A NEW NON-PATHOGENIC TETRAGENOUS AMEBA. I

The non-pathogenic tetragenous ameba described here multiplies in the intestine of man, without producing any noticeable symptoms of dysentery. This non-pathogenic ameba is distinguished morphologically and biologically from Entamœba tetragena Viereck and Entamœba coli Lösch emend. Schaudinn. The vegetative forms of the non-pathogenic tetragenous ameba may be divided into two groups, large and small. The large vegetative form is found in the normally evacuated or the diarrheal stools obtained after purgation. The small form is found in the soft stools. This form may be transformed into cysts, or may reproduce itself by binary fission. Cysts are found in relatively large numbers in the soft and formed stools. Experimentally, the new tetragenous ameba may be readily distinguished from the pathogenic variety. The vegetative forms of the dysentery ameba produced the disease in 91 per cent of cases when introduced per anum, and in 50 per cent where the cysts were introduced per os. Similar infection experiments undertaken on twenty-three young cats, i.e. ten experiments with the vegetative form of the non-pathogenic tetragenous ameba per anum, and thirteen experiments with the cysts per os, failed to produce pathological conditions. The intestines of the animals macroscopically and histologically failed to show any of the changes of dysentery. Hence we may conclude that the new type of tetragenous ameba described here is non-pathogenic for man and for young cats.     

THE NATURAL HISTORY OF HUMAN POLIOMYELITIS : II. ELIMINATION OF THE VIRUS

Studies on the elimination of virus in human paralytic poliomyelitis during the first 2 weeks of the disease, revealed the following:— 1. The nasal (not nasopharyngeal) secretions collected from 22 patients on cotton plugs over a period of 3 days and the saliva and oral secretions expectorated during a similar period by 20 patients failed to yield virus. 2. In 10 of the patients whose secretions (nasal, oral, or both) were investigated, virus was isolated from single specimens of the lower intestinal contents. 3. No virus was found in large amounts of urine (up to 200 cc.) obtained from 12 patients, 6 of whom had paralysis of the bladder. 4. In the present tests virus was found 4 times more often in the stools of patients under 8 years of age (64 per cent of 11 cases) than in older individuals (17 per cent of 12 cases). This difference was found to obtain when our data were combined with those which could justifiably be selected from the literature, the total figures indicating that virus has been isolated from 50 per cent of 58 children under 8 years of age and from 12 per cent of 60 older individuals. 5. No support was found for the hypothesis that poliomyelitis virus in the stools originates from swallowed nasal secretions and saliva or oral secretions.     

ETIOLOGY OF YELLOW FEVER : VIII. PRESENCE OF A LEPTOSPIRA IN WILD ANIMALS IN GUAYAQUIL AND ITS RELATION TO LEPTOSPIRA ICTEROH?MORRHAGI? AND LEPTOSPIRA ICTEROIDES.

By the inoculation of guinea pigs intraperitoneally with the emulsions of kidneys from wild rats and mice captured in Guayaquil, it was found that 67 per cent of the wild rats tested harbored in their kidneys a leptospira which produced in guinea pigs symptoms and lesions identical with those produced by Leptospira icterohamorrhagia derived either from patients suffering from infectious jaundice in Japan or Europe, or from wild rats caught in New York. Immune sera were prepared in rabbits by injecting different strains of the Guayaquil leptospira. These sera had a marked agglutinating and disintegrating influence upon the homologous strains, and also, but often to a less pronounced degree, upon the strains of Leptospira icterohœmorrhagiœ from other sources. Pfeiffer''s phenomenon was also found to be positive, and protection was demonstrated against infection with virulent cultures of strains of Leptospira icterohœmorrhagiœ. The same sera had no effect, or at most a very slight one, upon Leptospira icteroides. Guinea pigs inoculated with icteroides strains were not noticeably protected by the use of the immune sera prepared with the Guayaquil rat strains. Guinea pigs inoculated with killed cultures of the Guayaquil strains of leptospira proved to be resistant to a subsequent infection with heterologous as well as homologous strains of Leptospira icterohœmorrhagiœ. It is concluded, therefore, that the leptospira isolated from the kidneys of wild rats and mice in Guayaquil belongs to the group of Leptospira icterohœmorrhagiœ, and differs from Leptospira icteroides in its immunity reactions. No positive transmission was obtained with kidney material from bats and an opossum.     

