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Thymectomy in newborn pigs
Thymectomy in Newborn Pigs CARLOS ?z~- ~"4,NA, M.D., G E O R G E A. HALLENBECK, M . D , Ph.D, and ROY G. ';,~:i'~:~')~::.~ER, M.D,, M.R.C.P., Mayo Clinic
Thymecmmy performed soon after birth has been shown to impair certain immunologic responses of nnce, ' s...,4 rats, ~' ~:,,:~4 guinea pigs,~ rabbits,~. ~o hamsters~a,:~ and chickens:~; except for one repot, :~= the consensus is that it fails to do so in the d(~. ~' ~8..~:!Although the role played by the thymus in the immunologic development of some species extends up to or b-eyond the time of birth; it has been thought to occur in the dog considerably earlier during intrauterine tire2' ~s There is indirect evidence, based on morphologic studies, to suggest that this might atso be the case for man? 7 It is not known, however, whemer immune responses of large animals other than the dog are affected by thymectomy performed soon after birth. Since the pig is normally in some respects immunologically incompetent at birth?' TM '~' :''"~:''2s. ~7 studies in this species of the effects of neonatal thymectomy on rejection of allogmffs of skin, on production of circulating antibodies, on certain hematologic findings and on the question o f post-thymectomy "wasting" were thought to be worth while. MATERIALS AND METHODS Pregnant s o w s - s o m e Of common breeds (Poland China, Dumc4ersey, Chester White, Berkshire) a n d some of crosSbreeds-were housed in separate pens, fed a commercial pig feeding mixture, and Offered water ad libitum. After birth the:young pigs were alI0wed to nurse from thNr mothers and were weaned at the age of 5 weeks. A total of 189 animals from 24 different litters were used. Their ages at the From the Sections of Sm'gical Resea~h and Experimental arid Anatomic .Pathology, Mayo Clinic ~and,Mayo Foundation, Rochester, Minnesota.: Dr, Pestana :,z a resident in surgery in the .Mayo G~aduate School of Medicine .(University Of.Minnesota), Rochester. This investigatibn"was .supported in pa~by a grant from the:L0uisW.~d Maud Hi|lFamily Foandatlon. Submi(ted for publicatio~March 5; 71965. 306
time of thymectomy ranged from 2 to 48 hours; more than hNf of the operations were done within the first 24 hours of life.
Technique of Thymectomy The thymus of the pig is~ composed of a bilmeral cervical portion that extends from ~he floor o f the mouth to the thoracic inlet and a single intrathoracic part that lies anterior and to the left of the pericardium. Initially, the cervical portion Of the thymus was removed and an attemp! was made to destroy the intrathoracic portion by roentgen ray irradiation. After it was learned that the thoracic part of the thymus Su~ived the doses of radiation used, a technique w ~ developed for complete surgical removal of both cervical and thoracic portions of the organ. Newborn pigs were anesthetized with ether and placed supine on a plastic board. Body temperature was maintained by an electric heating pad placed under the animal Aseptic technique was used in all operations. ,'-::or removal of the cervical part of the thymus, a midline incision was made from the level of the thyroid cartilage to the sternal manubrium. Retraction of the edges of this incision exposed the larynx, the infrahyoid muscles and the thyroid gland; hhymic tissue laybila~terally between these s!mcmres and the sternomastoid muscles, With the use of Adson dissecting forCeps and mosquito forceps, the thymus was separated gently from contiguous Structures and was removed in one or Several pieces by simple traction, with no need for hemostasis (Fig. I)~ Special cm-e was taken t o remove completely ! h e cephalic end of the thymus which reaches the floor of the mouth and is wrapped around the twelfth cra:nial nerve (Fig. 2). When total thymectomy was to be pertbrmed, a tube w a s inserted through a tracheostomy and controlled positive pressure respiration ,gas begun. Theincision w ~ extended caudally JSR -- VoL V, No. 7 - July t965
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pieces using a fine forceps (Fig. 3). The sternal incision was closed with three interrupted sutures of cotton that passed around the bone; the skin was closed with a continuous suture of 3-0 silk. The tracheostomy tube was removed and the tracheostomy Was closed with two sutures of 5-0 silk~ If one or both pleural spaces had been opened inadvertently, air was aspirated from the chest at the completion of the operation by means of a syringe and a 21 gauge needle.
Technique of Skin Grafting
Figure 1,
Removal of cervical portion of Ihymus~
F{gure g, Cephalic pole of thymus reaches floor of mouth and is wrapped ~round twelfth nerve.
F i g u r e :i.
Aspect of thoracic portion of thymus,
and the sternum was split. The thoracic portion of the thymus was dissected gently from the surface of the pericardium a n d removed in
Ages of pigs at the time of skin grafting ranged from 1 to 6 days. The skin donors were unrelated pigs, 2 t o 4 months of age, Donors were anesthetized with ether. The skin of one flank was clipped, scrubbed with a hexachlorophene solution (pHisoHex) and shaved. U n d e r aseptic conditions, circular full thickness grafts of the Skin were Cut with a cylindrical cutting tool 2 cm..in diameter,i removed with scissors, and stored in a sterile saline solution at room temperature for from 15 minutes to 3 hours until they were grafted. Skin from a nonpigmented area was chosen in order that subsequent color changes could be easily recognized. The recipients w e r e also anesthetized with ether and placed prone on,a pla:~tiC board. Body temperature was maintained by a n electric heating pad placed under the animal Areas of skin in the interscapular and subscapular regions were clipped, scrubbed with warm hexachlorophene solution and shaved~ Tw0 circular incisions tO the, depth o f thesubcutane0us uss e were made w i t h the round cutting to01, c,ne on each side and about 5 mm.,fr0m the, mid!ine( Each piece~0f Skin was removed with a knife, and one of them was save d for auto-: grafting. Hemostasis on t h e resulting raY;/surfaces was accomplished w i t h p r e s s u r e m a i n rained for one t o tfireeminutesr Onlyrarely! was ligation Of a n individual vessel required; All fat attached to the deep surface o f theskin to be grafted was cut away until the roots of the hairs became Visible. The grafts ;were Sutured in place with from 4 t o !2 interrupted sutureg Of 5-0 Silk. A pressure dressing of gauze i:0ver~d three or four turns0f Elastoplast adhesive tape: the left side and au~ografts On the right;
