Multiple host-switching of Haemosporidia parasites in bats

1475-2875-6-157 1475-2875 Research Multiple host-switching of Haemosporidia parasites in bats Duval Linda linda@pasteur-kh.org Robert Vincent v.robert@mnhn.fr Csorba Gabor csorba.gabor.hnhm@gmail.com Hassanin Alexandre hassanin@mnhn.fr Randrianarivelojosia Milijaona milijaon@pasteur.mg Walston Joe jwalston@wcs.org Nhim Thy nhimthy@yahoo.com Goodman M Steve SGoodman@wwf.mg Ariey Frédéric fariey@pasteur-kh.org

Muséum National d'Histoire Naturelle, USM 504 et UMR 5202, 55-61 rue Buffon, 75231 Paris Cedex 05, France

Institut Pasteur du Cambodge, Unité Epidémiologie moléculaire, 5, Boulevard Monivong, BP 983, Phnom Penh, Cambodia

Institut de Recherche pour le Développement, UR 77, 213 rue La Fayette, 75480 Paris Cedex 10, France

Hungarian Natural History Museum, Department of Zoology, H-1083 Budapest, Ludovika tér 2, Hungary

Institut Pasteur de Madagascar, Unité Paludisme, BP 1274, Antananarivo 101, Madagascar

Wildlife Conservation Society, Cambodia Program, #21, St 21 Sangkat Tonlé Bassac, Khan Chamkarmorn, Phnom Penh, Cambodia

Phnom Tamao Zoo and Wildlife Rescue Center, Department of Forestry and Wildlife, 40 Norodom Boulevard, Phnom Penh, Cambodia

Care for Confiscated Wild Life, WildAid, St 99, Phnom Penh, Cambodia

Field Museum of Natural History, 1400 South Lake Shore Drive, Chicago, Illinois 60605, USA

WWF, BP 738, Antananarivo 101, Madagascar

Malaria Journal 1475-2875 2007 6 1 157 http://www.malariajournal.com/content/6/1/157 18045505 10.1186/1475-2875-6-157 02 11 2007 29 11 2007 29 11 2007 2007 Duval et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

There have been reported cases of host-switching in avian and lizard species of Plasmodium (Apicomplexa, Haemosporidia), as well as in those infecting different primate species. However, no evidence has previously been found for host-swapping between wild birds and mammals.

Methods

This paper presents the results of the sampling of blood parasites of wild-captured bats from Madagascar and Cambodia. The presence of Haemosporidia infection in these animals is confirmed and cytochrome b gene sequences were used to construct a phylogenetic analysis.

Results

Results reveal at least three different and independent Haemosporidia evolutionary histories in three different bat lineages from Madagascar and Cambodia.

Conclusion

Phylogenetic analysis strongly suggests multiple host-switching of Haemosporidia parasites in bats with those from avian and primate hosts.

Background

Plasmodium falciparum (Apicomplexa, Haemosporidia), the most dangerous of human malaria parasites, is responsible for at least one million deaths a year 1. It has been suggested that its exceptional virulence, compared to the three other species of human Plasmodium, is due to its relatively recent host-shift from birds to humans and the short period for the latter to adapt to the parasite 2. Given the heavy burden of P. falciparum on human populations around the tropics 3, there is a critical need to better understand the origin and evolution of this parasite and related organisms. Plasmodium falciparum belongs to a group that also infects a considerable range of birds, squamates, crocodilians, chelonians and non-human mammals 4. These parasites are known to be virulent, invasive pathogens in a variety of wild animals and contribute to the parasite burden of natural populations, including several threatened species 5. Host switching by these parasites could be the trigger for emerging virulent diseases 678.

Madagascar and Cambodia are two biodiversity "hot spots" 9. The faunas of these areas, which are, in evolutionary terms, distant from one another, provide an attractive system for characterizing haemosporidian parasite species 1011 and for evaluating host-parasite co-evolution and exchange. Madagascar was part to the Gondwana continent and was separated 160 million and 90 million years ago from Africa and from India, respectively; whereas, Cambodia originated from the Laurasia continent.

The paper presents the result of the screening of over 500 bats belonging to seven families from different field sites in Madagascar and Cambodia. Haemosporidia parasites were isolated in the bat families, Hipposideridae, Vespertilionidae and Megadermatidae. Molecular sequences of bat haemosporidian parasites were not previously available. Herein, the phylogenetic analyses with cytochrome b mitochondrial gene sequences illustrate the first documented example of a cross-class host-switching of haemosporidian parasites between birds and mammals.

