Short Stumpy Forms Research Articles

Growth of Pleomorphic Trypanosoma brucei rhodesiense in Irradiated Inbred Mice

It was shown that irradiation (650 rad) of 7 inbred strains of mice did not block the ability of Trypanosoma brucei rhodesiense to transform from the long slender (LS) to the short stumpy (SS) form or alter the plateau in parasitemia. In addition, it was observed that significant differences in parasitemia levels, in the rate of transformation from the LS to the SS form, as well as in the survival times occurred between the irradiated C3HeB/FeJ and several of the other strains. These differences in the nonspecific ability to control parasitemia appeared to be characteristic for each inbred strain of mice. The resistant strains generally had lower parasitemia than the susceptible strains. However, it was also shown that there is not a one-to-one correlation between the innate ability of a mouse strain to control its initial parasitemia, and the strain's ability to clear the parasitemia or increase its survival time. It was therefore concluded that the hypothesis which states that the ability of an animal to increase nonspecifically the rate of transformation, and therefore to lower the parasitemia, allowing intact animals to respond immunologically and survive longer is either incorrect or incomplete. The results further show that the ability of mice to clear their initial parasitemia by an antibody response is not necessarily correlated with their survival time. Therefore, this study suggests that factors other than an antibody response and the nonspecific control of parasitemia are important in resistance.

Polyamine depletion following exposure to DL-alpha-difluoromethylornithine both in vivo and in vitro initiates morphological alterations and mitochondrial activation in a monomorphic strain of Trypanosoma brucei brucei.

DL-alpha-difluoromethylornithine (DFMO), a specific irreversible inhibitor of ornithine decarboxylase (ODC), rapidly depletes cells of intracellular putrescine. When administered to animals and humans, DFMO cures acute infections of trypanosomiasis. In order to determine if the mechanism of drug action is related to initiation of transformation and biochemical alterations subsequent to polyamine depletion, trypanosome morphology and mitochondrial activation were studied in a monomorphic strain of Trypanosoma brucei brucei. Exposure of trypanosomes to DFMO in vivo in infected rodents or in vitro in culture resulted in a depletion of intracellular putrescine and a cessation of cell division without specific cytotoxicity. These events were followed by a transformation of the long slender bloodstream form to a short stumpy form via an intermediate morphology. Putrescine, the product of the ODC reaction, abrogates this effect. When introduced into SDM-79 medium, the intermediate form is capable of further transformation to an "insect" procyclic trypomastigote whereas the long slender form and short stumpy form are not. Short stumpy forms are incapable of binary fission and have lost their infectivity for the vertebrate host. In addition, the mitochondrial marker enzyme, NAD diaphorase, was found only in the short stumpy and intermediate forms. We hypothesize that the short stumpy phenotype may not be a viable stage in the natural transformation of the trypanosome from its mammalian host to the insect vector.

Growth and antigenic variation of Trypanosoma brucei, T. rhodesiense and T. gambiense in subcutaneous millipore chambers

The inability to cultivate infective bloodstream forms of the African trypanosomes in cell-free media has complicated studies of the biology of trypanosomes and the pathogenesis of trypanosomiasis. We attempted to overcome this problem by subcutaneous implantation in mice of Millipore chambers that isolate trypanosomes from cells but permit diffusion of soluble substances across their membranes. Chambers were inoculated with 5 × 10 4 to 5 × 10 5 per ml Trypanosoma brucei, T. rhodesiense or T. gambiense; the trypanosomes multiplied rapidly, persisted for as long as five weeks, and remained infective, even when the original inocula were freed of donor cells by ion-exchange. The presence of anti-trypanosomal IgG and IgM in the sera and chambers of recipient mice proved that trypanosomal and mammalian products crossed the membranes. Chamber trypanosomes also expressed two important aspects of normal in vivo biological behaviour: (i) differentiation from long slender to short stumpy bloodstream forms and (ii) antigenic variation. Death of trypanosomes was associated with the presence of IgM antibody in the chambers. This model provides a system for study of an entire population of trypanosomes in an extravascular, cell-free environment.

Studies on the influence of non-steroidal anti-inflammatory drugs upon trypanosomiasis in goats and sheep.