ON THE INHIBITORY INFLUENCE OF EOSIN UPON SPORULATION

Sporulation of B. anthracis, B. subtilis, B. cereus, B. ruminatus, B. mesentericus, B. anthracoides and B. megatherium does not take place in an agar medium containing eosin "Gelb" in a concentration exceeding 0.5 per cent. In a concentration of 0.1 per cent. most of these bacteria fail to produce spores. The greatest sensitiveness is shown by B. cereus and B. mesentericus. In a bouillon medium sporulation is likewise inhibited by eosin, but after a longer time—seven weeks or more—sporulation still occurs where the concentration of the dye equals one-tenth per cent. Sporulation of B. tetani, B. anthracis symptomaticus, B. botulismus, B. œdema maligni, B. enteritidis sporogenes, and B. putrificus does not take place in a medium containing eosin "Gelb" in concentrations exceeding 0.03 per cent. With these organisms, no difference was noted in the final effect, depending on the medium employed. No permanent loss of power to produce spores ensues with the bacteria tested even after long sojourn in the eosinized media. It may be stated that on the whole the inhibitory action of eosin was more pronounced upon the anaerobic than upon the aerobic species of bacteria employed in these experiments.     

REPRODUCTION IN VITRO OF ENTAM?BA TETRAGENA AND ENTAM?BA COLI FROM THEIR CYSTS

1. The vegetative forms of amebæ may be developed in vitro from the cysts. 2. The cysts of Entamœba tetragena and Entamœba coli each produce only a single vegetative individual. It has not been observed in vitro that a number of daughter amebæ, corresponding to the number of nuclei present in the cyst, emerge from the mother cyst, as claimed by Casagrandi and Barbagallo, Schaudinn, and a number of other investigators. 3. The daughter nuclei in the cystic stage correspond to the gamete nuclei of other protozoa. The syngamic nuclear union in the case of these two amebæ does not take place in the tetranuclear stage of the cyst, as claimed by Schaudinn and Wenyon, but during reproduction. 4. The process of autogamy of both amebæ is not restricted to two nuclei; it may involve more than two, which unite to form a syncaryon. In the case of all other protozoa, autogamy is restricted to two nuclei. 5. In order to distinguish the two types of autogamy, one has been termed simple autogamy and the other polynuclear autogamy. The latter may again be classified according to the number of nuclei involved, as trinuclear and tetranuclear autogamy. 6. The majority of cysts treated for 5 minutes in a 2 per cent solution of hydrochloric acid survives. 7. It was possible to demonstrate in vitro the developmental cycle of Entamœba tetragena. 8. No evidence of heterogamy has been observed in vitro in Entamœba tetragena or Entamœba coli. 9. The vegetative form of Entamœba tetragena, at a certain stage of its life cycle in vitro, shortly after its formation, has not only one or two, but may have as many as three or four nuclei.     

OCCURRENCE OF POLIOMYELITIS VIRUS IN AUTOPSIES,PATIENTS, AND CONTACTS

1. Poliomyelitis virus has been recovered in monkeys from 50 per cent of spinal cords, 10 per cent of olfactory bulbs, 50 per cent of tonsil-adenoid tissue, and from 26 per cent of the colon contents of autopsies; from the stools of 20 per cent of patients, and of 5 per cent of the contacts examined in this series. 2. Other materials as indicated in Table I were tested without success. 3. In autopsies with positive cords, tonsil-adenoid tissues, or colon contents were positive in 73 per cent. 4. 22 per cent of stools from patients with paralysis yielded virus and 19 per cent of the stools from patients without paralysis yielded virus. 5. 20 per cent of the stools from males and 22 per cent of the stools from females yielded virus. 6. 40 per cent of 35 stools from patients under 16 years yielded virus while 8 per cent of 38 stools from patients above the age of 15 yielded virus. 7. 71 per cent of the cords of 35 autopsies under 16 years yielded virus while 31 per cent 16 years and over yielded virus. 8. Repeat stools from 5 positive cases, 1 month after the first positive stool, were negative. The stool of one contact was positive the 2nd month after first recovery but was negative the 3rd month.     

