John C. Torrey

A pernicious anaemia-like blood condition produced in monkeys with B. welchii toxin

In a recent article by Moench, Kahn and Torrey 1 a report was given of an analysis of the fecal flora in 33 cases of pernicious anæmia. The most striking feature revealed by these examinations was the constant presence of unusually large numbers of B. welchii in the stool specimens and presumably throughout the large intestine. These findings were a confirmation and extension of the earlier observations of Herter 2 and also of Simond. 3 It was suggested that the absorption of B. welchii toxin from the intestinal tract might conceivably be the essential etiological factor in the production of the clinical syndrome which characterizes pernicious anæmia, and that our findings justified further inquiry by experimental methods. In the present communication we wish to report in a preliminary way our observations on the effect of a potent B. welchii toxin on the blood of monkeys when injected intravenously. Several strains of B. welchii, isolated from different cases of pernicious anæmia, were compared as regards toxin production, employing the Bull and Pritchett 4 method of production, and injecting graded amounts of the filtered toxin into the wing vein of pigeons. Our strain “Navital” proved the best suited for our purposes but it was deemed advisable to enhance its virulence by animal passages. Accordingly it was passed through six successive rabbits by intravenous inoculation, and then through four pigeons by injections into the breast muscles and recovery from the lesions on the death of the bird. The virulence of the whole culture was finally such that 0.02 cc. caused death within 18 hours. Sterile filtrates of the culture were found to be, however, only slightly more toxic than the unpassed strain.

The relation of the intestinal flora to the scurvy of guinea pigs and of infants

The question recently has been raised as to whether scurvy in guinea pigs and in human beings is due to constipation and to the putrefactive activity of the bacteria in the intestinal tract. In order to determine this point a study of the intestinal flora was made in guinea pigs on a normal diet, on a diet which produced scurvy, and again on a diet which cured this disorder. For this purpose cultures and grain stained films were made from the feces, as well as from the different levels of the small and large intestine immediately after chloroforming the animals. This study on guinea pigs is portrayed in Table I. It will be seen that proteolytic bacteria other than subtloid types were not found in the intestinal tract; that the bacteria which were cultivated were merely those found in the outer world, for example, on dried foodstuffs. Similar organisms, indeed, were cultivated from the hay and oats which were fed these animals. Attention may be called to the fact that with none of the diets did B. coli develop, and that it was isolated from only one animal. The number of viable bacteria will also be noted as remarkably few, generally less than 1,000 per milligram of material. Among these there were hardly any that were actively proteolytic. Furthermore, a point of prime importance, there was no change in the flora on adding orange juice to the diet, although the scorbutic symptoms disappeared. It is our opinion, therefore, that most of the bacteria entering the intestinal tract are destroyed, and that the pabulum is not such as to encourage the growth of native intestinal bacteria. A similar study was carried out on the stools of three infants suffering from scurvy.

The Intestinal Flora of Rachitic Rats Before and After Treatment with Ultra-violet Rays

Zucker and Matzner 1 showed that when rats develop rickets as the result of being placed on a standard rickets-inducing ration, high in calcium and low in phosphorus, the feces become more alkaline and that on the administration of cod liver oil the reaction veers quickly to the acid side. These observations have been amply substantiated. Grayzel and Miller 2 showed later that the reaction of the intestinal contents of dogs is acid throughout almost the entire length of the gut, but that this reaction tends to become alkaline when rickets is brought about. As in the case of the rat the reaction reverts to normal on the administration of cod liver oil or treatment with ultra-violet irradiation. Our investigation was carried out on white rats weighing approximately 50 gm., i. e., about 4 weeks of age. Its main purpose was to ascertain whether rickets leads to a change in the flora of the intestinal tract and whether, after the animal is treated with ultra-violet rays and healing of the rachitic lesion is brought about, there was any associated modification of the flora. The rats were fed the rickets-inducing ration of McCollum and some were irradiated with a mercury vapor lamp. Immediately after the animals were killed by trauma they were autopsied and segments of the intestinal tract were tied off; that of the small intestine extending from the duodenum to the lower ileum and that of the large intestine extending from somewhat below the caecum to the beginning of the rectum. The content of each of these segments was squeezed out and the H-ion reaction obtained as soon as possible by a colorimetric method (Clark and Lub indicators). The material was then suspended to a standard density in normal saline solution, gram-stained films prepared and cultures made by methods adequate to determine quantitatively and qualitatively the character of the flora.

