BIOLOGY OF BACTERIUM LEPISEPTICUM : IV. VIRULENCE OF DIFFUSE AND MUCOID TYPES AND THEIR VARIANTS.

As a preface to a general discussion of these experiments, we wish to refer again briefly to the titrations of virulence described in this paper. None of them was carried out in an altogether proper manner,- it was necessary to employ rabbits of mixed breed, to use small numbers, and in some instances, to compare the results of titrations done at different times of the year. Furthermore, many tests were made in the mouse, a foreign host, and by means of artificial injection into the peritoneal cavity. Hence, if our results had shown fluctuations and wide differences, we should have experienced difficulty in interpreting their significance. Fortunately, however, the control measures which we were able to employ,-namely the utilization of animals of similar age and weight, bred at the Rockefeller Institute under uniform environmental conditions, and free of previous exposure to Bact. lepisepticum, together with the technique of intranasal instillation of similar doses,-were sufficient to give consistent and relatively uniform effects. These we have regarded as presumptive evidence of the following: (1) that of the three types of Bact. lepisepticum described, "D" strains are the most virulent or pathogenic, mucoid forms less so, and "Grdquo; types little or not at all virulent; (2) that different strains of the same type are of similar virulence; and (3) that passage of type-pure strains through non-toxic, nutrient media, or through animals, does not modify their virulence. The experiments described in the present communications were planned to investigate certain microbic factors which seem to influence the spread of Bact. lepisepticum infection. Accordingly, we studied the types encountered in nature, their behavior in the rabbit host, and other qualities which might possibly be related to their parasitic activities. At the outset we discovered that very little is known about the "Pasteurella" or "hemorrhagic septicemia" organisms. Every small, blunt bacillus with bipolar granules and certain quite definite fermentative reactions is placed in this group and is further designated by prefixing the name of the animal from which it was obtained. Differentiation on the basis of serological studies has been extremely difficult and as yet not sufficiently detailed and complete to be of use. We have, therefore, called our strains "Bact. lepisepticum," because they belong to the Pasteurella group and were obtained from rabbits, but at the same time have recognized the possibility that similar forms may also be present in other animal hosts. The "D" and mucoid types may or may not be mutually related. Each has its distinctive growth characteristics and antigenic properties, but these differences are not sufficient to exclude the possibility that the two types may have resulted from the action of bacteriophage or so called "mutation" phenomena upon a single "parent" strain. We have attempted to explain the difference in natural prevalence of "D" and mucoid types in terms of virulence and vegetative capacity (1, a, d). Thus we find that "D" types are at present rarely encountered in rabbits, while the mucoid forms are widespread. The former prove to be highly virulent and slightly vegetative; the latter, less virulent and readily vegetative. The same inverse relationship between virulence and prevalence obtains in human diphtheria (7) and pneumococcus infections (8);-virulent Type I and II pneurnococci and diphtheria bacteria are relatively uncommon in carriers, while the less pathogenic diphtheria forms and Type III and IV pneumococci are widespread. It is possible, therefore, that in these respiratory diseases the virulent types of bacteria are transients, unable to survive in a community except at epidemic times, while the less pathogenic types are more adapted to a parasitic existence in the tissues of a considerable percentage of a population (9). Just what properties of bacteria are responsible for their ability to kill or to survive indefinitely in the surface tissue of an animal has not been determined. We find that physical properties such as agglutinability in acid buffer solutions, stability in suspension, and potential, bear no consistent relationship to virulence and vegetative capacity. Qualitative fermentation and indole reactions, likewise, gave no significant results. Experiments relating to comparative growth rates and nutritional requirements have, however, shown the following differences: Virulent "D" types were found to require a medium with relatively low oxygen tension and these multiplied with extreme rapidity; mucoid types were less exacting and grew more slowly; while "G" variants needed no accessory substances for good growth. Hence, there may prove to be some relation between food supply, metabolic activity, and pathogenicity; bacteria may be parasitic or saprophytic because of their own specific nutritional requirements, and may be virulent in one host and not in another because of chemical differences in the available food. It will be interesting to test the validity of these suggestions by further experiment. We may summarize the results of this investigation as follows. A "D" and a "mucoid" type of Pasteurella organisms have been recovered from rabbits; the former is relatively rare, is virulent, and not vegetative; the latter is very common, less virulent, and readily adaptable to a vegetative existence. The virulence of the different strains of each type appears to be about the same and not to be affected by passage through animals or non-toxic, nutrient media. Although each possesses distinctive and constant characteristics, we feel that the possibility of mutual relationship has not been entirely excluded. Investigation of bacterial properties associated with virulence has as yet given no definite and positive results.