Maurice Brodie

Effect of X-Ray on Experimental Encephalitis in Mice Inoculated with the St. Louis Strain.

Since perivascular lymphocytic infiltration is the essential lesion in encephalitis and since lymphocytes are highly sensitive to irradiation, it seemed logical that small doses of X-ray should be effective as a therapeutic measure. The treatment by X-ray of 3 cases of encephalitis in human patients has been reported. 1 Since then 4 other cases have been treated. One was an early case and recovered completely in a short time. Two others were more protracted, having had the disease for 3 months before treatment was instituted. In these 2 latter cases various groups of muscles were showing progressive paralysis. The patients showed marked improvement after a series of X-ray treatments and are symptom-free to date. The fourth case, one of 6 months duration with early Parkinsonian syndrome, is still under treatment but shows improvement. Although the clinical evidence is very suggestive the findings are empirical and since the clinical studies can not be controlled, it was decided to carry out the treatments under experimental conditions, Mice are known to be highly susceptible to the St. Louis strain of virus. 2 The infective dose has been standardized and the incubation period is known. 3 If mice that were treated with X-ray after inoculation with the virus recovered or had the period of incubation or the duration of the disease prolonged, it seemed certain that the X-ray had a definite effect upon the disease. A series of normal mice exposed to varying amounts of X-ray to determine how much they could tolerate, showed no apparent ill effects after a series of 10-minute treatments (each equivalent to 1/2 a human erythema dose) given daily for 10 days. Preliminary experiments showed that the small X-ray doses given 48 to 72 hours apart that proved so effective in human beings were not effective in mice because of the fulminating character of the disease. Therefore more prolonged treatments at 24-hour intervals were given. Each treatment was 135 K.V., 5 ma., distance 15 inches, filters 3 mm. aluminum, time 5 minutes, equivalent to one-quarter of a human skin erythema dose.

Virus Isolated from Nasal Washings during Acute Poliomyelitis in New York City in 1935.

Conclusion Poliomyelitis virus was isolated from the nasopharyngeal secretions of a bulbar case on the 9th day of illness. The authors wish to acknowledge their appreciation to Dr. Morris Schaeffer of the Bureau of Laboratories, New York City Department of Health, for his valuable suggestions during this work.

Route of Transmission of St. Louis Encephalitis Virus in Mice.

Histopathological studies by Webster and Fite 1 suggest that the virus isolated from the St. Louis outbreak of encephalitis, travels by way of the central nervous system in mice. The present report confirms and extends these findings. In order to determine the portal by which mice could be infected, they were given virus by the nasal and gastro-intestinal routes. Three series of mice each consisting of 4 to 6 animals, were given a concentrated suspension of the virus in their food over 2 to 3 days. Not one of these mice became infected. Three animals, upon whom a laparotomy was performed, were injected intrastomachally with 0.2 cc. of a thick virus suspension. None of these showed evidence of the disease. Mice could be infected regularly with the intracerebral virus, within an incubation period of 6-8 days, by placing 0.03 cc. of a 1% suspension into one nostril, while 0.1% failed to give regular infection. The virus was transmitted serially by the intranasal route for 28 passages, using a 10% suspension, but its infectivity, by this route, did not increase. After 28 passages the virus was infective by the intranasal route, at approximately a 1:250 dilution. In order to trace the route of infection from the nasopharynx, the blood, parenchymatous tissues and various areas of the cerebrospinal axis were tested for the presence of virus after intranasal instillation of virus at various stages of the incubation period and of the disease. Two experiments were carried on simultaneously; one in which various organs and areas of the cerebrospinal axis were suspended in distilled water and one in which 10% normal monkey serum, which enhances the infectivity of the virus, was employed as diluent. In testing any area, a suspension made of a pooling of 2 to 3 organs in that area was injected intracerebrally and intraperitoneally Control animals receiving intranasal inoculations of the same virus were included in east test and always showed symptoms.

