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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22952922PONE-D-12-1708810.1371/journal.pone.0044187Research ArticleBiologyEvolutionary BiologyEvolutionary GeneticsEvolutionary SystematicsZoologyHelminthologyParasitologyMedicineInfectious DiseasesNeglected Tropical DiseasesParasitic DiseasesZoonosesVeterinary ScienceVeterinary DiseasesVeterinary ParasitologyParasite EvolutionPhylogenetic Evidence That Two Distinct Trichuris Genotypes Infect both Humans and Non-Human Primates Two Trichuris Genotypes Infect PrimatesRavasi Damiana F. 
1
O'Riain Mannus J. 
1
Davids Faezah 
2
Illing Nicola 
2

*

1 
Department of Zoology, University of Cape Town, Rondebosch, South Africa

2 
Department of Molecular and Cell Biology, University of Cape Town, Rondebosch, South Africa
Parkinson John Editor
Hospital for Sick Children, Canada
* E-mail: xrobinson@example.com Interests: The authors have declared that no competing interests exist.

Conceived and designed the experiments: MJO NI DFR. Performed the experiments: DFR FD NI. Analyzed the data: NI DFR. Contributed reagents/materials/analysis tools: MJO. Wrote the paper: NI DFR MJO.

2012 28 8 2012 7 8 e4418713 6 2012 30 7 2012 © 2012 Ravasi et al2012Ravasi et alThis 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 properly credited.Although there has been extensive debate about whether Trichuris suis and Trichuris trichiura are separate species, only one species of the whipworm T. trichiura has been considered to infect humans and non-human primates. In order to investigate potential cross infection of Trichuris sp. between baboons and humans in the Cape Peninsula, South Africa, we sequenced the ITS1-5.8S-ITS2 region of adult Trichuris sp. worms isolated from five baboons from three different troops, namely the Cape Peninsula troop, Groot Olifantsbos troop and Da Gama Park troop. This region was also sequenced from T. trichiura isolated from a human patient from central Africa (Cameroon) for comparison. By combining this dataset with Genbank records for Trichuris isolated from other humans, non-human primates and pigs from several different countries in Europe, Asia, and Africa, we confirmed the identification of two distinct Trichuris genotypes that infect primates. Trichuris sp. isolated from the Peninsula baboons fell into two distinct clades that were found to also infect human patients from Cameroon, Uganda and Jamaica (named the CP-GOB clade) and China, Thailand, the Czech Republic, and Uganda (named the DG clade), respectively. The divergence of these Trichuris clades is ancient and precedes the diversification of T. suis which clustered closely to the CP-GOB clade. The identification of two distinct Trichuris genotypes infecting both humans and non-human primates is important for the ongoing treatment of Trichuris which is estimated to infect 600 million people worldwide. Currently baboons in the Cape Peninsula, which visit urban areas, provide a constant risk of infection to local communities. A reduction in spatial overlap between humans and baboons is thus an important measure to reduce both cross-transmission and zoonoses of helminthes in Southern Africa.

Partial funding was provided by grants from the National Research Foundation (South Africa) and University of Cape Town to M. J. O'Riain and N. Illing. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
Helminths are the most common parasites infecting humans in developing countries and can cause malnutrition, anaemia, growth retardation, and increased susceptibility to other infections [1]. Many pathogens that infect humans and domesticated animals can infect more than one host species [2], [3] and according to Taylor et al.
[3], 96% of the 287 helminth species found in humans are zoonotic. As urbanization and human population growth forces humans and wild animals into closer and more frequent contact, there are increasing concerns that cross infection of parasites between different primate hosts may lead to the emergence of new diseases in both humans and non-human primates [4]–[6]. This itself is a subject of debate, as although some diseases such as measles and HIV have emerged recently in urbanized human populations, molecular dating suggests that other diseases such as infection by tapeworms, leprosy, and treponematosis have Paleolithic origins [7].

The helminth Trichuris trichiura (whipworm) is considered the third most common roundworm to infect humans with an estimated 600 million people infected worldwide [8]. Infection is direct and caused by the ingestion of embryonated eggs from contaminated hands, food, soil or water. After T. trichiura eggs have been swallowed, the larvae hatch in the small intestine before travelling to the large intestine where they grow into adult whipworms [9]. The diagnosis of T. trichiura is typically done by microscopic visualization of the characteristic lemon shaped eggs in the faeces. The eggs measure 50–55 µm by 22–24 µm, are dark brown in colour and present "plug like" prominences at each pole [10].

Three Trichuris species, namely T. trichiura, T. suis, and T. vulpis are considered zoonotic parasites which are a threat to human health [3]. In the Cape Peninsula (Western Cape Province, South Africa), where informal settlements of humans are characterized by overcrowding and inadequate sanitation, T. trichiura is the predominant helminth, with prevalence averaging 51% in children [11]. In parallel, a high prevalence (66%) of Trichuris sp. has been recorded in the local population of chacma baboon (Papio ursinus) [12]. This geographically isolated and protected baboon population lives in close proximity to urban populations [13], many of which have inadequate sanitation, which raises the risk of zoonotic infection from contaminated water and soil.

