Co-infection and cross-species transmission of divergent Hepatocystis lineages in a wild African primate community

https://doi.org/10.1016/j.ijpara.2013.03.002Get rights and content

Highlights

  • Malaria-like parasites of primates can evolve into significant human pathogens..

  • Primates in Kibale National Park, Uganda were screened for malaria-like parasites.

  • Multiple, species-specific lineages of the parasite Hepatocystis were discovered.

  • Two Hepatocystis lineages co-infect hosts and one moves between primate species.

  • Certain malaria-like parasites can cross primate species barriers in nature.

Abstract

Hemoparasites of the apicomplexan family Plasmodiidae include the etiological agents of malaria, as well as a suite of non-human primate parasites from which the human malaria agents evolved. Despite the significance of these parasites for global health, little information is available about their ecology in multi-host communities. Primates were investigated in Kibale National Park, Uganda, where ecological relationships among host species are well characterized. Blood samples were examined for parasites of the genera Plasmodium and Hepatocystis using microscopy and PCR targeting the parasite mitochondrial cytochrome b gene, followed by Sanger sequencing. To assess co-infection, “deep sequencing” of a variable region within cytochrome b was performed. Out of nine black-and-white colobus (Colobus guereza), one blue guenon (Cercopithecus mitis), five grey-cheeked mangabeys (Lophocebus albigena), 23 olive baboons (Papio anubis), 52 red colobus (Procolobus rufomitratus) and 12 red-tailed guenons (Cercopithecus ascanius), 79 infections (77.5%) were found, all of which were Hepatocystis spp. Sanger sequencing revealed 25 different parasite haplotypes that sorted phylogenetically into six species-specific but morphologically similar lineages. “Deep sequencing” revealed mixed-lineage co-infections in baboons and red colobus (41.7% and 64.7% of individuals, respectively) but not in other host species. One lineage infecting red colobus also infected baboons, but always as the minor variant, suggesting directional cross-species transmission. Hepatocystis parasites in this primate community are a diverse assemblage of cryptic lineages, some of which co-infect hosts and at least one of which can cross primate species barriers.

Introduction

Hemoparasites of the apicomplexan family Plasmodiidae include some of the most globally important human pathogens. Over 20 species of Plasmodium, which cause malaria, are known to infect non-human primates and at least four species, Plasmodium inui, Plasmodium simium, Plasmodium cynomolgi and Plasmodium knowlesi, are known to be transmissible to humans (Coatney et al., 2003, Cox-Singh et al., 2008). Human malaria parasites, including the particularly virulent Plasmodium falciparum, have zoonotic origins in non-human primates (Escalante et al., 1998, Rich et al., 2009, Duval et al., 2010, Krief et al., 2010, Liu et al., 2010, Prugnolle et al., 2011). However, the specific primate species from which each human malaria agent originated remains a matter of debate. Although the closest relative of P. falciparum was previously considered to be P. reichenowi, found in chimpanzees (Pan troglodytes), recent studies have discovered more closely related Plasmodium variants in other African apes and monkeys (Rich et al., 2009, Duval et al., 2010, Krief et al., 2010, Liu et al., 2010, Prugnolle et al., 2011).

Studies of Plasmodium and its relatives in non-human primates have been highly informative but influenced by the geography of sampling. Samples from captive animals close to humans may bias results towards zoonotic and reverse zoonotic transmission (Liu et al., 2010, Prugnolle et al., 2011). Broad surveys of primate hosts have yielded valuable data on geographic patterns of infection, but inferences about local transmission are difficult to make from such studies (Liu et al., 2010, Ayouba et al., 2012). Focused investigations of particular host species are useful for measuring local prevalence and parasite diversity but less so for examining transmission among species (Krief et al., 2010, Liu et al., 2010). Evaluating the propensity for Plasmodium and its relatives to cross species barriers in nature would require studying well-defined ecological communities where multiple host species are potentially exposed to the same vector populations (Prugnolle et al., 2011).

This study reports results from the well-characterized primate community of Kibale National Park, Uganda (Kibale, hereafter) and the infection of its constituent host species with parasites of the genera Plasmodium and Hepatocystis. Like Plasmodium, Hepatocystis is a hemosporidian parasite within the family Plasmodiidae that infects primates (Desser and Bennett, 1993). Unlike Plasmodium, Hepatocystis is transmitted by biting midges of the genus Culicoides, rather than anopheline mosquitoes (Garnham, 1966). At least four species of Hepatocystis are known to infect African monkeys: Hepatocystis kochi, Hepatocystis simiae, Hepatocystis bouillezi and Hepatocystis cercopitheci (Seethamchai et al., 2008). Hepatocystis can cause parasitemia for up to 15 months without causing clinical signs (Vickerman, 2005), although early studies documented anemia and visible merocyst production, followed by scarring of the liver (Garnham, 1966). Primates can be infected repeatedly and prevalence increases with age (Vickerman, 2005). Hepatocystis is not known to infect humans.

Microscopy and molecular methods were used to assess infection in six species of monkeys in Kibale: black-and-white colobus (Colobus guereza), blue guenons (Cercopithecus mitis), grey-cheeked mangabeys (Lophocebus albigena), olive baboons (Papio anubis), red colobus (Procolobus rufomitratus), and red-tailed guenons (Cercopithecus ascanius). “Deep sequencing” was then used to assess co-infection of individual hosts with multiple parasite variants. To our knowledge, this is the first study of cross-species transmission of malaria-like parasites in a well-defined community of wild primates where host species overlap spatially and temporally and would thus encounter the same vector population.

Section snippets

Study site and sampling

Kibale is a 795 km2 semi-deciduous, mid-altitude forest in western Uganda (0°13′–0°41′ N, 30°19′–30°32′E; Chapman and Lambert, 2000) within the Albertine Rift, an area of exceptional biological diversity and conservation value (Plumptre et al., 2007; Fig. 1). Kibale’s primate community has been studied for over 40 years, such that a wealth of information exists on the ecology of its 13 constituent species (Chapman et al., 2005). Plasmodium infection has previously been documented in Kibale’s

Microscopy and PCR

Microscopy identified intraerythrocytic parasites in 75% of blood smears across six primate species in Kibale (Table 1). Parasites displayed trophozoite and gametocyte forms that were morphologically similar in all host species (Fig. 2). Prevalence ranged from 22.2% in black-and-white colobus to 91.3% in baboons (Table 1). Mean intensity (percentage of infected red blood cells) across all species was 0.058%, ranging from 0.052% in baboons to 0.090% in black-and-white colobus. PCR yielded

Discussion

This study examined a community of wild primates in Uganda where ecological relationships among host species are well defined and where spatio-temporal overlap would make cross-species transmission of vector-borne parasites possible. Microscopy and PCR indicated rates of infection with Hepatocystis ranging from approximately 20% to 90% across host species. This finding is consistent with published data from wild greater spot-nosed guenons in Cameroon, which documented a prevalence of

Acknowledgments

The authors gratefully acknowledge the Uganda Wildlife Authority and the Uganda National Council for Science and Technology for granting us permission to conduct this research. The authors also thank P. Omeja and L. Kilby for assistance with logistics; J. Byaruhanga, P. Katurama, A. Mbabazi, A. Nyamwija, E. Nyamwija, J. Rusoke and K. Cameron for assistance in the field; A. McCord for assistance in the laboratory; D. Mills for assistance with image preparation; J. Bleecker for assistance with

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