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Termination of pregnancy occurred in the rat after infection with Trypanosoma lewisi early in gestation. Rats were inoculated on Day 2 of pregnancy and the uteri were excised and examined on Days 10 and 14 of gestation. There were no detectable differences in size in the fetuses of infected and control females at Day 10, but by Day 14 young of infected females were being reabsorbed, and their weight was markedly less than that of control young. A bioassay of estrogens and progestogens based on the decidual cell response indicated that there was a sufficient hormone level to maintain pregnancy beyond Day 10, so the mechanism of action by which fetal death was produced did not appear to be hormone depletion. The crucial changes in fetal weight occurred between Days 12 and 14 of gestation in rats infected with T. lewisi on Day 2 of gestation. Trypanosoma cruzi caused little or no pathologic change in the pregnant laboratory rat throghout the period of gestation and infected females gave birth to apparently normal young.

A short review is given about recent observations of infections in pregnant laboratory mice with strains of Trypanosoma musculi (= T. duttoni ) isolated in southern Europe. When being infected during the highly sensitive period of mbryogenesis, the female is completely unable to inhibit the reproduction of the trypanosome. Like an excessively growing benign tumor, the normally well-adapted nonpathogenic blood parasite population becomes, under these conditions, harmful to the host. The placenta acts as a “focus” of the trypanosome reproduction. Unusual, incomplete multiple fissions occur as big rosette formations in the placental blood. The possible microecological conditions of the accelerated trypanosome growth are briefly discussed. An American strain of this trypanosome species shows the same behavioral pattern in pregnant hosts.

A study has been made of changes in alcohol dehydrogenase (ADH) isozymes in livers of rats infected with the following trypanosomes: Trypanosoma cruzi, T. rhodesiense, T. congolense, and six geographical isolates of T. lewisi. A control group of the same age was injected with saline, and another left uninjected. Liver homogenates were electrophoresed in noble agar gels followed by formazan staining according to the method of Ursprung and Leone (1965). Of a total of 25 replicates, 15 showed that the level of rat liver ADH isozymes was similar in the uninjected and saline injected animals; 6 replicates showed a slightly higher level of ADH isozymes in uninjected than saline injected control rat livers, and 4 replicates had a higher level in saline injected than in the uninjected. With either 1-octanol or ethanol as substrate, a marked elevation of ADH activity was observed beginning with day 7 post inoculation in rat livers infected with T. cruzi, T. lewisi, and T. congolense, or as early as day 4 post inoculation with T. rhodesiense. The elevation persisted 59 days or more in rat livers infected with T. lewisi, but rats infected with the pathogenic species usually died 5 to 14 days after infection. The elevation of ADH isozymes has also been demonstrated spectrophotometrically for the first 5 minutes of reaction time, using acetaldehyde as substrate. Also observed in livers infected with pathogenic species and certain strains of T. lewisi were: (1) ADH isozyme pattern changes involving an elevated level of certain isozymes only, and (2) abnormal isozyme mobilities.

