Infectivity and route of penetration in rats after oral and intraperitoneal inoculations of bloodstream and in vitro-cultured metacyclic forms of Trypanosoma lewisi

Morphological changes of Trypanosoma lewisi blood trypomastigotes cultured in Schneider's Drosophila medium (SDM), supplemented or not with uric acid (SDM + UA), were compared to those that occurred in a control medium (M-199). No difference in trypanosome morphology and numbers was observed between SDM + UA and SDM cultures; there was little transformation into metacyclic stages in M-199. No difference was observed between the capacity of SDM- or SDM + UA-cultured metacyclic stages to infect rats. The infectivity of bloodstream forms was always higher than that of the SDM- or SDM + UA-cultured forms, whether inoculated orally or intraperitoneally. The oral inoculation of rats with tritium-labeled culture and bloodstream forms showed that the metatrypanosomes from the cultures remained longer in the salivary glands and tongue of the animal than the blood trypanosomes.     

Properties of ablastin--a factor in the serum of rats infected with Trypanosoma lewisi which inhibits the parasites' division

The present study investigated the nature of ablastin, a factor present in the serum of rats infected with Trypanosoma lewisi and which inhibits the parasites' division. Ablastin was unstable to dialysis at pH 1X8, was not adsorbed from serum by trypanosomes and could not be induced in vivo by means other than a natural infection. Attempts to purify ablastin from serum by conventional chromatographic techniques were unsuccessful. Removal of over 90% of the immunoglobulins from ablastinic serum did not reduce the ablastin titre. It is concluded that ablastin is unlikely to be an immunoglobulin as has been previously suggested.     

Cell surface saccharides of Trypanosoma lewisi. I. Polycation-induced cell agglutination and fine-structure cytochemistry.

Trypanosoma lewisi bloodstream and culture forms were agglutinated differentially with low concentrations of the cationic compounds: ruthenium red, ruthenium violet, Alcian blue chloride, 1-hexadecylpyridinium chloride, lanthanum chloride, and cationized ferritin. The bloodstream form trypanosomes gave the highest agglutination levels with each of the compounds tested. Ruthenium red was the most effective inducer of cell agglutination among the several cations used. Trypsin-treated bloodstream forms were agglutinated less in the presence of ruthenium red than untreated controls. Ruthenium red-induced cell agglutination also was lowered with chondroitin sulphate and dextran sulphate, but not with alpha-D-glucose, alpha-D-mannose or with several methyl glycosides. Treatment of the bloodstream trypanosomes with alpha-amylase, dextranase, or neuraminidase had little effect on agglutination levels obtained with ruthenium red. Fine-structure cytochemical staining with ruthenium red, ruthenium violet, and Alcian blue-lanthanum nitrate was used to ascertain the presence and distribution of presumptive carbohydrates in the trypanosome cell surface. The extracellular surface coat of the bloodstream forms stained densely with each of the polycationic dyes. Trypsin treatment removed the surface coat from bloodstream trypanosomes; however, the surface membranes of the organisms were stained densely with the several dyes. Similar surface-membrane staining was obtained with the cationic compounds and the culture forms, which lack a cell surface coat. Cationized ferrin was used at the fine-structure level to visualize the negative surface charge present in the cell surface coat and external membrane of the several trypanosome stages. Results obrained from the agglutination and cytochemistry experiments indicate that complex polysaccharides are present in the surface membranes and cell surface coat of T. lewisi bloodstream forms. Similar conclusions also pertain to the surface membranes of the T. lewisi culture from trypanosomes. The carbohydrates probably represent glycopeptide and glycoprotein structural components of the surface membrane of this organism.     

Trypanosoma lewisi Infections in Normal Rats and in Rats Treated with Dexamethasone

SYNOPSIS. Administration of dexamethasone to rats infected with Trypanosoma lewisi resulted in the development of exceedingly large populations of trypanosomes which were fatal to their hosts. The elevated levels of parasitemia in treated rats early in infections were thought not to be a result of an increased reproductive rate. However, trypanosomes in treated rats 2 days postinfection did have a higher coefficient of variation in total length and a greater percentage of dividing forms than those observed from infected rats which were not given the drug. The course of infection may be markedly altered not only in intensity but also in length by this corticosteroid. It is suggested that dexamethasone administered at the levels recorded to rats infected with T. lewisi inhibits the production of ablastin and trypanocidal antibodies.     

Cultivation in a semi-defined medium of animal infective forms of Trypanosoma brucei, T. equiperdum, T. evansi, T. rhodesiense and T. gambiense.

