Illllrlrl‘ l i | | 1 014 03—1 .4 I UDLDCD POLIOMYEUTIS ‘.N THE EASTERN COTTON RAT Thesis for the Degree of M. S. MICHIGAN STATE COLLEGE Walter N. Mack I940 IIHEE-ifi IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII IIHIHHIIHHIIHIIHWIIHIIUIMIIHllllll llllll‘l‘lll é ., 3 1293 00612 949 221+”? (M W“) POLIOMYELITIS IN THE EASTERN COTTON RAT An Attempt To ProPagate A Second Strain Cf Poliomyelitis Virus In Sigmodon hispiius hispiius And hispidus littoralis Rats. by Walter R. Mack ’- A THESIS Submitted to the Graduate School of Michigan State College of Agriculture ani Applied Science in partial fulfilment of the requirements for the degree of Master of Science Ibpartment of Bacteriology 191:0 THESIS POLIOMYELITIS IE THE EASCCRL COTTCN RAT An Attempt To Propagate A Second Strain of Poliomyelitis Virus in The Sigmodon hispidus hispiius and hispiius littoralis Eats. .4 «fiver? “ ,0 -L l):"-" \4: “33' k) .5 Ii «sen... v .5 . .. at \u .u ,i‘ 4. j‘lu'PEJII \- I , n Pray 9* 4: f"'*“'m AVIS.» V H 1.4 .2: .131 21.5 This Opportunity is taken to acknowledge my indebtedness to Dr. S. D. Kramer and Dr. H. E. COpe of the Kichigan State Department of Health for their COOperation in making availeble the meter- ial for this study ani to Dr. H. J. Stafseth of Michigan State College for his helpful suggestions. 1| 1M:-Af.sfi . J. . ...~. 9.7.9.31 ....1 9A.. a. vet? '. Lit “\n. .. u ,. a .0 II III IV V VI CONTESTS Introduction ........ ... .............. . ............. Page :{etllodSoooo00.90.09.009.ooooooooo oooooo o oooooo oooooPage Experimental 1. Pool Spinal Cords of Monkeys.................Page [\J Fecal Specimen S.M. Strain...................Page 3. Kessel Strains..... ............. .. ..... ......Page h. The effect of hyperpyrexia induced by short wave iniuction thermy on the transmissibil- ity of thr virus of Poliomyelitis............Page 5. The Susceptibility of immature rats to the virus of Poliomyelitis.. .......... ...........Page Discussion.... .......... .................... ....... Page mmaryOOOOl. ...... 0.0.0.000... 0000000000 OOOOOOOOOOPage References... 0000000000 .000. ........ ......OOOOOOOOOPaée 18 19 20 2h 25 IKTRODUCTIOK Experimental work in poliomyelitis was initiated by Land- steiner and POpper in 1909 (1) who finally found an experimental animal which developed symptoms of this disease. A monkey, in- oculated intraperitoneally with a suspension of the spinal cord of a child who had succumbed to the disease, develOped symptoms of poliomyelitis. The infection was not carried through subse- quent monkey passages as further intraperitoneal inoculations of this monkey's spinal cord failed to produce symptoms. At a later date Flexner and Lewis (2), (3) were able to infect monkeys with this virus by inoculating them intrecranially. Serial passages were accomplished by these workers. Strauss (h) in this country also reported similar findings from monkey inoculation. Leiner and von Wiesner (5) in Vienna, and Lendsteiner and Levaditi (6) in Paris, all independently succeeded in transferring the disease to an experimental monkey. Since 1909 a great variety of animals of different species have been inoculated in the hepe that a less expensive means of prOpagation of this virus could be found. All attempts to infect other animals with the virus were unsuccessful (8) until Armstrong (9), (10) in September of 1939 reportei that he had successfully transferred a strain of poliomyelitis virus into the Eastern Cotton rat. Sigmodon hispidus hispidns. This strain, recovered from an eighteen year old boy from Lansing, Michigan, had been passed ser- ially through fifteen monkeys with the deveIOpment of clinical and histological poliomyelitis. The fourth monkey passage cord was -2. inoculated into the cotton rat, and twenty-five days later the rat developed nervousness and, on the following day, had.paralysis in both rear legs. Eleven cotton rats were inoculated with the suspension of this animal's spinal cord and brain and in twenty- nine days one of these rats was paralyzed. The inoculum was a five per cent suspension of the brain and cord in the dosage of 0.06 cc. intracerebrally, 0.06 cc. intranasally. and 0.25 cc. sub- cutaneously. The spinal cord.and brain from the seventh rat passage was ground to a five per cent suspension and inoculated into mice. One of the five mice developed paralysis (11). This animal's cord and brain, when passed into twenty-four mice, brought down twelve of the animals with paralysis of one or more extremities in from three to twelve days. Successive transfers of the spinal cord and brains of mice have been made, and mouse spinal cord and brains, when in- oculated into monkeys. have brought down these animals with typical experimental poliomyelitis. This study was undertaken in an attempt to determine whether other strains of poliomyelitis virus could be prOpagated in the cotton rat. Not all the experiments tried will be presented be- cause many different trials were made on the same strain of virus with the same result. Five individual strains will be discussed in