No. 4616

FEBRUARY 17, 1912.

A LectureON

EXPERIMENTAL POLIOMYELITIS.Delivered at the National Hospital for the Paralysed and

Epileptic, Queen-square, on Feb. 6th, 1912,

BY FREDERICK E. BATTEN, M.D. CANTAB.,F.R.C.P. LOND.,

PHYSICIAN FOR OUT-PATIENTS AT THE HOSPITAL, ETC.

GENTLEMEN,--The earlier experimental work on the trans.mission of poliomyelitis from man to monkeys is now so wellknown that it is not proposed in this lecture to refer to it inany detail, but the following brief summary of the facts of itstransmission may well be mentioned.

EARLIER EXPERIMENTAL WORK.In 1909 Landsteiner and Popper succeeded in transmitting

poliomyelitis to two monkeys ; they did not succeed in trans-mitting the disease to a series of monkeys. In the same yearKnöpfelmacher produced the disease in a monkey by injectinginto the peritoneum an emulsion of the spinal cord from acase of poliomyelitis. In the same year Strauss and Huntoonin America succeeded in producing poliomyelitis in a monkeyby intraperitoneal injection.Flexner and Lewis in 1910 produced the disease in monkeys

by intracerebral injections, and were the first observers totransmit the disease through a series of monkeys. Landsteinerand Levaditi showed that the virus would pass throughporcelain filters and also that glycerine did not destroy thevirus. Working on the same lines and at the same time,Leiner and Wiesner in Vienna, Roemer and Joseph in

Marburg, Krause and Meinicke confirmed these observationsand added much to the knowledge of the nature of thevirus.

NATURE OF THE VIRUS.

(a) Filterability.-It was shown by Flexner and Lewis,and Landsteiner and Levaditi that the virus would pass a

porcelain filter. The latter observers noted that it would

pass the Berkefeld, Chamberland, and Reichel filters, but

that, after such a passage through the filter, the virus lostsome of its virulence, and the incubation period was alwaysprolonged, and the monkey either did not die or only diedsome days after the development of the disease.

(b) Glyoerine resistanoe.-It has been shown by Landsteinerand Levaditi that the virus will resist the action of glycerineeither concentrated or with 50 per cent. of water withoutimpairing its virulence. The virus has been kept for 142days in pure glycerine without affecting its virulence. Inthis respect the virus resembles that of rabies and vaccinia.

(e) Resistanoe to drying.-Flexner and Lewis, Landsteinerand Levaditi found that prolonged drying even to 24 days ata temperature of 220 C. did not diminish the virulence, and inthis respect differed from rabies. Leiner and Wiesner,however, found that drying of thin films for four hours at atemperature of 370 C. did destroy the virulence. Leiner andWiesner have shown that the virus is killed by exposure to atemperature of 550 C. for half an hour, and it is not killed byexposure to a temperature of - 8° C.

(d) Resistance to disinfectants. -It has been shown byLandsteiner and Levaditi that 0’2 per cent. solution ofpotassium permanganate will kill the virus in one hour at atemperature of 390 C., and that 6 per cent. peroxide at thesame temperature will destroy the virus in 45 minutes. Thevirus is not killed by to 1 per cent. of carbolic acid.

(e) Cultwation of organism.-The organism has not yetbeen cultivated, although Flexner and Lewis found someevidence that the organism multiplied in mixtures of brothand monkey serum.The organism has not’been stained or seen under the micro-

scope, although Levaditi found a small round and oval

corpuscle which he was able to stain by L6ffler’s method asmodified by Borrel. Roemer made a similar observation, andexamined them under the ultra-microscope.THE RETENTION OF THE VIRUS IN THE ANIMAL BODY.The question how long the virus remains in the body of the I’animal is difficult to answer. It has been found in the sninal I

cord 33 days after the onset of the disease. On the otherhand, Leiner and Wiesner found that it had disappeared fromthe cord six days after the onset of the paralysis. In other

parts of the body-viz., the nasal mucous membrane-it hasbeen found six months after the onset (Osgood and Lucas).PREPARATION OF EMULSION AND METHOD OF INFECTION.

