Trypanosome Isolates Research Articles

Kenyan purple tea anthocyanins and coenzyme-Q10 ameliorate post treatment reactive encephalopathy associated with cerebral human African trypanosomiasis in murine model

Human African trypanosomiasis (HAT) is a tropical disease caused by two subspecies of Trypanosoma brucei, the East African variant T. b. rhodesiense and the West African variant T. b. gambiense. Melarsoprol, an organic arsenical, is the only drug used to treat late stage T. b. rhodesiense infection. Unfortunately, this drug induces an extremely severe post treatment reactive encephalopathy (PTRE) in up to 10% of treated patients, half of whom die from this complication. A highly reproducible mouse model was adapted to assess the use of Kenyan purple tea anthocyanins and/or coenzyme-Q10 in blocking the occurrence of PTRE. Female Swiss white mice were inoculated intraperitoneally with approximately 104 trypanosome isolate T. b. rhodesiense KETRI 2537 and treated sub-curatively 21days post infection with 5mg/kg diminazene aceturate (DA) daily for 3days to induce severe late CNS infection that closely mirrors PTRE in human subjects. Thereafter mice were monitored for relapse of parasitemia after which they were treated with melarsoprol at a dosage of 3.6mg/kg body weight for 4days and sacrificed 24h post the last dosage to obtain brain samples. Brain sections from mice with PTRE that did not receive any antioxidant treatment showed a more marked presence of inflammatory cells, microglial activation and disruption of the brain parenchyma when compared to PTRE mice supplemented with either coenzyme-Q10, purple tea anthocyanins or a combination of the two. The mice group that was treated with coenzyme-Q10 or purple tea anthocyanins had higher levels of GSH and aconitase-1 in the brain compared to untreated groups, implying a boost in brain antioxidant capacity. Overall, coenzyme-Q10 treatment produced more beneficial effects compared to anthocyanin treatment. These findings demonstrate that therapeutic intervention with coenzyme-Q10 and/or purple tea anthocyanins can be used in an experimental mouse model to ameliorate PTRE associated with cerebral HAT.

Isolation and Phylogenetic Relationships of Bat Trypanosomes from Different Biomes in Mato Grosso, Brazil

In the order Chiroptera, more than 30 trypanosome species belonging to the subgenera Herpetosoma, Schizotrypanum, Megatrypanum, and Trypanozoon have been described. The species Trypanosoma cruzi , Trypanosoma cruzi marinkellei, and Trypanosoma dionisii are the most common in bats and belong to the Schizotrypanum subgenus. Bats from 2 different biomes, Pantanal and Amazonia/Cerrado in the state of Mato Grosso, Brazil, were evaluated according to the presence of trypanosome parasites by means of hemoculture and PCR in primary samples (blood samples). A total of 211 bats from 20 different species were caught and the trypanosome prevalence, evaluated through hemoculture, was 9.0% (19), 15.5% (13), and 4.8% (6) in the municipalities of Confresa (Amazonia/Cerrado biome) and Poconé (Pantanal biome). Among the 123 primary samples obtained from the bats, only 3 (2.4%) were positive. Phylogenetic analysis using trypanosomatid barcoding (V7V8 region of SSU rDNA) identified all the isolates and primary samples as T. c. marinkellei. The sequences of the isolates were segregated according to the bat host genus or species and suggest that co-evolutionary patterns exist between hosts and parasites. Further studies in different Brazilian regions and biomes need to be conducted in order to gain real understanding of the diversity of trypanosomes in bats.