detail, and references will be made to tables I to IV which out- line the experiment. METHODS In the majority of experiments the source of poliomye- litis virus was spinal cords of monkeys taken directly from the monkey, or after storage at three degrees centrigrade in either fifty per cent buffered glycerine or Locke's glycerine. In the experiment with the D.G. strain, (table I), the inoculum used was a pool of fragments of eight monkey spinal cords, all of which had been stored at three degrees centrigrade in buffer- ed glycerine. All cords used were from animals whose clinical symptoms and cord lesions conformed to those of experimental poliomyelitis. Samples of the cord used were weighed and then ground in a mortar with sterile sea sand and saline; in a few cases ten per cent testicular extract (13) was used as suspensoid. The concentration of the spinal cord.suspension used was generally ten per cent, but in a few cases a five per cent concentration was made. In one experiment, human feces, from which the virus had previously been obtained in monkeys, was treated with ether, con- trated (16) and used as inoculum. Blood plates were seeded with the spinal cord suspensions to determine bacterial contamination. In a few cases, bacteria were found in the spinal cords and brains of mice that were transferred from animal to animal. In the rats, however, no trouble with bacterial contamination was encountered. The cord suspensions were centrifuged at a moderate rate of speed for a few minutes to throw down the detritus. The supernatant suspension was then poured off and used as inoculum. The virus was kept at three to four degrees centrigrade when not being used, and was never left standing at room temperature unless in the process -h. of manipulation or use. Virus suspensions more than twenty-four hours old were not used. The injections of the virus were made by various routes i.e., intracerebrally, intracerebrally after trauma (using both needle damage and Sawyer and Lloyd's two per cent starch method (12), intranasally, subcutaneously, and intra~ peritoneally. The cotton rats used were shipped from Florida and South Carolina. Sigmodon hispidus hispidus and a closely related sub- species, Sigmodon hispidus littoralis, were used. It was found in our laboratory that the littoralis rat was also susceptible to poliomyelitis, Lansing strain. The animals were kept in stock for a few days before being placed on test. After inoculation, each aninal was observed daily, and any animal that did not appear normal was placed under special observation and watched through- out the day. When an animal develOped a generalized weakness, a peculiar gait, excitability or inactivity, or a weakness in any extremity, it was either watched for exacerbation or sacrificed for passage of the virus. The animals found dead in their cages at any time other than the day following the inoculation had their cerebro-spinal axis removed, ground to a five per cent suspension, and inoculated into new animals. If the nervous tissue appeared soft from postmortem changes or bacterial invasion, the material was filtered through a Seitz filter and the filtrate inoculated into new rats. The method used to transfer the virus was as follows: the animals were sacrificed with chloroform anaesthesia and, with ster- ile precautions, their brains and spinal cords were removed. A ~5- sagittal section of each brain and spinal cord was removed and placed in a ten per cent formalin solution for histological study. The remaining spinal cord and brain, or both, were then weighed and a five or ten per cent suspension made by grinding the weighed material in a mortar and then slowly adding the required.amount of sterile saline. The ground suspension was then placed in a sterile test tube and centrifuged at 2,000 r.p.m. for five minutes. After centrifugation the supernatant fluid was inoculated into new animals. Six to twelve mice were inoculated intracerebrally with 0.03 cc. of the virus for each group of new rate used. The mice were used as controls to indicate the presence of choriomeningitis virus (1h), (15) which might have been present in the monkey or rat cords. In a few instances guinea pigs were used, receiving 0.1 cc. of the material intracerebrally. The amount of inoculum used in the rats varied according to the size of the animal and amount on hand. From 0.03 to O.h cc. were given intracerebrally, averaging about 0.1 cc. For intranasal infection, usually one drOp per nostril was administered. The amount of the intraperitoneal inoculum ranged from O.h cc. to 1.0 cc. The material for histological study was imbedded in paraffin, sectioned, and stained with haematoxyline and eosin or with Mallory's phyloxive methylene blue. -6- EXPEILEZIT I Pooled Monkey Cords - D. G. Strain The most potent strain of poliomyelitis virus, D.G., isolat- ed during the 1939 Detroit, Michigan, epidemic by Kramer, et.al., (16), was used as inoculum. A total of 103 rats, forty-eight mice and ten guinea pigs were used in this series of experiments. Two rats were inoculated with a pool of fragments of five monkey cords (numbers 22, 28, 61. 