in Dogs Infeted ith Virulent Cnine Distemper Virus MAX J. G. APPEL,'* WILLIAM R. SHEK,tt AND BRIAN A. SUMMERS2 Jmes A. Bker Institute for Animl Helth, Deprtment of Mirobiology,1 nd Deprtment of Pthology,2 Ne York Stte College of Veterinry Mediine, Cornell University, Ith, Ne York Reeived 8 Jnury 1982/Aepted 12 April 1982 Immune lymphoyte-medited ytotoxiity (ILMC) s evluted in dogs fter intrnsl exposure to one of the folloing three virulent strins of nine distemper virus: Cornell A75/17, Ohio R252, nd Snyder Hill. Cytotoxiity s tested ith peripherl blood lymphoytes s effetor ells nd primry dog testile ells tht ere mthed for histoomptibility s trget ells. A strong orreltion s found beteen ILMC nd the ourse of the infetion. Dogs tht suumbed to enephlitis ith ny of the strins hd little or no ILMC, heres dogs tht reovered hd the highest tivity. In the intermedite rnge ere dogs ith delyed or redued ILMC hih developed persistent but sublinil entrl nervous system infetions. A signifint differene in onset, pek, nd durtion of ILMC s observed in dogs infeted ith different strins of nine distemper virus. ILMC responses begn t 14 dys postinfetion (p.i.), rehed pek t 21 to 28 dys p.i., nd returned to preinoultion levels by 63 to 7 dys p.i. in nine distemper virus A75/17- nd R252-infeted dogs. In ontrst, ILMC in nine distemper virus Snyder Hill-infeted dogs begn t 1 dys p.i., peked by 14 to 17 dys p.i., nd pprohed preinoultion levels by 28 dys p.i. Antivirl immunity s mesured by ILMC ppers to be ritil ftor in determining the outome in nine distemper virus-infeted hosts. Furthermore, for ertin virl biotypes, delyed ILMC response orrelted ith persistent infetion of the entrl nervous system. Cnine distemper virus (CDV), morbillivirus losely relted to mesles virus (3), uses ftl infetion ith entrl nervous system (CNS) involvement in dogs nd other rnivores. The Snyder Hill (SH) strin (2) uses n ute enephlomyelitis ith predominntly gry-mtter hnges, heres strins Ohio R252 (18, 19) nd Cornell A75/17 (2 use delyed enephlomyelitis ith predominntly hitemtter lesions nd demyelintion reminisent of CNS lesions in multiple slerosis. Regrdless of the virus strin, some dogs reover spontneously, heres others suumb. In ddition, strin R252 or A75/17 my indue persistent infetion ithout linil signs of CNS disese (18, 19, 2. Thus, the outome of infetion depends upon both virl nd host ftors. A ruil host ftor tht limits the spred of virus is the immune response. In nine distemper, orreltion hs been found beteen survivl nd the pbility of dogs to produe CDVneutrlizing ntibody (2, 15). Hoever, both humorl nd ellulr immune tivity re import Present ddress: Deprtment of Avin nd Aquti Animl Mediine, Ne York Stte College of Veterinry Mediine, Cornell University, Ith, NY tnt for effetive resistne to virl diseses. In previous ommunition, e demonstrted tht dogs vinted ith ttenuted CDV mount lymphoyte-medited y- strong virus-speifi, totoxi response ginst 1Cr-lbeled, CDV-infeted dog testile ells. Using the sme system, e report here strong orreltion beteen suppressed immune lymphoyte-medited ytotoxiity (ILMC) nd ftl or persistent infetion of dogs exposed to virulent CDV. MATERIALS AND METHODS Virus strins. Three virulent strins of CDV ere used for dog inoultions; SH nd Cornell A75/17 strins ere isolted in the Jmes A. Bker Institute for Animl Helth, Ith, N.Y. The Ohio R252 strin s kindly supplied by A. Koestner. All three strins ere mintined in frozen spleen suspensions tht ere hrvested from speifi-pthogen-free dogs 5 dys postinfetion (p.i.). The ttenuted Rokborn strin of CDV s used for the infetion of trget ells. For virus neutrliztion tests, the Vero-ell-dpted Onderstepoort strin of CDV s employed. Animl inoultion, experimentl design, nd preprtion of trget nd effetor ells. Speifi-pthogenfree mle begles from the Bker Institute olony ere used. Peripherl blood leukoytes ere typed for dog leukoyte ntigens (DLA) by Frnes D. Cnnon (2). Donloded from on June 13, 218 by guest 592

