Abstract Genetic recombination between pathogens derived from humans and livestock has the potential to create novel pathogen strains, highlighted by the influenza pandemic H1N1/09, which was derived from a re-assortment of swine, avian and human influenza A viruses. Here we investigated whether genetic recombination between subspecies of the protozoan parasite, Trypanosoma brucei, from humans and animals can generate new strains of human pathogen, T. b. rhodesiense (Tbr) responsible for sleeping sickness (Human African Trypanosomiasis, HAT) in East Africa. The trait of human infectivity in Tbr is conferred by a single gene, SRA, which is potentially transferable to the animal pathogen Tbb by sexual reproduction. We tracked the inheritance of SRA in crosses of Tbr and Tbb set up by co-transmitting genetically-engineered fluorescent parental trypanosome lines through tsetse flies. SRA was readily transferred into new genetic backgrounds by sexual reproduction between Tbr and Tbb, thus creating new strains of the human pathogen, Tbr. There was no evidence of diminished growth or transmissibility of hybrid trypanosomes carrying SRA. Although expression of SRA is critical to survival of Tbr in the human host, we show that the gene exists as a single copy in a representative collection of Tbr strains. SRA was found on one homologue of chromosome IV in the majority of Tbr isolates examined, but some Ugandan Tbr had SRA on both homologues. The mobility of SRA by genetic recombination readily explains the observed genetic variability of Tbr in East Africa. We conclude that new strains of the human pathogen Tbr are being generated continuously by recombination with the much larger pool of animal-infective trypanosomes. Such novel recombinants present a risk for future outbreaks of HAT.

Author Summary Genetic recombination allows transfer of harmful traits between different strains of the same pathogen and enables the emergence of genetically novel pathogen strains that the host population has not previously encountered. This can be particularly important when a pathogen acquires a virulence trait that allows it to spread beyond its normal host population. Here we show that this happens among the single-celled parasites—trypanosomes—that cause human African trypanosomiasis (HAT) or sleeping sickness carried by the tsetse fly. Genetic recombination readily occurs between the human and animal parasites when they are co-transmitted by the tsetse fly, creating new pathogen genotypes or strains. There is a single gene that confers human infectivity and each of the genotypes that inherits this gene is potentially capable of infecting humans. In this way new strains of the human pathogen can be generated by recombination between the human-infective and animal-infective trypanosomes. Such novel recombinants present a risk for future outbreaks of HAT.