The protozoa Crithidia and Trypanosoma contain within a mitochondrion a mass of DNA known as kinetoplast DNA (kDNA) which consists mainly of an association of thousands of small circular molecules of similar size held together by topological interlocking. Using kDNA from Crithidia acanthocephali, Crithidia luciliae, and Trypanosoma lewisi, physicochemical studies have been carried out with intact associations and with fractions of covalently closed single circular molecules, and of open single circular and unit length linear molecules obtained from kDNA associations by sonication, sucrose sedimentation, and cesium chloride-ethidium bromide equilibrium centrifugation. Buoyant density analyses failed to provide evidence for base composition heterogeneity among kDNA molecules within a species. The complementary nucleotide strands of kDNA molecules of all three species had distinct buoyant densities in both alkaline and neutral cesium chloride. For C. acanthocephali kDNA, these buoyant density differences were shown to be a reflection of differences in base composition between the complementary nucleotide strands. The molar ratios of adenine: thymine:guanine:cytosine, obtained from deoxyribonucleotide analyses were 16.8:41.0:28.1:14.1 for the heavy strand and 41.6:16.6:12.8:29.0 for the light strand. Covalently closed single circular molecules of C. acanthocephali (as well as intact kDNA associations of C. acanthocephali and T. lewisi) formed a single band in alkaline cesium chloride gradients, indicating their component nucleotide strands to be alkaline insensitive. Data from buoyant density, base composition, and thermal melting analyses suggested that minor bases are either rare or absent in Crithidia kDNA. The kinetics of renaturation of 32P labeled C. acanthocephali kDNA measured using hydroxyapatite chromatography were consistent with at least 70% of the circular molecules of this DNA having the same nucleotide sequence. Evidence for sequence homologies among the kDNAs of all three species was obtained from buoyant density analyses of DNA in annealed mixtures containing one component kDNA strand from each of two species.

1. 1. Treatment of rats with the diabetogenic agents alloxan and streptozotocin caused a significant increase in populations of T. lewisi when hyperglycemia was moderate, but not in severe hyperglycemia. 2. 2. Greater trypanosome parasitemias were typically seen in insulin-treated rats, and leukocyte counts of the treated animals were higher than controls. 3. 3. Pharmacological doses of phenformin also caused significant increases in peak numbers of circulating trypanosomes, even though this oral hypoglycemic did not reduce blood sugar levels as effectively as insulin. 4. 4. Although glucagon injections produced only transient hyperglycemias, rats treated with this hormone displayed significantly greater numbers of circulating T. lewisi than comparable controls. 5. 5. In vitro studies of T. lewisi isolated 6 days post-infection indicated that insulin, glucagon and phenformin treated organisms apparently consume more glucose than do controls. 6. 6. Insulin may also cause decreased lactate production in T. lewisi .

Contrary to the reports of others, the surface coat of Trypanosoma brucei brucei was not removed by extensive washing in the various media investigated; in fact, other than a deformed profile when washed in saline, ultrastructure was little affected by column separation and washing in any of these media. Washing in saline increased the agglutinability but reduced the activity and infectivity of the organisms. Washing in bicine-buffered saline-glucose did not impair activity or infectivity but increased agglutinability, albeit to a lesser extent than after washing in phosphate-buffered saline-glucose. The inclusion of 0.1% serum or plasma in the washing medium (phosphate-buffered saline-glucose or bicine-buffered saline-glucose) increased the activity of the T. b. brucei and did not increase agglutinability or impair infectivity. T. congolense and T. vivax were more susceptible to ionic strength changes than were T. b. brucei or T. lewisi, in that not only was their activity impaired, but they began to aggregate on standing in lower ionic strength buffers.

Ablastin formed by animals in response to infections by rodent trypanosomes possesses the characteristics of an antibody. Partial resistance to Trypanosoma lewisi is demonstrable in animals previously injected with live Trypanosoma musculi . Antisera from T. musculi infected mice do not inhibit reproduction by T. lewisi bloodstream forms in vitro as efficiently as homologous antisera collected at similar times during infections, indicating a degree of specificity. Ablastin activity in antisera is not altered by treatment with 2-mercaptoethanol or by heatng at 60 °C for 3 hr. Sephadex G-200 gel filtration of early and late antisera from T. lewisi infected rats and assays with bloodstream forms cultured at 37 °C detect ablastin activity in the second major fraction eluted from the columns. Ablastin appears to be an antibody of the immunoglobulin G species. Immunosuppressant procedures utilized in studies of the host responses to rodent trypanosomes are reviewed and include: chemical agents, irradiation, splenectomy, reticuloendothelia blockade and thymectomy, and treatment with antilymphocyte and antithymocyte sera. Evaluation of the results of the application of these procedures to rodent parasite systems indicates ablastin is an antibody and supports the concept that the inhibition of trypanosome reproduction is separate and distinct from the first trypanocidal event responsible for the decreasing parasitemias observed during the infections. Recent studies concur and suggest that the first crisis in the infections is mediated by the combined actions of a thymus-dependent ablastin and a thymus-independent trypanocidal antibody.