Methods

Field sites and sample collection

Screened material was obtained in Madagascar and Cambodia. In Madagascar, specimens were obtained at eight different field sites in the provinces of Mahajanga, Toliara and Antananarivo. The fieldwork was conducted between November 2001 and late 2004, in collaboration with the Institut Pasteur de Madagascar and WWF-Madagascar. In Cambodia, two field sites were sampled in the provinces of Kampot and Mondolkiri between December 2004 and May 2006, in collaboration with the Institut Pasteur du Cambodia and Wildlife Conservation Society. The field research was conducted with local and national permits from the Direction des Eaux et Forêts of Madagascar and the Ministry of Agriculture, Forestry and Fisheries in Cambodia. A total of 530 bats, belonging to seven families (Pteropodidae, Rhinolophidae, Hipposideridae, Megadermatidae, Emballonuridae, Vespertilionidae, and Molossidae) were trapped in the wild. A small sample of blood was obtained and the host was released without injury or in some cases retained as a voucher specimen.

Microscopic examination

Thin blood smears were made for each animal. They were fixed in methanol and stained by incubation with 10% Giemsa for 10 minutes. The smears were examined by microscopy for haemosporidian parasites. Parasites isolated from bats were referred to as Haemosporidia sp. Positive blood smears were catalogued in the Département 'Régulations, Développement et Diversité Moléculaire', Muséum National d'Histoire Naturelle, Paris, France.

DNA extraction and amplification

DNA was extracted from blood samples using the phenol/chloroform technique 12. The 709 bp cytochrome b fragments were amplified using PCR and nested-PCR. The PCR reaction was carried out in a total volume of 25 μl under the following condition: 1 μl of each of the primers: PLAS 1 (5'-GAGAATTATGGAGTGGATGGTG-3') and PLAS 2 (5'-GTGGTAATTGACATCCWATCC-3'), 1 mM of each dNTP, 1 U of Taq polymerase (Solis), 3 mM MgCl₂. The PCR conditions were: 5 min at 94°C, 30 sec at 94°C, 30 sec at 55°C and 1 min 30 sec at 72°C for 40 cycles and a final 10 min extension at 72°C. The nested-PCR was carried out using 1 μl of the PCR products and performed with the following primers: PLAS 3 (5'-GGTGTTTYAGATAYATGCAYGC-3') and PLAS 4 (5'-CATCCWATCCATARTAWAGCATAG-3'). The conditions were: 5 min at 94°C, 30 sec at 94°C, 30 sec at 55°C, 1 min 30 sec at 72°C for 40 cycles and a final 10 min extension at 72°C. The PCR products were sequenced using PLAS3 and PLAS4 primers by Macrogen (Korea). All of these primers were designed by the research group. They are specific to haemosporidian parasites and do not amplify others Apicomplexa parasites or host DNA. Relevant sequences were selected for the phylogenetic analysis. There are numerous avian parasite sequences available and some representative sequences were chosen based on geographical localizations.

Phylogenetic analyses

The nucleotide sequences (709 bp) were translated into amino acid sequences to minimize homoplasy due to saturation of synonymous mutations since some taxa have diverged over hundreds of millions years. The sequences were aligned using CLUSTAL W 13. Reference sequences of at least 219 amino acids without ambiguous positions were retrieved from GenBank. In the case of identical sequences in amino acids, only one sequence for the analysis was kept. Maximum Likelihood (ML) was performed using Phyml 14 and Parsimony analysis (P) was performed using Phylip package (version 3.62) 15. Nodal robustness was evaluated by non-parametric bootstrap (100 replicates). Bayesian analyses were conducted with MrBayes V3.1 software 16 using gamma distribution, 1 000 000 generations (average standard deviation of split sequences is below 0.01), with tree sampling every 100 generations and a burn-in of 2500 trees. The aim of the study was to analyse Haemosporidia phylogeny, so Theileria annulata, Babesia gibsoni and Toxoplasma gondii which belong to the phylum Apicomplexa but are non-haemosporidian parasites were choose as out-groups. The phylogenetic study was run using cytochrome b sequences. This is the most abundant marker in GenBank for a variety of haemosporidian parasites and widely used in phylogenetic studies because reference sequences from other genes are lacking.

Results

530 blood samples were collected in Madagascar and in Cambodia belonging to seven families of bats. Haemosporidian infections were identified in three families: Hipposideridae, Vespertilionidae and Megadermatidae (Table 1 and Table 2). All the cytochrome b sequences that were isolated from these bats were previously unpublished and the parasite taxa, the host names, the collection localities and the GenBank accession numbers are given in Table 3. The three phylogenetic methods used, Parsimony (P), Maximum Likelihood (ML) and Bayesian analyses produced the same tree topology. The phylogeny is presented in Figure 1.