Isolation and In Vitro Culture of Trypanosomes FromLeptodactylus ocellatusFrom the Atlantic Forest in a New Experimental Culture Medium

The purpose of this study was to verify the in vitro development of Trypanosoma sp. isolated from Leptodactylus ocellatus frogs under a new protocol using a biphasic medium composed of Novy, McNeal, and Nicolle (NNN) blood agar medium as a solid phase and liver infusion, brain heart infusion, and tryptose (LIBHIT) medium as a liquid phase. Blood forms, collected by cardiac puncture or after the maceration of different organs, were inoculated in culture tubes containing the biphasic medium composed by NNN and LIBHIT. Trypanosomes were observed 4 days postinoculation; most bloodstream trypomastigotes had differentiated into epimastigotes and amastigotes by this time. Trypomastigotes were again observed in older cultures (7 days). Parasites were successfully subcultured for 8 mo in this medium and successfully cryopreserved. The present study provides a new protocol medium for the isolation and culture of anuran trypanosomes.

Evolutionary Insights from Bat Trypanosomes: Morphological, Developmental and Phylogenetic Evidence of a New Species, Trypanosoma (Schizotrypanum) erneyi sp. nov., in African Bats Closely Related to Trypanosoma (Schizotrypanum) cruzi and Allied Species

Parasites of the genus Trypanosoma are common in bats and those of the subgenus Schizotrypanum are restricted to bats throughout the world, with the exception of Trypanosoma (Schizotrypanum) cruzi that also infects other mammals and is restricted to the American Continent. We have characterized trypanosome isolates from Molossidae bats captured in Mozambique, Africa. Morphology and behaviour in culture, supported by phylogenetic inferences using SSU (small subunit) rRNA, gGAPDH (glycosomal glyceraldehyde 3-phosphate dehydrogenase) and Cyt b (cytochrome b) genes, allowed to classify the isolates as a new Schizotrypanum species named Trypanosoma (Schizotrypanum) erneyi sp. nov. This is the first report of a Schizotrypanum species from African bats cultured, characterized morphologically and biologically, and positioned in phylogenetic trees. The unprecedented finding of a new species of the subgenus Schizotrypanum from Africa that is closest related to the America-restricted Trypanosoma (Schizotrypanum) cruzi marinkellei and T. cruzi provides new insights into the origin and evolutionary history of T. cruzi and closely related bat trypanosomes. Altogether, data from our study support the hypothesis of an ancestor trypanosome parasite of bats evolving to infect other mammals, even humans, and adapted to transmission by triatomine bugs in the evolutionary history of T. cruzi in the New World.

Identification of novel trypanosome genotypes in native Australian marsupials

In the present study, the occurrence and molecular phylogeny of trypanosome parasites were studied in both wild and captive marsupials from Western Australia and Queensland. Blood samples were screened by PCR at the 18S rDNA locus, and the glycosomal glyceraldehyde phosphate dehydrogenase gene.Overall, 5.3% of the blood samples were positive at the 18S rDNA locus. All positives belonged to wild-captured Western Australian individuals, where trypanosome-specific DNA was detected in 9.8% of the screened samples from wild marsupials, in common brushtail possums, and woylies. The detection rate of trypanosome DNA in these two host species was 12.5% and 20%, respectively.Phylogenetic analyses based on two loci, indicated that the possum-derived trypanosome isolates were genetically distinct, and most closely related to the Australian marsupial trypanosomes H25 from a kangaroo, and BRA2 from a bush rat. This is the first study to genetically characterise trypanosome isolates from possums.The analysis of the woylie-derived isolates demonstrated that this marsupial host can harbour multiple genotypes within the same geographical location and furthermore multiple genotypes within the same host, indicative of mixed infections. All the woylie-derived genotypes grouped with trypanosomes found in Australian marsupials, suggesting that they are more likely to belong to an endemic or Australasian trypanosome species.This is the first study to genetically characterise trypanosome isolates from possums (Trichosurus vulpecula). Although the clinical significance of these infections is currently unknown, the identification of these novel sequences may support future investigations on transmission, threats to endangered wildlife, and evolutionary history of the genus Trypanosoma.