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Postoperative Care and Obsemations ~ c h animal received 75,000 units of penicfl~n and 022. gin. of streptomycin intramuscularly, daily, ~for 10 days after ~rlymeetomy or 8 days after skin grafting. The dressings were removed on the eighth day after grafting, and the p i g s w e r e anesthetized lightly with ether. Specimens were taken from autografts and allografts for microscopic exarnination. From every animal that died during the first two months oflffe, sections were also taken of the full leng~ of ~ e autograft and al!ogra_ft, together with stm'ounding skin: Since baby pigs have a high mortality rate, this afforded additional material: for microscopic study at different stages from I to 57 days after grafting. The tissues obt~ned were fixed in 10% neutral formalin ~ d embedded in paraffin wax. MuP tiple sections, 8 ~ c r o n s hxck, were cut and stained ~t.h hematoxylin and eosin for examination By light microsc0py. AnimNs surMving beyond two months of age were observed for development Of the "wastiai-" g s yna"rome until they Were. 6 or 12 months of age, whenthey were sacNflCed. Postmortem examinations were.performed in all animals. Microscopic sections .were 'taken from ~ e
t o See whether any thymic tissue remained in tg0se mnimNs ha which surgical removal of the nation o f seeti0ns f r o m regions m o s t likely af their relative ; special attentwelfth nerves
in antibody fiter has been reported to occur if administration of antigen is delayed until the age of 6 weeks. ~ To avoid these variables introduced by age, all immunizations were carried out in s u l l y i n g animals after ~ e age of 2 months. Salmonella pullorum antigen* ,:,'as administered as described by E. R. Miller and coworkers2~; that is, one intrapentoneal iNecfion of antigen was given daily for three consecutive days. The dose was 1 ml. of diluted antigen per 100 ml. of estimated blood volume per day. Blood volumes were estimated on the basis of body weight, according to the scale of Hansard and co-workers) ~ Diluted antigen was prepared fresh every day; 1 ml. of unstained concentrated antigen with a turbidity corresponding to 50 on the McFm'land Nephelometer scale was used for every 50 ml. of sterile normal saline containing 1t4 % phenol. The final mixture ,,,,'as equilibrated to a p H o f 8.2 to 8.5 with a few drops of 1.5 normal sodium hydroxide. Eight days after the last injection, the animals were bled, and tests for agglutination were made. Diluted Salmonella pulloram antigen was used in equal amounts with progressive dilutions of pig serum in saline. The tubes were incubated at 37? C. for 12 to 24 hours and agglutination ",'as assessed microscopically. At this time blood ~also w a s obtained f o r determination of hemoglobin concentration and for total and dhS~erential leukocyte counts. RESULTS Forty pigs that served as controls were not operated on. Another 63 animals were addedto the control ~ o u p because they had incomplete thymectomy: 56 underwent what pr0~ed to be ineffecti~,e:r0entgen ray irradiation O f the even ~though t o ~ :thymectomy h a d . been at-
tom~
Dete~inat
of
Seventeen a n i m u s died ~whfie thymectomy was being attempted~: Complete thymectomy was accomplished surgically in 69 pigs, Skiri=Gra~ing. ' : Many pigs died, after corn=
*Ob~ned from Vineland Poult~ Labs., VJnela,'~d~ NewJersey.
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grafts was based on 97 animals, 70 of which were controls and 27 of which were thymectomized. Autografts were successful in all 97 pigs. These grafts became vascularized, remMned normal in color, felt soft and pliable, and had growth of hair thin was evident at 3 weeks. Microscopic sections showed conservation of the normal structures and v ~ o u s stages of healing by the eighth postoperative day. All 97 allografts were rejected, regardless of the status of the thymus. Initially the allografts became revascul~ized, felt soft and pliable, and had normal color, but as early as the third postoperative day some of the grafts began to appear reddened.i Seventy-seven pigs, 56 Controls and 21 thymectomized, survived at least 8 days after grafting, and, in all cases, altografts were:red or dark purple and were either edematous or entrusted at that time (Fig. 4). Biopsies showed that microscopic evidence of the initial phases of rejection was present in 5 of 9 allografts examined three or four days after grafting. More severe changes, including necrosis of areas of epithdium, were present in all o f 6 grafts examined after six'or Seven days, Biopsies of all 77 all0grafts on the eighth day after grafting revealed inflammation, necrosis and infiltration with small round cells; complete shedding of the epithelium had occurred i n specimens from 23 control and I2 thymectomized pigs. Thus first-set aitogeneic skin grafts were rejected in about eight days,both ~n control and thymectomized newborn pigs. Formation of Antibody in' Response to Salmonella pullorum. Eight thymectomized pigs and 12 ; controls received. Salmonella pullorum antigen when 2 months O f : ~ e , ! a n d their agglutination titers were determined Sub-
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Table 1. Antibody Production* by Pigs. 2 Months o/Age: Response to Salmonella pullorum Antigen
!28, ~28; 128 2 5 6 t 2 5 6 , 256, 256,
256
256, 256
512, 512 i024, 1024
512, 512. 512 I024, 2048,.:2048
* Antibody ~titer is e x p r e s s e d a s ~the, r e c i p r o c a l of the h i g h e s t dilution of serum yielding a p o s i t i v e agglutlnat ion.
sequently. •Pigs in both groups responded by formation of antibody in a similar range of titers (Table t). Absence of we nng ~ After, Neonatal Thymeetomy. Ten control andsixthymectomized pigs w e r e sacrificed w h e n 6, months o f age and two Control and :two :thYmeCtomized animals :were observed for t year..~dl animals grew normally. Thymectom!zed pigs sh0wed no evidence of "wasting"; indeed; they could not be distinguished:from controls On gross mspec= ti0n: Furthermore, n o dLfferenees c o t d d be detected between the two gr with respect to the histology o f and spleens. ,
"
,
.
......
%
.....
werei0btained from6 control and "8 thymectomized pigs 24 tO .48 hours after birth:and at::2 months of age. Blood hemoglobin c6nC6nt~a{i0n declined :: insi~nificai
mectomized pigs were 13;400 andi6,9OO,:re: ?: 30 per cubic mil|imeter all two months of age ~P~ 0.02); Thus;fin pigs, thymectomy ~ e a r Figure 4- Allograft fleft) ~and autograft (rigilt) eight clays after grafting.
dur;mg the: first :try.6"months* of life: iii ~ontiol animals.
