David Richard Lincicome

Schistosoma mansoni: glycogen content and utilization of glucose, pyruvate, glutamate, and citric acid cycle intermediates by cercariae and schistosomules.

Abstract We investigated the metabolism of three larval stages of Schistosoma mansoni: the developing cercaria infecting Biomphalaria glabrata, the freeliving cercaria, and the schistosomule collected after penetration through rat skin membrane. The metabolism of the developing cercaria was studied by exposing infected snails to 14C-glucose and thereafter determining the distribution of 14C in the emerging cercariae. In one experiment, the radioactivity in the cercariae increased to a high peak on day 6 after 14C exposure, declined rapidly to day 12, and less rapidly to day 30. In a second experiment, increase and decrease were more moderate and the peak was reached on day 13. Protein, nucleic acid, lipid, and glycogen fractions were labeled with 14C. Radioactivity was highest during the first few days after infection and declined rapidly thereafter in most fractions, but the nucleic acid fraction retained a relatively high level of radioactivity for 19 days. When 14C-labeled cercariae were maintained free-living overnight, most of the radioactivity of the glycogen fraction disappeared. Free-living cercariae metabolized exogenous glucose to a very slight degree shortly after emergence, but activity increased after 18 hours of free-living existence and deletion of endogenous glycogen. Pyruvate was utilized very rapidly under both conditions resulting in production of CO2 from all three carbons and incorporation of a small fraction of the pyruvate carbon into the principal macromolecules. α-Ketoglutarate and acetate, and, to a small extent, glutamate, glutamine, and some of the intermediates of the citric acid cycle were also catabolized. The metabolic activity of the schistosomule appeared to differ considerably from that of the cercaria—the most pronounced difference being a great reduction in the level of pyruvate catabolism. We concluded that the developing cercaria is primarily engaged in synthesis and storage of glycogen. The free-living cercaria is geared to the production of energy either from its stores of glycogen or from an exogenous substrate such as pyruvate. The schistosomule loses its adaptation to rapid production of energy and, presumably, is again primarily engaged in synthesis.

CHEMICAL BASIS OF PARASITISM.

Examination of definitions of the words “parasites” and “parasitism” in dictionaries, reference volumes, specialized moncgraphs, and research papers will show that there are variations in meanings and philosophic concepts of these terms. Such have existed probably since the inception of these words, since there are now several additional terms in use whose parameters overlap to such an extent that to distinguish among all of them is hopeless. Thus such terms as mutualism, symbiosis, commensalism, parasitism, phoresis, all in varying degrees refer to the same phenomenon. Cameron (1958) was of the opinion that “no scientific definition of parasitism is possible in our present state of knowledge.” I think this was a fair judgment. Today, however, metabolic and biochemical studies of host relationships of organisms varioiisly referred to as parasites, symbionts, mutualists, commensals, erc., are bringing about significant changes in views with respect to the nature of the whole phenomenon designated under all the terms I have just employed. Rogers (1962) in his book on “The Nature of Parasitism” says that “it is often implied that an understanding of virulence or pathogenicity is basic to an understanding of parasitism.” He points out that a major effort in research has been concerned with immunity and pathology and the natural history of forms. Pragmatic considerations have overshadowed the development of fundamental understanding of host and parasite relationship at the cellular or molecular level. It must be remembered that these terms were born in a time when there were essentially no data on the physiological basis of association. Smyth (1962) in his monograph on “Introduction to Animal Parasitology” recognizes the confusion of parameters amongst commensalism, phoresis, symbiosis, mutualism, and parasitism, and suggests a basis for classification. Commensalism and phoresis, he states, represent only loose associations and are for shelter, defense, or obtaining food. This is the limit of dependence. On the other hand, symbiosis, mutualism, and parasitism indicate much more intimate ecologic associations where the organisms themselves have a permanent attachment in some degree to the environment and where the metabolism of the individual is geared to or regulated by the host in some manner. The difference therefore between commensalism and phoresis on the one hand and parasitism, mutualism, and symhiosis on the other is one of dependence of metabolism. A good many years ago when I first became interested in the philosophic nature of parasitism this metabolic basis of dependence formed the framework for a series of investigations which has continued to the present (Lincicome, 1953). I shall have more to say of this later. Since commensalism and phoresis are ecologic descriptors identifying certain intimate relationships that have no metabolic dependence at their core, there is no justification for the retention of thcse terms in the field of parasitology. Entariioebn coli of the human bowel is not a commensal in these terms. It is dependent for some metabolic product, although the nature of this has not been