Monitoring the pleomorphism of Trypanosoma brucei gambiense isolates in mouse: Impact on its transmissibility to Glossina palpalis gambiensis

Substantial differences have been observed between the cyclical transmission of three Trypanosoma brucei gambiense field isolates in Glossina palpalis gambiensis (Ravel et al., 2006). Differences in the pleomorphism of these isolates in rodent used to provide the infective feed to Glossina, could explain such results, since stumpy forms are preadapted for differentiation to procyclic forms when taken up in a tsetse bloodmeal. To assess this possibility, mice were immunosuppressed and inoculated intraperitoneally with the three isolates (six mice for each trypanosome isolate); then parasitaemia and pleomorphism were determined daily for each mouse. The three T. b. gambiense isolates induced different infection patterns in mouse. The parasitaemia peak was rapidly reached for all the isolates and maintained until mice death for two isolates, while the third isolate rapidly showed a falling phase followed by a second parasitaemia plateau. The proportion of the stumpy forms varied from 15% to 70% over the duration of the experiment and according to the isolate. One isolate, which displayed the highest proportion of stumpy forms and reached the stumpy peak at the onset of the falling phase of parasitaemia, was used to study the relationship between the proportion of stumpy forms and transmissibility to tsetse fly. The results indicated that the transmissibility of trypanosomes was not correlated to the proportion of non-dividing stumpy forms.

Phylogenetic analysis reveals the presence of the Trypanosoma cruzi clade in African terrestrial mammals

Despite the impact of some trypanosome species on human and livestock health, the full diversity of trypanosomes in Africa is poorly understood. A recent study examined the prevalence of trypanosomes among a wide variety of wild vertebrates in Cameroon using species-specific PCR tests, but six trypanosome isolates remained unidentified. Here they have been re-examined using fluorescent fragment length barcoding (FFLB) and phylogenetic analysis of glycosomal glyceraldehyde phosphate dehydrogenase gGAPDH and 18S ribosomal RNA (rDNA) genes. Isolates from a monkey ( Cercopithecus nictitans) and a palm civet ( Nandinia binotata) belonged to the Trypanosoma cruzi clade, known previously only from New World and Australian terrestrial mammals, and bats from Africa, Europe and South America. Of the four other isolates, three from antelope were identified as Trypanosoma theileri, and one from a crocodile as T. grayi. This is the first report of trypanosomes of the T. cruzi clade in African terrestrial mammals and expands the clade's known global distribution in terrestrial mammals. Previously it has been hypothesized that African and New World trypanosomes diverged after continental separation, dating the divergence to around 100 million years ago. The new evidence instead suggests that intercontinental transfer occurred well after this, possibly via bats or rodents, allowing these trypanosomes to establish and evolve in African terrestrial mammals, and questioning the validity of calibrating trypanosome molecular trees using continental separation.

Detection of mutant P2 adenosine transporter (TbAT1) gene in Trypanosoma brucei gambiense isolates from northwest Uganda using allele‐specific polymerase chain reaction

To assess the application of allele-specific PCR (AS-PCR) as a fast, cheap and reliable method for detecting mutant TbAT1 associated with melarsoprol relapse in Trypanosoma brucei gambiense isolates from northwest Uganda. A total of 105 trypanosome isolates were analysed using SfaN1 restriction fragment length polymorphism (RFLP) and AS-PCR, the former used as the gold standard. Sensitivity, specificity, positive and negative predictive values of AS-PCR as well as agreement between the tests were determined. Eleven trypanosome isolates had mutant TbAT1 while 94 exhibited the wild-type TbAT1 genes. There was a highly significant agreement between SfaN1 RFLP and AS-PCR with kappa and intra-class correlation values of 1.0. The sensitivity and specificity of AS-PCR were both 100%, while the positive and negative predictive values were found to be equal to 1.0. Cost and time analyses were performed and AS-PCR was 4.3 times cheaper than SfaN1 RFLP, in addition to the less time required for its execution. AS-PCR should be the test of choice for screening for mutant TbAT1 in the ever-increasing numbers of field trypanosome isolates.

