Kinetoplast DNA and molecular karyotypes of Trypanosoma evansi and Trypanosoma equiperdum from China

We compared 12 stocks of Trypanosoma evansi and 1 recently isolated stock of Trypanosoma equiperdum from different regions of China by analysis of kinetoplast DNA (kDNA), nuclear DNA and molecular karyotypes. The T. equiperdum stock was remarkably similar to the T. evansi stocks, except for the possession of kDNA maxi-circles, suggesting a very close evolutionary relationship between T. evansi and T. equiperdum. The maxi-circles of the Chinese T. equiperdum stock were approximately 14.3 kb in size, i.e., about half the size of those of Trypanosoma brucei. This stock is thus similar to an old laboratory stock of T. equiperdum, which also has maxi-circles with a sizeable deletion. Both T. equiperdum and T. evansi kDNA mini-circles hybridised with a T. evansi-specific mini-circle fragment isolated from a Kenyan T. evansi stock. Our results extend the generality that T. evansi and T. equiperdum mini-circles are microheterogeneous rather than homogeneous. Molecular karyotypes obtained by pulsed field gradient gel electrophoresis provided a more sensitive way of distinguishing the T. evansi stocks than isoenzymes or restriction fragment length polymorphisms in kDNA mini-circles, genes for ribosomal RNAs and variant surface glycoproteins. Our results fit the general idea that T. evansi stocks worldwide have a single origin.     

Molecular profiles of<Emphasis Type="Italic"> Trypanosoma brucei</Emphasis>,<Emphasis Type="Italic"> T. evansi</Emphasis> and<Emphasis Type="Italic"> T. equiperdum</Emphasis> stocks revealed by the random amplified polymorphic DNA method

A total of 20 random primers (10-mers) were used to amplify RAPD markers from the genomic DNA of four Trypanosoma brucei stocks from East and West Africa, four T. evansi stocks from Africa, Asia and South America and one T. equiperdum stock from Asia. Between 65 and 88 reproducible fragments ranging from 0.25 to 2.15 kb were generated from these stocks depending on the stock/primer combination. The similarity coefficient (SC) among the stocks of T. brucei from Kenya, Nigeria, Tanzania and Zambia ranged from 62.9% to 74.0% (average: 67.6%). The SC among the stocks of T. evansi from Kenya, China and Brazil was 76.4%–95.5% (average: 86.4%), while the SC between T. evansi stock from China and Brazil was 95.5%. For T. evansi and T. equiperdum, the SC among the stocks ranged from 81.2% to 94.4% (average: 87.6%). As for the SC among the stocks of T. brucei and T. evansi, it was found to be from 54.7% to 80.3% (average: 68.0%) and the SC among stocks of T. brucei and T. equiperdum was from 59.4% to 76.9% (average: 68.1%). Our results indicate that the stocks of T. evansi from China and from Brazil are more closely related to the stock of T. equiperdum from China than to the stocks of T. evansi isolated from Kenya and to the stocks of T. brucei. In addition, our results further support the hypothesis that T. evansi stocks from China and Brazil could have arisen from a single lineage. The possible evolution of T. evansi and T. equiperdum is also discussed.     

High immunological response against a Trypanosoma equiperdum protein that exhibits homology with the regulatory subunits of mammalian cAMP-dependent protein kinases

Previously, we have identified a protein in Trypanosoma equiperdum that possesses homology with the regulatory (R) subunits of the mammalian cAMP-dependent protein kinase (PKA). The recombinant T. equiperdum PKA R-like protein was expressed in bacteria and purified to homogeneity. Mice polyclonal antibodies were raised against the recombinant R-like protein to serologically evaluate its humoral immune response. High titers of specific sera antibodies were obtained against the parasite R-like protein by indirect enzyme-linked immunosorbent assay (ELISA), and immunoblots revealed that this protein was specifically recognized by the hyperimmune mice sera. Cellular proliferation assays using splenic B cells from the immunized mice showed higher values when the recombinant T. equiperdum R-like protein was employed than when concanavalin A was utilized as an unspecific mitogen. Two healthy horses that were experimentally infected using either T. equiperdum or Trypanosoma evansi showed a curve response characterized by the appearance of anti-T. equiperdum PKA R-like protein antibody production in sera using indirect ELISA. The recombinant parasite PKA R-like protein was also recognized by sera from naturally trypanosome-infected horses using western blotting. These findings demonstrated that the T. equiperdum PKA R-like protein is an antigen that exhibits cross-reaction with T. equiperdum and T. evansi.     

