The elimination of Trypanosoma brucei gambiense? Challenges of reservoir hosts and transmission cycles: Expect the unexpected.

The World Health Organisation has set the goal for elimination of Human African Trypanosomiasis (HAT), caused by Trypanosoma brucei gambiense (gHAT), as a public health problem for 2020 and for the total interruption of transmission to humans for 2030. Targeting human carriers and potential animal reservoir infections will be critical to achieving this ambitious goal. However, there is continuing debate regarding the significance of reservoir host animals, wild and domestic, in different epidemiological contexts, whilst the extent and duration of the asymptomatic human carrier state is similarly undefined. This paper reviews the status of the knowledge of latent infections in wild and domestic animal reservoir hosts towards the goal of better understanding their role in the transmission dynamic of the disease. Focus areas include the transmission cycles in non-human hosts, the infectivity of animal reservoirs to Glossina palpalis s.l., the longevity of infection and the stability of T. b. gambiense biological characteristics in antelopes and domestic animals. There is compelling evidence that T. b. gambiense can establish and persist in experimentally infected antelopes, pigs and dogs for a period of at least two years. In particular, metacyclic transmission of T. b. gambiense has been reported in antelope-G.p.palpalis-antelope and pig-G.p.gambiensis-pig cycles. Experimental studies demonstrate that the infectiveness of latent animal reservoir infections with T. b. gambiense is retained in animal-Glossina-animal cycles (antelopes and pigs) for periods of three years and human infectivity markers (human serum resistance, zymodeme, DNA) are stable in non-human hosts for the same period. These observations shed light on the epidemiological significance of animal reservoir hosts in specific ecosystems characterized by presently active, as well as known "old" HAT foci whilst challenging the concept of total elimination of all transmission by 2030. This target is also compromised by the existence of human asymptomatic carriers of T. b. gambiense often detected outside Africa after having lived outside tsetse infested areas for many years - sometimes decades. Non-tsetse modes of transmission may also play a significant but underestimated role in the maintenance of foci and also preclude the total elimination of transmission - these include mother to child transmission and sexual transmission. Both these modes of transmission have been the subject of case reports yet their frequency in African settings remains to be ascertained when the context of residual foci are discussed yet both challenge the concept of the possibility of the total elimination of transmission.

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 nai;ve 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.

A survival analysis of trypanosomosis diagnostic-test performance under natural infection challenge.

Little is known about the time-to-first detection and the time difference (TD) between first parasitological and first serological diagnosis of Trypanosoma spp. infections under natural infection challenge in cattle. The objective of our study was to estimate these measures of "longitudinal aspects" of diagnostic performance and to investigate potential biological factors. Emphasis was on diagnosis at the genus level (Trypanosoma spp.). Twelve N'Dama, 12 Gobra zebu and 12N'DamaxGobra (F1) crossbred cattle (all animals non-infected at the start of the experiment, six male and six female animals in each cohort) were exposed to natural high tsetse challenge in the Niamina East area in The Gambia [Acta Trop. 71 (1998) 57]. The animals were investigated parasitologically (detection of trypanosomes by buffy-coat technique), serologically (detection of T. brucei, T. congolense and T. vivax antigen by enzyme-linked immunosorbent assay (ELISA)) and clinically (packed-cell volume, PCV) over a period of 180 days. The time-to-first detection of trypanosomes, trypanosomal antigen (cut-off as suggested by test supplier) and drop in PCV (subject-based cut-off values) were recorded as outcomes of interest. Thus, incidence was both parasitologically (I(p)), serologically (I(s)) and clinically (I(c)). Recurrent events were not considered. The TD between first parasitological and first serological detection was established as I(s) time minus I(p) time. The effect of breed and sex on the time-to-first detection and on TD was investigated using Cox (proportional hazard) regression and ANOVA, respectively. We found that time-to-first parasitological detection of trypanosomosis in N'Dama animals was significantly longer than in the two other breeds (Cox regression, P=0.002). A similar but less-strong (P=0.063) effect of breed on time-to-first detection of trypanosomal antigen was found, whereas no breed effect was observed for clinical detection (P=0.432). Sex had no effect in all detection systems. The TD varied between -56 and 115 (mean 28). Marked differences among breeds and between sexes were not observed (ANOVA, P=0.8). We suggest that incidence studies are more suitable for detecting risk factors for animal trypanosomosis than prevalence-based (cross-sectional) studies because the latter often result in misinterpretation of factors that increase the survival time with infection as risk factors.