The emulsion is prepared by pounding up 1 gramme ofthe infected portion of the spinal cord with 20 c.c. of normalsaline solution ; Q. 5 c.c. of this emulsion is injected into thebrain and 4-5 c.c. into the peritoneal cavity (Roemer).The most certain method of producing infection is by

injection of the virus into the brain. Injection into theperitoneal cavity, the anterior chamber of the eye, the sub-cutaneous tissue, and intravenous injections have all beensuccessful in producing the disease.

Leiner and Wiesner showed that the virus could passthrough the gastro-intestinal mucosa, but other observers,Levaditi and Landsteiner, failed to obtain positive results,and suggest that infection only takes place if there is a

previous lesion of the intestinal walls. Leiner and Wiesner,in order to avoid the action of gastric juice, injected thevirus into certain portions of the intestines after opening theabdomen. Three of the four monkeys thus operated ondeveloped poliomyelitis. The fact that the virus can

penetrate the mucous membrane of the digestive tractraises the possibility of the transmissibility of poliomyelitisby milk.The virus will pass the nasal mucous membrane if injured

(Landsteiner and Levaditi), but Leiner and Wiesner haveshown that it will pass the uninjured mucous membrane. Nocase of natural contagion has been reported in a monkey,although infected and non-infected monkeys have been keptin close contact in the same cage.MODE OF PROPAGATION OF VIRUS WITHIN THE BODY.

(A) By the blood-stream.-Injection into the blood-streamgives rise to poliomyelitis. This shows that the blood- streamcan carry the virus.

(B) By the lymphatie system.-Leiner and Wiesner pro-duced poliomyelitis by injecting the inguinal glands with thevirus. Flexner and Lewis injected a monkey subcutaneouslywith the virus. The animal was killed while suffering frompoliomyelitis. From this monkey they then injected threemonkeys: (1) with the spinal cord emulsion ; (2) with thenodule formed at the place of injection ; and (3) with thelymphatic gland corresponding with the inoculated region.Only the monkey that was injected with No. 2, the nodule,remained unaffected.The virus has been obtained from the mesenteric glands of

a child suffering with poliomyelitis (Flexner and Lewis). Ithas been shown (Flexner and Lewis, Levaditi and Land-steiner) that the virus injected into a peripheral nerve givesrise to paralysis, the disease commencing in the limb corre-sponding to the nerve injected.

Leiner and Wiesner found that if the nerve is dividedimmediately after the introduction of the virus into the peri-pheral end the spread of the disease is prevented. In what-ever way the animal is infected, it is in the central nervoussystem, and especially the grey matter of the cord, that thevirus tends to locate itself.The disease cannot be transmitted by blood obtained from

paralytic men and monkeys. This statement, though in themain true, is not absolutely so, for it has been shown byFlexner and Lewis-if large quantities, i.e., 25 c.c., arewithdrawn from a monkey at the height of the disease,and are injected into a healthy monkey-that animal

develops the disease. From human blood the disease hasnot been produced. The cerebro-spinal fluid also is freefrom the virus. In this connexion, however, Flexner andLewis found that the cerebro-spinal fluid taken from a

monkey three days after it had been given a cerebral injec-tion and injected into a fresh monkey gave rise to polio-myelitis in six days. This shows that the virus can multiplyin the cerebro-spinal fluid before the outbreak of paralyticsymptoms.

INCUBATION PERIOD.

The incubation period is subject to very considerablevariations. The variations depend upon (1) the method ojinjection, (2) the amount of virus given, and (3) the methodof treating the virus before injection. Of 81 monkeysinfected by Lewis and Flexner, 47 became paralysed between

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the eighth and twelfth day, 18 became paralysed before theeighth day, and 16 after the twelfth day. Levaditi foundthe average incubation period from seven to ten days, andRoemer gives the incubation period as nine days.

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The following factors were found to govern the incubationperiod. The less virulent the material, the longer was theincubation period. Filtration diminished the virulence(Levaditi and Landsteiner, Leiner and Wiesner). Thus anunfiltered emulsion produced poliomyelitis in seven days ;the same quantity of filtered emulsion produced poliomyelitisin 27 days.The quantity given also alters the incubation period.