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Thymectomy performed soon after b i r t h - in mice, rats, hamsters, ' rabbits, guinea pigs and c h i c k e n s - m a y be followed b y tolerance to allogeneic skin grafts, diminished o r a b s e n t Production , of antibodies t o S o m e antigens, lymphopenia, the occurrence o f "wasting" disease,, or a combination of these. This phenomen o n has b e e n explained by t h e assumptions, first, that, cells .destined tO bec0meimmunologically competent and later toreproduce their kind must first be acted on in som e way by the thymusi and, second, that in the animals mentioned~ this -process has not yet occurred at bii'th. The observation tha t thymectomy in new -~ born pigs did not prevent rejection of Skin grafts orproductio n of. antibOdies and Was not followed byl '?wasting" o r lymphopenia could be explained b y any of the following theories: L .The thymus o f the pig.~in contrast to that of.rodents, and chickens, .has n o immunologic functions at all; 2,.It.hag immunolo#c ,fur~ctions, butit shares them~ with, S~-ne other a:~ ~e~ unknown tissue elsewhere in ~he animal 3;.,.'It has im,,,,~unologic f~2~ctkms, but they are performed before birth. Although the first the~,~. ~~-mains a logical possibility until either ; ~ ~'.c other two is .proved, it seems unlikely that the thymus has nothing tO doilwith immunologi c development
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occurs before birth in .the pig, is the same as that. proposed by Archer and co-workers" and Kelly 's for the dog, Their histologic Studies of dogs revealed small lymphocytes in the lymph nodes and Spleen before birth, in contrast to the situation in mice, chickens and rats wherein lymphoid tissue becomes populated with small lymphocytes a f t e r birth,-In the rabbit, whose response to neonatal thymectomy is inconsistent, these changes t ~ e place just about the time of .birth.~.'25 A morphologic study ' o f lymphoid tissue in .pig embryos is in progress in our laboratory, andpreliminary examinations have shown small lymphocytes to be present in the lymphoid tissues before bi~h. If we accept this last theory, we would expect newborn pigs to be immunologically competent, That this is indeed the case with respect to skin grafting was proved by rejection: of aIlografts a few days after birth with a sequence andduration, of events-comparable to those described for. rejection of Such grafts in adult a n i m a l s o f other species, including rabbits, guinea, pigs, mice, rats and cattleW This finding is of interest because newborn pigs havebeen considered in t h e pastlto be immunologically retativelyinc0mpetent~ Several authors have shown that the production of gamma globulin and:of serum antibodies in response to some antigens, including Salmonella puliorum, m does, not occui" until the age of .3 weeks: and is .n0t.:fully developed, until 6 :.weeks. of age?, ,3,)~, 22, S0,eG,~7 Immunity to common infegti0us;diSeaSes in.pig s during, the first few -weeks o f l i f e is normally ..furnished b y anti2 b0d!eslabSorbed from colostrum, However,. the. newb0rn pig's"immun0Iogic. deficiency, is :not abso!ute, ,for Kimi :Bradley .and.WatsonZ ° have days 0fbirth:.A~:similar. Situation exists in: new..
TfilChOm0fias foetus UP to the: 'ag~ o f 4
i-
the
0 f immunologic re-
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sponsivenes s varies from antigen to antigen, and, as Silverstein and associates ~ have remarked, inability t o demonstrate antibody response to certain antigens does not of itself establish overall immunologic incompetence. If it can be accepted, that the thymus participates in the development of immunologic com petence in the pig; it must follow that the thymus plays its role during fetal life because, despite the immuno!og~c deficiencies seen in normal newborn p~gs, thymectomy s!mrtly after birth does not alter subsequent immunologic development. SUMMARY The effects of neonatal thymectomy were studied in the pig. No differences were found in the pattern of rejection of allografts of skin or in the production of antibody in response to -Salmonella puilorum antigen between control animals and animals that underwent total thymectomy within 48 hours of birth. Atlografts were applied within the first week of life, and thymectomized and nonthymectomized animals rejected them in about eight days. Production of Serum antibodies, tested when the pigs became 2 months of age~ was found to be equal in nit animals, No "'wasting, was observed up to one ye~u• after the operation, and-both control and thymectomized •animals had higher Concentrations of.tymphocytes, in. the blood when: 2 : months, of age than at birth.. Various theories to account for t hese~ findings, are discussed, and some Comments aremade about the observation that newborn pigs can reject allografts within a:few days afterbirth :even though other' authors h a v e shown that~ they: are,:in s o m e Other-respects, relatively incompetent immunolo~cally.
REFERENCES 1. Archer, O. K.; Kelly, W. D;, Papermaster,. B. W.; and Good, R. A;::Furthermorph01ogicM and immunOlogical.studies 0f, tbe role 0fthe thymus in immun0biology (Abstr.), Fed. Proc:; 22:599,: 1963. 2, Archer,. 0 , K;,P!erce~ J~ c . , Papermaster( B. W.; and Good,. R: A,: Immunology:. Reduced-antibody:reSp0nse in. thymect0rnized rahbits~. Nature, London 195:19I-.!93~ 1962, 3. Archer;O. K.,.Sutherland, D.:E. R., and Good~ R, A:: Appendix of,t}~e rabblt: A homologtie of the bursa in the chicken? Nattire~ London,; 200:337-339, ~1963. 4; Arnason, B, G., Jankovic, B. D , Waksman, B. H.,a_nd Wennerstea,-C.: Role o f the thym:os in immune?re-
311 actions in rats. I1. Suppressive effect of thymectomy at
