Current Status and Perspectives of Cysticercosis and Taeniasis in Japan

This mini-review describes recent epidemiological trends in cysticercosis and taeniasis in Japan. Some of the topics discussed herein were presented at the first symposium on "Current perspectives of Taenia asiatica researches", that was held in Osong in Chungbuk Province, South Korea, in October 2011 and organized by Prof. K. S. Eom, Chungbuk National University School of Medicine. To better understand the trends in the occurrence of cysticercosis and taeniasis in Japan, clinical cases reported in 2005 have been updated. In addition, the current status of Taenia asiatica infections successively occurring in Japan since 2010 is also discussed.     

Current Status of Human Taeniasis in Lao People's Democratic Republic

Human taeniasis was investigated in Lao People''s Democratic Republic (Lao PDR) between 2000 and 2011 as part of the nation''s helminthiasis survey. A total of 55,038 inhabitants, including 29,846 school children, were examined using the Kato-Katz and scotch-tape anal swab method, and morphological observation of adult worms. Molecular identification of Taenia tapeworms was performed by multiplex PCR or DNA sequence analysis of the mitochondrial cox1 gene. Taenia eggs were present at a rate of 1.5% (845/55,038) in the subject population. Adult tapeworms were identified as T. solium or T. saginata by analyzing the collectable stool specimens (n=126). Three specimens identified as T. solium were found in Luang Prabang, while the remaining 123 specimens, which were T. saginata, were found in Bokeo, Bolikhamxay, Champasak, Houaphan, Khammouane, Luang Namta, Luang Prabang, Oudomxay, Phongsaly, Saysomboune, Saravane, Savannakhet, Xayaboury, Xekong, Xieng Khouang Province, and Vientiane Municipality.     

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.     

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.     

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.     

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 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.     

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).     

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.     

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.     

Recent Situation of Taeniasis in Mongolia (2002-2012)

Epidemiological situation of taeniasis in Mongolia was assessed based on mitochondrial DNA identification of the parasite species. Multiplex PCR was used on a total of 194 proglottid specimens of Taenia species and copro-PCR and loop-mediated isothermal amplification (LAMP) assays were utilized for detection of copro-DNA of 37 fecal samples from taeniasis patients submitted to the Mongolian National Center for Communicable Diseases (NCCD) from 2002 to 2012. In addition, 4 out of 44 calcified cysts in beef kept in formalin since 2003 were evaluated for histopathological confirmation of cattle cysticercosis. All proglottid specimens and stool samples were confirmed to be Taenia saginata by multiplex PCR and by copro-PCR and LAMP, respectively. Cysts collected from cattle were morphologically confirmed to be metacestodes of Taenia species. T. saginata taeniasis was identified from almost all ages from a 2-year-old boy up to a 88-year-old woman and most prominently in 15-29 age group (37%, 74/198) followed by 30-44 age group (34.8%, 69/198 ) from 15 of Mongolia''s 21 provinces, while cattle cysticerci were found from 12 provinces. The highest proportion of taeniasis patients was in Ulaanbaatar, the capital of Mongolia.     

Differential diagnosis of Taenia asiatica using multiplex PCR

Taenia asiatica and T. saginata are frequently confused tapeworms due to their morphological similarities and sympatric distribution in Asian regions. To resolve this problem, a high-resolution multiplex PCR assay was developed to distinguish T. asiatica infections from infection with other human Taenia tapeworms. For molecular characterization, the species specificity of all materials used was confirmed by sequencing of the cox1 gene. Fifty-two samples were analyzed in this study, comprising 20 samples of T. asiatica genomic DNA from China, Korea, and the Philippines; 24 samples of T. saginata from Belgium, Chile, China, Ethiopia, France, Indonesia, Korea, Laos, the Philippines, Poland, Taiwan, Thailand, and Switzerland; and 10 samples of T. solium from Cape Verde, China, Honduras, and Korea. The diagnostic quality of the results obtained using PCR and species-specific primers designed from valine tRNA and NADH genes was equal to that based on the nucleotide sequencing of the cox1 gene. Using oligonucleotide primers Ta4978F, Ts5058F, Tso7421F, and Rev7915, the multiplex PCR assay was useful for the differentially diagnosing T. asiatica, T. saginata, and T. solium based on 706-, 629-, and 474-bp bands.     

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.     

Progesterone induces mucosal immunity in a rodent model of human taeniosis by Taenia solium

