Wendy C. Gibson

Sensitive detection of trypanosomes in tsetse flies by DNA amplification

African trypanosome species were identified using the Polymerase Chain Reaction (PCR) by targeting repetitive DNA for amplification. Using oligonucleotide primers designed to anneal specifically to the satellite DNA monomer of each species/subgroup, we were able to accurately identify Trypanosoma simiae, three subgroups of T. congolense, T. brucei and T. vivax. The assay was sensitive and specific, detecting one trypanosome unequivocally and showing no reaction with non-target trypanosome DNA or a huge excess of host DNA. The assay was used to identify developmental stage trypanosomes in the tsetse fly. The use of radioisotopes was not necessary and mixed infections could be detected easily by incorporating more than one set of primers in a single reaction. The use of crude preparations of template made the process very rapid. The methodology should be suitable for large-scale epidemiological studies.

Numerical analysis of enzyme polymorphism: a new approach to the epidemiology and taxonomy of trypanosomes of the subgenus Trypanozoon.

Publisher Summary The subgenus Trypanozoon contains several kinds of trypanosome, which are morphologically indistinguishable but differ in their behavior. At various times specific or subspecific status has been accorded to each kind, but, in recent years, those cyclically transmitted by tsetse in Africa have been separated into subspecies of Trypanosoma brucei , while the others, transmitted non-cyclically outside the African tsetse belts, have retained separate species status. However, although such new terminology may be more correct taxonomically, the practical problems of distinguishing the trypanosomes remain. Enzyme electrophoresis is being used on an increasingly wide scale for the intrinsic characterization. However, as the number of enzyme systems being used for identification grows, comparison of stocks becomes increasingly difficult and intricate, particularly when an enzyme occurs in a multibanded form after electrophoresis. After using 12 enzyme systems to characterize 160 Trypanozoon stocks, the results have been analyzed to establish the degree of relatedness of each of the 59 zymodemest encountered. The detailed analysis is presented in this chapter.

A high prevalence of mixed trypanosome infections in tsetse flies in Sinfra, Côte d'Ivoire, detected by DNA amplification.

The prevalence of various species and subgroups of trypanosomes in the Sinfra area of Côte dIvoire was determined using the polymerase chain reaction (PCR). Using this technique to amplify specific satellite DNA targets, it was possible to identify developmental-stage trypanosomes in the midguts and the proboscides of tsetse without expansion of parasite populations. The predominant tsetse species in the area was Glossina palpalis, while G. pallicera and G. nigrofusca were also present. Microscopical examination of 811 non-teneral flies revealed an infection rate of 14% in midguts and/or proboscides. Three subgroups of Trypanosoma congolense (Savannah, Forest & Kilifi), T. simiae, T. godfreyi, West African T. vivax and T. brucei ssp. were identified using PCR. T. congolense Forest was the most abundant of the Nannomonas trypanosomes. Approximately 40% of all infections were mixed, and there was a significantly higher prevalence of apparently mature T. brucei ssp. trypanosomes than has previously been reported. The present study demonstrates that PCR facilitates the easy identification of mature trypanosome infections in tsetse, providing a reliable estimation of trypanosomiasis challenge.

Further analysis of intraspecific variation in Trypanosoma brucei using restriction site polymorphisms in the maxi-circle of kinetoplast DNA

We have compared the maxi-circle kinetoplast DNA of 21 Trypanosoma brucei sp. stocks by analysis of restriction sites for nine restriction endonucleases. The analysis shows most of these stocks to have a maxi-circle sequence similar to that of 11 previously analysed stocks, with a difference of less than 3% between any two stocks. However, seven stocks stand out from the rest with at least two sites lost or gained for six of the nine restriction enzymes used. These seven distinctive stocks fall into two groups with some shared and some unique polymorphisms. One group had already been designated the kiboko group on the basis of isoenzyme patterns, but the relationship between nuclear markers and maxi-circle type is less clear-cut for the other group, designated sindo. Both groups seem to be in a wild animal-tsetse fly transmission cycle, with occasional infections in domestic stock, and may be reproductively isolated from the main T. brucei sp. population. The existence of the kiboko and sindo sub-groups shows that the maxi-circle is not shielded from evolutionary change. The lack of difference observed between the maxi-circles of the majority of T. brucei sp. stocks, including the gambiense and rhodesiense variants, must therefore reflect their close homology. Two geographical trends occur in T. brucei as a whole: (a) a trend in maxi-circle size, with increasing length of the variable region from West to East Africa, and (b) a greater frequency of certain restriction enzyme polymorphisms in East African stocks as compared to West African stocks.

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.