A Test for Diagnosis of Thrombo-Angiitis Obliterans.∗

In a previous report, one of us (Goodman) had outlined the evidence for the belief based upon epidemiological studies of others that thrombo-angiitis obliterans was a late manifestation of typhus fever. Since that time, Zinsser has pointed out in studies of Brills disease that typhus infection may remain latent in the body for an indefinite length of time. In a previous communication we have pointed out that a formalized Rickettsia suspension seemed to give a positive skin reaction in individuals with a previous typhus infection. Therefore we attempted this skin test in a number of cases of thrombo-angiitis obliterans in order to determine whether or not they had had a previous infection of typhus fever. Accordingly, 14 males clinically diagnosed as thrombo-angiitis obliterans, some with and some without gangrene, were given intracutaneously 0.1 to 0.2 cc. of formalized Rickettsia suspension as used previously. At the same time for control, an injection of a similar amount of Proteus X19 filtrate was also likewise injected at a different site. A group of 12 controls included 2 with arteriosclerosis. The site of the injections was examined as in the previous study after 24 to 48 hours following the injection. The results are enumerated in Table I, where a negative reaction indicates no erythema or induration, and a + reaction a wheal or raised, reddened, indurated area 1/2 cm. to 1 cm. in diameter, and a ++ reaction from 1 cm. to 3 cm. The cases diagnosed clinically as thrombo-angiitis obliterans show a positive skin test with Rickettsia organisms which suggests the possibility of previous typhus fever infection. Moreover, a number of the cases diagnosed as thrombo-angiitis obliterans came from typhus infected areas. Of course the series is small and likewise further controls are required by skin-testing other vascular conditions as well as skin tests with further control suspension, such as tunica vaginalis and guinea pig testicle.

Infection of Monkeys with the Virus of Poliomyelitis in Human Spinal Cords.

With 4 out of 5 human cords taken from acute cases of poliomyelitis at autopsy the virus was successfully transmitted to Macacus rhesus monkeys. All 5 individuals had died of respiratory paralysis in the acute stage of the disease. Histopathological study showed intense infiltration and considerable nerve cell destruction. The first of these, strain B, was obtained from a child who died of bulbar paralysis, 5 days after the onset of paralysis, during an outbreak in Montreal, in 1931. More than 3 years later, during which interval the cord and tonsils were left in glycerine, at ice box temperature, a single intracerebral and intraperitoneal injection of each tissue was given to a monkey. The cord produced paralysis in 13 days and the animal was prostrate 4 days later, while the tonsil gave complete paralysis within 6 days. The second strain, 1049, from a case occurring in New York in 1933, and had been in glycerine for a year when tested, was given in combined intracerebral and intraperitoneal inoculations over 3 consecutive days and produced a paralysis 5 days later. Repeated intracerebral and intraperitoneal injections of liver, spleen, mesenteric and submaxillary glands, and kidneys failed to show the presence of virus. The third strain, J, from a case who had died of bulbar involvement on the 6th day of progressive paralysis, had been in glycerine a year prior to inoculation. A combined intracerebral and intraperitoneal injection brought on paralysis in 8 days. Of special interest was the rapid rise of infectivity during 3 serial passages in monkeys and then its sudden drop. In Table I, the M.C.P. (minimal completely paralyzing dose) of this strain during these monkey transmissions is compared with the infectivity of the fixed monkey strain.

A Skin Test indicating a Previous Typhus Infection.

The recent studies of Zinsser 1 concerning the possible relationship of Brills disease to a previous typhus fever infection suggests the importance of determining by a simple test a previous infection with typhus fever. Inasmuch as many such patients are of foreign birth, and in whom infection had probably occurred some years previous, the clinical history is not always readily obtained. Likewise, a Weil-Felix reaction usually becomes negative with, or soon after convalescence and therefore cannot be used for this purpose. The present work described the use of formalized Rickettsia suspension for this purpose. Fleck and Kurkowski 2 found that filtrates of Proteus X 19 were ineffective. Our tests were carried out with a formalized Rickettsia suspension made from tunica vaginalis scrapings kindly furnished us by Zinsser of Harvard University. Accordingly, a series of 12 males who had had typhus fever, the diagnosis having been established 6 months previously in one case, and in the remainder from one year to 8 years were given the test about to be described. These cases were obtained through the courtesy of Dr. J. Rosenbluth, of the Bureau of Communicable Diseases of the New York City Department of Health. In all these cases the diagnosis had been confirmed by a positive Weil-Felix reaction with the exception of one in which the Weil-Felix had not been carried out. For the control, 10 adults with no previous history of typhus fever infection were given a similar skin test. The test consisted of an intracutaneous injection of 0.1 to 0.2 cc. of formalized Rickettsia suspension. The patients were observed 24 to 48 hours after the injection. None of the controls gave any reaction, whereas all the 12 with the previous history of typhus infection showed an erythematous indurated area from 1 cm. to 4 cm. in diameter.

