The persistence of genetic homogeneity among Trypanosoma brucei rhodesiense isolates from patients in north-west Tanzania.

Trypanosomes isolated during 1991 from nine patients with Rhodesian sleeping sickness in north-west Tanzania were genetically characterized by electrophoresis of ten enzymes. Eight isolates were allocated to a known zymodeme (Z306); another had an enzyme profile (Z379) not previously encountered. An example of Z306 has been previously isolated in 1971, nearby in a part of Rwanda adjacent to the border with Tanzania; in addition, a closely related isolate, in Z307, was collected in 1959 from a patient in north-west Tanzania. The new zymodeme (Z379) was 94% similar to Z306, and both had a close similarity of 89% to Z307. All these isolates belonged to the zambezi strain group of related zymodemes, and evidence is presented that other examples of the group have been collected from man in Tanzania since 1959. Such apparent long term genetic stability is similar to circumstances further south in an endemic area of Zambia, where 12 examples of Z306 and two of Z307 were acquired over a period of 12 years from patients. The similar genetic homogeneity among trypanosomes in endemic parts of both Tanzania and Zambia contrasted markedly with the heterogeneity described to the north of Tanzania in that different strain groups circulate in epidemic areas of Kenya and Uganda.

Absence of the LiTat 1.3 (CATT antigen) gene in Trypanosoma brucei gambiense stocks from Cameroon.

Antibodies to the variable antigen type (VAT) designated LiTat 1.3 are common in sera from parasitologically confirmed patients with gambian sleeping sickness. For this reason, LiTat 1.3 has been considered a suitable antigen for detecting Trypanosoma brucei gambiense in the Card Agglutination Test for Trypanosomiasis (CATT; Testryp-CATT, Smith Kline-RIT). However, surveys in the T.b. gambiense endemic focus of Fontem in Cameroon have suggested that expression of LiTat 1.3 might be rare or absent. We show here that the gene for LiTat 1.3 was indeed absent from some T.b. gambiense stocks isolated from this focus, and a LiTat 1.3-like gene was present in others. The divergent gene differed from the cloned version of LiTat 1.3. In addition, antibodies to LiTat 1.3 could not be detected in rabbits infected with either of the two kinds of T.b. gambiense from the Fontem area. We suggest that the absence of LiTat 1.3 expression in this focus may have important implications for the epidemiology and control of sleeping sickness, especially if heavy reliance is placed on the CATT.

A simplified method for identifying subspecies and strain groups in Trypanozoon by isoenzymes.

To characterize trypanosomes from the subgenus Trypanozoon, 272 stocks in 111 zymodemes were analysed by the polymorphisms seen in a rationalized range of nine enzymes, resolved by electrophoresis, mostly on cellulose acetate. Several highly polymorphic or invariant enzymes used previously were omitted, while two new enzymes, NHD and SOD were included; the isoenzymes seen for SOD were interpreted as two separate enzymes, SODA and SODB. Isoenzyme band patterns were analysed by two complementary numerical methods to elucide taxonomic relationships within the subgenus; groups of zymodemes corresponding to subspecies and strain groups were defined, which agreed closely with previous studies. Except for one zymodeme, Trypanosoma evansi could not be clearly distinguished from the bouaflé strain group. This strain group had enzymic features that overlapped to some extent those of the busoga group. Trypanosoma brucei gambiense and the zambezi, kakumbi, kiboko and sindo groups were clearly defined. Eight zymodemes could not be classified. A rapid identification system using a limited number of enzymes is presented.

Characterization by isoenzyme electrophoresis of Trypanozoon stocks from sleeping sickness endemic areas of south-east Uganda.

An epidemic of sleeping sickness, which started in 1976 in a focus within the county of Luuka in Central Busoga, has spread to cover the three districts of Busoga and large parts of the neighbouring districts of Tororo and Mukono. Forty-three isolates of the subgenus Trypanozoon from Busoga and Tororo (27 from man, 9 from cows, 2 from pigs and 5 from tsetse flies) were compared by thin-layer starch-gel electrophoresis for seven enzymes. Thirty zymodemes were identified; 17 of them were found circulating in the human population. The zymodemes seen previously in Busoga were still circulating together with several new ones. Of the 16 isolates from cattle, pigs and tsetse flies, only two had the same profile, indicating a high degree of diversity. Two zymodemes from cows and a pig were identical to those found in man, implicating domestic stock in the transmission of human disease in south-east Uganda. A computer analysis of the results produced six main zymodeme groups. One comprised only isolates from man; two were composed of isolates from man, domestic animals and tsetse; and three consisted of stocks from domestic animals only. These groups quite probably indicate the different cycles of transmission involving man, tsetse fly and domestic stock.

The substitution of procyclic for bloodstream form Trypanosoma brucei gambiense in isoenzyme studies.

Examination of 10 enzymes from 8 stocks of Trypanosoma brucei showed that procyclic forms could be substituted for bloodstream forms in isoenzyme studies. T. b. gambiense procyclic forms cultured in vitro offer a better source of material for genetic investigations because this species is usually of low infectivity and virulence to laboratory rodents. Using 6 stocks of T. b. gambiense and 2 stocks of T. b. brucei, enzyme patterns of bloodstream and procyclic forms were identical for isocitrate dehydrogenase, malic enzyme, two nucleoside hydrolases (utilizing inosine and deoxyinosine respectively), phosphoglucomutase and superoxide dismutase. Procyclic forms appeared to have greater threonine dehydrogenase activity than bloodstream forms. Consistent differences between bloodstream and culture forms were observed for alanine aminotransferase, aspartate aminotransferase and malate dehydrogenase. These agreed with known differences in the metabolism of procyclic and bloodstream forms.