2 VOL. 37, 1982 CELLULAR IMMUNITY IN CANINE DISTEMPER 593 Testiles ere surgilly removed hen dogs ere 3 months of ge nd used to prepre CDV Rokborninfeted fibroblst trget ells for ILMC ssys. Beteen 7 nd 9%o of the trget ells ere infeted s shon by immunofluoresene. Infeted trget ells nd uninfeted testile ells ere stored in liquid nitrogen s desribed previously (22). After DLA typing nd preprtion of trget ells, ll dogs ere inoulted intrnslly t 4 months of ge ith pproximtely 5 x 13 dog lung mrophge (DLM) infetious doses of virulent virus in dog spleen suspensions in miniml essentil medium ith Erle slts. A totl of 7 dogs ere exposed to CDV SH, 12 ere exposed to A75/17, nd 6 ere exposed to R252. Body temperture s reorded dily, nd disese progress s monitored by eight loss, peripherl blood lymphoyte ounts, nd observtion of linil signs. Heprinized blood smples for the preprtion of peripherl blood lymphoytes to be used s effetor ells in ILMC ssys ere tken tie before infetion, on dys 7, 1, 14, 17, nd 21 PI, nd in eekly intervls therefter until ILMC s detetble t insignifint levels (<1%o). Effetor lymphoytes ere prepred from 1 to 3 ml of heprinized blood, the volume depending upon the level of lymphopeni of CDV-infeted dogs. Phgoyti ells ere removed ith rbonyl-iron poder, nd lymphoytes ere bnded in Fioll-Hypque (Phrmi, Fine Chemils, In., N.J.) s desribed previously (22). ILMC ssy. The ILMC ssy s desribed in detil previously (22). Briefly, peripherl blood lymphoyte effetor ells ere tested for their bility to lyse CDV Rokborn-infeted trget ells t the times speified bove. Trget ells ere removed from liquid nitrogen, thed, shed, suspended in MLA medium (22) supplemented ith fetl lf serum, nd pled into flt-bottomed, 96-ell miropltes (Linbro, Div. of Flo Lbortories, In., Rokville, Md.) for tthment overnight. Cell onentrtion s djusted to pproximtely 5 x 13 tthed trget ells per ell. Trget ells ere lbeled ith N[51Cr]4 s desribed previously (22). Lymphoyte preprtions ere dded to trget ells in miroplte ells t effetor/trget ell rtios of 1:1 in.2 ml of medium. Cells ere inubted for 6 h t 37 C nd 5% CO2 in ir. Immeditely therefter, superntnt fluids ere ompletely removed ith the Titertek olletion system (Flo Lbortories). The remining ells ere dissolved in 1% sodium dodeyl sulfte nd lso hrvested ith Titertek. Rdiotivity s ounted in Bekmn 4 Gmm Counting System (Bekmn Instruments, In., Fullerton, Clif.). Mesurement of lymphoyte-medited ytotoxiity. The totl 51Cr relesed during the ssy period s omputed s follos: (totl 51Cr relesed) = [superntnt - (sodium dodeyl sulfte-dissolved ells + superntnt)] x 1. The perentge of spontneous relese s the totl 51Cr relesed in the bsene of effetor lymphoytes. Cytotoxiity used by the presene of lymphoytes s omputed s follos: (lymphoyte-medited ytotoxiity) = [(totl 5 Cr relesed) - (spontneous relese)]. If the lymphoytemedited ytotoxiity s used by nonimmmune lymphoytes, it s termed nturl lymphoyte-medited ytotoxiity. After exposure to CDV, the perentge of ILMC s omputed s follos: ILMC = [(lymphoyte-medited ytotoxiity) - (nturl lymphoyte-medited ytotoxiity)]. Virus neutrliztion. Virus neutrliztion tests ere mde ith the Onderstepoort strin of CDV in Vero ells in flt-bottomed, 96-ell miropltes (Linbro) s desribed previously (5). Virus isoltions. Dogs ere euthnized either hen moribund or eletively up to 7 dys p.i. The ltter group, hih s linilly stble, inluded both persistently infeted nd reovered dogs. A fe dogs ere kept for linil observtion beyond 7 dys nd ere not neropsied (see Tble 1). Brin tissues ere sterilely removed from euthnized dogs. Virus isoltion ttempts from brin tissues ere mde in the folloing to ys. (i) Brin tissues ere ground nd suspended in miniml essentil medium ith Erle slts. Seril dilutions ere inoulted into primry DLM ultures s desribed previously (. Virusindued synytil formtion 5 dys fter inoultion nd diret immunofluoresene ere used s riteri for the presene of CDV. (ii) Brin tissues ere mined in miniml essentil medium ith Erle slts nd mehnilly stirred for 1 h t 2 C. Free ells ere filtered through heeseloth, shed three times in miniml essentil medium ith Erle slts, nd inubted t 37 C in 5% C2-95% ir in 4%o MCoy medium-4o Leibovitz medium-2% fetl lf serum (GIBCO, Grnd Islnd, N.Y.)