Spatially and Genetically Distinct African Trypanosome Virulence Variants Defined by Host Interferon‐γ Response

We describe 2 spatially distinct foci of human African trypanosomiasis in eastern Uganda. The Tororo and Soroti foci of Trypanosoma brucei rhodesiense infection were genetically distinct as characterized by 6 microsatellite and 1 minisatellite polymorphic markers and were characterized by differences in disease progression and host-immune response. In particular, infections with the Tororo genotype exhibited an increased frequency of progression to and severity of the meningoencephalitic stage and higher plasma interferon (IFN)- gamma concentration, compared with those with the Soroti genotype. We propose that the magnitude of the systemic IFN- gamma response determines the time at which infected individuals develop central nervous system infection and that this is consistent with the recently described role of IFN- gamma in facilitating blood-brain barrier transmigration of trypanosomes in an experimental model of infection. The identification of trypanosome isolates with differing disease progression phenotypes provides the first field-based genetic evidence for virulence variants in T. brucei rhodesiense.

Cyclical transmission of Trypanosoma brucei gambiense in Glossina palpalis gambiensis displays great differences among field isolates

Six sets of teneral Glossina palpalis gambiensis (Diptera: Glossinidae) were fed on mice infected with six different isolates of Trypanosoma brucei gambiense (each mouse was infected with one of the isolates), previously isolated from patients in the sleeping sickness focus of Bonon, Côte d’Ivoire and in Makoua, Congo. All the tsetse flies were dissected 42 days post-infection and midgut and salivary glands were examined for trypanosomes by microscopical examination. No infection was observed with the reference stock whereas each of the five recently isolated trypanosome isolates was able to infect tsetse flies, with rates of infection varying between 9.7 and 18.2% depending on the isolate. Three isolates displayed only immature infections with 9.7, 17.3 and 18% of the flies showing trypanosomes in their midgut. One isolate gave both immature (12.1%) and mature infections (6.1%). Finally, the last isolate involved only mature infections in 9.7% of the Glossina species examined. These substantial differences in the cyclical transmission of T. b. gambiense in the same fly species could have important implications for the epidemiology of the transmission of Human African Trypanosomiasis.

Detection of T.b. rhodesiense Trypanosomes in Humans and Domestic Animals in South East Uganda by Amplification of Serum Resistance‐Associated Gene

The human serum resistance-associated (SRA) gene was identified in 28 (80%) of the 35 T.b. rhodesiense trypanosomes from parasitologically confirmed sleeping sickness cases, using the primers designed by Radwanska and in 27 (77.1%) of the same 35 T.b. rhodesiense trypanosomes using the primers designed by Gibson. However, about 20% of the 35 T.b. rhodesiense trypanosomes could not be detected by SRA-polymerase chain reaction (PCR) even when an aliquot of the first PCR was used in the second PCR, indicating that the gene may be absent in those trypanosomes or the trypanosomes could be having another variant of SRA not detectable by these primers since three variants of SRA genes have so far been identified or the amount of trypanosomal DNA extracted from infected blood was too low to be detected. The trypanosome isolates that are SRA gene negative may indicate the presence of some T.b. rhodesiense trypanosomes with modified or lack SRA genes or simple loss of the SRA gene from the expression site in which it resides during antigenic variation. Analysis of trypanosomes derived from domestic animals showed that 79 (90.8%) of the 87 trypanosomes isolated from cattle were positive by Trypanosoma brucei (TBR)-PCR, indicating that they were Trypanozoon while 8 (9.2%) of the trypanosome isolates which were negative by TBR-PCR could be T. vivax, T. congolense, or T. theileri. When subjected to SRA-PCR, 10 (11.5%) of the 87 trypanosomes isolates derived from cattle were positive, indicating that there could be T.b. rhodesiense circulating in cattle, which is similar to the percentage of T.b. rhodesiense previously obtained in cattle in Serere, Soroti district.

