WEE HOST RANGE {2F AN ENE’ES‘UC (EWCEPATHQGENEC GRPHAN “ £39” ViRUS ESQiATE-D FRQM HEALTHY QARY CAfiLE 'fi‘hasis far fho Dsgrse :5 F41. D. MICHEAN S?A'§‘E BNWERSEEV Afifias Mflamaéfi 505532357 '59523 ms This is to certify that the thesis entitled THE HOST RANGE OF AN ENTERIC CYTOPATHOGENIC ORPHAN "ECBO" VIRUS ISOLATED FROM HEALTHY DAIRY CATTLE presented by Abbas Mohamed Soliman has been accepted towards fulfillment of the requirements for Ph. D. degree inMicrobiology 8: Public Health Mm Major professor Date ‘1' 8 W195? 0-169 LIB R .«1 R ‘r’ E 9 Mn‘lngm 5:30 5 University I W cm. I L“? #3 THE HOST RANGE OF AN ENTERIC CYTOPATHOGENIC ORPFULN "ECBO" VIRUS ISOLATED FROM HEALTHY DAIRY CATTLE By ABBAS MOHAMED SOLIMAN A THESIS Submitted to the School for Advanced Graduate Studies of ‘Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1958 ACMGWBIENT The author wishes to express his deep appreciation and grati- tude to Dr. w.‘N. Mack, Professor, Department of Microbiology and Public Health for his guidance, patience and interest that made this work possible. I also feel greatly indebted to Dr. L. C. Ferguson, Head, Department of Microbiology and Public Health, Dr. R. D. Earner, Professor, Department of Veterinary Pathology, Dr. W. J. Mathey, Associate Professor, Department of Veterinary Pathology, and Dr. L. '1', Tiffany, Department of Microbiology and Public Health, for their valu- able, helpful suggestions, criticism, and the part they played in the preparation of this thesis. THE HOST RANGE OF AN ENTERIC CYTOPATHOGENIC ORPHAN "ECHO" VIRUS ISOLATED FROM HEALTHY DAIRY CATTDE By ABBAS MOHAMED SOLIMAN An Abstract Submitted to the School fbr Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1958 Approved by 110' W M\ Abbas M. Soliman An Abstract A virus isolated from the stools of "healthy" dairy cattle was tested fer its host range. The virus was fbund to multiply in the amnionic cavity of White Ieghorn embryonated eggs, and after several passages, the agent was adapted to the allantoic sac. ‘When the virus was injected intracerebrally into adult mice, hamsters, white rats, cotton rats, guinea pigs, cats, dogs, chicks and chickens, no evidence of disease was found in these animals. Suckling mice and hamsters were fbund to be susceptible to the virus. Both animals developed paralysis fbllowed by death. The virus could be maintained serially in both suckling mice and hamsters. Two calves were exposed to this virus. The first animal was given the virus per gs. Althtngh the animal died after h days, it was thought that death was not produced by the virus. The virus was recovered from the animals intestinal content. A second calf was injected intraveneously with the virus. This animal showed no symptoms of disease although the virus could be recovered from.the those for 13 days following inoculation. Neutralization tests were done on paired sera from cats, dogs, chickens and one calf. Only the calf‘s post-inoculation serum sample showed evidence of antibodies to this virus. . Antiserum, for a different enteric virus isolated from.catt1e, did not neutralize the virus under study; indicating that there are probably several antigenic different enteric viruses in cattle. TABLE OF CONTENTS PAGE ".3 f?- .. 3 (J: I. INTRODUCTION .um HISTORY. . . . . . . . . 1 II. LIERATUIEREVIEW............2 III. MATERIALS AND "muons . . . . . . . . . . 15 IV. RESULTS . . . , . . . . . . . . . . . . . 23 Tables . . . . . . . . . . . . . . . . 32 v. DISCUSSION. . . . . . . . . . . . . . . . hS VI.smmar.................52¢ . v ‘ privflf-Q S FELEAu—nmvlweooeooooeone...ocoo6 PART I INTRODUCTION AND HISTORY Interest in the enteric viruses was stimulated by a study of the human digestive system in which successive investigations indicated that an uncertain number of unknown viruses exist. Poliomyelitis and the Ooxsackie group of viruses were known, for several years, to be harbored in the digestive tract. Recently, another group of viruses was found in the human digestive tract but these viruses were orphans, that is the diseases they produced, if any, were not known. This group of viruses was called enteric cytopathogenic human orphan (ECHO) viruses because they were enteric cytopathogenic agents from humans and were orphans. A search for viruses in the digestive tract of animals revealed that cattle also have their grou) of orphan viruses which were called Ehteric Cytopathogenic Bovine Orohans, or ECBO viruses. \ Let? II LI'I‘SI-L’L‘I' L1... as. 'JLLW Pcplioip‘elltis is CharacteIi zed b5, ' gt i.,I‘-Ji_11i',cz.: tine]. .‘33‘q)‘=;-:7,:3;Is with possible involve: lent oi the cen tral neer us system. .Iicjcxzan (1,13) publishe t--e Iirst extensive review of its infectious nature. Lendsteiner and Pepper LlyO9) and- leaner and Lewis (1909) transmitted t]. e di cease to 110111: e ’ iollow Ing .41‘!"1‘.'J€)"‘J.'LOIIC.L1 :i.noc*1 ation with :1 sci lune emulsion of the mine]. cord from. a. fetal c:~.se of polio- .zlv‘elitis. ..ueeessive I-roI'2-;ers Letiolimed the disease tiu‘oug‘n‘ :.-t1‘.cr ‘cu'xes in prirmtes. jfiiptozus, identical, to those occurring in man, developed in the nozflcetgr as a result of in tranase l iIIsLWall? .xti01 or oral schinist ‘etion of the. infective arterial. A vire Lia, similar to the‘ t 00:, erved in man, was fer-11d during the incubation period (horstmaml, 1952; bodien, 1932; gt a}, 1),} and 1? LL; norshnszm it al, 1231;) with development oi antibodies. Liven-s, et al, @234), triec Intr... Lesticul r 1.1.0CILL: .;_ i:-".rl'1e living rhea 1s and c:.*‘.'.o:.zolf;us :non‘zzegrs, mice, hanmwrs and guinea pigs, but “sued to pr.-duce typical puliem'elitis syzigteI-Is. Chimpanzces were found to be more susceptible to the different types of the poliovirus, while monl»:e;.'s of various genera were susceptible only to certain strains (Melnick and lediruo, 1951). The Atales ggoferyi morIcey is highly susceptible to infection I-ri'th ti'xrce strains of type I ieliovirus but is ' susceptible to two strains 01' bps II and. III polio- virus. I'Is;.::l.ptomtic infection with types II or III poliovirus conferred various degrees of protection against reinfection 1-:ith one strain of efyg I polioviius gJunggeblut, et a1, 1931;). However, at tin: s bot} chimpanzees and morJ-teys failed to show evidence :I‘ disease, ~. -he n the ') 2 . . . _ . v v .u . .0 ‘ - Q . V ‘ } _~ J ,'. ' v-.. .1. J.‘ ~f ‘ . ' \ '~. a‘._1:11‘.1a'1'.e<'. "11." strain, 113.3 used as ”he UCSL) agenL. ..--.h unis Lulam of virus, more re isbance to the dis ease was evident in the monkey as m compared with chimpanzees Liloprcwslzi, __"c_. 