Tropical Biomedicine 35(4): 1131–1139 (2018)

DNA barcoding relates species from a human and a man’s best friend to non-human primate sources

Brandon-Mong, G.J.1,2*, Ketzis, J.K.3, Choy, J.S.1, Boonroumkaew, P.4, Tooba, M.5, Sawangjaroen, N.4, Yasiri, A.6, Janwan, P.7, Tan, T.C.1 and Nissapatorn, V.7,8* 1Department of Parasitology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia 2Biodiversity Research Center, Academia Sinica, No. 28, Lane 70, Section 2, Yanjiuyuan Road, Nangang District, Taipei City, 115, Taiwan 3Ross University School of Veterinary Medicine, PO Box 334, Basseterre, St. Kitts and Nevis, West Indies 4Department of Microbiology, Faculty of Science, Prince of Songkla University, Songkhla, Thailand 5Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia 6Chulabhorn International College of Medicine, Thammasat University, Pathum Thani, Thailand 7School of Allied Health Sciences and 8Research Excellence Center for Innovation and Health Products (RECIHP), Walailak University, Nakhon Si Thammarat, Thailand *Corresponding authors e-mail: [email protected], [email protected]; [email protected] Received 2 January 2018; received in revised form 30 July 2018; accepted 30 July 2018

Abstract. , the whipworm of humans, is one of the most prevalent soil- transmitted helminths (STH) reported worldwide. According to a recent study, out of 289 STH studies in Southeast Asia, only three studies used molecular methods. Hence, the genetic assemblages of Trichuris in Southeast Asia are poorly understood. In this study, we used partial mitochondrial DNA (cytochrome c oxidase subunit 1 or COI) sequences for analysis. Trichuris grouped in a same clade with different hosts indicate the potential of cross infection between hosts. Based on COI, the adult Trichuris isolated from a Malaysian patient was most closely related to Trichuris isolated from Papio anubis (olive baboons) from the USA. The Trichuris isolated from the dog from Malaysia was genetically similar to a Trichuris species isolated from Macaca silenus (lion-tailed macaque) from Czech Republic. Both the human and dog isolated Trichuris grouped in clades with different hosts indicating the potential of cross infection between hosts. Specific PCR primers based on the partial COI of T. trichiura isolated from African green monkey and T. serrata were designed and successfully amplified using multiplex PCR of the pooled DNA samples. Our results suggest a complex parasite-host relationship, and support the theory of cross infection of Trichuris between humans and non-human primates as suggested in previous publications.

INTRODUCTION Most Trichuris spp. eggs cannot be distinguished from each other since all are In Malaysia, Trichuris trichiura is one of similar size, typically ‘lemon’ shaped of the most prevalent soil-transmitted and have clear, mucoid-appearing polar helminths (STH) with occurrence docu- plugs. Therefore, the genetic assemblages mented to be over 98% in some at risk of T. trichiura in humans and the zoonotic populations (Ahmed et al., 2011). Among cross-infection between humans and non- the 289 STH studies conducted within human in Southeast Asia are poorly Southeast Asia, only 3 (1%) used molecular understood. DNA barcoding, the use of a methods (i.e., PCR) to identify the species short fragment of mitochondrial DNA of the worm, whereas the remainder relied (cytochrome c oxidase subunit 1, cox1 or on egg identification (Dunn et al., 2016). COI) sequences for species identification

