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Cell-mediated immune protection in chickens against Pasteurella multocida
Research in Veter
Cell-meaiated immune ~rotectionin chickens against Pa lla mu,ltocida T. ~ H B A ueparrmenr , oj vererrnary ~rcroolorogy,co~regeof Agrlculfure, universrfy of Vsaka Prefecture, Mozu-Umemachi, Sakai, Osaka 591, Japan
:ellular i mmunity i n chickens lmmune protc focida was; investiga'ted by i n against Paste; ng spleen cells and iments usi~ vivo and i n v .... . culture supernatants o t lmmunlsed cnlcltens. rntraperitoneal or intravenous transfer o f immune splenic cells into normal chickens induced transmission o f an as effective protection as that exhibited in immunised chickens. lmmune protection was also obtained by intravenous treatment o f chickens with culture supernatant fluid from immune splenic cells o f hormonally bursectomised chickens. The i n vitro expc:riment showed that intracellular bacterial prolifer&ationwas inhibited i n peritoneal maclrophages frorn immunised chickens, or from normal chickens wnq ..-..,itised with culture supernatant fluid of lmmune splenic cells, and the macrophages were ~rotected from disruption by infection. Peritoneal macrophages sensitised with culture supernatant flluid from unimmunised splenic cells, or peritoneal tlxarnvphages from unimmunised chickens, allowed considerable intracellular proliferation o f bacteria with almost complete breakdown of the macrophages within 24 hours after bacterial challenge. These data suggest that the protective immunity of chickens against P multocida was dependent on cell-mediated immunity by mediators such as the macrophage vating factlor from T lymphoc ytes.
.
tective 'ENDEN( . . . to. . . . . . imtnunity niultoclda on humoral lmmunlty in cnlcKens nas beerI frequently reported (Collins 1977). H owever, it has also been shown that tra nsfusion of ainti-serum prepared from chickens or turlteys into Arllrrrtnn mice:does not produce passive immunity (Heuulcalvtn and Watko 1965, Heddleston et al 1966, Bhasin and Bibebrstein 1968, Heddleston and Rebers 1968, Aler:ander and Soltys 1973) and it has been suggested ,c h ot l t a , both humoral and cellular immune mechanisms (Duia and Mahleswaran 1978) are irivolved in immune prot ect ion. . A previous study s howea tn at serum hyper. . ,,l,tal.. une 10 r rnurrocrda P-1059 S L I ~ I I I CVIIIPIF~FIJ Imm failed either to inhibit the proliferation o f microorga nisms in vitro or to produce protective passive imrnunity against infection (Ando and Baba 1969).
.
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It was also rep0rted that jrotection is not redl in burse:ctomised immune chickens iin contras thymectc (Ba ba et a1 1 . . 3mised - . imlmune . . chi1ckens . . . a n a tnat [j-glucuronidase, acld phsophatase a] lysozyme activity is increased in macrophages fro immunised chickens (Yamaguchi and Baba 1974). The purpose of this study was to immuni chickens against P multocida and to determine the protective immunity in vivo and in vitro by trar~sfer of their spleen cells o r the culture supernatant f luid of such splenic cells and to verify thereby the existence mediated immune chickens focida. Materials and methods
Chicken.s : White..-.,1.I.,all exper imints. FcWile eggs obtained 1kom the same source u,ere incubiated a t 37''C.
Hormonal oursecromv Hormlonal bur sectomies were performed by allantoic injection of te:stosterone propi01nate Each 6:gg was injected with 2 mg testosterlone propi~ onate in 01.1 ml cor embryon,al days of ' incubatioIn. I".
\
urganrsr The vi~rulentP multocida srrajn r-low, serotype 8:A, was used. It had a minimal lethal dose (MLD) of 560 viabl e cells when introduced into seven-week-old -L:-,.--urllcnern bv the intravenous route.
Vaccine ,and immu nisation
. Forrna - ------.lin-killed vacclne
,
. was used..
-
(FKV)
p
multocida grown for 18 hours in brain-heart infu!$ion broth by shaking cultures was centrifuged and washed twice in phosphate buffered saline (pes). The bacteria were then suspended in PBS containing U ' rnL per cent formalin t o give 1 0) mg ~ ml-1 (wet weig ht). The suspension was incubate:d for two days at 3'7OC and store:d at 5°C. A
,
Baba
ruur-wetx-UIU C I I I C K C ~ S were immunisea suocutaneously with 0.5 ml FKV. Seven days later i1 second dose of 0.2 ml FKV was administered intra venously. Two weeks after the second immunisinl lse the chickens were used for preparation of spleen
'reparatior rld challen
ger of sphenic lymptioid cells
(ere prepa.red from spleens o,f Cell susp . *.:,cnlcKen8 . . nmunised ana unimmun~sea at sevel eeks old (two weeks after ttie second immunisa on). The spleens were finely m~incedwitkI scissors i~ Ad medium 199 and then pipetted .u..p a nd down :w times. The cell suspensions were filtered througlh ylon gauze to remove aggregated cells and tissu e ~bris. The cells were washed three times b Y zntrifugation at 500 rprn for 10 minutes with coltd medium 199. These washed splenic cells were layerel over Lymphoprep (Nyegaard) and were centrifuge1 at 1300 rpm for 30 minutes. After centrifugation., cells of the middle lavers (the lvmvhocvte riclh . petting, a nd- washec3 action) were remo! vice with c,old mediu iperitonea Ily (at five Chickens . . . were injt . eeks) or lntravenously (at nine weeKs) wlrn I x lu :[Is of splenic lymphocytes per bird. The chicken s ven an intraperitoneal transfer of spleen cells wer e oculated intraperitoneally with 200 MLD of th e -1059 strain of P multocida 18 hours after the cellI1 ansfer. C hickens given cells intravenc~ u s l y werie oculated i ntravenou sly with I(30 MLD of the P-105'9 rain 24 hours. after-.. t he cell tra nsfer. . The protective ettects were assessea oy observln;g le birds for 20 days after chal lenge and g [em with c ontrol binds (Tables 1 and 2). -3
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. . a .