-ntibiotis-29.2% glutmine. Beteen 6 nd 8 dys fter initition, ultures usully hd formed monolyers. They ere then pssged, nd over slips ere seeded ith seondry ells. At 5 to 7 dys lter, hen monolyers ere formed gin, over slips ere stined for diret immunofluoresene, nd the presene of free virus s tested by isoltion in DLM ultures s desribed previously (. If results ere negtive t tht time, ultures ere pssged seond time nd monitored for presene of virus s desribed. CNS pthology. One-hlf of the brin (sgittl setion) s fixed in 1%o buffered Formlin nd routinely proessed for light mirosopy. Setions ere tken just rostrl to the orpus llosum, through the interthlmi dhesion, t the rostrl olliulus, obliquely through the opti ortex, nd medilly (sgittl plne) through the erebellum nd medull. Slides ere stined ith hemtoxylin-eosin. RESULTS Clinil signs. Dogs ith elevted body temperture (bove 39.5 C) ere generlly depressed, nd norexi or dirrhe or both s folloed by eight loss. Fever, eight loss, nd lymphopeni re listed in Tble 1. All infeted dogs beme depressed nd norexi by 3 or 4 dys p.i. They beme linilly stble beteen 14 nd 21 dys p.i., ith the exeption of dogs ftlly infeted ith CDV SH, hih deteriorted rpidly, beme moribund 14 to 17 dys p.i., nd ere euthnized. Dogs tht suumbed to CDV A75/17 or R252 infetion beteen 22 nd 42 dys p.i. beme depressed nd noreti 5 to 1 dys erlier. CNS signs (onvulsions, txi, spsms, myolonus, presis, prlysis, or blindness) ourred beteen one nd severl dys Donloded from on June 13, 218 by guest

3 594 APPEL, SHEK, AND SUMMERS INFECT. IMMUN. l 2 = I+ u) u 42.( U2 -.)2 : =ạ I+ = _ - CIS )._ ) IL) - S = L2 bw z P4. C 8) s.._.2 C) * _.W C)C ew> s Co z C r. u =.ef.e A. 2._ '4- Co OCo I- * * Em e E~,.5gE= Co >< o~ -* 2 Z Z2 C_ E.E 1 E~~. -C _ tn W 1 tnew I4 rs O4- G 1 r m -6 -d ).2,o oo ML.*VF C-p.2 - ~~~~~~. en en en A -. )> Cs 4 en\ q m _I ".._ m-,f ) : o r--to _ ) F. T'- N- 6 en %O4en ) -.@. s r r' H. - S *Au"4 o v 2 s - *t. Donloded from on June 13, 218 by guest - e- re _t % Co. 4 (A ) -g >2.e.g " fi e4 n el m *'n f T-~ V- -4 V- t1 - V1 1- r- eq 4 l E t N- u D. 4E

4 VOL. 37, 1982 before dogs beme moribund. One dog (no. 12) developed fil myolonus on dy 35 p.i., hih remined unhnged until dy 49 p.i., hen he s euthnized (Tble 1). ILMC. The level nd durtion of ILMC depended on the linil progression of disese nd the strin of CDV. Dogs tht reovered nd hd miniml resolving CNS lesions mounted muh greter ILMC responses thn those tht suumbed to infetion. Little or no ILMC s deteted in the ltter group (see Fig. 1A, 2A, nd 3A). Of six dogs infeted ith A75/17 tht hd no or lo levels of ILMC response on dy 21 p.i., three (no. 14, 16, nd 19) ere moribund beteen dys 22 nd 32 p.i. One dog (no. 15) tht beme moribund on dy 42 p.i. hd only lo ILMC responses on dys 14, 21, 28, nd 42 p.i. In one linilly stble dog (no. 9) tht * S *4z }\ 5. 2/ : CELLULAR IMMUNITY IN CANINE DISTEMPER 595 developed inflmmtory demyelinting enephlitis, lo levels of ILMC ere found on dys 28, 35, nd 56 p.i., hen he s euthnized. One dog tht developed fil myolonus on dy 35 p.i. but tht s otherise linilly stble begn to respond on dy 28 nd hd inresing ILMC levels up to dy 49 p.i., hen he s euthnized (Fig. 1A). To CDV R252-infeted dogs tht beme moribund t dys 28 nd 31 p.i. nd three CDV SH-infeted dogs tht beme moribund beteen dys 12 nd 17 p.i. hd no mesurble ILMC. Dogs hih stbilized fter infetion ith the strins of CDV tht use delyed enephlomyelitis (A75/17 nd R252) hd ILMC responses tht persisted signifintly loer (P <.5) thn those deteted in survivors of CDV SH infetion (Tble 2). Six dogs infeted ith CDV A75/17 S Dys Post Infetion S. A Donloded from on June 13, 218 by guest * 21 - zi l Dys Pos rinetion FIG. 1. (A) ILMC response of 12 dogs fter intrnsl exposure to CDV A75/17. Upper line, Men vlues from six dogs (, ) tht reovered; loer line, men vlues from six dogs (O, *) ith enephlitis tht beme moribund or tht hd persistent CDV in the CNS beteen 28 nd 56 dys p.i. (B) Neutrlizing-ntibody titers in the ser of 12 dogs fter intrnsl exposure to CDV A75/17. Upper line, Men vlues from six dogs (, ) tht reovered; loer line, men vlues from six dogs (l, *) ith enephlitis tht beme moribund or tht hd persistent CDV in the CNS beteen 28 nd 56 dys p.i.