binh on reactions of delayed (cellular) hypersensitiv!ty and the ci~ulafing sm~l. tymphocyte. J: Exper. Med. I 16:177-186; t962. 5. Brown, H., Spoor. V. C., Quinn, L. Y., Hays, V. W, and Catron, Dr V.: Studies on colostrum~acquired immunity and active antibody production in baby pigs, J. Anlma! So., 20:323~328, 1961. 6. Dalmasso, A. P., Martinez, C., and Good, R. A.: Further studies of suppression of the homograft reaction by thymectomy in the mouse. Proc, Soc, Exper. Biol. & Med., I 11:143-146, 1962. 7. Fennestad, K. L., and Borg-Petersen,~C.: Antibody and Plasma cells i n bovine fetuses infected with Leptospirc, Saxkoebing. J. Infect.. Dis., 1!0:63-69, 1962. 8. Fichtelius, K.-E., Laurell. G . and Philipsson~ L.: The influence of thymectomy on antibody formaOon, Acta path~ et m!erobioL scandinav;, 5 ! :81-86, 196I, 9. Finstad, J.¢ Papermaster, B. W.; and Good, R. A.: Evolution of the immune response, II. Morphologic studies on ~the origin o f the thymus and Organized lymphoid tissue. Lab. invest,, 13:490-512, t964. 10, Good. R, A., Dalmasso, A. P., Martinez, C,;:Archer; O, K,, Pierce, J, C;, and Papermaster~ B; W,: The role of the thymus indevelopment of immunol0gie capacity in rabbits and mice: J. Exper, Med,, 116:773-796, 1962: 1l, Good, R,A., and Finstad, J,: Phylogeny Of immune reactions. Immunological, Capacity 0£ p01yodon, and other fishes (Abstr3. Fed. Proc.,23:285, ! 964~ 12. Hansard, S.L., Sauberlich. H. E,, and Comar; c . L.: Blood volume of swine. Pr0e. Sot. Exper. Biok:& Med. 78:544-545,. 195 I. 13~ Hoedein, A. B.: The influence of eolostrum o n antibody ~sponse inbaby pigs. J. ImmunoL, 78: 112-t 17, I~57. 14. Jank0v~e, B. D.. Isvancskl, M., Milosevic, D.; and Popeskovie, L.L Immunology: Delayed hypersensitive. reactions in burseetomized chickens. N a t u ~ London, 198:298~299, 1963 15, Jankovic/B. D.,.Waksman, B. H,, and Arnason~ B:.G.: Rote of the thymus in immune i'cactionsin rats: L T h e immunologic responseto, bovine s e ~ m albumin (anti-: body formation, Arthus ~reactivity, anddelayedhypersensitivity) in rats., thymect0mized~or" sPlenectomized at varioias times after:.birth~ J, Exper, Med:, .t16:]59~: 176, t962. 16. Kaeberle, M,, and Segre, D.:Response of baby pigs.to heterologous globulin (Absti~)~."aaet. Pme~.;:62:75~ t962. I7~ Kay; H. E; M., PlayfaJri J:H. L~:Wolfendale;M ,and Hopper. P. K.: Development of the.thymus in :Ihe htiman foetus andits 'relation to immun61~giea~potenfiat: Nature, London.. !96:238-240,: 1962. 18, Kelly, W : D.: ~he thymus ~and :lymphoid m0rphogenesis i n t h e ~dog (Abstr.). :~Fed.,Proc., 22:600;1963. 19. Kerr; W, R.~ arid.Rbbertson; M,::.Passiveiy and'actiVely acquired antibodies for Trichom0n~ ~foecos"~in: very y0ungcalves, J; Hyg,,52:253'~263,i 1954~ 20. Kim; Y, Bi,:Bradley, S:.G.;:aod.Watson,D] W~!Dev¢]5 opment.of.immunoglobulinS in germfree andc6fiWn; tional eolostmm;deprived piglets (Abstr)irFed.':Proc.2 23 !346,~1964. 21 .. ~mpkin, G : H 2 Quoted bY Bil|iegham;: R: E;; Breht~ L , and Medawa~,. P;. B;:-Quantlmtive studies on tisstie transplantaiion, immunily:!; l l t i . Actively:. acci~ired toierance.R0y. S o e L 0 n ~ m Phil, TrY; S~ B~.239:3574i4, t956:
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22. Miller, E. R., Harmon, B. G., Ulh'ey, D. E., Schmidt, D. A., Luecke, R. W., and Hoefer, J. A.: Antibody absorption, retention arid production by the baby pig, J, A n i ~ SJ., 21:309-314. 1962. 23. Miller, ~ R., Ullrey, D. E , Ackerman, 1., Sehmidt~ D. A , Hoefer, J. A., and Luecke, R. Wc Swine hematology from birth to maturity. L Serum proteins. J. Animal Sc., 20:31-35, 1961. 24. Miller, J. F. A. P.: Immunity and the thymus. Lancet, 1:43-45, I963. 25. Moral, H. Z., van ErkeL G. A., and Fernando, N~ V. P.: The lymphoid tissue in the spleen and thymus in newborn and f e ~ rabbits (Abstr,). Fed. Proc., 22:600, I963. 26. N0rdbrlng, F,~ and Olsson, B.: Eleetrophoretic and immunological studies on sera of young pigs-L Influence of ingestion of cotost~-tm on protein pattern and antibody titre in sera from suckling pigs and the changes throughout lactation. Acta So¢. reed. upsal, 62:t93-212, t 957. 27. Olsson, B:: Studies On the formation and absorption of antibodies and immune globulins in piglets. 1: The formation of H ~ u t i n i n s in piglets after vaccination with Salmonella par'atyphi A. Nord. Vetefinaermed., 1t:250-273, 1959. 28. Roos a, R. A,, Wilson, D., and Defendi, V.: Effect of neonatal thymectomy in hamsters {Abstr.); Fed. Proe., 22:599, 1963. 29. Schinckel, P. G , and Ferguson, K. A.: Skin transplan. ration in the foetal lamb. Austin/Jan J. Biol. Sc., 6: 533-546, 1953.
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30. Sherman, J. D., and Dameshek, W.: '*Wasting disease" foffowlng thymectomy in the hamster. Nature, London, 197:469-471, 1963. 31. Silve~teh% A. M , Uhr, J~ W,, Kraner, K. L , and Lukes, R. 3.: Fetal response to antigenic slimulus, It. Antibody p~duclion by the fetal Jamb. J. Exper. Med~, 1 1 7 : 7 9 9 - 8 1 2 , 1963, 32. Ti~rey, N. L , Beattie, E. L ; J r , and E¢onomou, S, G.: The effect of neonatal thymectomy in 1he dog. J. Surf. Res., 5:23-30, 1964. 33, Van de Water~ J. M. and gatzman, H.: Studies ofth~ immune mechanism in thymectomlzed pup.~. J. Surg. Res. 4:387.390, I964, 34. Waksman, B. H , Arnason, B. G,, and Jankovic, B. D,: Role of the thymus in immune reactions in rats. ItI. Changes in the lymphoid organs of thymectomized rats. J. Exper. Meal., 1t6:187-206, I962. 35. Warner, N. L , and Szenberg, A.: lmmuno|ogy: Effect of neonatal thymectomy on the immune response in the chicken. Nature, London, t96:784.785, 1962, 36. Warner, N. L., Szenbe~, A., and Bur~el, F. M.: The immunological role of different lymphoid organs in ~he chicken, l, Dissociation of immunological responsiveness. Australian J. Exper. BioL & Med. So. 40:373. 387, 1962. 37. Woodruff, M. F. A.: The Transplantation of Tissues and Organs. Springfield, Illinois, Charles C Thomas, '.960, 777 pp.
