Four Additional Cases of Diphyllobothrium nihonkaiense Infection Confirmed by Analysis of COX1 Gene in Korea

Most of the diphyllobothriid tapeworms isolated from human samples in the Republic of Korea (= Korea) have been identified as Diphyllobothrium nihonkaiense by genetic analysis. This paper reports confirmation of D. nihonkaiense infections in 4 additional human samples obtained between 1995 and 2014, which were analyzed at the Department of Parasitology, Hallym University College of Medicine, Korea. Analysis of the mitochondrial cytochrome c oxidase 1 (cox1) gene revealed a 98.5-99.5% similarity with a reference D. nihonkaiense sequence in GenBank. The present report adds 4 cases of D. nihonkaiense infections to the literature, indicating that the dominant diphyllobothriid tapeworm species in Korea is D. nihonkaiense but not D. latum.     

A Case of Diphyllobothrium nihonkaiense Infection as Confirmed by Mitochondrial COX1 Gene Sequence Analysis

Diphyllobothrium nihonkaiense has been reported in Korea as Diphyllobothrium latum because of their close morphologic resemblance. We have identified a human case of D. nihonkaiense infection using the mitochondrial cytochrome c oxidase subunit I (cox1) gene sequence analysis. On 18 February 2012, a patient who had consumed raw fish a month earlier visited our outpatient clinic with a long tapeworm parasite excreted in the feces. The body of the segmented worm was 2 m long and divided into the scolex (head) and proglottids. It was morphologically close to D. nihonkaiense and D. latum. The cox1 gene analysis showed 99.4% (340/342 bp) homology with D. nihonkaiense but only 91.8% (314/342 bp) homology with D. latum. The present study suggested that the Diphyllobothrium spp. infection in Korea should be analyzed with specific DNA sequence for an accurate species identification.     

Taenia asiatica and Taenia saginata: genetic divergence estimated from their mitochondrial genomes

We conducted a differential identification of Taenia asiatica and Taenia saginata, through the mapping of mitochondrial genomes and the sequencing of the cox1 and cob genes. The entire mitochondrial genomes of T. asiatica and T. saginata were amplified by long-extension PCR and cloned; each was approximately 14 kb in size. Restriction maps of T. asiatica and T. saginata mitochondrial genomes were then constructed using 13 restriction enzymes. The resulting restriction patterns enable us to estimate their genetic divergence at 4.8%. The actual sequence divergence was computed 4.5% from the cox1 gene, and 4.1% from the cob gene. These results support the designation of T. asiatica as a separate species from T. saginata.     

Infection of Taenia asiatica in a Bai Person in Dali,China

We report here a human case of Taenia asiatica infection which was confirmed by genetic analyses in Dali, China. A patient was found to have symptoms of taeniasis with discharge of tapeworm proglottids. By sequencing of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene, we observed nucleotide sequence identity of 99% with T. asiatica and 96% with T. saginata. Using the cytochrome b (cytb) gene, 99% identity with T. asiatica and 96% identity with T. saginata were found. Our findings suggest that taeniasis of people in Dali, China may be mainly caused by T. asiatica.     

Molecular Approaches to Taenia asiatica

Taenia solium, T. saginata, and T. asiatica are taeniid tapeworms that cause taeniasis in humans and cysticercosis in intermediate host animals. Taeniases remain an important public health concerns in the world. Molecular diagnostic methods using PCR assays have been developed for rapid and accurate detection of human infecting taeniid tapeworms, including the use of sequence-specific DNA probes, PCR-RFLP, and multiplex PCR. More recently, DNA diagnosis using PCR based on histopathological specimens such as 10% formalin-fixed paraffin-embedded and stained sections mounted on slides has been applied to cestode infections. The mitochondrial gene sequence is believed to be a very useful molecular marker for not only studying evolutionary relationships among distantly related taxa, but also for investigating the phylo-biogeography of closely related species. The complete sequence of the human Taenia tapeworms mitochondrial genomes were determined, and its organization and structure were compared to other human-tropic Taenia tapeworms for which complete mitochondrial sequence data were available. The multiplex PCR assay with the Ta4978F, Ts5058F, Tso7421F, and Rev7915 primers will be useful for differential diagnosis, molecular characterization, and epidemiological surveys of human Taenia tapeworms.     

