INVISIBLE MICROORGANISMS.

By M. DORSET, M. D.,

Assistant Chief of Biochemic Division, Bureau of Animal Industry.

A REVIEW OF THE SITUATION.

Fifty years ago the nature of infectious diseases was just beginning to be understood. The then existent beliefs concerning the origin of infectious diseases rested almost entirely upon a theoretical basis, and it was not until the beginning of the era of experimental medicine, in which movement Pasteur was the great leader, that any headway was made in the solution of those problems which were finally so brilliantly worked out by Pasteur and his collaborators of that period. The existence of bacteria and other microscopic forms of life had been recognized for more than a century when, in 1850, Pollender and Davaine made their observations upon anthrax, their work being the first experimental evidence of the fact that certain forms of bacteria occupied a causal relation to particular infectious diseases.

During the period between 1850 and 1875 the controversy regarding the spontaneous generation of microorganisms was the all-absorbing topic among biologists, and although this debate did not include the etiology of infectious diseases it involved questions, such as the cause of fermentation, which were so closely allied that the methods employed in the study of the one were in great part applicable to the other.

Thus it was that the foundations of our present-day bacteriological technique were laid, and when, in 1881, Koch gave to the workers in bacteriology his solid-culture media and his "plate method," the principles of sterilization and the methods of staining were already well understood. It is no wonder, therefore, that the decade following Koch's announcement of the "plate method" of isolating bacteria in pure culture was notable for the great advances made in experimental medicine, and especially in that branch of it which concerns the etiology of infectious diseases.

Notwithstanding the many notable advances in that period, however, the causative agents of a certain number of human and animal diseases completely escaped discovery. New and supposedly favorable culture media were prepared; microscopes were improved in every detail of construction; new staining methods were devised; and, in fact, everything was done which might aid in determining the infectious agents in such diseases as measles, scarlatina, smallpox, rabies, bovine pleuro

pneumonia, foot-and-mouth disease, rinderpest, and others of almost equal importance, and yet, as has just been stated, even at the present time we are almost entirely in the dark with regard to the etiology of the above-mentioned diseases.

In the year 1898, however, a number of important observations were made which were of such a nature that they seemed to give us a glimpse of an unknown world of infinitesimally small creatures whose existence had previously only been surmised.

It is with the idea of describing somewhat briefly the original discoveries and those which have followed them that this paper is written.

FOOT-AND-MOUTH DISEASE.

Foot-and-mouth disease is one of the most contagious maladies known to veterinary science, and has caused enormous losses to stock owners in Europe. Primarily it affects cattle, though few, if any, of the domestic animals are exempt, and is characterized by fever, loss of appetite, and emaciation, the last two symptoms being in large part due to the development of vesicles in the mouth, which cause swelling and tenderness of the buccal mucous membrane. Vesicles appear on the udders of cows and on the skin surrounding the hoofs. The mortality from this disease among affected cattle probably averages 1 or 2 per cent, though it may be very much greater. The chief loss, however, is to be noted in the general condition of the herd, which is, as a rule, 30 per cent poorer than before the attack of the disease.

In 1898 Loeffler and Frosch presented to the German Government a report of the results which they, as a commission, had obtained from their investigations of foot-and-mouth disease. They showed that the fluid contained in the buccal vesicles of affected cattle was extremely infectious, and that a very small amount of such material, when in a fresh state, sufficed to bring about typical attacks of foot-and-mouth disease in cattle and hogs, and this notwithstanding the fact that they were unable, even after the most careful search, to ascertain the existence of any microorganism whatever in certain lots of this fluid, either by staining or by culture experiments. Knowing, however, by experience that the infectious agent was present, and being desirous of obtaining some method of producing immunity, they conceived the idea that the filtered lymph from an animal in the acute stage of footand-mouth disease might be used for this purpose with advantage, the object of the filtration being to separate all the corpuscular elements from the soluble portion, the latter to be used for the production of immunity. With this idea in view, therefore, a certain quantity of virulent lymph, to which was added 39 parts of water, was passed repeatedly through a filter (Berkefeld) of infusorial earth. Before the filtration, however, a considerable amount of a culture of Bacil

lus fluorescens was added to the diluted virus in order that that bacillus might serve as a check on the efficacy of the filter. The filtrate was repeatedly tested, but at no time did B. fluorescens or any other microorganism which could be recognized by cultural methods appear.

