Global profiling of miRNA and protein expression patterns in rabbit peritoneal macrophages treated with exosomes derived from Taenia pisiformis cysticercus.

Infection of Taenia pisiformis cysticercus is very frequently found in lagomorphs and causes serious economic losses to rabbit breeding industry. T. pisiformis cysticercus has evolved numerous strategies to manipulate their hosts. The release of exosomes is of importance in the interaction between host and parasite. However, the mechanism by which T. pisiformis cysticercus evades the host immune system for long-term survival within the host remains unclear. Using small RNA sequencing and TMT labelling proteomic, we profiled the expression patterns of miRNAs and proteins in rabbit peritoneal macrophages treated with T. pisiformis cysticercus exosomes. Seven differentially expressed (DE)-miRNAs and six DE-proteins were randomly selected to validate the accuracy of the sequencing data by qRT-PCR or western blot. Functions of DE-miRNAs and proteins were analyzed using public data bases. And DE-miRNAs-DE-proteins correlation network were established. CCK-8 assay was used to evaluate the effect of exosomes on macrophages proliferation. Cell cycle of macrophages, isolated from T. pisiformis-infected rabbits, was determined using flow cytometry. A total of 21 miRNAs were significantly differentially expressed, including three worm-derived miRNAs. The expressions of miRNAs and proteins were consistent with the sequencing results. DE-miRNAs targets were related to cell proliferation and apoptosis. Exosomes treatment resulted in a decrease of macrophages proliferation. In vivo, T. pisiformis cysticercus significantly induced S phase cell arrest. Moreover, DE-proteins were related to production of interferon-gamma and interleukin-12, and immunoregulation. Correlation network analysis revealed a negative correlation relationship between DE-miRNAs and DE-proteins. Among them, novel334 and tpi-let-7-5p have potential regulatory effects on IL1ß and NFκB2 respectively, which imply that novel334-IL1ß/tpi-let-7-5p-NFκB2 axis may be an important way that T. pisiformis cysticercus modulates host immune response through exosomes. Further understanding of these potential regulatory mechanisms will contribute to clarify the mechanism of escape mediated by T. pisiformis exosomes.

Comparison of mitochondrial genetic variation of Taenia hydatigena cysticerci from China and Mongolia.

Parasitic infection is one of the many challenges facing livestock production globally. Cysticercosis tenuicollis is a common parasitic disease in domestic and wild ruminants (intermediate host) caused by the larval stage of Taenia hydatigena that primarily infects dogs (definitive host). Although genetic studies on this parasite exist, only a few describe the genetic variation of this parasite in Mongolia. Our aim was thus, to identify the mitochondrial differences in ovine isolates of Cysticercus tenuicollis entering China from Mongolia and comparison with existing Chinese isolates from sheep and goats based on the recently described PCR-RFLP method and mitochondrial genes of NADH dehydrogenase subunit 4 (nad4) and the NADH dehydrogenase subunit 5 (nad5). Sixty-nine isolates were collected during routine veterinary meat inspections from sheep that originated from Mongolia, at the modern slaughterhouses in Erenhot City, Inner Mongolia. Additional 114 cysticerci were also retrieved from sheep and goats from northern (Inner Mongolia Autonomous Region, Ningxia Hui Autonomous Region, and Gansu Province), western (Tibet Autonomous Region), and southern (Jiangxi Province and Guangxi Province) China. The PCR-RFLP approach of the nad5 showed nine mitochondrial subclusters A1, A2, A3, A5, A8, A9, A10, A11, and B of T. hydatigena isolates from sheep and goats from Mongolia and China. Meanwhile, haplogroup A1 RFLP profile was more widespread than other variants. These data supplements existing information on the molecular epidemiology of T. hydatigena in China and Mongolia and demonstrate the occurrence of similar genetic population structures in both countries.

Molecular characterization of Cysticercus tenuicollis isolates from sheep in the Nile Delta, Egypt and a review on Taenia hydatigena infections worldwide.

