In Vitro Production of Metacyclic Forms of Trypanosoma musculi and Their Infectivity in CBA Mice1

ABSTRACT. Bloodstream forms of Trypanosoma musculi, cultured in Schneider's drosophila medium at room temperature, multiply and differentiate through a series of developmental stages into infective metacyclic trypomastigotes in 10 days. Oral inoculation with these culture forms into CBA mice produced a parasitemia similar to that produced by intraperitoneal infection with bloodstream forms except for a three-day longer prepatent period. Attempts to induce parasitemia with bloodstream forms given orally were unsuccessful.     

Studies on parasitemia courses and mortality in mice infected with genetically distant Trypanosoma cruzi clonets.

The biological characterization of bloodstream forms of eleven Trypanosoma cruzi cloned stocks, corresponding to two genetically similar clonets (19 and 20) and one distant clonet (39), according to multilocus enzyme electrophoresis analysis, showed dissimilar parasitemia in an experimental isogenic mouse model. While clonet 39 stocks gave low parasitemias, clonets 19 or 20 stocks gave high parasitemias, independently of the inocula (10(2) and 10(4) bloodstream forms) used. High parasitemia did not always associate with greater mortality. Statistical studies on mortality using a low inocula showed significantly higher mortality with clonet 39 stocks when compared to clonets 19 or 20 stocks. Finally, in order to confirm the identity of each stock studied, typing by molecular karyotype was performed before inoculating mice.     

Experimental infections of farmed eels with different Trypanosoma granulosum life-cycle stages and investigation of pleomorphism

Trypanosoma granulosum, a flagellate protozoon commonly found in the blood of the European eel Anguilla anguilla, was injected experimentally into uninfected eels purchased from a local farm. In order to investigate the infectivity of different stages in the life cycle, trypanosomes from various sources were used for inoculation. Infectivity was greatly reduced in in vitro culture stages inoculated at 20 C. Isolated bloodstream stages injected into groups of animals held at 12 and 20 C could be detected for over 70 days but did not appear to multiply. Naturally infected Hemiclepsis marginata, a piscivorous leech known to serve as vector, produced detectable, single-peak infections in eels held at 20 C. Infections were characterized by a prepatent stage and a phase of rising parasitemia. Peak infection intensities ranged between 1 and 7 x 10(4) trypanosomes/ml. Trypanosomes in the bloodstream of eels experimentally infected with leeches, divided at a very low rate during the early stages of infection. Small morphs present during the early phase of rising parasitemia were gradually replaced by larger trypanosomes. The overall length frequency distribution of trypanosomes was unimodal.     

Follow-up of patent and subpatent parasitemias and development of muscular lesions in mice inoculated with very small numbers of Trypanosoma cruzi.

A sequential analysis of patent and subpatent parasitemias, mortality, and histopathology during acute Chagas' disease experimentally produced by inoculation of 10 or 100 bloodstream forms of Trypanosoma cruzi Y strain in susceptible mice was carried out. Parasites were searched for comparatively using three different methods: direct counting, Ficoll-MI density flotation, and hemoculture. Ficoll-MI density flotation promptly discriminated with high reproducibility subpatent parasitemic states not detected in the blood samples analyzed by direct counting. Despite the high proportion of supposedly uninfected animals and depending on the postinfection time, the majority of the mice had bloodstream parasites at the subpatent level detected by Ficoll-MI, and all of them had muscular lesions during the acute phase. All Ficoll-MI-negative blood samples from infected mice were also negative by hemoculture. Normal mouse blood purposely contaminated with parasite quantities ranging from 200 to 2000/ml was tested comparatively by density flotation and hemoculture and showed frequencies of reisolation varying from 25 to 100%. Overall, these data showed that inoculum as low as 10 infective forms of Y strain is able to induce acute Chagas' disease in susceptible mice and that a subpatent parasitemic state of 600-1000 forms/ml is a common finding. The use of Ficoll-MI to detect subpatent parasitemia is discussed.     

