Axenic Culture Conditions Research Articles

Major cysteine peptidases of Entamoeba histolytica are required for aggregation and digestion of erythrocytes but are dispensable for phagocytosis and cytopathogenicity

Cysteine peptidases of Entamoeba histolytica (EhCPs) are considered to be important pathogenicity factors. It has been described that under standard axenic culture conditions, only three (ehcp-a1, ehcp-a2 and ehcp-a5) out of approximately 50 cysteine peptidase genes present in the E. histolytica genome are substantially expressed, thus representing the set of major EhCPs. In this study, transcriptional silencing of the major peptidase genes was used to characterize their physiological role in more detail. Analysing the transfectants a fourth major cysteine peptidase activity belonging to EhCP-A7 could be characterized. Neither cytopathic activity nor phagocytosis of erythrocytes was altered in CP-inactivated amoebae. However, a significant difference in haemolytic activity was observed. EhCP-A1 and EhCP-A7 apparently had no influence on haemolytic activity, whereas transfectants silenced for ehcp-a5 as well as those silenced for all major peptidases showed a significant reduction in their haemolytic activity. Furthermore, cells silenced for ehcp-a1 and ehcp-a7 and more effectively cells silenced in all major ehcps were impaired in digesting of phagocytosed erythrocytes. Moreover, amoebae silenced for all major peptidase genes lost the ability to form aggregates of erythrocytes prior to phagocytosis.

Synthetic nonamer peptides derived from insect defensin mediate the killing of African trypanosomes in axenic culture

Synthetic antimicrobial 9-mer peptides (designated as peptides A and B) designed on the basis of insect defensins and their effects on the growth of African trypanosomes were examined using two isolates of Trypanosoma congolense, IL1180 and IL3338, and two isolates of Trypanosoma brucei brucei, ILTat1.1and GUTat 3.1, under axenic culture conditions. Both peptides inhibited the growth of all bloodstream form (BSF) trypanosomes at 200-400 microg/mL in the complete growth medium, with peptide A being more potent than peptide B. In addition, these peptides exhibited efficient killing at 5-20 microg/mL on BSF trypanosomes suspended in phosphate-buffered saline, whereas procyclic insect forms in the same medium were more refractory to the killing. Electron microscopy revealed that the peptides induced severe defects in the cell membrane integrity of the parasites. The insect defensin-based peptides up to either 200 or 400 microg/mL showed no cell killing or growth inhibition on NIH3T3 murine fibroblasts. The results suggest that the design of suitable synthetic insect defensin-based 9-mer peptides might provide potential novel trypanocidal drugs.

Host cell-free growth of the Q fever bacterium Coxiella burnetii

The inability to propagate obligate intracellular pathogens under axenic (host cell-free) culture conditions imposes severe experimental constraints that have negatively impacted progress in understanding pathogen virulence and disease mechanisms. Coxiella burnetii, the causative agent of human Q (Query) fever, is an obligate intracellular bacterial pathogen that replicates exclusively in an acidified, lysosome-like vacuole. To define conditions that support C. burnetii growth, we systematically evaluated the organism's metabolic requirements using expression microarrays, genomic reconstruction, and metabolite typing. This led to development of a complex nutrient medium that supported substantial growth (approximately 3 log(10)) of C. burnetii in a 2.5% oxygen environment. Importantly, axenically grown C. burnetii were highly infectious for Vero cells and exhibited developmental forms characteristic of in vivo grown organisms. Axenic cultivation of C. burnetii will facilitate studies of the organism's pathogenesis and genetics and aid development of Q fever preventatives such as an effective subunit vaccine. Furthermore, the systematic approach used here may be broadly applicable to development of axenic media that support growth of other medically important obligate intracellular pathogens.

Piriformospora indica protects barley from root rot caused by Fusarium graminearum

The beneficial endophytic fungus Piriformospora indica colonizes barley (Hordeum vulgare L.) roots, which results in protection against diseases and abiotic stress and eventually in higher yield. Infection of the roots with pathogenic necrotrophic fungi of the genus Fusarium, in contrast, leads to necrotized roots and severe reduction of root and shoot biomass. Upon infestation with P. indica, roots were protected from Fusarium infections as evidenced by reduced root rot symptoms. Consistently, Fusarium quantification using quantitative polymerase chain reaction (Q-PCR) revealed a correlation between reduced root rot symptoms and the relative amount of fungal Dna. In vitro analysis of the interaction of P. indica and F. graminearum under axenic culture conditions did not reveal reciprocal growth inhibition suggesting that retardation of Fusarium in roots is mediated by a plant response rather than by antibiosis. Expression of pathogenesis-related genes strongly increased in response to F. gram inearum infections, but in contrast was diminished in the presence of P. indica, indicating that Pr proteins do not play a crucial role in the P. indica-mediated resistance response to Fusarium.

