Experimental Production of Rapidly Fatal Blastomycosis in Mice for Testing Chemotherapeutic Agents

For several years I have used the intravenous route of inoculation to produce rapidly fatal blastomycosis in mice for purposes of testing agents of possible chemotherapeutic value for this disease. Although no substance thus far studied has given evidence of promise in the treatment of this type of experimental infection, the method is described because of the present-~day interest in the field of chemotherapy. For rapid screening of possible chemotherapeutic agents experimental infections with a uniformly fatal outcome within a few days are desirable. Such infections have the limitation that they are acute and do not simulate the natural disease. They can, however, be used to detect the activity of a therapeutic agent having a direct inhibitory effect on the infecting agent. Infections in mice following the intraperitoneal inoculation of Blastomyces dermatitidis have been described by several workers. In many cases a lethal result was irregular and often occurred only after a considerable period (1, 2). Spring (1) found the mouse to be the animal of choice but stated that death was not to be expected. She also noted a difference in virulence for experimental animals of different strains of the organism. Baker (3,4) and Hitch (5) produced uniformly fatal infections in mice by intraperitoneal inoculation of relatively large doses of the organism. The time before death, however, was usually considerably longer and more variable than was the case following intravenous injection of certain of the doses used in the present study. Hitch (5) inoculated several mice intravenously with Blastomyces dermatitidis but abandoned the method because in his experience it always produced immediate death. Such deaths following intravenous inoculation have not been observed in my laboratory. Gilchrist and Stokes (6) produced pulmonary blastomycosis in dogs by intravenous injection of material containing the organism. Following intravenous inoculation of large doses of the organism into rabbits Gutierrez (7) found great numbers of miliary blastomycotic lesions in the lungs. Experiments were undertaken to determine the relationship of dosage to the . time of death following intravenous injection of the yeastlike and of the mycelial phase of the organism. Two different strains of Blastomyces dermatitidis were employed. Strain A had been isolated four years previously from the meninges of a patient who had blastomycosis of the central nervous system and strain B had been isolated three years previously from the skin of a patient who had cutaneous blastomycosis. Both strains had been maintained in the 87 88 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY yeastlike phase since isolation by weekly transfer on slants of dextrose agar containing 10 per cent of horse serum and incubated at 37° C. The mice used in this study were all young adults of the dilute brown strain (dba, subline 212) bred in the laboratory. In subsequent experiments, however, similar results have been obtained with several other strains of mice. Suspensions for inoculation were prepared in nutrient broth. The yeastlike phase was obtained from heavily inoculated three day old cultures on dextrose-serum-agar slants maintained at 37° C. The cells were dispersed by repeatedly aspirating and ejecting the suspension through a Pasteur pipet drawn to a fine point. The mycelial phase was obtained by scraping the mycelium from seven to eight day cultures on dextrose-agar slants incubated at 30° C. Care was taken to avoid the inclusion of agar in this material. A suspension of the mycelium was prepared by gently grinding it in a mortar with a small amount of broth and diluting to the desired density. The number of organisms in each suspension was estimated by inoculating 1 ml. of each of a series of tenfold dilutions into dextrose-agar pour plates, incubating these plates at 30° C.for seven days and counting the colonies on plates containing from twenty to 200 colonies. The significance of the colony counts of the suspensions prepared with the mycelial phase was subject to the limitation that the number of separate viable elements determined by plating would vary with the degree of fragmentation of mycelium resulting from grinding in the mortar. Various dilutions of the primary suspensions in nutrient broth were prepared for inoculation. Each dilution was injected intravenously into the tail veins of several mice (usually four) in 0.1 ml. amounts, using a tuberculin syringe and a 27 gauge needle. Since the organisms in the suspensions tended to settle rapidly, the tube containing material for inoculation was shaken previous to the inoculation of each mouse, the syringe immediately filled and the material injected at once. In each experiment a dose as large or twice as large as the largest dose given intravenously was administered intraperitoneal