. . n
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repararron ana rransjer oj spleen cell culrure rpernatanl fluids and challenge
Splenic lymphoid cells were obtained frcliri immunised or unimmunised hormonally bursectomised chickens as outlined above. The cells were adjusted to 1 x lo7 cell ml-I in Eagle's minimum sential metdium (ME M) which included 20 per cent :at-inactiv,ated norm~ a chicken l serum, 100 iu ml-I :nicillin a ]7d 100 pg: ml-I streptomycin. The cell . _. : . . into tissue culture bottles (20 ispension was plperted ml per bottle) and 100 pg ml-' washec3 FKV w a5 added to each bottle. After 24 hours inculbation in ;1 37"C, 5 per cent carbon dioxide incubat'or, culturc : - .was collected by centrifugation at 5°C at -sllpernatant 8C0 rpm for 10 minules and th e superna tant fluid!j filtered through a Sartorius mernbrane fillier of port: sii!e 0.45 prr1.
.....
-. . .
. . . lntraperironea~ly . were .injecrea (at weeks:) or intravenously (at seven weeks) with 1 cultur~ e supernatant fluid. For challenge, the ch receivi.ng cells transferred intraperitoneally inoculated intravenously with 100 MLD of the 1'-1059 strain five hours after the culture supernatant treatment. The intravenously transferred group we1re also inoculated intravenously with 100 MLD of the 1?-I059 strain three hourrs after thc:treatmen t. The protective:effects vvere assess,ed by observing the bil*ds for 20 days aftel. challenge: and com paring the two groups (Tables 3 a~ n d4). Lhl(:Kens
oneal macrophages r
ion
Mac:rophage preparatic)n follow^ed a pre\ descrilbed technique (BabaI 1975). Peri~tonealmacrophage!j were colllected twc3 days nnln, tin PBS ,, I . , ..,a, after t he administration of , solutic)n into thle peritoneal cavity ()f immunisea or unimnnunised ctlickens. A,bout 106 cells in 1 ml of Eagle' S MEM SUpplemente:d with 2( per cent heat, inacti! . ....... fatea normal chicken serum In a Lab-Tex. tissue cultur~ e chamber slide, we re allowed1 to incub;ste for three 1hours at 37 ° C in a 5 per cent: carbon d ioxide incubsLtor. h l r \ - nrlhnra.., L I W l I - a u i I c I c I I t C C I I ~W C I C I C I I I W V L U uy v v a a t l t l t ej """ cold I- tanks' sol1 tio on. The n 0.4 ml of antibiot ic free Eagle' s SEM in vvhich was suspended 40 pg (2() pg in the ex periment r;hown in I=ig 3) of live P-1059 strain o t r multocida was aaaea into each chamber. A Iter reincubation for three hours, extracellular b;~cteria were removed by washing four times with Eagle's iu MEM. Finally, 0 . 4 ml of the medium containinl3 10 .. penicil lin was placed into each chamber, f o l l o v ~ -hlf ~~ furthe r incubation. tively, Aftt:r two, five, eight 2 chaml:ler slides were stained with tilemsa sol ution. .- - ~ The ntlmber of i ntracelluliar bacteria. p e r 1 0 0nacruphage:i and the surviving nnacrophag :es were ccbunted micros,topically.
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Sensitisalron and rnJectron oJ rllacrophage culi with splenic lymphocyfe culture supernalant jl
Normal macrophages obtained from the unimmunised chickens in the same way as ind icated above were cultured in an equal volume of splec:n cell culture supernatant fluid from immunised o~r unimmur~isedchickens. Afte r non-adh erent cellsi were removc:d, the remaining ceIls were in cubated with an equal . . . lolume of fresh med ium for . 24 . . hours ,... an(. leach challenged with the live P-lu3r srraln or rr?rulro.;& For th is bacteria I were adcied at a concentrati 20 pg 1nl-I. Subsequent tr eatment a nd observ, were a!s above. Am,.
ckens agar
'ith transfc Ir of splen ic cells
bacterial challenge 18 and 24 hours after transfer of spleen cells from immunised or unimmunised chickens intraperitoneally into five-week-old recipients or intravenously into nine-week-old recipients respectively, are shown in Tables 1 and 2. l\bJULIJ
VL
TABLE 1: Mortality i n unimrnunised chic:kens given spleen cells from immunised or unirnmunised t:hickens st~bsequently challenged intraperitoneall)1 w i t h a cultrire of P m u l tocida Mortality in chickens givf Spleen cells' From Frorn immunised unimimunised chickc Number of chickens before challenge
ip treated with All t he recipielnts in botl ckens an(1 the spleen cells fronI u n i m m ~ : medium died control group trc.ated with within 1Five days. -However, chickens treated w ~ t hspleen cells rrom immunised chickens survived a t rates of 67 per cent and 63 per cent respectively, demonstrating protective. transfer by means of immune lymphocvteqThese survival rates reflect approxirrlately the same level of protective effect a s obtainedI in immu nised chickens in a previously reported stttdy (Baba et a1 1978). Protecl ion with trbe transfer ' of spleen supernl;rtant . ..
