Johannes H. Bauer

Ultrafiltration of Type I Antipneumococcal Sera

Elford, Grabar and Fischer 1 have reported that the “antibody activity” of antipneumococcal horse-serum is associated with the “larger complex protein in the serum.” Thus it was estimated by the method of optimal proportions that 75% of the antibody passed a 140mμ membrane, rather less than one percent passed an 80mμ membrane, while the filtrate from a 54mμ membrane showed no trace of antibody-activity. The membranes used in the present experiments were prepared by the method of Elford 2 with certain modifications described by Bauer and Hughes. 3 For filtration, Type I antipneumococcal horse-and rabbit-sera were diluted 1:5 in broth. Concentrated antipneumococcal horse-serum was diluted 1:10 in the same reagent. For evaluating the amount of antibody in the filtrate the quantitative precipitation method of Heidelberger, Sia, and Kendall 4 was used. Because of the difficulties in securing large amounts of filtrates complete analyses over a wide range of amounts of polysaccharide were impossible. Therefore an arbitrary amount (0.2 mg. per cc. of original serum) of the Type I acetyl polysaccharide∗ was employed for each filtrate. Determinations of nitrogen in the washed precipitates were carried out by the gasometric micro-Kjeldahl method of Van Slyke. 5 The end-points in filtration of the specific antibodies of Type I antipneumococcal horse- and rabbit-sera are shown in Fig. 1. The results are plotted in terms of the percentage of total specifically precipitable nitrogen recovered in the various filtrates against average pore-diameters. With antipneumococcal rabbit-serum no specifically precipitable protein passed through a membrane with average pore-diameter of 10.6mμ. A 13.8mμ filtrate contained 11.9% of the total specifically precipitable protein of the serum. Slightly greater amounts were then recovered as the pore-sizes were increased up to 73.0mμ. At this point the curve rose sharply until at 102.5mμ the filtrate contained 86.6% of the total amount of antibody.

Ultrafiltration of the Virus of Equine Encephalomyelitis

Summary The virus of equine encephalomyelitis, both the Eastern and the Western strains, was found to pass collodion membranes with an average pore diameter of 66 mμ, but was completely held back by those of 60 mμ. The results indicate that this virus has the same filtration end-point, and consequently the same particle size, as that of St. Louis encephalitis, which was shown by Bauer, Fite, and Webster, 8 and Elford and Perdrau 9 to be 20 to 30mμ.

Ultrafiltration of the Virus of Infectious Avian Encephalomyelitis

Conclusion The virus-particles of infectious avian encephalomye-litis as present in suspension of brain of chickens affected by the experimental disease were found to have a diameter of 20 to 30 mμ as determined by ultrafiltration through gradocol membranes.

Ultrafiltration of the Virus of Vesicular Stomatitis

Summary The filtration end-point of the virus of vesicular stomatitis, or the average pore diameter of the finest membrane passing the virus, was found to be approximately 140 mμ. Two im-munologically distinct strains of the virus, the “Indiana” and the “New Jersey” maintained either in tissue culture or in mouse brain, were studied, and the filtration end-point was found to be the same irrespective of the source or serological type of the virus. Our results confirm those of Galloway and El ford, who found that the virus passes through collodion membranes which have an average pore diameter of 160 mμ. but is held back by those of 130 mμ. From their results they estimated the particle size of the virus to be between 70 and 100 mμ.

Ultra-Filtration Experiments with the Encephalitis Virus from the St. Louis Epidemic

Webster and Fite 1 showed recently that the virus isolated from cases of encephalitis in the St. Louis and Kansas City epidemics of 1933, passes through Berkefeld N candles in a relatively high concentration. While these findings indicate that the virus particles are exceedingly small, it was considered of interest to determine their size more accurately by filtration through graded collodion membranes of known porosity. The membranes used in these experiments were prepared according to the method of El ford 2 with certain minor modifications adopted by Bauer and Hughes. 3 They were sterilized by steaming for one hour at 100°C. The average pore values were calculated according to Poiseulles law from data obtained by measurement of the thickness of the membrane, its water content, the rate of flow of water through a known area, and the pressure producing the flow. These measurements were made after the sterilization. The virus-containing material used consisted of fresh brain tissue from mice which had succumbed to the experimental disease. In most of the experiments 4 brains were ground in a sterile mortar, suspended in 100 cc. of a diluent of equal parts of hormone broth (pH 8.0), ascitic fluid, and distilled water. The suspension was centrifuged for 30 minutes at about 3000 r.p.m. The supernatant fluid was passed through a Seitz filter, and then through a collodion membrane with an average pore diameter of about 0.25μ. This stock filtrate was then passed through a series of membranes of different pore sizes. From 6 to 8 different membranes were used in each experiment. The effective filtration area for each membrane was about 5 sq. cm., and the amount of the virus suspension passed through such area was usually 10 cc. To reduce the amount of adsorption of proteins from the virus suspension by the membranes, 3 cc. of sterile broth was passed through each membrane before filtration of the virus suspension.