A number of calves were next inoculated with quantities of the filtrate which were the equivalents of from one-tenth to one-fortieth cubic centimeters of the original lymph. To the very great surprise of the experimenters, the calves that were inoculated with the filtered lymph sickened in the same length of time as did the control animals, which received a corresponding amount of unfiltered lymph, and, in addition, they exhibited all the typical symptoms of foot-and-mouth disease, such as high fever and vesicles in the mouth and on the feet. In order to confirm, if possible, this very unexpected occurrence, the experiments were repeated a number of times on calves and hogs, and always with the same result when fresh lymph was used. Here, then, was possibly a new fact for bacteriologists to reckon with, for Loeffler and Frosch had in their filtrate either an extremely powerful toxin or an organism which passed regularly through Berkefeld filters, which were fine enough to hold back B. fluorescens.

In order to show that they were not dealing with a toxin, Loeffler and Frosch compared the disease-producing power of their lymph with the most powerful tetanus toxin that had ever been reported, and found, according to calculations based upon the body weight of the animals and the amount of virus used, and taking into consideration the transference of the disease from an animal treated with filtered lymph to others, that if the disease produced by the filtered lymph was due to a toxin it was by far the most powerful ever known, its toxic value being 1:2,500,000,000,000. Such an extremely toxic substance is inconceivable, and, besides, we know of no disease which may be induced by one-fiftieth cubic centimeters of toxin and then be carried successively through six animals, using continually the same minute dose. It would therefore seem to be reasonably certain from the original experiments of Loeffler and Frosch that they had a living microorganism in their filtrates, and that this microorganism passed through the Berkefeld filter. More recently Loeffler has stated that, while the virus of foot-and-mouth disease passes through Berkefeld filters, it is retained by the finer-pored Kitasato filter, thus definitely disposing of the idea that the infecting substance may be in solution.

BOVINE PLEURO-PNEUMONIA.

Within a very short while after the publication of the researches of Loeffler and Frosch, the paper of Nocard and Roux upon the etiology of bovine pleuro-pneumonia appeared. The work of these French savants, done in collaboration with Borrel, Salimbeni, and Dujardin

upon the rabbits. Those rabbits which received the sacs inoculated with pleuro-pneumonia became emaciated, some of them even dying on the fifteenth or twentieth day. Those rabbits which received uninoculated sacs of bouillon remained perfectly well. As rabbits are entirely refractory to inoculations with the virus of pleuro-pneumonia made in the ordinary way, this experiment seems to show that they may be susceptible to the soluble toxins generated by this microorganism, although they are capable of overcoming the living virus, provided it is not protected in some such way as by the collodion sac. In other words, we have in the behavior of the rabbit toward pleuropneumonia an instance of an animal which is immune from the body of the microorganism, but susceptible to its toxins.

In a paper published about a year later Roux and Nocard announced that they had been able to filter the diluted pleuro-pneumonia virus through a Berkefeld, and also through a Chamberland F cylinder, but were unsuccessful in their attempts to filter it through a Chamberland B, thus indicating that an organism need not necessarily be ultra: microscopic because it will traverse with regularity a Chamberland filter and a Berkefeld filter, they having previously shown by their successful cultures that the pleuro-pneumonia virus is just within the limits of visibility.

Since the researches upon pleuro-pneumonia and foot-and-mouth disease were published there have been many efforts to apply the principles there introduced to other diseases which had baffled all previous attempts to determine their cause. The majority of such investigations dealt with animal diseases, for the reason that they could be reproduced experimentally when it was desired to do so.

YELLOW FEVER.

First of all, concerning yellow fever, mention should be made of the epoch-making experiments of Reed, Carroll, and their associates. These investigators had already shown by a series of daring and conclusive experiments that yellow fever is transmitted only through the bite of a mosquito (genus Stegomyia), when, a little later, they announced the results of some filtration experiments with a Berkefeld laboratory cylinder. A certain amount of blood was drawn from the elbow vein of a man who was suffering from a mild attack of yellow fever. The serum which separated from the clot was diluted with an equal volume of sterile distilled water and filtered slowly through a Berkefeld laboratory filter which had been previously sterilized. The clot which remained was beaten up with distilled water. One nonimmune man inoculated with 0.75 c. c. of the aqueous extract of this clot developed a mild but typical attack of yellow fever. Three nonimmunes inoculated with double the amount of the same material, except that it had been previously heated to 55 C. for ten minutes, remained perfectly well. These two experiments showed that the H. Doc. 743, 58-2—10