The whole mitochondrial genomes of T. asiatica and T. saginata were amplified and cloned [10]. Both of them were approximately 14 kb in size. The restriction enzyme map of T. saginata mtDNA was constructed from the restriction fragments: 8, 4, and 2 kb, BglII; 4.5, 3.8, 1.5, and 1 kb, HincII; 5, 4.5, 2, and 1.5 kb, HindIII; 9 and 4 kb, PvuII; 12 and 1.8 kb, XhoI. Enzyme sites were not available for BamHI, EcoRI, EcoRV, KpnI or PstI in T. saginata mtDNA. Between T. asiatica and T. saginata, the migration distances of the mtDNA fragments were observed from 46 fragments, including 12 fragments shared. The sequence divergence between T. asiatica and T. saginata was estimated as much as 4.8%. The full length of the mitochondrial cox1 and cob sequences were 1,620 and 1,068 bp in T. asiatica and T. saginata, respectively. The sequence difference between the 2 species was calculated as 4.6% in the cox1 and 4.1% in the cob genes. Two variant nucleotide positions (0.1% of total length) were detected in the cox1 gene among the 5 T. asiatica isolates from China, the Philippines and Korea, whereas 13 variant nucleotide positions (0.2 to 0.8% of total length) were detected in the 10 T. saginata isolates from China, Ethiopia, France, Indonesia, Japan, Korea, Laos, the Philippines, Taiwan, Thailand and Swiss [11].

The complete T. asiatica mitochondrial genome was 13,703 bp long and composed of 36 genes: 12 protein-encoding (3 subunit of cytochrome c oxidase, cox1, cox2 and cox3; 1 subunit of cytochrome b, cob; 7 subunit of NADH dehydrogenase, nad1, nad2, nad3, nad4, nad4L, nad5 and nad6; and 1 subunit of ATP synthase, atp6), 2 small and large subunit ribosomal RNA, 22 transfer RNA genes and a short non-coding region [8]. The tRNA genes were 61-69 bp long, and the secondary structures of 18 tRNAs had typical clover-leaf shapes with paired DHU arms. However, trnC, trnS1, trnS2 and trnR had unpaired DHU arms that were 7-12 bp in length. The tRNAs that transferred serine lacked a DHU arm. The non-coding region was composed of a short non-coding region of 72 nucleotides with a long non-coding region of 176 nucleotides separated by a trnL1/, trnS2/, trnL2/, trnR/, nad5 gene cluster. The sequence of the cox1 gene between T. asiatica and T. saginata differed by 4.6%, while the T. asiatica cytb gene differ by 4.1% and 12.9% from the cytb genes of T. saginata and T. solium, respectively [8].

The protein-coding sequences of T. saginata and T. asiatica contain 10,104 bps and 3,368 codons, while 10,048 bps and 3,349 codons in T. solium [9]. Twelve protein-coding genes of T. saginata and T. asiatica differed by 4.6%, while the overall difference between T. saginata and T. asiatica in the entire mtDNA sequence was 4.6%. Divergences in the mt genomes among the Taenia tapeworms ranged from 3.0% to 27.9%. Average pairwise similarity was about 95% in the functional regions of T. saginata and T. asiatica; and the most variable gene was nad5. Highly conservative regions were found in the subunits of cytochrome c oxidase, cytochrome b, 16S rRNA and the tRNAs. Predicted amino acid sequences of nad5 and cox1 genes exhibited 8.1% and 2.2% differences between T. saginata and T. asiatica, respectively. Two classes of functional regions can be identified in the mitochondrial genome of the 3 Taenia tapeworms: a slow-evolving region of non-synonymous substitution sites that includes tRNAs, rRNAs, and D-loop domains; and a fast-evolving region of synonymous substitution sites that includes atp6 and nad6. The overall sequence difference between T. asiatica and T. saginata was 4.6%, while that between T. saginata and T. solium was 11%. The degree of divergence in mtDNA sequence was estimated using the genetic distance of the cob gene between sister species, congeneric species and confamilial genera [12].

Species-specific forward primers were designed based on the nucleotide sequences of valine transfer RNA and NADH dehydrogenase subunit 2 from Taenia species [11]. They were designed to amplify different sized products: (1) Ta4978F, specific for T. asiatica (5'-GGG TTT AAG TTA TAA ATG TGA TGT-3'; nucleotides 4978 to 5001 from GenBank accession number AF445798); (2) Ts5058F, specific for T. saginata (5'-ACT ACA TTT GGT TTG TTT TTG TAG-3'; nucleotides 5058 to 5081 from AY684274); and (3) Tso7421F, specific for T. solium (5'-CTA GGC CAC TTA GTA GTT TAG TTA-3'; nucleotides 7421 to 7444 from AB086256). The reverse primer was from highly conserved region common to all of these tapeworms: Rev7915 (5'-CAT AAA ACA CTC AAA CCT TAT AGA-3'; nucleotides 5659 to 5685 from AF445798, nucleotides 5657 to 5683 from AY684274, and nucleotides 7870 to 7895 from AB086256) [11].