67, and 102). At the end of the third day, one of the two animals was sacrificed, the spinal cord and brain removed. ground to a ten per cent suspension, and inoc- ulated into two new rats. This method of rapid passage was repeat- ed four times. One of the animals of the fourth rapid passage ser- ies, after a ten day incubation period, was found to have flaccid paralysis of both rear extremities. The animal was sacrificed, the central nervous system removed and inoculated in a ten per cent suspension into one monkey, four rats, two guinea pigs, and eight white mice. A careful dissection of the involved extrem- ity, failed to reveal any local cause for the loss of function. No significant symptoms deve10ped in the new animals. The micro- sc0pic eXamination of the brain exhibited scattered areas of necrosis and infiltration with macrOphages. The cord was edemic, the motor cells took the stain irregularly and appeared degenerated. In the next attempt, fragments of eight monkey spinal cords, all from experimental poliomyelitis confirmed by examination of sections, were pooled, and ground and made into a ten per cent emulsion, and inoculated into eight cotton rats, four hispidus and four littoralis. Two animals, (see table I) numbers U and 8 (one of each sub-species), were sacrificed at the end of three days, their central nervous systems removed, and inoculated into new animals. These animals did not have symptoms of poliomyelitis but were sacrificed after three days for rapid passage of the virus. A ten per cent suspension of the spinal cord and brain from rat number 8 was inoculated into two rats, numbers In and 15. In turn, rat number 15 was found to be unstable in the rear extremities at the end of three days and was sacrificed. Rats, numbers 3M and 35 were inoculated with a suspension of the spinal cord and brain from this animal. On the twenty-fifth day, rat number 3h developed a weakness in the right rear leg. It was sacrificed and the spinal cord and brain was inoculated into four animals, numbers 72 to 75 inclusive. Of these four animals none develOped any abnormality and so they were discarded after the lapse of the usual observation period of thirty days. Some of these animals received one, and others, two reinoculations of freshly ground cord suspensions as indicated in table I. The spinal cord and brain of rat number M of the previous group were inoculated into three animals, numbers 16, 17, and 18. Rat number 17 received intracerebrally, in addition to the virus, 0.06 cc. of a two per cent starch solution (12). This rapid pass- age on every third day was carried out until four sets of three rate each were inoculated. One of the fourth passage group of rats, rat number 31, develOped a weakness in the left rear leg after two reinoculations and twenty-nine days of observation. When examined at autopsy, no abnormalities of the limbs could be found; however, the intestine contained tape worms. The central -g- nervous system in a ten per cent emulsion was inoculated into rats numbers 85, 86, and 87. The histopathology of rat number 31 presented a hemorrhage in the ventricles of the brain, and some vascular congestion and edema. 0f the three rats receiv- ing the spinal cord and brain of rat number 31, rat number 85 develOped a tremor on the third day and a definite weakness of the right front leg and foot. The animal fell to the right side, although there was not flaccid paralysis of the limb. The viscera were normal in this animal when examined at autOpsy. Upon section- ing, the cord exhibited no cellular reaction, but the motor cells appeared to take the stain poorly. Both the brain and spinal cord were congested and edemic. Since no abnormalities were evident at autopsy and in view of the suggestive symptoms, the central nervous system was ground and made into a ten per cent saline suspension and inoculated into twelve rats, numbers 95 to 106 inclusive. Each animal was given 0.2 cc. intracereorally, 0.5 cc. intraper- itoneally, and 0.03 cc. intranasally. On the fourth day, rats numbers 99, 101, and 103 develOped similar symptoms, consisting of tremor and weakness of one or more extremities. Rat number 99 was sacrificed and.passed into three new rate, two guinea pigs and six mice. All of this group remained.we11 except rat number 118, which on the fourth day appeared ill and weak, and was sacrificed and inoculated into three rats, numbers 165 to 16?, none of which deve10ped any symptoms. The spinal cord and brain of rat number l01, prepared into a ten per cent suspension, was inoculated into three rats, numbers 120, 121, and 122, two guinea figs and six mice. Rat number 122 of this group developed tremor and weakness in the rear legs. At postmortem examination a nutmeg liver and atrOth of the spleen was found. The spinal cord and brain were inoculated into three rats, numbers 137, 138, and 139. Rats numbers 137 and 133 both developed tremor and weakness in their extremities and were sacrificed on the third day. Sections