Whipworms found in parasitological surveys in non-human primates are typically assumed to belong to the species T. trichiura, the whipworm known to infect humans [14], [15]. However, due to the limited external characters of parasites, delimiting species using morphological criteria alone is not accurate [16]. Morphological parameters cannot be used to distinguish between adult females of T. trichiura and T. suis
[17]. Furthermore there is disagreement on whether spicule length in males is longer [17] or shorter [18] in T. trichiura compared to T. suis. Detailed morphological analyses of Trichuris sp. recovered from non-human primates are rare. Ooi et al.
[14] compared the morphology of Trichuris sp. worms collected from macaques (Macaca fuscata) and baboons (Papio papio) to T. trichiura collected from humans, using light and scanning electron microscopy, and concluded that it was not possible to distinguish between these Trichuris sp. on morphological grounds. It thus remains uncertain whether T. trichiura reported in captive and wild populations of non-human primates are one or more different species.

Molecular techniques are increasingly used as the main tool in the identification of species [19]. Cutillas et al.
[20] used the internal transcribed spacers (ITS) of the ribosomal DNA to prove the existence of two separate Trichuris species in murid and arvicolid rodents. This sequence has also been shown to be a reliable marker to distinguish between T. suis isolated from swine or wild boar, T. vulpis isolated from dogs [21], and T. trichiura isolated from the non-human primates Colobus guereza kikuyensis and Nomascus gabriellae
[17].

In this study the ITS1-5.8S-ITS2 region of ribosomal DNA was sequenced from adult Trichuris sp. worms isolated from five baboons from the Cape Peninsula, and from two adult T. trichiura isolated from a human patient in Cameroon to determine the genetic relationship between Trichuris sp. infecting baboons in the Cape Peninsula and humans. By combining this dataset with Genbank records for Trichuris sp. isolated from other humans, non-human primates and pigs from several different countries in Europe, Asia, and Africa, we show that two genetically distinct Trichuris genotypes infect humans and non-human primates.

Methods
Sample Collection

Trichuris sp. specimens were collected from chacma baboons (Papio ursinus) that range in the Cape Peninsula, an area of 470 km2 at the south western tip of the African continent, which stretches from the city of Cape Town to the Cape of Good Hope section of the Table Mountain National Park (Fig. 1). Chacma baboons are the only non-human primate species found on the peninsula and are currently protected by legislation. Historically they occurred throughout the peninsula, but agricultural and urban development resulted in a dramatic reduction in suitable natural habitat and a concomitant increase in the frequency and extent of close contact between extant troops and densely populated suburban areas [22]–[24].

10.1371/journal.pone.0044187.g001Figure 1 Home ranges of the baboon troops in the Cape Peninsula, South Africa, from which Trichuris sp. specimens were collected.
Urban areas are shaded in grey and the border of Cape of Good Hope section of the Table Mountain National Park is outlined (dashed line).

We sampled three of the existing 16 Peninsula baboon troops. The Cape Point troop ranges entirely in the Cape of Good Hope section of the Table Mountain National Park (Fig. 1). This troop's home range overlaps with the Cape Point visitors centre and thus troop members experience frequent, close contact with both local and overseas tourists. The Groot Olifantsbos troop resides in the northernmost section of Cape of Good Hope and their home range overlaps with a residential suburb (Scarborough) that is frequently raided by all troop members (Fig. 1). The Da Gama Park troop is immediately northeast of the Groot Olifantsbos troop and it too overlaps with a residential area bringing troop members into frequent close contact with humans, their waste and a variety of domestic animals [22], [24] (Fig. 1). Data on Trichuris sp. prevalence were obtained from 21 and 114 faecal samples collected from the Cape Peninsula and Da Gama Park troops, respectively, between July 2006 and May 2008. Faecal samples were processed using a modified formalin ether sedimentation technique [25] and Trichuris sp. eggs were identified and counted. Prevalence was calculated as the percentage of samples with positive Trichuris sp. identification.

Opportunistic necropsies were performed whenever dead baboons were brought to the University of Cape Town, South Africa, by the relevant conservation authorities. Adult specimens of the nematode Trichuris sp. were collected from the large intestines of three adult females of the Da Gama Park troop (samples DGI, DGII and DGIII), one adult male from the Cape Point troop (sample CP), and one infant female from the Groot Olifantsbos troop (sample GOB). The five worms were stored separately in 70% ethanol and were washed extensively in a saline solution of 0.9% sodium chloride, prior to DNA extraction. Two archived adult specimens of T. trichiura were obtained from a previous study [26] collected from a human patient in 2000, from the city of Kumba, Cameroon, following administration of a dose of the antihelminthic drug pyrantel. These specimens were stored at −80°C in a single vial at the University of Nottingham.

DNA Extraction and Sequencing of the ITS1-5.8S-ITS2 Region
It was not possible to extract DNA from Trichuris sp. eggs collected in this study as they had been preserved in formalin. DNA was extracted from whole specimens of Trichuris sp. obtained from baboons at the University of Cape Town according to the Tissue Protocol of the QIAamp DNA Mini Kit (Qiagen, Venlo, Netherlands) with the following modification: after overnight incubation at 56°C, two steel beads were added to the lysate solution and subjected to strong shaking for 4 min in a Gyromixer (Fast and Fluid Management, Sassenheim, Netherlands) to completely dissociate the tissue.

DNA from specimens of T. trichiura isolated from a human patient was extracted according to a standard protocol [27] at the University of Nottingham to prevent the possibility of any cross-contamination between these DNA samples and DNA samples purified from Trichuris sp. isolated at the University of Cape Town. The two worms were homogenized and DNA was sequentially purified, twice with phenol:chloroform:isoamyl alcohol (25∶24:1) and once with chloroform only. The DNA was then precipitated with 3M sodium acetate pH 5.2 and 100% ethanol at −20°C overnight, pelleted and washed with 70% ethanol. After a final centrifugation the DNA was air dried, resuspended in 1× TE buffer and quantified using a NanoDrop® Spectrophotometer (NanoDrop Technologies, USA). DNA was stored at −20°C.

