Concomitant Bacteria Research Articles

Enzymic proteolysis by Entamoeba histolytica; biochemical characteristics and relationship with invasiveness.

The proteolytic activity of extracts ofEntamoeba histolyticahas been further investigated. Casein and gelatin, but not haemaglobin, were hydrolysed. Activity was observed in the pH range 5·8 to 8·5 with optimal activity at pH 7·5 to 7·9. Activity was optimal at 37° C. and sulphydryl groups were not required. Concomitant bacteria showed no proteolytic activity. Hyaluronidase, collagenase and lecithinase could not be detected.An inhibitor of proteolytic enzyme was present in sera of all animals tested and in egg yolk. All culture media prepared from eggs were inhibitory, but inspissation or dilution of serum inactivated the serum inhibitor. Purified trypsin inhibitors from lima and soy bean were not active against the amoebic enzyme. Proteolytic enzymes were not secreted extracellularlyin vitro.High proteolytic activity was found in two out of five invasive, freshly isolated, strains ofE. histolyticaand after two series of liver passages of a single strain. The significance of these observations is discussed. It is concluded that the present evidence does not convincingly demonstrate that high proteolytic activity is required for tissue invasion by amoebae, but may accompany another factor.

Isolation of Trichomonas vaginalis from fungi and bacteria.

The study of the physiology, nutrition, antigenic structure and pathogenicity of single strains of Trichomonas vaginalis, and of their lack of sensitivity to certain antibiotics depends on the possibility of isolating these parasites “in vitro” from other associated microorganisms.The trichomonads are easily isolated from concomitant bacteria when specific antibiotics are used (Adler and Pulvertaft, 1944; Johnson and Trussell, 1945; Williams and Plastridge, 1946). To eliminate bacterial contamination from the broth cultures of Trichomonas vaginalis or T. foetus, these authors noted that the addition of 100 I.U. of penicillin and 100 µg. of streptomycin is sufficient. However a method for the rapid separation of T. vaginalis from bacterial as well as other vaginal organisms has not hitherto been described.T. vaginalis, particularly during pregnancy of the carriers, is often associated with species of the genus Candida. These yeast-like fungi are not sensitive to the commonly available antibacterial antibiotics. Anaerotropism (Magara et al., 1953) and motility of the trichomonads facilitate their isolation from fungi.

The influence of environmental factors on the size of the cysts of Entamoeba invadens Rodhain, 1934.

In dibacterial cultures of E. invadens the size of the cysts was significantly influenced by different species of concomitant bacteria, and by the composition of the media. These factors appear to influence cyst size as a result of their effect on the growth rate of the amoebae; a higher multiplication rate was associated with a decrease in the size of the cysts.I wish to thank Dr A. Bishop for criticizing the manuscript, and Mr W. McConnachie, jun., for assistance with the statistical methods. The work has been financed by the Medical Research Council.

Isolation of Coccidioides Immitis (Stiles) from the Soil

The epidemiological studies on coccidioidal granuloma, recently summarized by Beck, suggest the soil and the vegetation as the probable source of the fungus infection. The peculiar geographic distribution of the disease both in man and animals to the central and southern sections of the State of California with a predominance of the cases in Kern and Los Angeles Counties supports this contention. Although repeated attempts have been made to find the organism in nature the examinations, made with the aid of the customary bacteriologic technique, have thus far yielded negative results. The relatively slow growing atypical fungi are, as a rule, rapidly overgrown by the concomitant bacteria present in the soil and vegetations. Little progress could be made until a procedure had been developed which would concentrate, possibly enrich the stage of the mold (chlamydospore) supposed to exist in nature. In the course of detailed metabolism studies it was found that Coccidioides immitis satisfies its nitrogen and carbon requirements from relatively simple compounds. The medium, which gives selective cultures, has the following composition: Ammonium chloride 1 part, sodium acetate 1 part, equal mixture of KH2PO4 and K2HPO4 0.4 part and magnesium sulfate 0.01 part; distilled water 100 parts. While the majority of bacteria grow slowly or very poorly, Coccidioides, certain Blastomyces and Aspergillus varieties develop exceedingly well in the nutrient solution. Bacterial multiplication may be entirely suppressed and the purification of the fungus greatly facilitated by the addition of acriflavine in a concentration of 1:25,000. For several years the medium has been employed in the examination of clinical specimens (sputum, pus, blood, autopsy specimens, etc.) suspected to contain the mold. Samples of sputum, which contained very few typical double contoured fungoidal cells yet teeming with the usual respiratory and buccal flora, gave pure cultures of Coccidioides immitis in the dye culture medium.

Antibodies Against Vaccinia Virus Demonstrable in Vitro

Summary Sera from rabbits hyperimmunized by intravenous and intraperitoneal injections of vaccinial virus tissues (brain, testicle and skin) caused fixation of alexin in the presence of a suitable dilution of vaccinial brain or skin suspension but not in the presence of non-vaccinial tissue suspensions. The evidence indicates that this fixation is a process specific for vaccinial virus tissue, regardless of the tissue in which the virus is propagated and that it is not due to the immune reactions of concomitant bacteria.

STUDIES ON THE PHYSICAL AND CHEMICAL PROPERTIES OF THE VIRUS OF FOOT-AND-MOUTH DISEASE

A strain of foot-and-mouth disease virus was recovered from a cow at the height of the disease, and was propagated through at least 261 passages in the guinea pig. Considerably over 2000 animals proved susceptible to the virus, and the virus could be transferred at will back to cattle and hogs, and then again returned to guinea pigs. No natural immunity was discovered in guinea pigs. Secondary lesions were easily and regularly induced, thus making this strain particularly favorable to experimental purposes. In general, the guinea pig may therefore be regarded as the animal of choice for laboratory studies. The guinea pig could be infected by different methods of injection in different sites, but constant and regular production of primary and secondary lesions-or generalization of the disease-followed intradermal "tunneling" in the manner described, combined with subcutaneous inoculation of the posterior hairless pads of full grown animals. As we have indicated, the virus was peculiarly epitheliotropic, which in turn gives support to the opinion that its portal of entry may be limited. The active agent could purify itself of chance concomitant bacteria in the first passages, in a susceptible animal-a character possessed by filter-passing viruses in general. The virus was active in dilutions of 1:10,000,000. This shows not only the minuteness of the active agent, but also the necessity for a change of technique from that employed with larger sized infectious agents. Apart from this, the dilution factor is important in interpreting mere preservation of the virus rather than multiplication, when only early successive subplants in culture experiments are positive. Furthermore, some samples of virus were not so active-a factor of twenty-five existed between the weakest and strongest samples among fifteen titrated. This indicates that comparative tests, as, for example, of survival in different media, should be made with the same specimen. In any case, the rate and energy of action of the virus were proportional to its concentration, thus differing from the behavior of certain enzymes. The incitant is not sedimented by centrifugation. Non-centrifugability, a property of some other filter-passing, infectious agents, is not an indication of the fluid character of the virus, as we have already explained. In view of the evidence presented and other tests to be reported later, failure of deposition is related to the minute size of the incitant. The method of centrifugation has also failed to remove "virucidal bodies" in the meaning of Frosch and Dahmen.