The results ot bacterial c:hallenge f'ive hours after intraperitoneal transfer of spleen cell culture supernatant fluids from immu nised and unimmu nised chickens into recipients are snown In No siignificant di fferenc:es existec I the number of survi ving chic1
9
Nurnber of days unti11 death
Tot,
Spleen cell transfer: intraperltoneally Challenge: 18 hours after intr?iperitonealtrz~nsferof cells
TABLE 2: Mortality i n unimmunised chickens given spleep cells frorn immunis,ed or uninnmunised t ibsequently challenged intrt da ith a culture
TABLE 3: Mortality i n unimmunlsea cnlcKens inrrapertra~neally given spleen cell culture supernatant from immunised or unimmunised chickens subsequently challenged intravel10usly w i t h a culture of Prnuttocida
in chickens gvc!n -Mortality . No cells
-
tronn immunised chickcms
F unimmun~sed c hckens ~
Nurnber of -L.. :kens before challenge
Lllll
Nurnber of days unti 1 death
-
?nsgcven
Mort Splec From
.-.-.-
.>
chickens
Noce .,
,
ed chickens
Number of chrckens before challenge )f days h 1 2 3
Tot,
Ioral morral~rv
Spll?encell trans1fer: intravenously Chaillenge: 24 hours after intravenous transfer of cells
Challenge, f~ve hours after ~ntraperctonealtransfer of culture supernatant
13
11
11 -
221
Baba
TAIJ L t 4: Mottallty tn unmmnunlsecl chlckens intravenously an spleen cell culture supernatant from immunised or unnunised chickens subsequently challenged intravenously h a culture of Pmultocid
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S
~ncnlc~enswvt Spleen cells Frorn Fronn nunised irnmuni~sed unirn~ chicke!ns chickens MO~~IIIY
-
I
-da rn
Nurnber of chic:kensbefore challlenge
r
Nunnoer or aavs l death iune
ChaIlenge: thra? hours after intravenclus transfer of culture SUP2rnatant
. .. -...'raceiiuiargrowth oj-Y multociaa ana a~ ~ r v i v n l rates of norr;nal or imr;nune perit oneal mac in vitro culttrre -. . . - . I'he in vltro resistance ot peritoneal macrophages immunised and unimmunisied chickc rltocida is shown in Fig 1. 'eritoneal macrophages from normal L -.I. i chickens showed an increasc- :111 UIC IIUIIIUCI UI ~ntracellularbacteria and rapid disruption of cells after bacterial challenge. This resulted in the disruption of approximately. 80 .per cent and 88 per cent of the ----macrophages after five and 24 hours respectively. I'eritoneal macropha.ges from i mmunised l chickens shc)wed little intracell~ ~ l a rprolif eration 01 ? bacteria anci 97 per cc:nt survive:d even afiter 24 hou rs.
FIG 1: lntracellular prc lifera at ion of P multocida non-trnrnune peritoneaI rnacrophagc?sandtheir st in vitro culture
e spleen cell supernatant
I
Int raceNular growth of P multocida and survival ratles of norn~ amacrophages l exposed to the culture .--..-*--.SUDrrrrururrr> o f anlipen stimulated splenic
-
jpleen ce!Ils of im and unirnmunised .. :monally 1oursrcromlsea CnlcKens were cultures, . anc1 peritoneal macrophages from normal chickens sen sitised in vitro with culture supernatant fluid for 24 hours and then challenged with bacteria to exa mine the survival rate of macrophages and intracelllular bacte#rialprolifieration. F:ig 2 shou~s that ser siti is at ion of norma I chicken :s with irnmune ~leen cell itoneal rn~acrophagc
L
on-lmmune spleen cell culture supernatant
-m .-
$
V,
\
Medium
.
Hours alter lntectlon FIG 2: SLlrvival rates Iof normal rn supernatants of splenic lymphold ~rnrnunlsel d chlckens
sxposed to culture nrnunised and un-
tocida
ckens agar culture supernatant t l u ~ d resulted in survival ot 90 per cent of macrophages, 24 hours after bacterial challenge. However, sensitisation with non-immune spleen cell culture supernatant fluid resulted in d 93 per cent of mat:rophages disruption of Irs respect ively after bacterial at five, 10 a challenge. .
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were disrupted five hours after bacterial ch allenge. The results of P rnultocida multiplic ation are shown in Fig 3. Unsensitised macrophages showed an increase in intracellular bacteria from five hours arrer bacterial challenge until 10 hours later. In peritoneal macrophages sensitised with non-immune spleen cell culture supernatant, there was little intracellular bacterial multiplication for five hours after bacterial double by ch: illenge but se to apprc L .
