any,‘ |Hl|~| HIM, i I I WW — _1___ — __—— _._—— — —____ _—— :.,l,m‘:'ior\ M“ VACCiNAL WMUNTY 'l ‘. ifw‘iiT iiélCTEFlHW 58087315, WFECTICN f\ CUREAHGE Thesis for Degree of M. S. J. P. Torrey I926 .7 ‘ u‘ . . . . ..Ir..”.0h'0 “lurk... . l‘ a . ‘ In 3+1...) ‘ iuui‘I‘I‘l‘ I I 5“”. A I . I ..7'. In .I'lvlél I'm. » n0|ll¥. riatH.C...-n4¢0¢“ I . '1'. FIT“... . n .n. c. .1?” a ‘I III.‘ Y1 I, I. t lid 1 .1. .n.‘ |I “ l‘ ‘ I . h .4-xnl. ‘I ‘ v I ~' , V v '. O,‘ _. i o .1 .‘ 0 CC; .‘Qr C I V u ‘ g c _ \IN% I y b n L § ‘ . II.V v .y . THES‘S .THE DUdATION OF VACCINAL ILZMUNITY IN GUINEA-PIGS AGAINST BACTERIUM ABORE§ INFECTION THE DURATION OF VACCINAL IMMUNITY IN GUINEA-PIGS AGAINST BACTERIUM ABORTUS INFECTION [THESIS ‘Submittod to the Faculty of the Michigan State College in partial fulfillment of the requirements for the dbgree of Master of Science. By 'J. P. Torrey 'June 1326 THESQS 'CONTENTS ‘Introduction Review of Literature Method of Investigation Preparation and use of'Vaccine Distribution of Pigs Method of Exposure Preparation of Infectious Material Method of Determining Infection Experimental Data General Discussion Summary Acknowledgment References I ~- *4 ( '2: Is iilfl C a " -THE DURATION OF VACCINAL IMMUNITY IN GUINEA-PIGS 'AGAINST QéOTERIUM ABORTUS INFECTION. .INTRODUCTION The pathogenicity of Bacterium abortus for guinea-pigs has been recognized for about fifteen years. During this time these animals have been used for both diagnostic and experimental work. The lesions caused in guinea-pigs by infection from this organism are very out- standing and clear, so that it is not difficult to diagnose. Schroeder and Cotton (1) were the first eXperimenters to describe the lesions in the organs of guinea-pigs due to £233. abortus infection. The guinea-pigs had been fed milk containing this organism. The gross anatomical lesions were described as extreme enlargement and edema of the lymph glands gnnerally; the appearance of small glistening nodules in the lungs which seem to be caused by the enlargement of minute lymph glands that were ordinarily too small to be visible; the conversion of the minute nodules in the lungs into larger, necrotic areas; an enormous enlargement of the spleen, often to thirty or forty times its normal volume; an irregular thickening of the capsule of the Spleen, through which its surface becomes marked with white areas varying in size from mere points to several centimeters in diameter; an enlargement and degeneration of the liver, which organ becomes thickly beset on surface and section with irregular, pale yellow or dirty white areas that seem to be due to an enormous proliferation of connective tissue and a consequent crowding out and obliteration of the liver cells proper; a diquse, parenchymatous nephritis that reaches stages in which dense, fibrous nodules are formed in the cortex of. the.kidneys; and, in male guinea-pigs, a degeneration of the testicles, commonly beginning in the epididymis and often in the conversion of one or both testicles into structureless cysts filled with creamy pus. The lesions in.§ggt. abortus infected guinea- pigs have been described microscOpically by Seyfarth (8). The nodules in the lungs were scattered throughout the entire organ, and consisted of accumulations of lymphoid cells and groups of epitheloid cells. The nodules were Just beneath the pleura or about the blood vessels or bronchi. The interstitial tissue was extensively involved with small nodules. The structure of the nodules was similar to if not identical with, the tuberculous nodules. The spleen was found to contain greatly dilated blood sinuses, and there was marked hyperplasia of the endothelial cells in and around the malpighian corpuscles, with an active cellular proliferation of the splenic pulp. The epithelioid cells were observed in groups chiefly in relation to the malphighian bodies and directly beneath the capsule. Polymorphonuclear leukocytes were numerous. An occasional giant cell was distinguished. 3. £222. abortus which has been grown on artificial media does not always produce lesions when injected into guinea-pigs. Some of the virulence seems to be lost when grown under laboratory conditions. Organisms of low virulence have been used to produce immunity but the results are not always certain as there is some danger of an increase in the virulence of organisms wnich have been passed through animals. Huddleson (2) has developed a non-virulent strain of 2222. abortus which seems to produce a fairly high degree of immunity. The use of this organism in a vaccine to protect animals against infection from virulent cultures of £223. abortus has made it necessary to determine how long this immunity will last. The necessity for knowing the duration of this immunity has prompted the carrying out of this experiment. It is the purpose of this eXperiment to show how long immunity to 2223, abortus infection will last in guinea-pigs by injecting them with a non-virulent strain of 2222. abortus then feeding these guinea-pigs a virulent strain of oovine 2222. abortus. The presence or aosence of characteristic lesions in the spleen, liver, lungs, or testes of other organs and the iBOlation of 2221. abortus there-from determines whether or not the animal is immuned. 4. REVIEW or LITERATURE The literature reveals very little research pertaining to the duration of immunity in.animals which have been treated by a vaccine or other means of immunization. This is not only true ongggt. abortus but of other vaccines Is well. The duration of immunity in animals vaccinated with 2222 abortus has been stated in same instances but it has not been proved by experimental work. Certain investi- gators have drawn conclusions concerning the duration of immunity from.a few experiments which had been planned for other purposes. There has been no work done to show the duration of immunity acquired by injecting guinea-pigs with a non- virulent strain of gagt. abortus. The only other experimental work of this nature with guinea-pigs has been done by Calmmtte, Negro, and Boguet (4). They injected bacilli of a non-virulent strain of Bacterium tuberculosum into the blood circulation of guinea-pigs and rabbits and obtained only temporary immunity as was shown by subsequent infection with virulent organisms. Immunity seemed to last for about five months in guinea-pigs and about six months in the rabbit. The immunity gradually became weaker and seemed to disappear as soon as the tubercle bacilli were entirely eliminated from the body or reabsorbed. These workers drew the conclusion that the émmunity may be prolonged by renewing periodically the vaccinal injection 5. of the lymphatic organs. Schroeder’amd Cotton.(5) have immunized guinea- pigs with a strain of 2222. abortus which had host much of its original virulence for guinea-pigs and which caused lesions in them only when injected in very large doses. Thirty-three guinea-pigs were injected with the organism. Eightybone days later 12 of them with twelve controls were injected subcutaneously with a virulent bovine strain of 2222. abortus. Nine of the treated pigs showed no micro- scopic lesions, two showed slight lesions, and one showed marked lesions. 0f the 12 check, nine showed marked lesions and 5 slight lesions. 145 days after treatnr: nt 10 of the immunized pigs with 12 checks were exposed to a virulent strain of 2222. abortion. Five of the treated pigs showed no lesions, four showed slight lesions and l more marked lesions. Seven of the twelve check showed extensive lesions, four showed slight lesions, and one showed no lesions. The author concluded from his experiment that a high degree of resistance against virulent abortion germs is obtained by injecting them with attenuated abortion germs, and that this immunity begins to decline sometime after the third month following the immunizing injection. 