Host responses to infection were examined by observing the ultrastructural changes in liver from rats infected withTrypanosoma lewisi from day 1 to day 49 of infection. Trypanosomes were present in large numbers in the sinusoids on the 7th day of infection. By day 14, trypanosomes were seen in kupffer cells. The endoplasmic reticulum showed distended cisternae from day 4 of infection to day 14 of infection. Mitochondria in liver from infected rats increased in number and were swollen only on day 7 of infection. The hepatocytes showed focal areas of lipid from day 7 to day 28 of infection. Residual lysosomes in kupffer cells were found from the 14th to 21st day of infection. Initially, glycogen was dispersed but as the infection advanced, it became aggregated by day 7 of infection. By the 49th day of infection, glycogen again appeared dispersed. At the 35th, 42nd and 49th days of infection, the appearance of the liver was similar in general to that of normal liver.

No protection against Trypanosoma cruzi was afforded to mice by previously inoculating multiple doses of T. lewisi blood stream forms, strain RU or culture forms of the IMT strain. Since these results conflicted with those obtained by other authors, we secured a sample of the strain used by them. This strain, labeled “ T. lewisi ” and which we called “F”, was verified to be actually T. cruzi . It was characterized by its morphologic features in axenic cultures, its capability to infect HeLa cells where the intracellular cycle was completed, by its infectivity for white mice which had amastigotes in their heart muscle, and in which the strain has been maintained by serial passages, and by the completion of its biologic cycle in triatoma bugs. The necessity for carefully testing doubtful strains, and the criteria to be adopted in these cases are discussed.

1. 1. Folic acid deficiency was studied in rats infected with Trypanosoma lewisi and Trypanosoma rhodesiense. 2. 2. Irrespective of the day of inoculation, folic acid-deficient and pair-fed control rats showed greater parasitemias than complete diet rats throughout the infections. 3. 3. The average coefficients of variation in body length of T. lewisi cells indicated that the formation of the reproduction-inhibiting antibody (ablastin) was delayed 11 to 16 days in deficient animals compared to pair-fed and normal control animals. 4. 4. In all dietary groups, regardless of the time of inoculation, coefficients of variation in body length of T. rhodesiense cells ranged from 17 to 31.

The value of comparing processes in related species is emphasized; by this approach it is possible to consider the ablastin phenomenon, not as a unique process, but as having metamorphosis evolutionary affinity with conversion factors in other species of trypanosomes.

1. 1. Boby-weight gains and food consumption were studied in folate-deficient rats infected with Trypanosoma lewisi. 2. 2. Body-weight gains in infected rats, inoculated on Day 21, and fed complete diets showed a 2–64% increase over uninfected rats; those of infected rats fed a folate-deficient diet showed an increase of 1%–60%. 3. 3. Body-weight gains in infected rats, inoculated on Day 56, and fed complete, pair-fed and deficient diets ranged up to 44%, 47% and 27% respectively. 4. 4. Irrespective of diet or day of inoculation, infected rats showed statistically significant differences in food intake when compared with uninfected controls.