Biological characterization of Trypanosoma cruzi strains from different zymodemes and schizodemes.

The development in C3H mice of thirteen strains of Trypanosoma cruzi belonging to different zymodemes and schizodemes was studied. Host mortality, virulence, histiotropism, parasitemia and polymorphism of the parasites were recorded. The strains were grouped into: a) high virulence--causing 100% mortality and characterized by predominance of very broad trypomastigotes in the bloodstream at the end of infection; b) medium virulence--causing no mortality and with a predominance of broad trypomastigotes; c) low virulence--causing no mortality with blood forms not described due to the very low parasitemia. During 18 months maintenance the parasitemia curves were kept constant for all strains except one. A direct correlation between either zymodeme or schizodeme and experimental biological properties of T. cruzi strains was not found. However, the parasitemia was subpatent and patent for strains from zymodeme C and the others respectively. Furthermore the high virulence seems to be related to one of two schizodemes found within zymodeme B strains. All strains presenting patent parasitemia independent of shizodeme and zymodeme showed a myotropism towards heart and skeletal muscle with variable inflammatory intensity. The present study confirmed the heterogeneity found by isoenzyme and k-DNA patterns among the strains of T. cruzi isolated from chagasic patients in Bambuí, Minas Gerais State, Brasil.     

Early invasion of brain parenchyma by african trypanosomes

Human African trypanosomiasis or sleeping sickness is a vector-borne parasitic disease that has a major impact on human health and welfare in sub-Saharan countries. Based mostly on data from animal models, it is currently thought that trypanosome entry into the brain occurs by initial infection of the choroid plexus and the circumventricular organs followed days to weeks later by entry into the brain parenchyma. However, Trypanosoma brucei bloodstream forms rapidly cross human brain microvascular endothelial cells in vitro and appear to be able to enter the murine brain without inflicting cerebral injury. Using a murine model and intravital brain imaging, we show that bloodstream forms of T. b. brucei and T. b. rhodesiense enter the brain parenchyma within hours, before a significant level of microvascular inflammation is detectable. Extravascular bloodstream forms were viable as indicated by motility and cell division, and remained detectable for at least 3 days post infection suggesting the potential for parasite survival in the brain parenchyma. Vascular inflammation, as reflected by leukocyte recruitment and emigration from cortical microvessels, became apparent only with increasing parasitemia at later stages of the infection, but was not associated with neurological signs. Extravascular trypanosomes were predominantly associated with postcapillary venules suggesting that early brain infection occurs by parasite passage across the neuroimmunological blood brain barrier. Thus, trypanosomes can invade the murine brain parenchyma during the early stages of the disease before meningoencephalitis is fully established. Whether individual trypanosomes can act alone or require the interaction from a quorum of parasites remains to be shown. The significance of these findings for disease development is now testable.     

Trypanosoma brucei: Antigenic analysis of bloodstream,vector, and culture stages by the quantitative fluorescent antibody methods

