Suggested dosage rates of melarsoprol in the treatment of mice experimentally infected with Trypanosoma brucei gambiense.

One group of BALB/c mice infected with a highly virulent strain of Trypanosoma brucei gambiense were treated intraperitoneally with three series of three injections (each injection of 10 mg/kg) of Mel-B separated by seven days of rest, while a second group was treated once by a single injection All the Mel-B treated mice in both experiments were negative for parasites when examined using either the wet blood film or buffy coat methods, but were intermittently PCR positive during the sampling period. We encourage the use of a repeat negative PCR test over a one month period in combination with corroborative clinical and parasitological investigation to be suggestive of cure in experimental animals previously infected with trypanosomosis. In view of the exorbitant costs of Mel-B and its extreme toxicity, we recommend that Mel-B be given as one course of two injections (each equivalent to 10 mg/kg) separated by 2 d of rest in experimentally infected rodent models.

Interleukin 4 is a crucial cytokine in controlling Trypanosoma brucei gambiense infection in mice.

The role of interleukin 4 (IL-4) was studied in relation to host defense during Trypanosoma brucei gambiense IL3253 (IL3253) infection in mice. BALB/c/A-+/+ (BALB/c), BALB/c/A-nu/nu (nude) and C.B-17/Icr-scid/scid (SCID) mice were infected intraperitoneally with 5 x 10(3) bloodstream forms (BSFs) of the trypanosome. The BALB/c mice showed high resistance to IL3253 infection with sporadic parasitemia. The nude mice were also able to control IL3253 infection and experienced low, but persistent parasitemia. However, the SCID mice, which have no functional T- and B-cells, showed high susceptibility to IL3253 infection with more than 1 x 10(8) BSFs/ml. Serum IL-4 levels in the infected BALB/c mice were increased on days 12-18 post-infection (PI). In BALB/c mice depleted of CD4+ T-cells by monoclonal antibody (mAb) treatment, parasitemia was persistent, ranging from 1 x 10(4) to 1 x 10(6) BSFs/ml and was significantly higher than that of the other groups. IL-4 was not detected in the serum of CD4+ T-cells-depleted mice. On the other hand, anti-IL-4-treated IL3253-infected BALB/c mice relapsed significantly longer than the control mice (p < 0.01). These findings suggest that the CD4+ T-cells may control the levels of parasitemia in IL3253 infection through the IL-4 pathway.

Towards developing a diagnostic regimen for the treatment follow-up of Trypanosoma brucei gambiense.

BALB/c mice infected with a high virulent strain of Trypanosoma brucei gambiense IL3707 were treated intraperitoneally (i.p.) with either Melarsoprol (Mel-B) or PSG(+) buffer as controls. The mice were subsequently monitored regularly for parasites by direct microscopic examination of their tail blood or buffy coat and by polymerase chain reaction (PCR). Mel-B was found to be an effective drug for treatment against T.b. gambiense because at the end of the first treatment schedule, all treated mice were negative for parasites even by PCR, while all the control animals were positive. Three of the five Mel-B treated mice, while parasitologically negative, were PCR positive between 53 and 80 days post infection (DPI), indicating that they still harbored an infection. All treated mice were subsequently negative for parasites even by PCR at 88 DPI. A combination of conventional microscopic examination and PCR offers a good prediction of cure following treatment of trypanosomosis.

Susceptibility of severe combined immuno-deficient (SCID) mice to Trypanosoma brucei gambiense and T. b. rhodesiense.

Susceptibility of severe combined immunodeficient (SCID) mice to 7 isolates of Trypanosoma brucei gambiense and 2 isolates of T. b. rhodesiense was examined in terms of their infectivity, course of parasitaemia, packed cell volume (PCV) and survival period in comparison with that of normal immunocompetent (BALB/c) mice. All isolates of T. b. gambiense and T. b. rhodesiense caused high (> 1 x 10(8) parasites/ml) parasitaemia in the SCID mice, the survival periods ranged from 5 to 47 days. On the other hand, 5 of 7 isolates of T. b. gambiense developed chronic infection in the BALB/c mice with sporadic but persistent parasitaemia with less than 5 x 10(6) parasites/ml. All the mice tested in this group survived more than 60 days after infection. In contrast, the 2 remaining isolates of T. b. gambiense and both isolates of T. b. rhodesiense showed high virulence in the BALB/c mice and killed all of them within 30 days after infection. The results demonstrate that the SCID mice, in which functional B- and T-cell-mediated immunities are congenitally lacking, are highly susceptible for 'low-virulence' T. b. gambiense. This makes SCID mice useful tools for the isolation of parasites from T. b. gambiense sleeping sickness patients and the propagation of large amounts of such parasites.