COMPOSITION AND SYNTHESIS OF LIPIDS IN RESTING AND PHAGOCYTIZING LEUKOCYTES

The lipid content of rabbit polymorphonuclear leukocytes, obtained from peritoneal exudates, constituted 8.7 ± 2.9 per cent of the dry weight of these cells; 60 per cent of all lipids were phospholipids, 20 per cent triglycerides, and the remainder cholesterol and cholesterol esters and a small amount of non-esterified fatty acids (2 to 4 per cent). The composition of the fatty acids in leukocytes, as determined by gas-liquid chromatography, was slightly different from rabbit serum and red blood cells, but markedly different from the dietary fat. The synthesis, turnover, and composition of lipids in rabbit leukocytes at rest and during phagocytosis in vitro were compared. Lipid content and composition were not affected by the phagocytic process. However, active phagocytosis resulted in an increase in the rate of turnover of lipids. This stimulation of lipid metabolism was more marked in triglycerides and cholesterol esters than in phospholipids. It is suggested that the increased turnover of lipid during phagocytosis may reflect a general metabolic stimulation accompanying this process, rather than a specific synthesis of phospholipid for the production of new cell membrane.     

THE OCCURRENCE OF BACILLUS INFLUENZ? IN THE NORMAL THROAT

1. The frequency of occurrence of Baculus influenzœ in the throats of normal individuals has been as great in the period subsequent to the epidemic of influenza as it was during the period of the epidemic. 2. Bacillus influenzœ may persist in the throats of healthy carriers for a considerable period of time. 3. A considerable proportion of individuals not exposed during the epidemic has been found to carry Bacillus influenzœ in their throats. 4. Oleate hemoglobin agar has been found to be a very satisfactory selective medium for cultivation of Bacillus influenzœ from the throat. To give optimum results it should have a reaction of from pH 7.2 to 7.5.     

A PATHOLOGICAL STUDY OF SYPHILITIC AORTITIS AND ITS SEROLOGY

1. Syphilitic aortitis is a productive inflammatory process, the earliest and most constant feature of which is a perivascular round cell infiltration in the adventitia. 2. The typical gross picture of luetic aortitis is often obscured by a superimposed, diffuse atherosclerosis. In the early cases the aorta appears fairly normal, presenting only the characteristic histological changes. 3. A pure aortic insufficient valve, with the exception of an infectious endocarditis, is always luetic. 4. Cardiac hypertrophy is not a complication of luetic aortitis. When present it is usually associated with a nephritis. 5. The demonstration of Spirochœta pallida, even in advanced specimens of syphilitic aortas, is doubtful. 6. An antigen prepared from alcoholic extract of guinea pig heart with the original Wassermann technique should be preferred in diagnosing luetic aortitis. 7. Positive complement fixations in patients suffering with syphilis for a period of about fifteen years or longer suggest the probability, at least, of histological luetic changes in the aorta in 80 to 90 per cent of the cases. 60 per cent of these die from aortitis. 8. About 94 per cent of patients suffering with aortitis give positive Wassermann reactions.     

A METHOD FOR THE QUANTITATIVE DETERMINATION OF FECAL BACTERIA

From results of which the foregoing data form a part, it appears that the amount of bacterial nitrogen in the feces is a valuable index to intestinal conditions, and the method herein described is a simple and satisfactory one for making this determination. It involves three serial centrifugalizations of a two gram sample of the fresh feces brought into suspension in 0.2 per cent. hydrochloric acid. The bacterial suspension finally obtained is concentrated and extracted by alcohol, and nitrogen is determined in the precipitated material. The complete data on a given stool can be obtained in about five days, and one operator can take care of three or four stools in duplicate in one day. On an absolutely uniform diet of simple and easily digested food during a period of three to four weeks, the average amount of bacterial nitrogen in two subjects was found to be 53.9 per cent. of the total fecal nitrogen, and this percentage, though higher than that obtained by workers heretofore, is probably more nearly a true value for bacterial nitrogen, because no ether extraction was employed. The average daily amount of dry bacteria, calculated on the basis of the nitrogen values, is 8.27 grams.     