2.1., 1934). lurnel, nd thermisra LlQ'Bl') re 3orted that monkeys, convalescent i'rom paralytic attacks induced by a certain strain of polionrjelitis virus, were highly res stcmt to further infection by; the same strain oi‘ virus. h’oz.ever, in chiipanzees cula ted orally, a second In? ‘c tion was initiated e; the same strain when a period 01‘ a gear separated the we exposures Llielz lick and lic‘rstnaml, 19117; Hone, 228$ 1950). Altamira-11g (3.939) ada, ted the type II lensing strain of pcliovirus to cotton rats and then to mice, thus enhancing e3. perinent. -l or}: with this virus. Schlesinger, it a}; Ll)1:.3) succee .ed in adapting a ‘Ififpe II train (‘I-Liddle Fast Force - LEE-I) to mice. Casals, 3:. a_l_, (1)51) found that newborn mice were more susceptible than adult mice to LEE-I strain. Suckling; hamsters u re found to be susceptible to the same strain and develop para 3.-":‘is Seller-sing intramuscular injection (Zioyer, gig 3.3:, 1952)- i‘hr mg. alternate passage in tissue culture and intracerebrally in mice, Iirech (193-11) was able to adapt the Type I, Mahoney strain, and the '1';1e Ill, Laen strp in, to the mouse intracerebrallg. Sinilarlg', Iionr z:s1:i, et al (3.931”) prepaea ted the Mahone; and the Sickle strains in nulce and cotton rats. Stanl If, 91': Q (195M ada:_>ted 'Ijqies I and- III intracerebrm 1; in the ”Prince Eienry", but not in white Swiss mice. Li, and habel (1931) adapted the lime III Leon strain to mice :‘ntraspinally. later Li and :‘c‘; 1. ei‘i‘er (13 33).“1 -i'ied and acapted the method for use with the neutralization 'Lest. Syrian ha: sters inoculated int'I"..'Icerehshells,r 1:5. in the Saul-:ett strain (‘I‘jrpe III) developed paralysis within S - a (7.13:5 (seas-rm, Chang, and Brueckner, 195k). Schwartzman (1950) reported that hamsters, treated with cortisone prior to intraoeritoneal inoculation with MEF-l strain, showed an increased percentage mortality rate and paralysis. All known strains of the poliovirus belong to three types: Type I(Brunhilde), Type II (Lansing), and Type III (Leon) which are serologically distinct. The specific antiserum of any type fails to cross neutralize a heterologous type (The Committee on Typing of the National Foundation for Infantile Paralysis, 1951). For classification and identification, the serum neutralization test was performed in vitro (Netter and Levaditi, 1910; Shaughnessey, Harmon and Gordon, 1930, Paul and Trask, 1935), in white mice (Haas and Armstrong 19h0, Hammon and Izumi, l9h2), and in tissue culture (Robbins, Enders, Weller and Florentino, 1951). A specific complement fixation test for poliomyelitis has been described by Casals, Olitsky, and Anslow (1951). Casals and Olitsky (1951), used type II virus as antigen. A promksing virus antigen has been prepared from tissue cultures (Svedmyr, Enders, and Holloway, 1°53). However, the test is still in the experimental stage. For many years, the in 31252 cultivation of the poliovirus received a great deal of attention. Levaditi (1913), Flexner and Nogouchi (1913), and Long, Olitsky and Rhodes (1930), attempted to cultivate the virus in monkey spinal ganglia, rabbit kidney and human ascitic fluid, but results were irregular and controversial. Sabin and Olitsky (1936), were probably the first to propagate the poliovirus in vitro on nerve tissue in Tyrode's solution. Enders, S Weller and Robbins (19h9) introduced the use of extraneural tissue for the propagation of poliovirus. Later, Weller, Robbins and Enders (19h9) were able to grow the virus on tissue cultures of skin and subcutaneous tissues from children. Smith, Chambers, and Evans (1951) found that adult human testicular tissue would support growth of the poliovirus. Soon various adult tissues such as kidneys, lungs, and spleen were found capable of growing the virus when properly prepared in tissue cultures (Weller, Enders, Robbins and Stoddard, 1952). A strain of neoplastic cells, "HeLa" (Gey, Coffman and Kubicek, 1952), was found to propagate the virus with evident cytopathogenicity (Scherer, Syverton, and Gey, 1953). The agent was also grown in monkey testicular cells (Scherer, Butorac and Syverton, 1951) but was not observed to multiply on testicular tissues from mouse, guinea pig, hamster, cow or dog (Robbins, weller, and Enders, 1952; Evans, Chambers, Smith and Byatt, 19511) . ' The virus growth in these tissues from humans or animals, normal or abnormal in origin (tumors), was accom)anied by cytopatho- genicity which is a Visual criterion of virus multiplication. Kidneys from all primates were thought to share this property but Kaplan and Melnick (1955) found that the Cgbus capucina kidney was an exception. Tissue cultures derived from the kidney of this monkey supported the growth of Type II (Y5K) strain without evident cytopathogenicity. After 119 serial passages in suckling hamsters, Roca-Garcia, Moyer and Cox (1952) maintained the MEF-I strain for hl generations in the yolk sac of the embrypnate cnick, Cabasso, Stebbins, Dutcher, Moyer, and Cox (1952) adapted the same strain to the allantoic sac. Without previous passage of the virus in suckling hamsters, Durham and Ewing (1953) reported apparent adaptation to the chick allantoic sac after cortisone treatment of the chick embryo. Li and Schaeffer (195h) reported the multiplication of a number of substrains of modified poliomyelitis virus (Type I Mahoney strain) grown on monkey skin or kidney tissue culture grafted onto the chorio- allantoic membrane. Dalldorf and Sickles (l9h8) injected the processed stool from a child ill with poliomyelitis into one day old white mice. A virus was isolated which they believed to be the poliovirus. A complete inves~ tigatian of this virus, however, revealed that it was not the polio- virus but an unknown agent. The virus was called "Coxsackie" by the two workers because the child from whom the virus was isolated was from Coxsackie, New Yerk. Soon after the isolation of the Coxsackie virus, Melnick, Shaw and Curnen (19b9), Curnen and Melnick (1951), Hummeler, Kirb, Pa., and Ostapiak (195h), Tyrrell and Snell (1956), demonstrated that the virus could also be found in cases of aseptic meningitis. From cases of pleurodynia, a syndrome described by Sylvest early in 1933; Curnen, Shaw and Melnick (19h9), Curnen (1950), Kilbourne (1950), Shaw, Melnick and Curnen (1950) were able to isolate an agent which proved to be identical to the Coxsackie virus. Coxsackie virus was also found to be responsible for herpangia by Melnick and Ledinko (1950), Shaw, Melnick and Curnen (1950, Huebner, Armstrong, Beeman and Cole (1950), and by webb, Wolf and Hewitt (1950), Since then, Coxsackie virus has been found associated with a variety of illnesses ranging from aseptic meningitis to "summer flu" and also, in a large number of healthy carriers (Melnick and Agren, 1952). The Coxsackie viruses have been repeatedly isolated from the stools of paralytic policmyelitis patients along with the poliovirus (Melnick and Ledinko, 1951; Rhodes, EE.El°’ 1950; Melnick, 1951). On the basis of their antigenic relationship and pathogenicity for suckling mice and hamsters, the Coxsackie viruses have