Prevalence of human taeniasis was reported since 1915 in Korea. A great number of surveys were executed by many researchers thereafter but most of the surveys did not cover the whole country. Since 1971 nationwide surveys were conducted every 5 years revealing taeniid egg positives of between 0.02 and 1.9% [13]. During the period, T. saginata had been considered a dominant species over T. solium. The epidemiological profile of these Taenia species in humans remained unclear up to recent until Jeon et al. [14] reported distribution pattern of Taenia tapeworms in Korea. Morphological examination as well as partial nucleotide sequences of mitochondrial cox1 and ITS2 (internal transcribed spacer 2) were analyzed for 68 specimens from university or institute museum collections deposited since 1935 [14]. The specimens were identified as 3 T. solium, 51 T. asiatica, and 14 T. saginata (Table 1) [11,14]. Each province in Korea exhibited 1, 2, or 3 kinds of tapeworms. The distribution ratio of T. asiatica: T. saginata calculated from both morphological and molecular data was approximately 3.5 : 1. Interestingly this ratio is not much different from the estimation by Eom and Rim who predicted 4 : 1 according to the eating habit of raw foods [13]. Twenty-nine of the 68 examined specimens were preserved in 10% formalin. Most of the formalin-preserved samples yielded weak or no PCR amplification, but secondary PCR using the PCR products obtained from the first round of PCR amplifications as a template produced made it possible to use PCR product for direct sequencing. The results clearly indicate that all 3 human Taneia tapeworms are distributing together in Korea [14]. T. asiatica is dominating in Korea and the local Korean peoples get this tapeworm by eating undercooked livers and visceral organs of pigs.

During the period of 1998 and 2002, a total of 19,894 inhabitants belonging to 3 ethnic minorities in Guangxi Province were surveyed for Taenia tapeworms. Total 927 (4.7%) persons discharged proglottids of tapeworms. In 2002, 108 patients were treated and 117 adult tapeworms were obtained from them. Most of worms (n = 108) were found to be T. saginata, and 9 to be T. solium. Nine cases were mixed infection with both worms. Six adult tapeworms collected from 6 persons of the Zhuang minority residing in the southern part of China (Luzhai) were comparatively analyzed and were turned out to be T. asiatica (Table 1) [11,15]. Experimental infections with eggs from the isolate into the pigs produced cysticerci, each with hookletless scolex and with wart-like formations on the external surface of the bladder wall. There were rostellar protrusions on the scolices of the adult worms. Random amplified polymorphic DNA analysis using 3 arbitrary primers produced bands identical to those of the Korean T. asiatica [15]. This minority people likes to eat raw pork and raw pig liver mixed with sour sauce and salted garlic. The Luzhai people have eating habit of raw pig liver in 8.5% (77/902). Sometimes they eat the fresh raw pig liver without any seasoning right after slaughtering the pig. They live very closely with domestic animals sharing the same house. On the first floor, domestic animals and latrines usually share the same room; the second floor the host living room locates. The domestic animals always clean human feces by eating. This is very different from the customs of the Han people, the majority group, who cook meat thoroughly in boiling oil (Korea Association of Health Promotion and Korea International Cooperation Agency, 2004; Final report on the Korea-China collaborative project of control strategies for helminthiasis in pilot areas, 2000-2004).

Reports of other researchers give more information on the distribution of T. asiatica by finding out 2 cases from Lanping County, Yunnan Province which was confirmed by morphological observation and experimental infection in intermediate host animals [16]; Dali of Yunnan Province and Duyun of Guizhou Province by mitochondrial CoxI analysis of the worms [17]; and 3 cases from Tibet of Sichuan by DNA genotyping [18].

In December of 1998, Dr. Yosuke Yamane provided 3 Taenia tapeworms to the author's laboratory for a Christmas gift. The worms were kept in 10% formalin and each was attached with questionnaire sheet for detailed information asking about food and identification data. Two specimens were labeled as Taeniarhynchus saginatus, one was from inhabitant in Izumo City (male, 59-year-old) and the other from Yonago City (male, 41-year-old), and both of them were identified as T. asiatica by DNA genotyping (Table 1). PCR amplification and direct sequencing for the cox1 target fragment (349 bp in length corresponding to the positions 80-428 bp of the cox1 gene) were performed using the total genomic DNA extracted from formalin-preserved samples. PCR amplification was successful in these cases and generated high quality PCR products applicable to direct sequencing. This is the first report of this tapeworm from Japan (Details of this will be published elsewhere).