STUDIES ON TYPHUS FEVER : XII. THE PASSIVE IMMUNIZATION OF GUINEA PIGS,INFECTED WITH EUROPEAN VIRUS,WITH SERUM OF A HORSE TREATED WITH KILLED RICKETTSIA OF THE MEXICAN TYPE

Under uniform diet conditions the normal bile fistula dog will eliminate pretty constant amounts of cholesterol—about 0.5 to 1.0 mg. cholesterol per kilo per 24 hours. Diets rich in cholesterol (egg yolk) will raise the cholesterol output in the bile but compared to the diet intake (1.5 gm. cholesterol) the output increase in the bile is trivial (5–15 mg.). Calves'' brains in the diet are inert. Bile salt alone will raise the cholesterol output in the bile as much and often more than a cholesterol rich diet. Bile salt plus egg yolk plus whole bile give maximal output figures for bile cholesterol—60 mg. per 24 hours. Liver injury (chloroform) decreases both bile salt and cholesterol elimination in the bile. Blood destruction (hydrazine) fails to increase the bile cholesterol output and this eliminates the red cell stroma as an important contributing factor. Certain cholagogues (isatin and decholin) will increase the bile flow but cause no change in cholesterol elimination. The ratio of cholesterol to bile salt in the bile normally is about 1 to 100 but the bile salts are more labile in their fluctuations. The ratio is about reversed in the circulating blood plasma where the cholesterol is high (150–300 mg. per cent) and the bile salt concentration very low. Cholesterol runs so closely parallel to bile salt in the bile that one may feel confident of a physical relationship. In addition there is a suspicion that the bile cholesterol is in some obscure fashion linked with the physiological activity of hepatic epithelium.     

A SEROLOGICAL DIFFERENTIATION OF SPECIFIC TYPES OF BOVINE HEMOLYTIC STREPTOCOCCI (GROUP B)

Under uniform diet conditions the normal bile fistula dog will eliminate pretty constant amounts of cholesterol—about 0.5 to 1.0 mg. cholesterol per kilo per 24 hours. Diets rich in cholesterol (egg yolk) will raise the cholesterol output in the bile but compared to the diet intake (1.5 gm. cholesterol) the output increase in the bile is trivial (5–15 mg.). Calves'' brains in the diet are inert. Bile salt alone will raise the cholesterol output in the bile as much and often more than a cholesterol rich diet. Bile salt plus egg yolk plus whole bile give maximal output figures for bile cholesterol—60 mg. per 24 hours. Liver injury (chloroform) decreases both bile salt and cholesterol elimination in the bile. Blood destruction (hydrazine) fails to increase the bile cholesterol output and this eliminates the red cell stroma as an important contributing factor. Certain cholagogues (isatin and decholin) will increase the bile flow but cause no change in cholesterol elimination. The ratio of cholesterol to bile salt in the bile normally is about 1 to 100 but the bile salts are more labile in their fluctuations. The ratio is about reversed in the circulating blood plasma where the cholesterol is high (150–300 mg. per cent) and the bile salt concentration very low. Cholesterol runs so closely parallel to bile salt in the bile that one may feel confident of a physical relationship. In addition there is a suspicion that the bile cholesterol is in some obscure fashion linked with the physiological activity of hepatic epithelium.     

An endemiological study of enteric virus infections: poliomyelitis,coxsackie, and orphan (ECHO) viruses isolated from normal children in two socioeconomic groups

An endemiological study of enteric viruses was conducted among 136 normal children, living in households in two socio-economic groups, over a 29 month period in Charleston, West Virginia. A repeatable seasonal incidence of enteric virus excretion was noted with over 90 per cent of isolations occurring in the months of June to October. Of 592 stools examined in District I, a lower socio-economic group, 8.3 per cent yielded virus as compared to 3.1 per cent of 966 stools examined in District IV, an upper middle class district with good environmental sanitation. Among the 77 viruses isolated in tissue cultures of monkey kidneys, 44 per cent were ECHO or orphan viruses, 37 per cent Coxsackie viruses, and 19 per cent poliomyelitis viruses. Among poliovirus carriers, and 15 family contacts, 10 individuals had simultaneous heterotypic and type-specific antibody responses. The heterotypic ones were usually present at low levels and were transient in nature. Family infection with certain orphan viruses was also evident from antibody development which occurred following isolation of virus in the sentinel child.     