been clas- sified into A and B groups (Dalldorf, 1950). Most investigators agree that group A viruses are responsible for the herpangia syndrome (Cole gt_al., 1951; Huebner, 1951; Huebner, 93: 11:, 1952; Huebner, 1957) and that group B is the etiological agent of epidemic pleurodynia (Finn, weller and Mergan, 19h9) or the aseptic meningitis syndrome (Hummeler gt 31., 195h3 Tyrrell and Snell, 1956)- Group A viruses pnaduce typical lesions in the striated muscle tissue of baby mice and suckling hamsters. These resemble Zenker's hyaline degeneration and are observed with all Coxsackie viruses irrespective of their antigenic type (Dalldorf gt 31., 19h9; Dalldorf, 1950; Melnick and Godman, 1951). In suckling mice Group B viruses produce moderate muscular lesions, characteristic encephalopathy, inflammatory changes in dorsal fat and pads of the feet (Dalldorf, 1950; Rhodes, 1956). The suckling mouse may be infected by intracerebral, intraperitoneal or subcutane- ous injection of the virus, but rarely by oral administration. As the animal becomes older, susceptibility declines. Mature mice are refractory to infection with the virus (Kaplan and Melnick, 1951). Kilbourne and Horsfall (1951) renorted that treating adult mice with cortisone increased their susceptibility to infection with Conn. 5 strain of Coxsackie virus. Monkeys inoculated with Coxsackie viruses sometimes develop subclinical infections. When administered orally to various types of monkeys, including the cynomolgus, rhesus and cercopethicus, the Ohio strain produced subclinical infection in the cynomolgus only. Following a viremia, these animals finally produced neutralizing and complement fixing antibodies (Melnick and Ledinko, 1950). Co-existence of the Coxsackie and poliovirus creates a peculiar situation which some workers describe as a "sparing effect", others as an "interference" phenomenon. Dalldorf (1951) demonstrated the "soaring" effect of this dual infection with mice in which the "Nancy" strain of group B Coxsackie virus was inoculated and followed 5-8 days later with the "Lansing" strain of poliovirus. The survival rate was higher in those mice receiving both viruses. Stanley (1952) attempted to demonstrate "interference" between the Coxsackie and the poliovirus in monkeys and mice. He found, that if mice received group A Coxsackie virus first, followed by MEF-I strain of polio- virus, there was a decrease in the incubation period of the polio- virus. 0n the other hand, if group B Coxsackie virus was used, there was an increased incubation period. A similar effect was not demon- strated in monkeys. Sixteen antigenic types of the Coxsackie viruses have been recognized to date. Identification and classification of these viruses inere accomplished with the aid of cross neutralization test in mice (Sickles, Dalldorf, 19149; Silkin, Manire and Farmer, 1950) and the complement fixation test (Casals, Olitsky, and Murphy, l9h9). Using tissues of newborn mice, Slater and Syverton (1950) propagated the Coxsackie virus in tissue culture. The virus was also adapted to the chicken embryo (Huebner, Ransom, and Beeman, 1950; Codenne and Curnen, 1952) and was grown on chicken embryo culture in serum ultrafiltrate (Shaw, 1952). Stulberg, Schapira am! Eidam (1952) found that a tissue culture of fibroblasts derived from the footpads of newborn mice supported the growth of some strains of group B. Sickles, Mutterer, Florino, and Plager (1955) reported that group A strains, designated 11-19-13-15-18, are cytopathogenic for human uterine tissue culture in plasma clot, for the HeLa cells, but not for trypsinized monkey kidney and testicular cells. The ECHO Viruses (Enteric Cytopathogenic Human Orphans) are a group of human intestinal agents which were discovered as a result of refined tissue culture technic. In Egypt, Helnick and Agren (1952) isolated two agents by means of tissue culture. One proved to be a Coxsackie virus and the other failed to produce lesions in the central nervous system of rhesus or cynomolgus monkeys. The latter virus was also non-pathogenic for adult and suckling mice. Antisera for Types I, II and III poliovirus failed to neutralize its cytopathogenicity in tissue culture. This agent was antigenically distinct from similar agents isolated in the United States. Steigman, Kokko and Silverberg (1953) isolated a similar "unidentified" agent from the stools of a hospitalized child in Cincinnati, Ohio. Following this, a large number of strains was isolated. They were at first believed to have no significant role as 10 disease agents (Committee on ECHO Viruses, 19SS)-' However, since these viruses were found in the stools from patients with either polio- myelitis or Coxsackie infection, there was some aporehension regarding their pathogenicity. Occasionally, some of these agents could be isolated from patients with aseptic meningitis in which no poliovirus or Coxsackie virus was found (Enders, 1957; Winkelstein gt_al., 1957). They were also isolated from stool specimens from individuals not showing any evidence of disease (Alvarez and Sabin, 1956; Honig, 3t 3;, 1956; David and Melnick, 1°56; Enders, 1957). Laboratory animals, including adult and suckling mice, hamsters, guinea pigs and rabbits (Steigman, Kokko and Silverberg, 1953), monkeys (Alvarez and Sabin, 1956; Ormsbee and Melnick, 1957) were found to be non-susceptible to experimental infection. In tissue culture, the ECHO viruses are highly pathogenic to .cells of rhesus monkey (Magggga_mul§ta) kidney (Steigman, Kokko and Silverberg, 1953; Committee on ECHO Viruses, 1955; Duncan gt 31., 1955; Alvarez and Sabin, 1956). Kidney cells of the South American capuchin monkey (Cebus capucina) support the growth of type 10 virus, while cells from the African red grass military monkey (Erytherocebus 22233) propagate type 7. HeLa cells also supaort viral growth but are not as susceptible as rhesus monkey kidney cells (Committee on ECHO Viruses, 1955). Some types of ECHO viruses grow in cells derived from human embryonic skin and muscle, postnatal uterine and human kidney tissues (Hammon gt 51., 1957) and human amnion cells (Zitcer, Fogle and Dunnebacke, 1955). 