Quantitative direct fluorescent antibody methods were used in antigenic analysis of developmental stages of Trypanosoma brucei brucei strains, most of them having the same variant antigen B, which were derived from a cyclically transmissible stabilate. Antigen-B trypanosomes were used for initiation of cultures in modified Tobie's (Tm) medium and in Glossina morsitans morsitans organ cultures, and for the infective feed of G. m. morsitans. Antisera against antigen-B bloodstream forms and against Tm-grown culture forms were developed in rabbits by inoculations of disrupted organisms mixed (1:1) with complete Freund's adjuvant. The globulin fractions of the antisera were conjugated with fluorescein isothiocyanate, and processed on Sephadex G-25 and DEAE-cellulose columns. The DEAE fractions with 2.0 and 4.7 or 4.8 molar fluorescein:protein ratios were pooled and concentrated twofold.Examination of 109 flies at 30 or 31 days after the infective feed revealed about 18.3% midgut, about 10.1% proventricular, and about 3.7% salivary-gland infections. A salivary gland suspension from one of the infected flies gave rise to a parasitemia in a mouse, and trypanosomes from the first parasitemia were transferred by two 3-day syringe passages into another mouse. Smears were prepared of trypanosomes (antigens B-164, B-167) from the first parasitemias from these two mice, of intact B-antigen trypanosomes, of culture forms (CT) from Tm medium, and of procyclics (CG) from Glossina cultures as well as of midgut (GM), proventricular (GP), and salivary-gland (GS) forms from tsetse flies. All these forms were fixed by one or more of the three following methods: complete fixation (CoFix) by the formalin-NH₄OH-Tween 80 procedure; fixation before affixation to slides (F+); fixation after affixation to slides (F?). The best results with regard to fluorescence intensity and specificity were obtained by using the CoFix technique.Statistical analyses of the fluorescence means of the antigens subjected to direct and inhibition staining gave the following results: (1) CT, CG, GM, and GP forms were antigenically the same. (2) GM and GP trypanosomes from different flies were antigenically indistinguishable. (3) The surface antigen of the variant-B bloodstream trypanosomes was different from these antigens of culture, midgut, and proventricular forms. It differed also from those of metacyclics from two flies and of B-164 and B-167 bloodstream forms. (4) No antigenic differences were found, in preparations fixed by the F? method, between B-164 and B-167 bloodstream trypanosomes and the metacyclics from two flies, one of which served as the source of the salivary-gland trypomastigotes (GS-98) that gave rise to these two bloodstream form antigens. (5) Closer antigenic relationships were noted between B forms and B-164 and B-167 trypanosomes than between B and CT organisms in smears fixed by the F+ technique, but no such differences were discernible in preparations fixed by the F? procedure.     

Antigenic variation during the developmental cycle of Trypanosoma brucei

During the complex life cycle of Trypanosoma brucei, changes in the exposed surface antigens occur in both the mammalian host and the insect vector (Glossina spp.). These antigenic changes are associated with alterations of the variant surface glycoprotein (VSG) composition or with the loss of the VSG. In the bloodstream of the mammalian host, trypanosomes successfully evade destruction by the host's immune response by continuously expressing alternative VSGs, at low frequency, which are not destroyed by host antibodies. When ingested by the tsetse fly, the bloodstream trypanosomes rapidly lose their surface coat and surface membrane antigens are exposed which are normally covered in the bloodstream. In the salivary glands of the tsetse fly, the trypanosomes differentiate to the metacyclic stage, which reacquires a surface coat. The antigenic composition of the metacyclics is heterogeneous. The same metacyclic types are expressed regardless of the bloodstream antigenic type ingested by the tsetse fly. In the mammal the metacyclics differentiate to long-slender bloodstream forms but continue to express the metacyclic VSG for at least three days. The next VSGs expressed in the mammalian host appear to be influenced by the antigenic type ingested by the tsetse. The ingested antigenic type is often expressed in the first parasitemia following expression of the metacyclic antigenic types.     

Antigenic analysis by immunofluorescence of in vitro-produced metacyclics of Trypanosoma brucei and their infections in mice

The antigenic types in populations of Metacyclic trypanosomes of Trypanosoma brucei isolated from Glossina morsitans head-salivary gland trypanosome cultures and bloodstream forms in the early parasitemias produced from whole culture supernatant fluids containing metacyclic forms, were analyzed by the indirect fluorescent antibody test using clone-specific antisera. Metacyclic trypanosomes in cultures initiated with cloned bloodstream forms with heterogeneous with respect to their variable antigenic type (VAT). Trypanosomes comprising early parasitemias in immunosuppressed mice infected with metacyclics produced in cultures also had a range of VATs. Three of the VATs detected in the early parasitemias in mice have also been identified by other investigators in tsetse fly-transmitted populations of the same stock.     