The Entamoeba histolytica genome: primary structure and expression of proteolytic enzymes

BackgroundA number of studies have shown that peptidases and in particular cysteine peptidases constitute major pathogenicity factors in Entamoeba histolytica. Recent studies have suggested that a considerable number of genes coding for proteolytic enzymes are present within the E. histolytica genome and questions remain about the mode of expression of the various molecules.ResultsBy homology search within the recently published amoeba genome, we identified a total of 86 E. histolytica genes coding for putative peptidases, including 46 recently described peptidase genes. In total these comprise (i) 50 cysteine peptidases of different families but most of which belong to the C1 papain superfamily, (ii) 22 different metallo peptidases from at least 11 different families, (iii) 10 serine peptidases belonging to 3 different families, and (iv) 4 aspartic peptidases of only one family. Using an oligonucleotide microarray, peptidase gene expression patterns of 7 different E. histolytica isolates as well as of heat stressed cells were analysed. A total of 21 out of 79 amoeba peptidase genes analysed were found to be significantly expressed under standard axenic culture conditions whereas the remaining are not expressed or at very low levels only. In heat-stressed cells the expression of 2 and 3 peptidase genes, respectively, were either decreased or increased. Only minor differences were observed between the various isolates investigated, despite the fact that these isolates were originated from asymptomatic individuals or from patients with various forms of amoebic diseases.ConclusionEntamoeba histolytica possesses a large number of genes coding for proteolytic enzymes. Under standard culture conditions or upon heat-stress only a relatively small number of these genes is significantly expressed and only very few variations become apparent between various clinical E. histolytica isolates, calling into question the importance of these enzymes in E. histolytica pathogenicity. Further studies are required to define the precise role of most of the proteolytic enzyme for amoeba cell biology but in particular for E. histolytica virulence.

Growth-promoting effect on iron-sulfur proteins on axenic cultures ofEntamoeba dispar

A growth-promoting factor (GPF) that promotes the growth of Entamoeba dispar under axenic culture conditions was found in fractions of mitochondria (Mt), hydrogenosomes (Hg) and chloroplasts (Cp) obtained from cells of six different protozoan, mammalian and plant species. We were able to extract the GPF from the Cp-rich leaf cells of a plant (spiderwort: Commelina communis L.) in an acetone-soluble fraction as a complex of chlorophyll with low molecular weight proteins (molecular weight [MW] approximately 4,600). We also found that on treatment with 0.6% complexes of 2-mercapthoethanol (2ME), complexes of chlorophyll-a with iron-sulphur (Fe-S) proteins (e.g., ferredoxins [Fd] from spinach and Clostridium pasteurianum) and noncomplex rubredoxin (Rd) from C. posteurianum have a growth-promoting effect on E. dispar. These findings suggest that E. dispar may lack a sufficient quantity of some essential components of Fe-S proteins, such as Fe-S center.

Expression profiling by whole-genome interspecies microarray hybridization reveals differential gene expression in procyclic promastigotes, lesion-derived amastigotes, and axenic amastigotes in Leishmania mexicana

We examined the Leishmania mexicana transcriptome to identify differentially regulated mRNAs using high-density whole-genome oligonucleotide microarrays designed from the genome data of a closely related species, Leishmania major. Statistical analysis on array hybridization data representing 8156 predicted coding regions revealed 288 genes (3.5% of all genes) whose steady-state mRNA levels meet criteria for differential regulation between promastigotes and lesion-derived amastigotes. Interestingly, sample comparison of promastigotes to axenic amastigotes resulted in only 17 genes (0.2%) that meet the same statistical criteria for differential regulation. The reduced number of regulated genes is a consequence of an increase in the magnitude of the transcript levels in cells under axenic conditions. The expression data for a subset of genes was validated by quantitative PCR. Our studies show that interspecies hybridization on microarrays can be used to analyze closely related protozoan parasites, that axenic culture conditions may alter amastigote transcript abundance, and that there is only a relatively modest change in abundance of a few mRNAs between morphologically distinct promastigote and amastigote cultured cells. Leishmania may represent an alternative paradigm for eukaryotic differentiation with minimal contributions from changes in mRNA abundance.