A semi-defined medium for the cultivation of bloodstream forms of the African trypanosome brucei subgroup was developed. Out of 14 different strains tested, 10 could be cultured including Trypanosoma brucei, T. equiperdum, T. evansi, T. rhodesiense and T. gambiense. The presence of a reducing agent (2-mercaptoethanol or thioglycerol) was found to be essential for growth. The standard medium consisted of Hepes buffered minimum essential medium with Earle's salts supplemented with 0.2 mM 2-mercaptoethanol, 2 mM pyruvate and 10% inactivated serum either from rabbit (T. brucei, T. equiperdum, T. evansi and T. rhodesiense) or human (T. gambiense). Although a general medium could be defined for the long-term maintenance of trypanosome cultures, the initiation to culture nevertheless required particular conditions for the different strains. The cultured trypanosomes had all the characteristics of the in vivo bloodstream forms including: morphology, infectivity, antigenic variation and glucose metabolism.     

Life-Cycle of Trypanosoma conorhini. Influence of Temperature and Other Factors on Growth and Morphogenesis

SYNOPSIS. In continued observations on the in vitro growth and multiplication of the bloodstream trypanosome stage of Trypanosoma conorhini , a better medium was found for cultivating these forms at 37°C, but no subcultures could be obtained. The infectivity for mice of the blood type trypanosomes grown in vitro was comparable to that of the metacyclic trypanosomes. The only reproducing forms of T. conorhini found in the vertebrate were in the trypanosome stage.
It was also found that the in vitro reversion of the bloodstream trypanosome into crithidia, such as occurs in the invertebrate host and in the usual diphasic culture medium, is dependent on at least two factors: if incubated at 25–28° reversion did not occur in any of the liquid media tried (all containing blood serum and hematin or hemoglobin), unless total blood was part of the inoculum or washed red blood cells were added to the media; on the other hand, no reversion was seen, even in the presence of red blood cells if the cultures were incubated at 37°.     

Trypanosoma lewisi: Avidity and adsorbability of ablastin,the rat antibody inhibiting parasite reproduction

The relation of naturally acquired host IgG in the surface coat of bloodstream forms of Trypanosoma lewisi to ablastin was studied to determine whether, contrary to a long-held conclusion, the antibody is avid and adsorbable. It was found by immunofluorescence and agglutination tests with monospecific antisera to rat IgG that bloodstream forms collected from immunosuppressed hosts, in contrast to those from immunocompetent hosts, have little or no detectable surface IgO. Specificity of adsorption was also demonstrated in other immunofluorescence experiments in which bloodstream forms from immunosuppressed hosts adsorbed IgG from immune serum with ablastic activity only (previously adsorbed with trypanosomes from immunocompetent hosts to remove the trypanocidal antibodies), but did not adsorb IgG from normal rat serum. To determine whether this specific adsorption of IgG by the parasite could be correlated with a reduction in ablastic activity, immune sera were adsorbed with bloodstream forms from immunosuppressed hosts at packed cell/serum ratios of either 1.2 or 2.0, and the adsorbed sera were then tested for ablastic activity in vitro. With both cell/serum ratios, ablastic activity was reduced by 50%. In comparison, similar adsorptions of immune sera with trypanosomes from immunocompetent hosts resulted in reductions of ablastic activity of only about 9 and 27% with the low and high cell/serum ratios, respectively. It is concluded that the failure to effect significant adsorption of ablastin in earlier studies resulted from the use of ablastinsensitized trypanosomes from immunocompetent hosts.     

Immunosuppression and ablastin

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.     

Flow cytometric analysis of immunoglobulin and complement component C3 on the surface of Trypanosoma lewisi