A new method for isolating Trypanosoma brucei gambiense from sleeping sickness patients.

Low infectivity to laboratory mammals and low virulence make Trypanosoma brucei gambiense difficult to isolate and grow in amounts sufficient for biochemical characterization. We report the isolation of T.b. gambiense by feeding cryopreserved primary isolates to laboratory-reared Glossina morsitans morsitans, followed by rapid cultivation in vitro of procyclic forms dissected from infected tsetse fly midguts. This technique allows the characterization of hitherto unsampled populations and avoids selection due to long-term subpassage. Of 16 primary isolates from trypanosomiasis patients of the Fontem focus in Cameroon, 12 (75%) produced infections in tsetse whereas only 4 (25%) infected rats. Ten isolates were subsequently cultivated as procyclic forms in vitro; 2 failed to grow owing to bacterial contamination. In addition, 2 primary isolates from Côte d'Ivoire patients and a stock of low virulence from the Congo Republic were similarly grown. Only one primary isolate produced tsetse salivary gland infections, an observation consistent with the hypothesis that some populations of T.b. gambiense are intrinsically incompatible with G.m. morsitans.

Species-specific DNA probes for the identification of African trypanosomes in tsetse flies.

We have obtained 5 specific DNA probes for African trypanosomes of the subgenera Trypanozoon and Nannomonas. Each probe consists of one repeat unit of the major repetitive DNA (satellite DNA) of each species or intra-specific group. One probe hybridized with all members of subgenus Trypanozoon (except T. equiperdum which was not tested). In subgenus Nannomonas, one probe recognized T. simiae, but 3 probes were needed to identify all stocks of T. congolense available. Each of the 3 latter probes recognized trypanosomes from one of the 3 major groups of T. congolense previously defined by isoenzyme characterization, i.e. savannah, forest and Kenya coast types. As few as 100 trypanosomes could be unequivocally identified by dot blot hybridization and individual trypanosomes could be identified by in situ hybridization. We show how this simple methodology can be used in the field for the identification of immature and mature trypanosome infections in tsetse.

Trypanosoma congolense: the distribution of enzymic variants in east and west Africa.

A total of 114 stocks of Trypanosoma congolense originating from Kenya, Uganda, Tanzania, Zambia and Sudan, but including, for comparison, stocks from The Gambia, Liberia, Ivory Coast, Nigeria and Cameroun, were compared by isoenzyme electrophoresis for 6 enzymes. The zymodemes were grouped, both from a dendrogram and using a cladistic method, after calculating the dissimilarity, or distance, between profiles. Previous observations are broadly confirmed, the zymodemes clustering separately according to geographical origin and ecological zone. Thus, one group was composed almost entirely of East African stocks, and another of stocks from both East and West Africa, although each group was of savanna origin. A third group was composed of stocks from the humid, rain-forest zones of West Africa, and was particularly characterized by isoenzyme variants of superoxide dismutase and glucose-phosphate isomerase. Two stocks from the Kenyan coast formed a markedly separate group, which may be taxonomically distinct.

Two enzymically distinct stocks of Trypanosoma congolense.

Two stocks of Trypanosoma congolense, isolated from a sheep and a goat on the Kenyan coast, were found to be distinctly different from 112 other stocks of the same species after comparing, by isoenzyme electrophoresis, trypanosomes originating from various parts of Africa. The results show a marked genetic distinction that may be of epidemiological or taxonomic significance.

A preliminary comparison of Trypanosoma simiae and T. congolense by isoenzyme electrophoresis.

Trypanosoma simiae, although similar to T. congolense in morphology and mode of development in the tsetse vector, is regarded as a separate species mainly because it is highly pathogenic to the domestic pig and fails to infect rodents. To establish whether the two species are distinct biochemically, we compared by isoenzyme electrophoresis 2 isolates of T. simiae with 7 stocks of T. congolense, together with one of T. brucei. All isoenzyme patterns of the 6 enzymes examined differed in T. simiae from T. congolense and T. brucei stocks. This supports the designation of T. simiae as a separate species. However, a comparison involving a much larger collection of T. simiae is necessary in order to get conclusive evidence.

Isoenzyme characterization of some Trypanozoon stocks from a recent trypanosomiasis epidemic in Uganda.

The 1976-82 epidemic of human trypanosomiasis in south-east Uganda affected new foci to the north of the old endemic area bordering lake Victoria, and was associated with a different vector, Glossina fuscipes fuscipes; isoenzyme studies revealed that the epidemic involved different strains of pathogenic trypanosomes also. 58 Trypanozoon isolates from the epidemic area and from the adjoining endemic area of West Central Kenya were compared by thin-layer starch gel electrophoresis for 11 enzymes. Six different trypanosome zymodemes were circulating in man in the Ugandan epidemic, including the zymodeme found before 1976 in the old endemic area; all stocks examined from West Central Kenya belonged to this latter zymodeme. Trypanosomes identical to those found in man were found in cattle and a dog in Uganda, and in cattle in Kenya; these animals were presumably reservoir hosts of the human disease. Four isolates from triturated G. f. fuscipes collected in 1971 were identical but differed from all mammalian isolates examined.