5 5% APPEL, SHEK, AND SUMMERS INFECT. IMMUN. TABLE 2. ILMC in linilly stble dogs fter CDV infetion ILMC in dogs infeted ith folloing CDV virus strin: Dys SH A75/17 R-252 r--- No. positive/ % ILMC ± SEMb No. positive/ % ILMC + SEM No. positive/ % ILMC ± SEM %IM % LC±SM totl %IM±SEtotl totl 1 2/ ± 5.8 /6 <3 /4 <3 14 4/ ± / ± 3.89d / ± 1.11d 21 4/ ±.88 6/ ± 5.89d 4/ ± 6 9d 28 / ± / ± 7.7" 4/ ± 4.93d Number of dogs ith ILMC.1%/number of dogs tested. b.d Men ILMC for ll dogs tested. Different supersripts ithin ro denotes sttistilly signifint differenes in the levels of ILMC (P <.5 by the unpired Student's t test). tht beme linilly stble fter 2 to 3 eeks p.i. hd initil ILMC responses on dy 14 p.i.; mximl levels ourred on dy 21 p.i., folloed by grdul deline therefter until 7 dys p.i., hen levels hd rehed or pprohed preinoultion vlues (Fig. 1A). Similrly, four of six CDV R252-inoulted dogs tht linilly reovered developed pek ILMC levels on dy 28, ith grdul deline until dy 63 p.i., hen they pprohed preinoultion vlues (Fig. 2A). In ontrst, four dogs infeted ith CDV SH tht hd linilly reovered beteen 2 nd 3 eeks p.i. hd ILMC responses tht begn on dy 1 p.i., rehed pek on dys 14 nd 17, ere gretly redued by dy 21, nd pprohed preinoultion levels by dy 28 p.i. (Fig. 3A). Virus neutrliztion. Onset of CDV ntibody in dogs tht beme moribund s delyed, nd titers remined lo or bsent (Fig. 1B, 2B, nd 3B). CDV SH-infeted dogs tht lter reovered hd mesurble virus-neutrlizing ntibody by 1 dys tht beme mximl t 17 to 21 dys (Fig. 3B). Dogs tht reovered from CDV A75/ 17 or R252 infetion hd initil CDV ntibody by 14 dys; titers inresed up to 42 dys p.i. (Fig. 1B, nd 2B) nd remined onstnt therefter. Virus isoltion. Virus s isolted in DLM ultures from brin suspensions of dogs tht beme moribund ith ute polioenephlitis subsequent to infetion ith CDV SH nd noninflmmtory demyelinting enephlitis subsequent to infetion ith CDV A75/17 nd R252. Brin explnt ultures from these sme dogs beme virus positive. Virus s lso deteted in brin explnt ultures from dogs infeted ith CDV A75/17 nd R252 tht hd inflmmtory enephlitis. Hoever, brin suspensions from these dogs ere, ith one exeption, virus negtive in DLM ulture. Virus isoltion from reovered dogs remined negtive, irrespetive of the tehnique used (see Tble 1). Histopthology. Neuropthologil findings in dogs hih developed linil disese or persistent infetion of the CNS vried ith the virl biotype. Dogs hih suumbed to CDV SH infetion hd n enephlitis entered on grymtter res (erebrl ortex, bsl gngli, thlmus, nd hypothlmus). The retion reveled mirogliosis, neuronl stellitosis nd neuronophgi, nd nrro perivsulr uffs of lymphoid ells in the immedite viinity of neuronl injury. Speifi virl inlusions ere infrequently observed, minly in neuronl ytoplsm. Osionl smll glil foi ourred in myelinted tissue, but demyelintion did not our. Lesions in myelinted res indued by the A75/17 nd R252 strins of CDV ere predominntly subependyml or subpil nd most severe in the metenephlon. Myelin loss s typilly seen s vuolr hnge ith n ompnying stroytosis. Sometimes synytil stroytes ere present, nd virl inlusions ere most redily found ithin the nulei nd ytoplsm of stroytes. In some nimls, this retion s ompnied by n infiltrte of lymphoytes nd mrophges into the overlying leptomeninges nd into the demyelintive lesions beginning s perivsulr ggregtes. Aordingly, these hnges ere reorded s non-inflmmtory or inflmmtory demyelinting enephlitis (Tble 1). Miniml resolving lesions ere found in dogs tht hd reovered from experimentl exposure, regrdless of biotype. These lesions ere hrterized by spordi smll glil nodules hih ere sttered rndomly through the neurxis. They ere not present in ll setions exmined nd, iffeer levels hd been studied, ould hve been missed. DLA in reltion to disese. A distint pttern beteen DLA nd disese ould not be estblished. Most dogs tht beme moribund or tht hd persistent infetion hd DLAs tht ere identil to DLAs of dogs tht reovered (Tble 3). Hoever, dog no. 19, hih s the only dog E Donloded from on June 13, 218 by guest

