Differentiation of epimastigotes and production of infective metacyclic forms of Trypanosoma congolense were examined in a culture system which enabled manipulation of the population density of insect forms. Scanning electron microscopy of cultures revealed the attachment sites of epimastigotes in detail, showing them to be attached as ‘clusters’ or ‘bundles’ and having associated fibrillar structures. Dividing epimastigotes were observed either within individual bundles or in association with two bundles. Metacyclic forms were detected by an immunofluorescence antibody test (IFAT) using metacyclic variable-antigen type (M-VAT) specific monoclonal antibodies, by day 7 after seeding cultures. Trypanosomes expressing M-VATs appeared singly in bundles, observed by both IFAT and an immunogold labelling method. Statistical analysis using Poisson calculations suggested that, in general, the distribution of metacyclics expressing individual M-VATs was random throughout cultures.

Pathogenic animal trypanosomes affecting livestock have represented a major constraint to agricultural development in Africa for centuries, and their negative economic impact is increasing in South America and Asia. Chemotherapy and chemoprophylaxis represent the main means of control. However, research into new trypanocides has remained inadequate for decades, leading to a situation where the few compounds available are losing efficacy due to the emergence of drug-resistant parasites. In this review, we provide a comprehensive overview of the current options available for the treatment and prophylaxis of the animal trypanosomiases, with a special focus on the problem of resistance. The key issues surrounding the main economically important animal trypanosome species and the diseases they cause are also presented. As new investment becomes available to develop improved tools to control the animal trypanosomiases, we stress that efforts should be directed towards a better understanding of the biology of the relevant parasite species and strains, to identify new drug targets and interrogate resistance mechanisms.

The effect of trypanosome infection on vector survival was observed in a line of Glossina morsitans morsitans selected for susceptibility to trypanosome infection. The differential effects of midgut and salivary gland infections on survival were examined by exposing flies to infection with either Trypanosoma congolense which colonizes midgut and mouthparts or Trypanosoma brucei rhodesiense which colonizes midgut and salivary glands. A comparison of the survival distributions of uninfected flies with those exposed to infection showed that salivary gland infection significantly reduces tsetse survival; midgut infection had little or no effect on the survival of tsetse. The significance of these findings is discussed in relation to the vectorial capacity of wild flies.

The first step in studying the epidemiology of a disease is the accurate identification of the pathogen. Traditional reliance on morphological identification has given way to the use of molecular methods for the detection and identification of pathogens, greatly improving our understanding of epidemiology. For the African tsetse-transmitted trypanosomes, the growth of PCR methods for identification of trypanosomes has led to increased appreciation of trypanosome genetic diversity and discovery of hitherto unknown trypanosome species, as well as greater knowledge about the number and type of trypanosome infections circulating in mammalian hosts and vectors. Sequence data and phylogenetic analysis have provided quantitative information on the relatedness of different trypanosome species and allowed the new trypanosome genotypes discovered through the use of species identification methods in the field to be accurately placed in the phylogenetic tree.

An incomplete ring of electric nets was placed around uninfected cattle and cattle infected with Trypanosoma congolense. The numbers of fed and unfed Glossina pallidipes caught on the nets were used to estimate the attractiveness of infected and uninfected cattle to tsetse, and the feeding success of tsetse on the cattle. There was no difference in the attractiveness of infected and uninfected cattle to G. pallidipes. However, the feeding success of G. pallidipes on infected cattle was 75 % greater than on uninfected cattle. This suggests that certain effects of T. congolense on cattle behaviour or physiology act to increase the probability of transmission of the parasite by increasing the feeding success of the vector. The nature of the effects of T. congolense on cattle which caused this result is unknown, but several possibilities are discussed.

Restriction fragment length polymorphism (RFLP) analysis of both genomic and kinetoplast DNA from representative stocks from 3 Trypanosoma congolense subgroups (Savannah, Forest, and Kilifi), T. simiae and T. godfreyi, was used to investigate the relatedness of the different groups within subgenus Nannomonas, DNA probes for β-tubulin and the ribosomal DNA (rDNA) locus were isolated from a T. congolense Savannah genomic library; additional probes were generated by PCR amplification of mini-exon and glutamate and alanine rich protein (GARP) gene sequences. Our results provide evidence that at the molecular level the T. congolense Savannah and Forest groups are the most closely related groups within the subgenus Nannomonas: the Savannah and the Forest groups had mini-exon gene repeats of identical size, which shared homology, had mini-circles of the same size and had a high level of similarity (63%) when the banding patterns produced with a tubulin and rDNA probe were subjected to numerical analysis. All other pairwise combinations of groups have very low percentage similarities of < 10%, suggesting that the Kilifi group trypanosomes, are as distantly related to the T. congolense Savannah and Forest groups as they are to T. simiae or T. godfreyi. The conservation of the GARP gene between the Savannah, Forest and Kilifi groups provides the only evidence linking the Kilifi trypanosomes to the other groups in T. congolense. We find no evidence for the presence of the GARP gene in the T. simiae or T. godfreyi group trypanosomes.

