Eukaryotic expression and antigen epitope prediction of the LRRC15 protein in excretory secretory antigens of Taenia solium cysticercus / 中国血吸虫病防治杂志

OBJECTIVE@#To conduct eukaryotic expression of the leucine-rich repeat containing 15 (LRRC15), a differentially expressed protein in excretory secretory antigens of Taenia solium cysticercus, and predict its antigen epitope.@*METHODS@#The molecular weight, stability, amino acid sequence composition, isoelectric point and T lymphocyte epitope of the LRRC15 protein were predicted using the bioinformatics online softwares ExPASy-PortParam and Protean. The full-length splicing primers were designed using PCR-based accurate synthesis, and the LRRC15 gene was synthesized. The recombinant pcDNA3.4-LRRC15 plasmid was constructed and transfected into HEK293 cells to express the LRRC15 protein. In addition, the LRRC15 protein was characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting.@*RESULTS@#The recombinant pcDNA3.4-LRRC15 plasmid was successfully constructed, which expressed the target LRRC15 protein with an approximately molecular weight of 70 kDa. Bioinformatics prediction with the ExPASy-PortParam software showed that LRRC15 was a hydrophilic protein, which was consisted of 644 amino acids and had a molecular weight of 69.89 kDa and an isoelectric point of 5.6. The molecular formula of the LRRC15 protein was C3073H4942N846O953S28 and had an instability coefficient is 50.3, indicating that LRRC15 was an instable protein. Bioinformatics prediction with the Protean software showed that the dominant T-cell antigen epitopes were located in 292 to 295, 353 to 361, 521 to 526 and 555 to 564 amino acids of the LRRC15 protein, and the T-cell antigen epitopes with a high hydrophilicity, good flexibility, high surface accessibility and high antigenicity index were found in 122 to 131, 216 to 233, 249 to 254, 333 to 343, 358 to 361, 368 to 372, 384 to 386, 407 to 412, 445 to 450, 469 to 481, 553 to 564, 588 to 594, 607 to 617 and 624 to 639 amino acids. Following transfection of the recombinant pcDNA3.4-LRRC15 plasmid into HEK293 cells, SDS-PAGE and Western blotting identified LRRC15 proteins in cell secretory culture media, cell lysis supernatants and sediments. The LRRC15-His fusion protein was purified from the cell culture medium, and SDS-PAGE identified a remarkable band at approximately 70 kDa, while Western blotting successfully recognized the band of the recombinant LRRC15 protein.@*CONCLUSIONS@#The eukaryotic expression and antigen epitope prediction of the LRRC15 protein in the excretory secretory antigens of T. solium cysticercus have been successfully performed, which provides insights into further understandings of its biological functions.

Codon Usage Bias and Determining Forces in Taenia solium Genome

The tapeworm Taenia solium is an important human zoonotic parasite that causes great economic loss and also endangers public health. At present, an effective vaccine that will prevent infection and chemotherapy without any side effect remains to be developed. In this study, codon usage patterns in the T. solium genome were examined through 8,484 protein-coding genes. Neutrality analysis showed that T. solium had a narrow GC distribution, and a significant correlation was observed between GC12 and GC3. Examination of an NC (ENC vs GC3s)-plot showed a few genes on or close to the expected curve, but the majority of points with low-ENC (the effective number of codons) values were detected below the expected curve, suggesting that mutational bias plays a major role in shaping codon usage. The Parity Rule 2 plot (PR2) analysis showed that GC and AT were not used proportionally. We also identified 26 optimal codons in the T. solium genome, all of which ended with either a G or C residue. These optimal codons in the T. solium genome are likely consistent with tRNAs that are highly expressed in the cell, suggesting that mutational and translational selection forces are probably driving factors of codon usage bias in the T. solium genome.

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.

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. .

Genetic polymorphism in Taenia solium metacestodes from different Brazilian geographic areas

The aim of the present study is to investigate genetic polymorphisms in Taenia solium metacestodes from different Brazilian geographical areas and to relate them to antibody recognition in serum samples of neurocysticercosis (NC) patients. Metacestodes were obtained from the Distrito Federal (DF), Bahia, Minas Gerais (MG) and São Paulo (SP) regions of Brazil. Samples of human sera from 49 individuals with NC, 68 individuals with other helminthiasis and 40 healthy volunteers were analysed (157 individuals in total). Antigens were prepared and used in enzyme-linked immunosorbent assay and western blotting assays to detect specific immunoglobulin G antibodies. Genetic distances between metacestode populations were analysed using random amplified polymorphic DNA (RAPD) analysis. Our results show that there was a higher frequency of reactivity in the DF region in the sera from NC patients (p < 0.05), while discrimination between active and inactive NC was seen only in extracts from the MG and SP regions (p < 0.05). Using RAPD, the sample from the DF region presented a greater increase compared to the other regions. A relationship between genetic polymorphisms among T. solium metacestodes from different areas in Brazil and the differences in antibody detection in patients with NC were established.