Growth of Trypanosoma lewisi in the heterologous mouse host

Abstract Trypanosoma lewisi from the rat (homologous) host may be induced to grow abundantly in the mouse (heterologous) host by an homologous serum supplement to the mouse. The trypanosomes are found only about 24 hours after inoculation in the normal, healthy mouse, and up to a week or more in such mice that have been supplied with serum intraperitoneally. Restriction of the mouses diet and a supplement of rat serum combine to favor luxurious growth of the parasite. The action of serum may be wholly protective, or that of a blocking agent for the reticuloendothelial system, or it may be metabolic providing the trypanosome with necessary nutritional elements unavailable in heterologous hosts.

Experimental evidence for molecular exchanges between a dependent trypanosome cell and its host

Abstract Evidence in support of the general working hypothesis that dependent (parasitic) cells and organisms exchange chemical substances with their environment ( Lincicome, 1963 ) is presented in this study. The test system was the laboratory rat with its natural parasite Trypanosoma lewisi. The rat was made experimentally deficient in thiamine before introduction of the dependent cell (T. lewisi). The following categories of evidence were examined: (1) host body weight gains, (2) host food consumption, (3) host longevity, (4) oxygen consumption of host liver and kidney slices, and (5) transketolase activities of host liver and kidney homogenates. Accelerated body weight gains of T. lewisi infected rats, maintained on an adequate diet, ranged from 0–19% over uninfected rats. Weight advantages of infected thiamine-deficient rats over uninoculated thiamine-deficient ones were more consistent and ranged from 0–11%. No significant advantages were observed in weights of infected over noninfected inanition control rats. The amount of food consumed by uninfected thiamine-deficient rats decreased from about 6 gm per rat a day (initially) to about 1.5 gm (just prior to death). During the same period, food consumption of infected deficient rats decreased from 6 to 2.5 gm. Infected deficient rats lived approximately 46 days after initiation of the diet and 7 days longer than uninfected rats. The survival time of inoculated deficient rats ranged from 35 to 64 days; that of uninoculated rats was from 31 to 51 days. Liver slices from T. lewisi infected control diet rats consumed 4.5 μl of O2, which was not significantly different from the amount utilized by uninfected rat liver slices. The QO2 values for liver slices from thiamine-deficient uninfected rats decreased as the deficiency progressed from about 3.5 to 1.5 μl; liver from infected thiamine-deficient rats had QO2 values of about 4.5-1.9, but during the second week of infection the value for uninfected rat liver slices was 3 μl; that of liver from infected rats was about 4.3 μl. The QO2 values of liver slices from rats having 3-week-old infections were 3.3 μl and that of controls, 2.2 μl. Kidney slices from thiamine-deficient infected rats consumed 7.9 μl of oxygen per gram of tissue per hour during the second week of trypanosome infection; kidney slices of uninfected rats used 5.4 μl. Transketolase assays showed that this enzyme and its cofactors were more active in tissue homogenates from T. lewisi-infected rats than those from noninfected rats. Liver of infected rats, deficient in thiamine formed 23 mg of hexose per gram of tissue per hour as compared to 18 mg of hexose for liver of uninfected rats with a 23-day-old deficiency. The maximum difference observed was 6 mg of hexose. Kidney homogenates from infected rats and thiamine-deficient formed 2–4 mg more of hexose per gram of tissue than uninfected controls. The results of this study imply that T. lewisi cells stimulate growth of rats by increasing metabolic activities of host cells. Inasmuch as the lack of thiamine induced biochemical defects in rat tissue, and the presence of T. lewisi cells moderated the defects that thiamine alone can correct, theoretically T. lewisi supplies thiamine to the deficient host. This is considered evidence supporting the view that a dependent trypanosome cell exchanges molecular substances with its environment.

Growth of Rats infected with Trypanosoma lewisi.

Abstract Six experiments charting the growth of albino rats with and without infection with Trypanosoma lewisi were done. These extended over every period of a year. Starting body weights ranged from weanling (56 gm) to young adult (185 gm). Those rats infected with the trypanosome showed a consistently greater rate of growth than uninfected animals. Animals receiving a smaller initial dose of trypansomes sometimes had greater parasitemias that continued longer than those whose infections were begun with a larger inoculum of parasites, but there was no positive correlation with greater growth stimulation of rats.