Nine clones were derived from a drug-resistant Trypanosoma congolense stock (IL 2856) and characterized in mice for their sensitivity to isometamidium chloride and diminazene aceturate. All clones were derived from the stock without drug selection and expressed high levels of resistance to isometamidium chloride (50% curative dose [CD50] values ranging from 1·5 to 5·1 mg/kg) and intermediate to high levels of resistance to diminazene aceturate (CD50 values ranging from 5·1 to 21·0 mg/kg). By contrast, the isometamidium chloride and diminazene aceturate CD50 values for a drug-sensitive clone, T. congolense IL 1180, were 0·018 mg/kg and 2·3 mg/kg, respectively. For both drugs, there appeared to be significantly different levels in expression of drug resistance amongst the 9 clones derived from IL 2856. Isoenzyme analysis of 7 enzymes showed that all 9 clones expressed the same electrophoretic variants. Thus, all 9 clones were identical for these phenotypic markers. The clone which expressed the highest level of resistance to isometamidium in mice (IL 3270) was transmitted to Boran cattle via the bite of infected Glossina morsitans centralis. IL 3270 produced an infection rate in tsetse of 5·0%. The resulting infections in cattle were shown to be resistant to intramuscular treatment with 2·0 mg/kg isometamidium chloride and 14·0 mg/kg diminazene aceturate. This contrasts with doses of 0·25 mg/kg isometamidium chloride or 3·5 mg/kg diminazene aceturate which are deemed sufficient to cure fully sensitive infections. Finally, 9 clones (subclones) were derived from IL 3270 and characterized in mice for their sensitivity to isometamidium chloride. Seven of the subclones expressed a significantly lower level of resistance to isometamidium than the parental clone and amongst the subclones there was significant variation in resistance. Thus, expression of a high level of resistance to isometamidium appears to be unstable in the rodent host and at least a component of the genetic determinant(s) for this drug-resistant phenotype is (are) likely to be unstable.

An incomplete ring of electric nets was placed around oxen which were either uninfected, infected with Trypanosoma vivax, or infected with T. congolense. The numbers of fed and unfed Glossina pallidipes caught on the nets were used to estimate the attractiveness of the oxen to tsetse, and the feeding success of the tsetse on the oxen. Oxen infected with T. congolense attracted more G. pallidipes than the other groups of oxen. Taking into consideration daily variation in the abundance or activity of the flies, oxen infected with T. congolense were about 70% more attractive to G. pallidipes than were uninfected oxen or oxen infected with T. vivax. The latter two groups mostly attracted high numbers of G. pallidipes on days when the flies were especially abundant or active. The feeding success of G. pallidipes declined with increase in the rate at which oxen made anti-fly movements. Taking this movement rate into consideration, the feeding success of G. pallidipes on oxen infected with T. congolense was approximately 60% greater than on uninfected oxen or oxen infected with T. vivax. It is suggested that vasodilation induced by T. congolense may account for the difference in feeding success. The level of parasitaemia of T. congolense or T. vivax was not found to affect either the attractiveness of oxen or the feeding success on oxen. There was significant daily variation in the mean fat content of male G. pallidipes caught around the oxen but no effect of mean daily fat content on the proportion of males that fed. The mean haematin content of fed male G. pallidipes was positively correlated with the packed cell volume of the ox on which they fed.

Trypanosoma (Nannomonas) congolense comprises morphologically identical but genetically heterogeneous parasites infective to livestock and other mammalian hosts; three different genotypes of this parasite have been described previously. Restriction enzyme fragment length polymorphisms (RFLPs) in both kinetoplast DNA minicircle and nuclear DNA sequences, and randomly amplified polymorphic deoxyribonucleic acid (RAPD) patterns have been used here to demonstrate the existence of another type of T. (N.) congolense that is genotypically distinct from those that have so far been characterized at the molecular level. A highly repetitive, tandemly arranged DNA sequence and oligonucleotide primers, for use in polymerase chain reaction (PCR) amplification are described, which can be used for specific identification of the trypanosome and its distinction from others within the Nannomonas subgenus.