1131 (Hebert et al., 2003), may enable a better 161 samples from dogs (70), cats (50), pigs understanding of T. trichiura strains (33), apes (3 from Hylobates lar (White- infecting humans as well as the occurrence handed Gibbon) and 1 from Symphalangus of zoonosis. To our knowledge, DNA syndactylus (Siamang), and raccoons (4 barcoding of endoparasites is generally from Procyon lotor) were screened for small in scope with most studies performed Trichuris. From Thailand, a total of 135 on wildlife parasites that rarely infect fecal samples were screened with 55 from humans (Ondrejicka et al., 2014). monkeys, 40 from pigs and 40 from goats. Since limited molecular data on Trichuris are available in Asia, especially DNA extraction and PCR in Southeast Asia, this study was performed Genomic DNA of 5 adult Trichuris worms with the primary aims of: 1) DNA barcoding was extracted using a NucleoSpin Tissue Trichuris (worms and eggs) from human kit (Macherey-Nagel, Düren, Germany), and non-human animals in Peninsular following the manufacturer’s instructions. Malaysia; and 2) investigating the potential Genomic DNA of stool samples containing cross infection of Trichuris between Trichuris eggs was extracted using human and non-human animals, as well as FavorPrepTM Stool DNA Isolation Mini in between non-human animals. Kit (Favorgen Biotech Corp., Pingtung County, Taiwan), following the manufac- turer’s instructions. A universal primer MATERIALS set that targets ~400 bp of COI gene region of nema-todes was retrieved Sample collection from the literature (Guardone et al., 2013): A total of 5 specimens of adult Trichuris COIFmod (forward primer; TGRTTTTTT worms were collected for analysis. A GGICAYCCIGARG) and COIRmod (reverse Trichuris adult worm was isolated from a primer; CACTACATAGTADGTRTCRTG). middle-aged female patient suffering from For the AGM and cat adult Trichuris worms gastrointestinal symptoms and underwent and the Trichuris eggs isolated from a colonoscopy in January 2016. Two Trichuris dog, amplicons were produced by PCR in a adult worms (1 male and 1 female) were total volume of 25 µL with 2.5 µL of each isolated from a cat (Felis catus) in St. Kitts forward and reverse primer (10 µM), 12.5 using the same method previously reported µL of 2x PCR mastermix, 2.5 µL of ddH2O by Ketzis et al. (2015). Two Trichuris adult and 5 µL of DNA template. Thermocycling worms (1 male and 1 female) were isolated program: 95°C for 15 minutes; 40 cycles of from Chlorocebus sabaeus (African Green 95°C for 30 seconds; 54°C for 1 minute; 72°C Monkey; AGM) on St. Kitts, isolated using for 1 minute and a final extension of 72°C the same method previously reported by for 10 minutes. For the adult Trichuris Hawash et al. (2015) and provided under worm isolated from a human, amplicons a Materials Transfer Agreement with the were produced by PCR in a total volume of Behavioral Science Foundation, Caribbean 20 µL with 2.0 µL of each forward and reverse Primate Laboratory. All adult worms primer (10 µM), 10.0 µL of 2X ExPrime Taq were preserved in absolute ethanol and Premix (GENET BIO, Daejeon, Korea), 5.0 transported to the Department of Para- µL of ddH2O and 1 µL of DNA template. sitology, Faculty of Medicine, University of Thermocycling program: 95°C for 15 Malaya. minutes; 40 cycles of 94°C for 30 seconds; A total of 296 fecal samples were 48°C for 1 minute; 68°C for 1 minute and a collected from non-human animals in final extension of 68°C for 10 minutes. The Malaysia and Thailand and screened for amplicons were visualized on a 2% agarose Trichuris eggs using formalin-ether gel stained with ethidium bromide and sent sedimentation according to a previous to commercial sequencing company for study (Allen & Ridley, 1970). From Malaysia, Sanger sequencing.

1132 Sequence editing and analysis based on the sequences generated in this Sequences were edited (primers were study (see Table 1 for primer sequences). trimmed and ambiguous nucleotide was After testing all newly designed specific replaced with N using BioEdit) (Hall, 1999). primer sets on the DNA samples of Trichuris These sequences are available on BOLD worms isolated from the AGM and cat, database (Ratnasingham & Hebert, 2007) multiplex PCR was performed with total in the public project TBAV. Eight COI volume of 20 µL reaction; each PCR sequences from Callejón et al. (2015) and contained 0.5 µL of each forward and 9 COI sequences from Dolezalova et al. reverse primer, 7.5 µL of ddH2O, 14 µL of (2015) were included as reference se- EconoTaq® Plus Green 2x Master Mix quences. The COI gene region of a full (Lucigen Corp, Parmenter St. Middleton, mitochondria genome of T. discolor (Liu USA) and 2.5 µL of pooled DNA template et al., 2012c), T. muris (unpublished data (DNA samples of Trichuris worms isolated by Holroyd et al., 2015), T. ovis (Liu et al., from an AGM and a cat in St. Kitts). 2012c), T. suis (Liu et al., 2012b), and T. Thermocycling program: 94°C for 15 trichiura (Liu et al., 2012a; Liu et al., 2012b; minutes; 40 cycles of 94°C for 30 seconds, Hawash et al., 2015) were trimmed and also 50°C for 1 minute, 72°C for 1 minute; and included as reference sequences. All COI final extension of 72°C for 10 minutes. The sequences were aligned using ClustalW in amplicons were visualized on a 2% agarose Bioedit (Hall, 1999) with a final length of gel stained with ethidium bromide and sent 343 bp. All COI sequences were translated to a commercial sequencing company for into amino-acid and no stop codon was Sanger sequencing. Sequences produced by observed. Maximum Likelihood tree was the multiplex PCR were matched with the built and pairwise distance of the sequences sequences previously produced using the was calculated in MEGA 6 (Tamura et al., PCR with COIFmod/COIRmod. 2013). Trichuris grouped in a same clade with different hosts indicate the potential of cross infection between hosts. RESULTS