I
Medium
1 \
Immune spm l
..
~
v
ion of P mu FIGt 3: Intracellirlar prol~ferat phelges exposedI to culture supernatants frorn immun~sedand unimmunised chicker
Discuss In t vo experiment, normal chlckens acquirea prorecrive immunity by transfer of immune --I--a p n x l 1 :ells. This protection may be regarded a @A*.n i cells or t o anti1body proc1-uced by I~ransferrec cells. However, con:iidering tklat protec tion in bi ,omlse, cnlc,en, was no, IY/U) and th e relative ly early bacterial challenge after transfel: of spleeln cells, the antibody, even th ough ed, seems unlikely to be directly assoc:iated produc~ with protection. with spleen cell The results of iin vivo ex1 -itoneally culture supernal ant giver . , with challenge after five hours (lable 31, o r g~venintravenously, with challenge after three hours (Tab le 41, gave low and high degrees of protection respect ivel y. Although this may be ascribed to various rea sons, one of the factors may be a difference in thle cell population of the cultured spleen cells. For instance, while the spleen cells used in the experiment of Table 2 contain both T and B cells (unbursectomised . . . . . birds), most of the cells contained in the spleen. cells used in the experiment a~f Table r1 were T cells (bursectomised b irds). Assuming that protectiorI is primar ,ily provid~ .;ma c,.,.h T cell mediators ll.rm..h*L llYlllllllVhlllL as macror activating factor) contained in spleen cell ct supernatant, the difference in the absolute numt T cells existing in the spleen cells used in ct would be reflected in the respective activatic111 UL reticuloendothelial phagocytes in recipients and thereby a difference in protection from infectic)n. In consideration of the time of lapse from tra.nsfer .. of spleen cell culture supernatant to bacterial challenge in the Table 3 experiment in whic:h an intraperitoneal route was chosen and in thiIt of Table 4 in which an intravenous route was ch osen, the latter is considered t o induce more rapid sensiitisation and activation of thephagocytic system, prc>dueing a larger protective effect. Furthermore, an other factor may be the number of bacteria use(i for challenge. If there was no difference in the number or T cell mediators, the smaller number of bacteria used for challenge in the Table 4 experiment, which was one-tenth of that used in the Table 3 experiment. .....may haive resulte d in a dif 'ference inI the protc:ctive effect' Macr ophages ft.om non-ir nmune chickens sensitised \
1
1
.
1
-
-
,,
. A
JULll
spleen cell culture supernatan1
..
10 houirs, amounting eventually to the same le! (el of bacteri: 11 growth as in unsensitised macroph ages. Howevctr, the number of intracellular bacter ia in macroplhages sensitised with immune spleen cell culture supernata nt fluid, d ecreased a fter five a] hours t'o approxitnately ha1f the origi nal numbc
macromphoid cells Irma1
Baba .. - tn tne supernatant or spleen cell culture rrom bu rsectomised birds treated with P multocida, were eq'ually well protected from P multocida as pelritoneal macrophages from immunised chickens. This suggests an association of T cell mediators macrophages in ilmmunised th a c t i Z ickens. Sensitisatit crophages from .. .. no1n-immune chiIcl
..
fnrmal m, as m tne present experiment (~amacltlrnl and Ba~ba1974, Baba et a1 1978). Fronn these facts and the above results, it was conclucded that T lymphocyte-medliated imrr unity ..ln..r no;nrt playa o a major role in the protective resistance a& infection with P multocida in fowl cholera. I effected at cellular level, mainly by macroph activating factor activated immune macropha L.-,. wnlcn possess a markedly enhanced bactericiua~ activity
Referel -
ALEXANDER, A. M. & SOLTYS. M. A. (1973) Journal of Cornpararive Pathology 83, 191- 198 ANDO, T. & BABA, T. (1%9) Jupunese Journol of Veterrnary Science 31, supplement 26-27 BABA, T. (1975) Merhods of Experimental lmmunol-* 'JYY *, IG87- 1088 BABA, r., ANDO, 1r. a N U K I N Journal of 1Medical Micro/liology 11. 2181-288 I Avian Disecrses 12. BHASlhI,* - J. L. & BI BERSTEIN, 159-lba .- . - . - -
COLLINS, F. M. (1911) Lornett vererrnarian 67, IUJ-138 DUA. S. K. & MAHESWARAN, S. K. (1978) Avian Drseases 22. 748-764
HEDDLESTON, K.
L. & REBERS, P. A. (1968) Avian Diseases 12, 12s)-134 HEDDLIESTON, K. I REBERS, P. A. & RIT'CHIE, A. E. Journo11of fmmuno110gy 96, 124.-133 HEDDLlESTON, K . 1 & WATK(1, L. P. (1% 5) A vian D h 367-3; 16 r,c ,.n.,, TAD>-U.ARNON, L. Journa11 of Cotr~par alive Medicb 19 YAMAG UCHI, Y . 8r BABA, T. fedinas of the firrr
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... .
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lnrersectionol tongresr of / A M !