Multinuclear forms in a dyskinetoplastic strain of Trypanosoma evansi in mice

The production of short stumpy and multinuclear trypanosomes in a Chinese strain of dyskinetoplastic Trypanosoma evansi maintained in rabbits and mice is described. Production of multinuclear trypanosomes was increased following passage through a reptile (gecko), in which the trypanosomes did not multiply, and transfer back to mice. The multinuclear trypanosomes showed more nuclei than flagella indicating that disruption of the normal cell cycle had taken place and not simply inhibition of cleavage. A Chinese kinetoplastic T. evansi treated similarly rarely produced stumpy or multinuclear forms.     

Identification and characterization of cysteine proteinases of Trypanosoma evansi

Trypanosoma evansi is a causative agent of ‘surra’, a common haemoprotozoan disease of livestock in India causing high morbidity and mortality in disease endemic areas. The proteinases released by live and dead trypanosomes entail immunosuppression in the infected host, which immensely contribute in disease pathogenesis. Cysteine proteinases are identified in the infectious cycle of trypanosomes such as cruzain from Trypanosoma cruzi, rhodesain or brucipain from Trypanosoma brucei rhodesiense and congopain from Trypanosoma congelense. These enzymes localised in lysosome-like organelles, flagellar pocket and on cell surface, which play a critical role in the life cycle of protozoan parasites, viz. in host invasion, nutrition and alteration of the host immune response. The paper describes the identification of cysteine proteinases of T. evansi lysate, activity profile at different pH optima and inhibition pattern using a specific inhibitor, besides the polypeptide profile of an antigen. Eight proteinases of T. evansi were identified in the molecular weight (MW) ranges of 28–170 kDa using gelatin substrate-polyacrylamide gel electrophoresis (GS-PAGE), and of these proteinases, six were cysteine proteinases, as they were inhibited by L-3-carboxy-2,3-transepoxypropionyl-lecuylamido (4-guanidino)-butane (E-64), a specific inhibitor. These proteolytic enzymes were most reactive in acidic pH between 3.0 and 5.5 in the presence of dithiothreitol and completely inactive at alkaline pH 10.0. Similarly, the GS-PAGE profile of the serum samples of rats infected with T. evansi revealed strong proteolytic activity only at the 28-kDa zone at pH 5.5, while no proteolytic activity was observed in serum samples of uninfected rats. Further, the other zones of clearance, which were evident in T. evansi antigen zymogram, could not be observed in the serum samples of rats infected with T. evansi. The polypeptide pattern of the whole cell lysate antigen revealed 12–15 polypeptide bands ranging from 28 to 81 kDa along with five predominant polypeptides bands (MW of 81, 66, 62, 55 and 45 kDa), which were immunoreactive with hyperimmune serum (HIS) and serum of experimentally infected rabbits with T. evansi infection. The immunoblot recognised antibodies in experimentally infected rabbits and against HIS as well, corresponding to the zone of clearances at lower MW ranges (28–41 kDa), which may be attributed to the potential of these proteinases in the diagnosis of T. evansi infection. Since these thiol-dependent enzymes are most active in acidic pH and considering their inhibition characteristics, these data suggest that they resemble to the mammalian lysosomal cathepsin B and L.     