from the spinal cord and brain of these two animals were similar. Both had a small amount of hemorrhage in the gray matter of the spinal cord. An occasional round and plasma cell could be seen infil- trating the gray matter. Throughout the brain substance, scatter- ed hemorrhages were seen. The meninges covering the cerebrum and the subarachnoid spaces were filled with blood. The cords of these two animals were pooled, a ten per cent suspension made and inocu- lated into six rats, numbers lMO to 1H5 inclusive, two guinea pigs! and six mice. Some passages were made from this group of animals but did not appear to produce particularly significant results. Eat number 103 develoned symptoms similar to rats numbers 99 and 101 of the above group and was sacrificed on the fourth day. The stained sections of the spinal cord from this animal were very suggestive of poliomyelitis. The viscera were found to be normal with the exception of a nutmeg liver. A ten per cent suspension was made of the central nervous system of this animal and inoculated into rats numbers 123 to 125, two guinea pigs, and six mice. hone of these animals develOped any paralysis. In this experiment other passages were made, as can be seen from table I, but they proved to be of no significance. Reinocu- lations were performed once or twice on some of the animals as -10- indicated. Starch was used to traumitize the brain tissue in certain animals as indicated in the table. It is interesting to note that rat number 18h was able to withstand 0.2 cc. of the virus and 0.2 cc. of the starch solution intracerebrally. Pooled Monkey Cords D. C7 Siroin‘ Table I II an H T T 2 RAT I Reina, 2-1540 Reina, 24540 Reina. 7.4540 K!” 3 has Maohzz-‘E’I-eo RCI'IIoc,2.'27+0 Reina, 2.1140 fistilivercjds 33:3 " S «335—4119;...th— I“! 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S§Q\’c\\'_J I’d-RI I ‘ A7167 Ema ELF. 1.797 !RATI20H___ 0;“.5-16'50 O‘Iscm'Aea «Iv-Is-qo " @727 @723; Tremor \efl \eg 5-63“ 9 “Wm”, MUG. laced «Md 3 ‘ 2':5'9.- __ Tl” 6MK¢ K- ‘ Ma Ii“ '1 sm‘* Q\o§ 2.2.x Tm u\a\d~' ' ATE] 7 Ian . DIscaroco 6 ~:IL - +0 Reina: “inoculated. ' R P 'ra I'd' ssage : 5- Saar: sacrificed Enoc- hounded H H hisndus hispidus- HL‘. hispidus lI‘Horalis Disc: discarded *Number 0‘ monkey cord Pooled'QZ'ZB‘bi‘é'I-‘02'108'109'88 Reinocublion c owl numbers pool ed'ZZ'ZB'Q“67402408109’88‘ l -11- EXPERIMEKT II The use of virus from Fecal Specimen S.M. The source of the virus in this experiment was the stool of a child who had had contact with the Poliomyelitis virus. as reported by Kramer. et. a1.. (16). This stool specimen had been stored in the ice chest at four degrees centrigrade for six months and eleven days (reference in press). In this experiment twenty-nine rats and seven mice were in- oculated with this specimen. The stool was treated with ether and concentrated and inocu- lated into monkey number 123 (see table II) and four rats, numbers he to 51 inclusive. On the sixteenth day monkey number 123 develoP- ed flaccid paralysis of both rear extremities and was sacrificed. Sectioning of the spinal cord confirmed the diagno is of experimental poliomyelitis. The cord was ground and made into a ten per cent suspension in saline and inoculated directly into five rats, numbers 109 to ll} inclusive. Rats numbers 112. 113 and control rat number 11h were placed in a cage in a refrigerator at four degrees centri- grade. It was hoped that the virus would propagate in living tissue at that temperature. Experience had demonstratei that it could be so stored without loss of virulence. Rats numbers 109, 110, and 111 (see table II) did not show symptoms while rat number ll} on the third day was found on its side in the cage with all extremities spastic. This animal was sacrificed and the autOpsy presented a nutmeg liver and atrOphy of both.kidneys. The patholOgical pic- ture presented fatty degeneration of the liver. The brain contain— ed an abscess and the kidneys were degenerated and infiltrated with macrophages. The anterior horn cells of the cord were poorly stained while those of the posterior horn took the stain well. The cord and brain were ground and made into a ten per cent saline suspension and inoculated into five rats. One of these animals, rat number lh8, develOped symptoms and was sacrificed; lung worms were found in the pleural cavity as well as small hemorrhages in the lungs. The remaining animals of this group were discarded without symptoms after one month's observation. On the ninth day after inoculation, rat number ”8, referred to above, favored the left rear leg and was sacrificed. At autOp- sy the liver contained echinococcus cysts and the intestine was filled with tape worms. The spinal cord.upon sectioning was found to be normal except for a small amount of edema. A ten per cent suspension of the spinal cord and brain was inoculated into two rats, numbers 76 and 77, which did not show symptoms of polio- myelitis and were discarded after the observation period. On the third day after inoculation, rat number M9 developed a generalized