Stool Filtrates Research Articles

Visualization by immune electron microscopy of a 27-nm particle associated with acute infectious nonbacterial gastroenteritis.

A 27-nm particle was observed by immune electron microscopy in an infectious stool filtrate derived from an outbreak in Norwalk, Ohio, of acute infectious nonbacterial gastroenteritis. Both experimentally and naturally infected individuals developed serological evidence of infection; this along with other evidence suggested that the particle was the etiological agent of Norwalk gastroenteritis.

Biological Properties of Norwalk Agent of Acute Infectious Nonbacterial Gastroenteritis

Acute infectious nonbacterial gastroenteritis was induced in adult volunteers through three serial passages by oral administration of bacteria-free stool filtrates. This suggested that a replicating agent of subbacterial size was responsible for the observed disease. The biophysical properties of the agent, as assayed in volunteers, were consistent with those of a small virus, most closely related to the parvovirus group among known animal viruses. The agent appeared to have a diameter less than 66 nm and likely less than 36 nm. It appeared to lack a lipid containing envelope and was acid stable. It was stable to heating at 60° for 30 min. The agent appeared to be relatively host specific for man and conferred at least short-term immunity. Because of the high frequency of disease induced in unselected volunteers, widespread natural immunity to this agent may be absent or perhaps incomplete. Preliminary evidence suggested that the Norwalk agent replicated in an in vitro system, human fetal intestinal organ culture.

Transmission of acute infectious nonbacterial gastroenteritis to volunteers by oral administration of stool filtrates.

Raphael Dolin, NeU R. Blacklow, Herbert DuPont, Samuel Formal, Robert F. Buscho, Julius A. Kasel, Robert P. Chames, Richard Hornick, and Robert M. Chanock From the Laboratories of Infectious Diseases and Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland; the University of Maryland School of Medicine, Baltimore, Maryland; and the Walter Reed Army Institute of Medical Research, Washington, D.C.

STUDIES ON THE AGENT OF INFECTIOUS HEPATITIS

Two viral agents have been procured from patients with infectious hepatitisin two widely separated outbreaks of the disease by transfer of acute stage serum and stool filtrates to and passage in tissue cultures of rabbit liver cells in roller tubes and minced chick embryos in Simms-Sanders medium followed by passage in the amniotic cavity of the chick. Cultures of both agents, designated the Akiba and NL strains of virus, induced mild hepatitis without jaundice in the majority of volunteers tested after an incubation period of from 9 to 38 days. Although these agents have not been identified definitely as the virus of infectious hepatitis, the available evidence, as discussed, is compatible with the suggestion that such they are.

The Cause of Epidemic Diarrhea, Nausea and Vomiting. (Viral Dysentery?)

SummaryAmong the 32 volunteers who inhaled nebulized filtered garglings, symptoms developed in 17; of the 21 who inhaled nebulized stool filtrates, 11 developed symptoms. Of the combined groups of 53 volunteers, 28 (53%) developed symptoms as compared with 22 out of 240 (9%) of the rest of the student body in which the natural disease apparently occurred during the period of the tests. No attacks occurred in 6 volunteers who inhaled serum nor in 24 volunteers who were fed serum or filtrates of garglings or stools.The results, considering the unideal circumstances during which the tests had to be made, suggest that the causative agent of the disease is filtrable, airborne, enters through the respiratory tract and is present in the oropharynx and stool but not in the blood. Tests are now being made in a nonepidemic period with material from the November epidemic preserved at −70°C.

New Heat-Stable Agglutinogens in the Suipestifer Group

Observations on absorption of phage by heat-killed bacilli particularly those on the typhosus-paratyphosus B group and some strains of suipestifer suggested that further investigations along this line might yield information revealing new relationships in this highly complex group of organisms., , We here report the existence of differences in the suipestifer group first detected by characteristic phage absorption effects and confirmed by subsequent immunization and experiments on agglutinin absorption. The observations were made with a phage derived from chicken stool filtrates and propagated with a particular American strain of suipestifer, No. 26 in our collection. This phage lysed to a high titer numerous smooth cultures of the biochemically different types, namely American, Kunzendorf and one out of 4 strains of the Hirschfeld type, the other 3 being comparatively resistant. As already reported,, the strain Glässer-voldagsen was completely resistant even to the action of undiluted phage. We have since found one culture, No. 92 (American) in our collection which was poorly lysed but differed from other suipestifer strains in failing to absorb actively from the suipestifer 26 phage, particularly from some passages. Somewhat similar effects were observed with strains 80 and Glässer-voldagsen. Curiously enough, the same cultures, 80 and 92 were 2 of several strains, mostly of the Kunzendorf type, which on repeated occasions absorbed small quantities of the phage for paratyphosus B in contrast to the other suipestifer strains which did not at all absorb. These effects are indicated in Table I. In view of these results, it seemed desirable to determine whether serological characteristics could be found to correspond more or less with the qualitative differences shown in phage absorption.