II. BILE CHOLESTEROL : FLUCTUATIONS DUE TO DIET FACTORS,BILE SALT,LIVER INJURY AND HEMOLYSIS

Under uniform diet conditions the normal bile fistula dog will eliminate pretty constant amounts of cholesterol—about 0.5 to 1.0 mg. cholesterol per kilo per 24 hours. Diets rich in cholesterol (egg yolk) will raise the cholesterol output in the bile but compared to the diet intake (1.5 gm. cholesterol) the output increase in the bile is trivial (5–15 mg.). Calves'' brains in the diet are inert. Bile salt alone will raise the cholesterol output in the bile as much and often more than a cholesterol rich diet. Bile salt plus egg yolk plus whole bile give maximal output figures for bile cholesterol—60 mg. per 24 hours. Liver injury (chloroform) decreases both bile salt and cholesterol elimination in the bile. Blood destruction (hydrazine) fails to increase the bile cholesterol output and this eliminates the red cell stroma as an important contributing factor. Certain cholagogues (isatin and decholin) will increase the bile flow but cause no change in cholesterol elimination. The ratio of cholesterol to bile salt in the bile normally is about 1 to 100 but the bile salts are more labile in their fluctuations. The ratio is about reversed in the circulating blood plasma where the cholesterol is high (150–300 mg. per cent) and the bile salt concentration very low. Cholesterol runs so closely parallel to bile salt in the bile that one may feel confident of a physical relationship. In addition there is a suspicion that the bile cholesterol is in some obscure fashion linked with the physiological activity of hepatic epithelium.     

DIFFERENT AMOUNTS OF TRANSFORMED ATOXYL PRODUCED BY INCUBATING ONE PER CENT. AND TEN PER CENT. ATOXYL IN BLOOD

1. 10 per cent. atoxyl in blood incubated at 37° C. for 1 hour gives rise to a solution that is much more than ten times as toxic as a 1 per cent. solution of atoxyl similarly incubated. 2. When the comparison is made after incubation for 3 hours instead of for 1 hour, the toxicity of the 10 per cent. solution is but slightly greater than ten times that of the 1 per cent., provided the red blood corpuscles are not removed from the dilutions. 3. If the corpuscles are removed from both the 10 per cent. and the 1 per cent. atoxyl solutions immediately after incubation at 37° for 1 to 3 hours, the dilutions of the 10 per cent. atoxyl are much more than ten times as toxic as the corresponding dilutions of the 1 per cent. atoxyl. 4. After incubation with atoxyl at 37° for 1 to 3 hours, red blood corpuscles left at room temperature in dilutions made from the 10 per cent. and 1 per cent. solutions in blood increase markedly the toxicity of the dilutions made from the 1 per cent. atoxyl, but increase very slightly the toxicity of the dilutions made from the 10 per cent. atoxyl. 5. If one desires to produce a large amount of transformed atoxyl by incubating atoxyl in blood at 37° for 1 to 3 hours, strong solutions of atoxyl should be chosen in preference to weaker solutions.     

Faecal excretion of bile acids and neutral steroids on diets differing in type and amount of dietary fat in young healthy persons

The effect of different amounts and types of dietary fat on faecal steroid excretion in healthy volunteers was examined. In the first experiment with sixty volunteers four diets which differed in type and amount of dietary fat only were compared during a test-period of 5 weeks. Faeces were collected during 7 days before and at the end of the test period and analysed individually. In the second experiment with thirty-five volunteers, a low fat diet containing 22% of energy as total fat (4% as polyunsaturated fatty acids) was compared with a moderate fat diet (30% of energy as total fat; 11% as polyunsaturated fatty acids) during 13 weeks. Faeces were collected during the first 7 days of the test period and during 7 consecutive days near the end of the test period and pooled per dietary group. Excretion of faecal neutral steroids and bile acids was not significantly influenced by amounts of total fat ranging from 22% to 40% of daily energy or by differences in polyunsaturated to saturated fatty acids ratio from 0 . 2 to 1 . 7. The only exception was the excretion of bile acids in the second experiment which was 0 . 19 mmol/24 h (75 mg/24 h) higher on the low fat diet than on the moderate fat, high polyunsaturated fat diet (P less than 0 . 01).     

