Title. Author(s)SATO, Gihei; HIRATO, Katsushichi. CitationJapanese Journal of Veterinary Research, 2(4): Issue Date DOI.

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1 Title SOME OBSERVATIONS ON THE NON-SPECIFICALLY ENHANCED R INTRAPERITONEAL INFECTION WITH SALMONELLA ABORTUS-EQ Authr(s)SATO, Gihei; HIRATO, Katsushichi CitatinJapanese Jurnal f Veterinary Research, 2(4): Issue Date DOI /jjvr Dc URL Type bulletin File Infrmatin KJ pdf Instructins fr use Hkkaid University Cllectin f Schlarly and Aca

2 SOME OBSERVATIONS ON THE NON.. SPECIFICALLY ENHANCED RESISTANCE OF MICE AGAINST INTRAPERITONEAL INFECTION WITH SALMONELLA ABOR:L ' US-EQUI Gihei SATO* and Katsushichi HIRATO Labratry f Veterinary Hygiene and Micrbilgy, Faculty f Veterinary Jtl edicine, Hkkaid University, Sappr, Japan (Received fr Publicatin, Octber 7, 1954) Muse-prtectin test is very practical fr a~saying the ptency f varius vaccines. In this test, administratins f the immunizing injectins and f the challenge dses are ften made by the intraperitneal rute. Hwever, the nn-specifically enhanced resistance f mice that fllws the intraperitneal injectin f varius substances has been bserved by many wrkers. This phenmenn may be an imprtant surce f errrs in all muse-prtectin tests in which the challenge dse is injected by the intraperitneal rute. FEL.IX pinted ut that many wrkers engaged in experiments n typhid immunity use the faulty methd f injecting bth immunizing and challenge dses by the intraperitneal rute. The present authrs have studied n the mice-prtectin tests f the killed vaccine f S. abrtus-equi. In a previus paper, HIRATO et al. recgnized the high ptency f this vaccine in the case f injecting bth the immunizing and the challenge dse by intraperitneal rute. Hwever, it was left as an unslved prblem whether the nn-specific prtective mechanisms wuld play sme part in this prtectin test, and the authrs stated that sme additnal experiments are needed fr the establishment f this pint. ORSKOV and K~UFFMANN, PHILIPSON and 0RSKOV examined in detail the degree and duratin f this kind f nn-specific resistance. Frm thse studies, it is learned that ne intraperitneal dse f a vaccine f sme kinds f bacteria induces in mice a high degree f immunity t intraperitneal challenge with the ther kinds f bacteria and that the repeated injectins f the vaccine induce a lnger duratin f the same type f immunity. Frm these well established facts, it wuld seem tbat the same thing might interfere with the results f the muse-prtectin test f 'equine paratyphid vaccine. The present study was carried ut t * Present addre3s: Labratry f Epiztilgy in this faculty Jap. J. Vet. Res., Vl. 2, N.4, 1954

3 178 SATO, G. AND K. HIRATO ascertain the nn-specific rise f resistance in case f assaying the prtective value f this vaccine. MATERIALS AND METHODS Bacterial Strain: The strain f S. abrtus-equi used fr the preparat.in f vaccine and the test culture was a pretty fresh ne. A relatively ld stck culture f Escherichia cli var. cmmunir was used fr preparatin f a heterlgus vaccine. It had n cmmn antigenic cnstituents with S. abrtus-equi. Vaccine Preparatin: Heat-kiIIed vaccine was prepared frm saline suspensin f plain nutrient agar culture by heating at 60 C fr 40,..,...50 minutes. Chrme-vaccine Bacterial suspensin (1% frml saline) was mixed with equal vlume f 0.4% alum chrmate slutin. After having std fr 2 days at rm temperature, this suspensin was centrifuged and the sediment was washed tw times in nrmal saline. Muse-test: The same strain f white muse was used thrughut as described in previus papers n the muse-prtectin test f ~quine paratyphid vaccine. They were f female sex in almst all experiments, and weighed abut 15 g at the time f infectin. Mice were immunized by intraperitneal rute, with 0.5 ml saline suspensin cntaining respectively each vaccine dse f hmlgus r heterlgus bacteria and the challenge dse f S. abrtus-equi was given in the same manner after varius intervals. In rder t avid any fluctuatin f bacterial number f the challenge dse, the infectin was made simultaneusly against all grups f mice having different intervals after tbe immunizing injectins. Heart bld f mice which died was cultured t ascertain whether death had ccurred as a result f infectin. In the latter part f the experiments, bacilli in tail bld f infected mice were examined by cultural methd. Tw drps f bld were cultured n ENDO agar and a lpful f bld was enriched with brth cntaining sapnine and sdium citrate. Other detailed descriptins f experimental methds will be given in the next chapter. EXPERIMENTAL RESULTS 1. The Nn-specific Resistance f Mice t Fatal Infectin with a Large Dse f S. C!hrlus-equi. In the muse-prtectin test f equine paratyphid vaccine, the challenge dse is given after an interval f abut 10 days with a sufficient dse usually t cause septicaemic death. Firstly t investigate the effect f nn-specifically increased intraperitneal resistance, the authrs used tw kinds f killed vaccine. Each.grup f 3,...,4 mice was injected intraperitneally with 0.2 mg f heat-killed bacilli f abrtin r cli at perids ranging frm 1 t 14 days befre infectin as indicated in table 1. Intraperitneal challenge dse f 0.15 mg f abrtin bacilli was given t each immunized grup simultaneusly..,.