Polyvalency Demonstrated by Antiphages

A general procedure was described for the study of the specificity of absorption of bacteriophage by heat-killed bacilli.1, 2, 3 Polyvalent phages derived from chicken stool filtrates and propagated either against a strain of paratyphosus A or enteritidis were shown to consist of at least 2 prominent qualitatively different fractions, the one selective for suipestifer strains and another for the typhosus-paratyphosus B group. This effect was readily demonstrated by testing the quality of the residual phage with several serologically different sensitive organisms. The question naturally presented itself as to whether or not antiphages may be employed to indicate the presence in a phage of the several fractions.Accordingly 4 series of rabbits were injected intravenously with the 2 polyvalent phages for paratyphosus A and for enteritidis and also with the presumably monovalent paratyphosus B and suipestifer phages. After 4 injections of 2 cc. each, potent antiphages, practically lacking in agglutinins were r...

Further Observations on Specific Inhibition of Bacteriophage Action

Recently we described1 specific inhibition of bacteriophage by bacterial extracts (B. dysenteriae Shiga and B. paratyphosus B). To test further the specificity of the inhibition, experiments were made with phages and extracts of organisms within the salmonella group, namely, B. paratyphosus B and B. suipestifer. Both phages were derived from chicken stool filtrates and were specific in their reactions on the 2 cultures.The extracts were dissolved in saline with the aid of weak alkali and heat, neutralized, diluted with saline to make a 1-200 solution, and passed through Seitz filters. Suitable dilutions of the 2 phages in beef extract broth were prepared and mixed with equal quantities of the filtered bacillary extracts. Two tests were made with these mixtures; one after incubation overnight at 37°∗ and another following an additional interval of 12 hours at the same temperature. One-half cc. was removed from each one of the tubes, diluted with 4.5 cc. of beef extract broth and the homologous test organis...

Specific Inhibition of Bacteriophage by Bacterial Extracts

There is as yet no final explanation for the specificity involved in bacteriophage action. Burnet1 claims on the basis of experiments made with the Salmonella group of organisms that the action, in a general way, parallels the serologic definition of the heat stable factors. However, he recognized that several observations made by himself and others are not compatible with this point of view. One of these is the failure to demonstrate inhibition of phage action by means of extracts of bacteria containing specific carbohydrate material.∗In our experiments positive results were obtained using saline extracts prepared according to the method of Furth and Landsteiner,3 except that no alkali was employed. The 2 phages employed, antidysenteriae Shiga and anti-B. paratyphosus B were derived from chicken stool filtrates. They were specific in their reactions when tested against these 2 organisms; the anti-B. paratyphosus B phage, however, acted also on B. aertrycke.The tests were made by mixing serial dilutions o...

Method for Determination of Volatile Fatty Acids in Stools.

In a previous report it was shown that the volatile fatty acids, formic, acetid and butyric, could be steam distilled from a concentrated solution of magnesium sulphate at rates of 60 to 100% faster than from watery solutions. We wish to report here a method for the precipitation of stools and the application of the rapid method of steam distillation to the filtrates obtained. Stools were collected in a volume of 10% sodium hydrate equal to that of the stool. The alkali stops fermentation almost completely, deodorizing the stool, and favors emulsification. 100 gms. of alkali stool mixture is neutralized with strong sulphuric acid and 175 to 200 cc. of a 10% mercuric chloride solution added. This amount of mercuric chloride is soluble in 1.5% hydrochloric acid. The mercury is precipitated with solid calcium hydrate, which is added until the stool is strongly alkaline to litmus. The volume is made up to 500 cc. which is centrifuged in 100 cc. centrifuge tubes. Two 100 cc. portions of supernatant fluid are measured off and made acid to litmus with sulphuric acid. Excess mercury is precipitated with hydrogen sulphide, the excess being blown off with a current of air. After filtering 100 cc., portions of filtrate are distilled in the manner reported. These filtrates from stools are almost nitrogen free (95% being removed) and free from higher insoluble fatty acids and sugar. On distilling the stool filtrates the rates of distillation obtained closely resemble those obtained from mixtures of pure volatile fatty acids. When formic, acetic, propionic or butyric acids are added to stools they may be recovered to within 5 to 10%. Duplicate determinations usually agree to within 5%. From the observed rates of distillation, it is concluded that the principal volatile fatty acids in the stools are acetic and butyric.

Effect of Bile, Sodium Salts of Bile Acids, and Unsaturated Fatty Acids on Bacteriophagic Action.

Previous investigators record different observations on the effect of bile on bacteriophagic action. Hauduroy,1 Jong and Hauduroy,2 and d'Herelle3 found that bile was decidedly inhibitory, whereas Caublot,4 in observations extending over a period of two years, obtained no evidence of an inhibitory action of bile on bacteriophage.In the studies, recorded here, of the action of bile on bacteriophage, B. coli, B. typhosus and B. dysenteriae Shiga and Mt. Desert strains were used with bacteriophages obtained from stool filtrates and from culture filtrates. In no case was there any inhibition of the bacteriophage in the presence of bile, although the bile was added to broth medium in concentrations of 10 per cent and 50 per cent. Filtered bile was tested as well as bile sterilized by heat, and a peptone medium was substituted for beef infusion broth, but the end result was always the same, the bile never inhibited the bacteriophage.Bachmann and Aquino5 report that, in their experiments, bile stimulated the pro...