Table 1

Prevalence of Haemosporidia infection in bats from Madagascar

Species

Family

n sampled

n infected (%)

Eidolon dupreanum*

Pteropodidae

Rousettus madagascariensis*

Pteropodidae

Hipposideros commersoni

Hipposideridae

Triaenops furculus*

Hipposideridae

1 (4)

Triaenops rufus*

Hipposideridae

Emballonura tiavato*

Emballonuridae

Miniopterus gleni

Vespertilionidae

3 (23)

Myotis goudoti

Vespertilionidae

16 (24)

Miniopterus manavi*

Vespertilionidae

129

49 (38)

Chaerephon sp.*

Molossidae

Mormoproteus jugularis*

Molossidae

Otomops madagascariensis*

Molossidae

Chaerephon leucogaster*

Molossidae

Mops leucostigma

Molossidae

Total

440

* endemic species

Table 2

Prevalence of Haemosporidia infection in bats from Cambodia

Species

Family

n sampled

n infected (%)

Rhinolophus acuminatus

Rhinolophidae

Rhinolophus borneensis

Rhinolophidae

Rhinolophus malayanus

Rhinolophidae

Rhinolophus shameli

Rhinolophidae

Rhinolophus sp.

Rhinolophidae

Hipposideros armiger

Hipposideridae

Hipposideros larvatus

Hipposideridae

1 (8)

Hipposideros pomona

Hipposideridae

Megaderma spasma

Megadermatidae

4 (80)

Taphozous melanopogon

Emballonuridae

Glischropus sp.

Vespertilionidae

Murina cyclotis

Vespertilionidae

Murina tubinaris

Vespertilionidae

Myotis muricola

Vespertilionidae

Kerivoula hardwickii

Vespertilionidae

2 (20)

Kerivoula papillosa

Vespertilionidae

Tylonycteris robustula

Vespertilionidae

Chaerephon plicatus

Molossidae

Total

Table 3

Parasite taxa used in this study with host name, geographic location and GenBank accession number of the sequences used for the phylogenetic analysis

Parasites

Host

Geographic locality

GenBank accession number

Plasmodium falciparum

Homo sapiens

Tropical regions

AY069605

Plasmodium gonderi

Old world monkeys

Central Africa

AF069622

Plasmodium inui

Old world monkeys

India and southeast Asia

AF069617

Plasmodium knowlesi

Old world monkeys

Malaysia

AF069621

Plasmodium malariae

Homo sapiens

Tropical and subtropical regions

AF069624

Plasmodium ovale

Homo sapiens

Tropics of Africa and Asia

AF069625

Plasmodium vivax

Homo sapiens

Tropical and subtropical regions

AF069619

Hepatocystis sp.

Papio sp.

Ethiopia

AF069626

Plasmodium sp.

Mandrillus leucophaeus

Gabon

AF069623

Plasmodium atheruri

Atherurus africanus

Congo and Cameroon

AY099054

Plasmodium berghei

Grammomys surdaster

Central Africa

AF099049

Plasmodium chabaudi

Thamnomys rutilans

Central Africa

AF099050

Plasmodium vinckei

Grammomys surdaster

Congo

AY099052

Plasmodium elongatum

Passer domesticus

North America

AF069611

Plasmodium gallinaceum

Gallus gallus

Vietnam

DQ212189

Plasmodium relictum

Zeneida macroura

North America

AY099032

Plasmodium sp. 2

Ninox scutulata

Singapore

AY099035

Plasmodium sp. 1

Acrocephalus arundinaceus

Japan

AY099044

Plasmodium juxtanucleare

Gallus gallus

Asia

AB250415

Plasmodium cathemerium

Wide range Bird

Wallacean zones

AY377128

Plasmodium sp. 47

Agelaius phoeniceus

North America

AF465547

Plasmodium sp. 49

Zonotrichia leucophrys

North America

AF465549

Plasmodium sp. 50

Andropadus latirostris

Cameroon

AF465550

Plasmodium floridense

Anolis oculatus

Dominica

AY099059

Plasmodium mexicanum

Sceloporus occidentalis

California

AY099060

Plasmodium chiricahuae

Sceloporus jarrovi

Arizona

AY099061

Haemosporidia (FMNH 172853)