Thymecmmy performed soon after birth has been shown to impair certain immunologic responses of nnce, ' s...,4 rats, ~' ~:,,:~4 guinea pigs,~ rabbits,~. ~o hamsters~a,:~ and chickens:~; except for one repot, :~= the consensus is that it fails to do so in the d(~. ~' ~8..~:!Although the role played by the thymus in the immunologic development of some species extends up to or b-eyond the time of birth; it has been thought to occur in the dog considerably earlier during intrauterine tire2' ~s There is indirect evidence, based on morphologic studies, to suggest that this might atso be the case for man? 7 It is not known, however, whemer immune responses of large animals other than the dog are affected by thymectomy performed soon after birth. Since the pig is normally in some respects immunologically incompetent at birth?' TM '~' :''"~:''2s. ~7 studies in this species of the effects of neonatal thymectomy on rejection of allogmffs of skin, on production of circulating antibodies, on certain hematologic findings and on the question o f post-thymectomy "wasting" were thought to be worth while. MATERIALS AND METHODS Pregnant s o w s - s o m e Of common breeds (Poland China, Dumc4ersey, Chester White, Berkshire) a n d some of crosSbreeds-were housed in separate pens, fed a commercial pig feeding mixture, and Offered water ad libitum. After birth the:young pigs were alI0wed to nurse from thNr mothers and were weaned at the age of 5 weeks. A total of 189 animals from 24 different litters were used. Their ages at the From the Sections of Sm'gical Resea~h and Experimental arid Anatomic .Pathology, Mayo Clinic ~and,Mayo Foundation, Rochester, Minnesota.: Dr, Pestana :,z a resident in surgery in the .Mayo G~aduate School of Medicine .(University Of.Minnesota), Rochester. This investigatibn"was .supported in pa~by a grant from the:L0uisW.~d Maud Hi|lFamily Foandatlon. Submi(ted for publicatio~March 5; 71965. 306
time of thymectomy ranged from 2 to 48 hours; more than hNf of the operations were done within the first 24 hours of life.
Technique of Thymectomy The thymus of the pig is~ composed of a bilmeral cervical portion that extends from ~he floor o f the mouth to the thoracic inlet and a single intrathoracic part that lies anterior and to the left of the pericardium. Initially, the cervical portion Of the thymus was removed and an attemp! was made to destroy the intrathoracic portion by roentgen ray irradiation. After it was learned that the thoracic part of the thymus Su~ived the doses of radiation used, a technique w ~ developed for complete surgical removal of both cervical and thoracic portions of the organ. Newborn pigs were anesthetized with ether and placed supine on a plastic board. Body temperature was maintained by an electric heating pad placed under the animal Aseptic technique was used in all operations. ,'-::or removal of the cervical part of the thymus, a midline incision was made from the level of the thyroid cartilage to the sternal manubrium. Retraction of the edges of this incision exposed the larynx, the infrahyoid muscles and the thyroid gland; hhymic tissue laybila~terally between these s!mcmres and the sternomastoid muscles, With the use of Adson dissecting forCeps and mosquito forceps, the thymus was separated gently from contiguous Structures and was removed in one or Several pieces by simple traction, with no need for hemostasis (Fig. I)~ Special cm-e was taken t o remove completely ! h e cephalic end of the thymus which reaches the floor of the mouth and is wrapped around the twelfth cra:nial nerve (Fig. 2). When total thymectomy was to be pertbrmed, a tube w a s inserted through a tracheostomy and controlled positive pressure respiration ,gas begun. Theincision w ~ extended caudally JSR -- VoL V, No. 7 - July t965
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pieces using a fine forceps (Fig. 3). The sternal incision was closed with three interrupted sutures of cotton that passed around the bone; the skin was closed with a continuous suture of 3-0 silk. The tracheostomy tube was removed and the tracheostomy Was closed with two sutures of 5-0 silk~ If one or both pleural spaces had been opened inadvertently, air was aspirated from the chest at the completion of the operation by means of a syringe and a 21 gauge needle.
Technique of Skin Grafting
Figure 1,
Removal of cervical portion of Ihymus~
F{gure g, Cephalic pole of thymus reaches floor of mouth and is wrapped ~round twelfth nerve.
F i g u r e :i.
Aspect of thoracic portion of thymus,
and the sternum was split. The thoracic portion of the thymus was dissected gently from the surface of the pericardium a n d removed in
Ages of pigs at the time of skin grafting ranged from 1 to 6 days. The skin donors were unrelated pigs, 2 t o 4 months of age, Donors were anesthetized with ether. The skin of one flank was clipped, scrubbed with a hexachlorophene solution (pHisoHex) and shaved. U n d e r aseptic conditions, circular full thickness grafts of the Skin were Cut with a cylindrical cutting tool 2 cm..in diameter,i removed with scissors, and stored in a sterile saline solution at room temperature for from 15 minutes to 3 hours until they were grafted. Skin from a nonpigmented area was chosen in order that subsequent color changes could be easily recognized. The recipients w e r e also anesthetized with ether and placed prone on,a pla:~tiC board. Body temperature was maintained by a n electric heating pad placed under the animal Areas of skin in the interscapular and subscapular regions were clipped, scrubbed with warm hexachlorophene solution and shaved~ Tw0 circular incisions tO the, depth o f thesubcutane0us uss e were made w i t h the round cutting to01, c,ne on each side and about 5 mm.,fr0m the, mid!ine( Each piece~0f Skin was removed with a knife, and one of them was save d for auto-: grafting. Hemostasis on t h e resulting raY;/surfaces was accomplished w i t h p r e s s u r e m a i n rained for one t o tfireeminutesr Onlyrarely! was ligation Of a n individual vessel required; All fat attached to the deep surface o f theskin to be grafted was cut away until the roots of the hairs became Visible. The grafts ;were Sutured in place with from 4 t o !2 interrupted sutureg Of 5-0 Silk. A pressure dressing of gauze i:0ver~d three or four turns0f Elastoplast adhesive tape: the left side and au~ografts On the right;