The application of molecular biological tools to epidemiology of African trypanosomosis.

Difficulties have often been encountered in the field surveys due to a lack of definitive morphological characters, particularly where mixed infections are expected. To address this problem, some molecular biological techniques such as DNA probe hybridization, restriction fragment length polymorphism (RFLP) analysis, the polymerase chain reaction (PCR), analyses of ribosomal DNA, and pulsed-field gel electrophoresis (PFGE), have been applied to the analysis of field samples collected during epidemiological surveys of African trypanosomosis. Concurrent natural infection of different individual tsetse flies and mammalian hosts with different species of the trypanosomes have been demonstrated, through the use of a combination of specific DNA probe hybridization and the PCR. Molecular karyotypes of Trypanosoma brucei species were analyzed by PFGE in 45 - 2,000 kb range. There are distinctive differences in intermediate and mini-chromosomes among the strains. We have compared the nucleotide sequences of ribosomal DNAs of the parasites by PCR techniques. From this data new phylogenetic tree can be inferred. It is apparent that these technologies can provide powerful tools for identification and diagnosis of trypanosomes in their hosts and vectors, and for their more accurate phylogenetic classification.

Short communication: cultivation of bloodstream forms of Trypanosoma brucei and T. evansi in a serum-free medium.

Bloodstream forms of Trypanosoma brucei and T. evansi have been cultivated in an axenic culture system by using Iscove's modified DMEM-based HMI-9 medium supplemental with bathocuproinedisulphonic acid, L-cysteine, hypoxanthine, 2-mercaptoethanol, pyruvate, thymidine and 10% fetal bovine or adult horse serum. We developed a serum-free medium (HMI-244) in which serum in HMI-9 was replaced by fatty acid-free bovine serum albumin, bovine alpha 2-macroglobulin, bovine beta-lipoprotein, d-biotin, retinol, beta-alanine, L-anserine nitrate salt, L-ornithine hydrochloride, O-phosphorylethanolamine, sarcosine, taurine, adenosine-5'-triphosphate, 2'-deoxycytidine-5'-monophosphate, 2'-deoxyguanosine-5'-monophosphate, and 5-methyltetrahydrofolic acid. Maximum cell densities and population doubling times were 2.6 x 10(6) cells/ml; 10.6 hours and 2.2 x 10(6) cells/ml; 10.9 hours for T. brucei and T. evansi, respectively. Bloodstream forms continued to proliferate in the serum-free cultures for more than 90 days and the trypomastigotes retained their morphological characteristics and infectivity to mice. If validated, this serum-free medium may help reduce future interlaboratory variability in biochemical, immunological, molecular biological and drug sensitivity studies on these parasites.

Growth inhibitory effect of bovine lactoferrin on Toxoplasma gondii tachyzoites in murine macrophages: role of radical oxygen and inorganic nitrogen oxide in Toxoplasma growth-inhibitory activity.

To study the effector pathway of Toxoplasma growth-inhibitory activity induced by lactoferrin in murine macrophage, the role of reactive oxygen intermediates (O2-) and inorganic nitric oxide (NO) was examined. Production of O2- was diminished in cultures of macrophages supplemented with lactoferrin and the effect of lactoferrin was dose and time dependent. Production of NO was enhanced in cultures of macrophages supplemented with interferon-gamma, but not with lactoferrin. These findings suggest that this Toxoplasma growth-inhibitory activity induced by lactoferrin in macrophages is not mediated by O2- or NO molecules. A competitive inhibitor of the L-arginine dependent effector pathway, NG-monomethyl-L-arginine (NG MMA), virtually abolished the inhibitory effects induced by interferon-gamma. Similarly, the inhibitory activity induced by lactoferrin was also diminished in cultures supplemented with NG MMA. From these findings, it appears that the Toxoplasma growth-inhibitory activity induced by lactoferrin in macrophages may be mediated by an L-arginine-dependent effector pathway that does not involve NO production.

