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A simple technique is described for maintaining the life of adult schistosomes in vitroover a period of several weeks which, with frequent changes of medium, may be extended to 2½months. This is made possible by the use of small tissue culture flasks, which prevent bacterial contamination and desiccation of the media. The latter may be either horse, sheep or rabbit sera or human ascitic fluid.

The lethal action of sodium antimonyl Tartrate, Fouadin, Urea-stibamine and Meostibosan on Schistosoma japoni-cumis studied in vitroand the results support the prevalent belief that the trivalent antimony compounds are more effective in the treatment of schistosomiasis than pentavalent salts and our own clinical experience that permanent cure of Schistosomiasis japonicaresults more readily with tartar emetic than with fouadin.

The results in the experimental transmission of kala-azar thus far have been uniformly negative.1, 2Shortt, attempting transmission by means of sandflies, attributes his negative results to insufficient susceptibility of the experimental animals rather than to inability of the fly to expel the flagellates from its buccal cavity into the wound while feeding. We are inclined to believe that the negative results might have been due to the overlooking of one natural factor not previously considered, namely, the crushing of the fly by slapping the itching skin, where the fly is feeding. To ascertain the importance of this factor in the transmission of kala-azar, a method has been worked out, imitating as far as possible the natural conditions under which the flies feed and are crushed.The sandflies, P. major, are fed with the blood-parasite mixture on the feeding stage within 24 hours after hatching,3and are then kept in the ice box at 21° C. After 6 to 10 days, they are allowed to refeed and, while feeding, are crushed by slapping. This is occasionally done as late as the 18th day, if the flies have had a second feeding.1. The feeding stage.This stage is like the one for the blood-parasite mixture, except that the dish is only 1/2 inch in diameter. The flannel is cut a little smaller than the dish and is cut in 2 equal halves. It is then pressed and saturated with defibrinated rabbit blood, and covered with a piece of skin from a normal hamster. Strictest asepsis is necessary in these preparations, but the heavily contaminated external surface of the skin is not sterilized for reasons which are given below. The prepared feeding stage is placed on the electric heater set at 35° C., and the slapper is adjusted to the skin surface.

In the experimental attempts to transmit kala-azar by the bites of sandflies, it has been found desirable to substitute an artificial feeding on blood rich in Leishmann-Donovan bodies for the natural feeding upon infected animals, in order to obtain an abundant growth of flagellates within the flies. This blood is obtained by mixing the blood of a normal animal with the crushed spleen of a hamster heavily infected with kala-azar.For such artificial feeding we used the technique of Hertig and Hertig.1This technique was learned in a few days and in the course of 2 months during the summer of 1928, 565 flies were fed. During this work it became clear that this technique had a number of limitations. The flies had to be fed one by one, making the procedure tedious. During the etherization and handling of the flies, many were injured. A satisfactory engorgement was obtained with P. sergenti, while P. majoronly in rare instances could be fully engorged. In order to feed a larger number of flies with a minimum of injury and a maximum of engorgement, the following technique has been devised.Technique.Etherize a hamster heavily infected with kala-azar, pin its feet on a wooden board, and place its head and neck between the arms of a U-shaped wooden block. Remove the hair over the anterior surface of the neck and chest by pulling and sterilize the skin with alcohol and ether. With aseptic precautions, make a median incision through the skin extending from the anterior end of the lower jaw to the middle of the sternum. Pull apart the skin flaps and pin them to the arms of the U-shaped block, the upper surface of which is slightly raised above the level of the head and neck. Cut the carotid artery on one side and, as the blood wells up, quickly draw it into a sterile pipette. Practically all the blood that is obtained by bleeding can be drawn into the pipette. Defibrinate and centrifuge the blood.

1. In normal Chinese hamsters it was found that the maximum non-lethal doseof neostibosan is 1.0 g per kilo body weight; the maximum tolerated dose, 3.0 g per kilo body weight; and the universal lethal dose, 4.0 g per kilo body weight. 2. The sex and body weight of hamsters played no significant rôle. The maximum non-lethal dosewas found universal to animals weighing from 11 to 40 g. 3. There was no increase of tolerance after repeated injections of gradually increasing doses of neostibosan. A cumulative effect was clearly shown. A probable therapeutic dosewas found to be about 0.4 g per kilo body weight. 4. After an animal had been poisoned by a large dose of neostibosan, the repetition of the same or greater amount even after an interval as long as 2 months usually resulted in death.

Chinese hamsters infected with kala-azar seemed to have a lower tolerance for neostibosan than normal. hamsters, but the difference was not statistically significant. Five to 7 g of neostibosan per kilo body-weight constituted a course of treatment. The range of the effective dose was from 200 to 500 mg per kilo. The mortality rate during treatment was 31.6%. The apparent cure-rate in hamsters surviving the treatment was 100%, but the actual cure-rate was only 50%. An inoculation test is necessary to prove actual cure. Nine out of 14 apparently cured hamsters were refractory to a second inoculation with Leishmania donovani. Spontaneous recovery from and relapse of kala-azar were seen in Chinese hamsters.