11 Plaque morphology of types 1, 3, h, 5, 6, 7 and 9 was studied. All except type 7 could be differentiated from the poliovirus plaques by their diffuse irregular margins. Cells in some areas of these tissue cultures failed to show cytopathogenicity (Committee on ECHO Viruses, 1955). At the present time 19 strains of the ECHO viruses are known. Serologically, this group of viruses is not neutralized by poliomye- litis antiserum or by antisera for the Coxsackie viruses (Committee on the ECHO Viruses, 1955). However, the members of this group are neutralized by human gamma globulin and individual human serum. Complement fixing antigens for this group have been detected in tissue culture fluids (Committee on ECHO Viruses, 1955). For the sizes of the viral particles, the Committee on ECHO Viruses in 1955 approved the following measurements obtained by the gradocol membrane filtration: types 1, 2 and 3 are 11- 17 mu; tyae 10 is 60-90 mu. Alvarez and Sabin, (1056) found by the same method, that types 7, 8, 9 and 11 measured 300 mu and type 10 measured 60-90 mu. In 19h6 Olafson, McCallum and Fox described an explosive infection in cattle with a high morbidity and a low mortality rate. Affected animals showed anorexia, weakness, salivation and nasal discharge, ulceration of the buccal mucus membrane and diarrhea with sometimes a rise of temperature. Post mortem examination findings were: inflammation and ulcerations along the digestive system, congestion of subcutaneous tissues, hemorrhages on the eoicardium and vaginal mucus membrane. 12 Calves experimentally infected with the isolated virus did not always develop the symptoms described above. Mice, rabbits, guinea pigs and sheep were not susceptible to infection by any route of inoculation. Embryonated eggs inoculated by different routes did not support virus growth (Olafson, McCallum and Fox, l9h6; Olafson and Rickard, l9h7). Lee and Gillespie (1957) propagated this virus in tissue culture of bovine embryonic skin and muscle in chick embryo extract, and in trypsinized bovine embryonic kidney cortex tissue culture. In bovine skin and muscle tissue culture, twenty serial passages were made with this virus. In cultures of bovine kidney, 15 passages were made. The titer obtained in the former system was 10 LDSO and in the latter, 107 LD50. No cytopathogenicity was observed in either system. Moll and Finlayson (1958) described a febrile disease in calves characterized by cough, nasal discharge accompanied by considerable mucus in the stools. The virus isolated from the stools of these calves was demonstrated to be cytopathogenic in tissue culture but paralysis was not induced in suckling mice. The agent reported in this thesis is probably an ECBO (Enteric Cytopathogenic Bovine Orphan) virus. It was obtained from Miss Elva Funuse of the School of Public Health, University of Michigan. The virus was found to propagate with evident cytopathogenicity on tissue cultures originating from: cattle, and rhesus monkey kidney cells, and chick embryo body tissue cultures. In embryonate chicken eggs the agent multiplied when introduced onto the chorioallantoic membrane producing pock-like lesions. It also developed in the yolk and amninnic sacs of the fertile chicken egg. 13 The virus did not grow in HeLa cell tissue cultures, and when introduced into the allantoic sac of embryonate eggs, did not multiply. Suckling mice succumbed with paralysis upon intraperitoneal inoculation with viris propagated in eggs or tissue culture but not with the virus directly isolated from stools (Kunin and Minues, 1957). Antisera prepared by intravenous inoculation of rabbits with this virus neutralized the virus from tissue cultures and embryonate eggs. Serum from the host cattle did not protect chicken embryos but did delay their death (Kunin and Minuse, 1957). Antisera from 1b of the ECHO viruses, Vesicular Stomatitis-types New Jersey and Indiana, calf pnemonitis enteric virus, the Sabin calf 25 enteric cytopathogenic virus, bovine mucosal disease and bovine rhinotra- cheitis failed to neutralize this virus. Kunin and Minuse (1957) found that the viral particle has a sedimentation constant ranging between 150-200 Svd. Storage of the virus at hC for 5 days did not affect the titer while at -70C for few weeks, the titer was reduced. Infected amnionic fluid stored for several months at hC retained the titer. When this agent was inoculated into the amniotic sac of embryonate eggs from Barred Rock chickens, Kunin and Minuse (1957) found that the harvested fluid contained.what appeared to be melanin particles. Since the virus reported in this study was isolated as an "enteric agent" from cows, it was of interest to determine the infec- tivity spectrum of this agent. To also determine the effect of this Virus on cattle and to describe the symptoms seen in ex)erimentally infected animals. 1h PART III MATERIAL AND METHODS Embryonatelgggg Eggs used were from White Leghorn hens and.were incubated for 9 consecutive days prior to inoculation. The incubated eggs were inocu- lated by the allantoic sac route, each was given 0.1 m1 of the appro- priate dilution of inoculum. Before harvesting the allantoic fluid, all eggs were chilled at h C. Control eggs were, in every case, one- fourth the number of inoculated eggs and were inoculated with the same amount of phosphate buffer solution (pH 7.6). All test and control eggs were candled daily for 10 days following inoculation. As death of the embryos was the criterion of infectivity, all survivals were discarded after this period. All eggs with dead embryos were stored at h C until the allantoic fluid was harvested aseptically. A sterility test was performed on.the allantoic fluid removed from.every egg. ‘When proved sterile, the material was pooled and stored.at -20 C in.a screw cap Vial. ‘Eirus Titration The virus contained in the pooled allantoic fluid was titrated :in.embryonate hens' eggs. The virus suspension, in allantoic fluid, Twas quickly thawed under tap water and centrifuged at hOOO rpm for 15 Hminutes to remove coarse particles. The supernatant fluid was removed ffinom the sediment. Using the phosphate buffer solution as diluent, 30ma1 ten fold dilutions ranging between 10 1 and 10 were prepared 16 from the undiluted stored allantoic fluid. One tenth m1 of each dilu- tion was injected into the allantoic sac of six 9-day old embryonate eggs. Control eggs received 0.1 ml of the buffered solution only. From each dilution, two serum broth tubes were inoculated with 0.1 ml of the suspension and incubated for 5 days at 37 C to test fer sterility. The LDSO titer of the virus as determined by the method of Reed and Muench (1938) was 107'0 per 0.1 m1 of the allantoic fluid. This pool of allantoic fluid served as a virus source for all the eggs and animals tested in this work. seems: All mice used were Swiss white mice of the Webster strain. Nice 3 weeks old or more were considered as adults. Inoculations were intra- cerebral, intraperitoneal or subcutaneous, with 0.03, 0.5, 0.2 m1 of the virus respectively. Oral administration of the virus was in 0.1 m1 amounts. Uninoculated control mice were also included and all animals were observed for a period of 21 days subsequent to inoculation for the development of symptoms. Suckligg Mice Immature mice were one or two days of age when injected with the ‘Virus or material suspected of containing a virus. Those animals inocu- lated intracerebrally received 0.01 ml, and those inoculated intra- peritoneally were given 0.05 ml of the material. Control animals received no injections. All mice used as test maimals were observed for 21 days subsequent to inoculation for symptoms 0f disease. 