Miniopterus manavi

Madagascar

AY762070*

Haemosporidia (FMNH 172862)

Miniopterus manavi

Madagascar

AY762071*

Haemosporidia (FMNH 172918)

Miniopterus manavi

Madagascar

AY762074*

Haemosporidia (FMNH 175810)

Myotis goudoti

Madagascar

AY762075*

Haemosporidia (C285)

Kerivoula hardwickii

Cambodia

EF179354*

Haemosporidia (C289)

Megaderma spasma

Cambodia

EF179355*

Haemosporidia (C272)

Hipposideros larvatus

Cambodia

EF179356*

Haemoproteus majoris

Parus caeruleus

Sweden

AY099045

Haemoproteus sylvae

Acrocephalus arundinaceus

Sweden

AY099040

Haemoproteus sp. 1

Phylloscopus occipitalis

India

AY099043

Haemoproteus sp. 2

Phylloscopus occipitalis

India

AY099043

Haemoproteus sp. 3

Acrocephalus scirpaceus

Spain

AY099046

Leucocytozoon toddi

Accipiter francesii

Madagascar

AY684973

Toxoplasma gondii

AF023246

Theileria annulata

M63015

Babesia gibsoni

AB215096

FMNH: Field Museum of Natural History

C: Cambodia

* Previously unpublished sequences

Figure 1

Phylogeny of Haemosporidia inferred from cytochrome b amino acid sequences

Phylogeny of Haemosporidia inferred from cytochrome b amino acid sequences. Value above branches are Bayesian posterior probabilities [16] (value less then 0.5 not shown), below are bootstrap percentage obtained by maximum likelihood [14] (left of the slash, values under 50% not shown). In red are the previously unpublished bat sequences. See Tables 1 and 2 for sampling details. H. = Haemoproteus, L. = Leucocytozoon and P. = Plasmodium.

The results show the existence of two clades within Haemosporidia, separating mammal and sauropsid hosts (birds and lizards) (1.00 Bayesian posterior probabilities, 99 and 100 for ML and P respectively bootstrap support). In the first clade, the four malaria parasites afflicting humans, Plasmodium malariae, Plasmodium ovale Plasmodium vivax and P. falciparum form a polyphyletic group 17. Rodent Plasmodium are the sister group and P. falciparum still exhibits a deep branch. Interestingly, parasite isolated from the bat Hipposideros larvatus (Family Hipposideridae) (Figures 2 and 3) clusters with a Hepatocystis parasite obtained from a baboon (Papio sp.) and falls within the Plasmodium primate group (Bayesian posterior probabilities of 1.00 and bootstrap support of 99 and 96 for ML and P, respectively).

Figure 2

Haemosporidian parasite isolated from Hipposideros larvatus (C 272)

Haemosporidian parasite isolated from Hipposideros larvatus (C 272).

Figure 3

Haemosporidian parasite isolated from Hipposideros larvatus (C 272)

Haemosporidian parasite isolated from Hipposideros larvatus (C 272).

In the second clade, between two clades of Plasmodium, are Leucocytozoon and Haemoproteus, two genera infecting only sauropsid hosts, and are close sister taxa of bird and lizard Plasmodium 18. This renders the genus Plasmodium polyphyletic. Unexpectedly, the haemosporidian parasite isolated from the bat Megaderma spasma (Family Megadermatidae) (Figure 4) is included within the sauropsid Plasmodium clade. However, the bootstrap value is low (Bayesian posterior probabilities of 0.62). Remarkably, haemosporidian parasites isolated from the Malagasy endemic bats Myotis goudoti and Miniopterus manavi and the parasite isolated from the widespread Asiatic bat Kerivoula hardwickii (Figure 5) form a monophyletic cluster and fall within the sauropsid Plasmodium clade. All these bats are classically placed in the Family Vespertilionidae. This result is well support with 0.75 Bayesian posterior probabilities and 75 ML bootstrap.

Figure 4

Haemosporidian parasites isolated from Megaderma spasma (C 289)

Haemosporidian parasites isolated from Megaderma spasma (C 289).

Figure 5

Haemosporidian parasite isolated from Kerivoula hardwickii (C 285)

Haemosporidian parasite isolated from Kerivoula hardwickii (C 285).

Discussion

The mitochondrial genome is more conserved in apicomplexan parasites 19 than in others metazoan eukaryotes. Cytochrome b gene is, therefore, a good marker to establish phylogenetic relationships between parasites that diverged several millions years ago 18.