6 VOL. 37, CELLULAR IMMUNITY IN CANINE DISTEMPER 597 A o 5* so I, 4 u O 3 u E 2 E E 2' z 1, 14 ii i Dys Post Infedion I B Donloded from Dys Post Infeon (A) ILMC response of six dogs fter intrnsl exposure to CDV R252. Upper line, Men vlues from FIG. 2. four dogs (, *) tht reovered; loer line, men vlues from to dogs (O, *) ith enephlitis tht beme moribund 28 nd 31 dys p.i. (B) Virus-neutrlizing-ntibody titers in the ser of six dogs fter intrnsl exposure to CDV R252. Upper line, Men vlues from four dogs (, ) tht reovered; loer line, men vlues from to dogs (l, *) ith enephlitis tht beme moribund 28 nd 31 dys p.i. in this study ithout g nd but ith m ntigen, s the first dog tht beme moribund (dy 22 p.i.) fter exposure to hroni strin of CDV. DISCUSSION Cell-medited immune responses in CDV-infeted dogs tht hve been studied erlier inluded skin tests (11), lymphoyte blstogenesis (13), lymphoyte-medited inhibition of CDVindued synytil formtion (16), ntibody-dependent ellulr ytotoxiity (12), nd nturl lymphoyte-medited ytotoxiity nd ILMC in vinted dogs (22). We previously reported (22) tht ILMC in response to vintion s medited by T ells nd not by NK or ntibody-dependent, ellmedited ytotoxiity for the folloing resons. (i) The level of ILMC s unffeted by depletion of B ells or F reeptor-bering ells. (ii) CDV ntibody persisted in the ser of vinted dogs long fter ILMC deresed to insignifint levels. Although ILMC s CDV speifi, sine uninfeted nd SV5-infeted trget ells ere not lysed, ntibody neither bloked nor enhned ILMC, suggesting reognition of lymphoyte-defined CDV ntigens. (iii) Interferon enhnes NK ell tivity (26), but Tsi (S. Tsi, Ph.D. thesis, Cornell University, Ith, N.Y.) ould not detet interferon in the ser of vinted dogs. (iv) Finlly, ILMC s genetilly restrited, s is knon for virus-speifi, T-ellmedited ytotoxiity (8). on June 13, 218 by guest

7 598 APPEL, SHEK, AND SUMMERS INFECT. IMMUN. TABLE 3. Serologilly defined DLA of experimentl dogs Virus strin Dog DLA Results no. SH 1 b g k 1 n o Clinilly stble 2 b e g k 1 n o Clinilly stble 3 befgkl o Reovered 4 be gkl o Reovered 5 b fgkl o Moribund t dy 14 p.i. 6 befgkl no Moribund t dy 15 p.i. 7 b gkl no Moribund t dy 17 p.i. Cornell A75/17 8 b e g k 1 n o Reovered 9 b e g k 1 n o Persistently infeted 1 b g k l n o Reovered 11 b e g k 1 n o Clinilly stble 12 e g 1 n Persistently infeted 13 e g 1 n Clinilly stble 14 b e g k l n o Moribund t dy 3 p.i. 15 b g I n o Moribund t dy 42 p.i. 16 b g 1 n o Moribund t dy 32 p.i. 17 b g 1 n o Reovered 18 b g I n o Reovered 19 b f k m Moribund t dy 22 p.i. Ohio R252 2 g 1 n Clinilly stble 21 g 1 n Reovered 22 g 1 n Moribund t dy 28 p.i. 23 b gkl n Reovered 24 g 1 n Moribund t dy 31 p.i. 25 g 1 n Clinilly stble s un x u E E A * Donloded from on June 13, 218 by guest DAYS POST INFECTION Dys Post Infeion FIG. 3. (A) ILMC response of seven dogs fter intrnsl exposure to CDV SH. Upper line, Men vlues from four dogs (, ) tht reovered; loer line, men vlues from three dogs (O, *) tht died beteen 14 nd 17 dys p.i. ith ute enephlitis. (B) Virus-neutrlizing-ntibody titers in the ser of seven dogs fter intrnsl exposure to CDV SH. Upper line, Men vlues from four dogs (, ) tht reovered; loer line, men vlues from three dogs (l, U) tht died beteen 14 nd 17 dys p.i. ith ute enephlitis. ILMC eliited by virulent CDV, like tht eliited by the vine strin CDV Rokborn, s found to be genetilly restrited (dt not shon) nd to derese to insignifint levels t times p.i. hen CDV-neutrlizing ntibody s present t high levels (Fig. 1B, 2B, nd 3B). Trnsient, genetilly restrited ytotoxiity s lso reported in humns utely infeted