to several mice to compare the death time of two different routes of inoculation. Data on four experiments are summarized in table 1. It is evident that the time of death following intravenous inoculation was related to the dose. Even a small number of organisms administered by this route caused death. In experiments not listed in table 1 the intraperitoneal inoculation of small doses of the two strains used in this study did not cause any symptoms in mice. The intraperitoneal injection of relatively large doses of the organism, as shown in table 1, usually gave a fatal result, but the interval between inoculation and death was much longer and usually more variable than when a similar dose was given intravenously. It is evident that both the mycelial and the yeastlike form were virulent for mice. After inoculation by either the intravenous or the intraperitoneal route there were no immediate symptoms. In the case of the intravenously inoculated mice rapid and labored breathing was regularly observed from one to several days before death. RAPIDLY FATAL BLASTOMYCOSIS IN MICE 89 Necropsy was perforated on every mouse. In mice dying within a short period following intravenous injection of large doses of the organism the lungs were found to be affected with a massive embolic pneumonia which had converted them into a firm, gray, granulomatous mass. Their surfaces were granular and there was a small amount of clear, sticky pleural exudate. Sections of such lungs revealed numerous yeastlike forms of Blastomyces dermatitidis. Examination of the livers, kidneys and spleens of such mice rarely revealed TABLE 1 Effect of size of dose on time of death following inoculation of Blastomyces dermatitidis into mice STRAIN OF PHASE OF FUNGUS ROUTE OF INOCULATION ORGANISMS MICE INOCU- MICE THAT DAY OF FUNGUS LATED DIED A Yeastlike Intravenous 184,000 5 5 7-8 A Yeastlike Intravenous 92,000 4 4 7-12 A Yeastlike Intravenous 46,000 4 4 9-13 A Yeastlike Intravenous 9,200 4 4 11-15 A Yeastlike Intravenous 920 4 4 16-18 A Yeastlike Intravenous 92 4 4 23-32 A Yeastlike Intravenous 9 3 3 33-42 A Yeastlike Intraperitoneal 184,000 10 10 32-58 A Mycelial Intravenous 706,000 4 4 7-8 A Mycelial Intravenous 353,000 4 4 10-12 A Mycelial Intravenous 35,300 4 4 18-20 A Mycelial Intravenous 3,530 4 4 25-35 A Mycelial Intraperitoneal 706,000 4 4 27-33 B Yeastlike Intravenous 68,000 4 4 5-8 B Yeastlike Intravenous . 6,800 4 4 11-13 B Yeastlike Intravenous 680 4 4 15-17 B Yeastlike Intravenous 68 4 4 23-28 B Yeastlike Intraperitoneal 136,000 4 4 17-25 B Mycelial Intravenous 14,000 4 4 10-17 B Mycelial Intravenous 1,400 4 4 19-20 B Mycelial Intravenous 140 4 4 24-32 B Mycelial Intravenous 14 1 4 1* 38 B Mycelial Intraperitoneal \ 28,000 1 4 2* 28-40 * Surviving animals killed on the fifty-third day after inoculation. gross lesions but microscopic lesions were present fairly frequently. It was evident that the mice died because of the extensive pneumonic involvement. In mice dying at long intervals after the intravenous injection of small doses of the organism discrete large tubercles were present in the lungs. With the small doses few organisms were deposited in the lungs and death did not occur until the infected foci had grown to a degree that caused death from respiratory insufficiency. In such mice grossly visible tubercles of blastomycosis were occasionally present in the liver, kidneys or spleen. In mice dying following intraperitoneal inoculation there was usually exten- 90 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY sive growth of blastomycotic tubercles on the peritoneal surfaces with frequent involvement of the abdominal viscera and the lungs. Since Spring (1) has noted differences in virulence of different strains of Blastomyces dermatitidis for mice it is considered likely that not all strains of the organism may kill mice with the regularity of the strains used in the present study. The process following intravenous injection of Blastomyces dermatitidis into mice appears to be similar to that produced by the intravenous administration of Mycobacterium tuberculosis to this animal. The intravenous inoculation into mice of large doses of virulent human strains of Mycobacterium tuberculosis results in fatal infections due to extensive involvement of the lungs (8). Parsons (9) produced fatal infections in young mice by the intravenous injection of very large doses of Histoplasma capsulatum. In such infections the disease process was generalized and an outstanding feature was striking enlargement of the spleen, a condition not observed in experimental infections with blastomycosis in mice. SUMMARY Uniform and rapidly fatal infections in mice have been produced by the intravenous injection of the yeastlike and of the mycelial form of two different strains of Blastomyces dermatitidis. The time of death was directly related to the number of organisms administered. Death was due to embolic pneumonia.