We have reinvestigated whether surface immunoglobulin (sIg) on Trypanosoma lewisi is antibody directed toward parasite antigen by using flow cytometry to analyze parasites stained with fluoresceinated F(ab')2 fragments of antibodies to rat IgG and IgM. We have confirmed that IgG antibody to the parasites is present both in the serum of rats and on the surface of parasites between the fourth and twentieth days of infection, that the amount of sIg per cell increases as the infection progresses, and that considerably more IgG is present on parasites harvested from intact rats than on those from rats that had been immunosuppressed by whole body gamma-irradiation. In addition sIgM was detected on trypanosomes from intact, but not on parasites from irradiated rats. We have also made two observations suggesting that not all sIg is specific antibody made in response to T. lewisi. First, a low but significant amount of sIgG was detected on parasites throughout infection in irradiated rats; no sIgM was detected on these parasite. Second, when parasites harvested from immunosuppressed rats were incubated in normal rat serum, the amount of both sIgG and sIgM detected by flow immunofluorescence increased. Parasites harvested from intact animals bound IgM but not IgG from normal rat serum. These results suggest either that natural antibody to the trypanosomes is present in the serum of uninfected rats or that some rat immunoglobulins bind to structures on the trypanosome surface in ways that do not depend on usual antigen-antibody interactions. Finally, flow immunofluorescence was also used to detect complement component C3 on the surface of both intact and trypsinized bloodstream forms harvested from intact or immunosuppressed rats. The amount of sC3 per cell did not increase until late in the infection and consequently did not correlate with the increase of sIgG. Therefore, T. lewisi avoids destruction by the immune system although immune effector molecules, IgG, IgM, and C3, are on its surface.     

Effect of in vitro cultivation on the stability of resistance of Trypanosoma brucei brucei to diminazene, isometamidium, quinapyramine, and Mel B

A Trypanosoma brucei brucei stock resistant to diminazene aceturate, isometamidium chloride, quinapyramine sulfate, and Mel B was grown in vitro and its response to these drugs compared to that of a drug-sensitive trypanosome stock. There was little if any change of drug sensitivity after in vitro propagation as bloodstream forms for 120, 177, and 275 days and after in vitro transformation of bloodstream forms into procyclic, epimastigote, and finally metacyclic forms. Drug resistance was stable during in vitro maintenance in the absence of drugs in both culture systems. The response of resistant and sensitive T. b. brucei to diminazene in vitro correlated with their sensitivity pattern in vivo. Thus, in vitro techniques can be used to study drug resistance in trypanosomes.     

Establishment of a long-term culture system for rat colon epithelial cells

Summary The aim of this study was to establish a long-term culture system for rat colon epithelial cells. Colonic crypts were isolated by incubating a 4-cm-long rat colon segment cut longitudinally with an ethylenediaminetetraacetic acid [disodium salts]-containing buffer, taken up in conditioned medium from the normal rat kidney fibroblast cell line NRK (i.e., the supernatant of pure NRK cultures), directly plated on mitomycin C-treated NRK cells and subcultured with conditioned medium from NRK cells. Cells started to migrate out of the crypts shortly after plating them on NRK feeder layers. Some of the crypts fell apart during the isolation procedure, whereas the vast majority of them did it within 1 to 2 h after plating. The cells proliferated extremely slowly but continuously over a period of 4 mo and were epithelial because they expressed cytokeratin 19 and were stained by crystal violet at pH 2.8. In conclusion, the experimental system described in this study allows to maintain rat colon epithelial cells for up to 4 mo in culture and can be used to study the effects of a variety of tumor-modulating factors on growth and gene expression of normal colon epithelial cells in vitro.     

In Vitro Cultivation of Bloodstream Forms of Trypanosoma brucei,T. rhodesiense,and T. gambiense1

ABSTRACT. A series of new in vitro systems for the cultivation of bloodstream forms of Trypanosoma (Trypanozoon) brucei brucei, T. (T.) b. rhodesiense, and T. (T.) b. gambiense was developed. The standard system consists of a feeder layer of fibroblast-like cells derived from embryos of New Zealand White rabbits (REF) or a mountain vole, Microtus montanus (MEF), with HEPES-buffered Minimum Essential Medium (MEM), with Earle's salts, supplemented with 15% inactivated rabbit serum. These two and other feeder layers were cross-checked with different sera to test for growth support of bloodstream forms of the three trypanosome subspecies studied. Cultures could be initiated with bloodstream forms from mammalian hosts or from cryopreserved stabilates. Metacyclic forms from infected Glossina m. morsitans could also be used as inoculum; they transformed within 6 h to bloodstream forms. Maintenance of cultures and growth properties are described in detail. Experiments were undertaken to confirm that the cultivated bloodstream forms still possess some of the characteristic features of pleomorphic bloodstream populations. Cultivated bloodstream forms were always infective for mice, and a surface coat could be demonstrated by electron microscopy. They could also be cyclically transmitted through tsetse flies, and the metacyclic forms from these flies could be brought back into culture. In vitro cloning with single bloodstream forms and metacyclic forms could be achieved with high cloning efficiency. The consumption of glucose and the production of pyruvate and lactate were determined.     