A Mitogen-activated protein kinase controls differentiation of bloodstream forms of Trypanosoma brucei

African trypanosomes undergo differentiation in order to adapt to the mammalian host and the tsetse fly vector. To characterize the role of a mitogen-activated protein (MAP) kinase homologue, TbMAPK5, in the differentiation of Trypanosoma brucei, we constructed a knockout in procyclic (insect) forms from a differentiation-competent (pleomorphic) stock. Two independent knockout clones proliferated normally in culture and were not essential for other life cycle stages in the fly. They were also able to infect immunosuppressed mice, but the peak parasitemia was 16-fold lower than that of the wild type. Differentiation of the proliferating long slender to the nonproliferating short stumpy bloodstream form is triggered by an autocrine factor, stumpy induction factor (SIF). The knockout differentiated prematurely in mice and in culture, suggestive of increased sensitivity to SIF. In contrast, a null mutant of a cell line refractory to SIF was able to proliferate normally. The differentiation phenotype was partially rescued by complementation with wild-type TbMAPK5 but exacerbated by introduction of a nonactivatable mutant form. Our results indicate a regulatory function for TbMAPK5 in the differentiation of bloodstream forms of T. brucei that might be exploitable as a target for chemotherapy against human sleeping sickness.     

Trypanosoma cruzi: in vitro and in vivo antiproliferative effects of epigallocatechin gallate (EGCg)

The trypanocidal activity of catechins on Trypanosoma cruzi bloodstream trypomastigotes has been previously reported. Herein, we present the effect of epigallocatechin gallate (EGCg) on parasitemia and survival in a murine model of acute Chagas' disease as well as on the epimastigote form of the parasite. Upon intraperitoneal administration of daily doses of 0.8 mg/kg/day of EGCg for 45 days, mice survival rates increased from 11% to 60%, while parasitemia diminished to 50%. No side effects were observed in EGCg-treated animals. Fifty percent inhibition of epimastigotes growth was achieved with 311 microM EGCg 120 h after drug addition. No lysis, total culture growth inhibition or morphological changes were observed upon addition of 1-3mM EGCg at 24 h. This treatment also produced oligosomal fragmentation of epimastigotes DNA, suggesting a programmed cell death (PCD)-like process. All these findings point out EGCg as a potential new lead compound for chemotherapy of Chagas' disease.     

Antigenic Analysis By Immunofluorescence of In Vitro-Produced Metacyclics of Trypanosoma Brucei and Their Infections In Mice*

SYNOPSIS. the antigenic types in populations of metacyclic trypanosomes of Trypanosoma brucei isolated from Glossina morsitans head-salivary gland trypanosome cultures and bloodstream forms in the early parasitemias produced from whole culture supernatant fluids containing metacyclic forms, were analyzed by the indirect fluorescent antibody test using clone-specific antisera. Metacyclic trypanosomes in cultures initiated with cloned bloodstream forms were heterogeneous with respect to their variable antigenic type (VAT). Trypanosomes comprising early parasitemias in immunosuppressed mice infected with metacyclics produced in cultures also had a range of VATs. Three of the VATs detected in the early parasitemias in mice have also been identified by other investigators in tsetse fly-transmitted populations of the same stock.     

The efficacy of ascofuranone in a consecutive treatment on Trypanosoma brucei brucei in mice