8 VOL. 37, 1982 ith mesles virus (17). Unlike ser from vinted dogs, ser from dogs exposed to virulent CDV hs interferon tivity (S. Tsi, B. A. Summers, nd M. J. G. Appel, Arh. Virol., in press). Hoever, serum interferon tivity peks erly nd, in ontrst to ILMC, is negligible by 2 eeks p.i. Thus, ILMC deteted in the peripherl blood lymphoytes of dogs exposed to CDV is probbly T-ell medited. To our knoledge, this nd our previous publition (22) re the only reports of genetilly restrited, ntivirl T-ell-medited ytotoxiity in dogs. ILMC responses in dogs fter exposure to subute (A75/17 nd R252) nd ute (SH) virl biotypes of CDV ere ssyed t vrious times p.i. to gin further insight into the host-virus intertion. Dt presented in this pper lerly demonstrte tht the outome of infetion ith CDV is determined by the bility of the host to mount virus-speifi immune response nd the virus strin. A strong orreltion s found beteen suppressed ILMC nd ftl or persistent infetion. Dogs tht suumbed to CDV SH did not hve mesurble ILMC t ny time. Similrly, dogs tht beme moribund ith non-inflmmtory enephlitis fter CDV A75/17 or R252 infetion hd little or no ILMC (see Tble 1). Dogs ith inflmmtory enephlitis hd delyed or gretly redued or both levels of ILMC in omprison ith those of reovering dogs (see Fig. 1A). It ppered tht CDV A75/17- or R252- infeted dogs tht hd strong ILMC response by 21 dys p.i. reovered, heres dogs ith little or no ILMC t tht time subsequently beme moribund or remined persistently infeted. The ssoition of strong erly ILMC nd neutrlizing-ntibody responses (Fig. 1, 2, nd 3) ith the bsene of morbidity support the importne of virus-speifi immunity in reovery from nine distemper. This is not surprising, sine ytotoxi T-ells hve been shon to be highly effetive t limiting virus spred (1, 27). Alterntively, in ertin virl disese, e.g., lymphoyti horiomeningitis in mie (9), virusspeifi immunity is believed to be the use of pthologil hnges. It is similrly possible tht demyelintion ssoited ith the inflmmtory enephlitis tht ourred in dogs infeted ith CDV A75/17 nd R252 s exerbted by infiltrting ytotoxi lymphoytes t time hen ILMC beme positive. Hoever, demyelintion s not dependent on lymphoyte infiltrtion of the CNS, sine four of eight dogs ith demyelintion did not hve inflmmtory enephlitis. Moreover, Summers et l. (2 reently demonstrted tht primry demyelintion in CDV-infeted dogs s independent of lymphoyte infiltrtion. CELLULAR IMMUNITY IN CANINE DISTEMPER 599 The bsis for the vrition in the immune responses of reovered nd moribund or persistently infeted dogs is presently unknon. The bility to respond immunologilly to ertin ntigens nd suseptibility to vriety of diseses hs been linked in vrious speies to the mjor histoomptibility omplex (MHC) (25). The suseptibility of murine retrovirus-infeted ells to T-ell-medited ytolysis is ffeted by the trget ell hplotype (6, 21). Resistne of hikens to Mrek's disese is ssoited ith ertin MHC hplotypes (7). On the other hnd, e ere unble to demonstrte orreltion beteen DLA nd suseptibility to nine distemper. Hoever, these results re inonlusive beuse the regents used ere polyspeifi nd deteted only serologilly