17 sane 2.15.2 Only adult animals were used. These animals were raised in the Department of Microbiology and Public Health, Michigan State University. Animals receiving intracerebral inoculations were injected with 0.1 ml, animals intraperitoneally inoculated were given 0.5 ml, and the sub- cutaneously injected hosts received 0.5 m1 of the material. Some guinea pigs were given 0.5 ml of the virus 22.!- pg. sue me Adult white rats were obtained from a stock raised in the Depart- ment of Chemistry, Michigan State University. The animus were injected intracerebrally, intraperitoneally, subcutaneously or received the mate- rial orally with 0.1, 0.5, 0.5, and 0.5 m1 of the virus, respectively. Uninoculated controls were also included. All animals were observed for 21 days for symptoms ‘ of disease. gauge at: . Adult cotton rats (Sigmodon higpidus hespidus) were obtained from the Michigan State Department of Health Laboratories in Lansing. Animals were inoculated intraperitoneally, subcutaneously or received the material orally with 0.1, 0.5, and 0.5 m1 of virus respec- tively. These uninoculated controls and test animals were observed for 21 days for symptoms of disease. Hamsters Adult Syrian hamsters were obtained from the Leptospirosis Lab- oratory, Michigan State University, and received the same amounts of Virus as described above for the white rats. 18 Suckling one to two day old hamsters were injected with 0.01 ml of the virus intracerebrally or 0.5 ml intraperitoneally. Again, un- inoculated controls were included and observed for symptoms of disease for 21 days. Cats Six cats ranging in age between 2 and 1; months were used in this study. The animals were of mixed breeds and sex. History of previous exposure to the virus under study was unknown in these animals; there- fore, four of the animals were bled for serum samples prior to exposure to the virus. Each cat, while under anaesthesia, was inoculated intra- cerebrally with 0.5 ml of the virus. Two cats were observed as controls. 0n the second day following inoculation, one of the animals died presumably from trauma and was discarded. The remaining test animals were observed daily and the temperatures recorded. After the usual observation period of 21 days ,. the test animals were again bled to obtain a post-inoculation serum sample. £9.33 Twa dogs of mixed breed, 3-1; months of age, were obtained from the Department of Physiology, Michigan State University. Thirty-five ml of blood were obtained from the external saphenous vein of each dog. The site of intracerebral inoculation was shaved, cleaned with soap and water and tincture of metaphen applied to the areas of inoculation. Using a sterile 14 cm x 2 m needle as a trephine, 1.5 ml of the virus was introduced into the left cerebral hemisphere of each dog. During the 21 days observation period, temperatures were recorded 19 twice daily and at the end of the observation period the two dogs were bled. The second blood sample represents the post-inoculation serum sample. Chickens A number of‘White Leghorn chickens, 6-8 weeks of age were kindly supplied by the Department of Poultry Husbandry, Michigan State Univer- sity. Some were inoculated while others served as controls. Test chickens were first bled from the‘wing vein, then each was inoculated intracerebrally with 0.1 ml of the virus. Fellowing the observation period, the inoculated chickens were bled for the second time. Serum was secured from pre- and post-inoculation blood for serun.neutralisation tests. Bag! Chicks A group of one day old.White Leghorn chicks were kindly offered by the Department of Poultry Husbandry, Michigan State University. Test birds were inoculated intracerebrally with 0.03 ml of the virus. The control and test birds were observed for 21 days subsequent to inocu- lation for any symptoms of disease. 9.411.122 Two calves were exposed experimentally to the virus in an attempt to produce disease. The first was a five day old Jersey calf Obtained from the Dairy Department, Michigan State University. It was not in good physical con- dition when obtained but had a good appetite and normal temperature. The calf was observed for 7 days prior to being used as a test animal. 20 On the eighth day, a fecal sample was obtained and a blood sample was collected aseptically from the jugular vein prior to feeding the calf with 5 ml of the virus. On the third day following the feeding of the virus, the animal refused food, and had diarrhea. AThe temperature was normal. The following day, the animal had a subnormal temperature and subsequently died. The second animal was a 3 month old AberdeeneAngus calf obtained from the Department of Animal Husbandry, Michigan State University. The animal appeared to be in good physical condition. The calf was Observed for a period of one week during which the animal had a normal tempera- ture. At the end of the observation period, stools and a blood serum sample were collected before the calf was injected intravenously with 2 ml of the virus. Temperatures were taken daily and a stool sample was collected each day starting the fifth day after inoculation. Samples were collected for 15 days and were stored in the freezer for subsequent evaluation. ‘Preparation‘g£.8tool Suspension The frozen fecal specimen was allowed to thaw at room tempera- ture. A 10 per cent suspension of fecal material was prepared in phosphate buffered solution at pH 7.6. Penicillin and streptomycin, 500 units and 500 ugms respectively, were added to each milliliter of the suspension. A portion of the suspension was then centrifuged at hOOO rpm for 30 minutes at room temperature. If the supernatant fluid ‘was still turbid, centrifugation was continued for another 30 minutes. frhe supernatant fluid constituted the virus suSpension. 21 Serum.