The accumulation of the trypanocide isometamidium chloride (Samorin®, RMB Animal Health Ltd, UK) by a range of clones of Trypanosoma congolense with varying sensitivity to the drug, was measured by methods based on the fluorescence of isometamidium. Fluorescence microscopy and flow cytometry showed a reduction in drug accumulation by resistant clones. Fluorescence spectrophotometry demonstrated an inverse correlation between the intensity of cell-associated fluorescence and the level of resistance of the clones expressed in vivo. The addition of the metabolic inhibitor SHAM/glycerol to the incubation medium resulted in a reduction of this apparent difference in drug accumulation between the clones; those clones which were sensitive to isometamidium showed a reduction in fluorescence while a percentage increase in fluorescence was observed as clones became more resistant to the trypanocide. These observations may be of value for the in vitro detection of resistant T. congolense populations and may also be used to estimate the mean level of resistance in a given sample. The results also imply that decreased accumulation of isometamidium by drug-resistant clones of the parasite may be responsible for the reduction in sensitivity.

The nuclear DNA of Trypanosoma congolense contains a family of highly conserved 369 base pair (bp) repeats. The sequences of three cloned copies of these repeats were determined. An unrelated family of 177 bp repeats has previously been shown to occur in the nuclear DNA of Trypanosoma brucei brucei (Sloof et al. 1983a). Oligonucleotides were synthesized which prime the specific amplification of each of these repetitive DNAs by the polymerase chain reaction (PCR). Amplification of 10% of the DNA in a single parasite of T. congolense or T. brucei spp. produced sufficient amplified product to be visible as a band in an agarose gel stained with ethidium bromide. This level of detection, which does not depend on the use of radioactivity, is about 100 times more sensitive than previous detection methods based on radioactive DNA probes. The oligonucleotides did not prime the amplification of DNA sequences in other trypanosome species nor in Leishmania, mouse or human DNAs. Amplification of DNA from the blood of animals infected with T. congolense and/or T. brucei spp. permitted the identification of parasite levels far below that detectable by microscopic inspection. Since PCR amplification can be conducted on a large number of samples simultaneously, it is ideally suited for large-scale studies on the prevalence of African trypanosomes in both mammalian blood and insect vectors.

Trypanosome infection of mammalian hosts leads, within days, to a strong early response against a small, distinct number of parasite proteins. One of these proteins is the variable surface glycoprotein (VSG). Most of the others are apparently non-variable, intracellular trypanosome proteins. Two of these antigens I2 and I17 are now characterized at the molecular level. Both exhibit a highly repetitive amino acid sequence organization, but they show no sequence similarity either to each other or to any other proteins known to date. Preliminary serological analyses indicate that both allow the early, sensitive and specific detection of infections with different species of trypanosomatids, making them interesting candidates for the development of diagnostic tools for trypanosomiasis detection.

A murine model using Heligmosomoides polygyrus and Trypanosoma congolense has been developed for studying the effects of concurrent chronic gastrointestinal nematode and trypanosome infections. Female outbred mice were infected either with 500 infective larvae (L3) of H. polygyrus or with 104 bloodstream forms of T congolense or both. In concurrent infections, animals were dosed with both parasites simultaneously or the trypanosomes were injected 5 or 10 days after the mice were infected with the nematode. The course of infection was monitored by routine parasitological and immunological techniques for 30 days after the H. polygyrus infection. Concurrently infected mice were severely compromised, except when T. congolense was superimposed on a 10-day-old (adult) H. polygyrus infection. In H. polygyrus-infected mice, simultaneous or subsequent infection with trypanosomes did not markedly influence worm establishment or fecundity, but the female worms were slightly stunted. Surviving mice displayed a markedly reduced antibody response to H. polygyrus antigens and a slightly reduced antibody response to T. congolense antigens.