Primer design and multiplex PCR Adult worms Each specific primer set targeting COI Based on COI, the Trichuris of a female region of the Trichuris worm isolated from patient from Peninsular Malaysia (T_T05) AGM (amplicon length: 370bp), and the formed a clade with the T. trichiura isolated Trichuris worms isolated from a cat from humans in China (GU385218 and (amplicon length: 348bp) was designed NC017750), and Papio anubis in USA

Table 1. fecal samples analyzed for Trichuris spp. using formalin-ether sedimentation

Number of fecal Number of Trichuris Number PCR Animals samples analyzed positive samples amplified

Peninsular Malaysia Dog 70 3 1 Cat 50 0 0 Pig 33 0 0 Ape 4 0 0 Raccoon 4 0 0

Thailand Monkey 55 7 0 Pig 40 8 0 Goat 40 8 0

Total 296 26 1

1133 (KT449825) (Figure 1). T_T05 was most shape and internal structure) (Table 1). Due closely related to KT449825 and the to low yields of Trichuris eggs in 25 of the pairwise distance between them was 4.8% samples, only one dog fecal sample from a (Figure 2). The two T. trichiura from AGM Malaysian animal shelter was successfully (T_T03 and T_T04) were in a separate clade, PCR amplified (i.e., T_S06). The Trichuris but one that also included human sourced (T_S06) was genetically similar to a T. trichiura from Uganda (KT449826) and Trichuris species isolated from a non- non-human primate sourced T. trichiura human primate (Macaca silenus; JF690966) from the Czech Republic (JF690963) and from Czech Republic (Dolezalova et al., Denmark (KT449824) (Figure 1). The 2015) (Figure 1). The pairwise distance pairwise distance between T_T03 and between T_S06 and JF690966 was 0.6% T_T04 was 0.0%. The pairwise distance (Figure 2). between T_T04 and KT449826 was 0.3% (Figure 2). The two adult Trichuris serrata Primers for multiplex PCR (i.e., T_S01 and T_S02) did not group with Specific PCR primers based on the partial any other Trichuris spp. The pairwise COI of T. trichiura isolated from AGM and distance between T_S01 and T_S02 was T. serrata were designed and successfully 0.0% (Figure 2). amplified using multiplex PCR of the pooled DNA samples (see Table 2 for primer Trichuris eggs sequences). The sequences amplified by the Out of 296 animal fecal samples, 26 samples multiplex PCR were 100% matched with the were positive for Trichuris eggs based sequences previously amplified using the on morphological identification (e.g., size, PCR with COIFmod/COIRmod.

Figure 1. Revised as attached.

1134 Figure 2. Revised as attached on COI sequences.

1135 Table 2. Primers used for DNA barcoding of In the case of the dog, previous studies on Trichuris Trichuris in dogs in Peninsular Malaysia have relied on morphological identification Primer Sequence (5’-3’) of eggs in feces with the infections declared TSCOI_F GCATTAGAAAGTGATACGCC as Trichuris vulpis (Ngui et al., 2014; Zain TSCOI_R TCTTGTATTACCCGCATTCG et al., 2015; Jia-Chi et al., 2016), except for TTCOI_F AGTATAAGGTCTAGCGAGGC Tun et al. (2015) who only declared the TTCOI_R TACATTTTAGTGCTCCCAGC genus in the dog. In this study, the Trichuris isolated from a dog was not Trichuris vulpis, but genetically similar to a Trichuris DISCUSSION species isolated from Macaca silenus (lion- tailed macaque; an endangered species) The results of this study, with different which, like Papio anubis, does not occur in clades containing both human and non- Malaysia (Roos et al., 2014). human primate Trichuris, support the Considering that neither Macaca hypothesis of multiple Trichuris species silenus nor Papio anubis are wild primates or strains in humans and non-human in Malaysia (Azlan, 2006) and Southeast primates (Ravasi et al., 2012; Ghai et al., Asia (Roos et al., 2014), there are a few 2014; Dolezalova et al., 2015; Hawash et al., possibilities as to how these Trichuris 2015). Both Trichuris species or strains species were transmitted to the human and seen in the human and dog are first reports the dog in this study. Macaques and baboons for Malaysia and suggest the potential of infected with Trichuris may be legally or more cryptic Trichuris species to be illegally imported into Peninsular Malaysia identified in Malaysia. The genetic distance with the Trichuris eventually transmitted between T_T05 and the T. trichiura isolated to the human and the dog in this study. If from Papio anubis in USA (KT449825) was this happened, these Trichuris species, 4.8%, which is more than 2.0%. According which are not native to Peninsular Malaysia, to Ratnasingham & Hebert (2013), the could be considered as invasive alien genetic difference within a described species (IAS). International Union for species rarely exceed 2.0%. Hence, T_T05 Conservation of Nature (IUCN) defines might be a cryptic species. However, genetic IAS as non-native species that settled in a markers like mitochondrial COI gene and habitat and subsequently threatens native nuclear ITS2 (internal transcribed spacer biodiversity as an agent of change (Westphal 2) gene, sometimes are unable to identify et al., 2008). If the dog has become a true evolutionary young sister species, poten- host of this non-human primate Trichuris, tially because the genetic markers are this switching of hosts might be due to polymorphic or poorly differentiated (Avise, climate change (Brooks and Hoberg, 2007), 2000; Lukhtanov et al., 2015a, b, 2016). To which can cause shifts in host or parasite confirm whether T_T05 is a cryptic species, ranges, eventually resulting in disease more studies should be conducted, for emergence (Harvell et al., 2002). Another example whole genome sequencing, mor- possibility is these Trichuris species or phological and ecological studies. strains are native to Malaysia and to these Based on the results, T. trichiura hosts but not previously identified, until now, (TT_05) may pose a risk of cross-infection due to the lack of genetic analysis of between humans and non-human primates. Trichuris in the region. Further studies Papio anubis (olive baboon) is not in the are needed to determine which hypotheses checklist of wild primates in Malaysia is correct. (Azlan, 2006) nor even listed as a species in The PCR primers designed based on Southeast Asia (Roos et al., 2014). However, T. serrata and T. trichiura (from AGM) this primate does exist in Malaysian zoos could be used individually or together although no Trichuris were found in six (multiplex PCR) with more multiplex PCR olive baboons examined (Lim et al., 2008). primers to detect a wider range of Trichuris