The non-steroidal anti-inflammatory drug (NSAID) flurbiprofen caused a rise in parasitaemia in goats infected with Trypanosoma vivax, Trypanosoma congolense and Trypanosoma brucei. All trypanosome-infected goats treated with flurbiprofen showed many dividing trypanosomes. This also included the short-stumpy forms of T. brucei. In T. vivax-infected goats flurbiprofen treatment resulted in 100% mortality in the acute and chronic stages of the infection. The increase in parasitaemia of T. brucei infected goats, treated with flurbiprofen, was not associated with an increase in mortality. The increase in parasitaemia of T. congolense-infected goats, treated with flurbiprofen, tended to be associated with a somewhat higher mortality but this was statistically not significant. The significant rise in parasitaemia could be reproduced in T. brucei-infected sheep without, however, killing the animals. Two other NSAIDs were also studied. Suprofen caused a rise in parasitaemia and 100% mortality when given to goats in the acute stage of T. vivax infection. Results with flunixin meglumine, when tested in T. brucei infected goats, were not conclusive.

Morphological changes in trypanosoma brucei rhodesiense following inhibition of polyamine biosynthesis in vivo

The effect of α-difluoromethylornithine (DFMO) treatment on the morphology of African trypanosomes was investigated. For this purpose inbred mice were immunosuppressed and infected with a clone of the protozoan blood parasite Trypanosoma brucei rhodesiense. The mice were then treated with DFMO, an irreversible inhibitor of ornithine decarboxylase, which inhibits polyamine synthesis. DFMO treatment in the absence of host immunity resulted in arrest of cytokinesis of the trypanosomes and many binucleated cells could be seen in blood smears. If mice were infected with a highly virulent trypanosome clone (ETat 1.10), which does not normally transform from long slender (LS) to short stumpy (SS) forms, DFMO treatment caused SS transformation to occur on days 3–4. This morphological SS transformation was substantiated by the presence of diaphorase activity and nuclear and mitochondrial changes. The results suggest a possible involvement of polyamines in the transformation from LS to SS forms.

Humoral responses against Trypanosoma brucei variable surface antigen are induced by degenerating parasites.

An analysis was made of the inductive stimuli for anti-T. brucei variant surface glycoprotein (VSG) responses and the role played by humoral immunity in trypanosome wave control. The first wave parasitaemia was influenced by the rate of parasite differentiation from rapidly dividing slender forms to short lived stumpy forms. Remission of first wave parasitaemia was caused by a humoral immune response against external determinants of surface expressed VSG. Anti-VSG responses were accompanied by anti-trypanosome plasma membrane responses and were followed by non-specific responses. Responses appeared to be initiated by fragments of parasites on which VSG external determinants and plasma membrane antigens were accessible and were possibly accelerated and amplified by a trypanosome mitogen which was not VSG. The parasite fragments may have arisen as a result of degeneration of stumpy form but not slender form parasites.

Cyclic 3′,5′-adenosine monophosphate levels during the developmental cycle of Trypanosoma brucei brucei in the rat

Intracellular content of cyclic 3′,5′-adenosine monophosphate was determined in several strains of Trypanosoma brucei brucei during their growth in rats. In non-relapsing infections with the cloned monomorphic strain 110M and with the cloned pleomorphic strain YTatl, the amount of cyclic AMP per 10 9 trypanosomes increased from 30 to over 90 pmol as the parasitemia increased from patency to over 10 9 organisms per ml. This increase was not observed during non-relapsing infections with strain 427. During infections with strain YTatl in both immunocompetent and lethally X-irradiated rats, cyclic AMP content of the parasite increased from 20-20 pmol per 10 9 cells early in logarithmic growth to 65–70 pmol per 10 9 cells at peak parasitemia, then decreased as the transition to intermediate and short stumpy forms commenced. At crisis, basal levels were reestablished when long slender forms were the lowest percentage of the total population and intermediate and short stumpy forms predominated, suggesting a correlation between morphologic type and level of cyclic AMP per cell during fluctuations in parasitemia. Increases in intracellular cyclic AMP were measured during in vitro incubation of the parasite in medium containing potential effectors of the trypanosome cyclic AMP system. Sodium fluoride, adenosine and methyl xanthines stimulated increases in cyclic AMP content while isoproterenol, prostaglandin E 1, serotonin, histamine and several trypanocidal drugs were ineffective. The results are discussed in terms of the possible regulatory role of cyclic AMP in differentiation of trypanosomes.

Protein uptake and digestion in bloodstream and culture forms of Trypanosoma brucei.

