Recombinant expression and biochemical characterisation of two alanyl aminopeptidases of Trypanosoma congolense

Trypanosoma congolense is a haemoprotozoan parasite that causes African animal trypanosomosis, a wasting disease of cattle and small ruminants. Current control methods are unsatisfactory and no conventional vaccine exists due to antigenic variation. An anti-disease vaccine approach to control T. congolense has been proposed requiring the identification of parasitic factors that cause disease. Immunoprecipitation of T. congolense antigens using sera from infected trypanotolerant cattle allowed the identification of several immunogenic antigens including two M1 type aminopeptidases (APs). The two APs were cloned and expressed in Escherichia coli. As the APs were expressed as insoluble inclusion bodies it was necessary to develop a method for solubilisation and subsequent refolding to restore conformation and activity. The refolded APs both showed a distinct substrate preference for H-Ala-AMC, an optimum pH of 8.0, puromycin-sensitivity, inhibition by bestatin and amastatin, and cytoplasmic localisation. The two APs are expressed in procyclic metacyclic and bloodstream form parasites. Down-regulation of both APs by RNAi resulted in a slightly reduced growth rate in procyclic parasites in vitro.     

In Vitro Binding of Trypanosoma congolense to Erythrocytes*

Synopsis. Trypanosoma congolense Broden, an intravascular parasite, binds to vessel walls and erythrocytes of infected hosts. In an attempt to characterize T. congolense adhesion to host cells, an in vitro assay was devised. It was shown in the in vitro experiments that T. congolense binds to bovine, sheep, and goat erythrocytes, but not always to erythrocytes of rats, mice, rabbits, horses or humans. Only the anterior part of live trypanosomes adheres to erythrocytes, and the attachment site on the trypanosomes is destroyed by trypsin and chymotrypsin. Trypanosomes did not adhere to bovine erythrocytes that had been incubated with neuraminidase, sodium periodate and poly-L-lysine. The foregoing experiments suggest that the surface of T. congolense contains a protein-associated site which binds to sialic acid of some host cells. This surface site is most likely responsible for attachment to blood vessels in vivo.     

Trypanosoma congolense. 3. Serological responses of experimentally infected cattle

A complement fixation (CF) test for the diagnosis of Trypanosome congolense infection in cattle was developed and compared with the indirect fluorescent antibody (IFA) test.Serological investigations were made with cattle immunized with irradiated T. congolense and challenged with untreated T. congolense. Trypanosomal antibodies were demonstrated by the CF test. The results indicated good specificity and sensitivity between the CF method and the IFA test. The CF test also afforded easier reading of results than the IFA test; however, antigen preparation was more difficult in the former.The serological responses detected by the IFA and CF test appeared to be influenced by early variations of parasitemia, although no correlation appeared to exist between titer and parasitemia at later stages of the infection. Antibodies were detected in animals which received injections of irradiated T. congolense prior to challenge with viable organisms.     

In vitro proliferation of rabbit bone marrow-derived and thymus-derived lymphocytes in response to vaccinia virus

Peripheral blood leukocytes from rabbits immunized with vaccinia virus were incubated in vitro with vaccinia antigen, and resultant lymphocyte proliferation was measured by incorporation of tritiated thymidine into acid-insoluble material. Significant lymphocyte stimulation was observed at a time when antiviral antibody was being synthesized in vivo. The extent of proliferation by bone marrow-derived lymphocytes after culture with viral antigen was determined by simultaneous detection of complement receptor lymphocytes (CRLs have been shown to be B cells) and uptake of tritiated thymidine in these CRLs by radioautography. The results indicate that both bone marrow-derived and thymus-derived lymphocytes participate in the in vitro proliferative response of rabbit peripheral blood lymphocytes to vaccinia antigen.     