Dilution, again, has the same effect, and if diluted beyond acertain limit the virus will not produce poliomyelitis(Roemer). Thus an animal, injected intracerebrally with0 5 c.c. of a 5 per cent. spinal cord emulsion, became para-lysed on the seventh day and died on the eighth day.Another monkey, inoculated with the same quantity but tentimes less concentrated, became ill on the twelfth and diedon the thirteenth day. Another monkey, injected with thesame quantity 100 times diluted, survived without havingmanifested any paralytic symptoms.

Leiner and Wiesner found that inoculation of a very con-centrated virulent emulsion appeared to exercise rather anunfavourable influence on the development of poliomyelitis.The incubation period was slightly longer, and the course ofthe disease goes on as if in the nervous tissues there were, inaddition to the acting virus, certain preventive substaneewhich exercises a neutralising action on the virus.

WHAT HAPPENS DURING THE INCUBATION PERIOD 7Leiner and Wiesner ;showed that the virus spread to the

nervous system before it was possible to observe any morbidmanifestation in the central nervous system. Levaditi andLandsteiner found that the anatomical and pathologicalalterations commenced at a time very close to the outbreakof clinical signs. The virus of poliomyelitis can invade thenervous system and multiply there without causing for atime any apparent trouble or distinct lesion. The paralyticphenomena in a monkey may commence very suddenly-i. e.,the monkey may be quite well in the morning and completelyparalysed by the evening. It is a striking fact that in themajority of cases, in spite of the inoculation of the virusinto the brain, the disease commences by paralysis localisedto the lower extremities.

INFECTION OF ANIMALS OTHER THAN MONKEYS AND APES.Most observers have failed to transmit the disease to

animals other than monkeys and apes. Flexner and Lewistried the horse, ox, pig, rat, cat, and rabbit. Levaditi andLandsteiner tried the rabbit, guinea-pig, young dog, andsheep. Leiner and Wiesner tried young dogs, fowls, pigeons,and rabbits ; and Roemer rabbits, guinea-pigs, and mice. Allwithout success.Krause and Meinicke, Lentz and Huntemuller assert that

they transmitted poliomyelitis regularly to rabbits. Theserabbits succumbed with or without paralytic symptoms, andon histological examination the characteristic changes ofpoliomyelitis, more or less pronounced, were found. These in-vestigators think that the inoculation into rabbits of cerebro-spinal fluid, blood, brain, or spleen obtained from humancases of poliomyelitis leads to the death of these animals,and the inoculation of similar tissues obtained from rabbitsthat have succumbed into other rabbits will bring about theirdeath. When the injections were made into the blood andperitoneal cavity a greater number of positive resultswere made than when they were made into the brain. Theeffects could be produced in rabbits, not only with anemulsion of the organs mentioned, but also with Berkefeldfiltrates prepared from them. Levaditi once succeeded intransmitting poliomyelitis in a rabbit, and the spinal lesionwas even more marked than in a monkey.Marks has further investigated this question. He used

young rabbits; these were injected with an emulsion of thespinal cord from a poliomyelitic monkey&mdash;2’5-35c.c. wereinjected intravenously and intraperitoneally. The injectionhad no immediate effect on the rabbit. Some of the animalsinjected died between the eighth and fifteenth day afterinjection. When death occurred the symptoms came onsuddenly, the final stage being generally ushered in by con-vulsions and rigidity, and death took place in from 10 to 30minutes after the onset of the symptoms. The post-mortemexamination showed hyperasmia of the cortex, but no other

striking lesion, and microscopical examination failed to showany further characteristic change. Other rabbits were

injected with :emulsions from various organs of the rabbitswhich died, and some of these rabbits succumbed. Thevirus was thus passed through a series of six rabbits. Fromrabbits in the second, fourth, and sixth set of the series ofrabbits, monkeys were inoculated, and these monkeys all

developed typical poliomyelitis.These results leave no doubt that poliomyelitis can be