~eceivedfor puorrcar~onJune 28, 1983
Cell-meaiated immune ~rotectionin chickens against Pa lla mu,ltocida T. ~ H B A ueparrmenr , oj vererrnary ~rcroolorogy,co~regeof Agrlculfure, universrfy of Vsaka Prefecture, Mozu-Umemachi, Sakai, Osaka 591, Japan
:ellular i mmunity i n chickens lmmune protc focida was; investiga'ted by i n against Paste; ng spleen cells and iments usi~ vivo and i n v .... . culture supernatants o t lmmunlsed cnlcltens. rntraperitoneal or intravenous transfer o f immune splenic cells into normal chickens induced transmission o f an as effective protection as that exhibited in immunised chickens. lmmune protection was also obtained by intravenous treatment o f chickens with culture supernatant fluid from immune splenic cells o f hormonally bursectomised chickens. The i n vitro expc:riment showed that intracellular bacterial prolifer&ationwas inhibited i n peritoneal maclrophages frorn immunised chickens, or from normal chickens wnq ..-..,itised with culture supernatant fluid of lmmune splenic cells, and the macrophages were ~rotected from disruption by infection. Peritoneal macrophages sensitised with culture supernatant flluid from unimmunised splenic cells, or peritoneal tlxarnvphages from unimmunised chickens, allowed considerable intracellular proliferation o f bacteria with almost complete breakdown of the macrophages within 24 hours after bacterial challenge. These data suggest that the protective immunity of chickens against P multocida was dependent on cell-mediated immunity by mediators such as the macrophage vating factlor from T lymphoc ytes.
.
tective 'ENDEN( . . . to. . . . . . imtnunity niultoclda on humoral lmmunlty in cnlcKens nas beerI frequently reported (Collins 1977). H owever, it has also been shown that tra nsfusion of ainti-serum prepared from chickens or turlteys into Arllrrrtnn mice:does not produce passive immunity (Heuulcalvtn and Watko 1965, Heddleston et al 1966, Bhasin and Bibebrstein 1968, Heddleston and Rebers 1968, Aler:ander and Soltys 1973) and it has been suggested ,c h ot l t a , both humoral and cellular immune mechanisms (Duia and Mahleswaran 1978) are irivolved in immune prot ect ion. . A previous study s howea tn at serum hyper. . ,,l,tal.. une 10 r rnurrocrda P-1059 S L I ~ I I I CVIIIPIF~FIJ Imm failed either to inhibit the proliferation o f microorga nisms in vitro or to produce protective passive imrnunity against infection (Ando and Baba 1969).
.
r, I
,.-.!A
..a--.-
a-
It was also rep0rted that jrotection is not redl in burse:ctomised immune chickens iin contras thymectc (Ba ba et a1 1 . . 3mised - . imlmune . . chi1ckens . . . a n a tnat [j-glucuronidase, acld phsophatase a] lysozyme activity is increased in macrophages fro immunised chickens (Yamaguchi and Baba 1974). The purpose of this study was to immuni chickens against P multocida and to determine the protective immunity in vivo and in vitro by trar~sfer of their spleen cells o r the culture supernatant f luid of such splenic cells and to verify thereby the existence mediated immune chickens focida. Materials and methods
Chicken.s : White..-.,1.I.,all exper imints. FcWile eggs obtained 1kom the same source u,ere incubiated a t 37''C.
Hormonal oursecromv Hormlonal bur sectomies were performed by allantoic injection of te:stosterone propi01nate Each 6:gg was injected with 2 mg testosterlone propi~ onate in 01.1 ml cor embryon,al days of ' incubatioIn. I".
\
urganrsr The vi~rulentP multocida srrajn r-low, serotype 8:A, was used. It had a minimal lethal dose (MLD) of 560 viabl e cells when introduced into seven-week-old -L:-,.--urllcnern bv the intravenous route.
Vaccine ,and immu nisation
. Forrna - ------.lin-killed vacclne
,
. was used..
-
(FKV)
p
multocida grown for 18 hours in brain-heart infu!$ion broth by shaking cultures was centrifuged and washed twice in phosphate buffered saline (pes). The bacteria were then suspended in PBS containing U ' rnL per cent formalin t o give 1 0) mg ~ ml-1 (wet weig ht). The suspension was incubate:d for two days at 3'7OC and store:d at 5°C. A
,
Baba
ruur-wetx-UIU C I I I C K C ~ S were immunisea suocutaneously with 0.5 ml FKV. Seven days later i1 second dose of 0.2 ml FKV was administered intra venously. Two weeks after the second immunisinl lse the chickens were used for preparation of spleen
'reparatior rld challen
ger of sphenic lymptioid cells
(ere prepa.red from spleens o,f Cell susp . *.:,cnlcKen8 . . nmunised ana unimmun~sea at sevel eeks old (two weeks after ttie second immunisa on). The spleens were finely m~incedwitkI scissors i~ Ad medium 199 and then pipetted .u..p a nd down :w times. The cell suspensions were filtered througlh ylon gauze to remove aggregated cells and tissu e ~bris. The cells were washed three times b Y zntrifugation at 500 rprn for 10 minutes with coltd medium 199. These washed splenic cells were layerel over Lymphoprep (Nyegaard) and were centrifuge1 at 1300 rpm for 30 minutes. After centrifugation., cells of the middle lavers (the lvmvhocvte riclh . petting, a nd- washec3 action) were remo! vice with c,old mediu iperitonea Ily (at five Chickens . . . were injt . eeks) or lntravenously (at nine weeKs) wlrn I x lu :[Is of splenic lymphocytes per bird. The chicken s ven an intraperitoneal transfer of spleen cells wer e oculated intraperitoneally with 200 MLD of th e -1059 strain of P multocida 18 hours after the cellI1 ansfer. C hickens given cells intravenc~ u s l y werie oculated i ntravenou sly with I(30 MLD of the P-105'9 rain 24 hours. after-.. t he cell tra nsfer. . The protective ettects were assessea oy observln;g le birds for 20 days after chal lenge and g [em with c ontrol binds (Tables 1 and 2). -3
-A
I
.
