Trypanosome genetics: Populations, phenotypes and diversity

Trypanosome genetics: Populations, phenotypes and diversity

Veterinary Parasitology 181 (2011) 61–68 Contents lists available at ScienceDirect Veterinary Parasitology journal homepage: www.elsevier.com/locate...

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Veterinary Parasitology 181 (2011) 61–68

Contents lists available at ScienceDirect

Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar

Trypanosome genetics: Populations, phenotypes and diversity Andy Tait a,b,∗ , Liam J. Morrison a,b , Craig W. Duffy a,b , Anneli Cooper a,b , C. Mike. R. Turner a,c,1 , Annette Macleod a,b,1 a b c

Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, United Kingdom Henry Wellcome Institute of Comparative Medical Sciences, Garscube, Glasgow G61 1QH, United Kingdom Glasgow Biomedical Research Centre, 120, University Place, Glasgow G12 8TA, United Kingdom

a r t i c l e Keywords: Trypanosomes Genetics Populations Pathogenesis

i n f o

a b s t r a c t In the last decade, there has been a wide range of studies using a series of molecular markers to investigate the genotypic diversity of some of the important species of African trypanosomes. Here, we review this work and provide an update of our current understanding of the mechanisms that generate this diversity based on population genetic analysis. In parallel with field based studies, our knowledge of the key features of the system of genetic exchange in Trypanosoma brucei, based on laboratory analysis, has reached the point at which this system can be used as a tool to determine the genetic basis of a phenotype. In this context, we have outlined our current knowledge of the basis for phenotypic variation among strains of trypanosomes, and highlight that this is a relatively under researched area, except for work on drug resistance. There is clear evidence for ‘strain’-specific variation in tsetse transmission, a range of virulence/pathogenesis phenotypes and the ability to cross the blood brain barrier. The potential for using genetic analysis to dissect these phenotypes is illustrated by the recent work defining a locus determining organomegaly for T. brucei. When these results are considered in relation to the body of research on the variability of the host response to infection, it is clear that there is a need to integrate the study of host and parasite diversity in relation to understanding infection outcome. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Since the 1980s, we have known that the main species of African trypanosomes infecting humans and livestock are genotypically diverse, with the exception of Trypanosoma vivax, where the difficulties associated with growing this parasite species in the laboratory has limited the number of isolates available for analysis. The basis for this variation has been controversial, with one view that genetic exchange between parasites was common, leading to the

∗ Corresponding author at: Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Bearsden Rd., Glasgow G61 1QH, United Kingdom. Tel.: +44 0141 330 5750; fax: +44 0141 330 5422. E-mail address: [email protected] (A. Tait). 1 Joint last authors. 0304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2011.04.024

observed diversity (Tait, 1980; Gibson et al., 1980), while another view was that these parasites primarily expanded clonally with the observed variation being largely due to mutation (Tibayrenc et al., 1990). In the last few years, with the advent of genome sequence data and the development of methods for the genetic characterisation (‘genotyping’) of parasites directly from blood samples, the question regarding the role of genetic exchange in trypanosome populations has been further investigated. Particularly important in these new studies has been the ability to develop panels of highly polymorphic micro- and minisatellite markers from genome sequences, so that parasites can be rapidly genotyped by PCR based methods. In parallel with these developments, our understanding of the mechanisms of genetic exchange, based on laboratory crosses (reviewed by Gibson and Stevens, 1999), has expanded substantially, such that we are now in a position