propagated in certain individual rabbits, and also that thevirus is not confined to the central nervous system, but occursin equal amount in other organs. It is probable also tlatnot all strains of the virus can be transmitted to even a smallfraction of individual rabbits, and this may account forRoemer and Joseph’s failure in a long series. It is thusestablished that the virus of poliomyelitis can survive andprobably be propagated in domestic animals that do notshow any of the symptoms of poliomyelitis as it occursin man.Roemer has observed in guinea-pigs a disease similar to

poliomyelitis in man. It arose spontaneously in a guinea-pig,and was transmitted to a series of these animals by inocula-tion. All the animals were paralysed between the ninth andtwenty-third day after injection. No organism was visible ;the virus passed the Berkefeld filter and was well preservedin glycerine. The virus was present in the brain, spinal cord,liver, inguinal and prsevertebral glands, but was not presentin the blood, kidney, lungs, or urine. The cord showed the

typical lesions of poliomyelitis.IMMUNITY.

On clinical grounds there is good reason to believe that anindividual who has survived an attack of poliomyelitis isimmune to a second attack. Experimentally it has beenshown by Flexner and Lewis, Levaditi and Landsteiner, andRoemer, that monkeys which have survived the acute periodof infection are immune to a fresh dose of the virus. Roemerconsiders that immunity is only acquired after about thetwenty-fourth day, but other observers do not confirm thisobservation.

In one case Leiner and Wiesner have been able to re-

infect a monkey which was paralysed for 18 days. Thisis probably an exceptional instance. Leiner and Wiesnerperformed experiments to determine if the nervous systemof monkeys, killed after the acute stage had passed and therefractory stage reached, contained an appreciable quantityof the active virus. They found that they could transmit thedisease by using the spinal cord of a monkey killed on thetwenty-fourth day of the disease. This would seem to showthat the active virus and the antibodies can exist at the sametime and in the same individual.

PRODUCTION OF ARTIFICIAL IMMUNITY.

Levaditi and Landsteiner used the same method for render-ing a monkey immune against poliomyelitis as is used forproducing immunity against rabies. To a certain extentthis method was successful, but in some cases poliomyelitishas been produced by injection of the dried cords. Flexnerand Lewis, by giving injections of dilated virus, were ableto make monkeys immune to many times the lethal dose.

Flexner and Clark found the immunity principle to exist,both in the blood and cerebro-spinal fluid, soon after anattack of poliomyelitis. After one to two months the cerebro-spinal fluid lost its immunity principles, but it remained in theblood for years.Hexamethylenamine given by the mouth delays, and may

prevent, infection (Flexner and Clark). Kraus found thatsaline emulsion of brain and cord of a monkey, treated forfive days with 1-1 per cent. carbolic, is no longer infectiveown subcutaneous injection; 10 c.c. of virus thus sterilised,injected subcutaneously, protects animals against a subsequentsubdural infection. Serum which is viricidal in vitro doesnot confer protection when injected subcutaneously.

SERUM DIAGNOSIS.1. Experimental method.-Levaditi and Landsteiner, and

Muller have shown that it is possible to test the serum ofpatients and monkeys for their viricidal properties. For this

purpose a 5 per cent. emulsion of the spinal cord containingthe active virus is mixed with an equal quantity of the serumto be tested. The mixture must be made at a temperature of340 C., and stood at room temperature for several hours. Itis then injected intracerebrally in quantities of O’ 6-0,8 c.c.

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into a normal monkey. A control monkey receives the samequantity of the virus. The control monkey becomes infected,the other monkey remains free. This experiment has beentried with the blood of patients who have had herpes zoster,but the experiments were inconclusive, as the control monkeywas not affected (Miiller).Anderson and Frost found that the blood serum, in six out I

of nine suspected cases of abortive poliomyelitis, was z,viricidal against the virus of the poliomyelitis. Landsteiner,Levaditi, Leiner, and Wiesner have all shown that theserum is viricidal in vitro, but not in vivo. The serum

injected into animals has no preventive or curative effect.2. Deviation of the complement method. - This point has

been investigated by Wollstein, Roemer, Joseph, Levaditi,and Landsteiner. All these observers agree that the use ofan extract of organs containing the virus as antigen does notpermit the discovery of antibodies in the serum or cerebro-spinal fluid.