Immunological characterization and expression in Escherichia coli and baculovirus systems of a Trypanosoma vivax antigen detected in the blood of infected animals.

A monoclonal antibody (MAb)Tv27 employed in an antigen-detection enzyme immunosorbent assay (Ag-ELISA) for diagnosis of Trypanosoma vivax infection was shown to react with a T. vivax-specific protein of an approximate molecular weight of 10 kDa. This protein is diffusely distributed throughout the cytosol and nucleus of metacyclic forms, bloodstream forms, and procyclic-like elongated trypomastigotes, but is not detectable in epimastigotes of T. vivax. The T. vivax-specific antigen prepared from parasite lysates appeared to be of lower molecular mass than the form expressed in either Escherichia coli or in baculovirus-infected silkworm insect cells. In the recombinant baculovirus-infected cells, the protein was expressed mostly as an 18-kDa peptide with less abundant forms of 13 and 12 kDa, while the protein expressed in E. coli was approximately 14 kDa. Both the low- and higher-molecular-weight proteins are recognized by the MAb Tv27 in Western blots and in Ag-ELISA. Although the crude preparations of the protein produced by the insect cells are labile when kept for more than 2 hr at 24 degrees C, they retained reactivity at temperatures below 4 degrees C for several weeks. The proteins expressed in both the insect cells and E. coli captured anti-T. vivax antibodies in sera prepared from trypanosome-infected animals. Since the recombinant protein expressed in the baculovirus-infected cells is available in large homogeneous quantities, it would serve as a positive control in Ag-ELISA and is also usable for antibody detection assays.

Expression of Trypanosoma congolense antigens in Spodoptera frugiperda insect cells.

Transcripts which encode two metacyclic-form-specific variable surface glycoproteins (mVSGs) of Trypanosoma congolense IL3000 have been cloned into baculovirus expression vectors using a novel transfer vector, pAcL11. One of the recombinant baculoviruses (AcVSG1) expressed a mVSG as a glycoprotein with a signal peptide which was cleaved in this expression system, whereas the other one (AcVSG2) expressed an unprocessed protein. From 1 liter of culture containing 10(9) Spodoptera frugiperda cells infected with the recombinant baculoviruses, 10 and 30 mg of mVSG1 and mVSG2, respectively, were obtained. Monospecific polyclonal antibodies produced by immunization of mice with the recombinant proteins reacted specifically with the respective proteins and showed no cross-reactivities between mVSG1 and mVSG2 in immunoblot assays. The antibodies to each of the proteins stained only the surface of a proportion of intact fixed T. congolense IL3000 metacyclic forms. It was possible to determine from these studies that, on the average, the parasites expressing mVSG1 constitute approximately 45% of the metacyclic population of T. congolense IL3000 maintained in in vitro cultures, whereas those that express mVSG2 constitute approximately 20%.

Axenic culture of African trypanosome bloodstream forms.

In this article, Hiroyuki Hirumi and Kazuko Hirumi review recent technical developments of in vitro systems that support the growth of bloodstream forms of African trypanosomes in the absence of mammalian feeder layer cells, which were required in earlier methods.

Fluorescence analysis of the interaction of isometamidium with Trypanosoma congolense.

Isometamidium chloride (Samorin) is the only compound recommended for prophylaxis against bovine trypanosomiasis in sub-Saharan Africa. The fluorescence property of this compound was used to investigate the interaction of the molecule with in vitro-derived bloodstream forms of Trypanosoma congolense IL 1180. Incubation of isometamidium with trypanosomes at 37 degrees C for 180 min resulted in a gradual alteration of the lambda max. with time (from 600 to 584 nm) and an increase in the intensity of trypanosome-associated fluorescence of approx. 2-fold. The alteration in fluorescence was temperature-dependent and inhibited by the addition of N-ethylmaleimide. In contrast, with intact cells addition of digitonin caused a rapid increase in fluorescence intensity to approximately four times that observed with intact cells. Uptake of isometamidium was also determined using radiolabelled drug; the results indicated that the time course of the uptake process resembled the fluorescence profile and was temperature-dependent. The results therefore indicate that the alteration of fluorescence is due to interaction of isometamidium with an intracellular component(s) and that isometamidium is transported across the plasma membrane via a protein carrier. The data also indicate that the described fluorescence technique can be used to investigate the role of membrane transport in resistance to isometamidium.