The presence of a major division within haemosporidian parasites separating mammal and sauropsid hosts, suggests that parasites of these two vertebrate groups evolved separately. In the mammalian clade of parasites, except for P. falciparum, all primate Plasmodium species share a common ancestor, although host shifts occurred during the course of primate speciation. Indeed, wild primate populations are potential reservoirs for human malaria parasites 8and host shifts have occurred in Southeast Asia with, for example, Plasmodium knowlesi, which usually infect macaques, afflicting humans 20. Recently evidence on the origin of P. vivax as a macaque monkey malaria parasite in Southeast Asia has been proposed 721. In addition, Plasmodium simium and Plasmodium brasilianum, two species infecting South American platyrrhini primates, are genetically indistinguishable from P. vivax and P. malariae, respectively, and may have been associated with a human-platyrrhini host-switch 821. Further, haemosporidian parasite isolated from Hipposideros larvatus clusters with a baboon Hepatocystis. This association between bat and primate parasites has been previously proposed based on mitochondrial data, where a Hepatocystis species isolated from a bat (Cynopterus brachyotis, Family Pteropodidae) clusters with this baboon Hepatocystis 4. Needless to say, this result is not congruent with mammal phylogeny 22 and suggests a host switch from primates to bats.

In the sauropsid clade, the evolutionary history of Plasmodium that infects birds and lizards is not resolved. The parasite phylogeny clearly does not fit with the host phylogeny. Plasmodium parasites from birds and lizards are known to show little host specificity 23. Previous conclusions support that infrequent and unpredictable host shifts have occurred in the parasite-host sauropsid system 24. Surprisingly, the Haemosporidia isolated from Megaderma spasma in Cambodia falls within the sauropsid Plasmodium clade. However, phylogenetic relationships between the parasites of Megaderma and sauropsids are not completely resolved.

Furthermore, closely related haemosporidian parasites isolated from Myotis goudoti and Miniopterus manavi, two endemic Malagasy bat species and the haemosporidian parasite from the Cambodian Kerivoula hardwickii, all of which are placed in the family Vespertilionidae, fall within the sauropsid Plasmodium clade. This result clearly does not fit with vertebrate phylogeny and supports host switching from birds to bats. The haemosporidian vespertilionid parasites from Madagascar and Cambodia are monophyletic, which suggest that the host switching took place in the early evolutionary history of these bats and was followed by subsequent radiation and co-speciation. This is the first report showing host switching in haemosporidian parasites between birds and mammals (bats).

After rodents, bats are the largest order of mammals (at least 1,100 species, more than 20% of extant mammal species). The Chiroptera are very diverse and they are distributed almost worldwide and have extremely diverse life history traits and morphology. Based on recent molecular work, they are classified into four super-families that apparently diversified in different areas during the early Eocene as a "Big Bang" radiation 25 coincident with the peak of Tertiary insect diversity 26. In developing echolocation and different flight strategies, the ancestors of modern bats colonized various ecological niches 27, where birds and their associated blood parasites are thought to have been present, thus favoring host switching from birds to bats. Furthermore, Myotis goudoti and Miniopterus manavi often share common day roost sites in tree hollows, caves and rock shelters 28, which expose considerable numbers of densely packed individuals to the same potential blood parasite vectors.

Conclusion

The introduction of the 7 new genetic sequences from chiropteran hemoparasites does not alter the deep branching of P. falciparum within the mammalian clade 417. This result does not support a recent parasite transfer from birds to Homo sapiens, which has been used to explain the pathogenicity of P. falciparum in humans. Rather, the nearly exponential recent growth in human populations may have acted on P. falciparum selection patterns 29. However, the sequence data from blood parasites isolated from bats provide further insights into the possible evolutionary pathway of human malaria parasites. Those results show that P. falciparum has a different and independent evolutionary history than other human malaria parasites. This is consistent with recent studies providing genetic evidence that the four human parasites did not emerge from the same geographical region 30. Based on clear evidence presented herein of host switching between birds and bats, it is difficult to reject the hypothesis that P. falciparum has a non-primate origin. It may have emerged in humans associated with an ancient host switching and, as such, it could be one of the oldest "emerging" diseases in humans.

Authors' contributions

LD, VR, and FA designed the study. LD, VR, GC, MR, JW, TN, SMG and FA do the sampling, LD and TN process samples and LD, HA, VR, SMG and FA analysed the data. LD wrote the first draft of the manuscript then VR, SMG and FA critically reviewed the manuscript. All authors read and approved the final manuscript.

Acknowledgements

We thank Genevieve Milon and Richard E. L. Paul for support and advices.

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