7. PREPARATION AND USE OF VACCINE The vaccine was prepared by growing the non- virulent'strain of gggg. abortus for forty-eight hours on liver agar. The liver agar used was prepared as described by Huddleson (6). The growth was then washed off with sterile physiological salt solution and the suspension was standardized so that the turbidity corresponded to turbidity V of McFarland's Hephelometer. Five tenths of one cubic centimeter of this suspension was injected subcutaneously with a small syringe. After a period of one month, and each month there- after, four pigs from the vaccinated group were placed in a cage with two normal control pigs. The cages were separated by a metal partition so that there was no possibility of transmission of infection from one cage to another. The pigs were then exposed to infection by sprinkling the feed with virulent organisms each morning for six days. PREPARATION OF INFECTIVE MATERIAL The Virtdent organisms were grown on liver agar in test tubes for forty-eight hours, then washed off with tap water and sprinkled over the feed of the pigs. At the beginning of the experiment three slants were fed daily for eight days. Some of the vaccinated pigs seemed to show signs of infection or rather enlarged organs, so the dosage was out down to one agar slant a day for six days. It was 8. thought that the first amount was more than could be resisted with the degree of'immunity which had been obtained. Virulent organisms from four different sources were used in this experiment. All of these cultures were isolated from aborted fetuses sent in to this laboratory for bacteriological examination. ‘material from these fetuses was injected into guinea-pigs and six weeks later these guinea-pigs were autopsied and the organs smeared on agar plates. These plates were placed in ten per cent carbon dioxide for forty-eight hours. All of these pigs were heavily infected when they were autopsied and good growths were obtained on the plates. Culture D l was isolated December 1, 1923. Culture B 54 was isolated October 1924. The cow from which this fetus came was bred March 9,1924 and aborted October 24,1924. Culture 44 was isolated from an aborted fetus, the dam of which was bred Bay 14, 1924 and aborted November 28, 1924. The dam of the fetus from which culture c 111 was isolated was bred July 16, 1924 and aborted February 11, 1925. Method of Determining Infection The six exposed pigs were kept in the cage for six weeks. This was considered a sufficient time for lesions to develop after exposure. At the end of this period of time all pigs were bled from the heart in the following manner: The animal was anesthetised and firmly fastened on its back to an animal board, a small space over the left side of the thorax was clipped free from hair, 9. the space was disinfected with phenolized saline solution and a small hypodermic needle was inserted between the second and third ribs into the left ventricle of the heart. If the needle punctured the right ventricle the blood flow- ed very slowly or in drops. Two or three cubic centimeters of blood were drawn from each pig in this manner and slant- ed before a clot was formed. The samples were placed in the ice box until the next day. When taken out of the ice box sufficient serum.had formed above the clot to make agglutination tests. In some instances where serum did not form the clot had to be broken and centrifuged. The antigen for the agglutination tests was prepared by grow- ing a culture of 2222. abortus for forty-eight hours upon liver agar. The growth was washed off with carbol-saline solution (.5 per cent carbolic acid in .85 per cent solution of sodium chloride in water) and the suspensionwas stand- ardized so that the turbidity corresponded to about .7 of tube one of McFarland's Nephelometer. Two cubic centimeters of this suspension was placed in each of five Wassermann tubes and the following quantities of blood serum added; .08, .04, .02, .01, and.005. Dilutions of approximately 1:25, 1:50, 1:100, 1:200, and 1:400 were obtained. These tubes were placed in the incubator and read at the end of 24 and 48 hours. The results shown in the tables were taken from the forty-eight hour reading. as soon as the agglutination tests were completed the animals were killed/and autopsied, and search was made for any anatomical changes. An attempt was made to culture 10. .2221 abortus from the liver, spleen, lungs, kidneys, testes, urinary bladder, and gall bladder. These organs were smear- ed on gentian violet liver agar plates as described by Huddle- son (6). These plates were placed in jars containing ten per cent carbon dioxide, sealed and incubated at thirty seven degrees centigrade for about six days. All colonies resembl- ing £222. abortus colonies, which appeared on the plates were transferred to liver agar slants and incubated. Agglutination tests were run using each organism as an antigen. Positive cow's serum was used in the agglutination tests. Agglutina- tions in only the highest dilutions were recorded in the tables here shown. 11. EXPERDMENTAL DATA The data recorded give the results obtained each month for ten months. In order to make clear some of the results recorded it is necessary to state that about a month after this experiment was started the pigs became infected with an organism.which caused the death of several animals. Some of those which recovered held their heads to one side and when they were autopsied it was found that they carried an infection in their ears. When cultured on liver agar the organism produced a very profuse, slimy growth. Pigs dying from this infection showed large amounts of exudate in the thoracic cavity and sometimes in the abdominal cavity. The atypical lesions here recorded were attributed to this cause. This interpretation was based on the fact that this same type of lesions was found in normal-guinea pigs which had never been exposed to 2323' abortus and which did not react to the agglutination test. The lesions in the liver were quite different from those caused by 2223. abortus. They cnnsisted of irregular, dirty brown foci, sometimes five millimeters in diameter. Some were longer than wide and others were about the same dimensions each way. The edges were irregular and the surfaces was flat or not elevated above the surface of the liver. The spleen was sometimes enlarged but did not show this type of lesion. The lesions which were considered typical of Best. abortus lesions corresponded in.soue respects to those 12. described by Schroeder and Cotton (7) and in other respects to those described by Seyfarth (8). The external appearance of the body was normal in the majority of cases, this being particularly true with immunized pigs. Controls which were heavily infected were emaciated, the hair was rough, and the pig felt soft and flabby when handled. The spleen appeared to be the organ most constantly infected, as was shown by both anatomical changes and cultural findings. The spleen was usually enlarged, and directly under the capsule were minute nodules or foci which were grayish white in color and raised. The surface of the spleen appeared rough due to these raised areas. The spleen was not always enlarged, for in some pigs which showed lesions in other organs the spleen was normal. The liver was the next most commonly infected. It usually contained grayish white or pearly white foci, varying in size from that of a pin point to a millimeter in diameter. These foci were usually raised and glistening, showing very clearly through the capsule of the liver. En- largment of the liver was never noted. One or both testi- cles sometimes contained lesions or abscesses. The epidid- ymus was were often involved. Sometimes the testicles were atrophied.