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.     

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.     

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.     

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.     

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.     

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.     

Human Neurocysticercosis Case and an Endemic Focus of Taenia solium in Lao PDR

A male patient with neurocysticercosis was identified in Montai Village, Xay District, Oudomxay Province, Lao PDR in February 2004. He had a history of diagnosis for neurocysticercosis by a CT scan in Thailand after an onset of epileptic seizure in 1993. A pig in the same district was found to contain Taenia solium metacestodes (=cysticerci); the slaughtered pig body contained more than 2,000 cysticerci. In addition to morphological identification, molecular identification was also performed on the cysticerci by DNA sequencing analysis of the mitochondrial cox1 gene; they were confirmed as T. solium metacestodes. The patient is regarded as an indigenous case of neurocysticercosis infected in an endemic focus of T. solium taeniasis/cysticercosis in Oudomxay Province, Lao PDR.     

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.     

Partial characterization of a 29 kDa cysteine protease purified from Taenia solium metacestodes

A 29 kDa cysteine protease of Taenia solium metacestodes was purified by Mono Q anion-exchanger and Superose 6 HR gel filtration chromatography. The enzyme was effectively inhibited by cysteine protease inhibitors, such as iodoacetic acid (IAA) and trans-epoxy-succinyl-L-leucyl-amido (4-guanidino) butane (E-64) while inhibitors acting on serine- or metallo-proteases did not affect the enzyme activity. The purified enzyme degraded human immunoglobulin G (IgG), collagen and bovine serum albumin (BSA), but human IgG was more susceptible for proteolysis by the enzyme. To define the precise biological roles of the enzyme, more detailed biochemical and functional studies would be required.     

Protein and antigen diversity in the vesicular fluid of Taenia solium cysticerci dissected from naturally infected pigs

Cysticercosis caused by Taenia solium is a health threat for humans and pigs living in developing countries, for which there is neither a flawless immunodiagnostic test nor a totally effective vaccine. Suspecting of individual diversity of hosts and parasites as possible sources of the variations of the parasite loads among cysticercotic animals and of the limited success of such immunological applications as well as, we explored and measured both in nine cases of naturally acquired porcine cysticercosis. For this purpose, 2-Dimensional IgG immunoblots were performed by reacting the sera of each cysticercotic pig with the antigens contained in the vesicular fluid (VF) of their own cysticerci. We found an unexpectedly large diversity among the proteins and antigens contained in each of the nine VFs. Also diverse were the serum IgG antibody responses of the nine pigs, as none of their 2D- immunoblot images exhibited the same number of spots and resembled each other in only 6.3% to 65.3% of their features. So large an individual immunological diversity of the cysticercal antigens and of the infected pigs′ IgG antibody response should be taken into account in the design of immunological tools for diagnosis and prevention of cysticercosis and should also be considered as a possibly significant source of diversity in Taenia solium′s infectiveness and pathogenicity.     

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.     

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.     

Immunoblot findings of calcareous corpuscles binding proteins in cyst fluid of Taenia solium metacestodes

After collecting calcareous corpuscles from plerocercoid of Spirometra mansoni (sparganum), we evaluated the antigenic values of calcareous corpuscles binding proteins obtained from the cyst fluid of Taenia solium metacestodes. Immunoblot analysis revealed that cysticercosis patient sera strongly recognized 10 and 95 kDa calcareous corpuscles binding proteins. This result demonstrated that calcareous corpuscles are bound with major secretory antigenic proteins, which is possibly involved in the secretory pathways of the 10 and 95 kDa proteins presenting in the cyst fluid of T. solium metacestodes.     

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.     

Population genetic structure of Taenia solium from Madagascar and Mexico: implications for clinical profile diversity and immunological technology

Taenia solium is a cestode parasitic of humans and pigs that strongly impacts on public health in developing countries. Its larvae (cysticercus) lodge in the brain, causing neurocysticercosis, and in other tissues, like skeletal muscle and subcutaneous space, causing extraneuronal cysticercosis. Prevalences of these two clinical manifestations vary greatly among continents. Also, neurocysticercosis may be clinically heterogeneous, ranging from asymptomatic forms to severely incapacitating and even fatal presentation. Further, vaccine design and diagnosis technology have met with difficulties in sensitivity, specificity and reproducibility. Parasite diversity underlying clinical heterogeneity and technological difficulties is little explored. Here, T. solium genetic population structure and diversity was studied by way of random amplified polymorphic DNA in individual cysticerci collected from pigs in Madagascar and two regions in Mexico. The amplification profiles of T. solium were also compared with those of the murine cysticercus Taenia crassiceps (ORF strain). We show significant genetic differentiation between Madagascar and Mexico and between regions in Mexico, but less so between cysticerci from different localities in Mexico and none between cysticerci from different tissues from the same pig. We also found restricted genetic variability within populations and gene flow was estimated to be low between populations. Thus, genetic differentiation of T. solium suggests that different evolutionary paths have been taken and provides support for its involvement in the differential tissue distribution of cysticerci and varying degrees of severity of the disease. It may also explain difficulties in the development of vaccines and tools for immunodiagnosis.     

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.     

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.     

A seroepidemiological survey of Taenia solium cysticercosis in Nabo, Guangxi Zhuang Autonomous Region, China

We have observed the seropositive rate of Taenia solium cysticercosis in residents at Nabo Village, Tiandong County, Guangxi Zhuang Autonomous Region, China by enzyme linked immunosorbent assay. The village had been found to be a relatively high endemic area of porcine cysticercosis among roaming pigs. Of 202 persons examined four males aged 15, 25, 35 and 41 year-old exhibited absorbance (abs) at 0.18, 0.20, 0.35 and 0.55, respectively. In addition, two females whose ages were 35 and 39 years revealed specific antibody levels of abs 0.26 and 0.41 in their sera. Overall positive rate among the people was 2.97%. All of these persons agreed that they had ingested the pork infected with T. solium metacestode (TsM), while history of proglottid discharge was not noticed from all of them. Three males and one female complained of intermittent headache. Our findings reinforced not only that the prevalence of cysticercosis might be related with roaming pigs infected with TsM but also that behavioral and environmental practices in local community constituted risk factors for transmission of the infection.     

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.