Consecutive administration of ascofuranone without glycerol was found to have therapeutic efficacy against Trypanosoma brucei brucei infection in mice. A suspension of ascofuranone (25-100 mg/kg) was administrated intraperitoneally every 24 h for 1-4 consecutive days to trypanosome-infected mice and efficacy was compared with oral treatment. With intraperitoneal administration, all mice treated with 100 mg/kg ascofuranone for 4 consecutive days were cured. On contrary, with oral treatment a higher dose of ascofuranone (400 mg/kg) was needed for 8 consecutive days to cure the mice. With intraperitoneal treatment, parasitemia was strongly suppressed, with almost all long slender bloodstream forms of the parasite changed to short stumpy forms by day 3 and the parasites have been eliminated 4 days after the start of treatment. These ascofuranone-induced short stumpy forms were morphologically analogous to the stumpy forms 2 days after peak parasitemia of pleomorphic clone of T. b. brucei GUTat 3.1. However, the properties of ubiquinol oxidase activity, which is the target of ascofuranone, in mitochondria isolated from before and after treatment, were almost same. The enzymatic activities of ubiquinol oxidase were only decreased to approximately 30% within a day after treatment, and then kept at nearly the same level. In the present study, we have improved regimen for administration of ascofuranone without glycerol, and demonstrated that consecutively administrated ascofuranone showed trypanocidal effects in T. b. brucei infected mice. Our present results strongly suggest that consecutive administration of ascofuranone may be an effective chemotherapy for African trypanosomiasis.     

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.     

Effects of methylglyoxal bis(ganylhydrazone) on trypanosomatid flagellates: inhibition of growth and nucleoside incorporation in Trypanosoma brucei

Methyglyoxal bis (guanylhydrazone) (MGBG) at 0.5 mM had little effect in vitro on Blastocrithidia culicis, Crithidia oncopelti, and Leishmania spp., but completely inhibited growth of Trypanosoma brucei. Inhibition became irreversible after a 3-h exposure of T. brucei culture procyclics. Treated organisms remained motile, but failed to divide. Polyamines, spermidine, and spermine, did not reverse the anti-trypanosome action of MGBG (preloading of cells or concurrent administration). Two intraperitoneal injections of the drug at a concentration of 50 mg/kg body weight at a 1-day interval greatly reduced the parasitemia of T. brucei and T. congolense in rats. Trypanosome infections, however, relapsed and killed the animals in 6 days after treatment. It was evident from the results of tracer experiments with T brucei that MGBF significantly lowered incorporation of [3H]thymidine by culture pocyclics and of [3H]uridine by bloodstream forms; in both stages [3H]leucine incorporation was only slightly inhibited. It is suggested that MGBG interferes with nucleoside incorporation by Trypanosoma and that its mode of action is different in bloodstream and culture procyclics.     

Trypanosoma rangeli--in vitro metacyclogenesis and fate of metacyclic trypomastigotes after infection to mice and fibroblast cultures

High metacyclogenesis was induced when freshly-isolated Trypanosoma rangeli from humans were grown in a modified liver-infusion-tryptose medium and transferred into the medium overlaid on mouse fibroblasts at 27 degrees C in a 5% CO2 atmosphere. Such in vitro-generated metacyclic trypomastigotes could induce a significantly high and constant parasitemia in both ICR and SCID mice for a period of about a week but thereafter the parasitemia gradually decreased. Histological examination could not detect any tissue-forms of T. rangeli in various organs of SCID mice. On the other hand, two long-maintained stocks of T. rangeli produced lower metacyclogenesis and only latent parasitemia in both strains of mice. When these populations were incubated in fibroblast cultures at 37 degrees C in a 5% CO2 atmosphere, only trypomastigotes survived for two to three weeks without proliferation, while other forms, mainly epimastigotes, soon began to swell and degenerate. Electron microscopy showed that most surviving trypomastigotes had the basket-like conformation of the kinetoplasts. This is characteristic of the non-dividing trypomastigote stage of T. cruzi, and suggests that T. rangeli trypomastigotes may survive long periods in the blood without proliferation.     

A medium for the continuous cultivation of bloodstream forms of Trypanosoma lewisi at 37 C