Ten experiments were performed to compare mitotic activity in livers of rats infected with Trypanosoma rhodesiense and Trypanosoma lewisi. The host dietary groups were: (1) complete of full complement; (2) folic acid deficient, and (3) pair-fed or calorically restricted. Liver mitotic counts of T. lewisi-infected rats fed complete and pair-fed control diets showed a high degree of significance over that of control rats. From days 35 to 55, liver mitotic counts of T. lewisi-infected rats deficient in folic acid showed an average of 16 +/- 2 mitotic phases as compared to 9 +/- 2 for livers of uninfected animals. Liver mitotic counts of T. rhodesiense-infected and non-infected rats fed complete and pair-fed control diets showed that there were essentially no differences in the mitotic counts from that seen in liver from control, uninfected animals. On day 25 (day 5 of infection), folic acid-deficient infected rats showed a significantly higher number of dividing cells than the controls. Folate was measured in isolated, washed T. lewisi and T. rhodesiense cells. T. lewisi and T. rhodesiense contained 9.6 ng folate per 10(8) cells and 6.2 ng of folate per 10(8) cells respectively. Metabolic products of T. lewisi contained an average of 0.51 ng of folate per 10(8) trypanosomes. T. rhodesiense metabolic products contained 0.3 ng of folate per 10(8) trypanosomes.

Progressive changes in folic acid levels in sera and tissues of rats infected withTrypanosoma lewisi are described. The host dietary groups were: (1) complete or full complement; (2) folic acid-deficient, and (3) pair-fed or calorically restrieted. Serum folate values of rats on a folic acid complete diet and pair-fed diet and inoculated on Day 21, showed significantly higher values than the controls between Days 35 and 45. The average serum folate values in adequately fed control rats was 16±3 ng/ml as opposed to 20±3 ng/ml for trypanosome-infected rats. Animals inoculated on Day 56 had higher folate values than control rats from Day 65 to Day 80. Serum folate content of deficient rats infected on Days 21 and 56 was significantly higher than the controls on Days 30 to 60 and 60 to 100, respectively. The livers of rats fed a folic complete and a pair-fed diet and inoculated on Days 21 and 56 withT. lewisi showed no significant difference in folate content when compared with the uninfected rats. Livers of folic acid-deficient rats infected withT. lewisi on Days 21 and 56 showed statistically significant increases over the controls from the 10th day of infection. Infected animals had 200% more folate than controls. Irrespective of diet and time of inoculation (Day 21 or 56), the folic acid content of brains and spinal, cords of rats infected withT. lewisi were not significantly different from that of the controls.

A study has been made of erythropoietic and immune responses in rats subjected to a simulated altitude of 17,000 ft and infected with Trypanosoma lewisi. Observations were also made during the period of rebound (posthypoxic polycythaemia). The erythropoietic response was evaluated on the basis of reticulocyte and hematocrit values in peripheral blood, and the immune response on the basis of the level of parasitemia. When hypoxia and infection were combined, the infection was heavier than in the nonhypoxic animal, as judged by the level of the parasitemia, whereas the erythropoietic response to hypoxia was less intense. Rebound animals, on the other hand, dealt with the infection more effectively, as indicated by the lower level of parasitemia, and the more rapid disappearance of parasites from the blood.

1. 1. Glucose-6-phosphatase, glycogen and protein changes were studied in rats given living cells, cell derivatives (metabolic products, homogenates) of Trypanosoma lewisi and Trypanosoma rhodesiense . 2. 2. Marked reductions in glucose-6-phosphatase and glycogen were directly related to the degree of parasitemia. No essential differences in glucose-6-phosphatase or glycogen were seen in animals inoculated with physiological saline or derivatives of trypanosomes when compared with uninoculated controls. 3. 3. Liver and blood protein levels were greatly reduced in rats inoculated with living cells and derivatives of T. lewisi but not in animals inoculated with physiological saline. 4. 4. The livers of rats inoculated with living cells of T. rhodesiense showed statistically significant reductions in protein content when compared with the uninfected rats. The protein content of livers of rats inoculated with physiological saline and derivatives of T. rhodesiense were not significantly different from that of the controls.

A brief report is made of a conference held on the subject of ablastic immunity. Included are the background of the meeting, its organization, and its purpose, which was to summarize our knowledge of the subject and to consider future research plans. A list of some of the research problems discussed by the participants and possible experimental approaches for their resolution are presented. The report also serves as an introduction to a series of papers by the participants of the conference published concurrently.