‘ No lesions were observed in the kidney, however, despite the fact that the organism was cultured from this organ many times. In a few animals the lungs contained foci from which 2221. abortus was obtained occasionally. Since the time between injection and the first appearance of infection of immunized pigs is the important 15. factor in this experiment it will be necessary to pay particular attention to the dates recorded. 14m . . _ wmfi SH . HnSHOQH . . oomua H Heswmfiiw. smmmmao segue . . H . H . . . H as Hoo . . . . P oz“ mamas nausea 30%. z _ mm&. = _ new .Hoapwoo. .. H m H _ ashes. uosaa as Hoom+ . . . t . . _ connfl . a Sosa. opens eassma. . . . . a . . . . mmwumwuem. seam Heaps: = . oem. _ . . . . . . phone. m4 cease eoeflpmh . e . can .Hoepeoo. . . H . oomua . mMCacsfim. commsasm sooaamu 4 . . t . . + . . H MSPHDO. HebaH Gd Hoow. : . 00m. . . A . . . . : oz. Hmoacapm 3mm. . . z . com . a . u . 0mm . an . .maMSH Sofia. womCmHHooH . . . u . . h _ . swwdoce_ \ saaaspsep. . . . . H . _ . on . . some mmaaa dmwasawo _ . . . . . m.H . m .pmcama. nooaam .mopaa am. a . ewe. . _ . . . . by mmeuaehrt «com Heoaaapa. . . a . nae . a H - . ooe . mm . oom.H _ horas. Hesse: mmswao. . . . A . b _ . . . HGHSPHDO. “0:90 HmPHH. : . Hbm. . . a . . . . oz. :H wooMlopanx . . : . mm¢ . e . I . own . HH . . . w HwEhofl. . . . a. a. . L . . was no heave. . . . . . om.H . o>Hpmmoz. ho>«H : . . . . H Hoes .e \ . . an obdpamom.;mppsoem. mt) pawns Hmoapapd. N om\mH. 0mm” ¢m\m\aa are H¢m\mm\m” n . can u m u 1: u . . mpphopm.m . mpazmom . . Namesm . 14 . _ r . masseuse o mo . .ampopsa popes .moawm .moawm . . . . . . ammofioao . a H . no . .mxs pmH o. . ooc> .ooma .oom> . L». m .uomnowemm encamog ofiscusssl open.. . m . enowom mo .osomoo oaomop mam . v a as. spam; .ma open. .mma. .ps . .oa seausauwdssH seams mama mm soapoowsH op oemoawm awam H macaw .H mamas 15. The data in table 1 shows the results obtained the first thirty five days after vaccination. Blood tests were made at the end of 30 days, but five days el- apsed Before time of autopsy in order to perform an in- tradermal test on each pig. This group was fed three slants a day for eight days. Culture DI was the organ- ism used. The vaccinated pigs of this group made an aver- age gain of 70 grams and the controls made an average gain of 74.5 grams. Atypical lesions were found in the vaccin- ated pigs but §323f abortus was not cultured from them. Number 28 showed an enlarged spleen, but since an organism producing a slimy growth was cultured from this organ,the enlargment was attributed to this organism. Number 31 also showed an enlarged spleen which was adherent to the wall of the body cavity. It was an old lesion which had healed and since nothing was cultured from it the cause was not attributed to 8223; abortus. Both of the controls showed typical 2223. abortus lesions but 2222. abortus was cultured from only one pig. The failure to culture 2222. abortus from the other pig and the agglutination reaction of 1:200 cannot be accounted forl 16. . . . . . . . . . . am>fia. . . . . . . . . . 63w Qmmflfiw. — _ . . . . . . . . . .ofiofipwmu. _ . . . . _ . . . . . aosm. . . . . . n . . . . . mapsoem.m. . , . . . . oomufi . mo masons. moaofiawmp a“ . a H mam ” = H Gem . Hoapwoo. _ . w . . . spasm. mmmomes empemw. t a . . . . r). . . _ _ . Hashes x m . . . . n . _ . . . . ammaam. coeapm .am>HH . , a. . . . . oomua . seem. as hose eases . a H see u = ” 0mm H Hcppaoc_ _ . n . . _ mewsops.m. masses Hmofipse r, 1+. . . . n . 4 . . . NQOHOO. . I . . . _ oomna . assesses. Hesse: mammao .mm\om\an New ” = H can . = . . mmm . we . . . mgr. Harem . h u . H H e a . . . MMQHQGHW . . I . . . . oomuH . Hmacpaso. Hesse: mammac . : H 000 n = H mmm H a n . mom . be . a - mM- HHJW — A J IN 11 - . . mmswmc.m. Hashes masmso . . . . n . . . . oomua . Hayesase. seeps .hmsaa.em .mm\nm\au mes H = H com . z . . new . an . . . on. maowmma Hmowcwpw . w .¢ . . . L a . . . . use ma sowpomw . . . . H . . . . _ . mmflwcflwm. ICH OH 056 odam . i. I . . . . coaua _ Hehapflee. see op assess ease .mm\aa\H” Nae nem\n\man owe . em\am\o_ . owe . mm . . . oz. Hmsaow msmwso Hflw . u. oflMl. . H . w l . a“ o>apfimom_ . . _ m r... . . . . . .. . .oom . . . .m mam . mocxm oom> oom> > ” mewnoem.mw mpwwmmmw . .mmcmwmm. pmmWM.pmm we .oaomon” we. «somepnopomoe. mam. m n a“ . a _ . ., . . . .. . . ammowmuom. uofiampomm. macamwn omEopszm . mesa w m p2. open . am. open u mwdhl pa 0%. sofipcaassssn sepmd mama mm sofipoomaH op ammopsm mmflm .HH manna .HH mamas 17. The second group shown in table number 11 was autopsied at the end of sixty five days. They were fed three agar slants a day for eight days of cul- ture 8.54. The average gain for the vaccinated pigs was 29.5 grams and the average gain for the controls was 154.5 grams. None of the vaccinated pigs showed typical lesions and Bact. abortus was not cultured from any of them. Pig number 32 carried its head to one side and when autopsied an abscess was found in one ear. Pig number 33 showed a few small irregular shaped lesions in its liver. There was a wide variation in the agglutin- ation reaction of the vaccinated pigs, ranging from 1:100 to 1:500. Both of the controls showed typical lesions and 2223. abortus was cultured from both. Pig number 4 reacted only 1:200 to the agglutination test But heavy growths of 2222. abortus were obtained from the testicles spleen and liver. ‘2222. abortus was cultured from the spleen of'number 3. 18. . . nmchm. Hesse: Kym. _ . w . . H a . . oomuH . anew. :emHCm .seaHH. a . mam . e . owe .Honpsoo. . . m . t mapmoem.m. aH Hoow Hmowrhe. . . . t . . . . . smeHH ems. . . . . . . U . . _ . . sooHpm. ouHm Hashes x a. . . . . . . . . . oomufl . aoaw. womHCm .AmpHH 2H. : . mwm . z . was .Hoapsoo. . . m . . L mppaoem.m. msOHmeH HmOHcma. . . . L! L It . . _ m mmchsHy. Hesse: mfihMLo. . . H . . H: 4 . . oomuH . HessaHse. passe .aeHHsam. = . sow . = . mam . z . - . ooe . e . . !L oat, mHewummp mac. . . . IL, . - . . . _ oHoHpmmp. - Hashes. . . . . . in a . _ _ Scam. mammao segue. . . _ . . . . _ . oomuH . meHwOHoo. eHOHpmep so. = . mam . = _ mam _ = . u . m . m _ . _ ammHoLi mmmomem HHmsm. . . (r . . i . . . . mmemem . . . . . . . H . . . .mez . HehaaHso . Hughes. = . mes . z _ mam . e . - _ can _ n . _ I. 02.; mwswmo Haw. _ _ r . . _ . H11 — gmmnflpm - p u u u — - J-( — p . . EOHH . . . . . . _ . . _ . . dmhfipfifio . . . . a . . . . . . . oomnH . mmHsoHoe . Hesse: mm\em\m . ems sm\om\mH ens .em\am\a. . . mHe . H . . .smmHo amm,. mammao me _ . . L . pl . (L ThflHOPfiPflMOFH-u _ _ 4 .eMOCvnm... . u n J . — _ mapsoem.mx mpHSmmm.. .mmpopam .aopwm. .mopxm_ .moaam. .ecm> . .oom>. .oom> . . . mwsHeus. onOH . . mo . . .mma..pmH mo. enemas; we ..osomoeloaowoe.x mHm. w endowmhmm -oHsepeemy.meoammm emaopeza . opeq . e.ps. sheet. .pa_ ease .. .mma. .pe . .oz. sOHpmundssH ampmm mama mm QOHpomme op ammomxm mmfim. HHH manna .HHH mamas. 