weakness and favored the left side. On the fourth day the animal was sacrificed. The autOpsy presented an enlarged spleen and the brain appeared congestei. The tissue when sectioned, showed vascular congestion with some edema. The spinal cord and brain from this aninal were ground and made into a ten per cent saline suspension and 0.2 cc. intracerebrally, 0.5 cc. intraper— itoneally, and 0.0} cc. intranasally were inoculated into each of six new animals, rats numbers 61 to 65. Of these six anisals, rat number 62 and 65 develOped symptoms. The remaining rats were discarded without showing symptoms after the usual observation period. On the third lay, rat number 62 was was inactive and seemed to spare the right rear extremity; on the fourth day this animal was ill and very inactive and was sacrificed. When the animal was autOpsied, a large unorganized clot of blood was found in the extremity involved; at the base of the clot an echinococcus cyst was found. The liver also con- tained echinococcus cysts and the spleen was moderately enlarged. Sections of the brain showed edema and moderate vascular congestion with IOCalized areas of hemorrhage. A ten per cent saline suspension of the spinal cord and brain of this animal was inoculated into two rats which after a month's observation presented no symptoms and were discarded. The second rat of this group, rat number 66, did not deve10p a paralysis, but was irritable on the fourth, fifth, and sixth days. On the seventh day the right rear leg appeared to tire easily and the animal was sacrificed for passage. With the exception of the gall bladder, which was filled with stones, the viscera were nega- tive. MicrosCOpically, the spinal cord exhibited in one horn, motor cells staining poorly, otherwise, the spinal cord and brain appeared normal. A ten per cent saline suspension of the spinal cord and brain of this rat was inoculated into three rats. numbers 78 to 80, and seven mice. Rat number 80 received 0.2 cc. intracerebrally, 0.0} cc. intranasally, and 0.7 cc. intraperitoneally. 0n the thirteenth day after inoculation, the animal was sacrificed because of weak- ness and irritability. The spinal cord and brain were sectioned, and scattered hemorrhages were found in the brain tissue as well as in the ventricle spaces. The cells of the choroid plexus were ~11;- hyperplastic. The spinal cord was negative. This material was not inoculated into new animals, but was stored in glycerine. The other two rats, numbers 7; and 79 remained free of symptoms. Of the seven mice, number MPSI, also receiving the specimen from rat number 66, one mouse was found to have weak legs on the day after the Operation. It was sacrificed, and the cord and brain, after being suspended in saline, were inoculated into six mice, cage number H—lSS (see table II). 11 these aninals were discarded without showing symptOns of any kind after the usual observation period. The six remaining mice, cage number M—Sl, were also dis- carded without showing symptoms. .wdwutwcwucou wco woalmfw _. HEP .mvflwU‘fiJOpthu UOC‘umumwwd\DuQ£u.VOCH ..‘VUVLGUW‘Q u.uw,_Q c “Hg Javier nfikfinlz 00+ #6 #3: use...) .V: u mZHN: fizmt 1:5 W358 sellers. 21..» 90 3E. §-mqodlm.§é 0255 6 -2 .9.E.o?uk3...u>.~ «9.532 3.53 him .63 2: «:2» fig 924 0.39% W E a seesaw 9: “sum“. «F3 0¢-¢d «.n 63.. 3cm one a he on, o Ilsa {832. E3; «aim Ease a; t E m: E A 2.: , - t -Eamaral «moch vuswiuom conLWOL Inufiwé $2 :2— océ -m. .35 83 _mC§_ mesa are.“ 3:85 Nm 2: Q5 .Ex 0% -... 3% .omafi¢- a :39“ .38 #33253 3» 32x5. «33 5,3 sins: wigéa 9315 ”has £320 :1sz 2.333 owed 35 +3 3 E «45 $3. 3-3r3sadfi octmm- .mmquunlm 1:69 er+£uuciuem 9.03 mbcocfiEi 2.33:. £5 33.33 .423 fig? On: 03% II {III owewwfivfljdjwpll L. fig. .2 .m any, :6 ease} Seek... Lva‘ TEE). wings an 5 88» In 1 ,m a m . 3-3,.EH -xuflzms Sawmwsflm $33.5 $335 53:85 : a E a NE 35 025 more. 4035 I: c: a: x: 1: t: oeasuEJafi- «Slim on 272.6 33390 t. E a: ramm— e . h v ...u m wflwgunmuo www.mw 3% 35 A“! 50¢ «an: .38. a: «£53m haunt “.33 osgoecM _ Elk: @th 1| ,IL EEGKW \SQ L EXPERI I‘vffIlfT I I I Kessel Strains The infectivity for the cotton rat of three human strains of poliomyelitis, isolated in California by Kessel, were studied. The spinal cord of monkeys stored in glycerine were the source of the virus, designated as the Kahn, KcCsll, and Schultz strains. Thirty rats and five mice were used in this experiment. The Kahn strain, in a ten per cent suspension. was inoculated into four cotton rats numbers Ml, M2, M6. and MY, and five white mice. Each rat received 0.1 cc. intracerebrally, 0.7 cc. intrap peritoneally, and 0.0} cc. intranasally. Rat number Ml, in an observation period of three months, was at first slow and in- active, but later irritable. After this animal was discarded from the test. it was given 0.1 cc. intracerebrally of the stock Armstrong Lansing strain of virus, and deveIOped typical paralysis end.poliomyelitis in seven days. On the fourth day rat number #6 had some weakness in the left rear leg with an eversion of the foot. On the folloving day the symptoms were about the same. The left side of this animal was definitely weak on the sixth day, and the animal was sacrificed; the spinal cord and brain were made up into a ten per cent stspension and inoculated into nine rats. numbers 52 to 60 inclusive. Two of the nine rats, numbers 5M and 55. became inactive, and on the eighth day after the Operation, both rats were sacrificed. The spinal cords and brains of these aniuals were pooled in a ten per cent suspension and inoculated into three new rats. numbers 82. 