Autoradiography of Trypanosoma lewisi labeled in vivo with 3H-thymidine (3HTdR) shows that the shortest doubling time for labeled organisms is 8 hr in intact and immunosuppressed rats. The parasite doubling time increases progressively after the fourth day of infection to 12 hr in immunosuppressed rats and to 24 hr or more in intact rats. The number of days following infection during which the trypanosomes reproduce is prolonged in immunosuppressed rats. In vitro studies of ablastin using 3HTdR-labeled trypanosomes confirmed that cell reproduction halts in the presence of ablastin, but resumes when the parasites are removed from the antibody. Several lines of evidence have been obtained, indicating that the primary effects of ablastin may be on membrane function. Thus, the saturable component for glucose transport in reproducing and ablastin inhibited trypanosomes has an average Km value of 2.8 × 10−4M, but the average Vmax values for glucose transport are reduced from 3.15 nmole/min/1.25 × 107 reproducing parasites to an average of 1.8 nmole/min/1.25 × 107 nonreproducing forms. Glucose transport is competitively inhibited by 2-deoxyd-glucose (2DOG). The exit and counterflow of 16C-2DOG from previously loaded trypanosomes is restricted in the presence of antiserum.

1. 1. Chromatographic and chemical analyses show that up to four major sterols may be present in blood and culture forms of trypanosomes. 2. 2. Blood and culture forms of Trypanosoma lewisi and culture forms of T. rhodesiense biosynthesize C(24)-alkyl sterols de novo. In these trypanosomes, and in blood forms of T. rhodesiense, exogenous cholesterol is incorporated and esterified, but not further metabolized. 3. 3. Changes in sterol composition and metabolism occur in T. lewisi during “maturation” in vivo, and during in vitro culture. 4. 4. The role of sterol biosynthesis in cyclical mitochondrial biogenesis in trypanosomes is discussed, and also the evolutionary significance of the phytosterols identified in this study.

SYNOPSIS. Previously, the reproduction-inhibiting effects of ablastin could only be shown in vivo. The present report describes techniques for the in vitro demonstration and titration of this antibody. With a medium composed of Hanks' balanced salt solution, rat serum, lactalbumin hydrolysate, yeast extract and rat blood lysate, blood stream forms of Trypanosoma lewisi can be grown for approximately 24 hours at 37dC. Starting with the medium containing normal rat serum and inoculated with adult (inhibited) trypanosomes from infected rat blood, 50% or more of the parasites are in various stages of division after incubation overnight. Under similar conditions with ablastic rat serum, the parasites do not reproduce but remain as adults. If the medium is inoculated with reproducing trypanosomes from the blood, parasites in the presence of normal serum continue to reproduce, whereas those exposed to ablastin are almost completely converted to adult, non-reproducing forms. Similar results are obtained when the immune sera used are first adsorbed with living parasites to remove all trypanocidal antibodies. Ablastic serum inactivated at 56dC for 20 minutes does not lose its inhibitory activity indicating that ablastin is not complement dependent, and parasites grown on media at room temperature are not affected by the antibody suggesting that basic antigenic differences exist between blood stream forms at 37dC and culture forms at room temperature. Studies of the conversion of blood stream forms to culture forms indicate that the critical temperature range for the conversion lies between 28d and 30dC. The significance of these results is discussed, and possible applications of the techniques described to studies of the mechanism of ablastic action are considered.

(1910). Observations on the Life History of Trypanosoma Lewisi in the Rat Louse (Haematopinus Spinulosus) Annals of Tropical Medicine & Parasitology: Vol. 3, No. 5, pp. 553-564.