The Polymerase Chain Reaction mix used to amplify the ITS1-5.8S-ITS2 region was prepared with 10 µl of 10× PCR buffer, 2 µl of 10 mM dNTP mixture (0.2 mM each), 6 µl of 25 mM magnesium chloride, 5 µl of forward and reverse primers (0.5 µM each), 1.5 µl of DNA template, 0.5 µl of Taq DNA polymerase (2.5 units) and autoclaved distilled water to 100 µl. The conditions applied were: 3 min at 94°C, 35 cycles of 1 min at 94°C, 1 min at 55°C, 1 min at 72°C, with a final extension step of 10 min at 72°C. The primers used in this study, namely forward primer NC5 (5′-GTAGGTGAACCTGCGGAAGGATCATT-3′) and reverse primer NC2 (5′-GGTTAGTTTCTTTTCCTCCGCT-3′) correspond to the conserved ends of the ITS1-5.8S-ITS2 region [28]. The reverse primer NC2 was modified by adding two extra Gs to the 5′ end, to assist with TA cloning and to increase the melting temperature. A negative control was included in each set of PCR reactions.

Purification of amplicons from Trichuris sp. samples was done using the Wizard® SV Gel and PCR Clean-Up System (Promega, Madison, USA). One µl of amplicon was cloned into Escherichia coli (DH5α) using the pGEM®-T Easy Vectors System (Promega, Madison, USA). Single clones were screened for inserts by colony PCR using flanking primers SP6 (5′-ATTTAGGTGACACTATAGAA-3′) and T7 (5′-TAATACGACTCACTATAGGG-3′). Plasmids were purified using a Qiagen Plasmid Midi Kit (Venlo, Netherlands) and an individual clone from each specimen was sent for sequencing to Macrogen Inc. (Seoul, Korea), using the T7 and SP6 primers. The PCR product from the human T. trichiura samples was purified with a QIAquick Gel Extraction Kit (Qiagen, Venlo, Netherlands) and cloned into 5-alpha F'Iq competent E. coli (NEB, Ipswich, UK) using thepCRTM4-TOPO® vector system (Invitrogen, Paisley, UK). Positive clones were selected on LB agar plates containing 100 µg/ml ampicillin prior to verification by colony PCR using the NC5 and NC2 prime pair. The plasmids from five confirmed positive clones were purified using a Qiagen Plasmid Mini Kit and were sequenced using T3 and T7 primers at the Biopolymer Synthesis and Analysis Unit of the University of Nottingham.

Sequence and Phylogenetic Analyses
The nucleotide sequences were edited in BioEdit (Ibis Biosciences, Carlsbad, USA). Clones which shared 100% identity were merged. Nucleotide sequences for the ITS1-5.8S-ITS2 regions generated in this study were submitted to GenBank, and their accession numbers are listed in Table 1. MUSCLE software [29], [30] was used to align these sequences to other publicly available ITS1-5.8S-ITS2 or ITS1 or ITS2 sequences (listed in Table 1). Phylogenetic trees based on the MUSCLE alignments were constructed with either the Neighbour-Joining method, Maximum Likelihood, or Maximum Parsimony using the MEGA, version 5 [31] software. The evolutionary distances were computed using the Tajima-Nei method [32]. Bootstrap consensus trees were inferred from 1000 replicates in each instance [33], and were rooted with Trichuris isolated from cattle and sheep.

10.1371/journal.pone.0044187.t001Table 1 GenBank Accession Numbers for full length ITS1-5.8S-ITS2 and ITS1 and ITS2 regions used in this study.
GenBank ID	
Trichuris species	Host species	Country	Region	Living conditions	Reference	
AB367794	
Trichuris discolor
	
Capricornis crispus
	Japan	ITS1-5.8S rRNA-ITS2	domestic animal	Unpublished	
AB367795	
Trichuris discolor
	Cattle	Japan	ITS1-5.8S rRNA-ITS2	domestic animal	Unpublished	
JF680987	
Trichuris ovis
	
Ovis aries
	Ireland	ITS1-5.8S rRNA-ITS2	domestic animal	Unpublished	
AM993012	
Trichuris suis
	
Sus scrofa domestica
	China	ITS1-5.8S rRNA-ITS2	domestic animal	
[8]
	
AM993016	
Trichuris suis
	
Sus scrofa domestica
	China	ITS1-5.8S rRNA-ITS2	domestic animal	
[8]
	
FM991956	
Trichuris trichiura
	
Colobus guereza kikuyuensis
	Spain	ITS1-5.8S rRNA-ITS2	zoo	
[17]
	
GQ301555	
Trichuris trichiura
	
Homo sapiens
	Cameroon	ITS1-5.8S rRNA-ITS2		This study	
AM992981	
Trichuris trichiura
	
Homo sapiens
	China	ITS1-5.8S rRNA-ITS2		
[8]
	
FM991955	
Trichuris trichiura
	
Nomascus gabriellae
	Spain	ITS1-5.8S rRNA-ITS2	zoo	
[17]
	
GQ301554	
Trichuris sp.	
Papio ursinus, CP_GOB	South Africa	ITS1-5.8S rRNA-ITS2	wild ranging, urban	This study	
GQ301553	
Trichuris sp.	
Papio ursinus, DGI	South Africa	ITS1-5.8S rRNA-ITS2	wild ranging, urban	This study	
GQ301552	
Trichuris sp.	
Papio ursinus, DGII	South Africa	ITS1-5.8S rRNA-ITS2	wild ranging, urban	This study	
GQ301551	
Trichuris sp.	
Papio ursinus, DGIII	South Africa	ITS1-5.8S rRNA-ITS2	wild ranging, urban	This study	
GQ352554	
Trichuris trichiura
	
