Trypanosoma congolense: Specific transformation in vitro of leukocytes from infected or immunized cattle

An assay to measure the specific proliferation in vitro of peripheral blood leukocytes (PBL) in response to ultrasonicated trypanosomes was adapted for use in cattle. The kinetics of mitosis exhibited by PBL from cattle which had been treated following infection with Trypanosoma congolense paralleled the development of a delayed-type skin reaction elicited with ultrasonicated and Formalin-fixed T. congolense. Responses in both tests were maximal on the fourth day after initiation. Specific proliferation of PBL harvested from cattle which had been immunized with intact, nonviable trypanosomes was also elicited in vitro by trypanosomal antigen. Peripheral blood leukocytes taken from cattle immunized with 50 μg of variant-specific surface antigen (VSSA) from T. brucei and from cattle infected with T. congolense were not stimulated to divide in vitro by ultrasonicated trypanosomes.     

A culture and biochemical assay system for Trypanosoma lewisi

Trypanosoma lewisi was cultivated as forms which appeared to be physiologically similar to those found in vivo. The medium consisted of 1.0 g peptone, 1.0 g glucose, 10 ml rat serum, 10,000 units penicillin G, 10,000 μg streptomycin and 90 ml Hank's Balanced Salt Solution. It was supplemented with 8.0 × 10⁸ rat erythrocytes per milliliter. In the complete medium trypanosomes multiplied for 48–72 hr. Cultured forms were lethal to newborn rats and infective to adults.Adsorbed early immune serum inhibited the growth of the trypanosomes in vitro and the percentage of reproductives declined from 66 to 45%. The cultured trypanosomes were also susceptible to both trypanocidal antibodies.     

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.     

The ablastin phenomenon: inhibition of membrane function.

Autoradiography of Trypanosoma lewisi labeled in vivo with ³H-thymidine (³HTdR) shows that the shortest doubling time for labeled organisms is 8 hr in intact and immunosuppressed rats. The parasite doubling time increases progressively after the fourth day of infection to 12 hr in immunosuppressed rats and to 24 hr or more in intact rats. The number of days following infection during which the trypanosomes reproduce is prolonged in immunosuppressed rats. In vitro studies of ablastin using ³HTdR-labeled trypanosomes confirmed that cell reproduction halts in the presence of ablastin, but resumes when the parasites are removed from the antibody. Several lines of evidence have been obtained, indicating that the primary effects of ablastin may be on membrane function. Thus, the saturable component for glucose transport in reproducing and ablastin inhibited trypanosomes has an average K_m value of 2.8 × 10^?4M, but the average V_max values for glucose transport are reduced from 3.15 nmole/min/1.25 × 10⁷ reproducing parasites to an average of 1.8 nmole/min/1.25 × 10⁷ nonreproducing forms. Glucose transport is competitively inhibited by 2-deoxyd-glucose (2DOG). The exit and counterflow of ¹⁶C-2DOG from previously loaded trypanosomes is restricted in the presence of antiserum.     

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: 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.     

Diversity of trypanosome species in small ruminants,dogs and pigs from three sleeping sickness foci of the south of Chad

Despite considerable data generated on livestock trypanosomoses in tsetse-infested areas, little attention was paid for animal African trypanosomosis (AAT) in sleeping sickness foci. This study aimed to fill this gap by determining the diversity and prevalence of trypanosome species in animals from three Chadian human African trypanosomosis (HAT) foci. Blood samples were collected from 443 goats, 339 sheep, 228 dogs and 98 pigs of the Mandoul, Maro and Moissala HAT foci in the south of Chad. Capillary tube centrifugation (CTC) and specific primers were used to search trypanosomes. The prevalence of trypanosome infections was 6.3% for CTC and 22.7% for PCR. Trypanosomes of the sub-genus Trypanozoon had the highest prevalence (16.6%) while T. congolense savannah (1.9%) was least prevalent. Significant differences were recorded between the prevalence of trypanosome species (χ² = 8.34; p = 0.04) and HAT foci (χ² = 24.86; p ≤0.0001). Maro had the highest prevalence (32.7%) and Mandoul the lowest (17.4%). Significant differences were also recorded for T. congolense forest (χ² = 45.106; p < 0.0001) and all T. congolense (χ² = 34.992; p < 0.0001). Goats had the highest prevalence (26.9%) and sheep the lowest one (18.6%). Between animals, significant differences were recorded for trypanosomes of the sub-genus Trypanozoon (χ² = 9.443; p = 0.024), T. congolense forest (χ² = 10.476; p = 0.015) and all T. congolense (χ² = 12.152; p = 0.007). Of the 251 animals carrying trypanosome infections, 88.8% had single infections while 11.2% had more than one trypanosome species. The overall prevalence of single and mixed trypanosome infections were respectively 20.1% and 2.6% in animal taxa of all foci. This study highlighted a diversity of trypanosomes in animal taxa of all HAT foci. It showed that AAT constitutes a threat for animal health and animal breeding in Chadian HAT foci. In these tsetse infested areas, reaching the elimination of AAT requires the designing and the implementation of control measures against trypanosome infections.     