Alteration to one of three adenosine transporters is associated with resistance to Cymelarsan in Trypanosoma evansi

 Continuous exposure of Trypanosoma evansi bloodstream forms to Cymelarsan in vitro resulted in the induction of resistance to the drug over a period of 4.5 months. Induction of resistance to Cymelarsan was accompanied by increased resistance to both Arsobal and Berenil, but Cymelarsan-resistant trypanosomes remained sensitive to Suramin and Antrycide. Since resistance to arsenical drugs in trypanosomes has been linked to changes in adenosine uptake, the adenosine metabolism in drug-sensitive and drug-resistant clones was measured. The initial rate of [³H]-adenosine uptake was much higher in sensitive trypanosomes than in drug-resistant parasites, but the amount of radiolabel accumulated by each population was similar. Both adenine and inosine inhibited incorporation of [³H]-adenosine in each population, but in quite different ways. Adenine inhibited more than 80% of adenosine incorporation in drug-sensitive trypanosomes but suppressed less than half of this process in the resistant population. In contrast, inosine inhibited only 10–15% of adenosine incorporation in the sensitive parasites but was inhibitory to a much greater extent in resistant trypanosomes. Lysis of drug-sensitive trypanosomes by 1 μM Cymelarsan was inhibited by adenosine, adenine and Berenil but not by inosine. Furthermore, in drug-sensitive trypanosomes, adenosine uptake could be reduced in three stages by the sequential addition of inosine, Berenil and adenine, whereas in drug-resistant parasites a reduction in adenosine uptake was caused only by the addition of inosine and adenine. These observations provide evidence that the acquisition of resistance to Cymelarsan is accompanied by the loss of one of three adenosine transporters in T. evansi. In drug-sensitive trypanosomes, this transporter mediates the entry of adenine and also of Cymelarsan and Berenil into the parasites. Received: 10 July 1995 / Accepted: 16 September 1995     

Specific probes for Trypanosoma (Trypanozoon) evansi based on kinetoplast DNA minicircles

Trypanosoma evansi is difficult to distinguish from other members of subgenus Trypanozoon, save for its inability to develop cyclically in the tsetse fly and its characteristic kinetoplast DNA (kDNA). We have used cloned kDNA minicircle fragments as specific probes to distinguish T. evansi from other trypanosomes of subgenus Trypanozoon. Two probes were required, each specific for one of the subgroups of T. evansi previously described. Probe A reacted only with the major isoenzyme group of T. evansi stocks, which have minicircle type A and occur in South America, Kenya, Sudan, Nigeria and Kuwait. The probe did not hybridise with various Trypanosoma brucei spp. stocks, Trypanosoma vivax, Trypanosoma congolense or Trypanosoma simiae, nor with trypanosomes of the minor isoenzyme group of T. evansi stocks found in Kenya with type B minicircles. Probe B was specific for the latter. The probes were sensitive down to a level of 100 trypanosomes in a dot blot. These probes thus provide a simple means of distinguishing T. evansi from T. brucei spp. using comparatively few trypanosomes and without resort to tsetse transmission experiments.     

Kinetoplast DNA analysis of four Trypanosoma evansi strains.

Kinetoplast DNA (kDNA), the mitochondrial DNA of trypanosomes, is a network of thousands of topologically interlocked DNA minicircles and about 50 maxicircles. In this study, we have analysed the kDNA molecules of 6 strains of Trypanosoma evansi from different geographical areas. 2 strains were found to be dyskinetoplastic mutants and other 4 kinetoplastic strains absent of maxicircles. The electrophoretic analysis of the minicircles digested with various restriction endonucleases clearly shows that all of the kinetoplastic strains lack profound minicircle heterogeneity typical of T. brucei. However, a slight restriction fragment length polymorphism could be observed with 2 enzymes (Dde I and HinfI) within the minicircle population of each cloned strain. We propose that this sequence diversity is the result of point mutations. Further analysis of the minicircles by nucleotide sequencing revealed that the 4 minicircles of T. evansi strains share extensive regions of homology with each other but only about 50% homology with other species. This homogeneity of T. evansi minicircle sequences may provide a useful tool for classification and identification.     