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Postoperative Care and Obsemations ~ c h animal received 75,000 units of penicfl~n and 022. gin. of streptomycin intramuscularly, daily, ~for 10 days after ~rlymeetomy or 8 days after skin grafting. The dressings were removed on the eighth day after grafting, and the p i g s w e r e anesthetized lightly with ether. Specimens were taken from autografts and allografts for microscopic exarnination. From every animal that died during the first two months oflffe, sections were also taken of the full leng~ of ~ e autograft and al!ogra_ft, together with stm'ounding skin: Since baby pigs have a high mortality rate, this afforded additional material: for microscopic study at different stages from I to 57 days after grafting. The tissues obt~ned were fixed in 10% neutral formalin ~ d embedded in paraffin wax. MuP tiple sections, 8 ~ c r o n s hxck, were cut and stained ~t.h hematoxylin and eosin for examination By light microsc0py. AnimNs surMving beyond two months of age were observed for development Of the "wastiai-" g s yna"rome until they Were. 6 or 12 months of age, whenthey were sacNflCed. Postmortem examinations were.performed in all animals. Microscopic sections .were 'taken from ~ e
t o See whether any thymic tissue remained in tg0se mnimNs ha which surgical removal of the nation o f seeti0ns f r o m regions m o s t likely af their relative ; special attentwelfth nerves
in antibody fiter has been reported to occur if administration of antigen is delayed until the age of 6 weeks. ~ To avoid these variables introduced by age, all immunizations were carried out in s u l l y i n g animals after ~ e age of 2 months. Salmonella pullorum antigen* ,:,'as administered as described by E. R. Miller and coworkers2~; that is, one intrapentoneal iNecfion of antigen was given daily for three consecutive days. The dose was 1 ml. of diluted antigen per 100 ml. of estimated blood volume per day. Blood volumes were estimated on the basis of body weight, according to the scale of Hansard and co-workers) ~ Diluted antigen was prepared fresh every day; 1 ml. of unstained concentrated antigen with a turbidity corresponding to 50 on the McFm'land Nephelometer scale was used for every 50 ml. of sterile normal saline containing 1t4 % phenol. The final mixture ,,,,'as equilibrated to a p H o f 8.2 to 8.5 with a few drops of 1.5 normal sodium hydroxide. Eight days after the last injection, the animals were bled, and tests for agglutination were made. Diluted Salmonella pulloram antigen was used in equal amounts with progressive dilutions of pig serum in saline. The tubes were incubated at 37? C. for 12 to 24 hours and agglutination ",'as assessed microscopically. At this time blood ~also w a s obtained f o r determination of hemoglobin concentration and for total and dhS~erential leukocyte counts. RESULTS Forty pigs that served as controls were not operated on. Another 63 animals were addedto the control ~ o u p because they had incomplete thymectomy: 56 underwent what pr0~ed to be ineffecti~,e:r0entgen ray irradiation O f the even ~though t o ~ :thymectomy h a d . been at-
tom~
Dete~inat
of
Seventeen a n i m u s died ~whfie thymectomy was being attempted~: Complete thymectomy was accomplished surgically in 69 pigs, Skiri=Gra~ing. ' : Many pigs died, after corn=
*Ob~ned from Vineland Poult~ Labs., VJnela,'~d~ NewJersey.
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grafts was based on 97 animals, 70 of which were controls and 27 of which were thymectomized. Autografts were successful in all 97 pigs. These grafts became vascularized, remMned normal in color, felt soft and pliable, and had growth of hair thin was evident at 3 weeks. Microscopic sections showed conservation of the normal structures and v ~ o u s stages of healing by the eighth postoperative day. All 97 allografts were rejected, regardless of the status of the thymus. Initially the allografts became revascul~ized, felt soft and pliable, and had normal color, but as early as the third postoperative day some of the grafts began to appear reddened.i Seventy-seven pigs, 56 Controls and 21 thymectomized, survived at least 8 days after grafting, and, in all cases, altografts were:red or dark purple and were either edematous or entrusted at that time (Fig. 4). Biopsies showed that microscopic evidence of the initial phases of rejection was present in 5 of 9 allografts examined three or four days after grafting. More severe changes, including necrosis of areas of epithdium, were present in all o f 6 grafts examined after six'or Seven days, Biopsies of all 77 all0grafts on the eighth day after grafting revealed inflammation, necrosis and infiltration with small round cells; complete shedding of the epithelium had occurred i n specimens from 23 control and I2 thymectomized pigs. Thus first-set aitogeneic skin grafts were rejected in about eight days,both ~n control and thymectomized newborn pigs. Formation of Antibody in' Response to Salmonella pullorum. Eight thymectomized pigs and 12 ; controls received. Salmonella pullorum antigen when 2 months O f : ~ e , ! a n d their agglutination titers were determined Sub-
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Table 1. Antibody Production* by Pigs. 2 Months o/Age: Response to Salmonella pullorum Antigen
!28, ~28; 128 2 5 6 t 2 5 6 , 256, 256,
256
256, 256
512, 512 i024, 1024
512, 512. 512 I024, 2048,.:2048
* Antibody ~titer is e x p r e s s e d a s ~the, r e c i p r o c a l of the h i g h e s t dilution of serum yielding a p o s i t i v e agglutlnat ion.
sequently. •Pigs in both groups responded by formation of antibody in a similar range of titers (Table t). Absence of we nng ~ After, Neonatal Thymeetomy. Ten control andsixthymectomized pigs w e r e sacrificed w h e n 6, months o f age and two Control and :two :thYmeCtomized animals :were observed for t year..~dl animals grew normally. Thymectom!zed pigs sh0wed no evidence of "wasting"; indeed; they could not be distinguished:from controls On gross mspec= ti0n: Furthermore, n o dLfferenees c o t d d be detected between the two gr with respect to the histology o f and spleens. ,
"
,
.
......
%
.....
werei0btained from6 control and "8 thymectomized pigs 24 tO .48 hours after birth:and at::2 months of age. Blood hemoglobin c6nC6nt~a{i0n declined :: insi~nificai
mectomized pigs were 13;400 andi6,9OO,:re: ?: 30 per cubic mil|imeter all two months of age ~P~ 0.02); Thus;fin pigs, thymectomy ~ e a r Figure 4- Allograft fleft) ~and autograft (rigilt) eight clays after grafting.
dur;mg the: first :try.6"months* of life: iii ~ontiol animals.