Trypanosoma brucei brucei: in vitro production of metacyclic forms.

An in vitro method has been established to obtain metacyclic form populations of Trypanosoma brucei brucei. Trypanosome populations containing more than 98% of metacyclic forms were obtained from cultures which were: 1) initiated with bloodstream forms in primary cultures in the presence of Microtus montanus embryonic fibroblast-like cells (feeder cell layers); 2) maintained in glucose-free Eagle's minimum essential medium supplemented with 10 mM L-proline, 2 mM L-glutamine and 20% (v/v) fetal bovine serum at 27 degrees C without medium change for five days; 3) subcultured in the absence of the feeder cell layers but in the presence of Cytodex 3 beads; 4) maintained for an additional nine days with medium changes on days 5, 8 and 11; and 5) harvested on day 14 by means of diethylaminoethyl cellulose column chromatography prior to the appearance of other infective forms. Most of the trypanosomes obtained under these conditions were morphologically similar to metacyclic forms derived from tsetse fly vectors, coated with variable surface glycoprotein and were infective for mice. In the primary cultures procyclic forms, epimastigotes and metacyclic forms appeared by day 8. When the duration of the subculture was prolonged to 17 days or more at 27 degrees C, the metacyclic forms decreased in number while short trypomastigotes, long slender epimastigotes, and long slender trypomastigotes increased in number. These forms in such long-term cultures also appeared in diethylaminoethyl cellulose-isolated populations along with metacyclic forms.

Metacyclic form-specific variable surface glycoprotein-encoding genes of Trypanosoma (Nannomonas) congolense.

A complementary DNA expression library in phage lambda gt11 was synthesized using mRNA from in vitro-produced metacyclic forms of a clone of Trypanosoma (Nannomonas) congolense. The unamplified library was screened with antiserum from a goat immune to infection with metacyclic (m)-forms of T. congolense ILRAD Nannomonas antigen repertoire 2(ILNaR2). Of the 100 antiserum-reactive phage clones identified, 22 were analyzed further: 21 of the clones contained overlapping portions of a single transcript, while one other contained a different transcript. Northern blot analyses indicated that the sequences contained in the clones were transcribed only by m-forms of ILNaR2. Immunological and sequence analyses indicated that the two different cloned sequences encode m-form-specific variable surface glycoproteins.

In vitro cultivation of Trypanosoma congolense bloodstream forms in the absence of feeder cell layers.

Bloodstream forms of Trypanosoma congolense (2 clones: ILNat3.1 and IL3000, and 4 stocks: IL2079, IL2466, IL3266 and CP-81) were continuously cultivated in vitro at 34-36 degrees C in the absence of feeder cell layers, using HMI-93 medium which was modified from Iscove's modified Dulbecco's MEM (Flow Laboratories, Irvine, Scotland). The modification was done by supplementing the medium with 0.05 mM bathocuproine sulphonate, 1.5 mM L-cysteine, 0.5 mM hypoxanthine, 0.12 mM 2-mercaptoethanol, 1 mM sodium pyruvate, 0.16 mM thymidine, 20% (v/v) heat-inactivated young goat serum and 5% (v/v) Serum Plus (Hazleton Biologics, Lenaxa, KS, USA). Trypanosomes obtained from two different sources were used to initiate primary cultures: (1) metacyclic forms which were produced in vitro at 27 degrees C, and (2) bloodstream forms obtained from Balb/c mice which had been infected with the bloodstream forms transformed in vitro from the metacyclic forms. Metacyclic forms placed in 25 cm2 T-type (T-25) flasks rapidly attached to the bottom of the flasks and transformed to bloodstream forms during the initial 24 h and continued to proliferate. The bloodstream forms isolated from the infected mouse blood by means of diethylaminoethyl cellulose (DE52) column chromatography also continued to proliferate in the flasks. Cultures were maintained by replacing the medium every 24 h. Every 4-5 days, the attached bloodstream forms were resuspended in fresh medium by gentle pipetting and then were subcultured. The method was further simplified by initiating primary cultures directly with 10 microliters of the tail blood of infected mice in 24-well culture plates and then by subcultivating either in wells or in T-25 flasks. The shortest population doubling time, 9 h, was achieved by seeding subcultures with 10(6) bloodstream forms/ml. The bloodstream forms propagated in this system were morphologically similar to those seen in infected mouse blood, they were covered with a surface coat as examined by electron microscopy and they were infective to mice.