Thrombocytopenia and increased clotting time in rats acutely infected by Trypanosoma evansi

The aims of this study were to evaluate clotting disturbances in the acute infection by Trypanosoma evansi in rats and to investigate two possible causes. To address this issue, 21 2-month-old male rats were separated into three groups with seven animals each. The control group (group I) was composed of non-infected animals and group II was composed of 14 T. evansi-infected animals. This group was subdivided into two homogeneous groups. Animals from group II-a and from group II-b were euthanized at days 3 and 5 post-inoculation (PI), respectively. Hematological parameters were evaluated for monitoring of the disease. Infected animals showed anemia and leukocytosis 3 days PI and a significant decrease was observed in platelet count at days 3 and 5 PI. The prothrombin time (PT) and activated partial thromboplastin time (aPTT) were longer in the infected groups. The averages of the megakaryocyte count significantly increased (P < 0.05) in group II-b (5 days PI). Splenomegaly was observed in all T. evansi-infected rats at necropsy. Based upon the results, it is concluded that the acute infection by T. evansi in rats causes thrombocytopenia and longer prothrombin and activated partial thromboplastin times. In response to the thrombocytopenia, an increased number of bone marrow megakaryocytes were observed in the infected rats. Thus, the decrease in circulating platelets may be a consequence of splenic sequestration.     

Lipid peroxidation in cats experimentally infected with <Emphasis Type="Italic">Trypanosoma evansi</Emphasis>

The objective of this study was to evaluate the lipid peroxidation and the susceptibility of erythrocytes to in vitro peroxidation as indicators of oxidative damage in erythrocytes and their roles in the pathogenesis of anemia during experimental Trypanosoma evansi infection in cats. Animals were divided into two groups: control and infected with T. evansi. Seven cats were infected with 10⁸ trypomastigotes each, and parasitemia was estimated daily for 49 days by microscopic examination of smears. Hematological and biochemical parameters were evaluated for monitoring of the disease. Plasma lipid peroxidation (Thiobarbituric Acid Reactive Substances (TBARS)) and the susceptibility of erythrocytes to in vitro peroxidation were evaluated. Blood samples for analysis were collected at days 21 and 49 post-inoculation. TBARS level, indicated by MDA concentration, was higher in the infected group than in the control group in both analyzed periods, as well as the in vitro erythrocyte peroxidation (P < 0.001). The infected cats had variable degrees of regenerative anemia, which could be explained by the damage in erythrocyte membrane caused by lipid peroxidation.     

Tandem repeat protein as potential diagnostic antigen for <Emphasis Type="Italic">Trypanosoma evansi</Emphasis> infection

Trypanosoma evansi infection (surra) causes significant losses in livestock production in tropical and sub-tropical areas. The current ELISA recommended by OIE for diagnosis of the disease is based on trypanosome lysate antigen. However, antigenic variation and unstable nature of cell lysate antigen make it difficult to standardize the assay. Thus, there are needs to develop recombinant antigen-based ELISA that improve stability, sensitivity, and specificity of the test. Since tandem repeat (TR) proteins of trypanosomatid parasites generally possess high antigenicity, they have been considered to be the promising antigens for trypanosomosis and leishmaniosis. In this study, IgG responses against 14 recombinant TR proteins of trypanosomes were examined by ELISA. Serum samples were obtained from three water buffaloes experimentally infected with T. evansi. Since Trypanosoma congolense GM6 (TcoGM6) elicited highest IgG responses to all water buffaloes, we further bioinformatically and molecular biologically identified Trypanosoma brucei brucei GM6 (TbbGM6) and T. evansi GM6 (TeGM6) TR genes, respectively. As expected, predicted amino acid sequences of TbbGM6 and TeGM6 were identical while the nucleic acid sequence homology between TbbGM6 and TcoGM6 was 63.8%. All buffaloes became clearly positive in recombinant TbbGM6 (rTbbGM6)-based ELISA at 48 days post-infection, suggesting that rTbbGM6 is usable as a serodiagnostic antigen for chronic T. evansi infection.     