The mechanisms of ferritin uptake and digestion differ in bloodstream and culture forms of Trypanosoma brucei. Ferritin enters bloodstream forms from the flagellar pocket by pinocytosis in large spiny-coated vesicles. These vesicles become continuous with straight tubular extensions of a complex, mostly tubular, collecting membrane membrane system where ferritin is concentrated. From the collecting membrane system the tracer enters large digestive vacuoles. Small spiny-coated vesicles, which never contain ferritin, are found in the Golgi region, fusing with the collecting membrane system, and around the flagellar pocket. Acid phosphatase activity is present in some small spiny-coated vesicles which may represent primary lysosomes. This enzymic activity is also found in the flagellar pocket, pinocytotic vesicles, the collecting membrane system, the Golgi (mature face), and digestive vacuoles of bloodstream forms. About 50 percent of the acid phosphatase activity of blood forms is latent. The remaining nonlatent activity is firmly cell-associated and probably represents activity in the flagellar pocket. The structures involved in ferritin uptake and digestion are larger and more active in the short stumpy than in the long slender bloodstream forms. The short stumpy forms also have more autophagic vacuoles. No pinocytotic large, spiny-coated vesicles or Golgi-derived, small spiny-coated vesicles are seen in culture forms. Ferritin leaves the flagellar pocket of these forms and enters small smooth cisternae located just beneath bulges in the pocket membrane. The tracer then passes through a cisternal collecting membrane network, where it is concentrated, and then into multivesicular bodies. In the culture forms, acid phosphatase activity is localized in the cisternal system, multivesicular bodies, the Golgi (mature face), and small vesicles in the Golgi and cisternal regions. The flagellar pocket has no acid phosphatase activity, and almost all the acitvity is latent in these forms. The culture forms do not release acid phosphatase into culture medium during 4 days growth. Uptake of ferritin by all forms is almost completely inhibited by low temperature. These differences among the long slender and short stumpy bloodstream forms and culture forms are undoubtedly adaptive and reflect different needs of the parasite in different life cycle stages.

The action of trypanocidal arsenical drugs on Trypanosoma brucei and Trypanosoma rhodesiense

1. 1. Catabolism of glucose by monomorphic Trypanosoma brucei and pleomorphic T. rhodesiense is inhibited by trivalent organic arsenicals. The utilization of α-oxoglutarate and pyruvate by short stumpy forms of pleomorphic T. rhodesiense is inhibited by similar concentrations of arsenicals. 2. 2. In lysed cell systems, a dual inhibition of pyruvate formation and utilization is observed. In whole organisms, however, pyruvate oxidase is uninhibited and pyruvate kinase is demonstrated to be a major site of inhibition. 3. 3. The aromatic arsenicals do not inhibit muscle pyruvate kinase, which accounts in part for the specific trypanocidal action of the drugs. 4. 4. Trypanosome pyruvate kinase is inhibited by N-ethyl maleimide and p- chloromercuribenzoate , but not by iodoacetate, iodoacetamide, pentavalent arsenicals or inorganic arsenite. The isofunctional muscle enzyme is equally susceptible to inhibition by p- chloromercuribenzoate .

The metabolism of carbohydrate by pleomorphic African trypanosomes

1.1. Monomorphic Trypanosoma brucei and T. rhodesiense metabolize glucose via the glycolytic pathway, producing pyruvate as the major metabolic end-product with a small amount of glycerol and no carbon dioxide, acetate or succinate.2.2. Under in vitro conditions, pleomorphic strains of T. rhodesiense produce significant quantities of succinate, acetate and carbon dioxide.3.3. The time course of metabolite production has been used to evaluate the significance of acetate, succinate and carbon dioxide formation in vivo and indicates that succinate production is induced by in vitro incubation.4.4. Oxidative decarboxylases for pyruvate and α-oxoglutarate are confined to the short stumpy forms of pleomorphic infections. However, the tricarboxylic acid cycle has minimal activity in these organisms due to limiting levels of citrate synthase (E.C. 4.1.3.7) and succinate dehydrogenase (E.C. 1.3.99.1).5.5. l-Glycerol-3-phosphate oxidase is the principal terminal oxidase of both long slender and short stumpy forms of T. rhodesiense.

Metabolic transformation of Trypanosoma rhodesiense in culture.