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to use crosses and genetic analysis to determine the genetic basis of phenotypes of relevance to the disease and its transmission (Tait et al., 2002). Reverse genetic techniques with trypanosomes are well developed (Burkard et al., 2007; Ngo et al., 1998), at least for T. b. brucei, and allow studies of the effect of gene knockouts or gene silencing by RNAi on phenotype. These are powerful technologies but are dependent on initially identifying and defining a gene (or genes) of interest and then determining the phenotype caused by gene silencing or knockout. In contrast, forward genetic techniques, such as crosses, allow the investigation of the genetic basis of known phenotypes without prior knowledge of the genes involved. However, such studies are dependent on the occurrence of naturally occurring variation in phenotype for genetic analysis, highlighting the need to study not only genotypic but also phenotypic variation. In this context, we review our current knowledge and understanding of the basis for genotypic diversity in field populations, the mechanism of genetic exchange and the variation in phenotypes of relevance to the disease, coupled to recent parasite genetic analysis of one such phenotype. This review is mainly an account of recent findings on trypanosome genetics and diversity. 2. Trypanosome genetic diversity T. brucei is found throughout the tsetse belt of sub-Saharan Africa and comprises three morphologically identical sub-species: T. b. rhodesiense, T. b. gambiense and T. b. brucei, with the first two causing significant disease in humans and the third infecting livestock and wild animals throughout the region (Fevre et al., 2008). The human infective sub-species occur in discrete foci of disease with T. b. gambiense found in West and Central Africa and T. b. rhodesiense in East and Southern Africa (Hoare, 1972). A combination of iso-enzyme and molecular markers has shown that T. b. gambiense isolates can be divided into two discrete sub-groups, designated as Groups 1 and 2 (Gibson, 1986). Although T. b. brucei is found in livestock, it is generally not considered to be a major pathogen, in contrast to T. congolense and T. vivax which both cause major disease (Hoare, 1972). A combination of molecular and iso-enzyme studies have sub-divided T. congolense into three groups or clades (Young and Godfrey, 1983; Majiwa et al., 1986; Gashumba et al., 1988), designated as Savannah, Forest and Kilifi, and one could speculate that these are different species or sub-species. In addition, a number of other trypanosome species are important, but have not been the subject of recent work on diversity and have thus not been considered here. In the last few years, molecular markers have been developed to study the genotypic diversity of isolates from most of the important species. While some analysis has previously been undertaken, the commonly used approaches/markers have been Amplified Fragment length Polymorphisms (AFLPs; Masiga et al., 2000; Agbo et al., 2002; Masumu et al., 2006a; Simo et al., 2007), Mobile genetic element-Polymerase Chain Reaction (MGEPCR; Hide and Tilley, 2001; Tilley et al., 2003) and micro- and minisatellites (Biteau et al., 2000; MacLeod

et al., 2000; Koffi et al., 2007). Essentially, the AFLP approach analyses restriction fragment polymorphisms on a genome-wide scale and therefore has a high sensitivity for detecting differences among strains. However this technique has a number of disadvantages; specifically, it requires growth of the parasites, preparation of purified DNA and is difficult to interpret in genetic terms. The MGE-PCR method overcomes the need to prepare DNA, but resultant electrophoretic patterns cannot be readily interpreted genetically. By contrast, microand mini-satellites do not suffer from these disadvantages although, unless a very large number are used, they do not provide a genome-wide analysis. There is no ideal marker system, but methods should be selected based on the question being addressed and the nature of the material available for study. In the future, these methods/markers could be superseded by whole genome sequence analysis, as new technologies become available and their costs reduce. However, this will still require the ‘amplification’ of parasites in rodents, potentially limiting their application as well as raising the possibility of genotypic selection, which can occur during rodent amplification (McNamara et al., 1995; Jamonneau et al., 2003). Detection of diversity, using these molecular methods, has been undertaken primarily for the three sub-species of T. brucei but, more recently, for T. congolense (Savannah) and T. vivax. Within T. brucei, the general picture is that T. b. brucei is highly diverse, T. b. rhodesiense (depending on the focus) ranges from showing very low levels of diversity to high levels, whereas T. b. gambiense shows low levels of diversity within a focus, but strains from different geographical foci are very distinct. These broad conclusions are supported, irrespective of whether microsatellite, AFLP or MGE-PCR methods of analysis are used. Recent studies of T. congolense (Savannah), using molecular markers and multiple isolates, have shown high levels of diversity both within a single geographical region (Morrison et al., 2009a) and between regions (Masumu et al., 2006a). However, limited information is available on the diversity between different geographically separated populations of T. congolense (Savannah) or within the Kilifi and Forest ‘clades’ of T. congolense. Thus, our knowledge of diversity in T. congolense is relatively limited, apart from the iso-enzyme studies in the 1980s (Young and Godfrey, 1983; Gashumba et al., 1988) but, where this species has been studied, it appears to be highly diverse. Because of the difficulty of growing T. vivax in laboratory rodents or culture, knowledge of the strain diversity of this species has been limited (Gardiner, 1989; Fasogbon et al., 1990), except for a recent study using microsatellite markers amplified directly from infected blood isolated from horses and cattle in a single location (Duffy et al., 2009). The latter study showed that there was limited diversity as many isolates have identical genotypes. The findings from all the studies on diversity raise two main questions, what is the basis for the observed diversity and why are differences in diversity found in different foci of the same sub-species as well as between different species. One central question is whether this variation is generated by genetic exchange, and this aspect is addressed in the next section.