Domestic and wild mammals infection by Trypanosoma evansi in a pristine area of the Brazilian Pantanal region

The objective of this study was to estimate the Trypanosoma evansi infection rate and epizootical status of wild and domestic animals from the Brazilian Pantanal region using a standardized polymerase chain reaction (PCR). We used a simple DNA extraction method based on Chelex resin (BioRad, USA) on blood eluted from filter paper confetti. Primers directed to repetitive nuclear DNA sequences were used in the PCR, and could detect 30 fg of T. evansi DNA. The analytical specificity of the assay was evaluated using T. evansi, T. rangeli, T. cruzi, Leishmania braziliensis, Crithidia fasciculata and Herpetomonas muscarum DNAs as templates and the technique showed the expected 164 bp specific band solely for Trypanozoon trypanosomes. The application of the standardized PCR protocol in 274 field samples from domestic and wild mammals from the Rio Negro (Brazilian Pantanal region), showed a general infection rate of 10.2% while the traditional parasitological technique (direct search of the protozoan by the microematocrit centrifugue technique) was able to determine infection in only 1.1% of the animals. The peccaries and feral pigs were found to be the animals most frequently infected with T. evansi (24.4% and 30.7%, respectively). Both sampling and extraction methods used herein, showed to be simple and efficient to be applied in epidemiological surveys using PCR.     

Towards a New Reference Test for Surra in Camels

Current serological diagnosis of Trypanosoma evansi infection in camels is based on the native variable antigen type RoTat 1.2. The goal of this study was to develop a novel serological diagnostic test based on a nonvariable protein and freed from the use of rats or mice for its production. An enzyme-linked immunosorbent assay using a recombinant extracellular domain of invariant surface glycoprotein 75 (ELISA/rISG75) was developed and tested on a collection of 184 camel sera. The results were compared to those obtained from three established antibody detection tests based on variable surface glycoprotein RoTat 1.2: an ELISA for T. evansi (ELISA/T. evansi), a card agglutination test for trypanosomiasis (CATT/T. evansi), and an immune trypanolysis (TL) assay. The ELISA/rISG75 and the ELISA/T. evansi showed a sensitivity of 94.6% (95% confidence interval [CI], 87.8 to 98.2%, at 19% positivity cutoff value) and 98.9% (95% CI, 94.1 to 99.8, at 12% positivity cutoff value), respectively. The ELISA/rISG75 had 100% specificity (CI, 95.9 to 100%), while the ELISA/T. evansi showed 98.9% specificity (CI, 95.9 to 100%). The ELISA/rISG75 demonstrated an almost perfect agreement with the TL assay, the CATT/T. evansi, and the ELISA/T. evansi, with kappa scores of at least 0.94. The ELISA/rISG75, having a performance comparable to that of the gold standard (the TL assay) and being independent of antigenic variation, may become a new reference test for surra in camels. It opens avenues for the diagnosis of T. evansi infections in other hosts as well as for the development of a pan-Trypanozoon test for detection of Trypanosoma brucei brucei, T. b. gambiense, T. b. rhodesiense, T. evansi, and T. equiperdum.Trypanosoma evansi is the causative agent of surra in domestic animals such as camels, equines, cattle, buffaloes, small ruminants, and dogs. Wild animals such as capybaras can act as reservoir hosts. Although T. evansi is noninfective for healthy humans, a case of human infection by T. evansi in India in 2004 has been reported; however, this infection was due to a genetic mutation in the host''s APOL1 gene (20). Camels and horses are very sensitive to T. evansi, and death can occur within 3 months without treatment. Cattle and other ruminants infected by T. evansi suffer from immunosuppression, resulting in increased susceptibility to other diseases or vaccination failure (5, 17). T. evansi is mechanically transmitted by bloodsucking flies, such as Tabanidae and Stomoxys species. The disease occurs in Africa, Asia, and South and Central America and causes important economic losses (16).Control of surra still relies mainly on the observation of clinical signs and subsequent treatment of sick animals, which is inefficient and results in high morbidity and mortality of undiagnosed animals that in the meantime act as reservoirs. Definitive diagnosis of T. evansi infection is achieved by microscopic demonstration of the parasite, which method, however, suffers from limited sensitivity. The most sensitive parasite detection test is the mini-anion-exchange centrifugation method, but it is seldom used (4, 9). Indirect diagnosis is possible through detection of specific antibodies in the mammalian hosts. All currently available antibody detection tests, including an immune trypanolysis (TL) assay (21), an enzyme-linked immunosorbent assay for T. evansi (ELISA/T. evansi) (22), a card agglutination test for trypanosomiasis for T. evansi (CATT/T. evansi) (1), and a latex agglutination test for T. evansi (24), are based on the native variant surface glycoprotein (VSG) of the predominant variable antigen type (VAT) RoTat 1.2 of T. evansi. Production of these tests requires the mass culture of T. evansi homogeneously expressing RoTat 1.2 VSG in rats or mice. In addition, the results of all these serological tests may remain negative in animals infected with T. evansi type B in Kenya, as this type has been reported not to express the RoTat 1.2 VAT (13-15).On the cell surface of a bloodstream form trypanosome, among the VSGs and nonvariable surface proteins, there are an estimated 5 × 10⁴ invariant surface glycoprotein (ISG75) molecules (28). The ISG75 gene family is present and transcribed in the bloodstream form of all species and subspecies of the Trypanozoon subgenus, including Trypanosoma brucei brucei, T. b. gambiense, T. b. rhodesiense, T. evansi, and T. equiperdum. This multicopy gene family consists of two main groups that share at least 75% similarity among their cDNA and genomic DNA sequences (19). Within each group, there is at least 92% similarity among the cDNA sequences. A putative ISG75, regardless of its belonging to group I or group II, has a conserved topology: a large N-terminal extracellular domain, a single α-helix transmembrane domain, and a small cytoplasmic domain at the C terminus (19, 27). Since ISG75 is not subject to antigenic variation, its corresponding antibodies circulate during the course of infections in mice (29), and its extracellular domain can be produced in Escherichia coli by a standardized protocol (18), it is considered a highly relevant antigen for diagnosis of Trypanozoon infections.The purpose of this study was to develop an antibody detection ELISA for T. evansi infection in camels by using recombinant ISG75 (rISG75). This ELISA/rISG75 was tested against a panel of 184 camel sera in parallel with the currently used serological diagnostic tests, including the TL assay, the CATT/T. evansi, and the ELISA/T. evansi.     