Genetic Diversity of Taenia asiatica from Thailand and Other Geographical Locations as Revealed by Cytochrome c Oxidase Subunit 1 Sequences

Twelve 924 bp cytochrome c oxidase subunit 1 (cox1) mitochondrial DNA sequences from Taenia asiatica isolates from Thailand were aligned and compared with multiple sequence isolates from Thailand and 6 other countries from the GenBank database. The genetic divergence of T. asiatica was also compared with Taenia saginata database sequences from 6 different countries in Asia, including Thailand, and 3 countries from other continents. The results showed that there were minor genetic variations within T. asiatica species, while high intraspecies variation was found in T. saginata. There were only 2 haplotypes and 1 polymorphic site found in T. asiatica, but 8 haplotypes and 9 polymorphic sites in T. saginata. Haplotype diversity was very low, 0.067, in T. asiatica and high, 0.700, in T. saginata. The very low genetic diversity suggested that T. asiatica may be at a risk due to the loss of potential adaptive alleles, resulting in reduced viability and decreased responses to environmental changes, which may endanger the species.     

Molecular identification of Taenia tapeworms by Cox1 gene in Koh Kong, Cambodia

We collected fecal samples from 21 individuals infected with Taenia tapeworms in Koh Kong Province, Cambodia, and performed nucleotide sequencing of the cox1 gene and multiplex PCR on the eggs for DNA differential diagnosis of human Taenia tapeworms. Genomic DNA was extracted from the eggs of a minimum number of 10 isolated from fecal samples. Using oligonucleotide primers Ta7126F, Ts7313F, Tso7466F, and Rev7915, the multiplex PCR assay proved useful for differentially diagnosing Taenia solium, Taenia saginata, and Taenia asiatica based on 706, 629, and 474 bp bands, respectively. All of the Taenia specimens from Kho Kong, Cambodia, were identified as either T. saginata (n=19) or T. solium (n=2) by cox1 sequencing and multiplex PCR.     

Taenia asiatica: the Most Neglected Human Taenia and the Possibility of Cysticercosis

Not only Taenia solium and Taenia saginata, but also Taenia asiatica infects humans. The last species is not included in the evaluation of the specificity of the immunodiagnostic techniques for taeniasis/cysticercosis. There is currently no specific immunodiagnostic method for T. asiatica available. Therefore, due to the fact that molecular techniques (the only tool to distinguish the 3 Taenia species) are normally not employed in routine diagnostic methods, the 2 questions concerning T. asiatica (its definite geographic distribution and its ability to cause human cysticercosis), remain open, turning T. asiatica into the most neglected agent of human taeniasis-cysticercosis.     

State of the Art of Taenia solium as Compared to Taenia asiatica

Three species of tapeworms infect humans in their adult stage (Taenia solium, Taenia saginata and Taenia asiatica). The 3 are flat, opaque white or yellowish, and exceptional long segmented parasites, measuring 1 to 12 m in their adult stage. In this review, the development of the knowledge regarding the first species, mainly focused on understanding how the larval stage or cysticercus is transmitted to humans, is described. The second species is a cosmopolitan parasite that only causes taeniosis and not cysticercosis; therefore, it will not be included. Information on the third species, which is presently being produced, since this species was recognized as such only at the end of the 20th century, will be discussed at the end of this review.     

Morphologic and genetic identification of Taenia tapeworms in Tanzania and DNA genotyping of Taenia solium

Species identification of Taenia tapeworms was performed using morphologic observations and multiplex PCR and DNA sequencing of the mitochondrial cox1 gene. In 2008 and 2009, a total of 1,057 fecal samples were collected from residents of Kongwa district of Dodoma region, Tanzania, and examined microscopically for helminth eggs and proglottids. Of these, 4 Taenia egg positive cases were identified, and the eggs were subjected to DNA analysis. Several proglottids of Taenia solium were recovered from 1 of the 4 cases. This established that the species were T. solium (n = 1) and T. saginata (n = 3). One further T. solium specimen was found among 128 fecal samples collected from Mbulu district in Arusha, and this had an intact strobila with the scolex. Phylegenetic analysis of the mtDNA cox1 gene sequences of these 5 isolates showed that T. saginata was basal to the T. solium clade. The mitochondrial cox1 gene sequences of 3 of these Tanzanian isolates showed 99% similarity to T. saginata, and the other 2 isolates showed 100% similarity to T. solium. The present study has shown that Taenia tapeworms are endemic in Kongwa district of Tanzania, as well as in a previously identified Mbulu district. Both T. solium isolates were found to have an African/Latin American genotype (cox1).     