Homo sapiens
	Thailand	ITS1		Unpublished	
AJ781762	
Trichuris suis
	
Sus scrofa domestica
	Spain	ITS1	domestic animal	
[17]
	
AJ783398	
Trichuris suis
	
Sus scrofa scrofa
	Spain	ITS1	wild animal	
[17]
	
JF690949	
Trichuris sp.	
Chlorocebus aethiops
	Tanzania	ITS2	wild ranging?	Unpublished	
JF690950	
Trichuris sp.	
Chlorocebus aethiops
	Tanzania	ITS2	wild ranging?	Unpublished	
JF690944	
Trichuris sp.	
Chlorocebus sabaeus
	Czech Republic	ITS2	zoo	Unpublished	
JF690940	
Trichuris sp.	
Homo sapiens
	Czech Republic	ITS2		Unpublished	
JF690946	
Trichuris sp.	
Macaca fascicularis
	Czech Republic	ITS2	zoo	Unpublished	
AB586133	
Trichuris sp.	
Macaca fuscata
	Japan	ITS2	wild ranging	Unpublished	
JF690945	
Trichuris sp.	
Macaca silenus
	Czech Republic	ITS2	zoo	Unpublished	
JF690948	
Trichuris sp.	
Pan troglodytes
	Netherlands	ITS2	zoo	Unpublished	
JF690942	
Trichuris sp.	
Papio anubis
	Czech Republic	ITS2	zoo	Unpublished	
JF690941	
Trichuris sp.	
Papio hamadryas
	Czech Republic	ITS2	zoo	Unpublished	
JF690943	
Trichuris sp.	
Theropithecus gelada
	Czech Republic	ITS2	zoo	Unpublished	
JN181814	
Trichuris trichiura
	
Homo sapiens
	Uganda	ITS2		
[18]
	
JN181820	
Trichuris trichiura
	
Homo sapiens
	Uganda	ITS2		
[18]
	
JN181822	
Trichuris trichiura
	
Homo sapiens
	Jamaica	ITS2		
[18]
	
JN181826	
Trichuris trichiura
	
Homo sapiens
	Uganda	ITS2		
[18]
	
JN181827	
Trichuris trichiura
	
Homo sapiens
	Uganda	ITS2		
[18]
	
JN181829	
Trichuris trichiura
	
Homo sapiens
	Uganda	ITS2		
[18]
	
JN181831	
Trichuris trichiura
	
Homo sapiens
	Uganda	ITS2		
[18]
	
JN181834	
Trichuris trichiura
	
Homo sapiens
	Uganda	ITS2		
[18]
	
JN181838	
Trichuris trichiura
	
Homo sapiens
	Uganda	ITS2		
[18]
	
JN181840	
Trichuris trichiura
	
Homo sapiens
	Uganda	ITS2		
[18]
	
JN181842	
Trichuris trichiura
	
Homo sapiens
	Uganda	ITS2		
[18]
	
JN181848	
Trichuris trichiura
	
Homo sapiens
	Uganda	ITS2		
[18]
	
JN181850	
Trichuris trichiura
	
Homo sapiens
	Uganda	ITS2		
[18]
	
JN181852	
Trichuris trichiura
	
Homo sapiens
	Uganda	ITS2		
[18]
	
JN181858	
Trichuris trichiura
	
Homo sapiens
	Uganda	ITS2		
[18]
	
JN181860	
Trichuris trichiura
	
Homo sapiens
	Uganda	ITS2		
[18]
	
JN181797	
Trichuris suis
	
Sus scrofa domestica
	Uganda	ITS2	domestic animal	
[18]
	
JN181770	
Trichuris suis
	
Sus scrofa domestica
	Uganda	ITS2	domestic animal	
[18]
	
JN181785	
Trichuris suis
	
Sus scrofa domestica
	Uganda	ITS2	domestic animal	
[18]
	
JN181791	
Trichuris suis
	
Sus scrofa domestica
	Uganda	ITS2	domestic animal	
[18]
	
JN181804	
Trichuris suis
	
Sus scrofa domestica
	Uganda	ITS2	domestic animal	
[18]
	
AJ249966	
Trichuris suis
	
Sus scrofa domestica
	Spain	ITS2	domestic animal	
[17]
	
JF690951	
Trichuris suis
	
Sus scrofa
	Slovakia	ITS2	domestic animal	Unpublished	
 A question mark is indicated when information on living conditions was not explicit in Genbank records.

10.1371/journal.pone.0044187.g002Figure 2 Neighbour-joining phylogenetic tree [38] based on a MUSCLE alignment of ITS1 5.8S-ITS2 sequences from Trichuris spp. isolated from human, non-human primates and pigs.
Bootstrap values are indicated as percentages on the branches of the consensus tree which was inferred from 1000 replicates [33]. The tree was rooted with T. ovis and T. discolor. The evolutionary distances were computed using the Tajima-Nei method [32] and the scale represents the number of base substitutions per site. Branches corresponding to Clade DG are highlighted in blue, while branches corresponding to the Clades CP-GOB and T. suis are highlighted in green and pink, respectively.

Ethics Statement
Research reported here is original and adhered to the American Society of Primatologists Principles for the Ethical Treatment of Non-Human Primates. Data were collected according to protocols approved by the University of Cape Town and South African National Parks, and adhered to the legal requirements of South Africa. A permit was obtained from Cape Research Centre, South African National Parks for both the collection of baboon faeces and the performing of opportunistic necropsies on dead baboons delivered to the University of Cape Town by Conservation Authorities. The permit was valid from January 2007 to January 2012.