53, and 8h. The viscera and extremities of rats numbers 5M and 55 were normal when examined for gross pathology. The stained sections of the spinal cord and brains, however, showed some edema and vascular congestion. Ho cellular degeneration of the motor cells could be found. The three animals receiving a pool of two spinal cords and brains of rats numbers 5M and 55 all deve10ped symptoms. Rat number 82 did not present paralysis, but on the third day had a peculiar gait. When it was sacrificed, the liver was anemic and contained echinococcus cysts. When the brain was sectioned, it exhibited edema. vascular congestion, and small scattered hemorrhages. The cord exhibited only slight congestion, and small scattered hemorrhages. Rat number 8” also developed a transient tremor on the third day. The organs were normal upon examination and.sections of the spinal cord and brain presented only slight edema and some vascular congestion. The spinal cords d brains of these two animals, numbers 82 and 8h were pooled and inoculated into three animals numbers 92, 9', and.9u, of which none presented symptoms of the disease. Bet number 83 of the previous group was found on the fourth day to have a fine tremor and was inactive. When sacrificed, liver cysts were present. On microsc0pic examination, the brain and spinal cord were found.to be slightly congested, the former having an occasion- al small hemorrhage into the brain substance. Transfer of a suspen- sion of this spinal cord and brain into two new animals was made, rats numbers 107 and 108. Eeither ani.al deveIOped symptoms during the month‘s observation period. The McCall strain of virus was inoculated into two rats, numbers OM} and OHM. Each animal received 0.1 cc. intracerebrally, 0.7 cc. intraperitoneally, and 0.0} cc. intranasally. On the twentieth day, rat number 0&3 develOped a systemic weakness and was sacrificed. The liver of this animal contained echinococcus cysts, and white patches were found on the kidneys. The micro- sc0pic patholoay of the kidneys was that of an acute hemorrhagic and toxic nephritis. The nervous system of this animal was not inoculated into new animals. The other animal receiving tris strain of virus did not deve10p sufficient symptoms to warrant an autopsy, and was discarded after three months daily observation. The third California strain, Schultz, was inoculated into two rats, numbers 39 and hO, and monkey number 73. The rats were dis- carded without having shown symptoms after the usual observation time. Monkey number 73 on the eleventh day developed flaccid.paral- ysis of the right shoulder and was sacrificed. Examination of sections from the spinal cord of this monkey confirmed the diagnosis of experimental poliomyelitis. Two rats, numbers 67 and 68, were inoculated with 0.15 cc. intracerebrally, 0.7 cc. intraperitoneally, and 0.03 cc. intranasally with a ten per cent suspension of the spinal cord of monkey number 73, but did not deve10p any symptoms. x. .__4.._U:__ wJV.mf{ . 4 I .. n31.mo.{ unwini P. 41 . 4 ...»deO 3:; S Em {scan a... a. J . «nae: b gaffré fly, 1:15.. .2 fur/dud 953» 0.. .3 ... 35.3.15 ——u_—— .meu.__ mwnhcg _ . .. i .-.e Cd in .c13,s,oocH_. £23 caiaycaafim ot $5 m ”admixed: fivAJ Les) 2. a? 4 2.3.6 33390 x33. 5H arc... 10.3 m2; 3:... Q. .25 0+. 9mm, , ii .umdmnoy fwd; -m.m . -II . £33.39... | JoCH who: .-fnJ r3. . . $8 ¢ .1 0:. n n ....ch..|L.P toguvm at. _ is. _I i .... _ Iww 1 n. 34.:uocH .3 .0. (odd .fitazaad - 429—11 4.. ...». o m “MI . lrm .. I a . . . . .U GE 3. oFBeSumfi hawk gawk... a .23.. nausea 82a TEE «E: R 2.. mm 5. mt; mm :5 m: WEE .... J... Tm! T. a; F; .. .... - .... r . - 0... N4 42 38H la! 0+ nah «3.5.56 72m mQEm 3.5m .. Hegahsmi .m..n@go>4_ 1 mrdfla 199. VCOGM a... T ...3 4.3 2. «Mani-Win afieéfin as 5.00.; n: .31 a. n, dd; ...de .N. 4.5 ‘a L a ,0 -... Shaun... aqua w .....M 145390 :4 <00) town‘s} F4 .35 f2wg§.g I E 3% SHE we»: 2. E {I m —11 _d.. at hdw. OWN J ..CJ’DJOfd 0¢ fifi gr I 3%. H — 9'00 3 .2 NM wEOHv 3:in qwnmwx -13- ETEUMLTIV The effect of hyperpyrexia induced by short wave induction thermy on the transmissibility of the virus of Poliomyelitis. Four rats. all littoralis, were subject to a short wave induction current until the boiy temperature was increased. Two animals were exposed for ten minutes. and a body tempera- ture of 105.M degrees Fahrenheit obtained. The second two animals were exposed for eighteen minutes. and temperatures of lOS.M and 109.h degrees Fahrenheit were obtainel. The nor- mal temperature of the animals ranged between 101 degrees and 103 degrees