The predator­prey-transmitted cestode Taenia hydatigena infects a wide range of definitive and intermediate hosts all over the world. Domestic and sylvatic cycles of transmission are considered as well. The parasite has considerable economic importance, particularly in sheep. Here, the molecular characters of T. hydatigena cysticerci in sheep from the Nile Delta, Egypt were investigated for the first time. For this purpose, 200 sheep carcasses and their offal were inspected at the municipal abattoir, Dakahlia governorate, Egypt. Cysticerci of T. hydatigena were collected and molecularly characterized employing the mitochondrial 12S rRNA gene. Cysticerci were found in 42 (21%) sheep, mostly attached to the omenti, mesenteries and livers. After molecular confirmation, nine isolates were sequenced displaying six different haplotypes. Analysis of the T. hydatigena 12S rRNA nucleotide sequences deposited in GenBank revealed 55 haplotypes out of 69 isolates, displaying high haplotype (0.797) and low nucleotide (0.00739) diversities. For the Tajima D neutrality index, a negative value (−2.702) was determined, indicating the population expansion of the parasite. Additionally, global data summarized in this study should be useful to set up effective control strategies against this ubiquitous parasite.

Molecular characterization and haplotypes of sheep and goat isolates of Cysticercus tenuicollis in Turkey.

Taenia hydatigena, is a common parasite, lived mostly in dogs and wild carnivores in its mature stage, and the larvae, Cysticercus tenuicollis, is found on ruminants and pigs. The aim of the current study was to determine the genetic diversity in 20 isolates of the sheep and goats. After the isolation of total genomic DNA from C. tenuicollis isolates, genetic characterization of the mitochondrial NADH dehydrogenase subunit 1 gene region was amplified using specific JB11-JB12 primers in PCR and the PCR products were sequenced and haplotype and genetic diversity analyses were utilized. As a result, multiple nucleotide changes were determined in the sequence analyses of the isolates leading to detection of 16 and 15 different haplotypes in sheep and goat samples, respectively. These findings are important in terms of showing the diversity of nucleotide variation in C. tenuicollis in Turkey.

Cloning, expression, purification, and kinetic characterization of mitochondrial thioredoxin (TsTrx2), cytosolic thioredoxin (TsTrx1), and glutaredoxin (TsGrx1) from Taenia solium.

We report the complete coding sequences of mitochondrial thioredoxin (TsTrx2) and glutaredoxin (TsGrx1) from the cysticerci of T. solium. The full-length DNA of the TsTrx2 gene shows two introns of 88 and 77 bp and three exons. The TsTrx2 gene contains a single ORF of 423 bp, encoding 140 amino acid residues with an estimated molecular weight of 15,560 Da. A conserved C64NPC67 active site and a 30-amino acid extension at its N-terminus were identified. An insulin reduction reaction was used to determine whether it was a functional recombinant protein. The full-length DNA of the TsGrx1 gene shows one intron of 39 bp and a single ORF of 315 bp, encoding 105 amino acid residues with an estimated molecular weight of 12,582 Da. Sequence analysis revealed a conserved dithiol C34PYC37 active site, GSH-binding motifs (CXXC, Lys and Gln/Arg, TVP, and CXD), and a conserved Gly-Gly motif. The r-TsGrx1 kinetic constants for glutathione (GSH) and 2-hydroxyethyl disulfide (HED) were determined. In addition, cytosolic thioredoxin (TsTrx1), as reported by (Jiménez et al., Biomed Res Int 2015:453469, 2015), was cloned and expressed, and its catalytic constants were obtained along with those of the other two reductases. Rabbit-specific antibodies showed immune cross-reactions between TsTrx1 and TsTrx2 but not with TsGrx1. Both TsTGRs as reported by (Plancarte and Nava, Exp Parasitol 149:65-73, 2015) were biochemically purified to obtain and compare the catalytic constants for their natural substrates, r-TsTrx1, and r-TsTrx2, compared to those for Trx-S2E. coli. In addition, we determined the catalytic differences between the glutaredoxin activity of the TsTGRs compared with r-TsGrx1. These data increase the knowledge of the thioredoxin and GSH systems in T. solium, which is relevant for detoxification and immune evasion.

The epidemiology of Taenia spp. infection and Taenia solium cysticerci exposure in humans in the Central Highlands of Vietnam.