§§utralization Tests The serum neutralization tests were performed on the pre- and post-inoculation sera from dogs, cats, chickens, calf 2, and pre-inocu- lation serum.from calf 1 that died before obtaining the post-feeding serum sample. The post-inoculation sera were obtained at the end of the 21 day observation period. The test was also carried out on the pre- and post-inoculation sera from a calf infected with "virus diarrhea" virus. The concentration of the virus contained in the allantoic fluid was determined immediately prior to use. The LDSO, as determined via the Reed-Muench method (1938), was found to be 106°6 i.u. per 0.1 ml of allantoic fluid. To perform the neutralization test, serial 10 fold dilutions of the virus were made in phosphate buffer. The unknown serum was inacti- vated at 56 C for 30 minutes. From each dilution tube, equal parts of diluted virus and the test serum were thoroughly mixed and incubated at roam temperature for 30 minutes prior to inoculation., In order to be within the range of 100 per cent mortality in the lower dilutions and 3 through 10"7 100 per cent survivals in the higher dilutions the 10- dilutions were employed . From each dilution, five 9 day old embryonate eggs were inocup lated via the allantoic sac, each with 0.1 ml of the virus serum mixture. Virus and serum controls were also included. From each dilution tube, five 9 day old embryonate eggs were inoculated.via the allantoic see, each with 0.1 ml of the virus serum mixture, and incubated at 37 c 1' or 10 days. 22 The end point of virus activity was considered to be the highest dilution of the virus in which 50 per cent or more of the inoculated embryos were killed. Assuming the end point dilution to contain one infectious unit, the reciprocal of the dilution would indicate the concentration of infective doses or the virus titer in the original undiluted sample. The end point of viral activity in the virus-serum mixtures was the lowest dilution of the virus in which 50 per cent or more of the embryos survived. The neutralization index, which is the measure of reduction of viral activity by neutralizing antibody, was expressed as the difference between the virus titer and the virus- serum mixture titer. The average neutralization.index was computed by dividing the sum of neutralization indexes of either the pre- or post-inoculation sera by the number of serum samples from each respec- tive group of animals. PART IV RESULTS The first virus passages in embryonate eggs were made by way of the allantoic sac in White Leghorn eggs in order to obtain enough of the virus suspension to complete this work. The original virus, as received in this laboratory, had been adapted to the amnionic cavity of Barred Rock embryonate eggs. 0f h8 - 9 day old embryonate eggs, inoculated via the allantoic cavity, each with 0.1 ml of a 10'”1 dilu- tion of the original amnionic fluid, only 15 embryos (31.2%) died within 10 days. The allantoic fluid from these eggs was harvested and after sterility tests, was pooled and stored in the frozen state for further passage. In contrast to control eggs inoculated with buffered solution, but no virus, the infected embryos were dwarfed and showed less develop- ment. Edema of the chorioallantoic membrane, congestion and.focal hemorrhages were the most consistent findings. Cutaneous and subcuta- neous hemorrhages, edema and.malformation of the toes were seen in many embryos. It was observed that the allantoic fluid from the White Leg- horn eggs did not contain the melanin particles which were present when this virus was harvested from the Barred Rock eggs (Kunin and Minuse, 1957). On the contrary, the allantoic fluid was invariably clear except for traces of blood or excess of urates in some of the harvested eggs. A sample of the harvested.virus in the allantoic fluid was 2h serially passed through eggs from White Leghorn hens. After 10 serial passages, this virus was found to produce high mortality when passed by the allantoic route of White Leghorn embryos. Changes, as described, in the morphology of the embryos continued. The titer of virus in the pooled, frozen, allantoic fluid harvested from the tenth serial passage was determined. Again, White Leghorn eggs were used for the titration and 0.1 ml of the allantoic fluid was introduced into the allantoic cavity. The LDSO titer of the virus 107'0 in the allantoic fluid. These data are represented in Table I. To determine if the virus in the allantoic fluid would infect the embryos by the amnionic sac, 12 eggs were inoculated, each with 0.1 ml of the pooled allantoic fluid into the amnionic cavity. Nine of the 12 eggs embryos became infected and died (Table 1). Five control eggs were inoculated by the same route with 0.1 m1 of phosphate buffer solu- tion (pH 7.6) and all survived the 10 days incubation period. The pooled allantoic fluid containing the virus (10th serial passage) was used as source of virus for the experimental animal inocu- lations. ‘When adult Swiss mice were inoculated with the virus, all inocu- lated mice remained free of symptoms of disease. Table IV shows the routes of inoculation of the adult mice. To determine if this virus could be propagated in suckling mice, a sample from the allantoic fluid.was injected intracerebrally into a litter of 7 suckling mice. Five days after inoculation (Table II) 2 of the suckling mice were found paralyzed. One of the two mice de.eloped paralysis of both hind legs and was sacrificed. The second animal 25 showed paralysis of the right front leg, or a "wrist drOp" type of paralysis, quite similar to that seen in adult Swiss mice after being injected intracerebrally with the type II poliovirus. This mouse was also sacrificed. The brains were removed from these two suckling mice and a 10-1 buffered suspension (pH 7.6) was made by grinding the brains with a mortar and pestle. The suspension was then centrifuged at 3000 rpm for 30 minutes to remove coarse particles. A sample of the supernatant fluid.was used to inject l2 suckling mice intracerebrally and 7 intra- peritoneally. Two of the 12 mice (Table II) died without observed symptoms. However, 10 were seen to be paralyzed. All seven intra- peritoneally inoculated mice died after developing paralysis (Table II). The first evidence of infection was sluggishness and impaired movement of the affected limb. Paralysis usually developed in the animals one day later and was progressive. The paralysis developed in either the front or hind legs but no evidence of complete paralysis in all four limbs was observed in mice. The animals were sacrificed at the height of paralysis to harvest the brains. A second passage of this mouse brain material was introduced intraperitoneally in seven suckling mice all of which developed paralysis and died (Table II). To compare infectivity by the intracerebral and the intraperi- toneal injection, the virus was titrated by both routes in one or two day old mice. Paralyzed mice brains were aseptically harvested and finely ground. A 10 per cent suspension was prepared in phosphate buffer solution (pH 7.6). Penicillin and streptomycin, 500 units and 500 ugms, respectively, were added to each ml of the suspension. 