4 On the Nn-Specifically Enhanced Resistance 179 TABLE. I. Surm'val Rate f Mice Immunized with Heterlgus r Hmlgus Vaccines within 30 Days after Fatal Infectin with S. abrtus-equi GROUPS IMMUNIZED INTERVALS BETWEEN VACCINATION AND INFECTION WITH HEAT-KILLED BACILLI OF days S. abrtus-equi 0/4 E. cli 2/4 Cntrls Vaccine dse: 0.2 mg i. p. Challenge ds'e: 0.15 mg i. p. 1/4 0/4 1/4 4/4 3/3 3/3 0/4 0/4 0/3 0/3 0/4 A glance at table 1 shws that a certain prtective resistance appeared in a mice grup infected at an interval f 1 day after heterlgus vaccinatin. In case f hmlgus vaccinatin, the mice grup infected at a 2- and 4-day interval indicated lw survival rate. This result seems t indicate that mice acquire sme resistance against fatal infectin when they are immunized with heterlgus r hmlgus vaccine and these nn-specific intraperitneal resistances appear at an earlier perid in cmparisn with specific immunity, In the latter experiment, a'larger dse f vaccine was used than that in the previus experiment, and infected mice were bserved clinically fr 30 days after infectin. This was dne because, in natural cnditin, mice infected with al~rtin TABLE 2. Resistance f )l,iice at Varius Intervals after Intraperitneal Treatment t Infectin with a Large Challenge Dse f S. abrtus-equi INTERVALS BETWEEN V ACCINATION AND INFECTION 1/ Cntrls SORVIV AL DAYS OF MICE IMMUNIZED WITH 3, 4, 4, 5, 5. 4, 4, 5. S, S, S. 5,5, 5, 5. 6, 6, 6, s. 5, 6, 6, 7. 1, 4, 4, 5. 4, 4, 5, 7, -x. 4, 5, 6, s. ** 4, 5, S, S. 1, 5, 6, 8, Vaccine dse: 0,5 mg i. p. * This grup was given 2 dses f vaccine (0,2, 0.3) with a 7-day interval. *-X. This grup was jnjected with 2 dses f vaccine (0.5, 0.5) with a 7-day interval. Challenge dse: 0.15 mg i. p. S: Survived and healthy fr 30 days after infectin. s: Survived but suffered frm septicaemia.