. . a .
. . n
.
repararron ana rransjer oj spleen cell culrure rpernatanl fluids and challenge
Splenic lymphoid cells were obtained frcliri immunised or unimmunised hormonally bursectomised chickens as outlined above. The cells were adjusted to 1 x lo7 cell ml-I in Eagle's minimum sential metdium (ME M) which included 20 per cent :at-inactiv,ated norm~ a chicken l serum, 100 iu ml-I :nicillin a ]7d 100 pg: ml-I streptomycin. The cell . _. : . . into tissue culture bottles (20 ispension was plperted ml per bottle) and 100 pg ml-' washec3 FKV w a5 added to each bottle. After 24 hours inculbation in ;1 37"C, 5 per cent carbon dioxide incubat'or, culturc : - .was collected by centrifugation at 5°C at -sllpernatant 8C0 rpm for 10 minules and th e superna tant fluid!j filtered through a Sartorius mernbrane fillier of port: sii!e 0.45 prr1.
.....
-. . .
. . . lntraperironea~ly . were .injecrea (at weeks:) or intravenously (at seven weeks) with 1 cultur~ e supernatant fluid. For challenge, the ch receivi.ng cells transferred intraperitoneally inoculated intravenously with 100 MLD of the 1'-1059 strain five hours after the culture supernatant treatment. The intravenously transferred group we1re also inoculated intravenously with 100 MLD of the 1?-I059 strain three hourrs after thc:treatmen t. The protective:effects vvere assess,ed by observing the bil*ds for 20 days aftel. challenge: and com paring the two groups (Tables 3 a~ n d4). Lhl(:Kens
oneal macrophages r
ion
Mac:rophage preparatic)n follow^ed a pre\ descrilbed technique (BabaI 1975). Peri~tonealmacrophage!j were colllected twc3 days nnln, tin PBS ,, I . , ..,a, after t he administration of , solutic)n into thle peritoneal cavity ()f immunisea or unimnnunised ctlickens. A,bout 106 cells in 1 ml of Eagle' S MEM SUpplemente:d with 2( per cent heat, inacti! . ....... fatea normal chicken serum In a Lab-Tex. tissue cultur~ e chamber slide, we re allowed1 to incub;ste for three 1hours at 37 ° C in a 5 per cent: carbon d ioxide incubsLtor. h l r \ - nrlhnra.., L I W l I - a u i I c I c I I t C C I I ~W C I C I C I I I W V L U uy v v a a t l t l t ej """ cold I- tanks' sol1 tio on. The n 0.4 ml of antibiot ic free Eagle' s SEM in vvhich was suspended 40 pg (2() pg in the ex periment r;hown in I=ig 3) of live P-1059 strain o t r multocida was aaaea into each chamber. A Iter reincubation for three hours, extracellular b;~cteria were removed by washing four times with Eagle's iu MEM. Finally, 0 . 4 ml of the medium containinl3 10 .. penicil lin was placed into each chamber, f o l l o v ~ -hlf ~~ furthe r incubation. tively, Aftt:r two, five, eight 2 chaml:ler slides were stained with tilemsa sol ution. .- - ~ The ntlmber of i ntracelluliar bacteria. p e r 1 0 0nacruphage:i and the surviving nnacrophag :es were ccbunted micros,topically.
.... . ........
- A
-
I
c r .
1 ,
.
A
0.
Sensitisalron and rnJectron oJ rllacrophage culi with splenic lymphocyfe culture supernalant jl
Normal macrophages obtained from the unimmunised chickens in the same way as ind icated above were cultured in an equal volume of splec:n cell culture supernatant fluid from immunised o~r unimmur~isedchickens. Afte r non-adh erent cellsi were removc:d, the remaining ceIls were in cubated with an equal . . . lolume of fresh med ium for . 24 . . hours ,... an(. leach challenged with the live P-lu3r srraln or rr?rulro.;& For th is bacteria I were adcied at a concentrati 20 pg 1nl-I. Subsequent tr eatment a nd observ, were a!s above. Am,.
ckens agar
'ith transfc Ir of splen ic cells
bacterial challenge 18 and 24 hours after transfer of spleen cells from immunised or unimmunised chickens intraperitoneally into five-week-old recipients or intravenously into nine-week-old recipients respectively, are shown in Tables 1 and 2. l\bJULIJ
VL
TABLE 1: Mortality i n unimrnunised chic:kens given spleen cells from immunised or unirnmunised t:hickens st~bsequently challenged intraperitoneall)1 w i t h a cultrire of P m u l tocida Mortality in chickens givf Spleen cells' From Frorn immunised unimimunised chickc Number of chickens before challenge
ip treated with All t he recipielnts in botl ckens an(1 the spleen cells fronI u n i m m ~ : medium died control group trc.ated with within 1Five days. -However, chickens treated w ~ t hspleen cells rrom immunised chickens survived a t rates of 67 per cent and 63 per cent respectively, demonstrating protective. transfer by means of immune lymphocvteqThese survival rates reflect approxirrlately the same level of protective effect a s obtainedI in immu nised chickens in a previously reported stttdy (Baba et a1 1978). Protecl ion with trbe transfer ' of spleen supernl;rtant . ..