1136 spp. in the environment (e.g., water and soil Acknowledgements. The authors thank samples) and to better understand host Dr. Romano Ngui for providing Trichuris specificity. This also could be useful for (TT_05), Mr Mohamad Azlan Abdul Majid clinical diagnosis of Trichuris infection in and Ms Alicia Choo Jia Chi for laboratory humans. work. This study was initiated when G.J.B.M. and N.S were at the Department of Para- sitology, Faculty of Medicine, University CONCLUSION of Malaya and was supported by the University of Malaya Research Grant The results of this study, while limited (UMRG 544/14HTM) and University of since based on only one dog and one human Malaya High Impact Research Grant (H- sample, suggest a complex parasite-host 20001-00-E000062). The funders had no relationship. While T. vulpis has limited role in study design, data collection and zoonotic potential, the ability of dogs to analysis, as well as preparation to publish serve as hosts of non-human primate the manuscript. Trichuris and the ability of non-human primate Trichuris to infect humans, could change the role of dogs as a zoonotic REFERENCES source of infection for humans. Also, T. trichiura from Peninsular Malaysia may be Ahmed, A., Al-Mekhlafi, H.M. & Surin, J. able to infect both humans and non-human (2011). Epidemiology of soil-transmitted primates. More sampling of Trichuris from helminthiases in Malaysia. Southeast dogs, humans, and non-human primates Asian Journal of Tropical Medicine (captive or wild) in Malaysia and its and Public Health 42: 527-538. neighboring countries should be conducted Allen, A.V. & Ridley, D.S. (1970). Further to strengthen the hypothesis of the zoonotic observations on the formol-ether cross-infection of T. trichiura between concentration technique for faecal humans and non-human primates, as well parasites. Journal of Clinical Patho- as the cross-infection of a Trichuris sp. logy 23: 545-546. between dogs and non-human primates. Azlan, J.M. (2006). Mammal diversity and Understanding these transmission patterns conservation in a secondary forest in will ultimately be of benefit in developing Peninsular Malaysia. Biodiversity and and accessing interventions to decrease Conservation 15: 1013-1025. human, non-human primate, and dog Brooks, D.R. & Hoberg, E.P. (2007). How will infections. global climate change affect parasite- host assemblages? Trends in Para- Competing interests sitology 23: 571-574. The authors have no competing interests to Callejón, R., Cutillas, C. & Nadler, S.A. declare. (2015). Nuclear and mitochondrial genes for inferring Trichuris phylogeny. Author contributions Parasitology Research 114: 4591-4599. G.J.B.M. and V.N. designed the experiment. Dunn, J.C., Turner, H.C., Tun, A. & Anderson, C.J.S., P.B. and T.M. carried out sample R.M. (2016). Epidemiological surveys collection, G.J.B.M., J.K.K., P.J., T.T.C. and of, and research on, soil-transmitted N.S. performed laboratory (microscopic and helminths in Southeast Asia: a syste- molecular) work. G.J.B.M. and A.Y. designed matic review. Parasites & Vectors 9: 31. the primers. G.J.B.M. carried out sequence editing and analysis. All authors contributed to writing the manuscript and agreed in submitting for publication.

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