The role of Trypanosoma brucei MRPA in melarsoprol susceptibility

We previously showed that over-expression of Trypanosoma brucei MRPA, a member of the multidrug resistance protein family in T. brucei, reproducibly resulted in resistance to the anti-trypanosomal drug melarsoprol in vitro. MRPA is predicted to mediate efflux of melarsoprol as a conjugate with trypanothione, a glutathione–spermidine conjugate which is the major small thiol in trypanosomes. Here, we show that depletion of MRPA by RNA interference resulted in moderate hypersensitivity to both melarsoprol and melarsen oxide. Over-expression of MRPA alone is not sufficient to cause melarsoprol resistance in vivo, although it is sufficient in vitro. This discrepancy is not an effect of drug metabolism since over-expression of MRPA alone conferred resistance to melarsoprol and its principle metabolite, melarsen oxide, in vitro. Over-expression of MRPA was not detected in four melarsoprol-resistant trypanosome isolates from sleeping sickness patients.

Diminazene Aceturate Resistance on the Virulence of Trypanosoma brucei for Rats

Four groups (A, B, C and D) of 10 naïve rats were used to compare the virulence of isolates of a strain of Trypanosoma brucei before and after the development of diminazene aceturate resistance. Group A rats were uninfected (controls). Group B rats were infected with a trypanosome isolate unexposed to the drug, while groups C and D rats were infected with two different drug-resistant isolates of the same strain. Rats in the three infected groups each received 10(6) trypanosomes intraperitoneally. Prepatent periods were longer (P<0.05) in groups C and D than in group B. The parasitaemic periods in groups B, C and D were similar, but group C and D rats differed from group B rats in surviving longer, and in showing lower degrees of parasitaemia, anaemia and hepatomegaly. No differences were noted between group C and D rats. Thus, diminazene aceturate resistance appeared to reduce the virulence of T. brucei.

The susceptibility of &lt;i&gt;Trypanosoma congolense&lt;/i&gt; isolated in Zambézia Province, Mozambique, to isometamidium chloride, diminazene aceturate and homidium chloride

Resistance to trypanocidal drugs has been detected in various African countries and is a serious impediment to the control of livestock trypanosomosis. To determine whether drug resistant trypanosome strains are present in the Zambézia Province of Mozambique a study was initiated. To assess the effect of the farming system and the drug-use regimen on the development of drug resistance, trypanosome isolates were collected from cattle from subsistence and commercial livestock production systems. The susceptibility of seven isolates against isometamidium chloride, diminazene aceturate and homidium chloride was tested in mice using a multiple-dose test. In four of the seven isolates high levels of drug resistance to diminazene aceturate and isometamidium chloride were detected. In most cases the observed levels of drug resistance correlated with the drug-use practices in the particular livestock production system.

A new lineage of trypanosomes from Australian vertebrates and terrestrial bloodsucking leeches (Haemadipsidae)

Little is known about the trypanosomes of indigenous Australian vertebrates and their vectors. We surveyed a range of vertebrates and blood-feeding invertebrates for trypanosomes by parasitological and PCR-based methods using primers specific to the small subunit ribosomal RNA (SSU rRNA) gene of genus Trypanosoma. Trypanosome isolates were obtained in culture from two common wombats, one swamp wallaby and an Australian bird ( Strepera sp.). By PCR, blood samples from three wombats, one brush-tailed wallaby, three platypuses and a frog were positive for trypanosome DNA. All the blood-sucking invertebrates screened were negative for trypanosomes both by microscopy and PCR, except for specimens of terrestrial leeches (Haemadipsidae). Of the latter, two Micobdella sp. specimens from Victoria and 18 Philaemon sp. specimens from Queensland were positive by PCR. Four Haemadipsa zeylanica specimens from Sri Lanka and three Leiobdella jawarerensis specimens from Papua New Guinea were also PCR positive for trypanosome DNA. We sequenced the SSU rRNA and glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) genes in order to determine the phylogenetic positions of the new vertebrate and terrestrial leech trypanosomes. In trees based on these genes, Australian vertebrate trypanosomes fell in several distinct clades, for the mostpart being more closely related to trypanosomes outside Australia than to each other. Two previously undescribed wallaby trypanosomes fell in a clade with Trypanosoma theileri, the cosmopolitan bovid trypanosome, and Trypanosoma cyclops from a Malaysian primate. The terrestrial leech trypanosomes were closely related to the wallaby trypanosomes, T. cyclops and a trypanosome from an Australian frog. We suggest that haemadipsid leeches may be significant and widespread vectors of trypanosomes in Australia and Asia.