Continuous cultivation of Trypanosoma brucei blood stream forms in a medium containing a low concentration of serum protein without feeder cell layers.

Blood stream forms (BSF) of Trypanosoma brucei brucei GUT at 3.1 were propagated in vitro in the absence of feeder layer cells at 37 C, using a modified Iscove's medium (HMI-18). The medium was supplemented with 0.05 mM bathocuproine sulfonate, 1.5 mM L-cysteine, 1 mM hypoxanthine, 0.2 mM 2-mercaptoethanol, 1 mM sodium pyruvate. 0.16 mM thymidine, and 20% (v/v) Serum Plus (SP) (Hazleton Biologics, Lenexa, Kansas). The latter contained a low level of serum proteins (13 micrograms/ml). Each primary culture was initiated by placing 3.5-4 x 10(6) BSFs isolated from infected mice in a flask containing 5 ml of the medium (HMI-9) supplemented with 10% fetal bovine serum (FBS) and 10% SP. The cultures were maintained by replacing the medium every 24 hr for 5-7 days. During this period, many BSFs died. However, from day 4 onward, long slender BSFs increased in number. On days 5-7, trypanosome suspensions were pooled and cell debris was removed by means of diethylaminoethyl cellulose (DE52) column chromatography. Blood stream forms then were collected by centrifugation, resuspended in fresh medium at 7-9 x 10(5)/ml, and transferred to new flasks. Subcultures were maintained by readjusting the BSF density to 7-9 x 10(5)/ml every 24 hr. Concentrations of FBS were reduced gradually at 5-7-day intervals by alternating the amounts of FBS and SP in HMI-9 with 5% FBS and 15% SP, with 2% FBS and 18% SP, and finally with 20% SP (HMI-18). By this method, 2-3 x 10(6) VSFs/ml were obtained consistently every 24 hr. for more than 80 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for diploidy in metacyclic forms of African trypanosomes.

The DNA contents of bloodstream form trypanosomes (life cycle stages circulating in the blood of the vertebrate host) of four African Trypanosoma species and of metacyclic forms (the life cycle stage that is injected into the vertebrate by the tsetse fly during its bite) of the same four species were measured by cytofluorometry of individual cells or nuclei. The results showed unambiguously that the metacyclic forms cannot be considered to be products of meiosis containing only half of the DNA of bloodstream forms, in contrast to what was previously reported for Trypanosoma brucei [Zampetti-Bosseler, F., Schweizer, J., Pays, E., Jenni, L. & Steinert, M. (1986) Proc. Natl. Acad. Sci. USA 83, 6063-6064] during an attempt to localize the gametes in the life cycle after experimental evidence of sexual gene exchange in this parasite was reported.

Factors influencing the duration of isometamidium chloride (Samorin) prophylaxis against experimental challenge with metacyclic forms of Trypanosoma congolense.