defined ntigeni determinnts (2). Furthermore, non-mhc geneti ftors might be more importnt in determining the outome of infetion ith CDV. Glltin nd Longeneker (1) reently demonstrted tht genetilly determined non-mhc-linked resistne to Mrek's disese s determined by the level of suseptibility of lymphoytes to virl infetion. Both B- nd T-ells in lymphti tissues nd in buffy ot ells re infeted during the erly viremi phse in ll CDV-infeted dogs. Infetion of lymphoytes, lymphoid depletion, nd lymphopeni result in immunosuppression (1. The degree of immunosuppression, the bility to mount virus-speifi immune response, nd onsequently, the linil outome ould in prt be determined by the suseptibility of lymphoytes or lymphoyte subpopultions to CDV infetion. A signifint differene in onset, pek, nd durtion of ILMC s observed in dogs tht ere infeted ith different strins of CDV. The erliest onset nd the shortest durtion ere observed in previous study in dogs tht ere immunized ith modified live CDV Rokborn strin (22). In dogs tht survived CDV SH infetion, the onset nd pek ourred severl dys lter thn in vinted dogs, nd the durtion s pproximtely 2 eeks longer (Fig. 3A). In ontrst, dogs infeted ith either CDV A75/17 or R252 hd delyed onset nd pek ith grdul deline tht lsted for more thn 2 months (Fig. 1A nd 2A). The delyed ILMC in CDV A75/17- or R252- infeted dogs my be responsible for the persistent CNS infetion tht is knon to our. The time of onset of ILMC my be ritil for lerne of virus from the CNS or even for preventing infetion of the CNS. A mild enephlitis ith CDV invsion into the CNS is usully seen by 8 to 1 dys p.i. (23). If CDV persists in the nine CNS beyond dys 14 to 21 p.i., the dog seems ommitted to develop gry- or hitemtter disese. If, on the other hnd, prompt Donloded from on June 13, 218 by guest

9 6 APPEL, SHEK, AND SUMMERS response develops t this period, then virus spred ithin the CNS is inhibited, the infetion is borted, nd the niml reovers ith miniml resolving CNS lesions tht support our ontention of erly CNS infetion (23). Dogs ith insignifint ILMC develop polioenephlitis fter CDV SH exposure nd non-inflmmtory demyelinting enephlitis fter CDV A75/17 or R252 exposure. Dogs ith delyed, lo-level ILMC beome persistently infeted, linilly stble nd, if neropsied, sho pttern of inflmmtory demyelinting disese. Virus isoltion results ere influened by the immune response. Isoltion of infetious virus from brin suspensions in DLM ultures s omplished from moribund dogs ith ute or non-inflmmtory enephlitis nd ith little or no ILMC or virus-neutrlizing ntibody. This tehnique filed for virus isoltion from dogs ith inflmmtory enephlitis nd ith positive ILMC nd virus-neutrlizing ntibody. Persistent virus s probbly neutrlized in brin suspensions from dogs ith inflmmtory enephlitis. One ells from these dogs ere freed of the immune environment nd gron in vitro, free infetious virus developed. Conversely, brin ell ultures from reovered dogs remined CDV negtive. ACKNOWLEDGMENTS This ork s supported by Publi Helth Servie grnt NS from the Ntionl Institutes of Helth. The ssistne of Mry Beth Metzgr nd Ann Signore nd the DLA typing of dog leukoytes by Frnes D. Cnnon re gretly ppreited. LITERATURE CITED 1. Ad, G. L., D. C. Jkson, R. V. Blnden, R. Th Hl, nd N. A. Boern Chnges in the surfe of virus infeted ells reognized by ytotoxi T ells. I. Miniml requirements for lysis of etromeli-infeted P-815 ells. Snd. J. Immunol. 