ENLARGEMENT OF THE ADRENAL CORTEX IN EXPERIMENTAL UREMIA

The production of an experimental uremia in the albino rat by removal of both kidneys is followed by hypertrophy of the adrenal glands. In the case of male rats 90 days of age this adrenal enlargement amounted to 65 per cent and in the case of female rats 180 days of age 47 per cent. The increase in the size of the whole gland is due entirely to hypertrophy of the cortex. This increase in the volume of cortical tissue amounted to approximately 40 per cent for males and 61 per cent for females and was due in large part to an increase in the size of the cells. The content of water and material soluble in fat solvents was higher in the uremic than the control glands. However after subtraction of such storage materials a true hypertrophy of the cortex still remained. It amounted to 21 per cent. Histologically the stainable fat had a more irregular distribution and was present in lesser amount in the adrenals from the uremic animals. The capillaries of the medulla and reticular cortex were distended. The nuclei of both the cortical and medullary cells were swollen and stained faintly.     

HEMOGLOBIN PRODUCTION IN ANEMIA AS INFLUENCED BY THE BILE FISTULA

The presence of a renal bile fistula with escape of all bile into the urinary tract seriously impairs the capacity of an anemic dog to form new hemoglobin on standard diets. These bile fistula dogs will produce about one-half as much hemoglobin in anemia on standard diets as during earlier control periods without a bile fistula. Iron given by mouth to an anemic bile fistula dog will effect the production of about one-half the amount of new hemoglobin as in control periods. Iron given by vein to an anemic bile fistula dog will approximate the theoretical 100 per cent return of new hemoglobin. Obviously absorption is a very important factor in the utilization of iron by these dogs. The reaction to liver feeding is much like the reaction to iron feeding but we have no proof of inadequate protein digestion and absorption in these bile fistula dogs. In fact the uniform body weight and normal clinical state over periods of years speak for adequate absorption of protein digestion products. Evidence cited above supports our belief that inadequate hemoglobin production (protein formation) noted in these bile fistula dogs may be related to a disturbed liver function.     

CULTIVATION OF SPIROCH?TA GALLINARUM

1. Spirochœta gallinarum can be cultivated in suitable artificial media for many successive generations and probably for indefinite periods. The presence of fresh tissue and a certain amount of oxygen seems to be essential for its growth. No perceptible odor is produced in the cultures. 2. The maximum growth of Spirochœta gallinarum is reached on about the fifth day, but the phase of degeneration commences slowly and gradually, so that in this respect the gallinarum differs from the duttoni, kochi, obermeieri, or novyi, whose cultures are characterized by sudden onset of degeneration soon after the maximum growth is attained. 3. No rod formation resembling bacilli arises in the course of multiplication of Spirochœta gallinarum in cultures. Many round or oval bodies appear in old cultures, but no infection of animals or formation of spiral forms from these granules has been produced. The granules are probably the degeneration products derived from the periblast of the spirochœtœ. 4. Cultures of Spirochœta gallinarum, either old or young, do not contain a form which passes through a Berkefeld filter (V) that infects chickens or grows into spirochætæ. 5. Spirochœta gallinarum remains virulent for chickens after being in cultures for at least thirteen generations, but it may become avirulent under certain cultural conditions. The inoculation of chickens with the attenuated culture renders the birds refractory to the subsequent infection with a virulent strain. 6. When the spirochætæ are cultivated in the media containing rabbit kidney instead of chicken muscle, the individual specimens are somewhat thicker, but otherwise typical. 7. Spirochœta gallinarum multiplies in culture by transverse division. No positive evidence of a longitudinal division has been obtained.     

ETIOLOGY OF YELLOW FEVER : XIII. BEHAVIOR OF THE HEART IN THE EXPERIMENTAL INFECTION OF GUINEA PIGS AND MONKEYS WITH LEPTOSPIRA ICTEROIDES AND LEPTOSPIRA ICTEROH?MORRHAGI?.

1. Slowing of the heart occurred in monkeys and guinea pigs during the febrile period of the experimental infection due to Leptospira icteroides. A similar reaction took place in animals inoculated with Leptospira icterohœmorrhagiœ. 2. The mechanism of slowing was usually due to slowing of the whole heart. 3. Once incomplete heart block was seen. Changes in the ventricular complex occurred four times.