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3. Population genetics and molecular epidemiology In the early 1990s, there was considerable debate about the role of genetic exchange in field populations of African trypanosomes. Tibayrenc et al. (1990) proposed that most parasitic protozoa expand clonally with limited genetic exchange, while others (Tait, 1980; Gibson et al., 1980) took a different view, proposing that, in some populations of T. brucei, mating occurred regularly. These different hypotheses were based on the analysis of the observed frequency of different genotypes compared with those predicted if random mating was occurring as well as the analysis of linkage equilibrium (LE; the predicted frequency of the combinations of alleles at pairs of loci, assuming random mating). As the authors were using different populations of isolates and used different definitions of what constituted a population, there was no real resolution to this debate. Clonal populations will show multiple isolates with the same genotype, high levels of heterozygosity, departure from LE (LD; linkage disequilibrium) and frequencies of genotypes at single loci that do not agree with those predicted by random mating. In contrast, a panmictic population will have few, if any, identical genotypes, and the frequencies of genotypes at single loci and alleles at pairs of loci will be in agreement with the proportions predicted for a random mating population. Thus, the population structure can either be clonal or panmictic. A third population structure was proposed by Maynard Smith et al. (1993), who were working on bacterial genetics (of an epidemic population), in which one or a few genotypes expand clonally due to a particular selective advantage but mask an underlying random mating population. They re-analysed data from a T. brucei population (from several hosts) in the Lambwe valley (Kenya) and showed that this population had an epidemic population structure. This dataset partly resolved the different conclusions about the role of genetic exchange in the field, as an epidemic population combines aspects of clonal expansion with underlying genetic exchange. Subsequent analysis using micro and mini-satellite markers and geographically discrete populations has led to a further resolution of the debate. The T. brucei group of sub-species has been most intensively studied using microsatellite markers, with a focus on the human-infective sub-species. Studies of T. b. gambiense Group 1 (Morrison et al., 2008; Koffi et al., 2009) from disease foci in West and Central Africa have shown that populations isolated from geographically discrete areas are clonal with multiple isolates having the same genotype, LD and genotype frequencies that do not conform to those predicted by random mating. An interesting finding is that the genotypes from geographically distinct foci were very distinct, giving measures of genetic distance that are comparable with those between sub-species. The basis for this high level of divergence is not understood, but it could be postulated that, originally, there was a single population throughout the region which has subsequently sub-divided, with each focus evolving independently and so diverging. This proposal would be consistent with evolutionary theories of clonal organisms (Balloux et al., 2003). Phylogenetic studies could test this hypothesis but have not yet been undertaken. Studies on T. b. rhodesiense and T.

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b. brucei in Uganda have provided evidence that the former expands clonally with limited genetic exchange, while the latter has an epidemic population structure with genetic exchange playing a significant role in generating diversity (MacLeod et al., 2000). However, studies of a separate focus of human sleeping sickness in Malawi show that there is a much higher degree of diversity in these parasites than those from Uganda, and these data are suggestive of a role for genetic exchange in generating diversity in T. b. rhodesiense in Malawi (Duffy et al., unpublished data). Thus, the picture that emerges from these more recent studies is that genetic exchange plays a limited role in T. b. gambiense populations, a variable role in T. b. rhodesiense populations and probably a significant role in T. b. brucei, although there are relatively few extensive studies of the latter sub-species. Furthermore, these studies emphasize the sub-structuring of these populations by showing significant genetic isolation between geographically separate populations. Recent population analysis of T. vivax and T. congolense from a single discrete area in The Gambia provides strong evidence for different population structures in the two species. Genotyping of 84 isolates of T. congolense (Savannah) with seven microsatellite markers shows that they comprised 80 different multilocus genotypes (MLG), suggesting that genetic exchange might be occurring (Morrison et al., 2009a). However, when the data were analysed to test for random mating, there was evidence for LD, and the genotype frequencies show a deficit of heterozygotes. The population has none of the properties of a clonal population. To investigate the reasons for the observed LD, the population was tested for cryptic sub-structuring and found to be sub divided into four subpopulations with one of these in LE and the others showing approaches to LE. Thus, genetic exchange occurs in T. congolense (Savannah). The reasons for this sub-structuring are not clear, but are not associated with the species of host or time of sampling. A similar analysis was undertaken using isolates of T. vivax from the same set of samples (Duffy et al., 2009). A total of 31 isolates were genotyped with 8 microsatellite markers, and the combinations of alleles at each locus for each sample were used to generate MLGs. Only 9 MLGs were defined, and one of these was the same in 15 of the samples, suggesting a clonal population structure. A further test was conducted using standard population genetic analysis, which showed an excess of heterozygotes, a high level of LD and a lack of agreement between the observed genotype frequencies and those predicted for a randomly mating population, further confirming that this population is clonal. For both species, it will be important to analyse additional populations in other areas of Africa to establish whether the findings from The Gambia apply generally, as it is possible that in different epidemiological situations the role of genetic exchange might vary. Therefore, understanding the role of genetic exchange in field populations will not only provide information on how genetic diversity arises and on the consequent ability to adapt to selective pressures, but will also increase our understanding of the role of mutation versus recombination in the evolution of trypanosomes.