BACKGROUND: Vietnam is endemic for taeniasis and T. solium cysticercosis. Despite this, information on the epidemiological characteristics of the diseases in the Central Highlands of Vietnam are poorly described. The aims of this study were to determine the epidemiological characteristics of taeniasis (Taenia spp.) and T. solium cysticerci exposure in humans in Dak Lak province in the Central Highlands, Vietnam. METHODS: This cross-sectional study was carried out in six villages in three districts of Dak Lak. A total of 190 households were visited. From each household, between one and five individuals were asked to donate a single faecal and blood sample and respond to a questionnaire. Serum samples were subjected to lentil lectin purified glycoprotein enzyme-linked immunoelectrotransfer blot assay to detect antibodies against T. solium cysticerci. Multiplex real-time PCR was used to detect Taenia spp. infection in faecal samples. A fixed-effects logistic regression model was developed to identify factors associated with the probability of Taenia spp. infection or T. solium cysticerci exposure risk. The contribution of each of identified factor was quantified using population attributable fractions. RESULTS: The prevalence of seroexposure to T. solium in Dak Lak was 5% (95% CI 3% to 8%). Consumption of raw vegetables, sourcing drinking water from lakes, streams or ponds and the practice of outdoor defaecation were identified as primary risk factors for the prevalence of T. solium cysticerci exposure, while consuming undercooked pork and beef, pork tongue and observing Taenia proglottids in stool were associated with Taenia spp. infection. Consumption of raw vegetables attributed to 74% of T. solium cysticerci exposure-positive cases and consumption of undercooked beef attributed to 77% of taeniasis cases in these communities. CONCLUSIONS: The prevalence of T. solium seroexposure in Dak Lak is consistent with those reported in other regions of Vietnam. The identified risk factors associated with the prevalence of T. solium seroexposure and taeniasis infection in Dak Lak are modifiable and thus advocate for targeted community intervention programs to mitigating these risks.

A gel-free proteomic analysis of Taenia solium and Taenia crassiceps cysticerci vesicular extracts.

The taeniasis/cysticercosis complex is a zoonosis caused by the presence of the parasite Taenia solium in humans. It is considered a neglected disease that causes serious public health and economic problems in developing countries. In humans, the most common locations for the larval form are the skeletal muscles, ocular system, and the central nervous system, which is the most clinically important. Several glycoproteins of T. solium and Taenia crassiceps cysticerci have been characterized and studied for their use in the immunodiagnosis of neurocysticercosis and/or the development of synthetic or recombinant vaccines against cysticercosis. The aim of this study was to perform a gel-free shotgun proteomic analysis to identify saline vesicular extract (SVE) proteins of T. solium and T. crassiceps cysticerci. After solubilization of the SVE with and without surfactant reagent and in-solution digestion, the proteins were analyzed by LC-MS/MS. Use of a surfactant resulted in a significantly higher number of proteins that were able to be identified by LC-MS/MS. Novel proteins were identified in T. solium and T. crassiceps SVE. The qualitative analysis revealed a total of 79 proteins in the Taenia species: 29 in T. solium alone, 11 in T. crassiceps alone, and 39 in both. These results are an important contribution to support future investigations and for establishing a Taenia proteomic profile to study candidate biomarkers involved in the diagnosis or pathogenesis of neurocysticercosis.

Generation and characterization of monoclonal antibodies specific for 18 kDa antigen from Taenia solium cysticerci.

