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Memórias do Instituto Oswaldo Cruz

Print version ISSN 0074-0276

Mem. Inst. Oswaldo Cruz vol.105 no.2 Rio de Janeiro Mar. 2010

http://dx.doi.org/10.1590/S0074-02762010000200020 

SHORT COMMUNICATIONS

 

Paleoparasitological report on Ascaris aDNA from an ancient East Asian sample

 

 

Chang Seok OhI, II; Min SeoIII; Nam Jin LimIV; Sang Jun LeeI, II; Eun-Joo LeeV; Soong Deok LeeI; Dong Hoon ShinI, II, +

IInstitute of Forensic Medicine
IIDepartment of Anatomy, Seoul National University College of Medicine, 28 Yongon-dong, Chongno-Gu, Seoul 110-799, Korea
IIIDepartment of Parasitology, Dankook University College of Medicine, Chonan, Korea
IV
Gyeongi Institute of Cultural Properties, Suwon-si, Gyeonggi-do, Korea
VDepartment of Multiplex Contents, Andong National University, Andong, Korea

 

 


ABSTRACT

In this study, Ascaris DNA was extracted and sequenced from a medieval archaeological sample in Korea. While Ascaris eggs were confirmed to be of human origin by archaeological evidence, it was not possible to pinpoint the exact species due to close genetic relationships among them. Despite this shortcoming, this is the first Ascaris ancient DNA (aDNA) report from a medieval Asian country and thus will expand the scope of Ascaris aDNA research.

Key words: Ascaris - ancient DNA - cytochrome b - 18S rRNA


 

 

Successful recovery of ancient DNA (aDNA) from various parasite species has been reported by a number of investigators (Guhl et al. 1999, Sallares & Gomzi 2001, Iñiguez et al. 2003, 2006, Aufderheide et al. 2004, Liu et al. 2007). In the case of ancient Ascaris spp, one of the most widespread parasitic infections in humans (Loreille & Bouchet 2003), PCR-based analyses have been performed and reported by Loreille et al. (2001) and Leles et al. (2008). However, even though Ascaris spp have been identified by molecular studies of archaeological specimens, their genetic diagnosis has not been studied in greater detail so far (Anderson 2001, Peng et al. 2005, Leles et al. 2010). Moreover, since most aDNA studies on Ascaris have been limited in their geographical distribution to Europe and South America, it is desirable to secure Ascaris aDNA data from a much wider geographic and temporal range. In this regard, our report on the successful extraction and sequencing of Ascaris aDNA from Korean archaeological specimens, the first such report from an East Asian country, is a valuable addition to the existing pool of Ascaris aDNA.

A medieval tomb constructed during the Joseon Dynasty (1392-1910) was uncovered in Seocheon, Korea on August 19, 2008. Similar to other medieval tombs in Korea, infiltration of contaminants from the outside was prevented by intact encapsulation of the lime-soil mixture barrier. The human remains were wrapped in very well preserved clothing. During removal of that clothing in our lab, textile specialists wore sterilised gowns, masks, gloves and head caps under contamination-minimised condition (Fig. 1A-C). Parasitological samples spread upon the surface of hip bones were immediately collected during the removal of the textiles (Fig. 1D, E). This stringent condition helped prevent contamination by other sources such as animals, confirming that the parasite eggs found were truly of human origin.

 


 

The samples were rehydrated for microscopic examination according to previously reported methods (Callen & Cameron 1960). Briefly, the samples were treated with 0.5% aqueous trisodium phosphate solution then filtered with multiple layers of gauze. After spontaneous sedimentation, the upper turbid layer was discarded and precipitates were dissolved in 20 mL rehydration solution. A volume of 20 μL from each sample was observed by light microscopy (Olympus, Tokyo, Japan) and observations were repeated 10 times. The sizes of parasite eggs were measured and the number of parasite eggs per gram was estimated. We also performed microscopic and molecular studies on surface soil within a 1 m radius of the tomb, which were used as negative controls.

We (Labs A and B) extracted Ascaris aDNA from samples where Ascaris eggs were identified, using the method reported by Iñiguez et al. (2003). Primers for Ascaris cytochrome b (cyt b) and Ascaris 18S small subunit ribosomal RNA (18S rRNA) gene were made according to the method reported by Loreille et al. (2001). The laboratories used the same primer sets. aDNA was amplified by PCR in a 20-μL reaction mixture containing 1X High Fidelity PCR buffer, 2 mM MgSO4, 200 μM dNTPs, two units of Platinum® Taq DNA Polymerase High Fidelity (Invitrogen, USA), 10 pmol of each primer and 1 mg/mL BSA (New England Biolabs, USA). PCR was done as follows: pre-denaturation at 94°C for 10 min; 50 cycles of denaturation at 94°C for 45 sec, annealing at 50°C for 45 sec and extension at 72°C for 45 sec; and final extension at 72°C for 10 min. Electrophoresis was done using a 2.5% agarose gel to identify amplified products.