In Vitro Effects of Thiazolides on Giardia lamblia WB Clone C6 Cultured Axenically and in Coculture with Caco2 Cells

The thiazolides represent a novel class of anti-infective drugs, with the nitrothiazole nitazoxanide [2-acetolyloxy-N-(5-nitro 2-thiazolyl) benzamide] (NTZ) as the parent compound. NTZ exhibits a broad spectrum of activities against a wide variety of helminths, protozoa, and enteric bacteria infecting animals and humans. In vivo, NTZ is rapidly deacetylated to tizoxanide (TIZ), which exhibits similar activities. We have here comparatively investigated the in vitro effects of NTZ, TIZ, a number of other modified thiazolides, and metronidazole (MTZ) on Giardia lamblia trophozoites grown under axenic culture conditions and in coculture with the human cancer colon cell line Caco2. The modifications of the thiazolides included, on one hand, the replacement of the nitro group on the thiazole ring with a bromide, and, on the other hand, the differential positioning of methyl groups on the benzene ring. Of seven compounds with a bromo instead of a nitro group, only one, RM4820, showed moderate inhibition of Giardia proliferation in axenic culture, but not in coculture with Caco2 cells, with a 50% inhibitory concentration (IC50) of 18.8 microM; in comparison, NTZ and tizoxanide had IC50s of 2.4 microM, and MTZ had an IC50 of 7.8 microM. Moreover, the methylation or carboxylation of the benzene ring at position 3 resulted in a significant decrease of activity, and methylation at position 5 completely abrogated the antiparasitic effect of the nitrothiazole compound. Trophozoites treated with NTZ showed distinct lesions on the ventral disk as soon as 2 to 3 h after treatment, whereas treatment with metronidazole resulted in severe damage to the dorsal surface membrane at later time points.

Expression of alternative oxidase inhibits programmed cell death-like phenomenon in bloodstream form of Trypanosoma brucei rhodesiense

Trypanosoma brucei rhodesiense is one of the causative agents of African Trypanosomiasis. Programmed cell death (PCD) is fundamental in the development, homeostasis and immune mechanisms of multicellular organisms. It has been shown that, other than occurring in multicellular organisms, the PCD phenomenon also takes place in unicellular organisms. In the present study, we have found that under high-density axenic culture conditions, bloodstream form of T. b. rhodesiense depicts a PCD-like phenomenon. We investigated the association of the PCD-like phenomenon with expression of trypanosome alternative oxidase (TAO) under low-temperature stress conditions. We observed that bloodstream form of T. b. rhodesiense did not show any PCD but had up-regulated expression of TAO. Inhibition of TAO by the addition of ascofranone caused the development of PCD in bloodstream T. b. rhodesiense under low-temperature stress, implying that expression of TAO may contribute to the inhibition of PCD.

Evidence for a Trypanosoma brucei Lipoprotein Scavenger Receptor

African trypanosomes are lipid auxotrophs that live in the bloodstream of their human and animal hosts. Trypanosomes require lipoproteins in addition to other serum components in order to multiply under axenic culture conditions. Delipidation of the lipoproteins abrogates their capacity to support trypanosome growth. Both major classes of serum lipoproteins, LDL and HDL, are primary sources of lipids, delivering cholesterol esters, cholesterol, and phospholipids to trypanosomes. We show evidence for the existence of a trypanosome lipoprotein scavenger receptor, which facilitates the endocytosis of both native and modified lipoproteins, including HDL and LDL. This lipoprotein scavenger receptor also exhibits selective lipid uptake, whereby the uptake of the lipid components of the lipoprotein exceeds that of the protein components. Trypanosome lytic factor (TLF1), an unusual HDL found in human serum that protects from infection by lysing Trypanosoma brucei brucei, is also bound and endocytosed by this lipoprotein scavenger receptor. HDL and LDL compete for the binding and uptake of TLF1 and thereby attenuate the trypanosome lysis mediated by TLF1. We also show that a mammalian scavenger receptor facilitates lipid uptake from TLF1 in a manner similar to the trypanosome scavenger receptor. Based on these results we propose that HDL, LDL, and TLF1 are all bound and taken up by a lipoprotein scavenger receptor, which may constitute the parasite's major pathway mediating the uptake of essential lipids.