310
PESTANA ET AL. COMME~ 1
Thymectomy performed soon after b i r t h - in mice, rats, hamsters, ' rabbits, guinea pigs and c h i c k e n s - m a y be followed b y tolerance to allogeneic skin grafts, diminished o r a b s e n t Production , of antibodies t o S o m e antigens, lymphopenia, the occurrence o f "wasting" disease,, or a combination of these. This phenomen o n has b e e n explained by t h e assumptions, first, that, cells .destined tO bec0meimmunologically competent and later toreproduce their kind must first be acted on in som e way by the thymusi and, second, that in the animals mentioned~ this -process has not yet occurred at bii'th. The observation tha t thymectomy in new -~ born pigs did not prevent rejection of Skin grafts orproductio n of. antibOdies and Was not followed byl '?wasting" o r lymphopenia could be explained b y any of the following theories: L .The thymus o f the pig.~in contrast to that of.rodents, and chickens, .has n o immunologic functions at all; 2,.It.hag immunolo#c ,fur~ctions, butit shares them~ with, S~-ne other a:~ ~e~ unknown tissue elsewhere in ~he animal 3;.,.'It has im,,,,~unologic f~2~ctkms, but they are performed before birth. Although the first the~,~. ~~-mains a logical possibility until either ; ~ ~'.c other two is .proved, it seems unlikely that the thymus has nothing tO doilwith immunologi c development
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occurs before birth in .the pig, is the same as that. proposed by Archer and co-workers" and Kelly 's for the dog, Their histologic Studies of dogs revealed small lymphocytes in the lymph nodes and Spleen before birth, in contrast to the situation in mice, chickens and rats wherein lymphoid tissue becomes populated with small lymphocytes a f t e r birth,-In the rabbit, whose response to neonatal thymectomy is inconsistent, these changes t ~ e place just about the time of .birth.~.'25 A morphologic study ' o f lymphoid tissue in .pig embryos is in progress in our laboratory, andpreliminary examinations have shown small lymphocytes to be present in the lymphoid tissues before bi~h. If we accept this last theory, we would expect newborn pigs to be immunologically competent, That this is indeed the case with respect to skin grafting was proved by rejection: of aIlografts a few days after birth with a sequence andduration, of events-comparable to those described for. rejection of Such grafts in adult a n i m a l s o f other species, including rabbits, guinea, pigs, mice, rats and cattleW This finding is of interest because newborn pigs havebeen considered in t h e pastlto be immunologically retativelyinc0mpetent~ Several authors have shown that the production of gamma globulin and:of serum antibodies in response to some antigens, including Salmonella puliorum, m does, not occui" until the age of .3 weeks: and is .n0t.:fully developed, until 6 :.weeks. of age?, ,3,)~, 22, S0,eG,~7 Immunity to common infegti0us;diSeaSes in.pig s during, the first few -weeks o f l i f e is normally ..furnished b y anti2 b0d!eslabSorbed from colostrum, However,. the. newb0rn pig's"immun0Iogic. deficiency, is :not abso!ute, ,for Kimi :Bradley .and.WatsonZ ° have days 0fbirth:.A~:similar. Situation exists in: new..
TfilChOm0fias foetus UP to the: 'ag~ o f 4
i-
the
0 f immunologic re-
JSR - VoL V, No. 7 - July t965
sponsivenes s varies from antigen to antigen, and, as Silverstein and associates ~ have remarked, inability t o demonstrate antibody response to certain antigens does not of itself establish overall immunologic incompetence. If it can be accepted, that the thymus participates in the development of immunologic com petence in the pig; it must follow that the thymus plays its role during fetal life because, despite the immuno!og~c deficiencies seen in normal newborn p~gs, thymectomy s!mrtly after birth does not alter subsequent immunologic development. SUMMARY The effects of neonatal thymectomy were studied in the pig. No differences were found in the pattern of rejection of allografts of skin or in the production of antibody in response to -Salmonella puilorum antigen between control animals and animals that underwent total thymectomy within 48 hours of birth. Atlografts were applied within the first week of life, and thymectomized and nonthymectomized animals rejected them in about eight days. Production of Serum antibodies, tested when the pigs became 2 months of age~ was found to be equal in nit animals, No "'wasting, was observed up to one ye~u• after the operation, and-both control and thymectomized •animals had higher Concentrations of.tymphocytes, in. the blood when: 2 : months, of age than at birth.. Various theories to account for t hese~ findings, are discussed, and some Comments aremade about the observation that newborn pigs can reject allografts within a:few days afterbirth :even though other' authors h a v e shown that~ they: are,:in s o m e Other-respects, relatively incompetent immunolo~cally.
REFERENCES 1. Archer, O. K.; Kelly, W. D;, Papermaster,. B. W.; and Good, R. A;::Furthermorph01ogicM and immunOlogical.studies 0f, tbe role 0fthe thymus in immun0biology (Abstr.), Fed. Proc:; 22:599,: 1963. 2, Archer,. 0 , K;,P!erce~ J~ c . , Papermaster( B. W.; and Good,. R: A,: Immunology:. Reduced-antibody:reSp0nse in. thymect0rnized rahbits~. Nature, London 195:19I-.!93~ 1962, 3. Archer;O. K.,.Sutherland, D.:E. R., and Good~ R, A:: Appendix of,t}~e rabblt: A homologtie of the bursa in the chicken? Nattire~ London,; 200:337-339, ~1963. 4; Arnason, B, G., Jankovic, B. D , Waksman, B. H.,a_nd Wennerstea,-C.: Role o f the thym:os in immune?re-
311 actions in rats. I1. Suppressive effect of thymectomy at