5 180 SATO, G. AND K. HIRATO bacilli regularly get septicaemia, but they d nt always die. Therefre by bserving the survival rate nly, ne is apt nt t learn whether they are perfectly prtected frm septicaemia r whether they escaped frm the danger f septicaemic death. Septicaemic mice shw clinical signs such as hair rughening, arched back r lss f appetite and are easily discriminated frm healthy niice. The data in table 2 seem t establish that 0.5 mg f heterlgus vaccine scarcely prduced nn-specific resistance. Hwever, it shuld be nticed that half the number f mice survived in a grup which were immunized with a dse f 1 mg cli vaccine and infected 10 days later. Further, these survived mice did nt suffer frm bth the primary and secndary septicaemia similarly t mice hmlgusly vaccinated. Frm the abve bservatins, it may be stated that mice immunized with killed vaccine f abrtin r cli bacilli resist slightly against fatal infectin with a large dse sllch as 0.15 mg f abrtin bacilli at the early perid after vaccinatin, but 1 mg f heter-vaccine gives a cmplete prtective pwer t sme f the mice n the 10 th day after vaccinatin. 2. Effect f the Nn-specific Resistance f Mice n the Destructin f Intraperitneally Injected Bacilli The previus experiments seem t shw that mst f the mice treated specifically r nn-specifically d nt strngly demnstrate nn-specific resistance t fatal infectin with a large dse f abrtin bacilli. Hwever, it is prbable that severe infectin caused by a large challenge dse may cver the feeble rise in nn-specific resistance, s hw t detect such a feeble resistance shuld be cnsidered. Accrding t the previus investigatins, abrtin bacilli injected with a prper dse int the peritneal cavity enter int the bld stream rapidly, but the immunized mice tend t inhibit this invasin by destructin f injected bacilli. Therefre, if the number f bacilli in bld stream is decided by cultural methd at different intervals fllwing infectin, the degree f resistance f mice may be bserved indirectly. Infectin with 0.01 mg f abrtin bacilli is able t prduce a bacteremia fr 1 week almst at the rate f 100%, but des nt kill the mice rapidly. As shwn in table 3, each grup f 4 mice was injected with 0.01 mg f abrtin bacilli and tail blds f mice were cultured during the time frm half an hur t the 7th day after infectin. Then the percentage f the psitive cultures was calculated as fllws: Ttal nt!-mber f psitive cultures in each grup 100 Ttal number f bld cultures in each grup x (In the case f mice which died within 1 week after infectin in the further experiments, the results f bld cultures were estimated t be psitive frm the date f death t the 7th day). Thus the lwer the percentage f psitive cllltures the larger becmes resistance f the grup f mice against infectin.

6 On the Nn-Specifically Enhanced Resistance 181 TABLE 3. Shwing the Result f Tail Bld Culture at Varius Times after Inculatin f S. abrtus-equi int H mlgusly and Heterlgu8ly Vaccinated M'ice. MICE IMMUNIZED INTERVALS RESULTS OF BLOOD CULTURES. BETWEEN WITH HEAT-KILLED VACCINATION BACILLI OF AND INFECTION 1/ ~~s days S. abrtus-equi 0.2 mg E. cli 0.2, 0.2 S. abrtus-equi 0.2 E. cli 0.2 Cntrls 11 days J () 0 I ~ ~ ~ ~ 1 day 1 day 1 day I It ~ ~ ~ l () =11 () 0 f It () H = 1 H f H 0 00 I D () It It It ~.. : : () 0 I * : : : f () 0 = () = It = 1 It = H () Challenge dse: 0.01 mg i. p. : negative culture ( ): psitive in enrichment culture : 1-50 clnies : clnies 1: ver 100 D: death Calculatin f Percentage f Psitive Cultures: Ttal Number f Psitive Cultures in each Grup X 100 Ttal Number f Bld Cultures in each Grup PERCENTAGE OF POSITIVE CuLTURES 6/40 x 100 = 15% 40% 82.5;:;; 80'}{' 100% It may be seen frm table 3, that a dse f 0.2 rug f heter- r hm-vaccine prduced slight nn-specific resistance, and the increase f the dse t 0.4 rug f heter-vaccine develped an appreciable nn-specific resistance against S. abrtus ('qui. These results seem t indicate that the nn-sr;ecific intraperitneal resistance is induced n the 1st day after vaccinatin depending n the dse f heter-vaccine. By means f the abve assaying cnceptin, the results f the fllwing experiments have been simplified as is shwn in table 4.

