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Divergent views exist concerning the morphology of Bacterium tularense. McCoy and Chapin's (1912) original description depicted an apparently immotile, small, questionably encapsulated, pleomorphic organism occasionally presenting enlarged, irregular, and apparent involution fonns; sometimes with predominant large globular forms. Ohara, Kobayashi, and Kudo (1935) claimed to have demonstrated flagella on both Japanese and American strains, stated that motility was observed repeatedly, and described clubbed, comma-shaped, dumbbell, and triangular forms. They further stated, "The more virulent is the bacterium, the greater is the pleomorphism"; also, "Virulence, pleomorphism, and motion are closely related and vary together." Galli-Valerio (1938), after working with cultures isolated by Drbohlav in Czechoslovakia, stated that both coccoid and bacillary forms were absolutely immotile and that no flagella were demonstrable by the Casares-Gil stain. He also failed to find any enlarged, elongated, filamentous, or involution forms. Most European language reports and textbooks omit all reference to flagella, state that the bacterium is nonmotile, present inadequate descriptions of the extraordinary pleomorphism, give scant mention to encapsulation except as an occasional finding in tissues or in tissue smears, and classify the organism among either Pasteurella or Brucella. Since the most extensive studies on morphology were conducted by Ohara and his associates, we reviewed thoroughly the Japanese literature. Ota (1936) states that Kudo and Kobayashi, working in Ohara's laboratory, first demonstrated a single polar flagellum in 1934, using the silver deposition method of Nishigawa and Sugahara (apparently the same as the Saisawa-Sugawara method mentioned by Ohara). They also observed capsules. Ota confirmed this work. He demonstrated flagella also with Victoria blue (4R), Burri's India ink method, and by his own modification of Benian's Congo red method. In his experience the methods of Loeffler, Benian, Zettnow, Inouye, Yokota, and Uyeno either failed entirely or showed few poorly stained flagella. Under dark-field illumination, "Refractile flagella were demonstrated." With regard to motility, "I found some actively motile, definitely changing their position." Capsules were well demonstrated by Ota's modification of Benian's method, mercurochrome negative staining, and by Gin's India-ink carbol-thionine method. The methods of Johne, Wadsworth, Hiss, Welch, and Friedlander were said to stain them poorly or not at all. Ota stained Bacterium tutarense and Yato-byo bacteria, also their flagella, in tissues with the Levaditi method. Successful preparations were made from human lymph node and skin, guinea pig spleen, and rabbit liver. 1 In partial fulfillment of the thesis requirements for the degree of Doctor of Philosophy.

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Heretofore all attempts to demonstrate protective antibody in antitularense serums have failed whenever animals were challenged with a strain of high virulence. Previous test animals have been the mouse, guinea pig, hamster, and rabbit. Sources of immune or hyperimmune serums were goats, horses, sheep, rabbits, and man: Our considerable unpublished experience with these animals and serums is in good agreement with the reports of Francis and Felton (1) and Bell and Kahn (2). If injected with serum before challenge these animals will usually exhibit significant prolongations of the survival time beyond that of control animals, but no actual survivals against as little as 1 to 10 M.L.D. of a virulent challenge strain.The inability to demonstrate serum protection in these highly susceptible animals has contributed to a widespread but unfounded belief that immune and hyperimmune serums are ineffective therapeutic agents in human tularemia. Although reports by Foshay (3, 4) on the results of serum therapy in individual patients, as well as analyses of accumulated data derived from the independent observations of more than 600 clinical observers, have continued to demonstrate significant reductions in mortality and highly significant reductions in morbidity in comparison with comparable data from untreated patients, universal acceptance of the value of serum therapy has been impeded by a lack of suitable means to demonstrate protective antibody and to establish quantitative criteria for potency. The work here reported was undertaken to supply these deficiencies.The inability of the highly susceptible mouse, guinea pig, hamster, and rabbit to react with any useful degree of resistance before death occurs. following infection with a single virulent unit of Bacterium tularense makes these animals unsuitable test hosts for serum protection experiments.The reasons for this inability, obviously inoperative for many other infectious agents under similar conditions of testing, are unknown at present. The reactions of these animals to invasion are quite unlike that of man, for whom there is ample evidence that the great majority rapidly develop a high degree of resistance, resulting-in a disease that is characterized clinically by a low mortality and, pathologically, by tissue reactions that soon exhibit subacuteness and chronicity.During the course of the initial investigations on this disease, McCoy (5) Challenge strain. The strain of Bacterium tularense used was the highly virulent strain SCHU, which had been studied extensively and had been maintained frequently in animal passage since its isolation in 1941. Its subcutaneous LDIs titers for mice, guinea pigs, and rabbits varied from 9.1 to 9.7/0.5 ml. The LD50 titers were determined by the method of Reed and Muench (6). They are expressed as the logarithms of the dilutions, disregarding the minus sign.Challenge suspensions. Challenge suspensions were prepared from the second of 2 consecutive 16-hour subcultures on glucose glycerol cystine human blood agar. The growth was p...