Citation: Gibson W, Peacock L, Ferris V, Fischer K, Livingstone J, Thomas J, et al. (2015) Genetic Recombination between Human and Animal Parasites Creates Novel Strains of Human Pathogen. PLoS Negl Trop Dis 9(3): e0003665. https://doi.org/10.1371/journal.pntd.0003665 Editor: Paul Andrew Bates, Lancaster University, UNITED KINGDOM Received: December 1, 2014; Accepted: March 2, 2015; Published: March 27, 2015 Copyright: © 2015 Gibson et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited Data Availability: All relevant data are within the paper and its Supporting Information files. Funding: This work was funded by The Wellcome Trust www.wellcome.ac.uk through project grants to WG and MB (079375, 088099). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Genetic recombination can generate new pathogen strains to which host populations have no prior immunity. This can have disastrous consequences; for example, the human population is at risk of an influenza pandemic caused by recombination between viruses derived from humans and domestic livestock. Microbial genetic recombination facilitates the transfer of genes for virulence and drug resistance into new genetic backgrounds, potentially creating pathogen strains with novel phenotypes as well as accelerating the spread of drug resistance. Among eukaryote pathogens, the impact of sexual reproduction is hard to predict, because of the wholesale mixing of genes from different strains. Trypanosoma brucei is the protist parasite responsible for the vector-borne disease human African trypanosomiasis (HAT) or sleeping sickness. In East Africa the disease is a zoonosis caused by T. b. rhodesiense (Tbr) which is morphologically indistinguishable from the non-human infective subspecies, T. b. brucei (Tbb). Both subspecies may occur in the same range of wild or domestic mammalian hosts and there has been a long-standing controversy about their identification [1]. This was resolved by the discovery that human infectivity in Tbr was governed by expression of a single gene (Serum Resistance Associated, SRA) [2] and the presence of the SRA gene now serves as a convenient marker for Tbr [3–5]. Clearly, transfer of this single gene could potentially generate new strains of human-infective trypanosomes, and this has been demonstrated experimentally by transfection of the SRA gene into Tbb, resulting in a trypanosome with a human-infective phenotype [2]. Population genetics analyses have failed to find consistent genotypic differences between Tbr and Tbb, other than presence/absence of SRA, and the idea that Tbb and Tbr are freely interchangeable by transfer of SRA has become central to the interpretation of population genetics data for Tbr and Tbb [6]; evidence of genetic admixture between Tbr and Tbb from recent genome comparisons of the two subspecies also supports this interpretation [7,8]. Genetic exchange in T. brucei occurs in the insect vector, the tsetse fly (genus Glossina) [9] and recent results show that it has the hallmarks of conventional eukaryote sexual reproduction: meiosis and production of haploid gametes [10,11]. All subspecies of T. brucei, including Tbr, have been shown to express meiosis-specific genes [11]. Genetic crosses between Tbr and Tbb have been carried out in the laboratory [12–14], but analysis of the progeny was carried out before the significance of SRA was recognised and presence/absence of the gene was not determined. Potential human infectivity of hybrid progeny was tested by analysing resistance to lysis by human serum [15]; however, this is not such a reliable test for human infectivity as presence of the SRA gene. SRA appears to be a single copy gene that resides in one of the telomeric expression sites (ES) for variant surface glycoprotein (VSG) genes, such that, when this ES is transcribed, SRA is also expressed [2]. The ES containing SRA is unusually short in that it contains only three ES-associated genes (ESAGs 5, 6 and 7), with SRA located between ESAG 5 and the telomeric VSG gene [2]. Both the SRA gene and its immediate genomic environment are conserved in different Tbr strains [16]. The chromosome carrying SRA has not been identified, though from its size (1.6 Mb [2]), it appears to be one of the smaller diploid chromosomes described in T. brucei [17]. It is also uncertain whether all Tbr strains carry only a single SRA allele or have multiple ES with SRA. It is technically difficult to sequence T. brucei ES because of their telomeric location [18], and the few studies to date show within-strain similarity of ES in structure and gene content [19–21], making it difficult to distinguish between different ES in the same trypanosome strain. From an evolutionary perspective, it seems unlikely that Tbr would have only a single SRA gene, as that would make it dependent on only a single ES for infection in the human host; antigenic variation would be restricted to replacement of the VSG in this ES, and switching to expression of another ES, which lacked SRA, would be lethal for the parasite. Dependence on this one ES in the human host would lock the trypanosome into expression of the single transferrin receptor encoded by the ESAG 6 and ESAG 7genes co-transcribed with SRA [22]. Moreover, according to the hypothesis that allelic variation in ESAG 6 and ESAG 7is adaptive for uptake of different mammalian transferrins [23,24], the receptor encoded by alleles in the SRA ES should be specific for human transferrin. A further problem confronts the trypanosome on transmission from tsetse to human, because metacyclics, the infective forms inoculated with the fly’s saliva, express a restricted set of VSGs residing in specialized ES lacking ESAGs [25] and presumably also SRA. Without protection of the SRA protein to inactivate the trypanolytic effect of human serum, how is it possible for Tbr metacyclics to survive the transition from fly to human? Here we provide the definitive experimental proof that SRA is readily transferred between Tbr and Tbb during sexual reproduction, creating new genotypes of the human pathogen Tbr, because the SRA gene is now in a new genetic background consisting of an equal mixture of the parental Tbr and Tbb genomes. We show that SRA is present as a single copy on one homologue of chromosome IV in the majority of Tbr strains analysed and explore the implications for the epidemiology of HAT in East Africa.