7 182 SATO, G. AND K. HIRATO GROUPS IMMUNIZED WITH HEAT-KILLED BACILLI OF TABLE 4. Nn-specific Resistance f Mice which were Tested at Varius Intervals between Vaccinatin and Challenge Dse PERCENTAGE OF POSITIVE BLOOD CULTURES (Intervals between Vaccinatin and Infectin) 7 hrs days ----_ '- S. abrtus-equi E. cli Cntrls 100% 45.4% 15.1% 96.9% 100% 100% 78.7% 75.7% 100%* 42.7%** 100% Vaccine dse: 0.2 mg i. p. * This grup was injected with 2 dses f vaccine (0.1, 0.1) with a 7-day interval. ** This grup was injected with 3 dses f vaccine (0.2, 0.2, 02) with 2-day intervals. Challenge dse: 0.01 mg f S. abrtus-equi i. p. Three mice were used in each grup. Percentages f psitive cultures in table 4 indicate that 0.2 mg f killed abrtin bacilli induced a certain resistance f nn -specific type n the 3rd day after vaccinatin, but the same dse f heter-vaccine culd hardly prduce it at any interval. On the ther hand, 0.6 mg f heter-vaccine induced the nn-specific resistance n the 10th day after treatment. Results btained frm tables 3 and 4 shw that the nn-specific resistance caused by a small dse f 0.2 mg f heterr hm-vaccine was slightly demnstrated n the 1st day after treatment and increased resistance was develped with a large dse f 0.4 mg f heter-vaccine at the same interval. Mrever, a cnsiderably larger dse f heter-vaccine induced the nn-specific resistance n the 10th day after treatment. Intraperitneal treatment with hm-vaccine tends t induce pwerfully the nn-specific rise in resistance (prmunity) in earlier pint f time than the specific types f immunity. Table 5 indicates nn -specific resistance caused by varius vaccines f heterbacteria at different intervals after treatment. As will be nted frm this table, heat-killed vaccine f cli did nt induce the nn-specific resistance 4 hurs after treatment, but it sustained the nn-specific resistance frm the first t the 10th day after treatment. Hithert, repeated injectins f large vaccine dses were made. Hwever, it is very likely that difference f degree between the nn-specific resistance enhanced by the repeated vaccinatin and the same resistance induced by a single dse may be prduced. Therefre, in rder t bserve the exact effect f large vaccine dse n the rise f nn-specific resistance, it is necessary t emply a single injectin. In rder t avid the lss f mice with a single injectin f large vaccine dse, the killed bacilli shuld be detxified. I t is well established by Kji ANDO et al. that the txicity f the killed bacilli is evidently decreased by treatment with alum chrmate. A.ccrdingly, frmlized cli bacilli were treated with 0.4% alum chrmate slutin fr the decrease f the txicity. As indicated in table 5,

8 On the Nn-Specifically Enhanced Res'istance 183 chrme-vaccine injected repeatedly seems t be as effective n the inductin f nn-specific resistance as heat-killed vaccine, but frmlized vaccine is less effective. TABLE 5. Percentage f Psitive Bld C1tltures f Mice Inculated with S. abrtus-equi at Different IntM'vals after V'accinat'in with a Large Dse f Vm'i'us Va;ccines VACCINE Heat-killed Bacilli f S. abrtu,s-equi Frml Vaccine f E. cli Chrme-Vaccine f E. cli Heat-killed Bacilli f E. cn Cntrls DOSE 0.2, 0 2, 02 mg 0.2, INTERVALS BETWEEN LAST VACCINATION AND INFECTION 10 days hrs. PERCENTAGE OF POSITIVE CULTURES 4.5% 70.4~;;; 43.1% 43.1% 47.7% 50.0% 84.1% 97.7'}{, 79.5.% Interval f repeated vaccinatin is 2-days. Challenge dse: 0.01 mg i. p. Fur mice were used in each grup. It may be seen frm table 6 that 1 mg f chrme-vaccine injected at a time gives appreciably higher resistance t the mice than is fund in the mice grup which was given a dse f 0.2 mg. Mrever, as in table 7, in the case f single injectin f 1 rng f cli chrme-vaccine, pwerful nn-specific resistance was bserved even 3 weeks after vaccinatin. (Hwever, the bdy weight f the mice used in this experiment was larger than that in the frmer experiments, s the animals might be much mre resistant'. TABLE 6. DUratin f Nn-specific Resistance f llfice Vacc1,nated uith Single Small 0,' Large Dse f Chrme-vaccine. VACCINE Heat-killed Bacilli f S. abrtus-equi Chrme-vaccine f E. cli Cntrls DOSE (mg) PERCENTAGE OF POSITIVE BLOOD CULTURES (Intervals between Vaccinatin and Infectin) days 38.7r;6 932.% 95.5'} {' 88.7'}'; 100.0'}'; 48.5'}'; 63.6% 43.2% 59.1% 90.9'}''; Challenge dse: 0.01 mg f S. abrtus-equi i. p. Fur mice were used in each grup.