Studies in immunity to tetanus bacilli

So much emphasis has been placed on the part played by antitoxin in immunity that when we found this antibody in the sera of guinea pigs carrying tetanus bacilli in their intestinal tract, we assumed that those animals would be immune to all types of this organism. We felt all the more secure in this assumption since Tulloch 1 has shown that the toxin produced by one type of tetanus is the same as that produced by any of the other types in that they are all neutralized by one antitoxin. When put to a test we found that this assumption is not entirely correct, as is shown by the results outlined below. We were not satisfied with the methods used by others for the production of tetanus as the incubation period was too short and the animals usually died within two or three days after inoculation. After considerable search we have adopted aleuronot as an irritating agent, when injected with a small number of spores, as symptoms appear on the fifth day, and death follows on the seventh or eighth day after inoculation. All control animals as well as those used for testing the protective power of sera were bled, and their serum was shown to be free from antitoxin. In addition cultures of their feces were negative for tetanus-like bacilli. As has already been pointed out 2 guinea pigs that carry one type of tetanus bacilli for about five months show appreciable amounts of antitoxin in their sera. When tested it is found that these animals are immune only to the type of bacilli which they are carrying. When injected with any other types they die in the same length of time as the controls.

The types of tetanus bacilli isolated from stools in Peking.

We had not seen any confirmation of Tullochs seriological classification of tetanus bacilli and as we had a number of strains of this organism, isolated from the stools of man, we thought it worth while to see if they could be differentiated into groups by the agglutination reaction. Tulloch not only sent us transplants of his four type cultures but has been most kind in answering our questions and has sent us an impure culture of a strain of tetanus bacilli that did not agglutinate with his type sera. This culture he received from France in 1918 but since no data accompanied it and since it was the only culture that did not agglutinate with his type sera he did not mention it in his work. This culture, which we have called Type V, has interested us very much for, as will be seen in the table, it is a type that we have found most frequently in Peking. Agglutinating sera were easily prepared by the injection into rabbits of washed bacilli from actively growing 24 hour bouillon cultures. The first injection was subcutaneous and was followed at intervals of a week hy three intravenous inoculations. The animals withstood the treatment well and ten days after the last injection their sera agglutinated the type bacillus used in a dilution of 1/6000 or higher. The differentiation of the types is quite striking as the other organisms were not, as a rule, clumped in dilution as low as 1/100. The results of our tests are given in the table where we have included the findings of Tulloch for comparison. It will be noted that we have confirmed the seriological grouping of tetanus bacilli though we have not, in the small numbers of cultures studied, found Types I1 or IV.

Tetanus carriers in experimental animals.

The demonstration that human carriers of tetanus bacilli have antitoxin in their blood has opened up a number of problems, which we are attempting to solve on experimental animals. We had first to determine whether carriers could be produced in these animals and if so whether antitoxin would be formed. The results of this preliminary experiment we will report on here and subsequently we hope to give the results of our further studies. That we would succeed in establishing tetanus bacilli in the digestive tract of guinea pigs was indicated by our finding that 27 per cent of our stock animals were already carriers and that these animals had antitoxin in their serum. On the other hand, we have failed to find a rabbit that carried these organisms nor have we detected tetanus antitoxin in the serum of a number of rabbits examined. As we planned to use the animals for other experiments a large number of guinea pigs were selected whose stools were free from tetanus spores and whose serum showed no antitoxin. These animals were starved for a day and were then fed a large number of washed spores and bacilli from month old bouillon cultures which were added to their usual food. After the feeding the animals were placed in metal cages which were sterilized from time to time. In order to prevent, as far as possible, infection from the outside all bedding and, with the exception of green stuffs which were washed, all food was autoclaved before it was placed in the cages. The results of the stool examinations made on these guinea pigs, approximately four months after the feeding of tetanus cultures, are summarized in Table I.