Structural characterization and epitope mapping of the glutamic acid/alanine-rich protein from Trypanosoma congolense: defining assembly on the parasite cell surface

Trypanosoma congolense is an African trypanosome that causes serious disease in cattle in Sub-Saharan Africa. The four major life cycle stages of T. congolense can be grown in vitro, which has led to the identification of several cell-surface molecules expressed on the parasite during its transit through the tsetse vector. One of these, glutamic acid/alanine-rich protein (GARP), is the first expressed on procyclic forms in the tsetse midgut and is of particular interest because it replaces the major surface coat molecule of bloodstream forms, the variant surface glycoprotein (VSG) that protects the parasite membrane, and is involved in antigenic variation. Unlike VSG, however, the function of GARP is not known, which necessarily limits our understanding of parasite survival in the tsetse. Toward establishing the function of GARP, we report its three-dimensional structure solved by iodide phasing to a resolution of 1.65 Å. An extended helical bundle structure displays an unexpected and significant degree of homology to the core structure of VSG, the only other major surface molecule of trypanosomes to be structurally characterized. Immunofluorescence microscopy and immunoaffinity-tandem mass spectrometry were used in conjunction with monoclonal antibodies to map both non-surface-disposed and surface epitopes. Collectively, these studies enabled us to derive a model describing the orientation and assembly of GARP on the surface of trypanosomes. The data presented here suggest the possible structure-function relationships involved in replacement of the bloodstream form VSG by GARP as trypanosomes differentiate in the tsetse vector after a blood meal.     

Plasmodium berghei: The in Vitro immune response

Following primary in vitro Stimulation by Plasmodium berghei, IgM titers by the indirect fluorescent antibody test (IFAT) were negative in in vitro reconstituted syngeneic mouse spleen cultures containing T cells and macrophages, or B cells and macrophages, or macrophages alone, but IgM titers of 1:20 were obtained from cultures containing B cells, T cells, and macrophages. IFAT IgG titers were negative for cultures with T cells and macrophages together, or macrophages alone, but rose to 1:40 with cultures containing B cells and macrophages and 1,80 with cultures of B cells, T cells, and macrophages together. After a second in vitro challenge, IgM and IgG titers were similar to the stimulated cultures from immunized mice; the IgM titer reached only 1:20 but the IgG titer rose to 1:160. Total immunoglobulin was higher in the cultures from immunized mice than from in vitro primed cultures.     

Identification of total and differentially expressed excreted-secreted proteins from Trypanosoma congolense strains exhibiting different virulence and pathogenicity

Animal trypanosomosis is a major constraint to livestock productivity in the tropics and has a significant impact on the life of millions of people globally (mainly in Africa, South America and south-east Asia). In Africa, the disease in livestock is caused mainly by Trypanosoma congolense, Trypanosoma vivax, Trypanosoma evansi and Trypanosoma brucei brucei. The extracellular position of trypanosomes in the bloodstream of their host requires consideration of both the parasite and its naturally excreted-secreted factors (secretome) in the course of pathophysiological processes. We therefore developed and standardised a method to produce purified proteomes and secretomes of African trypanosomes. In this study, two strains of T. congolense exhibiting opposite properties of both virulence and pathogenicity were further investigated through their secretome expression and its involvement in host-parasite interactions. We used a combined proteomic approach (one-dimensional SDS-PAGE and two-dimensional differential in-gel electrophoresis coupled to mass spectrometry) to characterise the whole and differentially expressed protein contents of secretomes. The molecular identification of differentially expressed trypanosome molecules and their correlation with either the virulence process or pathogenicity are discussed with regard to their potential as new diagnostic or therapeutic tools against animal trypanosomosis.     

Trypanosoma congolense: isolation and purification.

Yields of Trypanosoma congolense grown in rats may be increased by placing the rats in a 37 °C environment for 1 hr prior to sacrifice. A further increase in the number of parasites recovered per rat may be achieved by replacement of blood removed by a lactated Ringer's solution with 5% glucose as the rat is being bled from the abdominal aorta. The Ringer's solution serves to maintain intravascular volume during the bleeding procedure and thereby prevents premature cardiac arrest. Erythrocytes in infected blood may be then lysed by raising and rapidly lowering the osmolarity of the blood. This permits separation of the trypanosomes from 95% of the erythrocytes by differential centrifugation. The remaining blood cell contamination may then be removed on a small DEAE-cellulose column. The purified trypanosomes are motile, infective, and intact as judged by electron microscopy. More than 10¹⁰ purified T. congolense can be obtained from three adult rats by these methods.     

Cytotoxic effect of unilamellar detergent dialysis liposomes on Trypanosoma brucei and Trypanosoma congolense bloodstream forms in vitro

Liposomes from egg yolk lecithin and egg yolk lecithin/ganglioside are cytotoxic for Trypanosoma brucei and Trypanosoma congolense bloodstream forms in vitro. The trypanocidal effect is influenced by the liposome age and concentration. This effect is diminished in the presence of whole blood in vitro and could not be observed in vivo. Freeze-fractured parasite membrane showed an intramembranous particle aggregation after incubation with liposomes. Liposomes from egg yolk lecithin kill trypanosomes more rapidly than do liposomes from egg yolk lecithin/cholesterol.     