5: Appel, M. J. G Pthogenesis of nine distemper. Am. J. Vet. Res. 3: Appel, M. J. G., E. P. J. Gibbs, S. J. Mrtin, V. ter Meulen, B. K. kim, J. R. Stephenson, nd W. P. Tylor Morbillivirus diseses of nimls nd mn p In E. Kurstk (ed.), Comprtive dignosis of virl diseses, vol. IV: vertebrte niml nd relted viruses. Ademi Press, In., Ne York. 4. Appel, M. J. G., nd. R. Jones Use of lveolr mrophges for ultivtion of nine distemper virus. Pro. So. Exp. Biol. Med. 126: Appel, M. J. G., nd D. S. Robson A mironeutrliztion test for nine distemper virus. Am. J. Vet. Res. 34: Bubbers, J. E., S. Chen, nd F. Lilly Nonrndom inlusion of H-2K nd H-2D ntigens in Friend virus prtiles from mie of vrious strins. J. Exp. Med. 147: Clnek, B. W Mrek's disese virus nd lymphom, p In S. Rpp (ed.), Onogeni herpesviruses. CRC Press, Bo Rton, Fl. INFECT. IMMUN. 8. Doherty, P. C., R. V. Blnden, nd R. M. Zinkerngel Speifiity of virus-immune effetor T ells for H- 2K or H-2D omptible intertions: implition for H- ntigen diversity. Trnsplnt. Rev. 29: Doherty, P. C., nd R. M. Zinkerngel T-ell-medited immunopthology in virl infetions. Trnsplnt. Rev. 19: Glltin, W. M., nd B. M. Longeneker Expression of geneti resistne to n onogeni herpesvirus t the trget ell level. Nture (London) 28: Gerber, J. D., nd A. E. Mrron Cell-medited immunity nd ge t vintion ssoited ith mesles inoultion nd protetion of dogs ginst nine distemper. Am. J. Vet. Res. 37: Ho, C.-K., nd L. A. Bbiuk Immune mehnisms ginst nine distemper. I. Identifition of K ell ginst nine distemper virus infeted trget ells in vitro. Immunology 37: Krkok, S., G. Cokerell, nd A. Koestner Effets of nine distemper virus infetion on lymphoid funtion in vitro nd in vivo. Infet. Immun. 11: Krkok, S., R. J. Higgins, nd A. Koestner Cnine distemper virus: revie of struturl nd funtionl modultions in lymphoid tissues. Am. J. Vet. Res. 41: Krkok, S., R. Olsen, A. Confer, A. Koestmer, nd B. MCullough Serologi response to nine distemper virl ntigens in gnotobioti dogs infeted ith nine distemper virus. J. Infet. Dis. 132: Krkok, S., nd A. L. Wlle Lymphoytessoited immune responses to nine distemper nd mesles viruses in distemper-infeted gnotobioti dogs. Am. J. Vet. Res. 4: Kreth, H. W., V. ter Meulen, nd G. Ekert Demonstrtion of HLA restrited killer ells in ptients ith ute mesles. Med. Mirobiol. Immunol. 165: MCullough, B., S. Krkok, nd A. Koestner Experimentl nine distemper virus-indued demyelintion. Lb. Invest. 31: MCullough, B., S. Krkok, A. Koestner, nd J. Shdduk Demyelinting tivity of nine distemper virus isolte in gnotobioti dogs. J. Infet. Dis. 13: Mollen, N., D. St. John, F. D. Cnnon, nd J. W. Ferrebee Lymphoyte typing in llogrfted begles. Trnsplnttion 6: Pfizeniner, K., G. Trinhieri, D. Solter, nd B. B. Knoles Mpping of H-2 genes ssoited ith T- ell-medited ytotoxi responses to SV4-tumor-ssoited speifi ntigens. Nture (London) 274: Shek, W. R., R. D. Shultz, nd M. J. G. Appel Nturl nd immune ytolysis of nine distemper virusinfeted trget ells. Infet. Immun. 28: Summers, B. A., H. A. Greisen, nd M. J. G. Appel Possible initition of virl enephlomyelitis in dogs by migrting lymphoytes infeted ith distemper virus. Lnet il: Summers, B. A., H. A. Grelsen, nd M. J. G. Appel Erly events in nine distemper demyelinting enephlomyelitis. At Neuropthol. 46: Svejgrd, A., M. Huge, C. Jersild, P. Pltz, L. P. Ryder, L. Stub-Nielson, nd M. Thomsen The HLA system. Monogr. Hum. Genet. 7: Welsh, R. M Nturl ell-medited immunity during virl infetions, p In. Hller (ed.), Current topis in mirobiology nd immunology. Nturl resistne to tumors nd viruses. Springer-Verlg, Berlin. 27. Zlnkerngel, R. M., nd P. C. Doherty Mjor trnsplnttion ntigens, viruses nd speifiity of surveillne T ells. Contemp. Top. Immunobiol. 7: Donloded from on June 13, 218 by guest