Xuhang Wu

Identification of Dirofilaria immitis miRNA using illumina deep sequencing

The heartworm Dirofilaria immitis is the causative agent of cardiopulmonary dirofilariosis in dogs and cats, which also infects a wide range of wild mammals and humans. The complex life cycle of D. immitis with several developmental stages in its invertebrate mosquito vectors and its vertebrate hosts indicates the importance of miRNA in growth and development, and their ability to regulate infection of mammalian hosts. This study identified the miRNA profiles of D. immitis of zoonotic significance by deep sequencing. A total of 1063 conserved miRNA candidates, including 68 anti-sense miRNA (miRNA*) sequences, were predicted by computational methods and could be grouped into 808 miRNA families. A significant bias towards family members, family abundance and sequence nucleotides was observed. Thirteen novel miRNA candidates were predicted by alignment with the Brugia malayi genome. Eleven out of 13 predicted miRNA candidates were verified by using a PCR-based method. Target genes of the novel miRNA candidates were predicted by using the heartworm transcriptome dataset. To our knowledge, this is the first report of miRNA profiles in D. immitis, which will contribute to a better understanding of the complex biology of this zoonotic filarial nematode and the molecular regulation roles of miRNA involved. Our findings may also become a useful resource for small RNA studies in other filarial parasitic nematodes.

Annotation of the Transcriptome from Taenia pisiformis and Its Comparative Analysis with Three Taeniidae Species

Background Taenia pisiformis is one of the most common intestinal tapeworms and can cause infections in canines. Adult T. pisiformis (canines as definitive hosts) and Cysticercus pisiformis (rabbits as intermediate hosts) cause significant health problems to the host and considerable socio-economic losses as a consequence. No complete genomic data regarding T. pisiformis are currently available in public databases. RNA-seq provides an effective approach to analyze the eukaryotic transcriptome to generate large functional gene datasets that can be used for further studies. Methodology/Principal Findings In this study, 2.67 million sequencing clean reads and 72,957 unigenes were generated using the RNA-seq technique. Based on a sequence similarity search with known proteins, a total of 26,012 unigenes (no redundancy) were identified after quality control procedures via the alignment of four databases. Overall, 15,920 unigenes were mapped to 203 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Through analyzing the glycolysis/gluconeogenesis and axonal guidance pathways, we achieved an in-depth understanding of the biochemistry of T. pisiformis. Here, we selected four unigenes at random and obtained their full-length cDNA clones using RACE PCR. Functional distribution characteristics were gained through comparing four cestode species (72,957 unigenes of T. pisiformis, 30,700 ESTs of T. solium, 1,058 ESTs of Eg+Em [conserved ESTs between Echinococcus granulosus and Echinococcus multilocularis]), with the cluster of orthologous groups (COG) and gene ontology (GO) functional classification systems. Furthermore, the conserved common genes in these four cestode species were obtained and aligned by the KEGG database. Conclusion This study provides an extensive transcriptome dataset obtained from the deep sequencing of T. pisiformis in a non-model whole genome. The identification of conserved genes may provide novel approaches for potential drug targets and vaccinations against cestode infections. Research can now accelerate into the functional genomics, immunity and gene expression profiles of cestode species.

Characterization and evaluation of a Sarcoptes scabiei allergen as a candidate vaccine

BackgroundSarcoptic mange caused by the mite Sarcoptes scabiei is a worldwide disease affecting both humans and animals. Here we report the molecular characterization and evaluation of a recombinant S. scabiei tropomyosin (SsTm) protein in a vaccination trial in rabbits.MethodsThe full-length cDNA was cloned in a bacterial pET vector, and the recombinant protein was expressed in BL21 (DE3) cells and purified. Using specific rabbit antiserum, tropomyosin was localized immunohistochemically in mite tissue sections. Vaccination trials with the recombiant SsTm was carried out in New Zealand rabbits.ResultsThe full-length open reading frame (ORF) of the 852 bp cloned gene from S. scabiei encodes a 32.9 kDa protein. The amino acid sequence showed 98.94%, 97.89% and 98.59% homology to Dermatophagoides farina and Dermatophagoides pteronyssinus group 10 allergens and Psoroptes ovis tropomyosin, respectively. Tropomyosin was localized immunohistochemically in mite tissue sections mainly in the mouthparts, legs and integument of the epidermis. The predicted cross-reactivity of SsTm indicated that it is an allergenic protein. While vaccination with the recombiant SsTm resulted in high levels of specific IgG (P < 0.01), a low IgE antibody response and no significant protection against S. scabiei challenge were observed. After challenge, specific IgG levels remained significantly higher than the control (P < 0.01), while changes of total IgE levels were not significant (P > 0.05). However, the lesion areas in the vaccination group decreased at the end of the experiment compared with controls.ConclusionsAlthough vaccination with recombinant SsTm did not efficiently control sarcoptic mange in rabbits, the immunogenic properties of tropomyosin suggest it may be developed as a vaccine with alternative adjuvants or delivery methods.

Detailed transcriptome description of the neglected cestode Taenia multiceps.