Growth and Immunologic Studies on the Culture Forms of Trypanosoma lewisi*

SYNOPSIS. Culture forms of Trypanosoma lewisi grown at 27 C in a diphasic blood agar medium resemble in structure the stage found in the invertebrate host. Cultures inoculated with approximately 1 × 10⁶ trypanosomes/ml attain maximum populations of 2–7 × 10⁷ organisms/ml after 5–6 days of incubation. The stationary phase persists 6–15 days. The decline of the population is of relatively long duration with approximately 1 × 10⁶ viable organisms/ml present after 90 days. Variations in growth were attributed to the preparation of defibrinated heated rabbit blood incorporated into the culture medium. With inocula of 3.0 × 10⁵ trypanosomes/ml there was a lag in growth not observed with larger inocula. Trypanosomes incubated at elevated temperatures had altered growth curves compared to organisms at 27 C. Agitation of cultures did not affect the growth or stationary phases, but hastened the population decline. Heated and unheated 5% (v/v) normal rat serum incorporated in the liquid phase of the medium altered the growth of the organisms. Heated serum caused a decrease in the population and an extended lag phase. The effects on growth were more marked with unheated serum suggesting that both heat-stable and labile components affect growth. Antisera from rats injected with live culture forms included in the liquid phase inhibited, while antisera from rats 24 days after infection with the blood stream forms had no effect on the growth of the culture forms. Antisera from rabbits immunized with sonicates of culture forms also altered the growth of the organisms in culture. Rabbit antisera prepared by immunization with sonicates of dividing and non-dividing blood stream forms had no effect on the in vitro growth. Antisera from animals immunized with rat blood and culture medium were also without effect. The immunologic implications of the data are considered and discussed.     

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.     

Binding of Trypanosoma congolense to the Walls of Small Blood Vessels*

SYNOPSIS The mesenteric microvasculature was studied in rats and rabbits infected with Trypanosoma congolense. By examining vessels in the living animals, trypanosomes were observed to adhere to vessel walls by their anterior ends. It was evident from stained preparations of the vessels that the microcirculation contained 4–1400 times as many trypanosomes as were free in the cardiac blood. Parasites were more numerous in very small vessels than in larger vessels, and they were clustered in groups within the small vessels. The localization of T. congolense in the microvasculature is demonstrated and it is shown that this localization is established by attachment of the organism to the vessel wall.     

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.     

Patterns in Age-Seroprevalence Consistent with Acquired Immunity against Trypanosoma brucei in Serengeti Lions

Trypanosomes cause disease in humans and livestock throughout sub-Saharan Africa. Although various species show evidence of clinical tolerance to trypanosomes, until now there has been no evidence of acquired immunity to natural infections. We discovered a distinct peak and decrease in age prevalence of T. brucei s.l. infection in wild African lions that is consistent with being driven by an exposure-dependent increase in cross-immunity following infections with the more genetically diverse species, T. congolense sensu latu. The causative agent of human sleeping sickness, T. brucei rhodesiense, disappears by 6 years of age apparently in response to cross-immunity from other trypanosomes, including the non-pathogenic subspecies, T. brucei brucei. These findings may suggest novel pathways for vaccinations against trypanosomiasis despite the notoriously complex antigenic surface proteins in these parasites.     

A Cell-surface Phylome for African Trypanosomes

The cell surface of Trypanosoma brucei, like many protistan blood parasites, is crucial for mediating host-parasite interactions and is instrumental to the initiation, maintenance and severity of infection. Previous comparisons with the related trypanosomatid parasites T. cruzi and Leishmania major suggest that the cell-surface proteome of T. brucei is largely taxon-specific. Here we compare genes predicted to encode cell surface proteins of T. brucei with those from two related African trypanosomes, T. congolense and T. vivax. We created a cell surface phylome (CSP) by estimating phylogenies for 79 gene families with putative surface functions to understand the more recent evolution of African trypanosome surface architecture. Our findings demonstrate that the transferrin receptor genes essential for bloodstream survival in T. brucei are conserved in T. congolense but absent from T. vivax and include an expanded gene family of insect stage-specific surface glycoproteins that includes many currently uncharacterized genes. We also identify species-specific features and innovations and confirm that these include most expression site-associated genes (ESAGs) in T. brucei, which are absent from T. congolense and T. vivax. The CSP presents the first global picture of the origins and dynamics of cell surface architecture in African trypanosomes, representing the principal differences in genomic repertoire between African trypanosome species and provides a basis from which to explore the developmental and pathological differences in surface architectures. All data can be accessed at: http://www.genedb.org/Page/trypanosoma_surface_phylome.     