binh on reactions of delayed (cellular) hypersensitiv!ty and the ci~ulafing sm~l. tymphocyte. J: Exper. Med. I 16:177-186; t962. 5. Brown, H., Spoor. V. C., Quinn, L. Y., Hays, V. W, and Catron, Dr V.: Studies on colostrum~acquired immunity and active antibody production in baby pigs, J. Anlma! So., 20:323~328, 1961. 6. Dalmasso, A. P., Martinez, C., and Good, R. A.: Further studies of suppression of the homograft reaction by thymectomy in the mouse. Proc, Soc, Exper. Biol. & Med., I 11:143-146, 1962. 7. Fennestad, K. L., and Borg-Petersen,~C.: Antibody and Plasma cells i n bovine fetuses infected with Leptospirc, Saxkoebing. J. Infect.. Dis., 1!0:63-69, 1962. 8. Fichtelius, K.-E., Laurell. G . and Philipsson~ L.: The influence of thymectomy on antibody formaOon, Acta path~ et m!erobioL scandinav;, 5 ! :81-86, 196I, 9. Finstad, J.¢ Papermaster, B. W.; and Good, R. A.: Evolution of the immune response, II. Morphologic studies on ~the origin o f the thymus and Organized lymphoid tissue. Lab. invest,, 13:490-512, t964. 10, Good. R, A., Dalmasso, A. P., Martinez, C,;:Archer; O, K,, Pierce, J, C;, and Papermaster~ B; W,: The role of the thymus indevelopment of immunol0gie capacity in rabbits and mice: J. Exper, Med,, 116:773-796, 1962: 1l, Good, R,A., and Finstad, J,: Phylogeny Of immune reactions. Immunological, Capacity 0£ p01yodon, and other fishes (Abstr3. Fed. Proc.,23:285, ! 964~ 12. Hansard, S.L., Sauberlich. H. E,, and Comar; c . L.: Blood volume of swine. Pr0e. Sot. Exper. Biok:& Med. 78:544-545,. 195 I. 13~ Hoedein, A. B.: The influence of eolostrum o n antibody ~sponse inbaby pigs. J. ImmunoL, 78: 112-t 17, I~57. 14. Jank0v~e, B. D.. Isvancskl, M., Milosevic, D.; and Popeskovie, L.L Immunology: Delayed hypersensitive. reactions in burseetomized chickens. N a t u ~ London, 198:298~299, 1963 15, Jankovic/B. D.,.Waksman, B. H,, and Arnason~ B:.G.: Rote of the thymus in immune i'cactionsin rats: L T h e immunologic responseto, bovine s e ~ m albumin (anti-: body formation, Arthus ~reactivity, anddelayedhypersensitivity) in rats., thymect0mized~or" sPlenectomized at varioias times after:.birth~ J, Exper, Med:, .t16:]59~: 176, t962. 16. Kaeberle, M,, and Segre, D.:Response of baby pigs.to heterologous globulin (Absti~)~."aaet. Pme~.;:62:75~ t962. I7~ Kay; H. E; M., PlayfaJri J:H. L~:Wolfendale;M ,and Hopper. P. K.: Development of the.thymus in :Ihe htiman foetus andits 'relation to immun61~giea~potenfiat: Nature, London.. !96:238-240,: 1962. 18, Kelly, W : D.: ~he thymus ~and :lymphoid m0rphogenesis i n t h e ~dog (Abstr.). :~Fed.,Proc., 22:600;1963. 19. Kerr; W, R.~ arid.Rbbertson; M,::.Passiveiy and'actiVely acquired antibodies for Trichom0n~ ~foecos"~in: very y0ungcalves, J; Hyg,,52:253'~263,i 1954~ 20. Kim; Y, Bi,:Bradley, S:.G.;:aod.Watson,D] W~!Dev¢]5 opment.of.immunoglobulinS in germfree andc6fiWn; tional eolostmm;deprived piglets (Abstr)irFed.':Proc.2 23 !346,~1964. 21 .. ~mpkin, G : H 2 Quoted bY Bil|iegham;: R: E;; Breht~ L , and Medawa~,. P;. B;:-Quantlmtive studies on tisstie transplantaiion, immunily:!; l l t i . Actively:. acci~ired toierance.R0y. S o e L 0 n ~ m Phil, TrY; S~ B~.239:3574i4, t956:
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22. Miller, E. R., Harmon, B. G., Ulh'ey, D. E., Schmidt, D. A., Luecke, R. W., and Hoefer, J. A.: Antibody absorption, retention arid production by the baby pig, J, A n i ~ SJ., 21:309-314. 1962. 23. Miller, ~ R., Ullrey, D. E , Ackerman, 1., Sehmidt~ D. A , Hoefer, J. A., and Luecke, R. Wc Swine hematology from birth to maturity. L Serum proteins. J. Animal Sc., 20:31-35, 1961. 24. Miller, J. F. A. P.: Immunity and the thymus. Lancet, 1:43-45, I963. 25. Moral, H. Z., van ErkeL G. A., and Fernando, N~ V. P.: The lymphoid tissue in the spleen and thymus in newborn and f e ~ rabbits (Abstr,). Fed. Proc., 22:600, I963. 26. N0rdbrlng, F,~ and Olsson, B.: Eleetrophoretic and immunological studies on sera of young pigs-L Influence of ingestion of cotost~-tm on protein pattern and antibody titre in sera from suckling pigs and the changes throughout lactation. Acta So¢. reed. upsal, 62:t93-212, t 957. 27. Olsson, B:: Studies On the formation and absorption of antibodies and immune globulins in piglets. 1: The formation of H ~ u t i n i n s in piglets after vaccination with Salmonella par'atyphi A. Nord. Vetefinaermed., 1t:250-273, 1959. 28. Roos a, R. A,, Wilson, D., and Defendi, V.: Effect of neonatal thymectomy in hamsters {Abstr.); Fed. Proe., 22:599, 1963. 29. Schinckel, P. G , and Ferguson, K. A.: Skin transplan. ration in the foetal lamb. Austin/Jan J. Biol. Sc., 6: 533-546, 1953.
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30. Sherman, J. D., and Dameshek, W.: '*Wasting disease" foffowlng thymectomy in the hamster. Nature, London, 197:469-471, 1963. 31. Silve~teh% A. M , Uhr, J~ W,, Kraner, K. L , and Lukes, R. 3.: Fetal response to antigenic slimulus, It. Antibody p~duclion by the fetal Jamb. J. Exper. Med~, 1 1 7 : 7 9 9 - 8 1 2 , 1963, 32. Ti~rey, N. L , Beattie, E. L ; J r , and E¢onomou, S, G.: The effect of neonatal thymectomy in 1he dog. J. Surf. Res., 5:23-30, 1964. 33, Van de Water~ J. M. and gatzman, H.: Studies ofth~ immune mechanism in thymectomlzed pup.~. J. Surg. Res. 4:387.390, I964, 34. Waksman, B. H , Arnason, B. G,, and Jankovic, B. D,: Role of the thymus in immune reactions in rats. ItI. Changes in the lymphoid organs of thymectomized rats. J. Exper. Meal., 1t6:187-206, I962. 35. Warner, N. L , and Szenberg, A.: lmmuno|ogy: Effect of neonatal thymectomy on the immune response in the chicken. Nature, London, t96:784.785, 1962, 36. Warner, N. L., Szenbe~, A., and Bur~el, F. M.: The immunological role of different lymphoid organs in ~he chicken, l, Dissociation of immunological responsiveness. Australian J. Exper. BioL & Med. So. 40:373. 387, 1962. 37. Woodruff, M. F. A.: The Transplantation of Tissues and Organs. Springfield, Illinois, Charles C Thomas, '.960, 777 pp.