THE pathogenic African trypanosome, Trypanosoma rhodesiense, has three morphological cell types in the mammalian bloodstream—long slender (LS), intermediate short stumpy (ISS) and short stumpy (SS). The terminal respiration of the LS forms is independent of cytochrome pigments1 and is effected by L-glycerol-3-phosphate oxidase2 located in discrete extramitochondrial particles3. A functional citric acid cycle is absent4 and there is a promitochondrion in the form of a single canal with no cristae5. Vickerman5,6 has shown that in the transition of LS to SS forms, cristae and NADH-tetrazolium reductase activity develop within the mitochondrial tubule which may be related to the acquired ability of these stages to oxidize a-oxoglutarate5,7,8. These results suggested that a respiratory switch to intramitochondrial oxidation had occurred. Bowman et al.9, however, demonstrated that this switch is unlikely to be complete, for the SS forms do not possess all the enzymes of the citric acid cycle nor were cytochromes detectable. In contrast the culture (epimastigote) form derived from the SS congener has developed a functional citric acid cycle10, a cytochrome mediated electron transport system11 and a single complex mitochondrion6. It seems that mitochondrial activation is initiated in the mammalian bloodstream but is not completed until transfer into culture. This report describes the sequence of metabolic changes associated with the acquisition of mitochondrial mediated metabolism during transformation into culture forms.

Granules and tubules in the cytoplasm of the sleeping sickness trypanosome: An electron microscope study

A method is described for selecting a single trypanosome so that it can be studied under phase contrast and then by electron microscopy in serial section. Illustrations are given of 18 serial sections through a short-stumpy trypanosome. Ribosomal particles which lie free in the cytoplasm of the long-thin form aggregate into polyribosomes; analogy with other cell types indicates active synthesis of intracellular protein. Larger ribosomal particles are attached to the outer surface of the endoplasmic reticulum. Similar analogy indicates the synthesis of protein for secretion. In the short-stumpy form the endoplasmic reticulum is distended with amorphous material. Similar material is present in “vacuoles” in the cytoplasm which we have shown to correspond to the Type II (phase positive) granules, the appearance of which precedes a crisis in the infection. Type I (volutin) granules which stain with Giemsa have been demonstrated, and have the appearance of lysosomes. The short-stumpy form carries a variable structure between the nucleus and the basal vacuole (an area associated with pinocytotic activity) which may consist of vesicles or distended tubules. We discuss these differences in the ultrastructure of T. brucei in relation to its cycle of development in the blood and to the excretion of exo-antigen.

Degeneration Phenomena of Trypanosoma gambiense

After treatment with arsenophenylglycin the number of trypanosomes in an infected animal immediately begins to decrease, and the parasites disappear from the peripheral circulation 6—7 hours after the time of injection, the short stumpy forms being the last to degenerate.One of the first changes noticed in the trypanosomes after treatment is the rounding up of their posterior extremities caused by the retraction of the protoplasm behind the kineto-nucleus. Meanwhile the tropho-nucleus becomes filled with granules of volutinose, which are extruded into the cytoplasm and pass along the axial filament towards the anterior extremity of the body. In dividing forms the granules form two longitudinal rows, being distributed along the two axial filaments. Finally, the granules lose their regular arrangement and become scattered throughout the cytoplasm, being more abundant, however, at the anterior end.The kineto-vacuole, when present, enlarges considerably and may become filled with chromatic substance derived from the kineto-nucleus.The axial filament begins to degenerate soon after treatment, often breaking up into a row of granules. Occasionally, however, chromatic substance from the kineto-nucleus passes along the axial filament, and it then appears as a thick black band extending along the middle of the cell eventually to the anterior extremity. Subsequently it breaks up into granules which dissolve in the cytoplasm of the cell.Chromatic bodies are given off from the kineto-nucleus both before and after treatment and do not seem to be a characteristic feature of degeneration.Sometimes the tropho-nucleus completely disappears before the cytoplasm breaks up into fragments, but at other times it preserves its form long after all other parts of the trypanosome, with the exception of the flagellum, have disappeared. These free nuclei resemble the “latent bodies” described by Salvin-Moore and Breinl, but as they, along with the fragments of cytoplasm, granules etc., are ingested by the leucocytes, it seems possible that they are merely one of the results of degeneration and not part of the life-cycle of this parasite.These investigations have been carried on partly with the aid of a grant from the Tropical Diseases Research Fund (Colonial Office).