Taenia solium and Taenia saginata: identification of sequence characterized amplified region (SCAR) markers

Cysticercosis is one of the most important zoonosis, not only because of the effects on animal health and its economic consequences, but also due to the serious danger it poses to humans. The two main parasites involved in the taeniasis-cysticercosis complex in Brazil are Taenia saginata and Taenia solium. Differentiating between these two parasites is important both for disease control and for epidemiological studies. The purpose of this work was to identify genetic markers that could be used to differentiate these parasites. Out of 120 oligonucleotide decamers tested in random amplified polymorphic DNA (RAPD) assays, 107 were shown to discriminate between the two species of Taenia. Twenty-one DNA fragments that were specific for each species of Taenia were chosen for DNA cloning and sequencing. Seven RAPD markers were converted into sequence characterized amplified region (SCAR) markers with two specific for T. saginata and five specific for T. solium as shown by agarose gel electrophoresis. These markers were developed as potential tools to differentiate T. solium from T. saginata in epidemiological studies.     

Taenia saginata and T. hydatigena: intramuscular vaccination of calves with oncospheres

This is a preliminary attempt to immunize calves against bovine cysticercosis (Cestoda) by intramuscular injection of artificially hatched homologous or heterologous oncospheres. In the first two experiments five calves were vaccinated with the oncospheres of Taenia saginata. These calves, together with five controls, were subsequently challenged orally with the eggs of T. saginata. At autopsy all the vaccinated animals harbored a colony of living cysticerci at the site of injection. Three of these calves were completely protected against the oral challenge, and in the other two, only one, and two cysticerci, respectively were found in the sites of election. The five controls harbored 1154, 1680, 820, 960, and 1820 living cysticerci, respectively, in the sites of election.     

Two Human Cases of Diphyllobothrium nihonkaiense Infection in Korea

Diphyllobothrium latum and Diphyllobothrium nihonkaiense are the 2 reported main causes of human diphyllobothriasis in the Republic of Korea. However, the differentiation of these 2 species based on morphologic features alone is difficult. The authors used nucleotide sequencing of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene to diagnose Diphyllobothrium spp. Two patients visited the emergency room at Kyungpook National University Hospital on 3 April and 12 April 2013, respectively, with fragments of parasites found while defecating. The parasites were identified as Diphyllobothrium spp. based on morphologic characteristics, and subsequent cox1 gene sequencing showed 99.9% similarity (1,478/1,480 bp) with D. nihonkaiense. Our findings support the hypothesis that D. nihonkaiense is a dominant species in Korea.     

Epidemiological understanding of Taenia tapeworm infections with special reference to Taenia asiatica in Korea.

In endemic areas of Taenia tapeworms in Korea, most of the reports showed that T. saginata was dominant over T. solium, but eating pigs is the dominant habit over eating cattle. Why do they have more T. saginata despite lower consumption of beef? This problem actually has long been recognized but until recently there has been no intensive trial to give a scientific explanation on this epidemiological enigma. By summing up the data published between the years 1963 and 1999, the ratio of armed versus unarmed tapeworms in humans was estimated at approximately 1:5. The ratio of pig-eaters versus cattle-eaters, however, was approximately 5:1. This inconsistency could be explained with the recently described T. asiatica, which infects humans through the eating of pig''s viscera. We re-evaluate the importance of the consumption of visceral organ of pigs, leading us to an improved epidemiological understanding of the T. asiatica infection together with co-existing T. saginata and T. solium in Korea.     

Sympatric Distribution of Three Human Taenia Tapeworms Collected between 1935 and 2005 in Korea

Taeniasis has been known as one of the prevalent parasitic infections in Korea. Until recently, Taenia saginata had long been considered a dominant, and widely distributed species but epidemiological profiles of human Taenia species in Korea still remain unclear. In order to better understand distribution patterns of human Taenia tapeworms in Korea, partial nucleotide sequences of mitochondrial cox1 and ITS2 (internal transcribed spacer 2) were determined, along with morphological examinations, on 68 Taenia specimens obtained from university museum collections deposited since 1935. Genomic DNA was extracted from formalin-preserved specimens. Phylogenetic relationships among the genotypes (cox1 haplotype) detected in this study were inferred using the neighbor-joining method as a tree building method. Morphological and genetic analyses identified 3 specimens as T. solium, 51 specimens as T. asiatica, and 14 specimens as T. saginata. Our results indicate that all 3 Taenia tapeworms are sympatrically distributed in Korea with T. asiatica dominating over T. saginata and T. solium.     