Cytoplasmic SIR2 homologue overexpression promotes survival of Leishmania parasites by preventing programmed cell death

The Silent Information Regulator (SIR2) family of genes have been cloned from a variety of species ranging from bacteria to man. In previous studies, we reported the characterization of a Leishmaniamajor gene encoding a protein with extensive homology to yeast SIR2p and expressed by different Leishmania species and parasite developmental stages and thus termed LmSIR2. Unlike the yeast SIR2p, LmSIR2p is mainly localized within the cytoplasm. In the present study, sequencing of a homologue encoding gene in another Leishmania species, Leishmaniainfantum, revealed 93% overall amino acid identity with L.majorSIR2 gene. Further, using L.infantum as a recipient for a plasmid vector (pTEX) which allows overexpression of LmSIR2p led to the accumulation of the protein in the parasite cytoplasm of both promastigote and amastigote forms and a striking increase in the survival of amastigotes, the vertebrate stage of the parasite, when maintained under normal axenic culture conditions. This phenotype was also observed when L.infantum parasites were transfected with a cosmid vector (CLHyg), isolated from a L.infantum cosmid library, carrying the L.infantumSIR2 gene (CLHyg-LiSIR2). In contrast, no effect was observed on survival of the promastigote forms (insect stage) under similar culture conditions. However, when the glucose was used as a unique source of energy under starvation conditions, the viability of promastigotes was significantly enhanced. Moreover, we showed that amastigote forms in the stationary phase of culture died with a feature of apoptosis as revealed by the appearance of YOPRO-1 positive cells and that expression of LmSIR2 protein substantially delays this phenomenon. Taken together, these results demonstrate the existence of SIR2-related proteins encoding genes in different Leishmania species and suggest that LmSIR2p could participate among other factors in the control of cell death.

Recombinant tumor necrosis factor alpha does not inhibit the growth of African trypanosomes in axenic cultures.

Mice whose tumor necrosis factor alpha (TNF-alpha) genes were disrupted developed higher levels of parasitemia than wild-type mice following infection with Trypanosoma congolense IL1180 or T. brucei brucei GUTat3.1, confirming the results of earlier studies. To determine whether TNF-alpha directly affects the growth of these and other bloodstream forms of African trypanosomes, we studied the effects of recombinant mouse, human, and bovine TNF-alpha on the growth of two isolates of T. congolense, IL1180 and IL3338, and two isolates of T. brucei brucei, GUTat3.1 and ILTat1.1, under axenic culture conditions. The preparations of recombinant TNF-alpha used were biologically active as determined by their capacity to kill L929 cells. Of five recombinant TNF-alpha lots tested, one lot of mouse TNF-alpha inhibited the growth of both isolates of T. brucei brucei and one lot of bovine TNF-alpha inhibited the growth of T. brucei brucei ILTat1.1 but only at very high concentrations and without causing detectable killing of the parasites. The other lots of mouse recombinant TNF-alpha, as well as human TNF-alpha, did not affect the growth of any of the test trypanosomes even at maximal concentrations that could be attained in the culture systems (3,000 to 15,000 U of TNF-alpha/ml of medium). These results suggest that exogenously added recombinant TNF-alpha generally does not inhibit the growth of African trypanosomes under the culture conditions we used. The impact of TNF-alpha on trypanosome parasitemia may be indirect, at least with respect to the four strains of trypanosomes reported here.

Influence of plant morphology on root exudation of maize subjected to mechanical impedance in hydroponic conditions

Mechanical impedance stimulates maize root exudation. The purpose of this work was to evaluate the direct effect of mechanical impedance on root exudation from the indirect effect involving root morphological modifications induced by mechanical impedance. Maize plants were grown in axenic hydroponic culture conditions for 4, 8, 12 and 16 days, and mechanical impedance was simulated by glass beads. At the end of the culture, exudation of plants in a nutrient solution was measured during 24 h. At harvest, plant growth and development parameters as well as carbon exudation were measured. The results demonstrated a major influence of mechanical impedance on root growth with a reduction in root elongation. Comparisons with previous studies in soil conditions have indicated that the glass-bead system realistically simulated mechanical impedance. The carbon exudation rate fluctuated from 0.2 to 1.2 mg C plant-1 day-1 and a fraction of this carbon (0.06 to 0.11 mg C plant-1 day-1) was recovered from glass beads in impeded conditions. The difference in exudation between both treatments for comparable plant morphologies lead to the conclusion that the mechanical impedance had a direct effect on exudation rate. Correlations between plant morphology and root exudation suggest that root morphology is probably involved in the modification of root exudation.