Clones of Trypanosoma congolense which express resistance to the widely used trypanocide isometamidium chloride accumulate less of the drug than clones which are sensitive to drug treatment. A mathematical model has been developed which was able to predict theoretical lines representing the uptake kinetics in trypanosomes which were sensitive to isometamidium, as well as for resistant trypanosomes in which reduced accumulation was a result of either reduced uptake or enhanced efflux of the drug. Data from drug uptake experiments were then fitted to these theoretical lines. While the value for drug efflux could not be separated from the dissociation constant of the trypanosomes for isometamidium, it was demonstrated that reduced accumulation is not a result of reduced uptake of isometamidium by drug-resistant trypanosomes.

Bloodstream forms of Trypanosoma congolense (2 clones: ILNat3·1 and IL3000, and 4 stocks: IL2079, IL2466, IL3266 and CP-81) were continuously cultivated in vitro at 34–36 °C in the absence of feeder cell layers, using HMI-93 medium which was modified from Iscove's modified Dulbecco's MEM (Flow Laboratories, Irvine, Scotland). The modification was done by supplementing the medium with 0·05 mM bathocuproine sulphonate, 1·5 mM L-cysteine, 0·5 mM hypoxanthine, 0·12 mM 2-mercaptoethanol, 1 mM sodium pyruvate, 0·16 mM thymidine, 20% (v/v) heat-inactivated young goat serum and 5% (v/v) Serum Plus™ (Hazleton Biologics, Lenaxa, KS, USA). Trypanosomes obtained from two different sources were used to initiate primary cultures: (1) metacyclic forms which were produced in vitro at 27 °C, and (2) bloodstream forms obtained from Balb/c mice which had been infected with the bloodstream forms transformed in vitro from the metacyclic forms. Metacyclic forms placed in 25 cm2 T-type (T-25) flasks rapidly attached to the bottom of the flasks and transformed to bloodstream forms during the initial 24 h and continued to proliferate. The bloodstream forms isolated from the infected mouse blood by means of diethylaminoethyl cellulose (DE52) column chromatography also continued to proliferate in the flasks. Cultures were maintained by replacing the medium every 24 h. Every 4–5 days, the attached bloodstream forms were resuspended in fresh medium by gentle pipetting and then were subcultured. The method was further simplified by initiating primary cultures directly with 10 μl of the tail blood of infected mice in 24-well culture plates and then by subcultivating either in wells or in T-25 flasks. The shortest population doubling time, 9 h, was achieved by seeding subcultures with 106 bloodstream forms/ml. The bloodstream forms propagated in this system were morphologically similar to those seen in infected mouse blood, they were covered with a surface coat as examined by electron microscopy and they were infective to mice.

The uptake kinetics of a 14C-labelled trypanocidal compound isometamidium chloride (SamorinR, RMB Animal Health Ltd, UK) was measured in drug-resistant and drug-sensitive Trypanosoma congolense. It was established that drug uptake was significantly more rapid and quantitatively greater in drug-sensitive parasites. There was clear evidence that drug uptake in both the resistant and sensitive trypanosomes was by a specific, receptor-mediated process. This specific drug transport was energy-dependent, being sensitive to metabolic inhibition with SHAM/glycerol. Significant differences in drug transport were observed which could be correlated with resistance to isometamidium. The optimal pH for drug accumulation was lowered in the resistant trypanosomes; this finding, along with an observed change in specificity for the related compound homidium bromide, suggested that the specific receptor for isometamidium is altered in the resistant trypanosomes, possibly resulting in a reduction in drug uptake. In addition to these alterations in drug uptake, efflux of isometamidium also appears to occur in the resistant trypanosomes. Both a reduction in incubation temperature and metabolic inhibition increased the level of trypanosome-associated isometamidium in the resistant parasites. This was in contrast to observations using drug-sensitive parasites. Furthermore, the addition of calcium flux-modulating agents to the incubation medium also resulted in an increase in accumulation by the resistant parasites.

The polymerase chain reaction was used to detect trypanosomes in samples collected from cattle, wild animals and tsetse flies in KwaZulu-Natal Province, South Africa. A total of 673 samples from cattle and 266 from tsetse flies in the study area located near the Hluhluwe-Umfolozi Game Reserve were analysed. Both Trypanosoma congolense and T. vivax were found as single or mixed infections in cattle and tsetse flies. Moreover, the T. congolense in the infections were found to comprise 2 genotypic groups: the Savannah-type and the Kilifi-type, which were present either as single or mixed infections in cattle and in tsetse flies.