The Possible Relationship Between Absorption of B. Welchii Toxin and Pernicious Anemia.

The experiments reported here are in continuation of our investigation of the possible relationship between the absorption of B. welchii toxin from the intestine, and the condition of pernicious anemia. In a previous communication 1 there was described a severe type of anemia produced in monkeys by the intravenous inoculation of small doses of potent B. welchii toxin. In all the animals, however, after 3 weeks treatment, an immunity became apparent which was not broken down even by a greatly increased dosage, although abnormalities of erythrocytes and a leucopenia persisted for some time. It was next attempted by the continued inoculation of dosages of B. welchii toxin just below the limit of toleration, to cause such serious injury to the hematopoietic system as might prove irreparable, and to induce a progressive degenerative condition resembling that of pernicious anemia. The method of toxin preparation was essentially as described heretofore, except that somewhat larger pieces of fresh sterile pigeon muscle were added to the casein digest broth. Employing our pernicious anemia B. welchii strain, “Navitol”, sterile toxins were obtained which were lethal for pigeons within 24 hours after intramuscular inoculation of 0.5 cc. Two young ring-tail monkeys (Cebus capucinus) were given a long series of intravenous toxin inoculations in dosage sufficient to keep the animals in a condition of more or less severe anemia for 9 to 10 weeks, in contrast with the 3 or 4 weeks period of the previous experiment. After about 10 weeks, however, in spite of greatly increased dosage, it was not possible to check the tendency of reversion toward normal, and we became convinced that intravenous inoculations of B. zerelchii toxin would not damage the hematopoietic tissues to such a degree that the condition was irreversible.

A new and comparatively rapid method for the detection of liquefying bacteria

The results obtained by Feldstein and Weil 1 with Ostwalds viscosimeter in an investigation of the interaction of ferment and anti-ferment suggested to the writer that this apparatus might be of service for the early detection of the liquefying propensities of those bacteria, which under the methods commonly employed, may not reveal this function for one to four weeks. The identification of Bacillus coli in the bacteriological examination of water requires several tests, all of which may be completed within four days, with the exception of that for the action of the bacillus in question on gelatin which calls for a fourteen-day incubation at 20° C. It is generally agreed, among sanitarians, that a shortening of the period of this test is highly desirable, but, although a number of expedients have been suggested, none have been found sufficiently simple and reliable to warrant adoption.

Agglutinins and precipitins in anti-gonococcic serum

In December, 1906, I described the action and method of production of an anti-gonococcic serum which gave evidence of being of therapeutic value in the treatment of gonorrheal arthritis. At the time announcement was made of the fact that the serum contained specific agglutinins and precipitins for gonococcus. Since then a detailed investigation into the nature of these anti-bodies has been carried on. The results of this study may be summarized as follows: 1. Rabbits and other laboratory animals, when inoculated with cultures of gonococcus, raise specific agglutinins and precipitins. 2. Normal rabbit sera contain a varying amount of agglutinin for gonococcus. 3. Strains of gonoccoci differ greatly in the titer of their agglutination with various gonococcic immune sera. 4. After one inoculation with a certain culture a large amount of agglutinin was produced for some strains, but none for others. Further inoculations caused an increase in the titer of the agglutination for certain strains but a drop in that of others. 5. Absorption experiments indicate that an anti-gonococcic serum may contain, in addition to the specific homologous agglutinins, several groups of agglutinins which act on the different cultures quite independently of one another. At least three groups were found, whose major or specific agglutinins are not removed by inter-absorptions. This indicates that as far as agglutination is concerned there are specific differences between these groups. The family gonococcus is, accordingly, heterogeneous rather than homogenous, and in that respect resembles the dysentery, colon and streptococcus families. In making a serum for therapeutic purposes, this fact should be borne in mind. 6. The passage of a culture of gonococcus through a guineapig caused a very marked decrease in its agglutinability. 7. With the exception of one serum, meningococcus agglutinated only in low dilutions of the anti-gonococcic sera.