9 184 SATO, G. AND K. HIRATO TABLE 7. Lng Duratin f Nn~specific Resistance f Mice Vaccinated with a Large Dse f Chrme~vaccine VACCINE Heat-killed Bacilli f S. abrtus-equi Chrme vaccine f E. cli Cntrls DOSE (mg) PERCENTAGE OF POSITIVE BLOOD CULTURES (Intervals between Vaccinatin and Infectin) % 6.8% 2.3% 90,9% Challenge dse: 0.01 mg f S. abrtus-equi i. p. Fur mice were used in each grup. Als ther kinds f heter-vaccine besides cli were used t bserve the nnspecific rise in resistance against abrtin bacilli. As shwn in table 8 mice treated with a large dse f different vaccines nn-specifically have been fund t prvide different degrees f resistance. TABLE 8. Nn-specific Resistance Prduced by VaccinaUn f Varius Kinds f Bacteria MICE IMMUNIZED INTERVALS BETWEEN PERCENTAGE OF WITH HEAT-KILLED V ACCINATION AND POSITIVE BLOOD BACILLI OF INFECTION CULTURES S. abrtus-equ,i 5 days 9.1% Staphylcccus aureus 47.7% S. senftenberg 69.7% E. cli 52.3% Cntrls 86.4% Vaccine dse: Three dses (0.25, 0'.25, 0.25) were injected at 2-day intervals. Challenge dse: 0.01 mg f S. abrtu8-equi i. p. Mice injected with killed abrtin bacilli shwed nn-specific resistance t hmlgus infectin n the 3rd day after vaccinatin, and als mice injected with a large dse f cli vaccine prduced a cnsiderably pwerful nn-specific resistance t infectin with abrtin bacilli 10 days after treatment. A single dse f cli chrme-vaccine, such as 1 mg, induced a surprising resistance t infectin fr 3 weeks under certain cnditins. Nn-specific resistance n the 1st day after vaccinatin seemed t be very slight and transient; several hurs after treatment it was nt bservable. Hwever, nn-specific resistance induced by heterlgus bacteria is weaker cmpared with the resistance develped by specific vaccinatin. 'j CONSIDERATION Accrding t 0RSKOV and KAUFF!\fANN, the nn~specific resistance is..

10 On the Nn-Specifically Enhanced Resistance 185 develped with great rapidity after vaccinatin and it fades befre the appearance f specific immunity. Thus this rapidly prduced nnspecific resistance was demnstrable during a shrt time, and they called it prnlunity. On the ther hand, PIIILIl)SO~ fund that the resistance enhanced by means f repeated injectins appears mre rapidly and is f jnger duratin than prmunity. These wrkers reprted that the nn-specific resistance shws the highest degree in the case f injecting bth the immunizing and challenge dse by the intraperitneal rute. The present experiments were designed t determine whether r nt the nn-specifically enhanced intraperitneal resistance has effect n the muse-prtectin test f equine paratyphid vaccine. Accrding t the results btained frm the abve experiments, in the case f specific immunizatin, a nn-specifically enhanced resistance against infectin with abrtin bacilli develped mre rapidly than specific resistance. Als in the nn-specific immunizatin the same resistance appeared at an early perid similar t specific hnmunizatin. Even if this kind f resistance which is bserved at an early tinle may be prduced in muse-prtectin test f the equine paratyphid vaccine, it des nt practically disturb the test. Therefre, it shuld be examined whether the nn-specific resistance which appeared frm early perid is cntinued fr a lng time r nt. This kind f resistance in nn-specific immunizatin is prduced strngly by a large inlmunizing dse and it seems t last in a cnstant degree frm beginning t the 10th day, even if it is slighter than the resistance in the specific immunizatin. Under certain cnditins, a single injectin f 1 mg f heterlgus chrme-vaccine demnstrated a cnsiderably pwerful resistance t infectin with abrtin bacil1i fr 3 weeks, thugh a sluw disslutin f the bacilli might be caused by treatment with alum chrmate slutin. (It is believed that the decrease f txicity f the killed bacilli treated with alum chrmate slutin is due t a slw disslutin f their substances in the tissue). Therefre, it is presunled that nl0usa-prtectin test which is perfrmed by emplying a large dse f equine paratyphid vaccine may be influenced by the nn-specifically enhanced intraperitneal resistance. Accrding t 0H.RKOV and KAUFF:\IAN;-';, and PffILlPRON, regarding the relatin f vaccine dse t the nn-specific resistance, it was rendered clear that the degree r the duratin f this resistance is prprtinal