19. Table III contains the data from the group of pigs autopsied the third month or at the end of 92 days. They were fed three agar slants per day of cul- ture Number 44. The vaccinated pigs of this group made an average gain of 29.2 grams, and the controls made an average gain of 98 grams. One of the vaccinated pigs, number 5, showed a small abscess in one testicle. The organism.cultured from this lesion was agglutinatedby positive cow's serum only in a dilution of 1:25 while the organism in the controls was agglutinated in a dil- ution of 1:500 by the same serum. It was, therefore, not considered s 2222. abortus lesion. ane of the other vaccinated pigs showed any lesions or was 2222. abortus cultured from any of them. The blood from pig number 3 was negative to the agglutination test. Both controls -showed an enlarged spleen and typical foci in the liver. Bact. abortus was cultured from all of these organs. 20. . . . son a as . omufl . mmnfiecae. Hashes c H mom . .eomopxo sea a H n . _ HasspHSO oz; mnempo Has. 41 . cemonxe pcz mm\¢H\H. . . ewe . me 9 J . . omua . mmzwocfim. - daemon. z . mmb .( , . P H . . .Hwhflvflso CZ» mficmho HHflu « u . . . . U saws: use. . on so «H H oomua ”nooHCm Soy . Hashes. : H 0m¢ ” a ” mam” H p o“ h h. . . mappopm.ma x m somamm A . . . . . H . . H026 .HM mam. . . . . . . . . . . . mficwpmmpu . . . u . . . . - ugmmfiom SCH“ — ‘ u OOHPHHOO HH . oomnfl . eosssflpm; oHowpmmp. g ” mom ” = H one” a H H w . . mppmopx.mL cw mmmompm HHmSmL . Hm . . «mm. c . u . mo¢ H mm — om "H _ mwQHUCHH . HQHPCQ— t a m — B u n - b b . .HdssWHso mat msnsho HHdL a . . . . . H . . nomapm. Hesse: mmswao. . . . mam. : . n . mew . mm . OomuH . BORN. Hm£#o .HmeH. : . Omb . z . . . . t . . msmpopm.ma qfi wocm memmmm. a . . . . . In nmmflom Hesse: e I m an H OCmuH H thW” hmflpo .Hmbaa. : H mwo ” a n. «fibn : H hi 5 n W ll . . mupnopm.m. mm «ocmiHmoMpbpd. . . . . . . ‘W . mmswcsfiw \ I a on . mans ” Hebehapo” finance. mm\cn\eu ens ” mm\m\m pom” amrefl\man h ea w . . oz. mammao Had. J.mocxm. . . . . AM .na obupamom. . . . w . .mom . .mopxd. .oow> .oom> .oom> _ mapuonw.m. maasmom. . emnopmm. nwwwwn pma WW. onomom. Mo mehowmp.op0Mmp H mam _ awafiecaw. ofidoa. - . w .. .p;. 02. .m3. when .mmari .mg .om _ MHwOHonom.uoHanemm. mQOfimpm omeowmmmb ,mw 91‘ m . >H manna .pH mamaa soaprHndasH hmpmm when NHH nofipommcH op demonxm mwam 21. The data in table IV were obtained from the pigs autopsied the fourth month or at the end of 112 days. They were fed one slant a day of culture 0III for six days. The vaccinated pigs of this group show- ed an average loss of 50.6 grams. The controls showed an average loss of 33.5 grams. It will be seen that .2222! abortus was cultured from two of the vaccinated pigs while only one of them showed typical EEEE‘ abortus lesions. The other vaccinated pigs showed no lesions inor was £323. abortus cultured from the organs. The antibody index of these pwo pigs was also low. Both controls showed lesions and £223. abortus was cultured from the spleen, testicles, and kidney of number 11 and from the spleen and'urinary bladder of number 12 in very large quantities. It is difficult to explain why two of the vaccinated pigs showed no lesions, no cultural findings and a low agglutination titre while the other two gave a high agglutination titre, 222:. abortus was cultured from both and one showed lesions. The control from which large numbers of £323. abortus organisms were cultured reacted «only 1:200 to the agglutination test. It would seem that pig number 12 had become a carrier of £533. abortus with the infection in the urinary bladder. This would ' correspond very closely to the typhoid carrier in the human e 22; Under these conditions it may be seen that the vaccinated pigs were not exposed to infection for a per- iod of six days but were being constantly exposed for six weeks to enormous quantities of virulent £231. abortus It seemed that the other two vaccinated pigs were able to resist this infection since no lesions were found and .EEEE' abortus was not cultured. The agglutinins in.both of these vaccinated pigs had practically disappeared but some degree of immunity remained. It is evident, there- fore, that the presence of agglutinins is not an indica- tion of immunity. This was the first group in which vaccinated non-exposed pigs were used. This gives a check on the vaccinated pigs before they were exposed to virulent organisms. It also gives a check on the virulence of the organism being used as a vaccine. Reither lesions nor 2223' abortus organisms were present in these pigs. At the end of 145 days the agglutinins had dropped to 1:50. It should also be noted that these pigs were vaccinated 20 days after those exposed the fourth month and 106 days after those used during the other months. 23. III _ . mmHMmua mmpmmu. . am e . . .o MMMW. nwon cw mmmempm. . H . H . . . v. .0! I H oomua . sweepaeo. masefl .psfipc: x. . _ H . . . . .mspsnpm.m; m Semapm Hmpfia. : . mom . . . . _ t , as Hose Heewema. . .. e . mme Hospeoo. . . m . _ mHowpmmpu e . H . H _ . . — F a _ . flmwdmm. . . . H . . . . . . mwam. . _ . . . . . . J . . macflpmmp ego. . _ nom.H . eeseeaswnea mmoempe .so>aa. ” n . . . H . u pmnfippnpflnomfl HMO “00m“ HfiOHEHrH— 2 — mmfi — p. — mmn uHOOHPHHOO- — — KL _ Gonna . mmcmwcww. Hesse: mammsoa . . . H . . H H . HGHLPHWO. HH4 .mUHm mQO. : . mmb . . bmw . . . . IL P _.._,w «H a... I, a I . . _ mmefleefiWt o eeflsa.e oxen“ . n e _ . e n ” man . mfi e .I . a .II p H 00H.H . Hmns+aswn namepom. : . 5mm H _ c H . . H U _ L mwchan¢ erMpo Haw“ . . . : H 010 . a . I . owm . 0H e u . . p u a . OON.H . HQRSPHGOH IHQEHCC. z . Om) H . . . . h _ H .L stage oat enemas Has. . o . e . new H a . I . one n «H a cam 14 . . - OONHH H HQIHDPHSOH HgsoHOHH- mm\mw \fi—p \. . J \ \ — u p .— H we ep shoout 0241 enemas Haw. . .w . Hoe ” um swim. can Hem\mm\m. I . new . e M a - . Mafiphopflenml” MPHLme. W a. . ..wOCunm_ — . u. . a .. rm”...w .. a l e .. 1-: 0 v I . omwnficeaae ofimnan . I pmwsm. hmwww_ mopxt . momyw. .oom> . .oom>” .oom> . . H OHOHQWLIOHhmwodm mflowmmfl om8ngw2fi. 0W£3.. ..:..pmH %ot.mhnmm£. MO .QHOMQD- mhnmmfl . mam . z e or. seen . .ee. ones . .eee. .oe . .os moapmuaseEEH ampwa when cma nofipommeH 0p emmommm mmfim > macaw .> memes. 24. Table V shows the results for the fifth month or at the end of 150 days. This group of pigs was fed one agar slant a day of culture 0111 for six days. The vaccinated pigs of this group showed a n average loss of 29.5 grams between time of exposure and date of auto- psy. The controls showed an average gain of 113 grams during this same period of time. The difference in the age of the pigs probably accounts for the difference in gain or loss. The vaccinated pigs had practically reach- ed thier maximun weight before time of exposure, while the controls were about half grown. They would, there- fore, increase in weight much more rapidly. There were no lesions present in the vaccinated pigs and 2333. abortus was not cultured from any of them. The agglutin- ation titre had dropped considerably in three of the vaccinated pigs. Both controls showed lesions in the testes and liver and number 8 showed enlarged Spleen. gagt. abortus was cultured from the testes, spleen, and ,urinary bladder of these pigs. 25 ~-----—- _ . mwSHOCHL} . . . . . . . . oomua . Hmsapaeo. ,Hesaoc. mm\om\m. one . e . mmo . e . I . mom . om » OZ» mgwwwwho HHANf IF p l» u * L p . cmhepano. Hesse: mmnwpo. . . . . . . . . QMBORM. .Hmflpo .ocwm. . . _ . . . . . asham. one 0p smashes. . . . . . . . oomua .weaeeeoep. seem .eoaeaeaaow_ e _ mam . e . nee . e _ I . awn . mm . emgeeaeo_ aflaeangep emcee. _ . t, t _ . t . t . Hesse: mammao. . . . . . . . . menacefim. nonpo .HaSHQQ. . . . . . _ . oomna _ assesses. am eoeaem .uoesa. mm\efi\m. eee . . . ems . . . I . one . as . . 02a «MM «00% Hwoflbflbk _ l» p F a II. . . mmcfieeflh. Hashes meamno. . . . _ . . . cones . assesses. geese .ueefia. mm\om\n. HmoH.mm\mm\m. mm» . em\mm\o. I . ooe . e . oz. .5.