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An electron microscope study of a variety of invertebrate and vertebrate cell types has supported the postulate that the microtubule is a universal cellular organelle. Microtubules of similar dimensions have been observed in the flagellum and beneath the plasma membrane of Trypanosoma lewisi, in the flagellum, manchette and mitotic spindle of the earthworm (Lumbricus terrestris) spermatid; and in fibroblasts, proximal convoluted and collecting tubule cells of the hypertrophying rat kidney. The specific occurrence and organization of the microtubules in cells undergoing morphological and developmental changes have suggested that these organelles are contractile and that they effectively contribute to the maintenance of cellular form. The possibility that microtubules may function as an intracellular transport system is also suggested.

1. The morphological and developmental anomalies of a pathogenic strain of Trypanosoma lewisi have been described to show the unusual character of the strain. 2. Especial attention is called to the spontaneous occurrence of ablepharoplastic forms of Trypanosoma lewisi. 3. It is pointed out that morphological anomalies were most pronounced in infections that showed unusual conditions of multiplication, and that such infections usually proved severe. 4. Finally, an appreciable correlation between the morphological and developmental characteristics and the virulence as manifested in these examples of infection with Trypanosoma lewisi has been suggested.

Four experiments employing old rats and two experiments using young rats as serum donors showed that starvation of these animals altered the ability of their sera to support growth of Trypanosoma lewisi in mice. The reduction of trypanosome growth was greatest (167-fold) with sera from old animals starved for 12–14 days. Reduction was sixford with sera from old animals starved for 7–9 days and with sera from young animals starved for 5 days.

Peritoneal macrophages from Wistar rat's inoculated and non- inoculated with 10 T lewisi trypomastigotes , were cultured and infected with 10 T gondii tachyziodes.

Intracellular forms of Trypanosoma lewisi have been found in the mid-gut cells of the flea, Nosopsyllus fasciatus. These stages have been found only infrequently, and in only two of the four strains of T. lewisi studied.Attempts to find similar stages in Trypanosoma nabiasi, evotomys and microti were not successful.The possible significance of these forms is discussed.The financial assistance of the Agricultural Research Council is gratefully acknowledged. The author is also indebted to Dr P. Tate for his enthusiasm, help and guidance throughout the course of the study.

The reproductive activity of Trypanosoma lewisi was strikingly enhanced and sometimes prolonged for 2 days when sublethal doses of adenine were given to rats. The drug was effective when given for 5 or 7 days in the diet and/or intraperitoneally, starting one or two days before infection through two days after infection. Peak parasitemia was also enhanced. In terms of host immunity, daily sublethal doses of adenine given to rats within two days before or after infection with T. lewisi significantly depressed a hitherto unrevealed natural immunity to reproduction of the trypanosomes, but only slightly modified the development of acquired immunity.

SYNOPSIS. Two isolates (“A” and “B”) of Trypanosoma lewisi from the same rat stock source were serially transferred in calorically-restricted mice supplemented daily with normal rat serum. The “A” strain was transferred consecutively through 300 mice over a period of more than 3 years and was voluntarily discontinued. The “B” strain died out spontaneously after 43 consecutive passages in mice. The developmental histories of these 2 isolates were analyzed and compared with respect to duration of the parasitemic period, interval to the next subsequent passage in mice, day of death of each host animal, proportion of host animals that died, intensity of parasitemia in mouse tail blood, interval required for development of the observed maximal parasitemia, and duration of maximal parasitemia. “A” appeared to have become progressively adapted to the mouse as judged by a decrease in parasitemic period with successive transfer associated with a progressive increase in trypanosome population, and declines in the interval required for development of the observed maximal parasitemia and in the duration of this maximal response. There did not appear to be any correlation of the percentage of animals that died with any other factor. The “B” strain did not appear to have adapted itself to mice as judged by the foregoing criteria.