The gene encoding a mature 18 kDa glycoprotein of Taenia solium cysticerci (Ts18) was cloned and bacterially expressed with a His-tagged fusion protein. Monoclonal antibodies (MAbs) against the recombinant Ts18 antigen were generated in vitro by routine murine hybridoma technique of fusing splenocytes, from BALB/c mice immunized with the vesicular fluid of T. solium cysticerci (TsVF), with mouse myeloma cells (SP2/0). The reactivity and specificity of these MAbs were evaluated by indirect ELISA and immunoblotting techniques. Three stable hybridoma clones, namely 3B11, 6C5, and 6G4, were screened using His-Ts18-based ELISA, and these showed two IgG1 isotypes and one IgM isotype. All MAbs reacted with His-Ts18 at molecular weight (MW) 12.8 kDa and the native antigen at MW 18 kDa in TsVF and whole larval extracts (WLE). In a dot blotting test, MAbs 6C5 and 6G4 showed no obvious cross-reactivity with heterologous vesicular fluids from other taeniid species, including Taenia saginata (TsaVF), Taenia pisiformis (TpVF), Taenia hydatigena (ThVF), Taenia multiceps (TmVF), and Echinococcus granulosus (EgVF). Immunofluorescent assays showed that MAb 6C5 specifically reacted with the Ts18 expressed from pEGFP-N1-Ts18-transfected HeLa cells. Immunolocalization analysis, using MAb 6C5 as a probe, indicated that Ts18 was present at high concentrations in the region of the larval sucker and spiral canal. The results indicate that the Ts18 protein is an abundantly secreted parasite protein and MAbs against it might provide a step forward for improving the diagnosis of porcine cysticercosis.

Identification of species and genetic variation in Taenia isolates from human and swine of North India.

Taenia solium is the major cause of taeniasis and cysticercosis/neurocysticercosis (NCC) in the developing countries including India, but the existence of other Taenia species and genetic variation have not been studied in India. So, we studied the existence of different Taenia species, and sequence variation in Taenia isolates from human (proglottids and cysticerci) and swine (cysticerci) in North India. Amplification of cytochrome c oxidase subunit 1 gene (cox1) was done by polymerase chain reaction (PCR) followed by sequencing and phylogenetic analysis. We identified two species of Taenia i.e. T. solium and Taenia asiatica in our isolates. T. solium isolates showed similarity with Asian genotype and nucleotide variations from 0.25 to 1.01 %, whereas T. asiatica displayed nucleotide variations ranged from 0.25 to 0.5 %. These findings displayed the minimal genetic variations in North Indian isolates of T. solium and T. asiatica.

Sequence analysis and molecular characterization of Wnt4 gene in metacestodes of Taenia solium.

Wnt proteins are a family of secreted glycoproteins that are evolutionarily conserved and considered to be involved in extensive developmental processes in metazoan organisms. The characterization of wnt genes may improve understanding the parasite's development. In the present study, a wnt4 gene encoding 491amino acids was amplified from cDNA of metacestodes of Taenia solium using reverse transcription PCR (RT-PCR). Bioinformatics tools were used for sequence analysis. The conserved domain of the wnt gene family was predicted. The expression profile of Wnt4 was investigated using real-time PCR. Wnt4 expression was found to be dramatically increased in scolex evaginated cysticerci when compared to invaginated cysticerci. In situ hybridization showed that wnt4 gene was distributed in the posterior end of the worm along the primary body axis in evaginated cysticerci. These findings indicated that wnt4 may take part in the process of cysticerci evagination and play a role in scolex/bladder development of cysticerci of T. solium.

A rare case of Cysticercus tenuicollis infection in a neonate lamb: Evidence of prenatal transmission.

Cysticercosis develops in lambs following a Cysticercus tenuicollis infestation, which is the larval stage of Taenia hydatigena. A 7-day-old lamb was examined for depression, anorexia, fever (40.5°C), congested mucus membranes, reluctance to move, and a hunched back. Upon necropsy, congestion was noted in the intestines and brain, and the heart had a loose consistency. Soft and pulpy kidneys were evident coupled with watery intestinal contents. Epsilon toxin (Clostridium perfringens type D toxin) was detected using enzyme-linked immunosorbent assay. A transparent cystic structure was incidentally found attached to the pancreas, within which a scolex was well demonstrated upon histopathology. Chronic active peritonitis was diagnosed at the cyst attachment site. C. tenuicollis was confirmed by polymerase chain reaction and genome sequencing. This report describes prenatal transmission of C. tenuicollis in the present lamb, although this condition is quite rare.

Genetic diversity and haplotypes of Cysticercus tenuicollis isolates from slaughtered sheep and goats in Elazig and Bingol provinces of Turkey.

