Roy T. Fisk

Protective Action of Typhoid Phage on Experimental Typhoid Infection in Mice

Asheshov, Wilson and Topley 1 have reported on the protective effect in mice of typhoid bacteriophage given separately and by routes different from those used in infecting the animals. The preliminary studies here reported confirm the findings of the above investigators and enlarge upon certain aspects of the problem. A recently isolated strain of Eberthella typhi obtained by blood culture was employed. It forms smooth colonies, is motile, produces typical sugar fermentation reactions and agglutinates specifically with anti-typhoid serum. Organisms from 18- to 24-hour-old cultures grown on veal infusion agar were suspended in mucin∗ according to the methods of Rake 2 and Miller, 3 and mice were infected by the peritoneal route, all inoculations being made with a quantity of 1 cc of the mucin-organism mixtures. The phage was originally isolated from sewage and is a crude phage which produces several different types of plaques. Filtrates which contained approximately 3.3 × 108 phage particles per cc were used for mouse injection. Typhoid infection in mice was acute. Blood stream invasion with resulting bacteremia usually occurred within 1 hour following intraperitoneal inoculation and all of the fatally infected control mice died within 24 hours. The minimal fatal dose of the culture varied in 7 virulence tests from 1000 to 10 bacilli, thus the quantity of organisms used in the phage experiments represented from 1000 to 100,000 fatal doses. The protective effect of phage was tested by various ways in 7 experiments, the results of which may be summarized as follows: (1) Phage injected immediately before the intraperitoneal inoculation of 1 × 106 typhoid bacilli protected all of 32 mice when injected intravenously and all of 19 mice when administered by the subcutaneous method. Intravenously injected phage also protected against 1 × 107 and 1 × 108 bacilli, but failed to protect against 5 × 108 organisms which may have been due in part to the primary toxicity of such a large number of organisms.

Observations on the Virus Recovered from 1934–35 Poliomyelitis Epidemic in Los Angeles:

Attempts were made to recover virus from 19 autopsy cases during the recent epidemic of poliomyelitis in Los Angeles, Seven of these were successful in 1934, four in 1935, as judged by the following results upon inoculation of the emulsified human cord into Macacus rhesus monkeys: (1) Occurrence of pyrexia, roughness of coat, hyperirritability and tremor with subsequent development of paralysis. (2) Transmission of the virus in successive animal passage of selected strains. (3) The histopathology was characteristic in that oedema and hemorrhage, perivascular and diffuse infiltration, and necrosis of nerve cells were apparent. Since the 1934–35 epidemic of poliomyelitis in Southern California has been described as being especially mild in its clinical manifestations with a corresponding low mortality rate and a low residual paralysis rate, it seemed desirable to compare the viruses recovered from this epidemic with other strains of poliomyelitis virus. To date these Los Angeles strains and the “M.V.” strain from the Rockefeller Institute have been compared with reference to symptomatology and pathology produced and comparative immunologic studies have been made in detail with one Californian strain, the “McK” recovered from an autopsy in 1935. Observations include (a) symptomatology as shown by incubation period, temperature changes, type and degree of paralysis, residual paralysis rate and death rate; (b) histopathology produced in the brain and cord, (c) immunity produced and (d) neutralizing substance developed. Symptomatology. Both pyrexia and paralysis appeared in monkeys on an average of 2 days later with the “McK” strain than with the “M.V.” strain. In comparing the paralysis and recovery rate in monkeys infected with these 2 strains, it was found that monkeys inoculated with the “M.V.” strain showed a partial paralysis rate of 3.7%, a complete paralysis rate of 96.3%, with 4.7% of the total number recovering, while the monkeys inoculated with the “McK” strain showed a partial paralysis rate of 78%, a complete paralysis rate of 22%, with 84% recovering.