Clinical aspects of cats experimentally infected with Trypanosoma evansi

The aim of this study was to evaluate clinical signs of cats experimentally infected with Trypanosoma evansi. Thirteen adult female nonbreeding Felix catus were separated into two groups: seven animals were infected with 10⁸ trypomastigotes each, and six animals were used as negative controls. Blood smears were performed daily for 56 days. Cardiorespiratory frequency was observed weekly, and blood samples for hematocrit analyses were collected at 15-day intervals. The protozoan was found in the blood 24 to 48 hours post-inoculation and irregular peaks of parasitemia were observed. Hematocrit significantly decreased in the infected group 7 days post-inoculation. Moreover, we observed the same clinical signs in this study that had previously been reported in other species commonly infected by T. evansi, including hyperthermia, lymphadenopathy, cachexia, and generalized edema. Based on these results, we conclude that domestic cats are susceptible to T. evansi infection, showing severe clinical alterations and mortality due to the chronic evolution of the disease.     

The typing of Trypanosoma evansi isolates using mobile genetic element (MGE) PCR

The mobile genetic element PCR (MGE-PCR) is a simple and sensitive technique that can be used to detect genetic variability in Trypanosoma brucei ssp. To investigate the reliability of MGE-PCR in genotyping Trypanosoma evansi, stocks that were isolated directly from camels and after their respective passage in mice were analyzed. Construction of a dendrogram using the MGE-PCR banding profiles revealed a clear distinction between T. evansi and T. brucei, as well as discriminating the T. evansi strains (T. evansi with minicircle types B and A). A minor host-dependent clustering shows a genetic difference of <15%. Changes in the banding profiles were observed after serial passage of T. evansi type B in mice, while those of T. evansi type A were identical. It is apparent that significant random insertion mobile element positional variation occurs when T. evansi isolates are introduced into a new host, a factor that needs to be considered when MGE-PCR is used to determine genetic variation in T. evansi isolates that have different host origins.     