26 The suspension was spun at LOOO rpm for one hour. The clear super— nets was then removed from the sediment and serial ten fold '1 and 10'8 were made in phosphate dilutions ranging between 10 buffer solution (pH 7.6). Four suckling mice per dilution were inoculated either intra- cerebrally or intraperitoneally. Mice inoculated by the first route received 0.01 ml, while those inoculated by the second route had 0.05 ml of the respective dilution. The virus titers in both cases were determined according to Reed and Muench's formula. Following intracerebral injection the virus titer was LD50 10 '6, and LDSO 105°S,as a result of intraperitoneal inocu- lation (Table III). The guinea pigs did not show any evidence of paralysis or disease during the test period of 21 days. Temperatures were recorded on each injected animal twice each day. None of the animals developed abnormal temperature. White rats were injected with the virus by various routes (Table IV). None of the 16 animals injected showed symptoms of disease during the 21-day holding period. Three cats ranging in age between 2 and h months old, but of un- known breed, were given a 0.5 ml intracerebral injection of the virus contained in allantoic fluid. After recovery from the anesthesia, none cievelOped evidence of infection, during the 21 day observation period. The two dogs used in this study were 3 and h months old and of unknown breed. Each was given 1.5 ml of the virus intracerebrally. Ihxring a 21 day observation period the temperature of these animals was 27 recorded twice daily. At no time during this period did either animal have a rise in temperature or have symptoms of illness. Six White Leghorn chickens, about 6 months of age, were given 0.1 m1 of the virus intracerebrally. Thirty-seven 1 day-old chicks of the same breed were given 0.03 ml of the virus intracerebrally. As can be seen in Table IV, none of these animals developed any signs of illness during the 21 day holding period. Like the adult mice, adult cotton rats and hamsters did not develop any evidence of disease after being injected with the virus. In Table V the amounts of virus given and the routes are shown. All animals remained symptom-free during the 21 day observation period. Suckling hamsters were found to be susceptible to the virus (Table VI). All suckling hamsters receiving the virus intracerebrally and intraperitoneally died. 0f the eight animals receiving the virus intracerebrally, two died without symptoms being observed, while six were seen paralyzed before death. Of those eight animals receiving virus intraperitoneally, seven were seen to have paralysis. The para- lysis seen in these animals differed from that seen in the mice. In the suckling hamsters some had complete paralysis of all four limbs. The animals' limbs were extended.posteriorly and paralysis appeared to be of the epastic type. Table VI 1130 shows the results of eXposing young calves to this 'virus. The first animal received orally 5 ml of the virus contained in allantoic fluid. Three days after receiving the virus this animal developed diarrhea. No blood or mucus was observed in the feces. The temperature of calf I remained within normal limits. 0n the fourth 28 day after receiving the virus, the animal was weak and could not stand alone. The diarrhea continued and the animal's temperature was found to be subnormal. The animal died during the night and.was autopsied. The emaciated animal had patches of lung congestion. The major pathological changes were found in the digestive system. The omasum, abomasum, intestines, and cecum all had patchy inflammation of the mucus membrane. The intestinal contents were liquid, but did not contain blood. The liver and the kidneys were congested and the gall bladder was distended. Congested areas of the omasum, abomasum and segments of the small and large intestines were frozen in sterile glass tubes and held for isolation of the virus. Calf 2 received 2 ml of the virus, in allantoic fluid, intra- venously. The temperature of the animal remained within normal limits throughout the experimental period. During the 15 day observation period no clinical evidence of infection was observed except some mucus in the feces. The mucus was minimal but was not observed in the feces prior to inoculation. The animal remained well throughout the test period. To determine whether either calf was shedding virus in the feces, the pre- and post-inoculation fecal samples and tissue samples were tested. The stool and tissue frozen Specimens were thawed at room temperature. A 10 per cent suspension was made in phosphate buffered solution (pH 7.6). Pooled samples of tissue from the alimentary tract of calf 1 were also made into a suspension by the same method. The Suspension was centrifuged at 3000 rpm for 1 hour. The supernatant IILuid‘was removed from the sediment and the former was treated with 500 I 29 units of penicillin and 0.5 mg streptomycin per ml to destroy the bacteria; cultures were also made to determine sterility. The specimens were finally inoculated into the allantoic cavity of White Leghorn embryonate eggs (Table VI). Control eggs were inoculated with the phosphate buffer solution. The eggs were incubated at 37 C and candled daily. Embryos which died within 2h hours of inoculation were not considered in the results. The results of inoculating eggs with the fecal samples and tissues from calf 1 are given in Table VII. The pre-exposure sample produced death in one embryo of 12 inoculated. The post-exposure fecal sample killed half of the embryos inoculated. Only 2 of 12 embryos inoculated with tissue of the gastrointestinal tract were killed. None of the control embryos was killed by the procedure. A pool was made of the allantoic fluid from those embryos that died subsequent to injection with the fecal sample and another pool from those dying after inocu- lation with tissue sample preparations. The allantoic fluid was then injected intraperitoneally into suckling mice. Five of 7 mice receiving the allantoic fluid from eggs inocu- lated with post-inoculation fecal sample were either paralyzed or dead within b-7 days. The mice receiving the allantoic fluid of tissue sample apparently were not affected. (Table VIII). In Table IX the results given are of eggs inoculated with the fecal samples from calf 2. There was no evidence of virus in the feces of the animal prior to inoculation, as the pro—inoculation feces did :not kill any of the 5 eggs inoculated with this material. The post- inoculation fecal samples at first did not contain virus, as was 3O evidenced by survival of the eggs receiving these samples. The embryos receiving the fecal sample of the seventh day following inoculation began to die. This pattern continued throughout the 15 day period that the feces where collected and tested for infectivity of the virus. To make certain that the embryos were being killed by the virus, the allantoic fluid from embryos that died following inoculation with post-inoculation fecal sample pools of the allantoic fluid from the eggs previously receiving the fecal preparations was combined in five pools. From each pool a number of suckling mice was inoculated intraperitoneally. Each mouse was injected with 0.05 ml. All five pools (Table x) caused paralysis and death in the mice. The tyne of paralySis seen in the mice, after re-isolation of the virus, was identical to that already described. Control animals in all cases remained in a normal condition and were disposed of at the end of the eXperiments. Serum Neutralization Tests Neutralization tests were completed with pre- and post-inocu- lation sera from the two dogs tested. The neutralization index of the dog 2 pre-inoculation serum.sample was O.h and the post-inoculation increased to 0.6. The serum of dog 1 shows a negligible increase between the pre- and post-inoculation specimens (Table XI). The results of 3 cats paired sera are presented in Table XI. Cat 2 is the only one that shows an appreciable increase in neutral- ization index in the post-inoculation serum when compared to that of the pre-inoculation serum. The results of six neutralization tests on the pre- and post- iJnoculation sera of chickens are illustrated in Table III . 