" BREEDS " OF VIRUS.It is essential before discussing breeds" of viruses to

understand how the words breeds, " " races," and " strains "should be used. The word breed" is used to indicate avariation in character of the virus. The word "races" "

should be used in the same sense. Flexner and Clark haveused the word "race" " to indicate increased or decreasedvirulence of the virus as applied to monkeys and man. Theword 6&deg; strain" is used to indicate a variation in the sourcefrom which the virus is obtained.

Levaditi has brought forward evidence to show that thereare differences between the viruses obtained from differentsources. So far two breeds are known-the virus of Land-steiner, Vienna, and the virus of Flexner, America.Levaditi has shown that the virus of Flexner is neutralisedin vitro by the serum of a monkey which has been renderedimmune to the Flexner virus. It is not neutralised by theserum of a monkey rendered immune to the Landsteinervirus. On the other hand, the virus of Landsteiner isneutralised by the serum of a monkey rendered immune tothe Landsteiner virus, but it is not neutralised by the serumfrom a monkey rendered immune to the Flexner virus.The Flexner virus is more virulent than the Landsteiner

virus. It remains to be proved if the English virus differsfrom the above two. No one in England has succeeded intransmitting the disease from an English strain to a monkey.

Flexner and Clark state that they have succeeded in

implanting upon monkeys all the ten strains of human viruswhich they have examined. That is to say, monkeys havebeen infected from ten human cases. These authorsstate that other experimenters have only been able to

implant about one half of the human strains of poliomyeliticvirus upon monkeys. In order to succeed in all instances itis necessary to inoculate emulsions of the human spinalcord, and preferably to make double inoculation into thebrain and peritoneal cavity. ’

The human strains of the virus not only infect monkeys lessreadily than do the modified or monkey strains, but theexperimental disease produced by them is less severe andless fatal. After the strains have once become whollyadapted to the monkey the paralytic disease appears in amore severe form, and the degree of infectivity rises, so thatexceedingly minute doses of a filtrate are capable of pro-ducing constant infection.

ELIMINATION OF THE VIRUS. ILevaditi and Landsteiner have found the virus in the Isalivary glands. It has not been found in the saliva.Flexner and Lewis have found the virus in the mucousmembrane of the nose of a monkey killed during the acutestages of the disease. Osgood and Lucas have found it inthis situation six months after the onset of the disease.Leiner and Wiesner showed that the nasal mucous membranebecame infected on the second day after injection. Thevirus has not been found in the urine, fasces, kidney, or

intestinal mucous membrane.Flexner and Clark found the virus to be present in the

tonsils and pharyngeal mucosa of a patient who had diedduring the acute stages of poliomyelitis. They obtainedsuccessful experimental results by preparing an emulsion ofthe tonsil with 0-5 5 per cent. solution of carbolic acid.

DISTRIBUTION OF THE VIRUS OUTSIDE THE BODY.So far the only evidence upon this subject is that fur-

nished by Neustaedter and Thro, who examined the dust in

rooms in which cases of poliomyelitis had been nursed.After many failures they succeeded in producing polio-myelitis in a monkey with the filtrate of macerated dust insalt solution. They conclude, therefore, that poliomyelitisis propagated by dust, and that the naso-pharynx is’probablythe point of entry.

Flexner and Clark have shown that flies can harbour thevirus in their bodies in a living and infective state for atleast 48 hours.

TYPES OF EXPERIMENTAL POLIOMYELITIS.All the usual types of poliomyelitis as seen in man have

been reproduced experimentally in monkeys. There is theordinary type with flaccid paralysis of one or more limbs, theascending type, the cranial type, the abortive type. The

"jumping" type has also been observed in monkeys, andRoemer gives the history of a monkey paralysed on thetwelfth day which showed considerable amelioration at theend of the sixteenth day ; but three weeks afterwards it hada relapse and died two days after being ill.

PATHOLOGICAL CHANGES FOUND IN EXPERIMENTALPOLIOMYELITIS.