Discussion Our experimental crosses of Tbr and Tbb demonstrate unequivocally that the SRA virulence gene can be transferred by genetic exchange, thus creating new genotypes of potentially human infective parasites. The genetic heterogeneity of field isolates of Tbr from different regions of East Africa, together with their similarity to some Tbb isolates, first suggested that there might be hybridization between these two subspecies [42–44], and later studies have provided extensive evidence of genetic admixture [6,8]. Our crosses involved Tbr of the northern (LUMP 1198, TOR11) and southern (058) types [27], judged to differ in severity of HAT [45], and Tbb of different genotypic groups. Tbb J10 and 1738 belong to the kiboko/kakumbi group, distinguished from other East and West African Tbb such as Lister 427 by unusual isoenzymes, kinetoplast DNA maxicircle polymorphisms and microsatellite profiles [6,26,29]. Kiboko/kakumbi group isolates have never been found in human patients and originate from areas of East Africa that have a rich, large mammal fauna [46,47]. The tight association of kinetoplast and nuclear DNA polymorphisms suggested that the kiboko/kakumbi group circulates in separate wild animal-tsetse transmission cycles, without frequent sexual reproduction with other Tbb/Tbr strains. Contrary to this, we have shown that kiboko/kakumbi strains readily mate with different Tbr, as do other Tbb strains from both East and West Africa. Thus, there do not appear to be any intrinsic genetic barriers that prevent mating of Tbr and Tbb. The accumulated data on location and copy number of SRA support the hypothesis that most Tbr strains have a single copy of SRA located in a VSG ES at the end of chromosome IV ([2,16] and this paper). As a consequence, SRA is only expressed when this ES is active, which means that the parasite is effectively restricted to use of this single ES in the human host. As noted above, a switch to another ES without SRA would be lethal for the trypanosome in a human host. This seems peculiar in a trypanosome that depends on antigenic variation for survival in the mammalian host and has multiple ES, especially considering that the SRA ES is truncated and lacks most ESAG’s [2]. How can we explain this? One possibility is that there are fitness costs associated with expression of SRA in other non-human mammalian hosts, though there is currently no evidence for this. Tbr is a zoonotic pathogen that arguably depends on a large population of non-human hosts for longterm persistence in endemic areas. Hence, the ability to easily switch off a single copy of SRA by swapping to VSG expression from another ES might be advantageous. Although it has been suggested that there are fitness costs associated with resistance to human serum in Tbr in tsetse [48], this seems unlikely; bloodstream form ES are silenced during trypanosome development in the insect vector, with activation of another set of specialized ES lacking ESAG’s in the infective metacyclics in the salivary glands [49]; therefore SRA is probably not expressed in the fly. Our results suggest a more plausible hypothesis based on the dynamic between Tbb and Tbr. SRA is a truncated VSG gene [50,51] and is assumed to have evolved once, since the sequence and local genomic environment of SRA is conserved among different Tbr strains [16,27]. We do not know when this event occurred, but SRA would only have become advantageous when it allowed extension of T. brucei’s host range to include hosts with the trypanolytic factor, Apolipoprotein L1 (APOL1), in their serum [52]; this probably dates the evolution of SRA, and hence Tbr, to somewhere in the last 10 million years or so, when the ape lineages with APOL1 diverged [53,54]. Although Tbr might subsequently have been subject to selective pressure for gene or ES duplication, depending on how significant the size of the host population with APOL1, any increase in copy number of SRA would have been rapidly diluted by mating with Tbb. Currently, there are likely to be more Tbb than Tbr strains circulating in East Africa, considering the relative numbers of infected human and non-human hosts and the restricted distribution of Tbr. Hence the probability of mating between Tbr strains will be far lower than between Tbb and Tbr, except possibly in the midst of an epidemic. This may explain the duplication of SRA in TOR11 and other isolates TOR1 and TOR4 from the same HAT outbreak (S1 Table). We can assume that these isolates represent one Tbr strain that arose either by hybridization between Tbr strains or as a mutated strain with duplication of the SRA ES. Since Tbr typically has only a single copy of SRA in a bloodstream form ES, metacyclics presumably do not express SRA when inoculated into the human host and will therefore not be protected from lysis by APOL1. Indeed, we were unable to demonstrate expression of SRA by RT PCR of RNA prepared from tsetse salivary glands infected with Tbr 058. In vitro experiments comparing the resistance of Tbr and T. b. gambiense (Tbg1) to lysis by human serum showed that few Tbr metacyclics, but the majority of Tbg1 metacyclics, grew in medium containing human serum [55] and these authors hypothesized that survival of Tbr metacyclics in the human host depends on them being deposited in the skin tissue rather than bloodstream during tsetse bite, so that they are not directly exposed to the trypanolytic factor in the blood [55]. In support of this hypothesis, the absence of APOL1 in human tissue fluid needs to be verified. In conclusion, new human infective strains of the human pathogen Tbr can be generated by recombination of Tbr with the much larger pool of animal-infective trypanosomes, Tbb. Such novel recombinants present a risk for future outbreaks of HAT.

Acknowledgments We are very grateful to the IAEA lab in Vienna for provision of tsetse and to Dr Chris Helps for advice on qPCR.

Author Contributions Conceived and designed the experiments: WG LP MB. Performed the experiments: WG LP VF KF JL JT MB. Analyzed the data: WG LP KF JL JT MB. Wrote the paper: WG KF.