Standardised tests in mice and cattle for the detection of drug resistance in tsetse-transmitted trypanosomes of African domestic cattle.

Resistance to the drugs used to control African animal trypanosomosis is increasingly recognised as a constraint to livestock production in sub-Saharan Africa. The most commonly used tests for detection of trypanocidal drug resistance are tests using mice or ruminants, but these suffer from lack of standardisation and hence it may be difficult to compare the results of different investigators. Tests in mice are less expensive than tests in ruminants, but while tests in mice they may be useful as a general guide to resistance in a geographic area they should not be extrapolated to cattle on an individual trypanosome level. Moreover, the commonly used protocols are too laborious for their application to large number of trypanosome isolates on an area-wide basis. This paper presents guidelines for standardised testing of trypanocidal drugs in vivo, and introduces a simplified single-dose test for use in mice, which is convenient for use in areas with limited laboratory facilities. The single-dose test is appropriate for characterisation of geographic areas in terms of trypanocidal drug resistance using large numbers of trypanosome isolates, for making comparisons between areas, and for monitoring changes in trypanocidal drug resistance over time. Multiple-dose tests may be used to determine the degree of resistance of individual stabilates to be determined precisely in mice are also described, but for logistical reasons these will rarely be conducted on more than a few stabilates, and testing of a larger number of stabilates in the single-dose test will generally provide more useful information. Finally, we describe tests in cattle that may be used to determine the efficacy of recommended curative doses of trypanocidal drugs for the treatment of infection with individual trypanosome isolates, including Trypanosoma vivax, which is rarely infective for mice.

Multiple-drug resistant Trypanosoma congolense populations in village cattle of Metekel district, north-west Ethiopia.

Investigations were carried out to determine the prophylactic activity of isometamidium chloride in village populations of cattle naturally infected with trypanosomes in Metekel district, northwest Ethiopia. In a cross-sectional study in March 1997, 484 randomly selected cattle from four villages were examined for trypanosome infections by the dark ground/phase contrast buffy coat technique (BCT). The trypanosome prevalence was 17.2%. Trypanosoma congolense was the dominant species accounting for 47.6% of the overall infections. Fifty parasitaemic cattle from two villages were treated with isometainidium chloride (Trypamidium(R)) at a prophylactic dose of 1.0 mg/kg body weight (b.w.) and thereafter monitored on a monthly basis for parasitaemia. Trypanosomes were detected in six cattle within 1 month and in 18 cattle within 2 months of treatment. Twenty three percent (6/26) of cattle infected with T. congolense at the time of treatment were detected parasitaemic with this trypanosome species 1 month after treatment. Mice were infected with three T. congolense isolates obtained from cattle which were detected parasitaemic within one or 2 months after isometamidium treatment. The mice were subsequently treated with ranges of doses of isometamidium chloride or diminazene aceturate (Berenil(R)) and thereafter monitored for parasitaemia for a period of 60 days. Isometamidium chloride at doses of 0.5-4.0 mg/kg b.w. and diminazene aceturate at doses of 3.5-28.0 mg/kg b.w. failed to cure T. congolense infections in any of the animals. Three clones were derived from one of the isolates; each clone expressed high levels of resistance to both trypanocides when tested in mice. Based on these results it is concluded that the prophylactic activity of isometamidium is greatly reduced for some of the T. congolense populations present in the area, and in addition there is resistance to diminazene aceturate in this trypanosome species.

Financial analysis of animal trypanosomiasis control using cypermethrin pour-on in Kenya.