A case of T. asiatica was found from out of the 2 specimens examined by nucleotide sequencing of Cox1 and multiplex PCR (Table 1) [11]. The other was T. saginata. Leon commented that Taenia segments, which were identified morphologically as T. saginata, were examined for mitochondrial DNA through the courtesy of Drs. A. Ito and H. Yamasaki and finally resulted in 5 T. asiatica out of the 6 Filipino specimens [19]. Fan et al. [20] had a result of successful experimental infection in Small-Ear Miniature pigs with Taenia eggs from the Philippines. Hinz [21] already had stated that "In the Philippines the infections with T. saginata is clearly dominated in man but the extremely rare T. saginata cysticercosis in cattle and carabao constitutes a still unresolved epidemiological paradox for the Philippines. In general neither beef nor the carabao meat is often eaten by the population of the endemic area. In endemic area 92.6% of those asked, however, indicated that they ate raw pork (local food prepared as "kinilaw" or "sinugba"). The cycle of T. saginata in the endemic area did not follow the known cycle of man-Bovidaeman. In consideration of our results, we believe we are dealing with a T. saginata-like tapeworm [21]."

T. asiatica used to be called "Taiwan Taenia". Two of our collection demonstrated this tapeworm also with analysis of Cox1 sequencing (Table 1) [11]. Taiwanese Dr. P.C. Fan made Orchid Island (Lanyu Island) very well known among cestodologists and parasitologists while he was pioneering the research works on Taiwan Taenia, as one of the endemic areas of the tapeworm as well as the mainland Taiwan. This small island is a land of the Yami tribe who moved from South-East Asia via the Philippines 2 hundred years ago. The natural intermediate host of T. asiatica was also confirmed in Lanyu native pigs for the first time. Yeh et al. [22] described that "Food-borne parasitic zoonosis such as infections with Angiostrongylus cantonensis, Clonorchis sinensis, and T. saginata asiatica (Taenia asiatica) are not rare, but the former is seasonal and the latter 2 are ethnically and geographically associated". T. saginata and T. solium are also prevalent in Taiwan by consuming undercooked beef or pork.

Cox1 sequencing identified 2 specimens of our collection, both from Samosir Island in Indonesia, as T. asiatica (Table 1) [11]. This tapeworm species is already well known and the country is one of the most endemic areas of taeniasis/cysticercosis. The majority of the people are moslems, but christians predominate in East Indonesia and Hindus in Bali. The 3 major endemic areas of the taeniasis/cysticercosis in Indonesia are North Sumatra, Bali and Papua (former Irian Jaya). Endemic areas are also found in other islands, such as Timor, Flores, North Sulawesi, West Kalimantan and South Sumatra. Inhabitants of Bali eat pork and beef and cysticercosis is common. Approximately 23% of pork liver samples in Bali were found to contain T. asiatica metacestodes [23]. A total prevalence was as high as 13.0% (19/146) for T. solium taeniasis in Jayawijaya District, Papua. A 2003-2006 survey of 371 local people in Samosir Island, North Sumatra revealed 6 of 240 (2.5%) to be infected with T. asiatica: 2 of 58 (3.4%) and 4 of 182 (2.2%) cases in 2003 and 2005, respectively [24]. T. asiatica is well known in North Sumatra, especially Samosir Island in Lake Toba. T. solium and T. saginata are well known from Bali [24]. Indonesia is one of the countries which are endemic with all 3 species of human Taenia tapeworms: T. solium, T. asiatica and T. saginata.

A specimen of our collection was identified as T. saginata by Cox1 nucleotide sequencing but sympatric distribution of T. solium, T. saginata and T. asiatica is already reported from Thailand in 2007 by Anantaphruti et al. on the basis of mitochondrial DNA analysis [11,25]. This was the first report of T. asiatica in this country. During 2002-2005, the field investigation was conducted in Thong Pha Phum District of Kanchanaburi Province which was northwest area of Thailand close to Myanmar border. Karen, a tribe, was the most surveyed population where Mon and Thai minorities reside in the mountainous terrain. Total 6 specimens, most of them had scoleces when recovered, were turned out to be T. asiatica by Cox1 gene analysis. All of them had been considered as being T. saginata morphologically before DNA genotyping. The authors stated their study indicated that 53.3% (8/15) of taeniid specimens expected to be T. saginata were actually T. asiatica and that both T. asiatica and T. saginata were co-distributed in Kanchanaburi Province. A dual infection of T. solium and T. asiatica from a patient was also confirmed clearly. In the local area, raw or inadequately cooked beef, pork, or pig viscera, and fresh blood are commonly consumed by local people in the study areas [25].