Immune Response to Toxoplasma gondii

Peripheral blood leukocytes (PBL) from patients with toxoplasmosis were shown to be highly responsive to in vitro stimulation with Toxoplasma gondii extract as measured by incorporation of [³H]methylated thymidine. Analysis of Toxoplasma-specific proliferative cells in PBL by using monoclonal antibodies specific for human T cell subsets revealed that the Toxoplasma-specific proliferation response of PBL from the patients was mediated by Leu 1, Leu 3a positive cells, that is, helper/inducer T cells. Tests for the Toxoplasma-specific proliferation response may provide a readily available method for the diagnosis of congenital toxoplasmosis, especially during the newborn period.     

Trypanosoma congolense: mechanical removal of the surface coat in vitro

By shaking suspensions of Trypanosoma congolense in isotonic buffer the surface coat could be separated from the cell body. The release of radioactivity from trypanosomes, selectively labeled in the surface coat by diazoniobenzenesulfonate, was used to follow the kinetics of coat detachment. The proteins in the supernatants of shaken trypanosomes were analyzed by sodium dodecyl sulfate—polyacrylamide gel electrophoresis. The shaking conditions had to be carefully controlled to avoid complete rupture of trypanosomes. Otherwise the coat protein was rapidly degraded by endogenous proteases. The influence of several parameters on the yield of coat release and the degree of degradation of the coat protein was investigated, including the ratio of trypanosome suspension volume to shaking vessel volume, vessel surface, temperature, shaking frequency, and preincubation of the trypanosomes at 0 C. By combining these parameters an optimal scheme was developed which allowed the separation of more than 90% of the coat protein from T. congolense, the detached protein showing no degradation at all. These results could be confirmed by electron microscopy of shaken and unshaken trypanosomes.     

Quantitative studies on trypanosomes in tsetse tissue culture

Tissues from pupae of Glossina morsitans of various ages were cultured in modified Trager's medium. Cellular outgrowths were produced from explants of proventriculus, brain, and imaginal body wall and large vesicles were extruded from pieces of midgut of young pupae. Complete alimentary tract from older pupae displayed rhythmic contractions for up to 3 weeks. When Trypanosoma brucei and T. congolense in mouse blood were added to hanging drop cultures of tsetse tissues and incubated at 28 C, the organisms multiplied and changed into forms morphologically similar to those found in the tsetse fly midgut. The trypanosomes were maintained for 30 days by serial passage at 5-day intervals. The growth of T. brucei in the presence of different pupal tissues was studied. Of all the tissues tested the complete alimentary tract from pupae older than 21 days gave the best results. Growth also occurred when the trypanosomes were separated from the insect tissue by a semipermeable membrane. The trypanosomes failed to grow in (a) culture medium alone, (b) media containing extracts of alimentary canal and (c) medium in which alimentary tract had been cultured for 3 or 4 days.     

Plasmodium berghei: Specific stimulation of rat lymphocytes by soluble antigens released in vitro from infected erythrocytes

Soluble components released in vitro from rat erythrocytes parasitized with Plasmodium berghei have been shown to stimulate nonadherent spleen lymphocytes from rats convalescent from homologous infection. The active product is released from the infected erythrocytes either spontaneously during incubation, or as the result of artificial rupture of infected erythrocytes by freezing and thawing or sonication. This antigen produced precipitation lines with antiplasmodial antibodies from sera of convalescent and hyperimmune rats and from immunized rabbits.     

Identification of a 4-fluorobenzyl l-valinate amide benzoxaborole (AN11736) as a potential development candidate for the treatment of Animal African Trypanosomiasis (AAT)

Novel l-valinate amide benzoxaboroles and analogues were designed and synthesized for a structure-activity-relationship (SAR) investigation to optimize the growth inhibitory activity against Trypanosoma congolense (T. congolense) and Trypanosoma vivax (T. vivax) parasites. The study identified 4-fluorobenzyl (1-hydroxy-7-methyl-1,3-dihydrobenzo[c][1,2]oxaborole-6-carbonyl)-l-valinate (5, AN11736), which showed IC₅₀ values of 0.15?nM against T. congolense and 1.3?nM against T. vivax, and demonstrated 100% efficacy with a single dose of 10?mg/kg against both T. congolense and T. vivax in mouse models of infection (IP dosing) and in the target animal, cattle, dosed intramuscularly. AN11736 has been advanced to early development studies.     