11 186 SATO, G. AND K. HIRATO t the immunizing dse t sme extent. Als the present authrs btained similar results. Hwever, decrease f resistance bserved at a very early time after vaccinatin seems t be due t a txic effect f a large vaccine dse, as Pnu,u'sN stated in his reprt. Tpe present authrs detected the feeble nn-specific resistance by culturing the tail bld f mice after infectin. PHILIPSON reprted that the destructin f intravenusly r intraperitneally injected living bacilli is mre rapid in nn-specifically immunized mice than in the cntrls. 0RSKOV demnstrated the increased phagcytsis in the peritneal cavity f mice immunized nn-specifical1y. Therefre, the bld culture methd emplyed by the present authrs seems t be apprpriate fr bservatin f the nn-specific resistance. Als ther kinds f bacteria than cli, such as staphylccci r S. senftenberg, slightly induced nn-specific resistance t infectin with S. abrtus-equi 5 days after immunizatin. This result suggests that the resistance enhanced by cli vaccine must be whlly nn-specific. As indicated in table 4, prmunity in specific immunizatin is mre pwerful than in nn-specific immunizatin n the 3rd day after vaccinatin. Frm this result it may be stated that prm unity bserved in muse-prtectin test f equine paratyphid vaccine is relatively specific. As t this pint, 0H.SKOV and KAUFF,IA~N already indicated that the prmunity which appeared in muse-prtectin test f typhid vaccine was nt alway3 f cmplete nn-specificity. Cnsequently, it is reasnable t cnsider that nn-specifically enhanced intraperitneal resistance may have interfered with the authrs' previus experiment n the muse-prtectin test f equine paratyphid vaccine, which was perfrmed by injecting bth immunizing and challenge dse by intraperitneal rute. In rder t avid these cnfusins in case f muse-prtectin test f equine paratyphid vaccine, injectin f vaccine shuld be made by anther rute. Kiyshi ANDO et al. reprted that mice injected subcutaneusly with killed vaccine f a variant strain f S. typh'imurium were rendered slightly resistant t intraperitneal infectin with abrtin bacilli n the 10th day after vaccinatin. Nevertheless, frm the results btained by many wrkers, it is clear that subcutaneus vaccinatin induees nly slightly a nnspecific resistance against intraperitneal infectin with hmlgus r heterlgus bacilli, r did nt at all. Therefre, when the vaccine dse is very large, it is better t emply subcutaneus injectin than intraperitneal. Hwever, accrding t previus experiment f the present

12 On the Nn-Specifically Enhanced Resistance 187 authrs, subcutaneus treatment did nt always give prtective pwer t mice. Thus hw t immunize mice mst effectively is an imprtant prblem fr further investigatin. Mice immunized intra peritneally with killed vaccine f heterlgus bacteria shw an enhanced resistance t an intraperitneal infectin with S. abrtus-equi 1 r 2 days after vaccinatin and sustain it fr a perid extending t 10 days. This nn-specifically enhanced resistance is inferir t the resistance bserved in specific immunizatin. Hwever, in case f injecting a large dse f heterlgus vaccine, nnspecific rise in resistance smetimes becmes cnsiderably high and lasts fr 3 weeks. REFERENCES 1) ANDO, Kiyshi, Y.OCHI & S. SUDO (1934): Saikingaku-Zasshi, N. 464,51 (Japane3e). 2) AN DO, Kji, H. SHIMOJO & 1. TADOKORO (1952): Jap: J. Exp. Med., 22, ) FELIX, A. (1951): J. Hyg., 49, ) HIRATO, K., G. SATO, T. ONO & K. SHIMIZU (1953): Vet. Res., 1, 29 (Japanese with English abstract). 5) PHILIPSON, J. (1937): Acta. Path. Micrbil. Scand., Supp!., 32, 1. 6) 0RSKOV, J. & F. KAUFFMANN (1936): Z. Hyg. u. Infektkr., 119, 65. 7) 0RSKOV, J. (1940): Z. Immun.-Frsch., 98, 359.

STUDIES WITH HUMAN INFLUENZA VIRUS CULTIVATED IN ARTIFICIAL MEDIUM

Published Online: 1 June, 1936 Supp Inf: http://di.rg/1.184/jem.63.6.83 Dwnladed frm jem.rupress.rg n August 13, 218 STUDIES WITH HUMAN INFLUENZA VIRUS CULTIVATED IN ARTIFICIAL MEDIUM BY T. P. MAGILL,