Taenia specimens are not available in the authors' laboratory but reports are now available from other researchers. Somers et al. [26] reported that T. asiatica (T. saginata asiatica) was the most common species (55.4%) over the T. saginata (38.5%) and T. solium (6.2%) out of 65 Taenia samples collected from patients in a referral hospital in Hanoi, North Vietnam. Species identification was done with morphological and molecular techniques: PCR-RFLP of a mitochondrial 12S rDNA [26]. Willingham and his coworkers also reported in 2003 that surveys for human taeniasis in central and northern provinces indicated a prevalence of 0.2-7.2%. In addition to T. solium and T. saginata, T. asiatica is also known to be present in Vietnam [27]. Xuan and colleagues also commented that T. asiatica was recently detected in Vietnam [28].

A specimen of Taenia species from 51-year-old female Mongolian was given to our laboratory by Dr. Hong Sung-Tae (Seoul National University College of Medicine, 2003). Cox1 sequence analysis and multiplex PCR genotyping classified it as T. saginata (Table 1). Since Mongolian people do not prefer eating raw pork, most of the tapeworms found in the country is T. saginata (personal communication with Dr. D. Temuulen, Health Sciences University of Mongolia School of Biomedicine, 2009). In the years 2002-2006, surveys on taeniasis/cysticercosis was conducted in Mongolia; the 118 proglottids were confirmed to be T. saginata by mitochondrial DNA analysis using cytochrome c oxidase subunit 1 (Cox1) and cytochrome b genes. T. saginata taeniasis was widely distributed at least in 10 of 21 provinces. No variation in the nucleotide sequences of the 2 genes was observed among T. saginata. There was no evidence of T. solium taeniasis/cysticercosis nor T. asiatica taeniasis so far [29].

Total 15 specimens, 3 from Savanakhet and 12 from Khammouane, of Taenia species from our collection were analyzed by Cox1 sequence and multiplex PCR represented all of them as being T. saginata (Table 1) [11]. During the years between 2000 and 2008, helminthiases were surveyed nationwide which were funded by organizations KAHP, KOICA and KFIH. Total 37,090 subjects from 18 localities were examined for helminth eggs. Saravane revealed the highest prevalence (3.0% out of 2,869) of taeniasis over the average of 1.1% throughout the nation. Among 120 collected tapeworms by morphological and genotypical examination, 3 T. solium infections were identified from Luangprabang for the first time in the country. A male patient with neurocysticercosis was also found from Oudomxay in northern territory. A pig with T. solium metacestodes was also found from the same district. The official inspector had data of cysticerci as much as 0.59% (46/7826) in pigs, and 2.99% (44/1473) in cattle (2003-2004). Regarding the possibility of T. asiatica in Lao PDR, Phoumindr commented that "Since the neighboring countries like Thailand and Vietnam have it, T. asiatica probably exists in Laos" [30].

Total 21 Taenia specimens from Khokong in Cambodia were identified as being T. saginata by Cox1 sequencing and multiplex PCR (Table 1). A total of 280 stool samples were examined for helminth ova from 2007 to 2008. The overall prevalence of taeniasis was 21.7% (61/280). We performed molecular epidemiological survey on Taenia tapeworms by analysis of copro DNA in Kohkong. For DNA differential diagnosis of T. solium, T. saginata and T. asiatica eggs, multiplex PCR was used based on the nad5 gene analysis. Using oligonucleotide primers Ta7126F, Ts7313F, Tso7466F, and Rev7915, the multiplex PCR was useful for species identification based on the 706, 629, and 474 bp bands. Cambodia still remains unclear for T. asiatica.

T. solium (n = 1) and T. saginata (n = 4) were identified by Cox1 sequencing and multiplex PCR which were collected from Mbulu and Ijaka, Tanzania (Table 1). Fecal samples were obtained from inhabitants in Kongwa, Dodoma area in February, 2008. Total 929 subjects in Ijaka and Pembamoto with primary school students also were examined for helminth eggs using Scotch tape anal swab and Kato-Katz techniques. The overall prevalence of helminthic infection was 7.2% (67/929). The infection rate of taeniasis was 0.6% (6/929). Species identification of Taenia tapeworms was performed by multiplex PCR and nucleotide sequencing of Cox1 gene on the fecal samples containing eggs. T. solium and T. saginata were recognized from Ijaka. This area was newly recognized to be endemic for T. solium in Tanzania [31]. There was, however, no evidence of T. asiatica in Tanzania at the moment.

Other countries, besides the Asian countries which are expected to be endemic for T. asiatica, exhibited either T. saginata or T. solium or both in morphological observations or DNA genotyping in Ethiopia, Cape Verde, Chile, Honduras, France, Poland, Switzerland, and Belgium.