The results ot bacterial c:hallenge f'ive hours after intraperitoneal transfer of spleen cell culture supernatant fluids from immu nised and unimmu nised chickens into recipients are snown In No siignificant di fferenc:es existec I the number of survi ving chic1
9
Nurnber of days unti11 death
Tot,
Spleen cell transfer: intraperltoneally Challenge: 18 hours after intr?iperitonealtrz~nsferof cells
TABLE 2: Mortality i n unimmunised chickens given spleep cells frorn immunis,ed or uninnmunised t ibsequently challenged intrt da ith a culture
TABLE 3: Mortality i n unimmunlsea cnlcKens inrrapertra~neally given spleen cell culture supernatant from immunised or unimmunised chickens subsequently challenged intravel10usly w i t h a culture of Prnuttocida
in chickens gvc!n -Mortality . No cells
-
tronn immunised chickcms
F unimmun~sed c hckens ~
Nurnber of -L.. :kens before challenge
Lllll
Nurnber of days unti 1 death
-
?nsgcven
Mort Splec From
.-.-.-
.>
chickens
Noce .,
,
ed chickens
Number of chrckens before challenge )f days h 1 2 3
Tot,
Ioral morral~rv
Spll?encell trans1fer: intravenously Chaillenge: 24 hours after intravenous transfer of cells
Challenge, f~ve hours after ~ntraperctonealtransfer of culture supernatant
13
11
11 -
221
Baba
TAIJ L t 4: Mottallty tn unmmnunlsecl chlckens intravenously an spleen cell culture supernatant from immunised or unnunised chickens subsequently challenged intravenously h a culture of Pmultocid
-
S
~ncnlc~enswvt Spleen cells Frorn Fronn nunised irnmuni~sed unirn~ chicke!ns chickens MO~~IIIY
-
I
-da rn
Nurnber of chic:kensbefore challlenge
r
Nunnoer or aavs l death iune
ChaIlenge: thra? hours after intravenclus transfer of culture SUP2rnatant
. .. -...'raceiiuiargrowth oj-Y multociaa ana a~ ~ r v i v n l rates of norr;nal or imr;nune perit oneal mac in vitro culttrre -. . . - . I'he in vltro resistance ot peritoneal macrophages immunised and unimmunisied chickc rltocida is shown in Fig 1. 'eritoneal macrophages from normal L -.I. i chickens showed an increasc- :111 UIC IIUIIIUCI UI ~ntracellularbacteria and rapid disruption of cells after bacterial challenge. This resulted in the disruption of approximately. 80 .per cent and 88 per cent of the ----macrophages after five and 24 hours respectively. I'eritoneal macropha.ges from i mmunised l chickens shc)wed little intracell~ ~ l a rprolif eration 01 ? bacteria anci 97 per cc:nt survive:d even afiter 24 hou rs.
FIG 1: lntracellular prc lifera at ion of P multocida non-trnrnune peritoneaI rnacrophagc?sandtheir st in vitro culture
e spleen cell supernatant
I
Int raceNular growth of P multocida and survival ratles of norn~ amacrophages l exposed to the culture .--..-*--.SUDrrrrururrr> o f anlipen stimulated splenic
-
jpleen ce!Ils of im and unirnmunised .. :monally 1oursrcromlsea CnlcKens were cultures, . anc1 peritoneal macrophages from normal chickens sen sitised in vitro with culture supernatant fluid for 24 hours and then challenged with bacteria to exa mine the survival rate of macrophages and intracelllular bacte#rialprolifieration. F:ig 2 shou~s that ser siti is at ion of norma I chicken :s with irnmune ~leen cell itoneal rn~acrophagc
L
on-lmmune spleen cell culture supernatant
-m .-
$
V,
\
Medium
.
Hours alter lntectlon FIG 2: SLlrvival rates Iof normal rn supernatants of splenic lymphold ~rnrnunlsel d chlckens
sxposed to culture nrnunised and un-
tocida
ckens agar culture supernatant t l u ~ d resulted in survival ot 90 per cent of macrophages, 24 hours after bacterial challenge. However, sensitisation with non-immune spleen cell culture supernatant fluid resulted in d 93 per cent of mat:rophages disruption of Irs respect ively after bacterial at five, 10 a challenge. .
-
were disrupted five hours after bacterial ch allenge. The results of P rnultocida multiplic ation are shown in Fig 3. Unsensitised macrophages showed an increase in intracellular bacteria from five hours arrer bacterial challenge until 10 hours later. In peritoneal macrophages sensitised with non-immune spleen cell culture supernatant, there was little intracellular bacterial multiplication for five hours after bacterial double by ch: illenge but se to apprc L .