The T. trichiura worms used in this study were obtained from archived material from a previous study [26]. Verbal informed consent was obtained from the patient, and the guidelines for human experimentations from the Ministry of Health, Cameroon were followed. The study received clearance from the Cameroonian National Ethics Committee. The samples were analyzed anonymously.

Results

Trichuris sp. was one of seven nematode species found in the faeces of the Cape Peninsula baboon troops and had the highest mean (66%) and maximum (98%) prevalence of all nematodes recorded. Trichuris eggs from baboons in Da Gama Park measured in average 55.0 (±1.9) um×25.4 (±1.3) um (n = 10) and were found to be smaller than eggs from baboons in Cape Point (64.5 (±2.6) um×30.9 (±2.4) um, (n = 10)). However, a T-test revealed that these differences were not significant.

The ITS1-5.8S-ITS2 region was successfully amplified from genomic DNA from the specimens of Trichuris sp. collected from the Peninsula chacma baboons. The sequences (1180 bp) obtained from baboons of the Cape Point and Groot Olifantsbos troops were identical and were thus treated as one sequence, named Trichuris sp. Papio ursinus CP-GOB to indicate the troops from which the whipworms were isolated. The ITS1-5.8S-ITS2 sequences (1293 bp) from three specimens of Trichuris sp. obtained from three individuals of the Da Gama Park troop shared 98% identity with each other, and were named Trichuris sp. Papio ursinus DGI, DGII and DGIII respectively. The sequences of the ITS1-5.8S-ITS2 regions isolated from Trichuris sp. DGI-DGIII were clearly different from the CP-GOB sequence and shared only 54% identity.

10.1371/journal.pone.0044187.g003Figure 3 Neighbour-joining phylogenetic tree based on a MUSCLE alignment of ITS2 sequences from Trichuris spp. isolated from human, non-human primates and pigs.
Bootstrap values are indicated as percentages on the branches of the consensus tree which was inferred from 1000 replicates [33]. The tree was rooted with T. ovis and T. discolor. The evolutionary distances were computed using the Tajima-Nei method [32] and the scale represents the number of base substitutions per site. Branches corresponding to Clade DG are highlighted in blue, while branches corresponding to the Clades CP-GOB and T. suis are highlighted in green and pink respectively. Clones H5a and H5k, H7b and H7e, H8b and H8c, derived from the worms H5, H7 and H8 [18] which clustered to different clades respectively, are highlighted with red stars.

Since the sequence for the ITS1-5.8S-ITS2 region for T. trichiura isolated from humans was not available at the start of this study, we made use of archived material from a previous study in the Cameroon [26] to investigate the relationship between Trichuris sp. isolated from baboons in the Cape Peninsula and T. trichiura that infects humans. The T. trichiura ITS1-5.8S-ITS2 sequences (1400 bp) of five clones were identical. These sequences shared only 53% identity to the ITS1-5.8S-ITS2 sequence (AM992981) from T. trichiura isolated from a human patient in China that was subsequently made available in Genbank.

To our surprise, the Trichuris sp. Papio ursinus DGI-DGIII sequences shared 98–99% identity with the ITS1-5.8S-ITS2 sequence from T. trichiura isolated from a patient in China. Five variable number of tandem repeats (VNTR) were identified between these sequences, namely DG-VNTR1 ((CAG)n where n ranged from 7, 8 and 10), DG-VNTR2 ((GGC)n where n ranged from 3 to 4), DG-VNTR3 ((CAG)n where n ranged from 7 to 8), DG-VNTR4 ((GAC)n where n ranged from 4 to 5) and DG-VNTR5 ((GGC)n where n ranged from 1 to 2) (Fig. S1). Only six single nucleotide polymorphisms (SNPs) were identified which differed between the Trichuris sp. DG1-DGIII and T. trichiuria (AM992981) sequences (Fig. S1).

However, the sequence of T. trichiura isolated from the human patient in Cameroon was similar (91% identity) to the Trichuris sp. CP-GOB sequence (Fig. S1). An analysis of the sequence alignment highlights the greater variability between these sequences, with fewer VNTR differences, and many more SNPs and indels (Fig. S1).

Phylogenetic analysis with three different algorithms namely, Neighbour Joining, Maximum Likelihood, and Maximum Parsimony gave the same results (data not shown). Trichuris sp. DGI-DGIII clustered into the same clade (Clade DG) as T. trichiura isolated from patients in China, while Trichuris sp. CP-GOB clustered into the same clade (Clade CP-GOB) as T. trichiura isolated from a patient in Cameroon, as well as Trichuris sp. isolated from primates Colobus guereza and Nomascus gabriellae in a Spanish zoo 17 (Fig. 2). The separation of Trichuris spp. that infect humans and non-human primates into two genetically distinct clades had robust bootstrap support (Fig. 2). Interestingly, Trichuris sp. in Clade CP-GOB are more closely related to T. suis (sharing 79% identity), than they are to Trichuris sp. in Clade DG (Fig. 2). In addition to having strong bootstrap support, the branch lengths separating Trichuris sp. CP-GOB from T. suis were longer than the branch lengths separating Trichuris ovis from Trichuris discolour (Fig. 2). T. suis and Trichuris sp. CP-GOB are thus likely to be two different species that cluster in clade CP-GOB.