Immunization Studies with the Virus of Infectious Myxomatosis

Unsuccessful attempts to immunize the domestic rabbit against the virus of infectious myxomatosis by means of various vaccines have been reported by Sanarelli, 1 Moses, 2 and Hobbs, 3 In this study 8 rabbits immunized with phenolized and formalized vaccines prepared from the South American myxoma virus also demonstrated no immunity. Recently a strain of myxoma virus has been encountered in several rabbitries of Southern California (Kessel, Prouty and Meyer 4 ), and this strain has been employed in the accompanying series of vaccination experiments. All of the vaccines were made from virus in the form of fresh sterile blood collected from rabbits showing the symptoms of advanced myxomatosis, and were injected into the experimental animals by the subcutaneous route. Chemically inactivated vaccines were prepared by phenolizing and formalizing 10 and 20% solutions of the myxomatous blood. Heat was also employed as a means of inactivating the virus, and portions of the blood were heated both at 60°C for 30 minutes and at 45°C for 24 hours. Eight animals vaccinated with heat inactivated virus proved susceptible to subsequent inoculation of living virus. Attempts to immunize rabbits with phenolized and formalized vaccine have yielded more encouraging results, and are summarized in Table I. In addition to the above 29 control animals, 111 other unvaccinated rabbits have been inoculated with the California strain of virus in other studies in progress. Of these 150 unvaccinated animals, 5, or 3.3%, have shown partial resistance, and 2, or 1.3%, have recovered. No inoculated animals in this series have failed to develop symptoms. Thus a total of 7 unvaccinated animals, or 4.6%, as contrasted with 25.6% of vaccinated animals have exhibited either partial or complete resistance to inoculation.

The Occurrence of Anti-Rho Blocking Antibodies in Anti-Rh' Serums

Wiener1 has reported a new test for isoimmunization to the Rh agglutinogen. So-called “blocking antibodies“ were detected in serum specimens from Rh-negative individuals even though agglutinins were absent according to the usual methods of testing.

Further studies on the liver principle which is effective against burn shock

These experiments demonstrate that liver extract, which has significant antiburn shock activity, either when administered parenterally, as in previous work,1 or orally, as in the present study, is without comparable activity in the shock states which follow hind-leg ischemia or bacterial infection. It has been demonstrated that the mechanism of tourniquet shock in rats is complicated by several factors which are difficult to control. Before a final conclusion regarding the ineffectiveness of liver extract in this type of shock could be drawn, it would appear necessary that complicating factors be controlled. The negative results obtained with liver extract after acute exsanguination permit no conclusions to be drawn regarding liver activity in hemorrhagic shock. The fact that liver is effective in burn shock, but not in tourniquet or bacterial shock, is further evidence for the concept that there are different mechanisms responsible for various types of shock, although all may give rise to a similar terminal picture. It further indicates that therapeutic measures must be evaluated individually for each type of shock, and that results obtained with one type of shock cannot be justifiably transferred to other types of shock.

Experimental Rabies in White Mice. Studies on Passive Immunization I.

The subject of passive immunization against rabies has received relatively little attention, even though Fermi, 1 Pfeiler 2 and others have reported striking experimental demonstrations of the efficacy of anti-rabic serums. In the current report, which summarizes 12 protection tests performed in the past 2 years, the protective properties of such serums have been studied using white mice as experimental animals. A fixed virus strain, obtained through the courtesy of the Cutter Laboratories, was passed through rabbits and preserved at ice-box temperature, either fresh or in 50% glycerine, for periods which did not exceed 2 weeks. Details of preparation varied, but in effect the virus was ground to an initial dilution of 1/20 in normal saline and centrifuged 10 minutes at 2200 R.P.M. The supernatant fluid was then filtered through “Whatman No. 1′ paper and final dilutions of from 1/100 to 1/800 were made. The virus was injected intracerebrally, at first in amounts proportional to the body weights of the mice (0.005 cc./gm. or 0.001 cc./gm.), later in a constant dose of 0.02 cc. The serums were prepared by hyperimmunizing rabbits and goats with either 1/10 fresh fixed virus or 1/20 fixed virus preserved with 1/2% phenol. Serums were developed which would neutralize equal volumes of 1/200 fixed virus prepared as above to a final titer of 1/256, when the mixtures were incubated at 37° C. for 2 hours and tested by intracerebral mouse injection. Corresponding normal serums showed no in vitro neutralizing properties. The serums were filtered and inactivated before use. In the experiments reported the serums were injected intraperitoneally, the first dose being given at intervals ranging from 48 hours to 1 hour before the administration of virus. Doses for each serum injection varied from .05 cc./gm. body weight to .15 cc./gm. Serum was usually given only once, but at times as many as 2 subsequent injections were made, the maximum total of serum given to any mouse being .45 cc./gm. in a period of 5 days.