Fahrenheit. Immediately after exposure. each animal was given 0.15 cc. intracerebrally, and 0.6 cc. intraperitoneally of a ten per cent emulsion of the spinal cord of monkey number 88. (D. G. strain}. On the sixth day one of these rats develOped weakness of the right rear leg. On the ninth day the involved leg was in about the same condition as the previous three days, and the animal was sac- rificei. Cn autopsy the liver was very dark and firm in consistency. The cord and brain were removed and stored in glycerine while sections of this specimen were examined. The motor cells of the spinal cord stained poorly, and both spinal cord and brain showed some vascular congestion. The remaining three animals recovered from the exposure to the inductive current and Operation without symptoms of poliomyelitis for an observation period of two months. -19- E33719 IEIETT V The susceptibility of immature rats to the virus of polio- myelitis. Sabin and Olitsky in 1937 ani 1938 (2M‘, (25) determined that young mice receiving Vesicular Stomatitis virus succumbed to the disease with much more consistent incubation period and suscepti- bility. Therefore, six rats, two at the age of one week old, two tvo weeks old and two three weeks old were given proportionate dosages of freshly triturated spinal cord of monkey number 109 (3.0. strain}. Io. of Animal Age 197 1 wk. 196 1 wk. 195 2 wk. 19h 2 wk. 193 3 wk. .192 3 wk. Note: 10 w In TABLE IV Dosage Symptoms .03 IC Weak nth day. l-dr. IN do Weak 2nd day. .06 IC home .2 III do Jone .06 IC Hone do II one - Intracerebrally - Intranasally Table four gives the results of this experiment. Outcome Starvation on 5th day. Starvation on nth day. Discarded 1 mo. Discarded 1 mo. Iflscarded 1 mo. Discarded 1 mo. “20“ DISCUSSIOH Burnet and ialacnemara in 1931 (17') (13) and I‘Jeyer in 1932 (19) were able to show immunological differences between strains of poliomyelitis virus using the neutralizing and cross protection tests. In 1933, Paul and Trask (26) and again in 193 , Paul, Trask et. al., (27) found marked differences in some of the strains of virus they studied. All these workers used the monkey as their experiment- al animal. With the immunological differences of strains of virus in mind, it was h0ped that, first, a second strain of poliomyelitis virus might be found that would infect the cotton rat or second, a closely related immunological strain might be so modified as to propagate in this inexpensive experimental animal. In experiment number I, the method of rapid passage was carried out. Every third day the central nervous system of an animal was removed, triturated in a mortar, centrifuged, and inoculated in- to new animals. This method was Carried on until four transfers of the virus were obtained. Sawyer's method of traumatizing the brain with an inoculation of a two per cent starch solution was tried in experiment number I along with the rapid passage. Many of the animals were reinoculated two or three times with freshly triturated spinal cord of monkeys. waever, no direct results with reinoculations were obtained. The cotton rat receiving the fourth rapid passage transfer rat number 31. as well as the subsequent aninals, appeared to show symptoms of some significance. Although none of the animals develooed a flaccid paralysis, weakness appeared between three and ,. ?],.. seven days in some of the animals, and was carried through three or four passages with similar symptoms. The pathological lesiOns were suggestive of those found in the spinal cords and brains of rats which succumbed after inoculation with the Lansing strain of virus isolated by Armstrong (9). However, no definite neuronOphago is was found in the experimental animals' spinal cords. Lillie and Armstrong (21) report vascular congestion, polymorphonuclear infiltration, neuronOphagia, and cellular necrosis from the Lansing strain of virus in the rats' spinal cords. The symptoms in the experimental animals disappeared after the fourth transfer and could not be duplicated in other attempts. In experiment number II it was observed that strain S.M. produced flaccid paralysis in a Macacus rhesus monkey. The spinal cord of this monkey when suspended and inoculated into rats, failed to produce the disease. Rats received positive poliomyelitis feces. treated with ether and concentrated; glycerinized as well as fresh suspension of the spinal cord of monkeys, but did not develop typical sym- ptoms of the disease. Some of the animals were kept at three to four degrees Centigrade after inoculation with the virus. Both the symptoms and the pathological picture were negative. The virus stored in glycerine at thaL temperature has been shown by Rhoads (20) to survive for eight years without detectable deter- ioration. Three strains of poliomyelitis virus isolated in California were inoculated in to rats. Whether differences in strains might vary with the geOgraphic location could not determined from exper- iment number III. The Kahn strain produced enough symptoms in animals to warrant four passages but no paralyzed