Population sub-structuring among <Emphasis Type="Italic">Trypanosoma evansi</Emphasis> stocks

To investigate the population genetic structure of Trypanosoma evansi from domesticated animals, we have analysed 112 stocks from camels, buffaloes, cattle and horses using the tandemly repeated coding sequence (MORF2) and minisatellite markers 292 and cysteine-rich acidic integral membrane protein (CRAM). We recorded a total of six alleles at the MORF2 locus, seven at 292 and 12 at the CRAM loci. Nei’s genetic distance showed reduced allelic diversity between buffaloes and cattle stocks (1.2) as compared to the diversity between camels and buffaloes (3.75) and camels and cattle stock (1.69). The mean index of association (I _A = 0.92) significantly deviated from zero, and the average number of multilocus genotypes (G/N ratio) was 0.21. Twenty-four multilocus genotypes were defined from the combination of alleles at the three loci. The Kenyan sub-populations showed F _st = 0.28 and analysis of molecular variance showed significant divergence (22.7%) between the Laikipia, Kulal and Galana regions. The regional and host distribution of multi-locus genotypes significant population differentiation and high Nei’s genetic distances suggest existence of genetic sub-structuring within T. evansi stocks while the few multi-locus genotypes and deviation of association index from zero indicate the lack of recombination. In conclusion, this study reveals that some genetic sub-structuring does occur within T. evansi, which has a clonal population structure.     

Changes in the pathogenicity of relapsed diminazene aceturate (DA)-resistant Trypanosoma brucei brucei as the trypanosomes are transmitted from DA-treated hosts to another set of animals

This study investigated the changes in pathogenicity of relapsed diminazene aceturate (DA)-resistant Trypanosoma brucei brucei (TBB) as the trypanosomes are transmitted from DA-treated hosts to another set of animals. The Federe strain of T. brucei brucei which was known to be DA resistant was used to infect two groups of rats, one of which was treated at the peak of infection. There was temporary clearance of the trypanosomes from blood of the treated group and subsequent relapse of the infection. The relapsed trypanosomes were used to infect a group of rats (group A1), and the pathogenicity of the relapsed T. brucei brucei in this rat group was compared to that of a group infected with primary T. brucei brucei stock that was not treated with DA (group B1). Results showed that when compared with the B1 rats, the A1 group had a slower onset of parasitaemia, significantly lower (p < 0.01) level of parasitaemia all through the study, significantly longer (p < 0.01) time period to reach peak parasitaemia, significantly longer (p < 0.01) post-infection survival time and significantly higher (p < 0.05) packed cell volume from day 6 post-infection. Further sequential transmission of this same T. brucei brucei infection to another group of rats (A2 and B2) produced the same effects on the above parameters used to assess pathogenicity. It was concluded that beyond temporarily clearing trypanosomes from blood of treated animals, treatment with DA reduces the pathogenicity of DA-resistant T. brucei brucei, and this reduced pathogenicity is carried on as the T. brucei brucei is further sequentially transmitted to another set of animals.     

Changes in peripheral blood lymphocyte subpopulations and parasite-specific antibody responses in Trypanosoma evansi infection of sheep