In order to include the largest possible number of sequences available for Trichuris species isolated from humans and non-human primates, further phylogenetic analyses were performed using Genbank records for the ITS1 and the ITS2 regions for T. suis and Trichuris sp. isolated from human and non-human primates (Table 1). The restriction of the analysis to the ITS1 region expanded the dataset to include a T. trichiura sequence isolated from a human patient in Thailand, which was grouped into Clade DG, along with the T. trichiura from patients in China, whereas T. suis from S. scrofa domestica and S. scrofa scrofa
17 clustered with the other T. suis sequences (data now shown).

Phylogenetic analysis of aligned ITS2 Trichuris sequences showed that T. trichiura isolated from a patient in the Czech Republic, as well as several isolates from patients in Uganda, fell into Clade DG (Fig. 3). Trichuris sp. isolated from several species of primates kept in zoos in various countries in Europe and Asia, confirmed that Trichuris sp. from both Clade DG and Clade CP-GOB were able to infect non-human primates. All of the T. suis ITS2 sequences isolated from pigs around the world, formed a distinct cluster within clade CP-GOB (Fig. 3). The Trichuris sp. isolated from patients in Jamaica and Uganda 18 clustered with these T. suis sequences, with strong bootstrap support, and short branch lengths, suggesting that recent zoonotic infection may be taking place between pigs and humans living in close proximity in Uganda. Some of the clones in this study 18 had widely divergent sequences with 56–58% identity, even though they were sourced from the same worms, with one sequence clustering to Clade DG and the other sequence clustering to Clade CP-GOB (Fig. 3).

Discussion
We have isolated and analysed the ITS1-5.8S-ITS2 regions of five Trichuris specimens collected from five chacma baboons (Papio ursinus) ranging in three different troops in the Cape Peninsula, South Africa. Our results suggest that two distinct Trichuris genotypes infect these baboon troops, and that both Trichuris genotypes infect humans and non-human primates in Africa, Europe, and Asia. For the purposes of this study, we refer to these two distinct Trichuris genotypes as Trichuris sp. DG and Trichuris sp. CP-GOB, to signify the baboon troops that led to their discovery.

The ITS1-5.8S-ITS2 sequences of the three specimens of Trichuris sp. collected from the urban Da Gama Park troop were highly similar to each other (98% identity). The differences between them included three VNTRs and four SNPs. The sequences of the two specimens of Trichuris sp., collected from baboons in the neighboring troops of Cape Point and Groot Olifantsbos (Fig. 1) respectively, were identical. The Cape Point and Groot Olifantsbos troops live in the same region of the Cape of Good Hope section of the Table Mountain National Park. Although their home ranges do not directly overlap (Fig. 1), they are indirectly connected by the presence of other troops between them and the regular transfer of adult males between neighboring troops. The ITS1-5.8S-ITS2 sequence of Trichuris sp. found in the Da Gama Park troop differed from the Trichuris sp. found in the Cape Point and Groot Olifantsbos troops (54% identity).

We compared the ITS1-5.8S-ITS2 regions of our specimens to T. trichiura isolated from a patient in Cameroon and to a publicly available sequence of T. trichiura from a patient in China. The ITS1-5.8S-ITS2 sequences of T. trichiura recovered from humans did not cluster together, and shared only 53% identity. Instead, T. trichiura isolated from a patient in China clustered closely to Trichuris sp. DGI-DGIII while T. trichiura isolated from a patient in Cameroon clustered with Trichuris sp. CP-GOB, T. trichiura N. gabriellae and T. trichiura C. guereza
17.

The levels of genetic divergence were quite different between the clades in our phylogenetic analysis. Whereas the Trichuris sequences in Clade DG were all highly similar, with short branch lengths, the Trichuris sp. in Clade CP-GOB had much longer branch lengths. T. suis was found to cluster within Clade CP-GOB. Our identification of Trichuris sp. in clade CP-GOB as a separate genotype from T. suis is in agreement with Cutillas et al.
17 who concluded that T. trichiura isolated from N. gabriellae and C. guereza, were a distinct species from T. suis. Our analysis suggest however that these two genotypes (i.e T. suis and Trichuris sp. in clade CP-GOB) share a more recent common phylogenetic history than Trichuris sp. in clade DG.

An analysis based on only the ITS2 region allowed us to extend our analysis to a larger dataset of 34 publically available sequences, and confirm that two distinct Trichuris genotypes infect many species of non-human primates, including hamadryas baboons (Papio hamadryas), vervet monkeys (Chlorocebus aethiops), and chimpanzees (Pan troglodytes). In agreement with Nissen et al.
18, some of the ITS2 sequences derived from T. trichiura isolated from humans in Uganda, clustered tightly with the T. suis ITS2 sequences with little sequence divergence. However, Nissen et al.
18 reported that different clones from the same PCR amplification reaction from a single human-derived worm, clustered in two different clades (for example worms H5, H7 and H8). These ITS2 sequences share only 57–58% sequence identity, and are thus unlikely to be "heterozygote" worms as suggested by Nissen et al.
18, and may be a consequence of amplification of false PCR products due to cross-contamination of genomic DNA from the T. suis samples.


Trichuris sp. DG and Trichuris sp. CP-GOB may be ancient parasites that have moved between populations of humans and non-human primates for millions of years, having evolved in our hominid ancestors in Africa in the Palaeolithic era 7. It is possible that pigs subsequently became infected with Trichuris sp. CP-GOB as a consequence of domestication of animals by humans in the Neolithic era, or alternatively human became infected with T. suis, which subsequently infected non-human primates kept under captive conditions in zoos, or whom came into regular contact with humans.