Molecular Characterization of Taenia multiceps Isolates from Gansu Province,China by Sequencing of Mitochondrial Cytochrome C Oxidase Subunit 1

A total of 16 Taenia multiceps isolates collected from naturally infected sheep or goats in Gansu Province, China were characterized by sequences of mitochondrial cytochrome c oxidase subunit 1 (cox1) gene. The complete cox1 gene was amplified for individual T. multiceps isolates by PCR, ligated to pMD18T vector, and sequenced. Sequence analysis indicated that out of 16 T. multiceps isolates 10 unique cox1 gene sequences of 1,623 bp were obtained with sequence variation of 0.12-0.68%. The results showed that the cox1 gene sequences were highly conserved among the examined T. multiceps isolates. However, they were quite different from those of the other Taenia species. Phylogenetic analysis based on complete cox1 gene sequences revealed that T. multiceps isolates were composed of 3 genotypes and distinguished from the other Taenia species.     

Rodent model for long-term maintenance and development of the viable cysticerci of Taenia saginata asiatica

Although oncospheres of Taenia saginata asiatica can develop into cysticerci in immunodeficiency, immunosuppressed, and normal mice, no detailed information on the development features of these cysticerci from SCID mice is available. In the present study, the tumor-like cyst was found in the subcutaneous tissues of each of 10 SCID mice after 38-244 days inoculation with 39,000 oncospheres of T. s. asiatica. These cysts weighed 2.0-9.6 gm and were 1.5-4.3 cm in diameter. The number of cysticerci were collected from these cysts ranged from 125 to 1,794 and the cysticercus recovery rate from 0.3% to 4.6%. All cysticerci were viable with a diameter of 1-6 mm and 9 abnormal ones each with 2 evaginated protoscoleces were also found. The mean length and width of scolex, protoscolex, and bladder were 477 x 558, 756 x 727, and 1,586 x 1,615 microns, respectively. The diameters of suckers and rostellum were 220 microns and 70 microns, respectively. All cysticerci had two rows of rostellar hooks. These findings suggest that the SCID mouse model can be employed as a tool for long-term maintenance of the biological materials for advanced studies of immunodiagnosis, vaccine development, and evaluation of cestocidal drugs which would be most benefit for the good health of the livestocks.     

Molecular Analysis of Anisakis Type I Larvae in Marine Fish from Three Different Sea Areas in Korea

Anisakiasis, a human infection of Anisakis L3 larvae, is one of the common foodborne parasitic diseases in Korea. Studies on the identification of anisakid larvae have been performed in the country, but most of them have been focused on morphological identification of the larvae. In this study, we analyzed the molecular characteristics of 174 Anisakis type I larvae collected from 10 species of fish caught in 3 different sea areas in Korea. PCR-RFLP and sequence analyses of rDNA ITS and mtDNA cox1 revealed that the larvae showed interesting distribution patterns depending on fish species and geographical locations. Anisakis pegreffii was predominant in fish from the Yellow Sea and the South Sea. Meanwhile, both A. pegreffii and A. simplex sensu stricto (A. simplex s.str.) larvae were identified in fish from the East Sea, depending on fish species infected. These results suggested that A. pegreffii was primarily distributed in a diverse species of fish in 3 sea areas around Korea, but A. simplex s.str. was dominantly identified in Oncorhynchus spp. in the East Sea.     

Human Taeniasis in the Republic of Korea: Hidden or Gone?

History and current status of human taeniasis in the Republic of Korea, due to Taenia solium, Taenia asiatica, and Taenia saginata, are briefly reviewed. Until the 1980s, human taeniasis had been quite common in various localities of Korea. A study from 1924 reported 12.0% egg prevalence in fecal examinations. Thereafter, the prevalence of Taenia spp. ranged from 3% to 14% depending on the time and locality. Jeju-do, where pigs were reared in a conventional way, was the highest endemic area of taeniasis. An analysis of internal transcribed spacer 2 and mitochondrial cytochrome c oxidase 1 genes of 68 taeniasis cases reported from 1935 to 2005 in Korea by a research group revealed the relative occurrence of the 3 Taenia spp. as follows: T. solium (4.4%), T. asiatica (75.0%), and T. saginata (20.6%). However, national surveys on intestinal helminths conducted every 5 years on randomly selected people revealed that the Taenia egg prevalence dropped from 1.9% in 1971 to 0.02% in 1997 and finally to 0.0% in 2004. With the exception of 3 egg-positive cases reported in 2008 and 2 worm-proven cases in 2011, no more cases have been officially recorded. Based on these surveys and also on other literature, it can be concluded that taeniasis has virtually disappeared from Korea, although a few sporadic cases may remain hidden. Human cysticercosis is also expected to disappear within a couple of decades in Korea.