Determinación por medio de marcadores moleculares SSCP y RAPD de la diversidad genética en la especie Taenia solium en Colombia / Determination by SSPC and RAPD of genetic diversity in Taenia solium especies, main causative agent of teniosis and cysticercosis in Colombia

Utilizando las técnicas moleculares de SSCP y RAPD se pudo evidenciar rápida y claramente la variabilidad genética en Colombia de larvas del céstodo Taenia solium analizando fragmentos de genes de ADN mitocondrial y fragmentos aleatorios de ADN nuclear. El ADN estudiado se obtuvo de ocho aislados de cisticercos de cerdo provenientes de tres departamentos de Colombia: Antioquia, Nariño y Sucre. Los fragmentos obtenidos por PCR de los genes NADH deshidrogenasa 1 (ND1) y citocromo oxidasa c subunidad I (COI) al ser denaturados y analizados en geles no denaturantes de acrilamida, mostraron al menos tres patrones diferentes por cada gen analizado, verificando que estos genes conservados mitocondriales son polimórficos en T. solium colombiana. Por otra parte, los cebadores decaméricos de RAPD produjeron patrones polimórficos, corroboraron la diversidad genética entre los diferentes aislamientos analizados.

Taeniasis-cysticercosis in Southern Ecuador: assessment of infection status using multiple laboratory diagnostic tools

Taenia solium-taeniasis and cysticercosis were studied in the human and porcine populations of a rural community in the Southern Ecuadorian Andes. From the 1059 inhabitants, 800 serum samples and 958 stool samples could be collected. In addition, 646 from the estimated 1148 pigs were tongue inspected. Circulating antigen was detected by enzyme linked immunosorbent assay (Ag-ELISA) in 2.25 percent of the human population, whereas intestinal taeniasis was detected in 1.46 percent by the formalin-ether technique. Following treatment and recovery of tapeworm fragments these were all identified as T. solium. Porcine cysticercosis was diagnosed in 3.56 percent of the pigs by tongue inspection. In addition, enzyme linked immunoelectrotransfer blot (EITB) was performed on a subset group of 100 humans to confirm the results of the Ag-ELISA. One hundred serum samples from pigs were also analysed by EITB. It appeared that 43 and 74 percent of humans and pigs had antibodies against T. solium cysticerci, respectively. It is concluded that contrary to the high exposure of the human population to T. solium that is suggested by EITB, the number of active cysticercosis cases, diagnosed by Ag-ELISA, was low, which may indicate endemic stability. The further use of complementary diagnostic methods for a better understanding of the epidemiology of T. solium is suggested.

Fecal specimens preparation methods for PCR diagnosis of human taeniosis

Com o intuito de utilizar a Reação em Cadeia pela Polimerase (PCR) como método de diagnóstico diferencial da teníase humana, avaliaram-se alguns protocolos de preparação e extração de DNA de ovos de Taenia saginata presentes em amostras de fezes de paciente naturalmente infectado. O DNA obtido após extração com fenol/clorofórmio/álcool isoamílico ou DNAzol® teve que ser purificado antes da PCR para que fosse possível a amplificação dos fragmentos de 170 pb e 600 pb desejados. Com o kit QIAmp DNA stool mini kit® tal purificação não foi necessária. Os melhores resultados foram observados após o tratamento prévio das amostras com pérolas de vidro, tanto quando da utilização de fenol/clorofórmio/álcool isoamílico, quando de DNAzol® ou QIAmp DNA stool mini kit®.

Molecular identification of Taenia solium cysticercus genotype in the histopathological specimens.

PCR-based molecular diagnosis was done for identification of causative agents found in paraffin-embedded specimens that were resected from two suspected neurocysticercosis patients. DNA samples were extracted from tissues or sections and cytochrome c oxidase subunit 1 gene and cytochrome b gene were amplified for the detection of taeniid DNA. Two different genes were successfully amplified in both specimens, but the sizes of amplified products seemed to depend on the quality of DNA. Based on the nucleotide sequences of the PCR-amplified genes, the causative agents from two cases were identified as T. solium Asian genotype. When infection with T. solium is not confirmed by histopathological examination, molecular diagnosis will be more useful for definitive diagnosis.

Taeniasis and cysticercosis in Asia and the Pacific: present state of knowledge and perspectives.

Several topics on taeniasis and cysticercosis in Asia and the Pacific are overviewed. In Asia and the Pacific, three human taeniid species have been recognized: Taenia solium, Taenia saginata and Taenia asiatica. The first topic is on evolution of T. solium. Mitochondrial DNA polymorphisms of T. solium worldwide are discussed with emphasis of two specific genotypes: American-African and Asian. The second topic is recent major advances in sero- and molecular-diagnosis of T. solium cysticercosis in humans, pigs and dogs. The third is the present situation of T. solium taeniasis/cysticercosis in Papua (Irian Jaya), Indonesia. The forth is the present situation of T. solium cysticercosis and T. saginata taeniasis in Bali, Indonesia. The fifth is the present situation of T. asiatica taeniasis in Asia and the Pacific and in North Sumatra, Indonesia. The sixth is on the debate of the exact definition of T. asiatica. Because T. asiatica can not be differentiated from T. saginata morphologically, it is time to re-evaluate T. saginata in Asia and the Pacific. New and broad-based surveys across this region are necessary from epidemiological and public health perspectives, based on evidence.