Effet de la contrainte mécanique sur le système racinaire nodal et séminal du maïs

Hydroponic axenic culture conditions, in which the physical culture support of root growth consists of glass beads, are necessary to study physiological processes such as root exudation or absorption. The effects of the mechanical constraint induced by glass beads on the architectural evolution of the root system of young maize plants were examined. A reduction in length of all roots (seminal and nodal), an increase in seminal root diameter and a modification of order 2 root branching were observed. The root biomass was not affected (except for 16-day-old plants) but the total root area was reduced. The shoot growth was significantly decreased by the mechanical constraint.

Gelling agents for tissue culture of the seaweed Hizikia fusiformis

Callus and blade formation of the seaweed Hizikia fusiformis depended on the gelling agents used under axenic culture conditions. Excised cylindrical pieces (5 mm) of the hold fast were cultured on seven different gelling agents in seawater with added Provasoli's enrichment (PESI), at 40 µmol m−2 s−1 light intensity, 18 −C for 1 month. The highest percent of callus formation (47%), from holdfast pieces, was produced on solid medium composed of 2.0% high gel strength agar. No callus was formed in liquid medium. Blades, from holdfast pieces, were formed in PESI liquid medium at the rate of 45%, while the high level of axenic blade formation (30%) on solid support was observed on 0.5% high gel strength agar. Callus and blade were identified with the original strain, at the DNA level, using random amplified polymorphic DNAs.

Isoenzyme profile as parameter to differentiate pathogenic strains of Entamoeba histolytica in Brazil.

The isoenzyme profiles (IP) of 33 strains of Entamoeba histolytica isolated from patients and carriers of two regions in Brazil (Amazonia and Southeast) were determined. The enzymes phosphoglucomutase, glucose-phosphate isomerase, hexokinase and malic enzyme were considered. IP of the strains was correlated with culture conditions, time of maintenance in laboratory and clinical history of patients. The strains were maintained under polyxenic, monoxenic and axenic culture conditions: 27 polyxenic, 1 polyxenic and monoxenic, 1 polyxenic, monoxenic and axenic and 4 axenic only. The patients were symptomatic and asymptomatic. The symptomatic patients presented either non dysenteric (NDC) or dysenteric colitis (DC), associated or not with hepatic abscess (HA). One patient presented anal amoeboma (AM). The analysis of IP for isolates maintained in polyxenic culture showed non pathogenic IP (I) for strains from carriers and patients with NDC, while the strains isolated from patients presenting DC, HA and AM resulted in isolates II or XIX pathogenic IP. This parameter was not able to differentiate strains from carriers from symptomatic patients when these strains were found in axenic or monoxenic culture. All these strains displayed pathogenic IP (II), demonstrating the inability of this parameter to classifying for virulence since it showed identical IP for strains isolated from carriers or symptomatic patients.

Ultrastructural characterization of an extracellular matrix produced by Leishmania mexicana pifanoi

In a previous study it was shown that Leishmania braziliensis promastigotes secreted metabolic products to the culture media. These metabolites called “excreted factors” were chemically characterized as polysaccharides and glycoproteins that reacted with antileishmania sera. These observations together with the recent characterization of lipophosphoglycans and phosphoglycan lead us to investigate by ultrastructural procedues if leishmania is able to produce an extracellular matrix, in axenic culture conditions. In this study, Leishmania mexicana were cultured in minimum essential medium, supplemented with 10% fetal calf serum, at 22°C. Under these conditions, the parasites tend to associate forming small aggregates, called “rosettes” in which the flagellum is oriented inwards and the cell body outwards (Fig.1). In order to retain their association I supposed the existance of an adhesive substance that kept the parasites together. To test this possibility, rosettes were fixed with glutaraldehyde in cacodylate buffer 0.1 M, or with glutaraldehyde containing 1 mg of ruthenium red, that binds to glycoproteins or polyanionic polysaccharides of extracellular matrices.