The axenic cultivation of insect forms of Trypanosoma (Duttonella) vivax and development to the infective metacyclic stage

A method for axenic cultivation of epimastigote and metacyclic forms of Trypanosoma (Duttonella) vivax at 27 degrees C in vitro is described. Iscove's medium was supplemented with specific concentrations of foetal bovine serum, L-proline, L-glutamine hypoxanthine, adenosine, pyruvate, and 2-mercaptoethanol. Bloodstream form parasites rapidly transformed into epimastigote forms that grew as surface-adherent colonies in plastic culture flasks. Transformation of epimastigotes to metacyclic forms was first observed 9-12 days after initiation of cultures. Percentages of metacyclics varied: East African T. vivax ranged up to 40% and West African T. vivax ranged up to 24%. Subcultures were made at two-week intervals and maintained for several months. Transformation of bloodstream forms to epimastigotes depended on initial attachment to the bottom of culture flasks and the presence of L-proline. The number and maturity of metacyclic forms was influenced by the concentrations of foetal bovine serum, L-proline, L-glutamine, and 2-mercaptoethanol. Trypanosomes from cultures were cryopreserved, revived, and used to re-establish fresh axenic cultures. These results represent a significant advance in cultivation of T. vivax insect forms that should enable studies to be accomplished on metabolism, differentiation, and pharmacology of this parasitic protozoan, free from the influence of extraneous cells.     

Biochemical Requirements for Intracellular Invasion by Trypanosoma cruzi: Protein Synthesis1

The effects of irreversible inhibition of protein synthesis by pactamycin in either infective forms of Trypanosoma cruzi or mammalian host cells on cellular invasion by this human pathogen were investigated. Treatment of bloodstream forms of T. cruzi with pactamycin markedly reduced their ability to bind either fibroblast-like cells of monkey origin or myoblasts of rat origin. The number of amastigote forms that could be established intracellularly was also significantly decreased with respect to control values obtained when mock-treated (medium alone) trypomastigotes were incubated with the cells. Pactamycin treatment also reduced the infectivity of T. cruzi trypomastigotes for mice as evidenced by both significantly reduced parasitemia levels and mortality rates when compared with those of control mice infected with mock-treated parasites. Inhibition of protein synthesis in the host cells neither prevented cell infection by untreated trypomastigotes nor altered the percentages of infected cells or the magnitude of the infection in vitro. These results indicate that protein synthesis is a requirement for cell invasion by T. cruzi and that the parasite can establish itself and replicate within cells relying on its own protein synthesis ability.     

Infection of white rat peritoneal macrophages with Toxoplasma gondii, (Coccidia: Sarcocystidae) after Trypanosoma lewisi (Kinetoplastida: Trypanosomatidae) infection

Peritoneal macrophages from Wistar rats, inoculated and non-inoculated with 10(6) T. lewisi trypomastigotes, were cultured and infected with 10(6) T. gondii tachyzoites. Multiplication rates of this parasite were studied after 1, 24 and 48 h of infection but there were not significant differences between the number of parasites found inside of macrophages coming, either from T. lewisi infected or non infected rats. On the other hand, in vivo studies of Toxoplasma multiplication inside peritoneal macrophages, showed that there is an increase of parasite number in cells from T. lewisi infected rats, as compared with those macrophages from non infected rats. This effect was statistically significant and was more evident after four days of infection. Therefore, it has been demonstrated that in vivo, but not in vitro T. lewisi infections, causes an important decrease of the natural resistance to T. gondii of the white rats, which is manifested by the major invasion and multiplication of the parasite inside of peritoneal macrophages.     

The Axenic Cultivation of Insect Forms of Trypanosoma (Duttonella) vivax and Development to the Infective Metacyclic Stage

A method for axenic cultivation of epimastigote and metacyclic forms of Trypanosoma (Duttonella) vivax at 27°C in vitro is described. Iscove's medium was supplemented with specific concentrations of foetal bovine serum, L-proline, L-glutamine, hypoxanthine, adenosine, pyruvate, and 2-mercaptoethanol. Bloodstream form parasites rapidly transformed into epimastigote forms that grew as surface-adherent colonies in plastic culture flasks. Transformation of epimastigotes to metacyclic forms was first observed 9–12 days after initiation of cultures. Percentages of metacyclics varied: East African T. vivax ranged up to 40% and West African T. vivax ranged up to 24%. Subcultures were made at two-week intervals and maintained for several months. Transformation of bloodstream forms to epimastigotes depended on initial attachment to the bottom of culture flasks and the presence of L-proline. The number and maturity of metacyclic forms was influenced by the concentrations of foetal bovine serum, L-proline, L-glutamine, and 2-mercaptoethanol. Trypanosomes from cultures were cryopreserved, revived, and used to re-establish fresh axenic cultures. These results represent a significant advance in cultivation of T. vivax insect forms that should enable studies to be accomplished on metabolism, differentiation, and pharmacology of this parasitic protozoan, free from the influence of extraneous cells.     