The financial impact of use of cypermethrin pour-on (Ectopor(R)) in control of animal trypanosomiosis was determined in a trial undertaken by the Kenya Trypanosomiasis Research Institute (KETRI). This trial started in December 1990 and ended in February 1992. It was undertaken in two adjacent ranches in the coast province of Kenya. The trial site was in an area of high apparent density (AD) of tsetse flies, and at the start of the trial no cattle were kept in this area. Cypermethrin was applied fortnightly to the 1100 steers which were kept in pour-on ranch 'A' while another 100 steers were kept in control ranch 'B' to act as control sentinels. From the main pour-on group, 100 animals were identified as the pour-on sentinels and compared to the control sentinels which received no pour-on.Pour-on application led to a significant decrease in the tsetse AD in the pour-on ranch A to 90% of the initial AD in some areas. The animals treated with pour-on had a significantly higher mean packed-cell volume (PCV). The weekly prevalence of trypanosome infections in animals treated with pour-on was <4% with only one exception when it was <10%. In the control animals, the prevalence ranged between 10 and 50% (with a few exceptions when it was <10%). The incidence of tick-borne diseases was lower in the pour-on animals. The mean monthly weights of the pour-on animals was significantly higher, and at the end of the trial the pour-on animals had a mean weight gain of 136.70+/-16.7kg while the control animals had gained 97.16+/-22.6kg. The financial net return of using cypermethrin pour-on was positive and the financial rate of return of 122.6% indicated that use of the pour-on was highly beneficial despite the high cost of the product.

Impact of tsetse control on the age-specific prevalence of trypanosomosis in village cattle in southeast Uganda.

The prevalence of trypanosomosis, mean packed cell volume and anti-trypanosomal antibody levels in village cattle of different age groups (< 0.5 year, 0.5-2 years, > 2-5 years and > 5 years) in the areas with tsetse control were compared with those of corresponding age groups in areas without tsetse control in Tororo, southeast Uganda. The prevalence of trypanosomosis in cattle in the age groups of 0.5-2 years, > 2-5 years and > 5 years in the areas with tsetse control was significantly lower than in cattle in similar age groups in the areas without tsetse control (p < 0.5). Trypanosoma vivax was the most predominant Trypanosoma species in the areas with tsetse control, while T. congolense was the most predominant species in the areas without tsetse control. The mean Trypanosoma antibody levels in cattle in the age groups < 0.5 year, 0.5-2 years and > 2-5 years in the areas with tsetse control were significantly lower than those of the similar age groups in the areas without tsetse control (p < 0.5). The mean PCV values for cattle in the age groups 0.5-2 years, > 2-5 years and > 5 years from the areas with tsetse control were significantly higher than those of the similar age groups in the areas without tsetse control. Tsetse control appeared to have a considerable impact on the prevalence of trypanosomosis, distribution of Trypanosoma species, specific antibody levels and the packed cell volume of cattle in the different age groups.

Polymerase chain reaction and DNA probe hybridization to assess the efficacy of diminazene treatment in Trypanosoma brucei-infected cattle.

Four of eight Ankole longhorn cattle experimentally infected with Trypanosoma brucei were treated with 7 mg/kg diminazene aceturate (Berenil, Hoechst AG, Germany) at day 71 postinfection. The trypanocidal activity was monitored using polymerase chain reaction (PCR) and DNA probe hybridization. When extracted parasite DNA (without host DNA) was used, as little as 1 fg per reaction, which is equivalent to about 1-10% of the DNA in a single trypanosome, produced a specific product that was visible as a 177-bp band in an agarose gel. In infected cattle, specific PCR products could be amplified at as early as 1 day postinfection. PCR signals remained positive during infection, except in one sample, although aparasitemic phases occurred. In cases where treatment resulted in a significant clinical improvement, PCR signals disappeared at 3-4 days after the administration of the drug. By contrast, in cattle that showed clinical signs of CNS involvement after treatment, although aparasitemic, and died before the termination of the experiment, specific products could be amplified on several occasions following treatment. The PCR signals generated after treatment could be further enhanced by subsequent slot-blot hybridization with a T. brucei-specific DNA probe. We conclude that PCR coupled with DNA probe hybridization provides a highly sensitive tool for the assessment of therapeutic efficiency and disease progression in trypanosome infections, especially in chronic infections when the level of parasitemia is low or when trypanosomes are sequestered at cryptic sites.

