Cyclical transmission of Trypanosoma brucei rhodesiense and Trypanosoma congolense by tsetse flies infected with culture-form procyclic trypanosomes

Culture procyclic forms of Trypanosoma brucei rhodesiense and Trypanosoma congolense were fed to Glossina morsitans morsitans through artificial membranes. A very high percentage of the flies so fed produced established midgut infections, a proportion of which went on to develop into mature metacyclic trypanosomes capable of infecting mammalian hosts. The method offers a safe, clean way of infecting tsetse flies with African trypanosomes which reduces the need for trypanosome-infected animals in the laboratory.     

Trypanosoma congolense: susceptibility of cattle to cyclical challenge

Cattle primed by cyclical infection with Glossina morsitans morsitans infected with cloned derivatives of Trypanosoma congolense and treated with the trypanocidal drug Berenil after 3 or 4 weeks were immune to cyclical challenge with homologous clones 3 to 5 weeks later. In these animals, localized skin reactions (chancres) and parasitemia did not develop. The same results were obtained in cattle given a homologous superinfection without prior treatment. On the other hand, cattle subjected to a cyclical challenge with heterologous clones were completely susceptible as demonstrated by the development of chancres. Immunity to homologous challenge was achieved irrespective of the bloodstream variable antigenic types used to infect the tsetse. It was concluded that for a given serodeme the variable antigen composition of the metacyclic population which develops in the tsetse is constant and characteristic. Immunity to cyclical challenge was also obtained with uncloned stocks, providing the same stock was used for challenge. On the other hand, cattle immune to homologous cyclical challenge with cloned material were not always immune to cyclical challenge with parent stock, indicating that certain stocks consist of more than one serodeme. On the basis of these findings, it may be possible to use the chancre as a marker for serodeme analysis.     

Non-specific induction of increased resistance in mice to Trypanosoma congolense and Trypanosoma brucei by immunostimulants.

Administration of the immunostimulants Corynebacterium parvum, Bacillus Calmette-Guérin (BCG) or Bordetella pertussis prior to, or at the same time as, challenge with Trypanosoma congolense significantly increased survival times in mice, both of trypano-susceptible (A/J) and trypano-resistant (C57Bl) strains. The increased survival time was associated with significant alterations in parasitaemia, which included lengthening of the pre-patent period, a delay in the time taken to reach the first peak of parasitaemia and a reduction in the level of parasitaemia. Similar results were obtained when these strains of mice were challenged with Trypanosoma brucei following pre-treatment with C. parvum. Thus, by the use of immunostimulants it was possible to reduce the susceptibility of mice to trypanosomiasis and the hope is that this can also be achieved with domestic livestock.     

Trypanosoma congolense: host responses following tsetse transmitted infection of Kilifi isolates in goats

East African x Galla goats, when infected with Trypanosoma congolense isolates from the Kilifi area of Kenya by Glossina morsitans centralis, did not develop the characteristic chancre reaction at the bite sites, whereas bites of tsetse infected with the cloned T. congolense IL.1180 from Serengeti, Tanzania, resulted in chancres in the same goats. Histological changes could not be observed in skin biopsies collected 8 or 9 days after infection with Kilifi isolates. However, all goats became parasitemic about 10 days after challenge. It is concluded that the absence of chancre development is a characteristic feature of T. congolense parasites from Kilifi. The isoenzyme analysis of clones of two T. congolense Kilifi isolates and the T. congolense clone IL.1180 indicated that they belong to different zymodemes. Neutralizing antibodies to homologous metacyclic variable antigen types were detected in six out of seven (86%) of the sera from goats infected with a clone or stock of a T. congolense Kilifi isolate, 20 days after infection. Goats primed by tsetse transmitted infection with a stock or clone of T. congolense from Kilifi and treated with Berenil were, in three out of eight cases (37%), not immune to homologous challenge. It is suggested that the reduced immune response to metacyclic variable antigen types could be a result of the absence of cellular infiltration, i.e., chancre development in the skin at the tsetse bite site. It is concluded that the use of the chancre reaction as a marker for serodeme analysis of recently isolated stocks of T. congolense from Kilifi was not feasible.     

Susceptibility and resistance to Trypanosoma congolense infections

We have put emphasis on recent findings in experimental Trypanosoma congolense infections in highly susceptible BALB/c and relatively resistant C57Bl/6 mice. Based on various analyses, it has been shown that a major difference in resistance to T. congolense infections is expressed early in infection at the macrophage level. A novel plastic-adherent Thy1.2(+) suppressor lymphocyte, which in absolute synergy with a Thy 1.2(-) cell exerts its suppression via interleukin-10 and interferon-gamma opens up an exciting new field of research.     