Fall of Infectivity and Immunizing Power of Poliomyelitic Tissue at Intervals after Complete Paralysis.

Monkeys infected with the virus of poliomyelitis, develop after the height of paralysis, sub-normal temperature, difficulty in deglutition, inanition and bed sores and so it is extremely difficult to maintain them alive for long periods of time. However, by wrapping them in blankets, bottle feeding and other measures, it was possible to keep a small number alive for varying intervals after complete paralysis. The cord of these animals was tested for infectivity by titrating a 5% or 10% suspension made up of 6 to 8 segments. During the course of this work, the infectivity of cord removed at the height of paralysis varied between 0.000625 and 0.0025 cc. of a 5% suspension. The results are shown in Table I. Further tests were made with 23-day-old cords, using the double inoculation technique. 1 One animal was inoculated intracerebrally with 1 cc. of a 20% suspension and 10 days later it again received 1 cc. intracerebrally and also 6 cc. intraperitoneally. This animal developed no symptoms of the disease. A second monkey was given 23-day cord which had been washed as free as possible of antibody. The animal from which it had been obtained was, after bleeding, infused with 2 liters of Ringers solution. This, rather than isotonic gum acacia solution was used, for it was found that the latter sometimes reduced the infective power of spinal cord. One cc. intracerebrally and 6 cc. intraperitoneally of a 20% suspension and repeated in 11 days, gave no manifestations of poliomyelitis. It seems that the cord is no longer infective after 21 to 23 days. The infectivity of the cord declines rapidly for the first few days after complete paralysis and then more gradually until the end of the third week, when it is no longer infective.

Absence of Antiviral Substance in Normal Adults for the Virus of the St. Louis Encephalitis Epidemic.

Webster and Fite 1 ,2,3 have reported, that on the basis of serological tests, the virus isolated from the St. Louis encephalitis epidemic is not related to that of louping-ill, vesicular stomatitis, equine encephalomyelitis, acute anterior poliomyelitis, Japanese encephalitis (Type B), herpes, or chronic cases of von Economos disease. The present work confirms and extends some of these findings. To determine further whether or not there exists any relationship between the virus of the St. Louis epidemic and herpes virus, since there is a possible relationship between the latter virus and that of epidemic encephalitis (reviewed by report of Matheson Commission 4 ) both of these viruses were studied. Since neither rabbit, guinea pigs, nor rats could be infected with the encephalitis virus and since animals actively immunized against the latter were found not immune to herpes virus, it was evident that, as far as our tests went, there was no apparent relationship between the 2 viruses. In the following experiments, to test a serum for antibodies, the serum was mixed with equal quantities of virus suspension of various concentrations. The mixtures were incubated at 37°C. for 1 hour during which time they were agitated frequently. Then 0.03 cc. was injected intracerebrally into mice. Simultaneous virus titrations were carried out. A serum which failed to neutralize 1–10 minimal infective doses of virus was said to be devoid of neutralizing substances. A series of 16 cases of chronic encephalitis, of 1 to 14 years duration and all showing Parkinsonian syndrome, were tested for antibody content. One patient was a Montreal resident, the others attended the Bellevue Hospital Neurological Clinic. Nine of the group had a history of a definite acute onset. None of these serums had neutralizing substances.

Experimental Poliomyelitis in a Monkey without Demonstrable Lesions in the Central Nervous System

Conclusion 1. A Macacus rhesus monkey infected with the virus of poliomyelitis ran an unusually slow course, which simulated the diphasic type found in the human. 2. The histo-pathological findings of the cerebro-spinal axis were essentially normal, but the diagnosis of poliomyelitis was made by successful passage of cord emulsion into other monkeys.