Identity of trypanosome growth factors in serum. II. Active globulin components.

Abstract This paper describes the identification of the active fractions of rat serum that support the growth and survival of Trypanosoma lewisi in the heterologous mouse host. The relationship between dose of serum and the size of trypanosome challenge was first established. There was reciprocity between the amount of serum and the inoculum, so that at high serum dosages smaller inocula were sufficient for either a viable transfer back to the rat or for a microscopically demonstrable parasitemia in the mouse. At high inocula (> 6 × 107) organisms were detected on transfer back to the rat after 5 days. A problem arising in the fractionation of rat serum was the toxicity of certain preparations to the mouse. Since this effect was also dose dependent it was minimized by adjusting the inoculum size knowing the relationship between protein dose and inocula. Rat serum was then fractionated to determine which components retained the ability to promote T. lewisi parasitemias in the mouse. The proteins were active while the crystalloids were ineffective after their separation by ultrafiltration, dialysis, and Sephadex G-25. Precipitation of the globulins with (NH4)2SO4 and Na2SO4 yielded β and γ globulins which supported gross parasitemias in the mouse and albumin which did not. Further fractionation of the globulins by DEAE and Sephadex G-200 column chromatography, as well as ultracentrifugation, pointed to activity in the γ2 globulins and macroglobulins.

Oxygen uptake of liver and heart slices of Trypanosoma rhodesiense-infected mice

Abstract One hundred and nineteen male albino mice 18–20 gm in weight were used in six separate experiments to study the oxygen uptake of liver and heart slices of Trypanosoma rhodesiense -infected animals. Experimental mice were inoculated intraperitoneally (ip) with 100 T. rhodesiense cells and control mice received 1 ml of saline ip. Oxygen uptake was determined by conventional Warburg techniques in which 100-mg samples of liver slices or 50-mg samples of heart slices in 15-ml reaction vessels were used. Infected mouse liver slices consumed more oxygen than control, noninfected livers. Statistically significant increases in oxygen uptake occurred 3–7 days after inoculation and ranged from 20–51% more. No significant differences were observed between infected and control heart slices. Mean liver/body weight ratios showed significantly larger values for infected mice at 5 and 6 days after inoculation. No differences were apparent for mean heart/body weight ratios of infected and control mice.

Regulation of cell membrane permeability in Trypanosoma lewisi

Abstract Fluorescent properties of tetracycline (TC) were used to study permeability changes of T. lewisi in various surroundings under phase and fluorescence microscopy. Mitochondria and the mitochondrial elements of the kinetoplast fluoresced bright yellow under fluorescence microscopy when tetracycline entered the cells. Survival time of the organisms in saline was measured in hours, and in serum in days, either with or without TC. Survival time was proportionally shortened under conditions of fluorescence microscopy. This proportional shortening of the survival times was dependent on penetration of TC and the effect of the light necessary for fluorescence microscopy. The cells in saline became immediately visible and died rapidly, while those in serum became visible within an hour, followed by death. Serum serves as a protective permeability-regulating coat around the parasites. Thus, permeability changes which required hours or days under normal conditions could be measured in minutes with tetracycline. The protection afforded by different serum fractions of rabbit and rat sera was also measured. The beta globulins, and, to a lesser degree, the albumins, prevented the penetration of tetracycline. The presence of a serum barrier was also demonstrated by washing serum-coated organisms, by the use of fluorescein-labeled serum, and by leaching experiments. Anti- T. lewisi -antiserum does not prevent tetracycline penetration. The nature of the serum barrier and the probable role of the protective lipoprotein-containing serum fractions is discussed and their significance regarding chemotherapeutic studies pointed out.

A streptococcal decapsulation test for detection of hyaluronidase activity in animal parasites

Abstract 1. 1. A slide method for the qualitative demonstration of hyaluronidase activity in parasites is described. 2. 2. The method depends upon the principle of decapsulation of a Group C hemolytic streptococcus. The reaction volumes and materials are minute thus lending the technic to application in the examination of diverse types of parasites or particular glands or structures of individuals.

Trypanosoma duttoni: Oxygen uptake by liver slices of normal and pantothenate-deficient mice

SummaryEndogenously liver slices from mice infected with Trypanosoma duttoni and fed a control diet, consumed an average of 31 μl O2/mg N/hour which was significantly different from that (20 μl) utilized by uninfected mouse liver slices. Average endogenous