31 The results on chicken 2 serum show that only in this one animal was there any rise in the serum neutralization index. The results on the five remaining birds do not show a significant increase in the serum neutralization index. The results in Table XIII are those of the neutralization tests on calf l pre-inoculation serum.and calf 2 pre- and post-inoculation sera, and both sera from a calf infected experimentally with a strain of "virus diarrhea" virus. The latter sera were kindly supplied by Dr. G. H. Gillespie, New York State Veterinary College. Only calf 2 post-injection serum exhibits a significant neutralization index of 2.1 TABLE I 32 RESULTS OF lNflCULATING WHITE LEGHORH EMBRYGHATED EGGS WITH THE VIRUS Incubation Virus Test Dose period in titer animals in ml Route Results days LDSO __~ ,_ Embryonated 0.1 ms“) 50/56* 5 .. 6 107°0 Eggs Controls O/lh ' (b) Embryonated 0.1 Am.S. 9/12 3 - 1; Eggs Controls O/h (a) A.S.: Allantoic sac. (0) Am. 3.: Amnionic sac. *Numerator:Number Affected Denominator{thber Injected 33 TABLE II RESULTS OF INOCULATING SUCKLING MICE WITH THE VIRUS Amount Incubation in ml Inoculum Route Results period in days 0.01 A.F.(a) 1.0.(b) 2/7* hes 0.05 M.B.S.(c) I.P.(d) 7/7 h-S 0.01 M.B.S. I.C. 12/12 3-6 . 0.05 M.B.S. I.P. 7/7 h-6 Controls 0/10 (a)A.F.: Allantoic fluid. (b)I.C.: Intracerebral. (°)M.B.S.: Mouse brain suspension. ( d) I .P . : Intraperitoneal . fl'Numerator: Nunber affected fiffioifimor: Number irTJected 3h mommohca,uonemm,“nopmcmsonmn ompoomwo nonesz snowshoesz * .HaosOpuhoamuasH “.m.HAnv .HmanoaoomaucH u.o.Hva Wm“: <0 <0 m} in in a} .. . A333 a... mod 0.42 Heomh2H ho zomHmdmzoo HHH mqmda TABLE IV RESULTS OF INOCULATING ADULT WHITE MICE,GUINEA PIGS, AND WHITE RATS, WITH THE VIRUS 35 Test Amount animals in ml Route Results Adult 0.03 I.C.(a) 0/9 White (b) Mice 0.5 I.P. 0/9 0.5 s. 003) 0/9' 0.5 Oral 0/9 Controls O/lO Guinea pigs O.l I.C O/h 005 I.P. O/h 0.5 8.0. O/h 0.5 Oral o/u Controls. S O/h White 0.1 I.C. 0/1; Rate 0.5 I.P. o/h 0.5 3.0. O/h 0.5 Oral O/h Controls O/h (a) I.C.: Intracerebral. (b) I.P.: Intraperitoneal. (c) 8.0.: Subcutaneous * Numeratoerumber affected Denominatoerumber Injected TABLE V RESULTS OF INOCULATING CATS, DOGS, CHICKENS AND CHICKS -WITH THE VIRUS Test Amount Animals in ml Route Results Cats 0.5 I.C(a) 0/3* Controls 0/2 D0gs 1.5 I.C. O/2 Chickens 0.1 I.C. 0/8 Controls 0/3 Baby Chicks 0.03 1.0. 0/37 (1 day old) Controls 0/30 (a) I.C. Intracerebral. *Numerator:Number affected. Denominator:Numoer Injected. TABLE VI RESULTS OF INOCULATING ADULT COTTON RATS, ADULT AND SUCKLING HAMSTERS, AND CALVES WITH THE VIRUS Incubation Test Dose period in animals in ml Route Results days Adult Cotton (a) : Rats 0.1 I.C.(b) 0/h* 0.5 I.P.( ) O/u 0.5 5.0 C O/h 0.5 0 al O/h Controls O/h Adult Hamsters O.l I.C O/h 0.5 I.P. O/h 0.5 5.0. O/h 0.; Oral O/Ll Controls O/h Suckling Hamsters 0.01 I.C. 8/8 3 - 5 0.05 I.P. 8/8 h - 6 Controls 0/7 Calf (l) 5 Oral l/l Calf (2) 2 I.V.(d) 0/1 (a) I.C.: Intracerebral. (b) I.P.: Intraperitoneal. (c) 8.0.: Subcutaneous. (d) I.V.: Intravenous. ‘3flumerator:Number affected Denominatoerumber Injected 38 TABLE VII RESULTS OF INOCULATING 9 DAYS OLD EMBRYONATED EGGS BY THE ALLANTOIC SAC, WITH FECAL AND TISSUE SPECDKENS FROM UALF I Specimen Amount in ml Results Pro-feeding feces 0.1 l/12* Post-feeding feces 0.1 6/12 Pooled intestinal 0.1 2/12 tissues Controls 0/10 *Numergtor:Number affected Denominatoerumber Injected 39 TABLE VIII RESULTS OF INOCULATING SUCKLING MICE WITH ALLANTCIC FLUID FROM EGGS INOCULATED WITH FECAL AND TISSUE SPECIMENS FROM CALF I - InchEtion 1” Amount period in Specimen Inoculum in ml Route Results days Feces A.F. 0.05 I.P.(a) S/7* h—7 Pooled Tissues A.F. 0.05 I.P. 0/7 Controls 0/7 (a) I.P.: Intraperitoneal. eNumeratoerumber affected Denominator:Number Injected ho om\o . m} m\m m\m m\~ m\m m} m} m\m m\m m} m\m m} m\m ~m\a4 *m\o Spoons.“ .8852 «nepSHEocom copomuwm amnesz "nopmposzz * m\0 m\0 H.o .10 waonpcoo mooom compo? 85.” -oooa Hapoa meson :oflamasoocd -aaom mooom :Ofipmaooosfi .umwunanunanI ow HH OH m w P o Lm All mama mo coapmaoooca nevus when a N. 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Soc. 3.3;). .3101. and Med., :1, 325-529. L-s-bin, A. L., and 1715-11-31:], I’. R., 1936. Culiéir'tion oi‘ Poliorrzx-‘Clitis '«III‘I‘S i__z_1_ vitrn in Iiwnn Mm" onic m .rvous Laisrruc. r‘ ' idol. ‘13 11'?- (A0, 31;, BIZ-3)). 'Jcloror, '..'. -~'., -; r{.3313011, J. .=.'., :.nd .o', ’.‘—. C., 195,5. Stu-.ios on “.110 Propagation i__r_1_ vi-ro oi‘ i'olim-Lelitis ‘.i1‘1.‘.5‘.CS. 1‘17. u’iral L~E‘-.:1i,.;7--.;-1ical.:-on in a {table .T'brain 01' Lunch i-l:».li-'-;nant 3f oitholial Cells ( train HeLa) Lieriveu iron an E.,;Liemicid Jarcinoma of 'IIJIOI'VPili, J. 4:5). 1.0d., 9/, 025-739 V-“ - | -\‘-- ‘ . I‘-~ v. uI -\' " “1 \I -“ 4-~.'7.\I--' -* p szLCl‘Cl‘, .1. L., 1. utorac, o. and Val/01090., J. L., 1,;1. Colt... .10.; o... ‘ AW -:, 1- ~'-'- \ '.- " "r‘\1' "' ,v""‘ 1‘, ""‘.,- 1 ' .‘. III/ I' i C—L‘C- 'Ivl;I V-LS U *flhs 4n A'LUIL-UJ le .; U—LCLL—LLI‘ J. -4US Je 0 4I I‘LKL O liIOC . 1 \I , LLH. SC ‘110 singer, Li. J., Morgan, I. 11., and L‘Ili'tslqy, .l. 15., lyhj. 1.... unsion to Llodmts oi 1.3151 in; Sync of Poliorrwlitis ."irus " -.-: n . -'- f‘ as . {‘0 ’ 1"- mu-fl .41514’1 It'lle .UCLL‘Je ”c.1311. a)CiC;.CC, jU’ LI,[I)2-J_I J., . _'u) (31.2.2.1 5.1mm, 8., 1,",11'). Enhancing Ei'fect ci‘ cortisone ugon . olion;:e11tis 211130 tion ( rain IL‘F-I in JELISI‘J'TBS and Lie P100. .500. Exp. .I-liCl. mild HOLL, 7S, 83-5-8 U. (31:. 111.1210” 13. J., harm”, P. EL, zinc Cordon 1". 1., 1930. The -ch , "‘1 1.1.21 ion oi‘ Polio om'r rolitis Vina by Human Serum. J. Prev. 1 '7 “H :13". o ’ (Ii, 1,63" ‘4/ 12:11.7, 2:. J., 1/32. .111.LC.L\’-. ion of Coxsackie Virus in -21.:1‘302‘1- ed iggs and in Chick i'issue Jult ures. Prm. Joe. Li}:p.15iol. and Med., {9, Iily-If2O-o .,311’“7, i... J., lielniclg J. L., and 3 “nen, m. 3., 1990. lzuec ion of Le ‘00- 2:10?" work: rs with L;CI<.SC‘.C-IiG Viruses. Ann. Int. Med., 33, 32-40. LJiC.~.lCS, 6. 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