This paper reports on changes in the lymphocyte composition of the peripheral blood in sheep infected with Trypanosoma evansi. In addition, parasite-specific IgG₁ and IgM antibody responses were monitored using a double-sandwich enzyme-linked immunosorbent assay (ELISA) technique. Eight sheep were infected with 2 × 10⁶ T. evansi TREU 2143. The infection was characterised by chronicity and ended in self-cure in two of the sheep. These two sheep were designated group A, whereas the other six sheep, which remained parasitaemic until treated, were designated group B. Analysis of the peripheral blood lymphocytes (PBLs) by indirect immunofluorescence staining and flow cytometry revealed significant alterations in the numbers of T- and B-cell subsets detected in all infected sheep. In group A, whereas the numbers of CD8⁺ cells decreased, CD4⁺ cells showed marginal decreases, remaining at or above pre-infection figures and resulting in increase in the CD4:CD8 ratio. In group B, CD8⁺ cells showed few marginal decreases, being at or above pre-infection figures most of the time, whereas CD4⁺ cells decreased significantly from day 26 post infection (p.i.) such that the CD4:CD8 ratio decreased. Infection also resulted in significant increases (P < 0.001) as of day 26 p.i. in circulating B-cells in group B as shown by the numbers of sIg⁺, CD45R⁺, CD1⁺ and major histocompatibility complex (MHC) II⁺ cells. The increases, however, were moderate and biphasic in group A. T. evansi-specific IgM and IgG₁ antibody isotypes were detected in all infected sheep, but their levels were significantly higher in group A than in group B (IgM P <0.05; IgG₁ P <0.01). In addition, although an initially higher level of IgM response was subsequently replaced by a higher level of IgG₁ response in group A, this was never the case in group B until after drug treatment. Received: 16 May 1998 / Accepted: 17 June 1998     

Dominant IgM synthesis against the soluble form of the prevailing variant surface glycoprotein from TeAp-N/D1 Trypanosoma equiperdum throughout the experimental acute infections of horses with non-tsetse transmitted Trypanozoon parasites

ABSTRACT

Two horses were infected with distinct non-tsetse transmitted Trypanozoon Venezuelan stocks, namely TeAp-N/D1 Trypanosoma equiperdum and TeAp-El Frio01 Trypanosoma evansi. Preceding reports have revealed that a 64-kDa antigenic glycopolypeptide (p64), which is the soluble form of the predominant variant surface glycoprotein from TeAp-N/D1 T. equiperdum, can be used as a good antigen for immunodiagnosis of animal trypanosomosis. Here, the course of the experimental acute infection in both horses was monitored by evaluating total anti-p64 IgG and particular anti-p64 γ-specific IgG and μ-specific IgM isotypes in sera using indirect enzyme-linked immunosorbent assays. Both equines showed a maximum of whole anti-p64 antibody generation, which dropped to readings below the maximum but always above the positive cutoff point. Levels of specific IgG and IgM isotypes oscillated throughout the course of the experiments. Essentially, the γ-specific IgG response remained very close to the cutoff point, whereas the μ-specific IgM response displayed values that were mostly above the positive cutoff point, showing a major peak that coincided with the maximum of complete anti-p64 IgG production. These results showed that horses infected with non-tsetse transmitted Trypanozoon parasites developed an immune reaction characterized by a dominant IgM generation against the p64 antigen.     

Ultrastructural changes on various Trypanosoma spp. after a 30-year storage period in liquid nitrogen

 Four Trypanosoma species were examined for damage following prolonged storage in liquid nitrogen (–196°C). The stabilates were successfully recovered after a cryopreservation period of approximately 30 years. The structure of specimens was studied by means of light microscopy and scanning (SEM) and transmission (TEM) electron microscopy. All of the species tested –T. evansi, T. equinum, T. brucei, and T. congolense – proved to be infective to mice. However, as compared with controls, the trypomastigote bloodstream forms, which had been frozen and later recovered, showed clear differences. Formerly deep-frozen organisms usually appeared to have shrunk as a result of solution effects, which occur during freezing and thawing. Ultrastructural changes such as separation of the cytoplasm from the pellicle, the occurrence of large vacuoles in the cytoplasm and karyoplasm, a loss of cytoplasmatic ribosomes, membrane injuries, enlargement of the flagellar pocket, and denaturation of chromatin became obvious. The extent of the ultrastructural alterations appeared to be much greater after a cryopreservation period of approximately 30 years than those previously reported after a 13-year storage period. These changes, however, did not result in a complete loss of infectivity to mice. Received: 15 March 1996 / Accepted: 15 April 1996