Inbred Lewis rats infected with Trypanosoma lewisi tended to fall into two groups of high and low parasitemias. The levels of parasitemia were expressed as accumulative values for the first seven days (S 0 7 ) of infection. Splenectomy, performed two days preinfection, yielded values of S 0 7 beyond the range of the highest normals. An attempt was then made to demonstrate that immune splenic cells confer immunity in normal rats. Splenocytes were obtained from normal and immune rat spleens, and administered by tail vein into rats five days before challenge. Transplants of normal splenocytes did not seem to alter the pattern of the normal infection. The same number of cells from spleens of early convalescent animals (12 days of infection) reduced the levels of infection to the lowest S 0 7 values. Splenic cells taken just as the infection came under control on the seventh day (and accompanied by a few trypanosomes) were incapable of controlling the infection. In a few animals adoptively immunized, low levels of agglutinative antibody were detectable five days after cell transfer. In contrast to splenic cells taken on the Day 7, plasma from these animals did neutralize a challenging dose of simultaneously injected trypanosomes. These two points, considered with the effect of splenectomy in producing lowered γ globulin levels, suggested that “humoral” factors outweigh “cellular” ones in immunity to T. lewisi .

A vascular permeability-increasing factor, which has the characteristics of a protein, was demonstrated in extracts of Trypanosoma gambiense. The concentration of this factor required to produce a strong skin reaction experimentally appeared compatible with the numbers of trypanosomes (protein concentration) observed in the skin lesions. Trypanosoma lewisi was also shown to contain a vascular permeabilityincreasing factor, and produced a definite inflammatory reaction after the injection of living blood trypanosomes into the skin of rabbits. Early skin lesions (2 days) in rabbits produced by the intradermal injection of live T. gambiense were shown to contain greatly increased water content. It is suggested that a trypanosome toxin (a vascular permeability-increasing factor) causes edema of the skin, and at least partially accounts for the early pathologic changes in the skin.

The ability of certain species of trypanosomes (T. lewisi and T. equiperdum) to ingest previously stained oil drops which occurred simultaneously in the blood has been observed.Tryparsamide incorporated in dyed (Sudan IV) cod-liver oil.when administered orally, was found to be effective against T. equiperdum infections in mice.

In previous experiments it was shown that when trypanosomes are injected into the peritoneal cavity of a rat they are absorbed by the lymphatic system, pass readily and uninjured through lymph nodes and appear promptly in lymph from the thoracic duct.1 It was also discovered, from concurrent experiments not heretofore reported, that following the injection of large numbers of adult trypanosomes into the body cavity of a rat the parasites may be recovered almost immediately from the peripheral blood of the animal and that they then remain in the circulating blood throughout the natural course of the infection. These early observations were of particular interest since they ran contrary both to our expectation and also to the apparently widely accepted conception of the development of trypanosomes early in the incubation period; namely, that these parasites, following their transfer to a noninfected rat, undergo a rapid process of reproduction for from 3 to 7 days somewhere in the body before their appearance in the peripheral blood. Further experiments, reported here, give added proof that this view is untenable. As will be seen, Trypanosoma lewisi, throughout the incubation period of the infection, is a parasite of the circulating blood in which multiplication first commences.

SYNOPSIS. In rats infected with T. lewisi, 2 morphologically distinct forms of the trypanosome occur. By means of the immunodiffusion technic several antigenic differences between the 2 forms were observed. The time course in the antigenic and morphologic changes is somewhat different. The antigenic change from young to adult forms is gradual. It starts 12 days after infection and is complete in 14 days, 3 to 5 days after the morphologic appearance of the trypanosomes is that of adult forms. In addition, there appear to be 2 separate stages of young forms when they are analyzed for antigenic composition.

The influence of riboflavin deficiency on severity and duration of parasitemia was studied in Trypanosoma lewisi infected rats.