A drug incubation Glossina infectivity test (DIGIT) to assess the susceptibility of Trypanosoma congolense bloodstream forms to trypanocidal drugs (Xenodiagnosis).

Blood was collected from two Sahelian goats, experimentally infected with either a drug-sensitive cloned population of Trypanosoma congolense (IL 1180) or a multiple drug-resistant T. congolense stock (Samorogouan/89/CRTA/267) and incubated at 37 degrees C for 30 min and 12 h, respectively, in the presence of different drug concentrations (0.5, 1.0, 10.0 and 100.0 microg/ml blood) of diminazene aceturate or isometamidium chloride. After that, the trypanosome/blood/drug suspensions were offered to tsetse flies (2100 teneral Glossina morsitans submorsitans) through an in vitro feeding system, using a silicone membrane. All tsetse flies were dissected and examined for the presence of trypanosomes in labrum, hypopharynx and midgut 20 days after their infective blood-meals. Infectivity of the drug-sensitive cloned population was already completely abolished after incubation with 0.5 microg/ml of both drugs; however, 13.6-42.2% of tsetse having been fed on untreated blood had developed an infection. In contrast, no significant differences were observed in the infection rates between the experimental groups and their control groups when fed on blood infected with the multiple drug-resistant stock after incubation for 30 min with up to 10 microg/ml of diminazene or isometamidium. In consequence, tsetse appear to be a useful tool in the assessment of drug susceptibility of typanosome populations.

Use of a PCR assay for the specific and sensitive detection of Trypanosoma Spp. in naturally infected dairy cattle in peri-urban Kampala, Uganda.

The objective of this study was to compare the sensitivity and specificity of the polymerase chain reaction (PCR) with the hematocrit centrifugation technique (HCT) and the mini-anion-exchange centrifugation technique (m-AECT) for diagnosis of trypanosome infections in livestock. In a cross-sectional study, 486 cattle from 50 randomly selected farms in Mukono County, Uganda were investigated in June 1994. The direct parasitological techniques were performed in the field, resulting in 45 (9.3%) animals positive by HCT and 78 (16%) positive by m-AECT. The total prevalence (combined evidence of HCT and m-AECT) was 18.9%, with 78.2% Trypanosoma brucei only, 10.9% T. vivax and 10.9% mixed (T. bruceil T. vivax) infections. Trypanosomes of the subgenus Nannomonas were not detected. DNA was prepared by lysis from 181 randomly selected blood samples and amplified by PCR using species-specific oligonucleotide primers. Overall, the PCR gave positive results in 63 (34.8%) blood samples, with 76.2% positive only for T. brucei, 20.6% positive only for T. vivax and 3.2% positive for mixed (T. bruceil T. vivax) infections. The preliminary results from this study demonstrate that the detection rate of PCR is about two times higher than that of the direct parasitological techniques, suggesting a higher sensitivity. The higher proportion of T. vivax infections detected by PCR as compared to HCT/m-AECT is likely to be due to false parasitological classifications which might occur under field conditions.

Detection of trypanosome DNA in serologically positive but aparasitaemic horses suspected of dourine in Ethiopia.

A field study of horses was conducted in the province of Bale, Ethiopian highlands. A rapid questionnaire analysis indicated that dourine, known as "Dirressa", is a major health problem of equines in this area. A total of 121 horses suspected of dourine were examined by use of clinical, parasitological, serological and DNA based techniques. Incoordination of hindlegs (76%), swelling of external genitalia (48.8%) and emaciation (39.7%) were the most common clinical signs observed. Using the haematocrit centrifugation technique (HCT), no trypanosomes were detected in blood, genital washes or tissue fluids. By contrast, trypanosome specific DNA products were amplified by PCR and subsequently detected by DNA probe hybridization in blood samples of 29 horses (29/104), all serologically positive by CFT and/or ELISA. Positive PCR results were significantly associated with swelling of external genitalia (P< 0.05). There is strong evidence, although there was no direct detection of T. equiperdum, that dourine is highly prevalent in the area, a finding which is in accordance with earlier reports. It is concluded, that this PCR assay provides a very sensitive tool in the diagnosis of active infections of dourine in endemic areas where trypanocidal drug use is common.

