A. Tait
The question of whether Africa trypanosomes have a system of gene exchange has a long history, with a number of claims relating to the morphological observation of chromosomes, gametes and mating trypanosomes. Over the past 14 years, the use of enzyme electrophoresis as a means of characterizing these organisms (Godfrey & Kilgour 1976; Gibson et al. 1980) has led to evidence for a sexual cycle based on the nature of the enzyme variants and their frequencies in discrete populations of isolates (Tait 1980).
The central arguments for a sexual cycle are as follows:
1. the nature of the enzyme variant patterns
2. the frequency of the phenotypes observed which fit those predicted on a model of random mating
3. the frequency of all possible combinations of variants when two loci are examined in a single population or in clones from a single fly isolate
4. the frequency of variants observed when compared to asexual populations of other organisms.
The speaker reviewed this evidence by reference to specific examples and discussed its strengths and weaknesses, including cases where deviation from expected phenotype frequencies is observed. He briefly discussed the effect of a sexual cycle on various aspects of epidemiology, such as the definition of non-interbreeding populations and of subspecies.
R.E. Cibulskis
Isoenzyme electrophoresis provides a convenient means of exploring the genetic composition of a population over several loci. Data resulting from an isoenzyme survey can be subjected to various forms of analysis to yield information on sexual processes in natural populations of trypanosomes.
One approach has been to consider whether the diversity observed in a population could have been produced through an acceptable pattern of mutation. Previous arguments about mutation have been confined to the range of genotypes observed at a single locus, but they can be extended to consider several loci simultaneously by using a cladistic method. The method provides evidence that the diversity observed in three East African populations of T. brucei could not have been produced by mutation alone, but does not indicate to what extent genetic exchange currently operates.
Another approach has considered the relative frequencies with which different genotypes occur, i.e. whether or not they conform to Hardy-Weinberg expectations. In general, this analysis requires large sample sizes before it is of much value. A more powerful analysis is provided by considering loci in combination and using a randomization procedure to overcome problems of small sample sizes. It reveals significant associations between genotypes from different loci, suggesting a potential for distinct strains of T. brucei to evolve within a population.
Analysis of population infrastructure in Lambwe Valley, Kenya, indicates heterogeneity over comparatively short distances. A significant association is observed between host species and the multiple-laces genotype of T. brucei variants. The association appears to be stable over a 3-year period, suggesting that sex has little influence on the genetic structure of trypanosome populations, at least over this time scale.
W.C. Gibson
Among the diversity of Trypanozoon stocks found in nature, two types stand out on both behavioural and biochemical grounds, namely T. evansi and T.b. gambiense. Not confined to tropical Africa by dependence on a tsetse fly vector, T. evansi has been able to spread widely throughout tropical and subtropical regions of the world. Despite this wide distribution, T. evansi for the most part is remarkably uniform in terms of isoenzyme and kinetoplast DNA variation. In particular, all T. evansi stocks so far examined lack kinetoplast maxicircles and have lost the minicircle heterogeneity characteristic of Trypanozoon stocks in general. Only two biochemically distinct types of T. evansi have so far been identified, suggesting that T. evansi may have arisen from T. brucei on as few as two independent occasions and that subsequent mutations then created the minor variations observed within the two types. Since no genetic exchange is possible between T. evansi and T. brucei or among T. evansi stocks in the tsetse fly, T. evansi remains a small group of isolated mutant strains.
If T.b. gambiense is defined as all trypanosome isolates from man in areas of Gambian sleeping sickness, then such trypanosomes fall into two groups. Group 1 is easily and clearly demarcated and corresponds to the classical definition of T.b. gambiense. The trypanosomes have low infectivity and virulence for rodents, high resistance to human serum, a limited antigenic repertoire and particular isoenzyme and DNA markers. Group 2 is less easily defined and shares none of the above characters; its place in the epidemiology of Gambian sleeping sickness has yet to be investigated. The lack of variability among Group l stocks, even at the nucleotide level, is remarkable considering that they originate from several regions of Africa. It suggests that Group 1 T.b. gambiense consists of a handful of related trypanosome strains which have spread rapidly in recent times and so have not yet diverged noticeably. Neither do they appear to be changing genetically by recombination with T.b. brucei strains in the invaded areas. Thus both T. evansi and Group 1 T.b. gambiense would seem to be small, easily defined groups because each, although widespread, consists of only a handful of mutant T.b. brucei strains which do not exchange genetic information with the main body of T.b. brucei and thus evolve at a slow rate.