Dihydrofolate reductase activity was detected in extracts of rat-adapted bloodstream forms of Trypanosoma (Trypanozoon) brucei, T. (T.)rhodesiense, T. (T.)equiperdum, T.(Duttonella)vivax, T.(Nannomonas) congolense (section Salivaria); T.(Herpetosoma)lewisi, and T.(Schizotrypanum)cruzi (section Stercoraria). This enzyme was also detected in extracts of culture forms of T.(T.)rhodesiense, T.(H.)lewisi, and T.(S.)cruzi. The trypanosomal dihydrofolate reductases shared the general properties of the analogous enzyme from diverse genera in their requirement for dihydrofolic acid as substrate and reduced nicotinamide adenine dinucleotide phosphate as cofactor. They also showed the characteristic extreme sensitivity to the inhibitory action of 4-amino analogs of folic acid such as methotrexate. On the other hand, the trypanosomal reductases exhibited a pattern of susceptibility to inhibition by certain 2,4-diaminopyrimidines and related heterocyclic compounds which was different from the patterns of susceptibility to these agents that have been described for reductases from bacterial and mammalian sources. Superimposed upon this distinctive pattern, moreover, was a further differential response, whereby the drug sensitivities of the reductases of salivarian trypanosomes were almost identical and, as a group, clearly distinguishable from those of the stercorarian species. Those properties of dihydrofolate reductases from culture and bloodstream forms of T.(T.)rhodesiense, T.(H.) lewisi, or T.(S.)cruzi that were compared were closely similar.

When Acridine Orange was dispersed in cod-liver oil, its characteristic green fluorescence remained unaltered following absorption of this mixture into the peripheral circulation from the intestine. This result implies that there was little or no change in the lipid or its suspensoid as a result of undergoing this physiological process. The selective combination of this dye in aqueous solution with the cytoplasmic components of specimens of Trypanosoma lewisi, also present in the blood smears with the droplets of the green-appearing oil, resulted in an alteration of fluorescence as indicated by the rose red appearance of these organisms.The present findings on the absorption of lipid droplets, containing a fluorochrome, by trypanosomes occurring simultaneously in the circulation of rats confirm similar observations reported by Wotton & Halsey (1957). The possibility is suggested again that fats and oils may serve as pharmacological vehicles for orally administered trypanosomides for the elimination of trypanosomes.Part of this study was carried out in the department of Zoology and Anatomy of the University of Nebraska, Lincoln, Nebraska, and was supported in part by a grant from the Nebraska Heart Association for 1959. We would like also to extend our appreciation to the Division of Laboratories of the Nebraska State Health Department of Lincoln, Nebraska, for the use of their ‘Fluorex’, ultraviolet illumination microscope unit.

1. 1. Standard Warburg techniques were employed to determine the oxygen uptake of two isolates ( C and L ) of Trypanosoma lewisi (Costa Rican and the U.S.). The C cell developed greater parasitemias in rat tail blood during the first 2 weeks after inoculation. 2. 2. Oxygen uptake of both isolates progressively increased with time and was not related to rise in trypanosome populations in hosts. The C form (recently isolated) consumed 36 per cent more oxygen endogenously, 20 per cent more in glucose and 16 per cent more in serum than the L . 3. 3. The metabolic capacity (Max./initial Q O 2 ) of the C cell was greater endogenously and in glucose; the L was superior in serum. Efficiency (substrate Q O 2 ratios) of the L cell was superior.

Since the publication of Moore and Breinl's paper (1908) a new method of wet fixation has been introduced into the technique of protozoological research. It is generally stated that the old method of drying the blood films and fixing them afterwards in absolute alcohol, destroys the minute details of nuclear and protoplasmatic structure; that the preparations made in this way are wholly misleading and the structures do not correspond to those of the living Trypanosomes. This becomes evident when we compare the figures of Moore and Breinl and of Rosenbusch (1909), who studied Tr. lewisi with the aid of wet fixation in Flemming's liquid, with those of other authors (Prowazek (1905), Wenyon (1908) etc.) who studied the same subject. The structure of the nucleus is very dissimilar with the two methods. The big karyosome observed by the first authors cannot be found with dry fixation; then only one or more minute granules are to be seen.