BACKGROUND: The cestode Taenia hydatigena uses canids, primarily dogs, as definitive hosts, whereas the metacestode larval stage cysticercus infects a range of intermediate hosts, including domestic animals such as goats, sheep and pigs. OBJECTIVES: In this study, we aimed to determine the genetic differences and haplotypes of Cysticercus tenuicollis isolates obtained from sheep and goats slaughtered in the Bingol and Elazig provinces of Turkey. METHODS: C. tenuicollis isolates were collected from 44 sheep and 26 goats slaughtered in slaughterhouses in Bingol and Elazig. After the isolation of total genomic DNA from C. tenuicollis isolates, the genetic characterization of the partial mitochondrial cytochrome oxidase 1 (CO1) gene region (866 bp) was amplified using specific primers by polymerase chain reaction (PCR), the products were then sequenced, and haplotype and genetic diversity analyses were carried out. RESULTS: As a result of the haplotype network analyses, 34 different haplotypes were detected around the main haplotype (Hap02) arranged in a star-like configuration and separated from other haplotypes by 1-28 mutation steps and covering 22.85% (16/70) of all isolates. Twenty-seven polymorphic fields were detected, 77.77% (21/27) of which were parsimony-informative, and secondary haplotype and nucleotide diversity were observed. Additionally, we detected high intraspecies haplotype diversity (hd: 0.933) and high nucleotide diversity (π: 0.00383), with 27 different nucleotide variation positions among the haplotypes of the isolates. Tajima's D value was negative, indicating population expansion and/or selection purification. The significantly negative Fu's Fs values indicated recent population expansion or the presence of expected rare haplotypes. CONCLUSION: The results of this study confirmed that C. tenuicollis isolates clustered in one lineage and were closely related to the relevant reference sequences in different countries, confirming the circulation of C. tenuicollis in different geographical regions.

[Morphological and molecular identification of Cysticercus fasciolaris isolated from rodent hosts (Rattus norvegicus) in Buenos Aires province (Argentina)]. / Identificación morfológica y molecular de Cysticercus fasciolaris aislado de un roedor (Rattus norvegicus) de la provincia de Buenos Aires (Argentina).

In a rodent (Rattus norvegicus) survey in Buenos Aires province, metacestodes of tapeworms were found encysted in the liver of the host. The aim of this work was the morphological and molecular identification of this parasite. To achieve the molecular characterization of the parasite, ribosomal (28S) and mitochondrial (COI) DNA were amplified and sequenced. Based on both morphological and molecular data using bioinformatic tools, the metacestode was identified as Cysticercus fasciolaris. The adult form of this tapeworm (Taenia taeniaeformis) commonly infects felid and canid mammalian hosts. This is the first report on the molecular identification of Cysticercus fasciolaris in Buenos Aires province (Argentina).

Changes in cyst's nuclear chromatin resulting after experimental manipulation of Taenia crassiceps mice infections: biological implications.

During some estimations of the nuclear DNA content, based on determinations propidium iodide (PI) binding through fluorocytometry for Taenia crassiceps cysticerci, significant variation in the results were found. This initial observation led to a series of experiments designed to explain the variation. These changes could be induced by the diameter of the needles in the syringes used for the mouse to mouse transfer of the cysts. Nuclei from cysts transferred through 27-gauge needles showed 30% less PI staining than those transferred through 21 gauge needles, after 2 months infections. Reduction in PI capture induced by 27-gauge needle was reversible when the cysts were maintained in their mice hosts during 5 months. Moreover, variation in PI binding to cysticercal DNA was also found when comparing parasites grown in male versus female mice. The use of agents that homogenize the chromatin structure during PI staining, allowed demonstrating that variation were entirely due to differences in the chromatin relaxation/compaction. Additional experiments demonstrated that the higher compaction is accompanied by a reduced ability of cysts to grow in the peritoneal cavity of BALB/cAnN mice. Furthermore, proteomic analysis also showed that these changes in chromatin relaxation/compaction resulted in different levels and patterns of protein expression. Our results strongly suggest that chromatin is involved in several well characterized phenomena of the T. crassiceps murine model, and open new avenues for a detailed approach to understand such a complex host-parasite relationship.

Diagnostic epitope variability within Taenia solium 8 kDa antigen family: implications for cysticercosis immunodetection.