Impact of biological factors on the interpretation of bovine trypanosomosis serology.

A total of 457 cattle from dairy farms in Mukono County, Uganda, were investigated for Trypanosoma antibodies by ELISA. The objective of the study was to identify explanatory covariate factors for seropositivity among nine farm-specific and four animal-specific variables. We used logistic regression models for parasitological and serological outcome variables and then compared the adjusted odds ratios for explanatory factors between the models. Age is positively correlated with seropositivity but not with the detection of the parasite. Therefore, age group-specific cut-off values were established using mixture-distribution analysis. This procedure, as well as a mixture-distribution-derived cut-off value for the total sample, resulted in a greater relative efficiency of the ELISA as compared to conventional interpretation (cut-off value defined using non-exposed negative controls). The relevance of age and other biological factors for the serological status is briefly discussed.

Sleeping sickness in Zaire: a nested polymerase chain reaction improves the identification of Trypanosoma (Trypanozoon) brucei gambiense by specific kinetoplast DNA probes.

Blood samples collected in the sleeping sickness focus of Boma, Zaire, from human patients and domestic animals were analysed by polymerase chain reaction (PCR) for the presence of trypanosome DNA. The comparison of PCR and miniature anion exchange centrifugation technique (m-AECT) results clearly shows that in domestic animals mixed infections (Trypanozoon/Trypanosoma [Nannomonas] congolense) are more frequently diagnosed by PCR than by m-AECT. Trypanozoon positive blood samples were further analysed for Trypanosoma (Trypanozoon) brucei gambiense. For that purpose amplified minicircle kinetoplast DNA (minicircle kDNA) was differentiated in gambiense and non-gambiense by hybridization with DNA probes. To analyse blood samples, especially those with low parasite numbers, the amplification step had to be improved by a nested PCR. Subsequent hybridization was done with kDNA probes generated by PCR from blood samples which had been obtained from a human patient infected with T.(T.) b. gambiense and a pig infected with Trypanozoon. The hybridization results clearly show that at least two genotypes of Trypanozoon parasites occur in the sleeping sickness focus of Boma, Baz-Zaire. One obviously corresponds to T.(T.) b. gambiense and was present in humans and two domestic animals (pig, dog). The other genotype seems to be associated with T.(T.) b. brucei and could be detected only in the blood of domestic animals. This is the first time that field samples could be analysed by a technique which facilitates the molecular identification of T.(T.) b. gambiense without prior cloning, propagation, and/or isolation of the parasites. Therefore, this technique seems to be a promising tool to elucidate the significance of the animal reservoir for the epidemiology of the gambiense sleeping sickness in Africa.

Sequence and PCR-RFLP analysis of 14 novel BoLA-DRB3 alleles.

The genetic diversity of the bovine class II DRB3 locus was investigated by polymerase chain reaction (PCR) amplification and DNA sequencing of the first domain exon. Studying 34 animals of various cattle breeds, 14 previously unrecognized DRB3 alleles were identified. In three alleles, amino acid substitutions were observed that had not been previously found in bovine DRB3, but occurred at the same position in bovine DQB and in the DRB alleles of other mammals. For all newly identified alleles, the restriction fragment length polymorphism (RFLP) patterns of PCR products obtained with the enzymes RsaI, BstYI, and HaeIII were compared with patterns of 38 previously described alleles. Altogether, eleven novel PCR-RFLP types were defined. Twelve out of the 42 PCR-RFLP types identified so far were not found to be fully informative because they corresponded to more than one allelic sequence. PCR-RFLP may therefore be a rapid and useful method for DRB3 typing in cattle families, but for studies on outbred populations, sequencing and hybridization techniques are required.

Investigations on naturally occurring Trypanosoma evansi infections in horses, cattle, dogs and capybaras (Hydrochaeris hydrochaeris) in Pantanal de Poconé (Mato Grosso, Brazil).