Characterization of Trypanozoon stocks from the South Nyanza sleeping sickness focus in Western Kenya

220 Trypanosoma (Trypanozoon) brucei sp. stocks isolated between 1969 and 1983 from the Lambwe valley sleeping sickness focus in South Nyanza, Western Kenya, were characterized by isoenzyme electrophoresis using 12 enzymes. 12 different zymodemes of T. (T.) b. rhodesiense were isolated from patients during the 13-year period and identical stocks were also found in cattle, reedbuck (Redunca redunca) and tsetse (Glossina pallidipes). Cattle may have played an important role in maintaining and increasing peridomestic transmission of trypanosomes during the 1980 outbreak of sleeping sickness in the valley, even though they themselves suffered heavy mortality. Sleeping sickness in Lambwe valley is unlikely to have been introduced from elsewhere, since T. (T.) b. rhodesiense stocks isolated from the valley were different from those from neighbouring epidemic areas. Alternatively, the recent outbreak may have been caused by the increased transmission associated with an expanding tsetse population. The possibility that genetic exchange contributed to the biochemical diversity of the trypanosomes examined is discussed.

Quantitation of genetic differences between Trypanosoma brucei gambiense, rhodesiense and brucei by restriction enzyme analysis of kinetoplast DNA.

We have compared a total of 44 recognition sites for 12 restriction endonucleases on the 20 kilobase pair maxi-circle of kinetoplast DNA from nine Trypanosoma brucei stocks, four which are known to be infective to man (tow gambiense and two rhodesiense variants). In addition to five polymorphic sites, these DNAs differ in the size of a 5 kilo-base pair region which is cleaved only by one of the restriction enzymes tested and which varies in size over 1.5 kilo-base pairs. Our analysis shows that the maxi-circle sequences of these stocks are very similar, the maximal calculated difference between any two being 3%. A relatively large difference was found between a rhodesiense stock from uganda and one from Zambia, confirming the distinction between northern and southern East African rhodesiense stocks found by analysis of enzyme polymorphisms (Gibson et al. (1980) Adv. Parasitol. 18, 175-246). The gambiense variants could not be identified by unique restriction site polymorphisms, but contained the smallest maxi-circle found thus far in T. Brucei. Our results indicate that T. brucei stocks infective and not infective to man are so closely related as to preclude their differentiation by analysis of kinetoplast DNA. This analysis is useful, however, in providing quantitative information about relatedness of stocks.

Unravelling the phylogenetic relationships of African trypanosomes of suids

African trypanosomes of the subgenera Nannomonas and Pycnomonas have been recorded from both wild and domestic suids. However, complete descriptions of some of these trypanosomes with regard to host range, pathogenicity, transmission and distribution are still lacking. Neither the recently described Trypanosoma (Nannomonas) godfreyi nor Trypanosoma (Nannomonas) congolense Tsavo have been isolated from mammalian hosts, while Trypanosoma (Pycnomonas) suis remains the rarest of the Salivarian trypanosomes. The only isolate presumed to be of the latter species is maintained at the Kenya Trypanosomiasis Research Institute, Nairobi. We present here the results of characterization of this isolate by morphology, tsetse transmission, the use of species-specific DNA probes and DNA sequence analysis. Morphology in stained blood smears revealed a small trypanosome with a free flagellum. Experimental transmission through Glossina morsitans morsitans showed a developmental cycle typical of subgenus Nannomonas A positive identification was obtained with species-specific PCR primers for T. congolense Tsavo; moreover, the sequence of the SSU rRNA gene was almost identical to that of T. congolense Tsavo on database. In phylogenetic analysis of the SSU rRNA genes of Salivarian trypanosomes, T. congolense Tsavo grouped with T. simiae rather than T. congolense, suggesting that the name T. simiae Tsavo is more appropriate.

Trypanosoma brucei: The peptidases of bloodstream trypanosomes

Abstract Peptidases (EC.3.4.11 and EC.3.4.13.9) from bloodstream Trypanosoma brucei were examined by starch gel electrophoresis and substrate specificities and relative activities of six peptidases (S,B,E,A,F,D,) determined. The substrate specificities corresponded closely to those of the peptidases of human blood cells and tissues although human peptidase C appeared not to have a T. brucei equivalent.

A comparison of the isoenzymes, soluble proteins, polypeptides and free amino acids from ten isolates of Trypanosoma evansi

1. Soluble extracts from different strains of Trypanosoma evansi were compared by several analytical procedures. 2. No isoenzymic differences were detected. 3. Some clear intraspecies differences in protein isoelectric points, in polypeptide sizes and in free amino acid contents were found.