Complete In Vitro Life Cycle of Trypanosoma congolense: Development of Genetic Tools

Background

Animal African trypanosomosis, a disease mainly caused by the protozoan parasite Trypanosoma congolense, is a major constraint to livestock productivity and has a significant impact in the developing countries of Africa. RNA interference (RNAi) has been used to study gene function and identify drug and vaccine targets in a variety of organisms including trypanosomes. However, trypanosome RNAi studies have mainly been conducted in T. brucei, as a model for human infection, largely ignoring livestock parasites of economical importance such as T. congolense, which displays different pathogenesis profiles. The whole T. congolense life cycle can be completed in vitro, but this attractive model displayed important limitations: (i) genetic tools were currently limited to insect forms and production of modified infectious BSF through differentiation was never achieved, (ii) in vitro differentiation techniques lasted several months, (iii) absence of long-term bloodstream forms (BSF) in vitro culture prevented genomic analyses.

Methodology/Principal Findings

We optimized culture conditions for each developmental stage and secured the differentiation steps. Specifically, we devised a medium adapted for the strenuous development of stable long-term BSF culture. Using Amaxa nucleofection technology, we greatly improved the transfection rate of the insect form and designed an inducible transgene expression system using the IL3000 reference strain. We tested it by expression of reporter genes and through RNAi. Subsequently, we achieved the complete in vitro life cycle with dramatically shortened time requirements for various wild type and transgenic strains. Finally, we established the use of modified strains for experimental infections and underlined a host adaptation phase requirement.

Conclusions/Significance

We devised an improved T. congolense model, which offers the opportunity to perform functional genomics analyses throughout the whole life cycle. It represents a very useful tool to understand pathogenesis mechanisms and to study potential therapeutic targets either in vitro or in vivo using a mouse model.     

Modeling the control of trypanosomiasis using trypanocides or insecticide-treated livestock

Background

In Uganda, Rhodesian sleeping sickness, caused by Trypanosoma brucei rhodesiense, and animal trypanosomiasis caused by T. vivax and T. congolense, are being controlled by treating cattle with trypanocides and/or insecticides. We used a mathematical model to identify treatment coverages required to break transmission when host populations consisted of various proportions of wild and domestic mammals, and reptiles.

Methodology/Principal Findings

An Ro model for trypanosomiasis was generalized to allow tsetse to feed off multiple host species. Assuming populations of cattle and humans only, pre-intervention Ro values for T. vivax, T. congolense, and T. brucei were 388, 64 and 3, respectively. Treating cattle with trypanocides reduced R ₀ for T. brucei to <1 if >65% of cattle were treated, vs 100% coverage necessary for T. vivax and T. congolense. The presence of wild mammalian hosts increased the coverage required and made control of T. vivax and T. congolense impossible. When tsetse fed only on cattle or humans, R ₀ for T. brucei was <1 if 20% of cattle were treated with insecticide, compared to 55% for T. congolense. If wild mammalian hosts were also present, control of the two species was impossible if proportions of non-human bloodmeals from cattle were <40% or <70%, respectively. R ₀ was <1 for T. vivax only when insecticide treatment led to reductions in the tsetse population. Under such circumstances R ₀<1 for T. brucei and T. congolense if cattle make up 30% and 55%, respectively of the non-human tsetse bloodmeals, as long as all cattle are treated with insecticide.

Conclusions/Significance

In settled areas of Uganda with few wild hosts, control of Rhodesian sleeping sickness is likely to be much more effectively controlled by treating cattle with insecticide than with trypanocides.     

Trypanosoma congolense: surface glycoproteins of two early bloodstream variants. I. Production of a relapsing infection in rodents

A strain of Trypanosoma congolense has been cloned, passaged through the tsetse fly, and subsequently recloned. Relapsing infections have been induced in two rats by syringe passage of the cloned trypanosomes. The variant-specific glycoprotein of the initial cloned variant (VSG-1) and those from the two different variants produced in the two relapsing infections (VSG-2 and VSG-3) may be distinguished from each other by their isoelectric-focusing patterns. In this experimental system, cloned T. congolense, like Trypanosoma brucei, undergoes antigenic variation; the conversion of the VSG-1 into the VSG-2 isoelectric-focusing spectrotype was followed. These VSGs may be the products of sequentially expressed genes.     