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4. Mechanisms of genetic exchange Since 1986, it has been known that laboratory crosses can be undertaken between different strains of T. brucei (see Jenni et al., 1986), but it has only been relatively recently that the mechanism and other key features have been elucidated. It is now clear that mating takes place in the epimastigote stages in the salivary glands of the tsetse fly (Tait et al., 2007; Gibson et al., 2008) and that there is strong evidence for the occurrence of meiosis (MacLeod et al., 2005a). Earlier studies had shown that mating was not an obligatory part of the life cycle (Schweizer et al., 1988) and this aspect has been confirmed more recently using parental trypanosomes with fluorescent gene tags and genetic marker analysis of the progeny (Gibson et al., 2008). To date, some 12 crosses between different strains and sub-species have been undertaken, with genetic exchange being demonstrated between distinct strains of T. b. brucei, T. b. brucei and Group 2 T. b. gambiense, T. b. rhodesiense and T. b. brucei and T. b. rhodesiense and Group 2 T. b. gambiense (see Gibson and Stevens, 1999; MacLeod et al., 2007). Thus, there appear to be limited barriers to mating, even between sub-species under laboratory conditions, although this has not been tested with T. b. gambiense Type 1. However, to date, there is no evidence from the field to suggest that mating between the sub-species occurs (MacLeod et al., 2000), but this issue has not been systematically studied. As well as cross fertilisation between different parasite strains, self-fertilisation also occurs and, although this process was originally thought to only occur in the presence of cross fertilisation (Tait et al., 1996), recent data have shown that self-fertilisation can occur when a single strain is transmitted through tsetse flies (Peacock et al., 2009). The non-obligatory mating system and the ability to undergo self fertilisation suggests that trypanosomes are uniquely adaptable to environmental change and new niches; as they can propagate clonally without disrupting combinations of alleles, they can generate novel combinations of alleles by mating or become homozygous for ‘successful’ alleles by self fertilisation. Earlier studies had provided evidence that was consistent with the progeny of crosses being the equivalent of an F1 produced by the parental strains undergoing meiosis (Turner et al., 1990; Gibson, 1995). More recently, these conclusions have been rigorously tested by microsatellite marker analysis of >30 progeny clones from two independent crosses. Alleles at heterozygous loci on one parental strain, each located on one of the 11 housekeeping chromosomes, segregate into the progeny independently and in Mendelian proportions, thus strongly supporting the occurrence of meiosis (MacLeod et al., 2005a). All of the progeny analysed to date appear to be the products of a single round of mating. Furthermore, genetic maps of both T. b. brucei and T. b. gambiense Group 2 have been constructed using > 150 microsatellite markers and the resultant linkage groups align with the physical map of the genome provided by the T. b. brucei genome sequence (MacLeod et al., 2005b; Cooper et al., 2008). The construction and analysis of the genetic maps illustrated two further standard properties of the genetic system, namely crossing-over between pairs of homologous chromosomes