To study diagnostic epitopes within the Taenia solium 8 kDa antigen family, six overlapping synthetic peptides from an 8 kDa family member (Ts8B2) were synthesized and evaluated by ELISA and MABA with sera from patients with neurocysticercosis (NCC), from infected pigs and from rabbits immunized with recombinant Ts8B2 protein. The pre-immune rabbit sera and the Ts8B2 recombinant protein served as negative and positive controls, respectively. A similar analysis was done with the already described antigenic peptides from another member of the 8 kDa family, highly similar to Ts8B2, the CyDA antigen. Surprisingly, neither the Ts8B2 peptides nor the CyDA peptides were recognized by infected human and porcine sera. However, the entire Ts8B2 recombinant, as well as amino and carboxy-terminal halves were recognized by the positive serum samples. The observed lack of recognition of linear Ts8B2 peptides suggests that the principal serological response to the Ts8B2 family is focused on conformational epitopes in contrast to the previously observed antigenicity of the CyDA peptides. This differential antigenicity of 8 kDa family peptides could be related with parasite antigenic variability. The fact that rabbits experimentally immunized with Ts8B2 did make anti-peptide antibodies to peptides Ts8B2-6 and CyDA-6, located in the carboxy-terminal region demonstrated that the Ts8B2 peptides are not intrinsically non-immunogenic.

PCR test for detecting Taenia solium cysticercosis in pig carcasses.

Polymerase chain reaction (PCR) test was employed to detect Taenia solium DNA in muscle lesions for validation of the meat inspection results of slaughtered pigs. Two sets of oligonucleotide primers, one targeted against the large subunit rRNA gene (TBR primers) and the other targeted against cytochrome c oxidase subunit 1 gene (Cox1 primers) of T. solium were used in this study. On reactivity in PCR test, the TBR primers and the Cox1 primers yielded products of 286 and 984 bp, respectively, in cysticercosis positive cases. Both the sets of primers were found to be highly specific, since they did not yield any PCR product in negative controls. A total of 225 pig carcasses were screened for cysticercosis by meat inspection, out of which 25 carcasses with visible cysts (16 viable and 9 degenerated cysts) were also confirmed to be positive for cysticercosis in PCR test. However, out of the 35 carcasses with suspected lesions on meat inspection, only two were found to be positive for cysticercosis in PCR test. The detection limits for both the primer sets were analyzed. The TBR primer set could detect up to 10 pg of cysticercus DNA, whereas the Cox1 primer set could detect only up to 1 ng. It is evident from the study that PCR test is an efficient tool for validation of meat inspection results and also to rule out ambiguity in carcass judgment of suspected cases of porcine cysticercosis.

[Eukaryotic expression and antigen epitope prediction of the LRRC15 protein in excretory secretory antigens of Taenia solium cysticercus].

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.

Morphometry and genetic diversity pattern of Cysticercus tenuicollis, an important food-borne taeniid metacestode in goats in Bangladesh.

Cysticercus tenuicollis is a larval stage of Taenia hydatigena resulting in cysticercosis, and responsible for enormous economic loses, especially in livestock production. Here, we planned to determine the prevalence and explore genetic variation of C. tenuicollis isolated from goats based on small subunit ribosomal RNA (rrnS) and cytochrome oxidase subunit 1 (cox1). To do this, samples were collected from different slaughter houses of municipal areas such as Bramhapalli slaughterhouse, Jubileeghat slaughterhouse and Mesuabazar slaughterhouse at Mymensingh sadar, and tentatively identified by morphological and morphometrical analysis. To study genetic variation, DNA was extracted from C. tenuicollis, and amplified rrnS and cox1 genes using specific primers, and were sequenced. Among 1372 examined animals, 177 (12.9%) were infected with C. tenuicollis. Cysts were recovered from peritoneum (7.9%), liver (4.4%) and urinary bladder (0.6%) of the infected animals. Females (18.9%) and adults (20.7%) were significantly more susceptible than male (8.8%) and young (9.3%), respectively. Genetic analysis defined 8 distinct rrnS genotypes and 9 unique cox1 haplotypes among 20 C. tenuicollis isolates. The nucleotide diversities were 0.00283 and 0.00434 for rrnS and cox1 genes, respectively. Neighbor joining (NJ) trees of rrnS and cox1 gene were constructed and the studied sequences were clustered with reference sequences of T. hydatigena with strong nodal support (100%). To compare Bangladeshi isolates, a median joining network was constructed with the population from other geographical regions and hosts. This led to a clustering pattern, but the clusters were not built with unique geographical regions or hosts. In conclusion, this is the first study that describes the genetic variation of T. hydatigena population and suggests the existence of host-specific variants. Therefore, it is fundamental to dispose infected viscera, restrict dog movement and proper management of slaughter house for the prevention and control of cysticercosis.