Background The larval stage of Taenia multiceps, a global cestode, encysts in the central nervous system (CNS) of sheep and other livestock. This frequently leads to their death and huge socioeconomic losses, especially in developing countries. This parasite can also cause zoonotic infections in humans, but has been largely neglected due to a lack of diagnostic techniques and studies. Recent developments in next-generation sequencing provide an opportunity to explore the transcriptome of T. multiceps. Methodology/Principal Findings We obtained a total of 31,282 unigenes (mean length 920 bp) using Illumina paired-end sequencing technology and a new Trinity de novo assembler without a referenced genome. Individual transcription molecules were determined by sequence-based annotations and/or domain-based annotations against public databases (Nr, UniprotKB/Swiss-Prot, COG, KEGG, UniProtKB/TrEMBL, InterPro and Pfam). We identified 26,110 (83.47%) unigenes and inferred 20,896 (66.8%) coding sequences (CDS). Further comparative transcripts analysis with other cestodes (Taenia pisiformis, Taenia solium, Echincoccus granulosus and Echincoccus multilocularis) and intestinal parasites (Trichinella spiralis, Ancylostoma caninum and Ascaris suum) showed that 5,100 common genes were shared among three Taenia tapeworms, 261 conserved genes were detected among five Taeniidae cestodes, and 109 common genes were found in four zoonotic intestinal parasites. Some of the common genes were genes required for parasite survival, involved in parasite-host interactions. In addition, we amplified two full-length CDS of unigenes from the common genes using RT-PCR. Conclusions/Significance This study provides an extensive transcriptome of the adult stage of T. multiceps, and demonstrates that comparative transcriptomic investigations deserve to be further studied. This transcriptome dataset forms a substantial public information platform to achieve a fundamental understanding of the biology of T. multiceps, and helps in the identification of drug targets and parasite-host interaction studies.

Novel Insights into the Transcriptome of Dirofilaria immitis

Background The heartworm Dirofilaria immitis is the causal agent of cardiopulmonary dirofilariosis in dogs and cats, and also infects a wide range of wild mammals as well as humans. One bottleneck for the design of fundamentally new intervention and management strategies against D. immitis may be the currently limited knowledge of fundamental molecular aspects of D. immitis. Methodology/Principal Findings A next-generation sequencing platform combining computational approaches was employed to assess a global view of the heartworm transcriptome. A total of 20,810 unigenes (mean length  = 1,270 bp) were assembled from 22.3 million clean reads. From these, 15,698 coding sequences (CDS) were inferred, and about 85% of the unigenes had orthologs/homologs in public databases. Comparative transcriptomic study uncovered 4,157 filarial-specific genes as well as 3,795 genes potentially involved in filarial-Wolbachia symbiosis. In addition, the potential intestine transcriptome of D. immitis (1,101 genes) was mined for the first time, which might help to discover ‘hidden antigens’. Conclusions/Significance This study provides novel insights into the transcriptome of D. immitis and sheds light on its molecular processes and survival mechanisms. Furthermore, it provides a platform to discover new vaccine candidates and potential targets for new drugs against dirofilariosis.

Identification of neglected cestode Taenia multiceps microRNAs by illumina sequencing and bioinformatic analysis

BackgroundWorldwide, but especially in developing countries, coenurosis of sheep and other livestock is caused by Taenia multiceps larvae, and zoonotic infections occur in humans. Infections frequently lead to host death, resulting in huge socioeconomic losses. MicroRNAs (miRNAs) have important roles in the post-transcriptional regulation of a large number of animal genes by imperfectly binding target mRNAs. To date, there have been no reports of miRNAs in T. multiceps.ResultsIn this study, we obtained 12.8 million high quality raw reads from adult T. multiceps small RNA library using Illumina sequencing technology. A total of 796 conserved miRNA families (containing 1,006 miRNAs) from 170,888 unique miRNAs were characterized using miRBase (Release 17.0). Here, we selected three conserved miRNA/miRNA* (antisense strand) duplexes at random and amplified their corresponding precursors using a PCR-based method. Furthermore, 20 candidate novel miRNA precursors were verified by genomic PCR. Among these, six corresponding T. multiceps miRNAs are considered specific for Taeniidae because no homologs were found in other species annotated in miRBase. In addition, 181,077 target sites within T. multiceps transcriptome were predicted for 20 candidate newly miRNAs.ConclusionsOur large-scale investigation of miRNAs in adult T. multiceps provides a substantial platform for improving our understanding of the molecular regulation of T. multiceps and other cestodes development.