and regions of high and low recombination (hot and cold spots) along a chromosome. Thus, overall, T. brucei appears to have a conventional diploid, Mendelian genetic system in common with many other eukaryotes. It should be noted that no crosses have been reported with T. b. gambiense Group 1, and, to our knowledge, no crosses have been reported with either T. congolense or T. vivax. The recent report of the ability to replicate the whole life cycle of T. congolense in vitro (Coustou et al., 2010) offers the opportunity of testing whether crosses with this species could be undertaken. While many progeny clones in T. brucei crosses are diploid, a proportion of these are triploid or even tetraploid with the frequency varying between different crosses (Gibson and Stevens, 1999). For example, in the crosses between two T. b. brucei strains (TREU 927 and STIB 247), no triploids have ever been observed, but in a cross between T. b. gambiense and T. b. rhodesiense, 6/12 progeny were triploid (Gibson and Bailey, 1994). Originally, it was thought that this phenomenon only occurred in inter subspecies crosses, but, recently, both tetraploid and triploid progeny were reported in a purely T. b. brucei cross (Gibson et al., 2008). While this can be explained by some cells not undergoing meiosis, but still being stimulated to fuse, it is unclear why it occurs at quite a high frequency in some crosses. No triploid T. brucei have been described in the marker analysis of field isolates and, so, it appears that this does not happen in natural populations, although it could be that polyploidy trypanosomes are not viable in their natural hosts. However, it should be pointed out that similar deviations in ploidy are found in human conceptuses but result in miscarriage. Thus, this may be a general phenomenon but is only readily seen in trypanosomes, because of their tolerance of changes in ploidy. This phenomenon requires further investigation. 5. Phenotypic diversity The most extensively studied trypanosome phenotype has been drug resistance due to its importance for the control and treatment of both animal and human trypanosomosis. Variation in drug response to most of the commonly used trypanocidal drugs has been reported between different strains of the T. brucei group of subspecies and T. congolense. This diversity has been reviewed elsewhere (Matovu et al., 2001; Delespaux and de Koning, 2007), and most of the analysis of mechanisms has been undertaken using biochemical and molecular approaches rather than genetic analysis. Less attention has been focussed on strain variation in other phenotypes, although there are reports of differences in virulence (Masumu et al., 2006b; Holzmuller et al., 2008; Balmer et al., 2009), pathogenicity (Morrison et al., 2009b) and tsetse transmissibility (Welburn et al., 1995). It is important to identify the parasite genes that determine these traits and, from this begin to understand, the molecular determinants of the disease and its transmission. There have been relatively few reports on parasite strain variation in relation to the ability to infect and develop in the tsetse fly, yet, this could be a significant factor in determining which strains are likely to spread rapidly. A

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comparison between the transmission (by Glossina m. morsitans) of a set of T. b. rhodesiense and T. b. brucei isolates (Welburn et al., 1995) has shown that there were significant differences in the transmission index (TI = the ratio of mid gut to salivary gland infections). The results showed a clear statistically significant difference in the mean TI between the two sub-species with T. b. rhodesiense, giving lower values, but, interestingly, there was also variation between strains within each sub-species although these differences were smaller. As far as we are aware, there has not been further research on the basis for these differences. A similar finding of strain variation in tsetse transmission has been found for T. congolense (see Masumu et al., 2006c), although primarily in terms of mid-gut infectivity. Clearly, the outcome of an infection will be determined by both host and parasite factors, and the former have been studied extensively in many host–pathogen systems. This is also true for trypanosome infections, with studies implicating a range of host parameters that determine the outcome of infection (MacLean et al., 2004, 2007; Hill et al., 2005; Courtin et al., 2008). However, the focus of this review is on the parasite determinants and, so, the host component will not be considered in any detail. This is a relatively neglected area, despite its potential importance. There are several studies that implicate variation in the parasite as the determinant of clinical outcome (Jamonneau et al., 2000; Garcia et al., 2006), but as both parasite and host vary in such field studies, there is uncertainty as to the cause of the variation. Thus, much of the available data comes from studies using inbred mice as the host, making the assumption that the findings apply to the natural host. Examples from T. brucei include the study of two different strains of T. b. brucei showing significant differences in organomegaly, reticulocytosis and anaemia (Morrison et al., 2009b). The basis for this variation was investigated by analysing the response of the host to the infection with the two strains using microarray analysis of RNA from the spleens. The main pathways that were differentially regulated involved the innate immune system (IL10 signalling, LXR/RXR signalling and alternative macrophage activation), suggesting that strain specific modulation of these pathways is a key component of the pathogenesis. Differences in virulence between another pair of T. b. brucei strains have also been observed (Balmer et al., 2009) with significant differences found in host survival, thrombocytopenia, anaemia and hypoglycaemia measured in outbred mice. Interestingly, the less virulent line became virulent after multiple passage in mice and mixed infections with virulent and avirulent strains lead to the suppression of the virulent phenotype. In another study (Holzmuller et al., 2008), 10 strains of T. b. gambiense from Cote d’Ivoire demonstrated variation in virulence (defined by level of parasitaemia) and pathogenicity (defined by survival). Variation in virulence/pathogenicity has also been described in studies of the three different clades (Savannah, Kilifi and Forest) of T. congolense in both bovine and murine hosts, in which, importantly, the infection parameters for both hosts showed a good concordance (Bengaly et al., 2002a,b). Additionally, a further study of 31 strains of T. congolense (Savannah) from several locations in Zambia showed a range of infection parameters, with the strains