The prevalence of Mal de Cadeiras--Portuguese for Trypanosoma (T.) evansi infections in horses--as well as the prevalence of T.evansi infections in cattle, dogs and free-ranging capybaras (Hydrochaeris hydrochaeris) was investigated in Pantanal de Poconé (Mato Grosso, Brazil). In 0.3, 8.6 and 8.0% of the horses, dogs and capybaras, respectively, infection was detected using standard parasitological methods. A seroprevalence of 4.1, 2.3, 7.1 and 22.0% was found in horses, cattle, dogs and capybaras, respectively, using an enzyme-linked immunosorbent assay for the detection of T.evansi antigen (Ag-ELISA), whereas 9.6, 4.2, 18.6 and 14.0% of the animals investigated were reactive in a T.evansi antibody (Ab-) ELISA. Positive ELISA results ('high responders') were identified using computer-assisted mixture analysis (C.A.MAN). Agglutinating antibodies were detected by the T.evansi card agglutination test for trypanosomiasis (CATT/T.evansi) in 14.6%, 1.3%, 15.7% and 22.0% of the horses, cattle, dogs and capybaras, respectively. A moderate but significant (kappa test; p < 0.05) agreement beyond chance level was observed between Ab-ELISA and CATT results but generally not between antibody and antigen detection tests. Therefore, in an attempt to maximize the information yield of the three serodiagnostic tests, their results were numerically scored (negative = 0, intermediate = 1, positive = 2) and added up to a total score (TS) which was considered indicative for infection when TS > or = 4 (results of the Ag-ELISA received double weight). Estimates of seroprevalence according to TS were 13.2, 4.7, 30.0 and 24.0% for horses, cattle, dogs and capybaras, respectively. Identical isoenzyme profiles, known as zymodeme 58 (T.evansi MCAN/BR/86/H), were found in all T.evansi stocks isolated in the study area (six from dogs, one from a horse and one from a capybara). From the results of this study it can be inferred that Mal de Cadeiras is endemic in Pantanal de Poconé. Although clinical and parasitological findings support the possible role of the capybara as a reservoir host of T.evansi, dogs and cattle--due to their close contact with horses--should rather be regarded as efficient reservoir hosts for Mal de Cadeiras in the study area.

The relationship between rickettsia-like-organisms and trypanosome infections in natural populations of tsetse in Liberia.

A survey of natural populations of tsetse flies for rickettsia-like-organisms (RLO) has been carried out in Liberia. A population of G.p. palpalis showed a strong association between trypanosome and RLO infection; both infections were at low levels in this species suggesting that this population is highly refractory to trypanosome infection. A small sample of G. nigrofusca, considered the most effective vector of trypanosomiasis in Liberia, was found to have very high prevalence of RLO infection. The selection pressures which could determine RLO infection rate are discussed.

[Animal reservoir hosts of Trypanosoma brucei gambiense in Zaire: trypanosome infections in two foci in Bas-Zaire]. / Le réservoir animal de Trypanosoma brucei gambiense au Zaire: infections trypanosomiennes dans deux foyers du Bas-Zaire.

The prevalence of Trypanosoma spp. infections in domestic animals was estimated in a forest (Boma) and a savanna (Kimpese) sleeping focus in Bas-Zaire. The miniature anion-exchange centrifugation technique was used to determine the infection rates with T. congolense, T. vivax and T. brucei spp. in 505 animals. T. congolense predominated in both foci with the highest prevalence in pigs (76.2%), followed by sheep (31.3%), dogs (30.6%) and goats (7.4%). T. vivax was seen only on two occasions. In the forest zone, T. brucei spp. infections were frequent (pigs 16.5%, sheep 6.2%, dogs 3.4%, goats 1.1%) in contrast to the savanna area where only one T. brucei spp. infection was diagnosed. Twenty five primary isolations of T. brucei were done using different isolation and stabilization approaches. Isolates and stocks await behavioural, biochemical and molecular biological identification to discriminate T. b. brucei and T. b. gambiense of domestic animal origin.