Pentamidine transport and sensitivity in brucei-group trypanosomes.

Sensitivity to pentamidine of bloodstream forms and culture forms of Trypanosoma brucei brucei, strains of this subspecies, and strains of T. brucei rhodesiense characteristically differs in vitro. Analyses of transport parameters for pentamidine uptake in these organisms show differences that correspond with drug sensitivity. Long slender bloodstream forms of T. b. brucei have a high affinity for the drug and high rates of uptake at indicated by Km and Vmax values for [3H]pentamidine transport. Although pentamidine and stilbamidine resistance is associated with dyskinetoplasty, this condition does not itself confer resistance to pentamidine nor does it affect pentamidine transport. However, drug-resistant strains show lower rates for pentamidine transport as does T. b. rhodesiense, which is characteristically less sensitive to the drug. Of all the forms and strains studied, procyclic trypomastigotes were least sensitive to pentamidine and had a remarkable ability to exclude the drug.     

Comparative positions of kinetoplasts in Trypanosoma musculi and Trypanosoma lewisi during development in vitro

Abstract. The development of Trypanosoma musculi and Trypanosoma lewisi were studied in vitro in the presence of adherent splenic cells. Both parasites developed only when attached by their flagellar tips to adherent splenic cells. During the proliferation of T . musculi , the kinetoplast migrated towards the nucleus, and once in the vicinity of the nucleus, the nuclear division was triggered. The kinetoplast of T . lewisi did not migrate towards the nucleus, but remained at its original location. The nucleus and kinetoplast divided at the same time in both parasites, and parasites started dividing from their flagellar ends and T . musculi and T . lewisi daughter cells were formed within 48 h. The unavailability of the adherent splenic cells in vitro led the parasites to transform into round/oval nonviable forms.     

Elicitation of the reproduction-inhibiting antibody ablastin by serum exoantigens of Trypanosoma lewisi

Serum exoantigens of Trypanosoma lewisi were collected 5 days after infection from immunocompetent (untreated) rats and rats immunosuppressed by treatment with either hydrocortisone acetate or dexamethasone. Normal rats were then immunized with pooled, whole exoantigen-containing serum from 1 of these 3 sources plus alum as an adjuvant, and the immune sera produced were tested individually. All contained agglutinating (trypanocidal) antibodies to both antigenic variants of T. lewisi, but only about two-thirds showed precipitating activity with exoantigens in gels. More importantly, however, when these antisera were thoroughly adsorbed with living trypanosomes (from immunocompetent hosts) to remove agglutinating antibody only and then tested for ablastic activity in vitro, all showed significant (P less than 0.01) reproduction-inhibiting activity, comparable to that shown by ablastic serum collected from rats that experienced a natural infection. Antisera from control rats similarly immunized with normal rat serum were negative in all antibody tests. The exoantigens of T. lewisi are, therefore, a complex mixture of immunogens that are related to the known immune responses to the parasite and can elicit the formation of ablastic antibody with the same biological properties as that produced during a natural infection.     

Laboratory maintenance of Trypanosoma (Herpetosoma) rangeli Tejera, 1920

Two laboratory maintenance systems of Trypanosoma rangeli were compared. The maintenance by weekly subinoculations in Tobie's culture medium and the intrafemoral inoculation of Rhodnius prolixus with cultured flagellates, resulted in loss of infectivity of the metacyclic salivarian trypomastigotes for mice, ten months after maintenance in culture. With the system of cyclical passes through culture-Rhodnius-mouse-culture-Rhodnius, the infectivity of the metacyclic trypomastigotes for mice, was maintained during the three years of the experiment. The number and percentage of metacyclic trypomastigotes formed in the salivary glands of R. prolixus, previously inoculated intrafemorally or intracoelomically with culture forms of T. rangeli, did not show correlation with the inoculated dose, however the inoculated quantity demonstrated a direct relation with the mortality rate of the insects. The results indicate that T. rangeli requires an adequate maintenance system, so that under experimental condition the biological characteristics, normally expressed under natural conditions, are conserved.