Considering the high prevalence of Trichuris sp. in both humans (51%) and baboons (66%) recorded in the Cape Peninsula 11,12, the molecular analysis of T. trichiura isolated from human patients in the Cape Peninsula, and baboons both on the Cape Peninsula, and in the wild, far from urban contact, represents the next single most important step in this investigation of patterns of parasite infection. Additionally, the parallel phylogenetic analysis of other genetic loci such as β-tubulin and mitochondrial cytochrome oxidase subunit 1 gene is needed to refine the conclusion that two distinct Trichuris genotypes identified in this study are separate species.

Although several authors have concluded that morphological features are uninformative in distinguishing Trichuris sp. isolated from humans and non-human primates 14, and between T. trichiura and T. suis
17,18 these studies grouped all measurements of Trichuris isolated from humans and non-human primates and did not take into account the different genotypes of the Trichuris sp. DG and Trichuris sp. CP-GOB. Our finding of two distinct Trichuris genotypes suggests the need for further morphological analyses of adult worms coupled with genetic studies to determine whether the two genotypes reported in this study are distinct species, and whether there are statistically significant morphological features that can be used to distinguish them from each other, and from T. suis.

From a human health point of view, baboons could act as a reservoir for primate Trichuris sp. in areas with a high degree of contact between baboons and humans, and this remains an important area for future research in the Cape Peninsula. Likely areas for cross transmission of parasites suggested by various authors are: communal water holes 34, human refuse and food wastes, including animal parts 35,36 and soil 37. In the Cape Peninsula, baboons regularly access residential areas and have both direct and indirect (through faeces) contact with gardens and houses, exposing them to a variety of human pathogens 37. From a management perspective, the finding that baboons and humans may both serve as hosts to Trichuris sp. and hence could increase infection in one another, provides conservation authorities and town planners alike with a strong argument to restrict the spatial overlap between the human and baboon populations of the Cape Peninsula.

Supporting Information
Figure S1 MUSCLE alignment of ITS1-5.8S-ITS2 region of ribosomal DNA for Trichuris spp. isolated from a range of hosts. Sequence differences between T. trichiuris H. sapiens (Cameroon) and T. trichuris sp P. ursinus CP-GOB, are highlighted by black rectangles. Single nucleotide polymorphism differences (SNP) between T. trichuris sp P. ursinus DGI-DGIII and T. trichiuris H. sapiens (China) are indicated by red rectangles. Loci with Variable number of tandem repeats (VNTR) are indicated. The 5.8S region is indicated by a blue rectangle.
(PDF)

Click here for additional data file.