Congopain from Trypanosoma congolense: drug target and vaccine candidate

Trypanosomes are the etiological agents of human sleeping sickness and livestock trypanosomosis (nagana), which are major diseases in Africa. Their cysteine proteases (CPs), which are members of the papain family, are expressed during the infective stages of the parasites' life cycle. They are suspected to act as pathogenic factors in the mammalian host, where they also trigger prominent immune responses. Trypanosoma congolense, a major pathogenic species in livestock, possesses at least two families of closely related CPs, named CP1 and CP2. Congopain, a CP2-type of enzyme, shares structural and functional resemblances with cruzipain from T. cruzi and with mammalian cathepsin L. Like CPs from other Trypanosomatids, congopain might be an attractive target for trypanocidal drugs. Here we summarise the current knowledge in the two main areas of research on congopain: first, the biochemical properties of congopain were characterised and its substrate specificity was determined, as a first step towards drug design; second, the possibility was being explored that inhibition of congopain by host-specific antibodies may mitigate the pathology associated with trypanosome infection.     

Mechanisms of self-cure from Trypanosoma congolense infection in mice

The mechanism(s) of resistance to African trypanosomiasis caused by Trypanosoma congolense was investigated by using the Dinderesso/80/CRTA/3 isolate to which C57B1/6 are resistant (low parasitemia and self-cure) and BALB/c sensitive (high parasitemia and death). The resistance of C57B1/6 is similar to that found in some natural hosts of African trypanosomes such as certain indigenous West African cattle and wild Bovidae. The antibody response to epitopes exposed on the variant surface glycoprotein of a clone obtained from the Dinderesso/80/CRTA/3 isolate was measured by a complement-mediated lysis assay in C57B1/6 and BALB/c. After infections with 10(4), 10(5), or 10(7) motile organisms, antibody appeared in C57B1/6 4 to 8 days earlier than in BALB/c. Peak antibody titers were similar in both strains but were reached about 4 days earlier in C57B1/6. In this strain, antibody appeared during and controlled the first wave of parasitemia, whereas in BALB/c, parasitemia reached a plateau above 10(8) organisms per ml before antibody could be detected, and at this time the animals were dying. At peak antibody response, the proportion of immunoglobulin (Ig) M and IgG antibody was the same in both strains. The antibody response had the same kinetics in C57B1/6 and BALB/c after injection of 10(4), 10(5), and 10(7) lethally irradiated but intact parasites, but the peak titers were 10(3) to 10(4) times lower than after live challenge. The response to nonirradiated trypanosomes appeared to be T cell independent, because the antibody titers were the same in congenitally athymic nu/nu and normal C57B1/6, and no evidence for the induction of T cell activity could be demonstrated in the infected nude mice. A role for trypanolytic serum factors in resistance could not be demonstrated. The extent of immunosuppression after infection with nonirradiated organisms was compared in the two strains by measuring the in vitro response of their splenic lymphocytes to concanavalin A, pokeweed mitogen, and allogeneic cells and their ability to mount an in vivo response to an unrelated trypanosome challenge. Both strains were partially immunosuppressed during rising parasitemia, but as C57B1/6 controlled parasitemia, immunosuppression was gradually reversed, whereas in BALB/c it became worse. Several explanations might account for the resistance of C57B1/6 to the Dinderesso/80/CRTA/3 isolate of T. congolense. It appears that an early immune response is a decisive factor in this resistance.     

Trypanosoma congolense: paraoxonase 1 prolongs survival of infected mice

In vitro studies have suggested that a fraction of human high density lipoprotein (HDL), termed trypanosome lysis factor (TLF), can protect against trypanosome infection. We examined the involvement of two proteins located in the TLF fraction, apolipoprotein A-II (apoA-II) and paraoxonase 1 (PON1), against trypanosome infection. To test whether PON1 is involved in trypanosome resistance, we infected human PON1 transgenic mice, PON1 knockout mice, and wild-type mice with Trypanosoma congolense. When challenged with the same dosage of trypanosomes, mice overexpressing PON1 lived significantly longer than wild-type mice, and mice deficient in PON1 lived significantly shorter. In contrast, mice overexpressing another HDL associated protein, apoA-II, had the same survival as wild-type mice. Together, these data suggest that PON1 provides protection against trypanosome infection. In vitro studies using T. brucei brucei indicated that HDL particles containing PON1 and those depleted of PON1 did not differ in their lysis ability, suggesting that protection by PON1 is indirect. Our data are consistent with an in vivo role of HDL protection against trypanosome infection.     

Amino acid metabolism during flight in tsetse flies.