More than one quarter of human world's population is exposed to intestinal helminth parasites. The Taenia solium tapeworm carrier is the main risk factor in the transmission of both human neurocysticercosis and porcine cysticercosis. Sex steroids play an important role during T. solium infection, particularly progesterone has been proposed as a key immunomodulatory hormone involved in susceptibility to human taeniosis in woman and cysticercosis in pregnant pigs. Thus, we evaluated the effect of progesterone administration upon the experimental taeniosis in golden hamsters (Mesocricetus auratus). Intact female adult hamsters were randomly divided into 3 groups: progesterone-subcutaneously treated; olive oil-treated as the vehicle group; and untreated controls. Animals were treated every other day during 4 weeks. After 2 weeks of treatment, all hamsters were orally infected with 4 viable T. solium cysticerci. After 2 weeks post infection, progesterone-treated hamsters showed reduction in adult worm recovery by 80%, compared to both vehicle-treated and non-manipulated infected animals. In contrast to control and vehicle groups, progesterone treatment diminished tapeworm length by 75% and increased proliferation rate of leukocytes from spleen and mesenteric lymph nodes of infected hamsters by 5-fold. The latter exhibited high expression levels of IL-4, IL-6 and TNF-α at the duodenal mucosa, accompanied with polymorphonuclear leukocytes infiltration. These results support that progesterone protects hamsters from the T. solium adult tapeworm establishment by improving the intestinal mucosal immunity, suggesting a potential use of analogues of this hormone as novel inductors of the gut immune response against intestinal helminth infections and probably other bowel-related disorders.     

Immunolocalization of the 150 kDa protein in cyst fluid of Taenia solium metacestodes

The 150 kDa protein of cyst fluid (CF) of Taenia solium metacestodes was purified by ammonium sulfate fractionation and Superose 6 HR gel filtration chromatography. The purified protein consisted of three subunits (15, 10 and 7 kDa proteins), which were analyzed with the use of a 7.5-15% gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Immunofluorescence study was carried out by using immunize specific polyclonal antibody. Positive reactions were noticed at bladder walls, calcareous corpuscles, granules of cyst fluid and some host tissue surrounding the bladder wall of the metacestodes. These results suggest that the 150 kDa protein was secreted into host tissues, inducing immune responses in the host, and it may play important roles in the cellular physiology of the parasites.     

Historical Overview of Taenia asiatica in Taiwan

An overview of the epidemiological, biological, and clinical studies of Taenia and taeniasis in Taiwan for the past century is presented. The phenomenal observations that led to the discovery of Taenia asiatica as a new species, which differ from Taenia solium and Taenia saginata, are described. Parasitological surveys of the aborigines in Taiwan revealed a high prevalence of taeniasis, which might be due to the culture of eating raw liver of hunted wild boars. Chemotherapeutic deworming trials involving many patients with taeniasis were discussed. Praziquantel was found to be very effective, but sometimes complete worms could not be recovered from the feces after treatment, probably due to the dissolution of the proglottids. Atabrine, despite some side effects, can still be used, in properly controlled dosages, as the drug of choice for human T. asiatica infection if we need to recover the expelled worms for morphological examinations. Research results on the infection of T. asiatica eggs from Taiwan aborigines in experimental animals were also noted. Since the pig serve as the natural intermediate host of T. asiatica and the predilection site is the liver, a differential comparison of other parasitic pathogens that might cause apparently similar lesions is also presented.     

Genetic variation in the Cytb gene of human cerebral Taenia solium cysticerci recovered from clinically and radiologically heterogeneous patients with neurocysticercosis

Neurocysticercosis (NC) is a clinically and radiologically heterogeneous parasitic disease caused by the establishment of larval Taenia solium in the human central nervous system. Host and/or parasite variations may be related to this observed heterogeneity. Genetic differences between pig and human-derived T. solium cysticerci have been reported previously. In this study, 28 cysticerci were surgically removed from 12 human NC patients, the mitochondrial gene that encodes cytochrome b was amplified from the cysticerci and genetic variations that may be related to NC heterogeneity were characterised. Nine different haplotypes (Ht), which were clustered in four haplogroups (Hg), were identified. Hg 3 and 4 exhibited a tendency to associate with age and gender, respectively. However, no significant associations were found between NC heterogeneity and the different T. solium cysticerci Ht or Hg. Parasite variants obtained from patients with similar NC clinical or radiological features were genetically closer than those found in groups of patients with a different NC profile when using the Mantel test. Overall, this study establishes the presence of genetic differences in the Cytb gene of T. solium isolated from human cysticerci and suggests that parasite variation could contribute to NC heterogeneity.     

Four Cases of Taenia saginata Infection with an Analysis of COX1 Gene

Human taeniases had been not uncommon in the Republic of Korea (=Korea) until the 1980s. The prevalence decreased and a national survey in 2004 revealed no Taenia egg positive cases. However, a subsequent national survey in 2012 showed 0.04% (10 cases) prevalence of Taenia spp. eggs suggesting its resurgence in Korea. We recently encountered 4 cases of Taenia saginata infection who had symptoms of taeniasis that included discharge of proglottids. We obtained several proglottids from each case. Because the morphological features of T. saginata are almost indistinguishable from those of Taenia asiatica, molecular analyses using the PCR-RFLP and DNA sequencing of the cytochrome c oxidase subunit 1 (cox1) were performed to identify the species. The PCR-RFLP patterns of all of the 4 specimens were consistent with T. saginata, and the cox1 gene sequence showed 99.8-100% identity with that of T. saginata reported previously from Korea, Japan, China, and Cambodia. All of the 4 patients had the history of travel abroad but its relation with contracting taeniasis was unclear. Our findings may suggest resurgence of T. saginata infection among people in Korea.