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being classified into three groups: virulent, moderately virulent and low virulence (Masumu et al., 2006b). The parameters that differed between the groups were survival time, pre-patent period and packed cell volume (PCV). Thus, there was both within and between clade diversity. A key feature of T. brucei is the ability to penetrate the blood brain barrier, leading to one of the major clinical signs of the human disease, and field studies have reported (Jamonneau et al., 2004) infected patients who do not progress to stage 2-disease (i.e. penetration of the blood brain barrier). An in vitro system has been developed to measure the penetration of brain microvascular endothelial cells by T. brucei to study this process (Grab et al., 2004), and inhibitor studies have implicated a parasite secreted cysteine proteinase (brucipain) as the molecule that mediates penetration (Nikolskaia et al., 2006). From the perspective of parasite diversity, experiments were undertaken with T. b. rhodesiense (previously described as T. b. gambiense, in error) and two strains of T. b. brucei to show that the human-infective sub-species crosses the endothelial cells six times as effectively as the T. b. brucei strains. Interestingly, T. b. rhodesiense has some eight-fold higher brucipain activity, raising the possibility that this activity may be responsible for the increased ability of T. b. rhodesiense to penetrate the endothelial monolayer (Nikolskaia et al., 2006). Taken together, there is clear evidence for parasite strain/sub-species variation in a range of phenotypes that affect transmission, disease severity and disease treatment. Apart from considerable advances in our understanding of drug resistance (disease treatment), we have very limited knowledge of the genetic basis for the other phenotypes; yet they are likely to be important in terms of understanding the disease and its spread. Furthermore, identifying the parasite genes involved could, potentially have therapeutic implications. 6. Genetic analysis of phenotypic variation There is a considerable body of data on the variation in the host response to infection and the genetic basis of tolerance or ‘resistance’ to infection both in human and livestock disease (Kemp et al., 1997). In terms of human disease, a number of association studies have been undertaken and evidence for specific host genetic variants obtained (Courtin et al., 2008). In the case of animal trypanosomosis (using T. congolense), genetic analyses of both cattle and mice have been undertaken to identify loci that are major determinants of disease outcome (Iraqi et al., 2000; Hanotte et al., 2003; Hill et al., 2005). With the availability of single nucleotide polymorphism (SNP) arrays for humans, cattle and mice, the resolution of such mapping studies will be increased by using whole genome scans as an approach and it is likely that further the host gene variants responsible for disease outcome could be identified. The availability of genetic maps of T. brucei has opened up the possibility of undertaking analyses to identify the loci that determine phenotypes of relevance to disease transmission and pathogenesis/virulence. One study (from our laboratory) has been undertaken to map the loci that determine strain specific differences in organomegaly,