Trypanosoma lewisi has been maintained continuously at 37 C for more than 2 yr in Iscove's modified Dulbecco's medium with 10% fetal calf serum and a feeder layer of rat fibroblasts. In this medium the continuously reproducing hematozoic culture forms resemble bloodstream forms of T. lewisi in that they appear morphologically similar in Giemsa-stained preparations examined by light microscopy and have a surface coat that is absent in culture forms grown at ambient temperatures, when examined by electron microscopy. To determine whether these hematozoic culture forms also are similar functionally to bloodstream forms, comparative tests of the 2 forms were made of infectivity for the natural rat host, growth in vitro in the described culture medium, sensitivity to inhibition of reproduction by the rat antibody ablastin, and agglutinability by the 2 trypanocidal antibodies produced during a natural course of infection in the rat. Initially, differences between the 2 forms were minor, but after 16 mo in vitro greater differences began to emerge. Most marked was a reduction in infectivity by 22 mo, although sensitivity to ablastin, the single most important characteristic of bloodstream forms of T. lewisi, was still appreciable at this time. Nevertheless, despite this limitation, the culture system described supports hematozoic culture forms of T. lewisi for a considerably longer time than has been reported thus far.     

Trypanosoma vivax: adaptation of two East African stocks to laboratory rodents

Two Trypanosoma vivax stocks from East Africa have been adapted to rats and mice. Adaptation was induced by rapid passage at two- to four-day intervals in sublethally irradiated rats. After 200 such passages, the two stocks gave rise to parasitemias of 10(9)-10(10) trypanosomes/ml in peripheral blood, and the infection was fatal in 90% of the rats. By passaging the rat-adapted T. vivax into normal mice at two- to three-day intervals for over 200 passages, the two stocks also became pathogenic to mice. One of the stocks was also capable of maintenance in non-irradiated rats. The two stocks displayed a marked degree of pleomorphism in irradiated and non-irradiated rats and mice. In the early rising parasitemia, the organisms were predominantly short, with a well formed undulating membrane, a pointed posterior end, and a large terminal kinetoplast. As parasitemia approached its peak, the organisms transformed into long, slender forms with an inconspicuous undulating membrane, an elongated posterior end, and a sub-terminal kinetoplast. The short forms associated with the early, rising parasitemia were more infective for mice than the long forms encountered at peak parasitemia. Although the two rodent-adapted stocks retained their pathogenicity for goats, neither the original stocks nor their corresponding rodent-adapted stocks could be cyclically transmitted by tsetse flies. The availability of these stocks will greatly facilitate investigations on East African T. vivax which would otherwise be difficult to carry out in experimental rodents.     

Efficacy of dipalmitoylphosphatidylcholine liposome against African trypanosomes

We demonstrate here that dipalmitoylphosphatidylcholine (DPPC) liposome has an antitrypanosomal effect, especially against the bloodstream forms (BSFs) of African trypanosomes (Trypanosoma congolense, T. brucei rhodesiense, and T. brucei brucei). The DPPC liposome significantly decreased the in vitro percentage of viable and motile BSF African trypanosomes but only marginally reduced the percentage of viable and motile procyclic form (PCF) of trypanosomes. The DPPC liposome absorption was much more pronounced to BSF than to PCF trypanosomes. Administration of the DPPC liposome showed a slight but significant reduction in the early development of parasitemia in T. congolense-infected mice. These results suggest that parasites were killed by specific binding of the DPPC liposome to the trypanosomes. This work demonstrates for the first time that a liposome has antitrypanosomal activity.     

Evaluation of the rabbit as a model for Chagas' disease. I. Parasitological studies

In order to investigate the value of the rabbit as an experimental model for Chagas' disease, 72 animals have been inoculated by intraperitoneal and conjunctival route with bloodstream forms, vector-derived metacyclic trypomastigotes and tissue culture trypomastigotes of Trypanosoma cruzi strains Y, CL and Ernane. In 95.6% of the animals trypomastigotes had been detected at the early stages of infection by fresh blood examination. The course of parasitemia at the acute phase was strongly influenced by the parasite strain and route of inoculation. At the chronic phase parasites had been recovered by xenodiagnosis and/or hemoculture in 40% of the examined animals. The xenodiagnosis studies have shown selective interactions between the T. cruzi strains and the four species of vectors used, inducing significant variability in the results. The data herein present are consistent with the parasitological requirements established for a suitable model for chronic Chagas' disease.