The changes found in the brain and spinal cord in experi-mental poliomyelitis do not differ from those found in naturalinfection. In the spinal cord the lesions are usually moresevere and widespread than in the brain. The meningesusually show a more or less diffuse infiltration with roundcells. The layers immediately next to the white matter ofthe cord tend to show more cells than the layers next thedura mater. I

The greatest accumulation of cells is about the arteriesand veins, the sheaths of which are surrounded by cells. Theeffect of these cells on the lumina of the smaller vessels isconsiderable.The meningeal cellular invasion is only interstitial, and does

not give rise to an exudate upon the surface of the cord orbrain such as occurs in acute exudative inflammation.

- Bt&More[p7n/.&mdash;Andersen and Frost: Journal of the AmericanMedical Association. 1911, vol.lvii., p. 603. Flexner and Clark: Ibid.,1911, vol. lvii, p. 1685. Flexner and Lewis; Journal of ExperimentalMedicine, 1910, vol. xii., p. 227, and numerous notes in Journal of theAmerican Medical Association, 1909, 1910, 1911. Kn&auml;pfelmacher:Medizinische Klinik, 1909. Band xliv., p. 1671. Kraus : Zeitschrift fiirImmunitats forschung, 1911, Band ix., p. 117. Krause and Meinicke :Deutsche Medizinische Wochenschrift, 1909, 1910. Landsteiner andLevaditi: Comptes Rendus de la Societe de Biologie, 1909 and 1910.Leiner and Wiesner: Wiener Klinische Wochenschrift, 1909, 1910.

Levaditi : Presse Medicale, 1910. Levaditi : Journal of Royal Instituteof Public Health, 1911, vol. xix., p. 1. Marks: Journal of ExperimentalMedicine, 1911, vol. xiv., p. 116. Miiller: Deutsche MedizinischeWochenschrift, 1911, Band xxxvii., p. 1105. Neustaedter and Thro:New York Medical Journal, 1911. Osgood and Lucas: Journal of theAmerican Medical Association, 1911, Band lvii. p. 495. Roemer:Deutsche Medizinische Wochenschrift, 1911, Band xxxvii., p. 1209.Roemer : Die Epidemische Kinderlahmung, Berlin, 1911. Roemer andJoseph : Miinchener Medizinische Wochenschrift, 1910. Schreiber : LaPoliorny&eacute;lite Epidemique, 1911. Steinheil, Paris. Strauss and Huntoon:New York Medical Journal, 1909, Band xci., p. 64.

A NEW OBSTETRICAL AND GYNECOLOGICALSOCIETY.-A meeting will be held at the Grand Hotel,Birmingham, on Tuesday next, Feb. 20th, at 5.30 P.M., tofound an Obstetrical and Gynaecological Society embracingthe Midland, Eastern, and Western Counties of GreatBritain. The meeting will have the following agenda beforeit: (1) To consider the name of the proposed society ; (2) toform rules and by-laws ; (3) to arrange time and places ofmeetings ; (4) to elect officers and council for 1912-13 ; (5) todispose of such other business as may arise. At 7 P.m., afterthe meeting, a dinner will take place, the price of which,exclusive of wine, will be 7s. 6d. An early reply as tointention to be present or desire to join the society should bemade to Mr. H. Beckwith Whitehouse, 52, Newhall-street,Birmingham), who is acting as honorary secretary (pro tem.)to the movement. A letter has already been circulated, signedby Dr. Edward Malins, Dr. Christopher Martin, Dr. ThomasWilson, Dr. C. E. Purslow, Mr. Frederick Edge, Mr. J. Fnr-neaux Jordan, Mr. Smallwood Savage, Dr. John T. Hewetson,Dr. Ann E. Clark, Dr. Mary D. Sturge, and Mr. BeckwithWhitehouse, these representative gynaecologists practising inBirmingham believing that the time has arrived for theformation of a new Obstetrical and Gynaecological Societyembracing the Midland Counties, and, if sufficient support beobtained, including also the West of England and perhapsthe East. To this letter many favourable replies have alreadybeen received.