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reticulocytosis and anaemia (Morrison et al., 2010). The progeny from a cross between two strains that differed in these phenotypes were scored (in mice) for each of the parameters, and they were shown to segregate in a semi-quantitative manner. This information implied that these phenotypes are determined by allelic variation of several parasite genes and, so, a genetic linkage analysis was undertaken assuming that these were quantitative trait loci (QTL). Splenomegaly and hepatomegaly showed evidence for a highly significant QTL (LOD scores > 7) on chromosome 3, accounting for 66% and 64%, respectively of the phenotypic variance, thus demonstrating a parasite gene, or genes, determining a major component of these phenotypes. The identified region on chromosome 3 included >300 genes; therefore, further analysis is required to identify the individual genes involved. Several approaches are available including, fine-scale mapping with more markers, analysis of more progeny clones to identify crossovers within the designated region, analysis of stage-specific expression and, possibly, reverse genetics. While this locus was the most significant, additional significant QTLs were identified for reticulocytosis, anaemia and organomegaly on other chromosomes. Similar approaches to those described are needed to fully confirm the significance of these loci. The importance of this work is two fold. Firstly it establishes unequivocally that there are parasite loci determining pathogenesis and, secondly, that genetic analysis can be used to map such loci in trypanosomes. Such findings need to be considered in studies of the host genetic determinants, for which much of the work is based on using a single parasite ‘strain’. 7. Conclusions and future prospects The genotypic diversity within some of the major species and sub-species of trypanosomes varies substantially, ranging from low, in the case of T. gambiense Group 1 and T. vivax, to high, in the case of T. b. brucei and T. congolense. This variation reflects the role of genetic exchange in the populations studied. The recent sequencing of many protist genomes has led to the analysis of genes that are associated with meiosis and an argument that the presence of such genes indicates the existence of a sexual cycle – the simple ‘lose it or use it’ premise (Schurko and Logsdon, 2008). The meiosis-associated genes (‘meiotic tool box’) are present in all the trypanosome species and sub-species discussed here, but, clearly, the presence of these genes does not necessarily imply they are functional in the species with clonal population structures. Possible explanations for this apparent contradiction could be that (i) these species do undergo genetic exchange in certain epidemiological scenarios, (ii) these genes have alternative functions, or (iii) there has not been sufficient time for mutations and deletions to occur since the species started to expand clonally. In parallel with the population-based analyses, our understanding of the process of genetic exchange, at least for T. brucei, has advanced substantially to a point that genetic maps of the parasite have been constructed so that genetic analysis can be undertaken. There are still some significant unknowns, such as whether haploid gametes occur, the basis for the high proportion of non-diploid

progeny, the nature of the process that leads to cell fusion for the formation of progeny and the nature of the signals that initiate meiosis. The population-based identification of mating in T. congolense (Savannah) also raises a number of questions, such as whether this can be reproduced in the laboratory, whether the genetic system has a similar mechanism to that in T. brucei and whether the other clades of T. congolense also undergo genetic exchange. The basis for the variation in phenotypes, such as tsetse transmission, virulence and pathogenesis, is not well understood and, except for the penetration of the blood brain barrier, the genes involved largely remain unidentified. However, in terms of determining how disease is caused and the parasite is transmitted, it would be important to identify the key parasite mechanisms. Furthermore, knowledge of the parasite genes involved could provide avenues for new intervention and treatment strategies. The advances in trypanosome laboratory genetics offer an approach to identify such genes and offer promise for the future. An alternative approach would be to undertake association analyses between genotype and phenotype. For example, if large numbers of virulent and avirulent strains were sequenced, polymorphisms that specifically associated with one phenotype or the other would allow the identification of candidate genes. Such approaches have been successfully undertaken to identify specific genes responsible for non-infectious diseases in, for example, the dog (Karlsson et al., 2007) and cattle (Charlier et al., 2008). The advances in sequencing technologies would allow the genotyping/sequencing of large numbers of strains to be undertaken cheaply and rapidly and, so, this is a prospect for the future. Whether such analyses would be most effectively undertaken with populations of clonal parasite species or would require the use of panmictic populations is a matter for discussion. The demonstration of variation in virulence phenotypes in T. b. gambiense (see Holzmuller et al., 2008), for example, for which genotypic diversity is low, could allow rapid association with genotypic polymorphism but might suffer from a lack of resolution, as the haplotypes associated with the phenotype could be large due to the lack of recombination. In contrast, an analysis of T. congolense (with high levels of polymorphism) would reduce the size of the haplotypes but increase the levels of ‘polymorphic noise’, making any association more difficult to establish statistically. The studies of disease progression and pathogenesis suggest that parasite diversity plays a significant role in the outcome of infection and will need to be considered in relation to host diversity. We now have the tools and technologies to define the host and parasite loci that are involved and to identify the alleles that are responsible for different phenotypes. Thus, it is possible to envisage a host–parasite ‘interactome’ that would begin to define the multiple routes to symptomatic and asymptomatic infection. Similar approaches could also be taken to understand tsetse transmission, in order to define high and low transmission scenarios. Conflict of interest The authors declare that there is no conflict of interest.

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