Identification and Expression Profiling of Circulating MicroRNAs in Serum of Cysticercus pisiformis-Infected Rabbits.

Cysticercus pisiformis (C. pisiformis), the larval form of Taenia pisiformis, parasitize mainly the liver, omentum and mesentery of rabbits and cause huge economic losses in the rabbit breeding industry. MicroRNA (miRNA), a short non-coding RNA, is widely and stably distributed in the plasma and serum. Numerous data demonstrates that, after parasitic infection, miRNAs become the key regulatory factor for controlling host biological processes. However, the roles of serum miRNAs in C. pisiformis-infected rabbits have not been elucidated. In this study, we compared miRNA expression profiles between the C. pisiformis-infected and healthy rabbit serum using RNA-seq. A total of 192 miRNAs were differentially expressed (fold change ≥ 2 and p < 0.05), including 79 up- and 113 downregulated miRNAs. These data were verified by qRT-PCR (real time quantitative polymerase chain reaction) analysis. Additionally, GO analysis showed that the target genes of these dysregulated miRNAs were most enriched in cellular, single-organism and metabolic processes. KEGG pathway analysis showed that these miRNAs target genes were involved in PI3K-Akt, viral carcinogenesis and B cell receptor signaling pathways. Interestingly, after aligning clean reads to the T. pisiformis genome, four (miR-124-3p_3, miR-124-3p_4, miR-124a and novel-miR1) T. pisiformis-derived miRNAs were found. Of these, novel-miR1was upregulated in different periods after C. pisiformis infection, which was verified qRT-PCR, and pre- novel-miR-1 was amplified from the cysticerci by RT-PCR, implying novel-miR-1 was derived from C. pisiformis and has great potential for the diagnosis of Cysticercosis pisiformis infection. This is the first investigation of miRNA expression profile and function in the serum of rabbits infected by C. pisiformis, providing fundamental data for developing diagnostic targets for Cysticercosis pisiformis.

Novel isolate of Cysticercus tenuicollis-Kalar isolate has been revealed in Kalar, Iraq.

INTRODUCTION: Although Cysticercus tenuicollis is one of the most economic and veterinary important parasite in Iraq, scanty molecular characterization exists for this helminth. This study aimed to determine the prevalence and molecular description of C. tenuicollis isolates from sheep in Kalar district of Iraq. METHODOLOGY: A total of 2,906 slaughtered sheep were examined post-mortem. Up to 20 samples of C. tenuicollis was extracted and amplified using mitochondrial COX1 gene. RESULTS: The overall prevalence rate was 6.88%, and female sheep recorded higher rate of infection (24.35%) than male (6.16%) with significant difference (p<0.05). The molecular results showed 14 haplotypes for COX1 gene and the pairwise nucleotide variation among them was ranged from 0.2 to 2.6%. Twelve out of fourteen haplotypes of C. tenuicollis involving one to three base mutations were discovered in Kalar, Iraq for the first time and this could be a unique mutation internationally and did not registered previously. Eleven newly recorded haplotypes involved only one single mutation and the remaining one involved three mutations. Phylogenetic interpretation showed that Cysticercus tenuicollis-Kalar isolate were clustered in one clade, and closely related to isolates discovered in Nigeria, China, Turkey, Poland, and Iran. CONCLUSIONS: This study provided a new record data on prevalence and discovered novel strains of C. tenuicollis in the study area for the first time named Cysticercus tenuicollis-Kalar isolate. Novel haplotypes might consider endemic genetic characterization of this metacestode. The present data may be useful to provide a good molecular background for future preventive and control programs.