“ meow Hflowgma _ L . r L . . a.“ GPdemoW. I _ . . .wofummw. . . . . . mspaopw.m. mpaemom. . mmeousm. seems. .mcpwm.t.momxm. .oohp . .eom>. .eom> . masseuse. eaaoe_. .. so u .oee. ems sol oueeop. so .ocoaepiouoeoe . mam camoHonmm.Iomaewomm. meommmm emanpsawu open . m.pau open. .pz» opmmw u .wumu .pa.u .oz soapaNfiesssH sagas when OmH seawoomaH\op womenxm mufim H> macaw .He agree 25-A mefiuasu. — . a p p p . I — - u p . Om.H . HwhSpqpo. IHmEMCG. : . Ohm . .fiomoRNo +02 . e . I . mow . m a . OZ. mgfimmo HHdFI a F1 ll L lF F . . . cmarpsao. . beaten. . . . . . . . _ Emflatapo. so mowenamoEmm. . . . . _ . . . onmua _ mxfiw. Hemflm .Hmaaon. : . 0mm . rcmmomxo pom .mm\¢H\H. I . sow . u _ mSPLODn.m. mafiap cemaemr. . u u . u t a . .mmpmmp and. _ . . . . _ . . . . >®chw. mmpmmp Swoo SH. . . . . . . . . . Bosh. mmmomnp .Hmaacm. . . . . . . . _ oomua _ aoseefiem. an eeeaco .uoeaa. mm\em\e. New . e _ Hoe .Houeeeo. . . . L mSpHopn.m. QH «new Hmownmfia . . IL! L . a . . . . . . cmMpmHnm sewage. _ . . . . . . . . mquImé. .mwwmuwuunm mSOEHHDH.H. . . _ . . . . . . Ham EOHH. M0 Hst RPHPSO. . . . . .. . . . . ©®HD#H50.0wmflH0£P .opflwfixo. . . . . . . . . . amfigteoe. oeoeausse apes. mm\m~\e. some. mm\n\e. bee .Heupeoo. . . M |L b8wHfl. dmhmboo Hmbfimp IL 4 a L a . L . a mbwpwmoh. . . - ..mOQNH. . . . _ . . . .mspaonm.m_ mpaemmu. . mmpopse. poems. .mocxm ..mopxm. .oom> . .oom>. .ooe> . . masseuse. sawed. . so . .eaa. see so . oeoaoe. so . eeeeoe_ouoeoe . onOHohomp Icapmpommp mmnflmmq ofisopszs. mme,L m.an.. opens. .pee opmm.u .wwmu .pa.t QOHPflNHQSEBH MQPMd mhwa CDH SOAQOOWQH OP domomxm mmfim neeeespuoe. H> manna .H> mamas 26 Table VI shows the results obtained for the sixth month or at the end of 180 days. These pigs were fed one agar slant a day of culture 0111 for six days. The vaccin- ated pigs showed an average loss of 78.5 grams between time of exposure to virulent culture and autopsy. The con- trols made an average.gain of 81 grams. .EEEE' abortus was not cultured from any of the vaccinated pigs however, one of them, Number 17, showed typical pearly foci in the liver and an enlarged spleen. All of these pigs have a high agglutin- ation titre. An organism producing a slimy growth was cul- tured from the lungs of Number 18. The lungs were partially solidified and adherent to the body wall. It also carried an infection in its ear. Control Number 9 died about a month before this group was to be autppsied. Death was due to an infection with an organism previously described. The growth of the organism on the plates was so heavy that 3233. abortus could not be detected. Control Number 10 showed lesions in the liver and both testes,also an enlarged spleen. Bact. abortus was cultured from these organs. One of the vaccinated non-exposed pigs of this group showed no lesions or cultural findings and gave an agglutination titre of 1:50. The other pig showed lesion but an organism resembling .EEEE' abortus in some respects was cultured from the spleen. When agglutination tests were made on this organism it did not agglutinate. The blood of this pig reacted 1:500 to the agglutination test. 27 — - Gmmflpm— - a u — u a n u . . 80h“. . . . . . . . . . coaua . Imwmbwwew. notfia. mm\mm\m. saw . e . saw . e . I _ owe . mm . » mapeopm.mrl we “00% HMnthp. u t . IL rt, u u u . . masseuse. . . . . . . . . . omufl . Heeeeflee. Heston. mm\am\m. mam . e . sew . e . I . owe . em . . CZ» mflmwho HHd. . L _ . . . L . . MESH 9..on” . w . . . . . . . . . . GQHZVHSO . . . . . . . . . . . Emfimmmho. Hashes H m. . . . . _ . . . 00m. "H . QMHH. GmmHQm .hmmrHH. mN\NN\© . Nmm . r . mmw . : . I _ 0mm" . OH . _ maphopm.m_ QH aoom Hmofiphee L a . . ILI e, . It . . mumps. . . . . . . _ . - — EOHMH u u u a — a p p u . . deDPHDo. _ . . . . . . . . . Ewfiflgho. MEMOS N N. . . . . . . _ . oomufi . oaaH. seesaw .ueesfi. mm\em\e. eoe . mm\mm\e. New . em\am\e. I _ can . m . _ mapanpm.m. ma «cow Hhoacmb. g _ LI L r! . _ Em ®>Hvfimowr . . . ..m0CunH. . . . . . . mapaopm.m. measmem. . hmpopem. mopwm. .mogxm ..moaxm. .oom> . .ooe>. .oom> . . emaaeeaa. enmea. . as . .mae_ was we .oeosop. so .ouoeoe. apogee . mam . mmmonaom. IowamwommuxmaOHmmq.0fisopsede mama L m.p2. open.. .pms open . .mwdu .pa.t .oz coapmnwnessH ampmd mama Ham soapoowaH on dememxm one: mem HH> macaw .HHa mamas 27~A b moeaagsa. . _ . . . . . . . . Omud . HdHSHHwo. hm>HH. : . 5&0 . 1 domofixm #02. c . I . 50¢ . mm . » OZ» CH “pow HJOHCHPMP! _ II? a . . . . . mmmficcww. . . . . . , . . . oomufi . assesses. deepen .mm\em\e. ome _ eemoeao; peg. mm\ea\fi. I _ nae . we . » om. mQSMMc HHdIb . _ V VI a _ . . .Dwmflfiw 625. . . . . . . . . . .mmaua Bony. Hashes x m . . _ . . . . . . oomHH . umsepasw. moefimm .smswa . = . wwm . z . was. Hospmoo. . . «a . _ mapaepm.mt ca “00% Hmoflpmmtt a _ . . a . _ In . . Cmmflflm . . . . . . . . . . .omfifi QEHHD. . . . . . . . . . .623 mmwmmp. . _ . . . . . . . . EOHW. . . . . . . . . . . . mSPthfl.m. HSSAOQ . . . . . . . . . oomufi ,. we spaced. as seesaw .ueena .mm\mm\e. use . mmxmm\e. obs. Hostess. . . ma . . hpamme CH atom Hmoflcme . u . . . . siI . _ Em>fipfimom. . . ..mopvum . . . . . . msupopm.m. mpaemer. .thOpzm. seams. .momxm . .mcsam .oom> . .oohp. .ooda . . omeaeaaa. eases. . so . .oae. see we . spouse so .eoeeop. eeoaep . mam . OHMOHoamML Iomampommu msofimmn o.§opn:a L, open _ m.phu mesa L .93 mesa . .mmmu .palu .oz aoflpmmfizdssH seams mama Ham mofipommnH on ammocwm mamm nemsenpeee. HH> manna .HHa unmta 28. Table VII shows the results for the seventh month or 211 days. This group was fed one agar slant a day of culture 0111 for six days. The vaccinated pigs showed an average loss of 58 grams from time of exposure to vir- ulent culture to date of autopsy. The controls made an average gain of 47 grams. Three of the vaccinated pigs showed typical foci in the liver and two of these showed enlarged spleens. An organism resembling £221. abortus was cultured from.the urine of Number 9 and from the lungs of Number 10, but it would not agglutinate with positive Cow's serum. .222£° abortus was cultured from the spleen of Number 25. The blood of Number 25 reacted only 1:100 while the blood of Number 9 and 10 reacted 1:500. This cannot be accounted for unless Number 25 had Just recently become in- fected from the controls. The other vaccinated pig Number 24, showed no lesions nor was Bact. abortus cultured from it. Both controls showed typical foci in the liver and very large spleens. EEEE! abortus was cultured from the spleen of both pigs and in large numbers from the testes and urine of Number 13. .2321 abortus was cultured from the lungs of Number 14. This was uncommon in this experiment. Both vaccinated controls were free from lesions and the presence of Eagt. abortus. The agglutinations titre of Number 62 was 1:50, thus showing that at the endof six months the agglutinins had become greatly reduced. 29. p u — . mmpmwp. . . . . . . . . .o:m mooHCm. . . . .. . . . . _ seem. Hesse: x n . . . . . . . . oomufl _ souehaem. oooaeo .eoeafi . mm\om\e . mmoa. e . oeoa. z . I . tome . an » msphopw.m. aw woom Hm0wcthg a r a . . . s 1H . hmPfiH wmw. . . . . . . . . . mmpmmw. . . . . . . . . . mbmflfiwfl. . . . . . . . . _ gmmflpm— — p u u a a u — . EOFW. . . . . . _ _ . oomua . oesosaow. Hoeuoa . mm\ea\e . sow . e . was . e . I . mam . on _ mDWHonw.mr wflfimHDLHHdkrI . LL . . b _ . . IL . meHCCH%. . . . . . . . . OOmuH . HGHDPHSO. JHNBHOQ . = . me . t . 