Phylogenetic analysis of freshwater fish trypanosomes from Europe using ssu rRNA gene sequences and random amplification of polymorphic DNA

The taxonomy and phylogenetic relationships of fish trypanosomes are uncertain. A collection of 22 cloned trypanosome isolates from 14 species of European freshwater fish and 1 species of African freshwater fish were examined by molecular phylogenetic analysis. The small subunit ribosomal RNA (ssu rRNA) genes of 8 clones were sequenced and compared with ssu rRNA gene sequences from a wider selection of vertebrate trypanosome isolates by phylogenetic analysis. All trypanosomes from freshwater fish fell in a single clade, subdivided into 3 groups. This clade sits within a larger, robust clade containing trypanosomes from marine fish and various amphibious vertebrates. All 22 trypanosome clones were analysed by random amplification of polymorphic DNA. The resulting dendrogram shows 3 groups, which are congruent with the groups identified in the ssu rRNA gene phylogeny. Two of the groups contain the majority of trypanosome isolates and within-group variation is slight. These groups do not separate purported trypanosome species distinguished by morphology or host origin, and thus these criteria do not appear to be reliable guides to genetic relationships among fish trypanosomes. However, we suggest that the 2 groups themselves may represent different species of fish trypanosomes. The polymorphic DNA markers we have identified will facilitate future comparisons of the biology of these 2 groups of fish trypanosomes.

Randomly amplified polymorphic DNA analysis of Trypanosoma rangeli and allied species from human, monkeys and other sylvatic mammals of the Brazilian Amazon disclosed a new group and a species-specific marker.

We characterized 14 trypanosome isolates from sylvatic mammals (9 from primates, 1 from sloth, 2 from anteaters and 2 from opossum) plus 2 human isolates of Brazilian Amazon. These isolates were proven to be Trypanosoma rangeli by detection of metacyclic trypomastigotes in the salivary glands of triatomines and by a specific PCR assay. Polymorphism determined by randomly amplified polymorphic DNA (RAPD) revealed that most (12) of the Brazilian T. rangeli isolates from the Amazon differed from those of other geographical regions, thus constituting a new group of T. rangeli. Four Brazilian isolates clustered together with a previously described group (A) that was described as being composed of isolates from Colombia and Venezuela. Isolates from Panama and El Salvador form another group. The isolate from Southern Brazil did not cluster to any of the above-mentioned groups. This is the first study that assesses the genetic relationship of a large number of isolates from wild mammals, especially from non-human primates. A randomly-amplified DNA fragment (Tra625) exclusive to T. rangeli was used to develop a PCR assay able to detect all T. rangeli groups.