The duration of a single isometamidium chloride (Samorin) prophylactic treatment against Trypanosoma congolense ILNat. 3.1 and T. congolense IL 285 was examined in 24 Boran steers with regard to (1) the dose of drug, (2) the level of metacyclic challenge and (3) the influence of infection with an unrelated serodeme at the time of treatment. The cattle were repeatedly challenged at monthly intervals between 2 and 7 months following treatment, either by five infected Glossina morsitans centralis or by intradermal inoculation of 5 X 10(3) or 5 X 10(5) in vitro-derived metacyclic trypanosomes. A dose of 1 mg kg-1 afforded complete protection for 4 months and 0.5 mg kg-1 for 3 months against the two T. congolense serodemes examined, irrespective of the method or weight of challenge. In another group of cattle, which had an established infection at the time of treatment, the duration of chemoprophylaxis against an unrelated serodeme was the same as the other groups which had no previous experience of trypanosome infection. Antibodies to metacyclics did not appear in any of the cattle as long as the chemoprophylaxis was effective. An exception to this was the group challenged with 5 X 10(5) in vitro-derived metacyclic parasites, in which low antibody titres were detected. In all cases these proved to be non-protective. It was concluded that, under the experimental conditions employed, (1) there was a direct relationship between drug dosage and the duration of chemoprophylaxis, (2) the weight of metacyclic challenge did not affect the duration of chemoprophylaxis and (3) when used to treat an existing infection, isometamidium chloride exerted the same degree of chemoprophylactic activity.

Ro 15-0216: a nitroimidazole compound active in vitro against human and animal pathogenic African trypanosomes.

In vitro systems for the continuous cultivation of Trypanosoma brucei brucei, T. b. gambiense, T. b. rhodesiense, T. congolense and T. vivax were used to determine the antitrypanosomal activity of the 2-substituted nitroimidazole Ro 15-0216. For all trypanosome species, the concentration which inhibited parasite growth by 50% (IC50 value) was established: 0.0957 microgram ml-1 (T. b. brucei TC221), 0.1327 microgram ml-1 (T. b. gambiense STIB 754-A), 0.0450 microgram ml-1 (T. b. rhodesiense STIB 704-BABA), 0.0896 microgram ml-1 (T. congolense ILNat 3.1) and 0.0109 microgram ml-1 (T. vivax ILRAD 1392). The IC50 value of its major metabolite Ro 19-9638 was 0.0341 microgram ml-1 (T. b. rhodesiense STIB 704-BABA). Furthermore, minimum exposure times required to render T. b. brucei non-infective for mice as well as preventing their growth in vitro have been established to be three, four, six and ten hours at drug concentrations of 30, 10, 3 and 1 microgram ml-1, respectively.

Cell adhesion in Trypanosoma: in vitro studies of the interaction of Trypanosoma vivax with immobilized organic dyes.

Certain bloodstream forms of Trypanosoma vivax have been shown to attach to Amicon Matrex Gel Green A dye beads in a manner similar to the in vivo binding of T. vivax to the inner surface of the tsetse fly proboscis. We now report an in vitro assay for trypanosome-bead attachment and show that only the 9,10-anthraquinone portion of the dye molecule is involved in the binding of trypanosomes to beads and that bead-bound dyes with similar structures also support binding to differing degrees. The binding is dependent upon the amount of dye on the beads and this, and other evidence, suggests that an array of dye molecules, rather than individual molecules, may be the actual recognition site. Various external effectors, including temperature, soluble protein-dye complexes, and serum of mice with chronic T. vivax infections, reduce trypanosome binding, indicating that at least one immunogenic trypanosome macromolecule is involved. The trypanosome-bead interaction mimics the in vivo binding to tsetse proboscis and warrants closer examination as a model of trypanosome cell adhesion in the tsetse fly.

Trypanosoma (Nannomonas) congolense: properties of hexokinase and phosphofructokinase from cultured procyclic trypomastigotes and bloodstream forms.

The distribution and kinetics of two key glycolytic enzymes hexokinase (HK) and phosphofructokinase (PFK) were studied in animal-infective bloodstream forms (haematozoic trypomastigotes) and uninfective procyclic forms (insect trypomastigotes) of Trypanosoma congolense. The results show that in both forms of T. congolense HK and PFK are particulate and are probably localized in a membrane-delimited organelle, the glycosome. Hexokinases of bloodstream and procyclic forms of T. congolense are kinetically similar with respect to their affinity for glucose and ATP, the apparent Km for glucose being within the range, of 91 microM to 100 microM and that for ATP, 65 microM to 91 microM. Phosphofructokinase of both forms responds to its substrate in a complex manner: a plot of initial velocity versus substrate concentration displays intermediary plateau regions.