The lack of substantial evidence as to whether T. evansi evolved from T.b. brucei or whether both evolved from a common ancestor was noted. Another point raised was whether genetic exchange occurs between T.b. gambiense and T.b. brucei or T.b. rhodesiense. At the population level, certain enzyme characters specific to T. gambiense have been encountered in areas of T.b. gambiense and T.b. rhodesiense overlap, namely along Lake Victoria and T.b. gambiense does hybridize with T.b. rhodesiense from this area, yet classical T.b. gambiense and T.b. rhodesiense should not occur together in the same area. The more convincing approach should be one of experimental co-transmission through tsetse.
D.A. Barnes, J.C. Mottram and N. Agabian
The T.b. gambiense metacyclic variable surface glycoprotein (mVSG) genes L2 and U1 were characterized and their genomic environment physically mapped. The L2 VSG gene in an L2-expressing, substrain-B organism is present in three telomere-linked copies. In a 30-day L2 relapse, there is one telomere-linked copy. One of the copies in the L2 expressor appears to be a bloodstream-like expression-linked copy (ELC) in that it contains a very large barren region. This copy is not expressed. The other two copies appear to be highly related and are more metacyclic-like in that they have little or no S’ ‘barren region’. One of these two copies is identical to the one remaining copy in the L2 relapse. A PVU II polymorphism exists in all of the unexpressed copies of the L2 gene.
The U1 VSG gene in a U1-expressing, substrain-A organism was present in two copies. One copy appeared to be a bloodstream-like ELC and the other a metacyclic-like ELC. It is believed that the bloodstream-like ELC copy is the one that is transcribed because it is linked to an expression site-associated gene (ESAG) which was cloned from an exreession library. This ESAG is highly homologous to the two ESAG sequences published by Cully et al. (1985). It hybridizes to a 1.2 Kb transcript on northern blots of total U1 RNA. This clone does not hybridize to any transcript in L2 total RNA.
The two cDNA clones of U1 and L2 were used to probe Southern blots of genomic DNA from T.b. brucei, T.b gambiense, T.b. rhodesiense, T equinum and T. vivax. The U1 gene is only found in T.b. gambiense substrain-A organisms. A U1-related gene fragment hybridizes to the U1 cDNA probe. This fragment is found in many T. brucei subspecies, but neither the U1 gene nor the U1-related gene fragments were observed in the substrain-B organisms. The L2 gene is found only in T.b. gambiense substrain-A and -B isolates and was not observed in any other Trypanosama species.
Long-term intentions are, first, to develop markers which would be useful in the identification and genetic manipulation of T.b. gambiense isolates for epidemiological and/or diagnostic purposes and, second, to provide a basic research agenda describing and characterizing the metacyclic antigens and regulatory regions associated with expression of T.b. gambiense-like specific VSGs.
P.A.O. Majiwa
Chromosome-sized DNA molecules from different clones of T. congolense have been size-fractionated by OFAGE in order to obtain molecular karyotypes. The trypanosome clones used were deliberately chosen for purposes of comparison to represent: (1) isolates from different geographic areas, (2) distinct VAT repertoires (serodemes), (3) different VATs within a single antigenic repertoire.
The chromosomes of T. congolense were observed to fall into three broad size categories: (1) minichromosomes which are approximately 50 kb and constitute the smallest trypanosomal chromosomes observed so far; (2) medium-sized chromosomes which have a size range of 0.4 to 1 Mb and appear to vary in both size and number depending on the cloned isolate examined; and (3) large chromosomes greater than 1 Mb.
Molecular karyotypes obtained appeared to differ among clones from different antigenic repertoires, but were identical for variant clones from the same antigenic repertoire. Furthermore, sequences encoding VSGs could not be detected by Southern blot hybridizations in the genomes of T. congolense clones derived from heterologous antigenic repertoires.
All clones derived from T. congolense isolates from Kilifi so far examined by OFAGE appear to have distinctly different molecular karyotypes, quite unlike those observed among the other T. congolense clones described above. In these trypanosomes, the minichromosomes are generally bigger, about 50 kb, and the chromosomes in the region of 0.2 to 0.4 Mb are more numerous than in the other T. congolense clones. These trypanosomes have subsequently been compared with the others by molecular and biochemical techniques, and shown to differ in the following ways: molecular karyotypes, repetitive DNA sequences, kinetoplast DNA sequences, and restriction enzyme fragment size polymorphisms as revealed by conserved sequence probes.
It is concluded that T. congolense is comprised of genomically diverse trypanosomes that probably do not have the capacity to participate in the conventional exchange of genetic material. Such trypanosomes could constitute distinct species or subspecies.
It was pointed out that the Kilifi isolates were identical, to the T. congolense isolates from Matuga at the Kenya Coast described by Dr. Gashumba. However, the isoenzyme profiles of these isolates were different from those of the savannah- and forest-type isolates described by Christine Young. Furthermore, the T. congolense isolates of the forest type did not occur in East Africa.