Experiments carried out using teneral Glossina pallidipes indicate that flight can continue for at least 4 to 7 min after the thoracic proline reserves have fallen to low levels, suggesting that some other energy source is available. Earlier work suggests that alanine formed during flight is transported from the thorax to the abdomen where proline is resynthesized. Injection experiments using ¹⁴C alanine confirm that the transport mechanism does occur, that it is enhanced by flight, and that alanine is more rapidly incorporated into glutamate and proline in the abdomen than in the thorax. An analysis of published work shows that there is evidence for the involvement of residual blood meal amino acids even in the early stages of flight and supports the suggestion that they are of importance in prolonging flight. A decline in amino nitrogen during the early stages of flight is consistent with the action of glutamate dehydrogenase at this time. The poor flight durations in teneral flies may be due both to the low proline levels and to the absence of the residual blood meal. Very high energy consumptions are noted and appear to be related to the abnormally large musculature necessary for the fly's haematophagous and viviparous habits.     

Quantitative aspects of proline utilization during flight in tsetse flies

ABSTRACT. The proline concentration of the haemolymph in resting tsetse flies provides a reasonable indication of their total proline content. Estimates of pre-flight proline content obtained on this basis were compared with the proline content of flies that had been flown for different durations to provide an estimate of the rate of proline consumption at different stages of flight. The results indicate that the apparent ability of tsetse flies to continue flight after their proline reserves have been exhausted is an artefact of experimental procedure. It is concluded that the flight capacity of tsetse flies is effectively limited by the magnitude of their proline reserve, although this reserve is capable of being supplemented to some extent by the limited synthetic capacity of the fat body.     

Trypanosoma congolense: inheritance of susceptibility to infection in inbred strains of mice

Inbred strains of mice have shown marked differences in susceptibility to infection with Trypanosoma congolense, as judged by survival and levels of parasitemia. The underlying genetic basis of the susceptibility was examined with F1 hybrids and backcrosses derived from mouse strains of high and low susceptibility. The influence of H-2 haplotype on susceptibility was studied using H-2 congenic resistant strains of mice. F1 hybrids between the most susceptible strain (A/J) and the least susceptible strain (C57Bl/6) showed similar survival to that of the C57Bl/6 parent. This was reflected in a similar undulating pattern of parasitemia, although the level of parasitemia was consistently higher in the F1 hybrids than in the C57Bl/6. Backcrosses of the F1 hybrids with C57Bl/6 also had a similar pattern of parasitemia although there was a greater scatter in survival times so that a few animals survived longer than either of the parental strains. Backcrosses of F1 hybrids with A/J showed a range of survival times; approximately 25% of these animals died during the period when the A/J mice died, approximately 25% had a similar survival to that of C57Bl/6, while the remaining animals showed an intermediate duration of survival. All these backcrosses had a high initial peak of parasitemia; in about 70% of the mice the early parasitemia showed a distinct undulating pattern. F1 hybrids of A/J and C57Bl/6 with C3H/He mice, which are known to be of intermediate susceptibility, were also examined. The degree of dominance for low susceptibility was much less pronounced in these hybrid combinations than in the A/J × C57Bl/6 hybrids. The H-2 congenic resistant strains, all of which were on a C57Bl/10 genetic background, showed a similar pattern of parasitemia and survival. However, although the majority of all these strains survived for more than 100 days, there was a significant difference in survival between the C57Bl/10 mice and the H-2 congenic resistant strains. It was concluded that susceptibility of mice to T. congolense infection is likely to be under complex genetic control and that, at least in C57Bl/mice, H-2 haplotype has little influence on susceptibility.     

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.     

Trypanosoma congolense: thrombocytopenia in experimentally infected cattle

Hereford cattle infected with Trypanosoma congolense developed a thrombocytopenia which was most severe early in the course of infection when parasite levels in peripheral blood were highest. As the disease progressed, parasite levels gradually decreased and a corresponding increase in the number of thrombocytes occurred. In chronically infected animals, the number of thrombocytes was reduced during and shortly after periods of patency. However, during extended remission, thrombocyte values rose to normal or higher levels. Thrombocytes in surviving animals returned to within normal limits. When acutely ill, thrombocytopenic animals were treated with Berenil, a thrombo-cytosis developed rapidly. Accompanying the thrombocytopenia in infected animals was a consistent prolongation of partial thromboplastin times between the 6th and 14th weeks of infection. Plasma protamine paracoagulation tests were positive and fibrinogen levels were decreased in infected animals. No difference were found however in prothrombin times between infected and control animals.     