0mm . z . I . Own . ON . OZ. mflsmho HHd . a p _ . _ a . IL — mmCHUQHLH— - u a p a p a — .moz . assesses. gasses: . om\em\e . moo . mm\on\a. one . em\am\a. I . oan . am p 03. mGéMHo HHd . a a . . . a a .aooeaeaooa_ . . ..memxm. . . . . . weaponm.m. mpaomom . . mmeopse . poems. .moaxm _.moaxm. .oom> . .oom>. .oom> . maoaeeaa. enmofi _ _ so _ .mae. sea so .oeouep. so .oueaop..ueaoe . mam OHMOHOHmmMInHHmpomm..m20fimmm owBOpnmd.LI mpmq u m.pR. mama » .uRL mama . .wmw. .palu .oa oompmuwczaaH nopm4 when new mofipoomcH op HHH> moose .HHH> mamas ammonxm mwflm 29-h . . omefiooma. . . . . . . . . I . Hegeofiee. Iaeeuou. . . was _ I.eoeeawo- pez . e . I . eon . me . a WM» mfimflhoIHHHPI . ILL . . . . IU . . mmeaeeas. . . . _ . _ _ . _ I . assesses. fleeces. mm\m\o. see . eooeeuo- pea .mm\ea\a. I . mom . as . . em. enemas Has. . u _ _ . . In . . . UmmanHoo hfihmmfl. . . . . _ . . . . _ mmflflfl .de390hpw. . . . . . _ _ . DNuH . . QHOHmep .hobfifl. = . OHfl . z . an“ .HOMflGOO. . . 0H . . t on seem Hooneapau It . . . . _ . _ _ . flQWHMm. . . . . . . . u — . find mmgfi - - - n u p — . u . _ 800.8% — HGEIHO: N N. u u p u . . a . oomua . eoeaeHem.. seesaw .eoeafi. om\om\e. was . mm\oo\o. mom .Hoeoeoo_ . . ma . _ eepuomewm. aw,meom weenpae. . . t . . u _ .OFHWHmOM. . . ..momWfl. . . . . . . .Bbpopo.m. mpaemom. . homopem. poems. .monxm ..moaam_ .oom> . .oom> . .oom> . . masseuse. eases. . so . .mae. sad so .oueeep. so .ouomoe...uouoe . ma ”amofiouom_ Iemuopoemt snowmen sweepoeag, .oeo.. o.mar open u . a. open.. .mmaut .es.+ mm moapmofissssH popww omen new nowpoommH op oomoaam mwfim. Aeoeaaeooe. HHHe mwoao .HHH> mamas. Table VIII shows the results obtained for the eighth month or 245 days. These pigs were fed one agar slant a day for six weeks. The vaccinated pigs showed an average loss of 67 grams and the controls showed an average loss of 62 grams between the time of exposure to virulent culture and time of autopsy. Only one of the vaccinated pigs of this group, Number 57, showed the presence of les- ions, however, gégt. abortus was cultured from two, Num- bers 56 and 57. Number 56 showed signs of heavy infection since the organism was cultured from the spleen, kidneys, testes, and liver. The absence of characteristic lesions cannot be abccunted for. .2223' abortus was cultured from the spleen and testes of Number 57. The other two vaccinat- ed pigs showed no lesions or cultural findings. Number 29 was negative to the agglutination test. It seems evident that this pig either failed to pick up enough organisms to cause an increase in the agglutinins or the agglutinins had entirely disappeared and did not reappear when the pig was exposed to virulent culture. ‘ control Number 15 showed typical lesions in the liver and spleen. 15223. abortus was cultured from the lungs and spleen. Control Number 16 did not show any character- istic 2251' abortus lesions. The liver showed a few typical lesions, the spleen.was normal, the lungs were practically solidified and adherent to the thoracic wall, the testes were strephied and the pig was emaciated generally. A slimy organ- ism was cultured from all organs. Such a heavy growth was 31. produced that no other organism could be seen on the plates. The blood of this pig reacted 1:25 to the agglutination test. It would seem from this that £221. abortus was not present. Both vaccinated controls were negative to 2323. abortus culturally and anatomically. They reacted negativ- ely to the agglutination test, thus showing that agglutinins. will last about seven months after a single injection of liv- ing organisms. 32. — u — a _ p a — oomuH . HQMSPaSC. HQSHOE . a . me . r . Nflb . t . I . 0&8 . Hfi . DBL QOMHo Adm L . L PL . . L L . nopwa. downpoaps mopmop . . . . _ . . . . EOHH. H6890: MID . . . . . . . . OOmuH . GQHSPHDIOL domHflm .gooImpm . z . mmb _ z . mww . z . I . Ofifl . m¢ PmSHHODm.m. MM HOWMHQOHPhB L L L L LL . LL . LU - mmgwrvgfihfl- — n a a a u n — OOmuH . HfihDPHSO. HGEHOC . z . bow . a . fimm . = .. I . 0mm . 0* . oat enemas awe . I . _ _ . . . . mwGHU HM. HGSHOG KN nomHPm . . . . . I . . . oomua . assesses..oooaeo ego seems _ em\ma\e. see .mm\em\o. one . em\am\s. I . one _ an . QRL a“ Hmc% HmOthm . LL . L L . L +1 ammwmwmmem. . . . ..oeexm . _ . . . . mapaopm.m. mpaomox. . harness . poems ..mopxm ..mocxm. .oon> . .oom>. .oom> . emoaeeaa. eaMoa. . no . .oae .poa as .ouomoe. so .oaoaee..uomoe . mam omwOHOHom.IommopommL mmoamoH omsopmad L open L m.pz LI open L .pau open L .wme .92 _ .oz noupmufiaesaH momma whom Ham sofipoowaH op oomoexm mmflm NH moose .aH «J. AII Pam.a 32.A . . m humps: one Memo 0 t 1m m n . L . . . Ogfioflfll QOHOfimmP HGGHBQW _I .I .. H w. u : n . . . 809M. .mHOHpmmP J m m w M . . . . I moanoaoo a“ mmoompm oHo - . . m I . . . _ H gofiflwwh %0H~UHN OHHO hflqo . t - N¢® - COMOQNQ PO“ m. B u. - Hm¢ .00 . . . mwwuoean= o t o . In L LL L I Homepas memos . . . _ . I . . . u u HHQQHIAMQ p LuF1 - a u F n i H b . - UHHW HmPfiH- QHOGPQOP. . - a» p p p a m . n . mopmop. ago a“ mmoomom . . . . . . . a . . com H . Sony. Hasnon N m . . . n H n _ . . . " cohupaso nooacm .Aopaa . _ . L H oepueee.mw ea HeomHeeaeae H I H nos H g . Lee .Houeeeo. 1 sea . . . moose». .. . . H . . A m L . . and. medompmmp. _ . . . m . . . u nooanm. upon a“ mmoompm. . . n . . m L . . oom.H . EOHM. Hdfihofi K n. . . . H n . . . . . . cmhfivafio. QOOHQM .Qmmanmmm mH m CH m . _ PIIIIHH P mflwmond.mL Ga «00% Havamhmw\ \ H m ”mm\vm\©” mfim WHOHPHOO. . .SH . ANIMWHWHQWW. . . ..meWfi . . . H m L L newmmmwsam. opwwmmm. .hoaop:<. seems . .ooaxm..ooawm . .oos>o. .ooo> . .ooo>n .. . . . no .omw poH mo oaomop no oaomon .oao e w e we I . . . . . a H n 0 «fl _ « Houom. oesopeomL mammeoq easooeoa I openlrlmlumai. open. .LBIL Ben.L .mw4.n .pnn.on_ Aooewapmoo. NH moomo NH mnmde aoapoudnsseH nopud when Ham woapooHnH os oomoaxm swam .. .I A- ~ 0 a. .— .e. D a «I A o I l I o‘ _4 w _. .e e .. .. I .. .. 33. Table IX shows the data obtained the ninth month or 271 days. This group was fed one agar slant a day for six days of culture 0111. The vaccinated pigs showed an average loss of 75 grams between the time of exposure and autopsy. The controls showed an average loss of 54.5 grams. Two vaccinated pigs of this group showed typical 8223..§27 ggtgg’lesions. Both had enlarged spleens which were covered with white foci. The testes of Number 45 were completely atrophied. lgggt. abortus was cultured from the liver of Number 45 butnot from Number 59. The other two vaccinated pigs were free from lesions and nothing was cultured from them, It is interesting to note that all of these pigs re- acted 1:500 to the agglutination test while in most of the foregoing tests there were some which reacted only in low dilutions. Both controls were heavily infected. The spleen of Number 17 was about five times normal size and showed typical foci. There were abscesses in both testes. 2223! abortus was cultured from testes and spleen. Number 18 showed typical foci in the liver,an enlarged spleen and an abscess in one testicle. 8223. abortus was cultured from the liver,ttesticle and spleen. One of the vaccinated non-exposed pigs, Number 70 showed no lesions, no cultural findings and was negative to the agglutination test. The other pig, Number 66, had only one kidney. There was an old abscess in one testicle and the seminal vesicles were dark and watery.. Staphylococcus aureus was cultured from the urine. The blood reacted negatively to the agglutination test. 34. . I . mwwfionaw. assnoq . a . «mm. . douoawo no: . a . I . was .mm . . .qcmwpaso mm. mammno HHH . . . . . . . L . I . mwaaoedw. Hasnom . z . mam . ooooexe pom .mmweawa. I . was .em . . Zfimwfizfieo ouruL mgmnoLHd . . . . . . . L . m mfldwua and. . a a a . . . . d: . . mapmop1mwgfl. . . L L .— . . a . . u . oflHOOHpm— - u a p a a a a n . .ofihd. EOHH. uovmmv QPOQ fl.