Transmission experiments were conducted to compare the transmissibility of genetically different Trypanosoma congolense (Savannah subgroup) strains isolated from cattle in a trypanosomiasis endemic area of eastern Zambia. A total of 17 strains were compared. Three strains were extremely virulent with a short pre-patent period, high parasitaemia and a short median survival time (between 5 and 9 days) in mice. The remainder of the strains belonged to the moderate (6 strains) or low (8 strains) virulence categories with median survival times between 10 and 30 days and >30 days, respectively. Batches of 40 teneral Glossina morsitans morsitans (Diptera: Glossinidae) were offered a single bloodmeal on mice infected with one of those strains. Flies were dissected to determine their infection status 21 days later. The proportion of flies with procyclic and metacyclic infections differed significantly between trypanosome strains and were significantly higher in flies infected with extremely virulent strains (P=0·033 and P=0·016 for the differences in the procyclic infection rate of strains with moderate and low virulence, respectively and P=0·005 and P=0·019 for the differences in the metacyclic infection rate of strains with moderate and low virulence, respectively). On the other hand, moderately virulent strains had, in general, higher procyclic and metacyclic infection rates compared to low virulent strains. But the differences were not significant (P>0·05). The outcome of those experiments shows clear differences in transmissibility of trypanosome strains associated with their virulence. This observation confirms the theory for the evolution and maintenance of virulence in a parasite population and may explain the persistence of virulent trypanosome strains in a susceptible host population.

This experiment studied the effects of body condition, long- and short-term levels of nutrition and trypanosomosis infection on the productivity of N’Dama cows using a 2 x 2 × 2 factorial cross-over design. Pre-partum, 23 cows received supplements for 6 months (H), the other group of 20 for 2 months (L). Both groups grazed native pastures. Two days post partum, half the cows from each group were placed on a basal (B) or supplemented (S) plane of nutrition. The diet of concentrate, groundnut hay and andropogon hay was the same, only the quantities differed. Four weeks post partum half the animals in each group were inoculated with T. congolense organisms (I), the others acted as controls (C). The trial continued for a further 6 weeks.

Pre-partum nutrition (H, L had no effect on dry-matter intake (DMI) but pre-partum feeding (H) improved post-partum productivity, evident by higher dam live weights (P < 0·05), body condition (P < 0·001), calf birth weight (P < 0·05) and calf live-weight gain (P < 0·01). Post-partum nutrition had no effect on productivity. Trypanosomosis infection caused a reduction (P < 0·05) in total DMI. The decline in groundnut hay and concentrate intake was proportionally (P < 0·001) greater in the S-I group than in the B-I group. A low plane of nutrition pre-partum depressed milk yield but increased fat concentration (P < 0·05). Infection significantly reduced milk offtake (P < 0·05). The reduction in milk offtake (P < 0·01) and calf live weight (P < 0·05) were proportionally larger in the B-I than in the S-I group. Infection caused a decline in milk protein concentration (P < 0·05) and protein yield (P < 0·01) which was independent of dietary effects. Infection reduced (P < 0·01) the packed-cell volume but there were no interactions with diet. None of the cows was pregnant 150 days post partum but seven were cycling, 3(5) in the H-S-I group, 2(7) in the H-B-I group, 1(5) in the L-B-I group and 1(5) in the L-S-C group. These results suggest that S-I cows attempted to maintain milk yield at the expense of live weight whereas the B-I cows had insufficient live-weight reserves that could be mobilized. This suggests the nutritional balance and changes in weight at the time of infection might be more important than historical planes of nutrition.

Teneral tsetse flies, Glossina morsitans morsitans (Mall) aged less than 32 h were divided into two groups and infected initially with Trypanosoma congolense IL 1180. Flies of group A were maintained on healthy rats and those of group B fed exclusively on parasitaemic rats, to ensure regular re-infection. Flies in group A recorded mesoprocyclic, metacyclic and vectorial competence (VC) index values of 0.65 ± 0.09; 0.73 ± 0.10 and 0.47 ± 0.11, respectively. The corresponding values for group B flies were 0.76 ± 0.08; 0.79 ± 0.08 and 0.60 ± 0.10. In group A, the mesoprocyclic and the VC indexes of the females were significantly higher than those of the males. In group B, no such significant differences were registered between the sexes. For both sexes taken together, the mesoprocyclic and the VC indexes of the tsetse flies of group B were more important than those of flies of group A. Males of group B revealed a higher VC index than that of group A males; no significant differences in this index were observed in the females of both groups.