Bovine babesiosis: Cattle protected in the field with a frozen vaccine containing Babesia bovis and Babesia bigemina cultured in vitro with a serum-free medium

An attenuated live vaccine containing Babesia bovis and B. bigemina cultured in vitro with a serum-free medium was assessed for its clinical protection conferred of naïve cattle, under natural tick-challenge in a high endemicity zone to Babesia spp. Three groups of six animals were treated as follows: group I (GI) received a vaccine derived from parasites cultured with a free-serum medium; group II (GII) were immunized with the standard vaccine, with parasites cultured in a medium supplemented with 40% (v/v) bovine serum; and a control group (GIII) inoculated with non-infected bovine erythrocytes. Inocula were administered by IM route. Experimental animals were kept during 23 days after vaccination in a cattle farm free of ticks and Babesia spp. Thereafter, cattle were moved to a high endemicity farm for natural exposure to Babesia spp. transmitted by Rhipicephalus microplus ticks. Protection against clinical babesiosis was observed in bovines belonging to GI (100%) and GII (83.33%), while the control animals (GIII) were not protected, and showed severe clinical signs, closely related to babesiosis, were observed for at least three consecutive days during the challenge. These were fever, anemia, which were measured simultaneously, and circulating parasites were detected by optic light microscopy. All cattle showed B. bovis and B. bigemina in stained blood films during the challenge; B. bovis antibody titers were higher than those to B. bigemina in GI and GII, and lower titers were determined in GIII. The protective capacity of the vaccine derived from B. bovis and B. bigemina cultured in vitro in a serum-free medium was demonstrated.     

The antitheilerial effects of Theileria parva parva reaction and recovery sera in vitro

Peripheral blood leucocytes (PBL) of cattle were infected in vitro with the sporozoites of Theileria parva spp. The transformed cell lines were adapted to grow in sera from the PBL donors. The cattle were then infected with T. p. parva stabilate and either treated with parvaquone or the disease allowed to run its course. Sera harvested during severe disease reaction or early recovery were substituted for pre infection sera and caused the intracellular degeneration of the Theileria macroschizonts. Cell lines passaged in these sera died out as the parasites were eliminated. The antiparasitic effects of sera were short lived and were neither host nor parasite isolate restricted.     

Trypanosoma vivax GM6 Antigen: A Candidate Antigen for Diagnosis of African Animal Trypanosomosis in Cattle

Background

Diagnosis of African animal trypanosomosis is vital to controlling this severe disease which hampers development across 10 million km² of Africa endemic to tsetse flies. Diagnosis at the point of treatment is currently dependent on parasite detection which is unreliable, and on clinical signs, which are common to several other prevalent bovine diseases.

Methodology/Principle Findings

the repeat sequence of the GM6 antigen of Trypanosoma vivax (TvGM6), a flagellar-associated protein, was analysed from several isolates of T. vivax and found to be almost identical despite the fact that T. vivax is known to have high genetic variation. The TvGM6 repeat was recombinantly expressed in E. coli and purified. An indirect ELISA for bovine sera based on this antigen was developed. The TvGM6 indirect ELISA had a sensitivity of 91.4% (95% CI: 91.3 to 91.6) in the period following 10 days post experimental infection with T. vivax, which decreased ten-fold to 9.1% (95% CI: 7.3 to 10.9) one month post treatment. With field sera from cattle infected with T. vivax from two locations in East and West Africa, 91.5% (95% CI: 83.2 to 99.5) sensitivity and 91.3% (95% CI: 78.9 to 93.1) specificity was obtained for the TvGM6 ELISA using the whole trypanosome lysate ELISA as a reference. For heterologous T. congolense field infections, the TvGM6 ELISA had a sensitivity of 85.1% (95% CI: 76.8 to 94.4).

Conclusion/Significance

this study is the first to analyse the GM6 antigen of T. vivax and the first to test the GM6 antigen on a large collection of sera from experimentally and naturally infected cattle. This study demonstrates that the TvGM6 is an excellent candidate antigen for the development of a point-of-treatment test for diagnosis of T. vivax, and to a lesser extent T. congolense, African animal trypanosomosis in cattle.