Cloning of PCR products was performed using the pGEM-T Easy Vector system (Promega, USA) according to manufacturer's instructions. Sequencing of each clone was carried out with an ABI Prism 3100 automatic sequencer (Applied Biosystems, USA) and the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, USA). We repeated cloning and sequencing 2-5 times for each amplified product. Sequences were analysed using the BioEdit sequence alignment editor version 7.0.9.0 (http://mbio.ncsu.edu/BioEdit/bioedit.html). In Lab B, PCR products were directly sequenced and compared with the consensus sequence obtained in Lab A. Sequences obtained from the laboratories were also compared with public databases using BLAST searches (http://www.ncbi.nlm.nih.gov/BLAST). aDNA analysis was done according to criteria of authentication (Hofreiter et al. 2001). We did not perform any DNA work with modern Ascaris spp in our lab before the current study. We also set up our aDNA lab facilities in accordance with the suggestions of Hofreiter et al. (2001) (Supplementary data).

We observed fertile or infertile Ascaris and Trichuris trichiura eggs in the Seocheon sample under the light microscope (Fig. 1F-I). The average sizes of Ascaris eggs were 65 ± 1.2 μm (length) and 51 ± 3.4 μm (width). The estimated number of eggs was 6714.3 per gram. In PCR-based aDNA analyses, fragments of Ascaris 18S rRNA and cyt b mitochondrial genes were successfully amplified while negative controls (surface soil and extraction controls) did not show any amplified bands (Fig. 2).

In Lab A, cloning and sequencing was performed on the amplified fragments of 18S rRNA (176 bp) and cyt B (98 bp) genes. Among 113 clones of 18S rRNA gene (Asc6 and Asc7, Asc8 and Asc9, Asc10 and Asc11), 63 sequences were successfully obtained. For the cyt b gene (Asc1 and Asc2), 24 sequences were obtained from 25 clones. We found that eight clone sequences (SC89-B1, SC89-B2, SC89-C6, SC89-C7, SC1011-A8, SC1011-A10, SC1011-B10 and SC12-A13) had single nucleotide substitutions and three nucleotide substitutions were found in clone SC12-B3. When the consensus sequences of 18S rRNA and cyt b genes obtained in Lab A were compared with direct sequencing results in Lab B, the results were identical (Fig. 3). Next, the sequences were compared to those in GenBank. Our 18S rRNA gene sequence was 100% identical to that of Namur (Loreille et al. 2001), Ascaris suum (U94367), Baylisascaris transfuga (U94369) and Baylisascaris procyonis (U94368). They also showed similarities to 18S rRNA genes of Anisakis sp. (98%, U94365), Toxocara canis (97%, U94382) and Ascaris lumbricoides (97%, U94366). For the Ascaris cyt b sequence, our result showed 100% identity to Namur (Loreille et al. 2001); 97% to Ascaris lumbricoides gene (EF150653, Brazil; EF439710, Lubeck, Germany; EF439714 to EF439718, Walraversijde, Belgium; EF439722, Minas Gerais, Brazil); 96% to A. suum (X54253) and A. lumbricoides (EF439709, Lubeck, Germany; EF439712, Walraversijde, Belgium; EF439719, Walraversijde, Belgium; EF439720 and EF439721, Minas Gerais, Brazil; EF439723, Piauí, Brazil; EF439724, San Pedro de Atacama, Chile). However, some A. lumbricoides sequences (EF439711, Lubeck, Germany; EF439713, Walraversijde, Belgium) showed only 95% identity to our cyt b sequence. Considering that most previously reported Ascaris sequences are from European or South American countries, the results from this study improve the range of information collected on Ascaris aDNA by adding data from East Asia, thereby expanding the geographical scope.

However, even though our Ascaris eggs are likely of human origin, the Ascaris 18S rRNA gene sequence was more similar to A. suum than to A. lumbricoides. In addition, some previously reported mitochondrial cyt b gene sequences of A. lumbricoides in GenBank showed less similarity to our Ascaris sequence than did A. suum. Therefore, the aDNA analysis reported here falls short of confirming the Ascaris aDNA as A. lumbricoides. Many currently available nuclear or mitochondrial targets are not satisfactory for differentiating A. lumbricoides from A. suum (Anderson 2001, Peng et al. 2005, Leles et al. 2010), which stems from the close relationship between the two species (Barry & O' Rourke 1967, Crompton 1989, Zhu et al. 1999, Leles et al. 2010). This is again evident in our study because the differentiation between these two species was not achieved after genetic analysis.

 

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Received 10 August 2009
Accepted 9 February 2010

Financial support: National Research Institute of Cultural Heritage, Korea (08D011Y-00110-2008)

 

 

+ Corresponding author: drdoogi@snu.ac.kr

 

 

Supplementary data

 


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