Identification of a genetic marker for isometamidium chloride resistance in Trypanosoma congolense

Isometamidium chloride has remained a very important prophylactic and therapeutic drug against trypanosomosis in cattle since its introduction into the market in the 1950s with, unfortunately, a concomitant development of resistance in trypanosomosis endemic areas. Amplified Fragment Length Polymorphism (AFLP) was used to compare two isogenic clones of Trypanosoma congolense. The parent clone, sensitive to isometamidium, has a CD50 (the curative dose that gives complete cure in 50% of the animals) in the mouse of 0.018 mg/kg and its derivative exposed to increasing doses of isometamidium, has a CD50 that is 94-fold higher. Sixty-four combinations of eight Eco RI and eight Mse I primers were used in comparative AFLP analysis to detect subtle genetic differences between the two clones. Thirty-five polymorphic fragments of DNA that were observed only in the resistant clone were purified and then sequenced. The nucleotide sequences were used in searching the GeneDB T. congolense database to find surrounding sequences upstream of an open reading frame and downstream to a stop codon. The sequences of the open reading frames were subsequently compared to the sequences in the genomic databases. A predicted gene coding for an 854 amino acids protein was thus identified. The protein contains a putative ATP binding site, Walker B and LSGG motifs and eight predicted trans-membrane domains. The gene in the resistant strain of T. congolense has a triplet insertion coding for an extra lysine. Using polymerase chain reaction-restriction fragment length polymorphism, the insertion was sought in the genomes of 35 T. congolense strains isolated from different geographic origins and whose response to isometamidium chloride had been determined through single dose mouse tests. The presence of the insertion, specifying an extra codon was found to always be present in the genomes of T. congolense clones that were resistant to isometamidium chloride.     

Olfactory sensitivity in tsetse flies: a daily rhythm

The diurnal tsetse Glossina morsitans morsitans bites especially in early morning and late afternoon; around midday feeding is at a low. In laboratory apparatus that measures the amount of locomotion under constant conditions over the photophase, the flies display a similar patterning of activity levels. The profile of daily rhythms for G. morsitans reported in the literature includes a number of motor and sensory motor systems that fluctuate cophasically. Lacking is a study on the patterning of the senses' response levels. In this paper we present the first instance of a daily modulation in the sense of smell. We stimulated the antennae with concentration series of host-derived odours and measured the spiking rate of cells at different times during the photophase. The concentration-response curves suggest that the sensitivity of antennal olfactory cells flows in parallel with the other daily rhythms. This was also reflected in electroantennograms (EAGs). The electroantennography was extended to G. fuscipes fuscipes, whose level of spontaneous locomotor activity--instead of following a U- shaped pattern--rises gradually over the photophase. Again, the EAGs appeared to parallel the species' locomotor activity. What we believe happens is that the organism tones down the sensitivity of its odour receptors during periods of anticipated inactivity for reasons of economy.      

A Mensural Study of Division in Trypanosoma simiae and Trypanosoma congolense

SYNOPSIS. Dividing forms of Trypanosoma simiae and T. congolense in stained thin blood films taken from pigs infected by wild Glossina morsitans submorsitans were measured employing a technique which took account of the distance between the divided kinetoplasts, the positions of the nucleus or nuclei and the lengths of the original and developing flagella.
Analysis of these measurements showed that binary fission in these trypanosomes consisted of a gradual increase in the distance between the divided kinetoplasts along the long axis of the body; progressive outgrowth of a daughter flagellum from the blepharoplast associated with the posteriorly placed kinetoplast; migration of the nucleus toward the posterior end of the body; separation of the divided nuclei in the direction of the long axis of the body; and fission of the cytoplasm in an antero-posterior direction and finally separation into two individuals by a stepped, sliding motion.
No evidence to support syngamy or other type of germ cell reproduction was observed.     

Cell surface interactions between Trypanosoma congolense and macrophages during phagocytosis in vitro.

Trypanosoma congolense bloodstream forms preincubated with a high titer of anti-variant surface antigen (VSG)-specific antibody, a low amount of anti-VSG plus complement-active mouse serum (MS), MS alone, and trypsin were cocultivated with mouse peritoneal macrophages in vitro. Immunofluorescence as well as transmission and scanning electron microscopy revealed that upon attachment to the macrophages' surface, trypanosomes opsonized with anti-VSG/MS formed opsonized filopodia, which were rapidly internalized by the phagocytes. Although these cells attached as frequently as anti-VSG or trypsin-pretreated parasites, the rate of phagocytosis of anti-VSG/MS pretreated trypanosomes was reduced significantly. Trypanosomes pretreated with high antibody titers alone were lysed on the surface of the macrophages before phagocytosis was completed. Parasites opsonized with complement alone adhered only occasionally and were rarely phagocytosed. Trypsin-treated trypanosomes, which served as positive control cells, rapidly attached and remained intact until ingulfment by the macrophages was completed. Untreated control parasites did not attach to the macrophages and were not phagocytosed. Cocultivation of macrophages with anti-VSG/MS-opsonized trypanosomes caused internalization of the flagellum by membrane fusion. Filopodia formation by T. congolense is thus correlated with a marked reduction in phagocytosis even in the presence of only a sublytic antibody titer.