I
Medium
1 \
Immune spm l
..
~
v
ion of P mu FIGt 3: Intracellirlar prol~ferat phelges exposedI to culture supernatants frorn immun~sedand unimmunised chicker
Discuss In t vo experiment, normal chlckens acquirea prorecrive immunity by transfer of immune --I--a p n x l 1 :ells. This protection may be regarded a @A*.n i cells or t o anti1body proc1-uced by I~ransferrec cells. However, con:iidering tklat protec tion in bi ,omlse, cnlc,en, was no, IY/U) and th e relative ly early bacterial challenge after transfel: of spleeln cells, the antibody, even th ough ed, seems unlikely to be directly assoc:iated produc~ with protection. with spleen cell The results of iin vivo ex1 -itoneally culture supernal ant giver . , with challenge after five hours (lable 31, o r g~venintravenously, with challenge after three hours (Tab le 41, gave low and high degrees of protection respect ivel y. Although this may be ascribed to various rea sons, one of the factors may be a difference in thle cell population of the cultured spleen cells. For instance, while the spleen cells used in the experiment of Table 2 contain both T and B cells (unbursectomised . . . . . birds), most of the cells contained in the spleen. cells used in the experiment a~f Table r1 were T cells (bursectomised b irds). Assuming that protectiorI is primar ,ily provid~ .;ma c,.,.h T cell mediators ll.rm..h*L llYlllllllVhlllL as macror activating factor) contained in spleen cell ct supernatant, the difference in the absolute numt T cells existing in the spleen cells used in ct would be reflected in the respective activatic111 UL reticuloendothelial phagocytes in recipients and thereby a difference in protection from infectic)n. In consideration of the time of lapse from tra.nsfer .. of spleen cell culture supernatant to bacterial challenge in the Table 3 experiment in whic:h an intraperitoneal route was chosen and in thiIt of Table 4 in which an intravenous route was ch osen, the latter is considered t o induce more rapid sensiitisation and activation of thephagocytic system, prc>dueing a larger protective effect. Furthermore, an other factor may be the number of bacteria use(i for challenge. If there was no difference in the number or T cell mediators, the smaller number of bacteria used for challenge in the Table 4 experiment, which was one-tenth of that used in the Table 3 experiment. .....may haive resulte d in a dif 'ference inI the protc:ctive effect' Macr ophages ft.om non-ir nmune chickens sensitised \
1
1
.
1
-
-
,,
. A
JULll
spleen cell culture supernatan1
..
10 houirs, amounting eventually to the same le! (el of bacteri: 11 growth as in unsensitised macroph ages. Howevctr, the number of intracellular bacter ia in macroplhages sensitised with immune spleen cell culture supernata nt fluid, d ecreased a fter five a] hours t'o approxitnately ha1f the origi nal numbc
macromphoid cells Irma1
Baba .. - tn tne supernatant or spleen cell culture rrom bu rsectomised birds treated with P multocida, were eq'ually well protected from P multocida as pelritoneal macrophages from immunised chickens. This suggests an association of T cell mediators macrophages in ilmmunised th a c t i Z ickens. Sensitisatit crophages from .. .. no1n-immune chiIcl
..
fnrmal m, as m tne present experiment (~amacltlrnl and Ba~ba1974, Baba et a1 1978). Fronn these facts and the above results, it was conclucded that T lymphocyte-medliated imrr unity ..ln..r no;nrt playa o a major role in the protective resistance a& infection with P multocida in fowl cholera. I effected at cellular level, mainly by macroph activating factor activated immune macropha L.-,. wnlcn possess a markedly enhanced bactericiua~ activity
Referel -
ALEXANDER, A. M. & SOLTYS. M. A. (1973) Journal of Cornpararive Pathology 83, 191- 198 ANDO, T. & BABA, T. (1%9) Jupunese Journol of Veterrnary Science 31, supplement 26-27 BABA, T. (1975) Merhods of Experimental lmmunol-* 'JYY *, IG87- 1088 BABA, r., ANDO, 1r. a N U K I N Journal of 1Medical Micro/liology 11. 2181-288 I Avian Disecrses 12. BHASlhI,* - J. L. & BI BERSTEIN, 159-lba .- . - . - -
COLLINS, F. M. (1911) Lornett vererrnarian 67, IUJ-138 DUA. S. K. & MAHESWARAN, S. K. (1978) Avian Drseases 22. 748-764
HEDDLESTON, K.
L. & REBERS, P. A. (1968) Avian Diseases 12, 12s)-134 HEDDLIESTON, K. I REBERS, P. A. & RIT'CHIE, A. E. Journo11of fmmuno110gy 96, 124.-133 HEDDLlESTON, K . 1 & WATK(1, L. P. (1% 5) A vian D h 367-3; 16 r,c ,.n.,, TAD>-U.ARNON, L. Journa11 of Cotr~par alive Medicb 19 YAMAG UCHI, Y . 8r BABA, T. fedinas of the firrr
..
... .
fi
,.
lnrersectionol tongresr of / A M !
~eceivedfor puorrcar~onJune 28, 1983