Trypanosoma brucei: An evaluation of salicylhydroxamic acid as a trypanocidal drug

The chemotherapeutic potential of salicylhydroxamic acid (SHAM) was studied in adult rats infected with a strain of Trypanosoma brucei that kills the rats in about 100 hr. The median lethal dose, administered intraperitoneally in a carboxymethyl-cellulose suspension, is approximately 820 mg/kg body weight for male and 850 mg/kg for female rats. The apparent cause of death is severe depression of the central nervous system.Half-maximal inhibition of O₂ uptake by trypanosomes in vitro requires 15 μM SHAM, whereas 100 μM inhibits over 90%. This inhibitory effect on trypanosome respiration was used as a biological assay for the effective SHAM concentration in rat plasma. After administration of a sublethal SHAM dose to rats, the effective plasma SHAM concentration rose rapidly to about 500 μM and then fell to about 10 μM at 4 hr. Nevertheless, this dose did not significantly affect the survival time of rats infected with T. brucei. Even if, by repeated SHAM administration, the plasma SHAM concentration was kept at around 100 μM for more than 4 hr, no therapeutic effect was observed.These results show that O₂ uptake is not essential for the survival of trypanosomes in rats and they support the idea that bloodstream trypanosomes have an alternative pathway for glycolysis, allowing energy production in the absence of respiration.The possibility that SHAM or other inhibitors of trypanosome respiration could stilll be trypanocidal if used in conjunction with another inhibitor of glycolysis is discussed.     

Immunologic-mediated Protection of Trypanosoma congolense-Infected Mice by Polyribonucleotides

SYNOPSIS. When the synthetic polyribonucleotides polyinosinic acid-polycytidylic acid (poly I poly C) and polyadenylic acid-polyuridylic acid (poly A poly U) were tested against mice infected with varying numbers of Trypanosoma congolense the results varied with the method of passage of trypanosomes in mice. Thus, when 100 flagellates were passaged every 7th day and experiments were initiated with these trypanosomes from mice on the 7th day of their infection, the protective effects of poly I poly C and poly A poly U apparently varied independently of each other as assayed by the mean parasitemias and cumulative mortalities of infected mice. Poly I poly C-resistant and poly I poly C-susceptible variants (“R” and “S”, respectively) were isolated and maintained in mice by passage of 10⁶ trypanosomes every 4th day. Mice infected with these variants responded consistently to poly I poly C and poly A poly U injections in that mice infected with the “R” variant showed no response to either polyribonucleotide but those infected with the “S” variant consistently had a decrease in mean parasitemias and cumulative mortality when treated with poly I poly C, but not with poly A poly U. Using mice infected with the “S” variant, the protective effect of poly I poly C was dose-dependent and best protection was afforded when 1st injections of poly I poly C were given around the time of infection of the mice. The protective effects of the synthetic polyribonucleotides used in these experiments are probably due to their immunologic enhancing capacities and not to interferon. To support this, injections of Newcastle disease virus, a strong inducer of interferon in mice, did not protect against T. congolense in mice. It was not possible to determine whether serum from poly I poly C-treated mice had a greater neutralizing effect upon trypanosomes in vitro than serum from saline-treated mice since any effect of antibody was masked by a naturally occurring inhibitor in normal mouse serum which was reduced during infection. The protective effects of poly I poly C in T. congolense-infected mice were reversed by treatment with cyclophosphamide. This strong immunosuppressant, however, could not reverse the protective effects of poly I poly C against mice infected with Semliki Forest virus, strongly suggesting that the protective mechanisms stimulated by poly I poly C in these 2 infections were different. The response of mice infected with the “R” and “S” variants of T. congolense to synthetic polyribonucleotides is discussed in relation to antigenic variation of trypanosomes.     

Improved monoclonal antibody tumor/background ratios with exchange transfusions

Blood exchange transfusions were performed in nude rats with subcutaneous HTB77 human ovarian carcinoma xenografts in an attempt to improve specific monoclonal antibody (MoAb) tumor/non-tumor uptake ratios. Animals were injected intravenously with both ¹³¹I-5G6.4 specific and ¹²⁵I-UPC-10 non-specific MoAb. Twenty-four hours later 65–80% of the original blood was exchanged with normal heparinized rat blood and then these rodents were sacrificed. Exchange transfusion significantly (P < 0.05) decreased normal tissue activities of ¹³¹I (except for muscle) by 63–85%. while tumor activity decreased only 5%. Tumor to background ratios increased from 0.1–0.8 to 2.3–6.3. Exchange transfusions substantially enhance tumor/normal tissue antibody uptake ratios and, along with plasmapheresis, may be useful in enhancing antibody localization in vivo, particularly for therapy.     