Identification of Trypanosoma brucei circulating in a sleeping sickness focus in Côte d’Ivoire: assessment of genotype selection by the isolation method

Genetic studies of Trypanosoma brucei have been mainly based on rodent inoculation (RI) for isolation of trypanosome strains. However, Trypanosoma brucei gambiense is difficult to grow in rodents. The development and use of the Kit for In Vitro Isolation (KIVI) of trypanosomes has led to a better isolation success. However, some authors report a genetic monomorphism in T. b. gambiense, and the extensive use of the KIVI was suspected as being responsible for this low genetic diversity. In the present work, trypanosome stocks were isolated from both humans and pigs in an active sleeping sickness focus in Côte d’Ivoire. Two methods were simultaneously used for this purpose: KIVI and rodent inoculation. None of the human stocks grew in rodents. Some of the stocks originating from pigs could be isolated with both methods. Each of these stocks (from the same pig) showed a different isoenzymatic pattern according to the isolation method used. All the human stocks identified belonged to the major zymodeme 3 of T. b. gambiense group 1, whereas the stocks isolated from pigs belonged to a new group of zymodemes even if they were genetically closely related. These observations may have significant implications when analysing the population structure of T. brucei, and also raise again the question of the importance of the animal reservoir in Human African Trypanosomiasis (HAT).

Trypanocidal activity of conformationally restricted pentamidine congeners.

A series of conformationally restricted congeners of pentamidine in which the flexible pentyl bridge of pentamidine was replaced by trans-1,2-bismethylenecyclopropyl, phenyl, pyridinyl, piperazinyl, homopiperazinyl, and piperidinyl groups were synthesized. The compounds were evaluated for trypanocidal activity in vitro and in vivo against one drug-sensitive and three drug-resistant trypanosome isolates. The DNA binding affinity of the compounds was also studied using calf thymus DNA and poly(dA-dT). The nature of the linker influenced the DNA binding affinity as well as the trypanocidal activity of the compounds. trans-1,2-Bis(4-amidinophenoxymethylene)cyclopropane (1) was over 25-fold more potent than pentamidine against the drug-resistant isolate KETRI 243As-10-3, albeit with comparable DNA binding affinity. N,N'-Bis(4-amidinophenyl)homopiperazine (8) was the most potent trypanocide in vitro against all four trypanosome isolates studied, but N,N'-bis(4-amidinophenyl)piperazine (6) was the most effective agent in vivo against both drug-sensitive and drug-resistant trypanosomes.

Drug sensitivity of trypanosome populations from cattle in a peri-urban dairy production system in Uganda

Cattle from 50 farms in Mukono County, Uganda, were monitored for trypanosomes every second month over an 18-month period (1995–1996) by mini-anion exchange chromatography and haematocrit centrifugation techniques. Eighteen trypanosome isolates collected from cattle during this period were characterised in cattle, goats and mice for their sensitivity to homidium, isometamidium and diminazene; 10 of the isolates were selected randomly, 8 were from animals that had the highest serum isometamidium concentrations at the time the isolates were collected. All the isolates contained only Trypanosoma brucei and/or T. vivax. In naı̈ve Boran ( Bos indicus) cattle the isolates exhibited low pathogenicity and were sensitive to diminazene aceturate at 3.5 mg/kg body weight (bw) and isometamidium chloride at 0.5 mg/kg bw. In goats, 5 of 8 isolates were highly pathogenic, producing clinical signs indicative of central nervous system involvement within 60 days of infection; all such isolates contained T. brucei. However, all 8 populations were sensitive in goats to diminazene aceturate at 3.5 mg/kg bw. In contrast, 4 populations were refractory to treatment with isometamidium chloride at 0.5 mg/kg bw in at least 1 out of 3 goats each. Furthermore, 5 populations were refractory to treatment with homidium chloride at 1.0 mg/kg bw in a minimum of 2 out of 3 goats each. In mice, the 50% curative dose values for 11 Mukono isolates that contained T. brucei ranged from 0.30 to 1.89 mg/kg bw for diminazene aceturate, from 0.02 to 0.17 mg/kg bw for isometamidium chloride and from 0.90 to 4.57 mg/kg bw for homidium chloride. Thus, by comparison to reference drug-sensitive populations, all the stabilates were highly sensitive to diminazene and isometamidium, while some expressed low levels of resistance to homidium.