“ . . . L a . . . . . oooua. eoueeapw. oooeoee.fleeueu . e . «me . I . ooo .Houaeoo. . .oH . . L mpvhomdem’» N n “OOH—“gm L L L L L L L L a . . omswonfiu. moaoapoop ea . . . . . . e . . . . Homepaco. mmoompo .nepaa . . . I . . . . . . . omua. ea..na neon Hoeaeue< . e . Noe ._ . . one . . . I . moo .ne . a n in h F b h b P L h n u u HHOLQUHM- . a u u p a u a a _ . 809M . Hchofl . . . . . . . L . . oomaa. mouowmew. enemas Has . . . moo . I . mob . . . I . non . me. . L abhond.mrr IL L L 2L L L L L L L . n - HHdB N609 . u a u a . - a u _ n - OP “BORN mmgfl u a a a a - - - a . . mwflfiddfih. “~0th a.“ dooH . a . . . . . . . . . Houspdso. Headphpe.assnow . . . . . . . . . . oomua. on. w m a..aem . sm\ma\e . mam . om\om\e. nee .em\aa\e. I . see . so. a L L h L- b .- b b b P F Eon. . . tomes . I . . . . . . suppose.m. uaasmeu . .hmnopee .nopwe ..mepwm ..moawm . .oos> . .ood». .ooep . . .mwnflosau. canoa . . no . .ouw .ama Ho .euouon . no .onouon.onouon . man omonoamm.Iomaopocml.mnomamHIomSomuquL even _ m.wBLL oven L hm; . even . .wer . n _ .om wouuswaqsssu neand.mhen arm soapoeunn N mboma N Hands on dooonwm swam 35. . Table X shows the results for the tenth month or 506 days. There were only three pigs left to use in this group. They were fed one agar slant daily for six days of culture 0111. The vaccinated pigs made an average loss of 46.5 grams between time of exposure and autOpsy. The one control made a gain of 62 grams. This group of vaccinated pigs gave some very interesting results. The spleen of Number 54 was twice normal size, there were at- ypical foci in the liver and one lobe of the lungs was adherent to the body wall. No organisms were cultured from this pig and.tha agglutination reaction was 1:200. Number 45 presented a similar result with atypical foci in the liver and an abscess in one testicle. Nothing was cultured from this pig and the. agglutination reaction was 1:50. In contrast to these two pigs Number 42 showed no lesions but £223. abortus was cultured from the kidneys. The blood re- action was 1:500. The spleen of the control was three times. normal size and there were abscesses in both testes. [2222. abortus was cultured from the spleen, lungs, testes, kidney, and urine. Both vaccinated non-exposed pigs were negative culturally, anatomically and serologically. 36. GENERAL DISCUSSION This experiment was undertaken to determine how long immunity t°.§§23‘ abortus infection would last in guinea-pigs which had been vaccinated with a non-virulent strain. The monthly records give the results obtained. When the conditions under which this experiment has been run are noted it will be seen that the protection produced by vaccination with this organism was subjected to a more severe test then would take place under natural conditions. As will be pointed out later, in several eases the pigs were subjected to virulent organisms for a period of six weeks. If this experiment were run on cattle instead of guinea-pigs it would be comparable to placing immunized cattle in a box stall for six weeks with cattle which were eliminating virulent organisms constantly. This would be a condition seldom, if ever, approached in the field. When the small number of organisms required to cause infection in a guinea-pig are considered, the severity of this test is made more emphatic. According to Hagan (12) less than 100 organisms are sufficient to cauee infection, dependent some- what upon the resistance of the animal. It is somewhat difficult to make a definite state- ment as to the length of time this immunity will last, but it seems safe to state that protection is evident at the end 37. of six months. After this time there seems to be a gradual lowering of resistance to infection. Some protection, how- ever, is evident at the end of ten months, fer as is shown in table x one pig out of three vaccinated pigs showed in- fection. When the individual resistance of the pigs is con- sidered it would be natural to expect that some pigs would show a higher resistance than others. Topley, Wilson and Lewis(9) in their work on mice showed that there was a wide variation in animals. Out of 185 mice fed cultures of 'g. aertryche 92 died, 61 showed presence of organisms in the feces and 52 showed no symptoms. When these 52 were in- jected intraperitoneally along with 50 controls they showed a mortality of only 12.5 per cent while the controls showed a death rate of 76.6 per cent. The remaining 25 were inject- ed along with 25 controls and in 11 days all controls had died and only 5 of the immunized pigs had died. In 68 days 44 per cent of the 25 had died. so it is not unreasonable to suspect that some animals would fall short of a definite length of time and others would exceed this length of time. There is another factor which makes it difficult to say whether an animal has become infected and therefore has lost its immunity or power of resistance. The only means of telling whether an animal is infected or not are the lesions produced and the cultivation.of the organism. Even these dannot always be given as positive proof. Other organisms produce lesions which resemble those of Bact. fibertus 38. very closly and there is some chance of misinterpreting these results. It seems possible that lesions may be pro- duced when the animal is first exposed but later the animal's resistance is able to overcome the infection.and the organ- isms are thrown off or destroyed. Smillie (10) has shown that the number of living bacteria in the spleen of the guinea-pig which he had infeited with.§ggt. abortus was larger at three to fear weeks than later although the micro- scepic lesions tended to become more pronounced as the bacter- ia declined. 0% the other hand there are guinea-pigs which show no lesions but 2223. abortus can be dultured from their internal organs. It can be seen from their data that the controls were constantly eliminating large numbers of organ- isms in the urine. Meet of the time the drinking water and feed contained considerable amounts of feces and litter, not-with-standing the fact that the containers were emptied, cleaned and refilled daily. If the food and.water contained living organisms they would be taken into the system of the animals and pass to the various organs. If these organs were semared on media when containing these organisms, growth might be obtained. Besides giving some light on the topic for which this experiment was planned other interesting results can be pointed out from the data obtained. The question of the relation between the presence or absence of agglutinins and immunity is one which has caused much discussion. Most 59. immunologists are agreed that agglutinins are not an in- dication of the presence or absence of immunity while some still speak of them as such. Topley, Wilson, and Lewis (9) have shown by experimenting with mice that the presence of agglutinins is no measure of resistance. 0n the other hand, in cases where agglutinins were present an increasing re- sistance was associated with an increasing proportion of positively reacting mice as Judged by agglutination tests. Fleishner, meyer and “Shaw (11) in their work on cutaneous hypersensitiveness claim that a high degree of immunity was established in all of their animals as was evidenced by the constantly positive agglutination reac- tion. These data do not bear out this statement. As was shown by the non-exposed vaccinated pigs the agglutination reaction of most of these pigs was below 1:50 or negative before they were exposed to virulent culture. Yet there was immunity present as was shown by the absence of lesions and cultural findings. After they were eXposed there was a rise in the agglutination titre. The variation on the agglutina- tion titre cannot be accounted for. This varied from negative in one case to 1:500 in most of the pigs. No higher dil- utions than this were run. With the exception of one guinea- pig all the controls reacted 1:200 or more to the agglutina- tion test. It is not shown by these data how soon the agglutin- ation began to disappear in the vaccinated pigs, as no pigs which had been.exposed were killed under four months. 