 We would like to thank Jan Bradley and Ivonne Siebeke at the University of Nottingham for making archived T. trichiura specimens available for this study, and for performing the DNA analysis for us. We thank the South Africa National Parks for the permission to conduct work in the Table Mountain National Park. We are also grateful to Denise Brito and MCB3012Z 3rd year students at the University of Cape Town for their assistance during the laboratory phase of this work, Dr. Barbara Wicht for her assistance during the data analysis, and to Dr Darren Martin for bioinformatics advice. Peter Enyong based at the Medical Research Centre Kumba, is to be thanked for assistance in recruitment of patients for this study.
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References
1 
Hotez PJ , Brindley PJ , Bethony JM , King CH , Pearce EJ , et al (2008 ) Helminth infections: the great neglected tropical diseases . J Clin Invest 
118 : 1311 –1321 .18382743 
2 
Cleaveland S , Laurenson MK , Taylor LH  (2001 ) Diseases of humans and their domestic mammals: pathogen characteristics, host range and the risk of emergence . Philos T Roy Soc B 
356 : 991 –999 .
3 
Taylor LH , Latham SM , Mark EJ  (2001 ) Risk factors for human disease emergence . Philos T Roy Soc B 
356 : 983 –989 .
4 
Wolfe ND , Escalante AA , Karesh WB , Kilbourn A , Spielman A , et al (1998 ) Wild primate populations in emerging infectious disease research: the missing link? 
Emerg Infect Dis 
4 : 149 –158 .9621185 
5 
Daszak P , Cunningham AA , Hyatt AD  (2000 ) Emerging infectious diseases of wildlife–threats to biodiversity and human health . Science 
287 : 443 –449 .10642539 
6 
Jones-Engel L , Engel GA  (2006 ) Disease risk analysis: a paradigm for using health-based data to inform primate conservation and public health . Am J Primatol 
68 : 851 –854 .16900505 
7 Trueba G, Dunthorn M (2012) Many neglected tropical diseases may have originated in the Paleolithic or before: new insights from genetics. PLoS Neglet Trop D 6: in press.
8 
Liu GH , Gasser RB , Su A , Nejsum P , Peng L , et al (2012 ) Clear genetic distinctiveness between human-and pig-derived Trichuris based on analyses of mitochondrial datasets . PLoS Neglet Trop D 
6 : e1539 .
9 
Bethony J , Brooker S , Albonico M , Geiger SM , Loukas A , et al (2006 ) Soil-transmitted helminth infections: ascariasis, trichuriasis, and hookworm . Lancet 
367 : 1521 –1532 .16679166 
10 
Stephenson LS , Holland CV , Cooper ES  (2000 ) The public health significance of Trichuris trichiura
 . Parasitology 
121 : 73 –95 .
11 
Adams VJ , Markus MB , Adams JFA , Jordaan E , Curtis B , et al (2005 ) Paradoxical helminthiasis and giardiasis in Cape Town, South Africa: epidemiology and control . Afr Health Sci 
5 : 276 –280 .16246001 
12 Ravasi DF, O'Riain MJ, Adams VJ, Appleton CC (2012) A coprological survey of the protozoan and nematode parasites of free-ranging chacma baboons (Papio ursinus) in the south-western Cape, South Africa. S Afr J Wildl Res: in press.
13 
Hoffman TS , O'Riain MJ  (2011 ) The spatial ecology of chacma baboons (Papio ursinus) in a human-modified environment . Int J Primatol 
32 : 308 –328 .
14 
Ooi HK , Tenora F , Itoh K , Kamiya M  (1993 ) Comparative study of Trichuris trichiura from non-human primates and from man, and their difference with T. suis
 . J Vet Med Sci 
55 : 363 –366 .8357906 
15 Cogswell F (2007) Parasites of non-human primates. In: Flynn RJ, Baker, D G., editor. Flynn's Parasites of Laboratory Animals. Ames, Iowa: Blackwell Pub. 693–743.
16 
Criscione CD , Poulin R , Blouin MS  (2005 ) Molecular ecology of parasites: elucidating ecological and microevolutionary processes . Mol Ecol 
14 : 2247 –2257 .15969711 
17 
Cutillas C , Callejón R , De Rojas M , Tewes B , Ubeda JM , et al (2009 ) 
Trichuris suis and Trichuris trichiura are different nematode species . Acta Trop 
111 : 299 –307 .19467214 
18 Nissen S, Al-Jubury A, Hansen TVA, Olsen A, Christensen H, et al.. (2012) Genetic analysis of Trichuris suis and Trichuris trichiura recovered from humans and pigs in a sympatric setting in Uganda. Vet Parasitol: in press.
19 
McManus DP , Bowles J  (1996 ) Molecular genetic approaches to parasite identification: their value in diagnostic parasitology and systematics . Int J Parasitol 
26 : 687 –579.896.0613 
20 
Cutillas C , Oliveros R , De Rojas M , Guevara DC  (2002 ) Determination of Trichuris muris from murid hosts and T. arvicolae (Nematoda) from arvicolid rodents by amplification and sequentiation of the ITS1–5.8 S-ITS2 segment of the ribosomal DNA . Parasitol Res 
88 : 574 –582 .12107482 
21 
Cutillas C , de Rojas M , Ariza C , Ubeda JM , Guevara D  (2007 ) Molecular identification of Trichuris vulpis and Trichuris suis isolated from different hosts . Parasitol Res 
100 : 383 –389 .17004099 
22 
Van Doorn AC , O'Riain MJ , Swedell L  (2010 ) The effects of extreme seasonality of climate and day length on the activity budget and diet of semi-commensal chacma baboons (Papio ursinus) in the Cape Peninsula of South Africa . Am J Primatol 
72 : 104 –112 .19851982 
23 
Kaplan BS , O'Riain MJ , van Eeden R , King AJ  (2011 ) A low-cost manipulation of food resources reduces spatial overlap between baboons (Papio ursinus) and humans in conflict . Int J Primatol 
32 : 1397 –1412 .
24 Hoffman TS, O'Riain MJ (2012) Landscape requirements of a primate population in a human-dominated environment. Front Zool 9: in press.
25 
Allen AVH , Ridley DS  (1970 ) Further observations on the formol-ether concentration technique for faecal parasites . J Clin Pathol 
23 : 545 –547 .5529256 
26 
Turner J , Faulkner H , Kamgno J , Else K , Boussinesq M , et al (2002 ) A comparison of cellular and humoral immune responses to trichuroid derived antigens in human trichuriasis . Parasite Immunol 
24 : 83 –93 .11874563 
27 Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual: CSHL press.
28 
Gasser R , Nansen P , Guldberg P  (1996 ) Fingerprinting sequence variation in ribosomal DNA of parasites by DGGE . Mol Cell Probe 
10 : 99 –105 .
29 
Edgar R  (2004 ) MUSCLE: a multiple sequence alignment method with reduced time and space complexity . BMC bioinformatics 
5 : 113 .15318951 
30 
Edgar RC  (2004 ) MUSCLE: multiple sequence alignment with high accuracy and high throughput . Nucleic Acids Res 
32 : 1792 –1797 .15034147 
31 
Tamura K , Peterson D , Peterson N , Stecher G , Nei M , et al (2011 ) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods . Mol Biol Evol 
28 : 2731 –2739 .21546353 
32 
Tajima F , Nei M  (1984 ) Estimation of evolutionary distance between nucleotide sequences . Mol Biol Evol 
1 : 269 –285 .6599968 
33 Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution: 783–791.
34 
Hausfater G , Sutherland R  (1984 ) Little things that tick off baboons . Nat Hist 
2 : 55 –60 .
35 
Rolland RM , Hausfater G , Marshall B , Levy S  (1985 ) Antibiotic-resistant bacteria in wild primates: Increase prevalence in baboons feeding on human refuse . Appl Environ Microb 
49 : 791 –794 .
36 
Hahn NE , Proulx D , Muruthi PM , Alberts S , Altmann J  (2003 ) Gastrointestinal parasites in free-ranging Kenyan baboons (Papio cynocephalus and P. anubis) . Int J Primatol 
24 : 271 –279 .
37 
Muriuki SMK , Murugu RK , Munene E , Karere GM , Chai DC  (1998 ) Some gastro-intestinal parasites of zoonotic (public health) importance commonly observed in old world non-human primates in Kenya . Acta Trop 
71 : 73 –82 .9776144 
38 
Saitou N , Nei M  (1987 ) The neighbor-joining method: a new method for reconstructing phylogenetic trees . Mol Biol Evol 
4 : 406 –579.896.0613


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