animals were encountered. The other two strains tested were also negative, however, the Schultz strain infected a monkey. Results of inoculation with the virus after increasing the animals body temperature with short wave induction current was negative. In the last effort to propagate the virus, young rats born in the laboratory were inoculated with an emulsion of spinal cord of a monkey. Ages varied from one to three weeks. The rate one week old starved after removal from the mother, the remaining animals recovered from the Operation and at the end of one month were well, developing no symptoms. The cotton rate used in this work were wild rats captured in South Carolina.end Florida and shipped to the laboratory. The majority of animals were infested with parasites of one kind or another. This may explain some of the symptoms seen in the rats. However, many of the animals had symptoms and no parasites were found at postmortem examination. All surviving experimental animals were inoculated with Arm- strong's Lansing strain of virus. They uniformly deve10ped exper- mental poliomyelitis. This indicates that no immunity was deveIOp- ed following the inoculation of this material. Toomey and Tekacs in March, 19MO (22) report negative results in the attempt to infect s. hispiius and littoralis rats with nine strains of poliomyelitis virus in their laboratory. Seventy-nine rats were used by these workers in attempting to propagate the nine fl?3.. strains of virus. Parachivesco and Papazoiu in 19h0 (23) report failure. using irradiated mice inoculated with monkey passage spinal cord poliomye- litis virus. It appears that differences in strains of virus of poliomye- litis exist. So far only the Lansing strain of poliomyelitis virus will infect the cotton rat and white mouse. The reason for this variation remains an unsolved problem. -214. SUMfiARY 1. An attempt was made to establish other strains of poliomyelitis virus in the Eastern cotton rats Sigmodon hispidus hispiius and hispidus littoralis. 2. Eight methods known or suspected as contributing to successful infection with a virus disease in animals, were employed without success. 3. Five strains of the virus studied failed to produce typical symptoms or pathological lesions of the disease in the cotton rat. (1) (2) (3) (h) (5) (6) (7) (8) (9) (10) REFEREKCES Landsteiner, and Pepper, E. Uebertragung der Poliomyelitis acuta auf Affen. Ztschr. f. Immunitatsforsch. u. exper. Therap., Crig., 1909, 2, 377. Flexner, S., and Lewis, P.A.: The transmission of Acute Polio- myelitis to Monkeys: J. A. M. A., 1309, 53, 1639. First note. Flexner, S. and Lewis, P.A.: Transmission of Epidemic Poliomye- litis to Kankeys; a.further note, second note. J.A.M.A., 1909, 53: 1913- Strauss and Huntoon: N.Y. Med., Jour., 1910, XCI, 6h. Leiner and von Wiesner: Wien., klin., Wchnschr., 1909, XXII, 1698. Landsteiner and Levaditi: Compt., rend., Soc., de Biol., 1909, IXVII, 592. Peabody, F.W., and Draper G.: Dochez, A.P.: Monographs of the Rockefeller Institute Ho. M, June 2”, 1912. Poliomyelitis, International Committee: Williams and Vilkins Co., Baltimore, 1932, p. 107. Armstrong, Chas.: Public Health Reports Vol., 5h, Sept. 22, 1939. Armstrong, Chas.: Jour., Bact., 39; 63, Jan. 19MC. (11) Armstrong, Chas.: Public Health Report, Vb1., 5h, Ho. 52, Dec. (12) (13) (1h) (15) (16) (17) (18) (19) 29. 1939- Sawyer, ¥.A., and Lloyd, w.: Jour. Exper., Med: 5k, 533, 1931. Hoffman, 3.0., and Duran-Reynals: Science, Nov. 1M, 1939, vol. LXXII, Ho. 1872, p. 5C8. Traub, E.: Science: Vol. 81, p. 298, 1933. Traub, E.: Jour., Exper., Med., Vol. 63, 1936, p. 533. Kramer, S.D.: Public Health Reports; Vil. 5h, N0. M3, Oct. 27, 1939. Burnet, F.M. and Macnamara, J.: Immunoldgical difference between Strains of Poliomyelitis Virus: Brit. J. Exp. Path., 1931, 12, 57. Macnamara, 3.: Serum Therapy in Acute Poliomyelitis, Canad. Pub. Health. Jour., 1932, 23, 318. weyer, 3.3., Immunological differences between a Strain of Monkey Virus and Human Poliomyelitis Virus: Proc. Soc. Exp. Bio. and (20) (21) (22} -26- Med., 1931, 29. 289. Rhoads, C.P.: Jour. Exp. Mei.: April 1, 1929, Vol. XLIX, pp. 70l-70h. Lillie, P.D. and Armstrong, Chas.: Public Health Reports, Vol. 55, Jan. 19, 19MO, P. 115. Toomey and Takacs: Proc. Soc. Exp. Bio. and Med., vol. M3, {0. 3, March l9h0, P. 536. (23) Parachivesco, M. (Mme.) and Papazoiu (Mlle.), Compt. rend. (21+) (25) Soc. 3101., p. 503—508. iguo. Sabin, A.B., and Olitsky, P.K.: Influence of Host Factors on Heuroinvasiveness of Vesicular Stomatitis Virus, J. Exp. Med. July 1, 1937, Vol. 66. No. 1, P. 15-57. Sabin, A.B. and Olitsky, P.K.: Influence of Host Factors on Neuroinvasiveness of Vesicular Stomatitis Virus, J. Exp. Med. February 1,'1938, 701. 67, No. 2, P. sol-aha. (26) Paul, J.E. and Trask, J.D.: .A Comparative Study of Recently (27) Isolated Human Strains and Passage Strains of Poliomyrlitis Virus; J. Exper., Med., 58: P. 513-529, 1933. Paul, J.R., Trask, J.D: Beebe, A.H., and German, W.J.: Viruses of Poliomyelitis,: Jour. Exper. Med., 65: 687-7ou: 1937. ‘ii717177177711