Characterisation and analysis of thioredoxin peroxidase as a potential antigen for the serodiagnosis of sarcoptic mange in rabbits by dot-ELISA

BackgroundScabies caused by Sarcoptes scabiei is a widespread but a neglected tropical zoonosis. In this study, we characterised a S. scabiei thioredoxin peroxidase (SsTPx) and evaluated a recombinant SsTPx as a diagnostic antigen in rabbits.MethodsThe open reading frame of the gene encoding SsTPx-2 was amplified and the recombinant protein was expressed in Escherichia coli cells and purified. SsTPx was localized in mite tissue by immunolocalisation using the purified recombinant protein. Serodiagnosis assays were carried out in 203 New Zealand White rabbit serum samples by dot-ELISA.ResultThe open reading frame (489 bp) of the gene encodes an 18.11 kDa protein, which showed highly homology to that of Psoroptes cuniculi (98.77% identity) and belongs to the 2-Cys family of peroxiredoxins. SsTPx was mainly distributed in muscle tissues of mites, integument of the epidermis and the anterior end of S. scabiei. Although SsTPx cross-reactivity with psoroptic mites was observed, the SsTPx dot-ELISA showed excellent diagnostic ability, with 95.3% sensitivity and 93.8% specificity in mange-infected and uninfected groups.ConclusionsThis study showed that the purified SsTPx is a highly sensitive antigen for the diagnosis of mange infection by dot-ELISA. This technique is a rapid and convenient method that can be used worldwide for the clinical diagnosis of sarcoptic mange in rabbits, and is especially useful in developing regions.

Evaluating troponin C from Psoroptes cuniculi as a diagnostic antigen for a dot-ELISA assay to diagnose mite infestations in rabbits

The mite Psoroptes cuniculi is globally widespread and has a serious impact on commercial rabbit breeding. In China, diagnosis of P. cuniculi is currently based on conventional clinical methods that entail numerous disadvantages, including their failure to diagnose subclinical infections. Hence, alternative measures are required, and dot‐ELISA is one of the most promising strategies. We cloned and expressed the recombinant P. cuniculi troponin C gene for use as a basis for novel dot‐ELISA assay to detect P. cuniculi infections in rabbits. This amplified sequence encoded a 153 amino acid protein of 17·6 kDa and theoretical pI 4·18 without signal peptide. The recombinant troponin C of P. cuniculi is an outer membrane protein and may also be a new P. cuniculi allergen. Results of dot‐ELISA test showed that this novel assay had more than 90% sensitivity but low specificity in distinguishing infections with P. cuniculi or Sarcoptic scabiei, despite very high agreement between observers (97–99%; κ values ranged from 0·95 to 0·98 for inter‐ and intra‐observer variability test). This study showed that this novel method, at present, lacks diagnostic utility. Therefore, although simple serological assays such as dot‐ELISA show great promise as diagnostic tools, we suggest that troponin C is not a suitable diagnostic antigen candidate.

Vaccination with recombinant actin from scab mites and evaluation of its protective efficacy against Psoroptes cuniculi infection.

The mite Psoroptes cuniculi is globally widespread and has a serious impact on commercial rabbit breeding. Current treatment methods are based on chemotherapy. Because of the disadvantages of these methods, alternative measures are required, and vaccination is one of the most promising strategies. Here, we cloned and expressed the recombinant P. cuniculi actin gene (rPc‐act). Antiserum levels against rPc‐act in rabbits were used to locate actin distribution in mite sections. Challenge trials were carried out to evaluate the immunity protection of rPc‐act in rabbits, with antibody levels determined by ELISA. Sequence analysis of this gene fragment showed 89·26% and 84·91% identity to Sarcoptes scabiei and Mayetiola destructor sequences, respectively. Immunohistochemistry showed rPc‐act to locate widely throughout the mites, especially in feet and muscle tissues. Recombinant P. cuniculi actin with QuliA adjuvant was used to immunize six rabbits. Each animal was challenge‐infested with 25–50 adult mites. Although IgE levels showed no significant difference to controls, IgG levels were significantly higher, and clinical development showed no significantly different severity of lesions in vaccinated rabbits than in the controls. This study showed that rPc‐act is a muscular isotype actin and has no clinical protective efficacy against P. cuniculi.

Expression of Translationally Controlled Tumor Protein (TCTP) Gene of Dirofilaria immitis Guided by Transcriptomic Screening

Dirofilaria immitis (heartworm) infections affect domestic dogs, cats, and various wild mammals with increasing incidence in temperate and tropical areas. More sensitive antibody detection methodologies are required to diagnose asymptomatic dirofilariasis with low worm burdens. Applying current transcriptomic technologies would be useful to discover potential diagnostic markers for D. immitis infection. A filarial homologue of the mammalian translationally controlled tumor protein (TCTP) was initially identified by screening the assembled transcriptome of D. immitis (DiTCTP). A BLAST analysis suggested that the DiTCTP gene shared the highest similarity with TCTP from Loa loa at protein level (97%). A histidine-tagged recombinant DiTCTP protein (rDiTCTP) of 40 kDa expressed in Escherichia coli BL21 (DE3) showed immunoreactivity with serum from a dog experimentally infected with heartworms. Localization studies illustrated the ubiquitous presence of rDiTCTP protein in the lateral hypodermal chords, dorsal hypodermal chord, muscle, intestine, and uterus in female adult worms. Further studies on D. immitis-derived TCTP are warranted to assess whether this filarial protein could be used for a diagnostic purpose.