40. At the end of four months agglutination reactions were 1:50. At the end of seven months the agglutinins had entirely disappeared. Hagan states that a fall in the agglutinin titre begins about the tenth week of infection. Other interesting points presented in these data are the distribution of lesions and infections as determined by cultural findings. Meyer (13) found that the enlargment of the spleen was most striking and constant in guinea-pigs injected with body fluids or tissues infect- ed with bovine §agt, abortus. Smillie (10) also states that the spleen was the organ in which the bacteria were regularily present in.large numbers. The data in this paper conform very closly to these statements as is shown in table XI and XII. 41, TABLE XI Numbers of Immunizsd Guinea-pigs Showing Lesions and Cultural Findings '"'".l'""""""""" B. abortus fissions‘in c.01ffio.offi ’Days 'Pigs 'liver'spleenrlungs'kidneyrtestes'urinary 'galI 'bladder bladder FT 4 ' 35 4 92 4 '150 4 '180 4 '_ I '211 '243 4 4 ' Y '271 3 '303 39 Total B. abortus cultured from 2 Lise-414'; 4.4.4.4 fi'Afw-v'ilA 5522 5691 l 1 rl """ 'rv w'ATe-Lr' fies? 1 2 1 II'Q'J'I 4 4. 4 4 iii In fil- V'Afi 0 O 1 no 5 8 1 4. 1 1 2 2 ' 4 1271 ' :5 v 305 59 Total 424 £11 tus Lesions in livef'spleen lungs Eidney’testasrurinaryfirgall J ’1 “‘D B, abo' CU1tura1 Findings 'LBLfl H Li Lesions and ‘Number of Control Guinea-pigs Showing No.0 'Days 'Pigs ' 0.0 lllllllllllllllllllllll 1'1111 . r «G d d .d .1 0......" bfiv'r"'T'1'Y"'T'r-'T'fille III-"lulu- a d d 11 a m 1 O b r Instill-"lulu" d 9 .i 1. as .l 1. 9. 1i .3 M 1. .i 1. 1. t It!" "'fi'rs""'r'r'r'fil'fe"' m'I-"I.II" c s. 1 1 U """"" O b a IFIV'r'I'WIfi-IW'TIU'TI" BI'II"""II 11211221214 2221 1 'Ilj'V'j'Y'r-IT'V'T'fil-T' II 9n .i 9~ 9s .1 9~ n4 1. MW 1. 1i allu'll'fit'wl'I'T'r'T'TI-fi'lT' eels-III """ el' 23222222219 1 222222 OUT-'T'rl'r'iv""elr"'rII-TII lu'elelslellu'wl'" 1 as .9 as as no Au 1. as .i «a a no .5 9” as n. nu 3691581470t 569158 1 13.1 as 2fi9#.5fno. 1 al 1 'Il""""' Iii-"we ' I. T ""ee'"'el" '13 5 .2 .2 '2 '1 '211 '243 '271 '503 Total 19 43. In.the vaccinated pigs the liver showed the most changes, but East abortus was cultured from the spleen in a great number of cases. In the controls the spleen was more constant in.showing lesions and the presence ofggggg. abortus. The liver was second in number of lesions but the testes yielded a greater number of cultural findings. The liver, spleen and testes were the only organs showing lesions, yet 2233. abortus was cultured from practically all the organs except the gall bladder. Heyer has reported the culturing of g. melitensis from the gall baldder in large quantities but no reports show a record °f.2223' abortus being cultured from this organ. Some experimenters report difficulty in infecting guinea-pigs orally with Eggt. abortus. Hagan (12) claims that the susceptibility of guinea-pigs to infection through ingestion of Eggt. abortus is relatively slight. Schroeder and Cotton (14) were not able to infect guinea-pigs at all, lath bovine strains of abortus, when given orally to them. They obtained 58 per cent infection with a swine strain of ‘2223. abortus. This experiment does not confirm their resutls. 0f eighteen control pigs which were fed bovine strains of 2223' abortus all showed lesions and the organ- ism was recofered from them. Only in one case was gggt. abortus not recovered and this one showed typical lesions. This gives a rate of 94;? per cent infection. Very little emphasis can be placed on the comparison of weights in this experiment.~ The controls used were much younger than 'i'*. the vaccinated pigs, therefore, they made larger gains after being exposed to virulent cultures. The average initial weight of the vaccinated pigs was 567 grams. The average weight of the control pigs was 415.4 grams. The average weight of the vaccinated pigs before exposure was 720.17 grams. The average wtight of the vaccinated pigs after exposure was 697 grams. The average loss in weight of the vaccinated pigs was 23.17 grams. The average weight of the controls after exposure was 455.6 grams. 45. SUMMARY Guinea-pigs vaccinated with a non-virulent strain of £323. abortus are protected against infec- tion for a period of six months. Agglutinins are not an indication of immunity in guinea-pigs. Guinea-pigs can be easily infected by feeding virulent gagt. abortus organisms on their food. [2333. abortus infections are most common in the spleen of guinea-pigs. ACKNOWLEDGMENT The writer wishes to acknowledge his in- debtedness to Dr. I. Forrest Huddleson and Dr. Ward Giltner for assistance and suggestions received during this investigation. 46. (1) (2) (3) (4) (5) (6) (7) (8) 47. REFERENCES Schroeder, E.C. and Cotton, W.E. 1911. The Bgcillus of Infectious abortion found in Milk. Twenty-eighth Annual Report of the Bureau of Animal Industry. Huddleson, I.F. 1924 Studies on a Non-Virulent Living Culture of Bact. Abortus Toward Protective Vaccination of Battle Against Bovine Infectious Abortion. (Bang). Michigan Agri. Exp. Sta. Technical Bul. No. 65. Craig, F.J. and Kehoe, D. 1922. An Outbreak of Contagious Abortion in an IriBh Herd e Jour. Comp. Path. and Theraputics, Vol. 35, p. 711. Calmette, A., Negre, L. and Boquet, A. 1922. Attempts to Vaccinate Rabbits and Guinea- pigs against Infection with Tuberculosis. Annals of Pasteur Institute, Vol. 56, p. 625. Mc Farland, Joseph. 1907. The Nephelometer. Jour. Am. Med. Assoc., Vol XLIX, p. 1176. Huddleson, I.F. 1920. The Isolation of Bacterium Abortum from Milk. Tech. Bul. No. 49, Rixh, Agri. Exp. Sta. part IV 0 Schroeder, E.C. and Cotton, W.E. 1911. The Bacillus of Infectious Abortion Found in Milk. Twenty-eighth Annual Report of the Bureau of Animal Industry, p. 139. Seyferth, M.H. 1924. The abortion Reaction in the Testicle as an Indicator of the Hypersensitiveness of Infection. Joure Info DiSe, V01e 350 ppe 489-4970 ‘ (9) (10) (ll) (12) (15) (14) (15) ‘tOe Topley, w. 0., Wilson, J. and Lewis, E. R..- ~ 1925. Immunization and Selection as F actors in Herd Resistance. Jour. Hygiene, Vol. 25, pp. 421-456. Smillie, EeWe ‘ 1918. An Improvement in.the Method of Ital, olating and Recovering the Bacillus of Ca tle ‘ Abortus Through Guinea-pigs. Jour. Exp. Med., Vol. 28, p. 585". Fleischner, E.C., Meyer, K.F. and Shaw, E.B. 1919. A Resume of Some Experimental Studies on Cutaneous Hypersensitiveness. American Jour, of Die. 6f Children, Vol. 18, pp. 577-590. Hagan, WeAe 1922. Studies on the Disease of Guinea-pigs Due to Bacillus Abortus. Jour. Exp. Med, Vol. 56, pp. 697-709. Meyer, K.F., Shaw E. B. and Fleischner, E.C. 1922. The Pathogenicity of B. Melitensis and B. Abortus for Guinea-pigs. 'Jom ,Ifif. Dis. Vol. 51, pp. 159-196. SOhmeder, EeCe and Cotton, YfleEe 1924. Infection of Guinea-pigs via the Conjuctival Emeosa with Bacillus of Bovine Infectious Abortion. Joure AeVeI‘JeAe, V01. 18, pp. 211‘214e Schroeder, E.C. and Cotton, W.E. 1924. Recent Bureau of Animal Industry Ex- periment Station Bovine Infectious Abortion Studies. Jour. A.V.M.A., Vol. 66, pp. 550-561. . . . . h I n ,I . . 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