Threshold for fatigue crack propagation: Castro, D.E. DFVLR Report No TIB/B89-82183/XAB 1989, 104 pp (in German)

Gynochthodes umbellata is a rare medicinal plant used in traditional systems of medicine for curing various ailments. It is a woody climber belonging to the family Rubiaceae. Its over collection from the wild population resulted the rapid depletion of this species. There is an urgent need to conserve this species and also establish an alternative system for the production of bioactive compound in vitro without harming the wild population. The present investigation was carried out to analyse secondary metabolite (anthraquinone) production in callus cultures of G. umbellata. In vitro production of anthraquinone through callus cultures of G. umbellata was standardized using in vitro leaves, derived from the nodal explant cultures maintained in Murashige and Skoog solid medium containing 2 mg/l 6-benzylaminopurine and 3% sucrose. Leaves were harvested from the healthy shoots of axenic cultures and culture conditions were optimised for the establishment of callus. In vitro derived leaves (1–2 cm2) were inoculated onto Murashige and Skoog medium containing different individual concentrations of 2, 4-Dichlorophenoxyacetic acid (0.5–5 mg/l), indole-3-butyric acid (0.5–5 mg/l), indole-3-acetic acid (0.5–5 mg/l) and 1-naphthaleneacetic acid (0.5–5 mg/l). Of the different auxins tried for callus induction, 2, 4-Dichlorophenoxyacetic acid was found to be the most suitable auxin with different concentrations for the induction of friable yellow callus. 1-Naphthaleneacetic acid doesn’t show any callus formation while indole-3-butyric acid and indole-3-acetic acid produced greenish white compact callus. A maximum fresh weight (1.7953 ± 0.61 g) and dry weight (0.1149 ± 0.04 g) of the friable yellow callus and highest amount of anthraquinone content (18.1875 ± 0.58 mg/g dw) were recorded in MS medium supplemented with 1 mg/l 2, 4-Dichlorophenoxyacetic acid. From the present study it revealed that the callus culture is an effective method for the production of anthraquinone in vitro in G. umbellata. Anthraquinone production through in vitro leaf segment derived callus culture is a viable system for the production of this pharmacologically and industrially important natural plant pigment.

Serum lipoprotein and Trypanosoma brucei brucei interactions in vitro

Trypanosoma brucei brucei IL3201 and IL3202, which are dependent on serum high or low-density lipoproteins to multiply under axenic culture conditions, acquired lipoprotein-associated 3H-lipids without binding, accumulating or degrading apolipoproteins. Uptake by the T. b. brucei of lipoprotein-associated [1α, 2α(n)- 3H]cholesterol, [1α, 2α(n)- 3H]cholesteryl linoleate, [1α, 2α(n)- 3H]cholesteryl oleoyl ether and l-3-phosphatidyl [ N-methyl- 3H]choline, 1,2-dipalmitoyl, occurred at 37°C but not at 0°C, and tended towards saturation with increasing concentrations of 3H-lipid-labelled lipoproteins in the incubation mixture. The uptake processes did not discriminate between high- or low-density lipoproteins, did not require exogenous divalent ions and were not inhibited by the presence of acidotropic agents (chloroquine, ammonium chloride) in the incubation mixture. Uptake by T. b. brucei of lipoprotein cholesterol was likely to result mainly from desorption and diffusion processes, whereas specific binding sites were probably involved in the uptake by T. b. brucei of lipoprotein cholesteryl linoleate, cholesteryl oleoyl ether and possibly phosphatidylcholine. Exponentially growing T. b. brucei hydrolysed cholesteryl linoleate to cholesterol and had only a small capacity to re-esterify cholesterol, whereas committed non-dividing stumpy form T. b. brucei had a large capacity to esterify cholesterol. Conversion products of phosphatidylcholine were generated during or after uptake of this phospholipid by exponentially growing T. b. brucei.

Serum lipoproteins are required for multiplication of Trypanosoma brucei brucei under axenic culture conditions

Bloodstream form Trypanosoma brucei brucei IL3201 and IL3202, derived from two different serodemes, were adapted to grow in a semi-defined medium under axenic culture conditions. The organisms required low- or high-density lipoproteins, isolated from foetal bovine serum (FBS) in addition to components of lipoprotein-depleted serum, to multiply in vitro. Low- and high-density lipoproteins, isolated from a number of different species, were equally efficient at supporting in vitro growth of the parasites in medium supplemented with lipoprotein-depleted FBS. Chylomicrons and very-low-density lipoproteins did not support multiplication of the T. b. brucei and did not influence the capacity of low- or high-density lipoproteins to support parasite multiplication. Removal of the lipoprotein-lipids abrogated the capacity of low- or high-density lipoproteins to support T. b. brucei multiplication.