Development of Real Time PCR to Study Experimental Mixed Infections of T. congolense Savannah and T. b. brucei in Glossina morsitans morsitans

Tsetse flies are able to acquire mixed infections naturally or experimentally either simultaneously or sequentially. Traditionally, natural infection rates in tsetse flies are estimated by microscopic examination of different parts of the fly after dissection, together with the isolation of the parasite in vivo. However, until the advent of molecular techniques it was difficult to speciate trypanosomes infections and to quantify trypanosome numbers within tsetse flies. Although more expensive, qPCR allows the quantification of DNA and is less time consuming due to real time visualization and validation of the results. The current study evaluated the application of qPCR to quantify the infection load of tsetse flies with T. b. brucei and T. congolense savannah and to study the possibility of competition between the two species. The results revealed that the two qPCR reactions are of acceptable efficiency (99.1% and 95.6%, respectively), sensitivity and specificity and can be used for quantification of infection load with trypanosomes in experimentally infected Glossina morsitans morsitans. The mixed infection of laboratory Glossina species and quantification of the infection suggests the possibility that a form of competition exists between the isolates of T. b. brucei and T. congolense savannah that we used when they co-exist in the fly midgut.     

Trypanostatic activity of rat IgG purified from the surface coat of Trypanosoma lewisi

Host IgG is a component of the surface coat of Trypanosoma lewisi; it is specifically acquired during infection in the rat, concomitant with a rise in titer of trypanostatic (ablastic) activity of host serum. Host IgG was eluted from trypomastigotes at 7 to 9 days postinfection with a high salt-low pH buffer. Surface coats and trypanosome ultrastructure were not notably altered by the elution procedure, as determined by electron microscopy. Rat IgG was removed and purified from the trypanosome eluates on an immunoadsorbent column made with the IgG fraction of anti-rat IgG serum coupled to Sepharose beads. Concentrated column eluates, by comparison with a standard, were shown to be rat IgG by immunoelectrophoresis and SDS polyacrylamide gel electrophoresis. As a control, IgG from normal rat serum was purified by the same technique. IgG-negative trypanosomes harvested from immunosuppressed rats bound IgG purified from surface coats of trypanosomes, but not IgG purified from normal rat serum, as demonstrated by subsequent labelling with FITC-conjugated, rabbit anti-rat IgG. The IgG purified from surface coats inhibited the reproduction of T. lewisi in an in vitro assay, but purified, normal IgG did not. These data show that antigen-specific host IgG, adsorbed to the surface of T. lewisi, is ablastic antibody.     

The B Cell Adaptor Molecule Bam32 Is Critically Important for Optimal Antibody Response and Resistance to Trypanosoma congolense Infection in Mice

Background

Bam32, a 32 kDa adaptor molecule, plays important role in B cell receptor signalling, T cell receptor signalling and antibody affinity maturation in germinal centres. Since antibodies against trypanosome variant surface glycoproteins (VSG) are critically important for control of parasitemia, we hypothesized that Bam32 deficient (Bam32-/-) mice would be susceptible to T. congolense infection.

Methodology/Principal Findings

We found that T. congolense-infected Bam32^-/- mice successfully control the first wave of parasitemia but then fail to control subsequent waves and ultimately succumb to their infection unlike wild type (WT) C57BL6 mice which are relatively resistant. Although infected Bam32^-/- mice had significantly higher hepatomegaly and splenomegaly, their serum AST and ALT levels were not different, suggesting that increased liver pathology may not be responsible for the increased susceptibility of Bam32^-/- mice to T. congolense. Using direct ex vivo flow cytometry and ELISA, we show that CD4+ T cells from infected Bam32^-/- mice produced significantly increased amounts of disease-exacerbating proinflammatory cytokines (including IFN-γ, TNF-α and IL-6). However, the percentages of regulatory T cells and IL-10-producing CD4+ cells were similar in infected WT and Bam32^-/- mice. While serum levels of parasite-specific IgM antibodies were normal, the levels of parasite-specific IgG, (particularly IgG1 and IgG2a) were significantly lower in Bam32^-/- mice throughout infection. This was associated with impaired germinal centre response in Bam32^-/- mice despite increased numbers of T follicular helper (Tfh) cells. Adoptive transfer studies indicate that intrinsic B cell defect was responsible for the enhanced susceptibility of Bam32^-/- mice to T. congolense infection.

Conclusions/Significance

Collectively, our data show that Bam32 is important for optimal anti-trypanosome IgG antibody response and suppression of disease-promoting proinflammatory cytokines and its deficiency leads to inability to control T. congolense infection in mice.