Deying Yang

The mitochondrial genome of Baylisascaris procyonis.

Background Baylisascaris procyonis (Nematoda: Ascaridida), an intestinal nematode of raccoons, is emerging as an important helminthic zoonosis due to serious or fatal larval migrans in animals and humans. Despite its significant veterinary and public health impact, the epidemiology, molecular ecology and population genetics of this parasite remain largely unexplored. Mitochondrial (mt) genomes can provide a foundation for investigations in these areas and assist in the diagnosis and control of B. procyonis. In this study, the first complete mt genome sequence of B. procyonis was determined using a polymerase chain reaction (PCR)-based primer-walking strategy. Methodology/Principal Findings The circular mt genome (14781 bp) of B. procyonis contained 12 protein-coding, 22 transfer RNA and 2 ribosomal RNA genes congruent with other chromadorean nematodes. Interestingly, the B. procyonis mtDNA featured an extremely long AT-rich region (1375 bp) and a high number of intergenic spacers (17), making it unique compared with other secernentean nematodes characterized to date. Additionally, the entire genome displayed notable levels of AT skew and GC skew. Based on pairwise comparisons and sliding window analysis of mt genes among the available 11 Ascaridida mtDNAs, new primer pairs were designed to amplify specific short fragments of the genes cytb (548 bp fragment) and rrnL (200 bp fragment) in the B. procyonis mtDNA, and tested as possible alternatives to existing mt molecular beacons for Ascaridida. Finally, phylogenetic analysis of mtDNAs provided novel estimates of the interrelationships of Baylisasaris and Ascaridida. Conclusions/Significance The complete mt genome sequence of B. procyonis sequenced here should contribute to molecular diagnostic methods, epidemiological investigations and ecological studies of B. procyonis and other related ascaridoids. The information will be important in refining the phylogenetic relationships within the order Ascaridida and enriching the resource of markers for systematic, population genetic and evolutionary biological studies of parasitic nematodes of socio-economic importance.

Acaricidal activity of extract from Eupatorium adenophorum against the Psoroptes cuniculi and Sarcoptes scabiei in vitro

The possible acaricidal activity of Eupatorium adenophorum was analyzed using extracts created by water decocting, ethanol thermal circumfluence, and steam distillation. The toxic effect of each extract was tested against Psoroptes cuniculi and Sarcoptes scabiei in vitro. Ethanol thermal circumfluence extract had strong toxicity against mites, killing all S. scabiei at 0.5 and 1.0 g/ml (w/v) concentration, while 1g/ml extract was also found to kill all P. cuniculi within a 4-h period. Similarly, 0.25, 0.5 and 1.0 g/ml concentration of extract had strong toxicity against S. scabiei, with median lethal time (LT(50)) values at 0.866, 0.785 and 0.517 h, respectively. 0.5 g/ml and 1g/ml showed strong acaricidal action against P. cuniculi; the LT(50) values were 0.93 h and 1.29 h, respectively. The median lethal concentration (LC(50)) values were 0.22 g/ml for Scabies mite and 0.64 g/ml for P. cuniculi in 1h. The results indicated that E. adenophorum contains potent acaricidal ingredients; as a first step in the potential development of novel drugs, it may provide new acaricidal compounds for the effective control of animal acariasis.

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.

Genetic variability of Echinococcus granulosus from the Tibetan plateau inferred by mitochondrial DNA sequences

To analyse genetic variability and population structure, 84 isolates of Echinococcus granulosus (Cestoda: Taeniidae) collected from various host species at different sites of the Tibetan plateau in China were sequenced for the whole mitochondrial nad1 (894 bp) and atp6 (513 bp) genes. The vast majority were classified as G1 genotype (n=82), and two samples from human patients in Sichuan province were identified as G3 genotype. Based on the concatenated sequences of nad1+atp6, 28 different haplotypes (NA1-NA28) were identified. A parsimonious network of the concatenated sequence haplotypes showed star-like features in the overall population, with NA1 as the major haplotype in the population networks. By AMOVA it was shown that variation of E. granulosus within the overall population was the main pattern of the total genetic variability. Neutrality indexes of the concatenated sequence (nad1+atp6) were computed by Tajimas D and Fus Fs tests and showed high negative values for E. granulosus, indicating significant deviations from neutrality. FST and Nm values suggested that the populations were not genetically differentiated.

Clinical efficacy of botanical extracts from Eupatorium adenophorum against the scab mite, Psoroptes cuniculi.

This study evaluated the in vivo clinical efficacy of Crofton weed (Eupatorium adenophorum) extracts against the scab mite, Psoroptes cuniculi. A 30-day experiment was performed using New Zealand rabbits that were naturally infested with P. cuniculi on a farm. Rabbits were randomly divided into five groups (6 animals per group); animals in groups A, B and C were treated in each ear topically with 2 ml of 1.0, 0.5 and 0.25 g/ml (w/v) E. adenophorum ethanol extract, respectively. Animals in groups D and E were treated with ivermectin (by injection; positive controls) and glycerol with water only (by embrocation; negative controls), respectively. Each rabbit was treated twice with separate treatments on days 0 and 7. Rabbits were observed daily and detailed examinations were performed on days 0, 7, 14 and 30, to inspect the presence or absence of mites and scabs/crusts. Clinical infection and the degree of recovery were evaluated, and the rate of reduction in mites and clinical efficacy rate (%) were calculated. The clinical effect of treatment with E. adenophorum extracts was similar to treatment with ivermectin. Seven days after the initial treatment, the mean clinical scores (presence of scabs/crusts) decreased from 3.32, 3.08 and 3.17 to 0.37, 0.47 and 0.48 in the left ears of animals in groups A, B and C, respectively, and from 3.53, 3.73 and 3.67 to 0.40, 0.45 and 0.48 in the right ears of animals in groups A, B and C, respectively, which were similar to the observations recorded in the positive control rabbits. However, the clinical score for negative control rabbits did not decrease significantly (P>0.05) during the experiment, and this changed from 3.32 to 2.75 in the left ears and from 3.50 to 3.25 in the right ears, and there were no significant differences in clinical efficacy between left and right ears. After two treatments (7 days space), the rabbits in groups A, B, C and D had recovered completely 30 days after the last treatment and no recurrences of infection were observed. These results indicate that E. adenophorum contains potent compounds for the effective control of animal acariasis.

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.

Rapamycin preserves gut homeostasis during Drosophila aging

Gut homeostasis plays an important role in maintaining the overall body health during aging. Rapamycin, a specific inhibitor of mTOR, exerts prolongevity effects in evolutionarily diverse species. However, its impact on the intestinal homeostasis remains poorly understood. Here, we demonstrate that rapamycin can slow down the proliferation rate of intestinal stem cells (ISCs) in the aging guts and induce autophagy in the intestinal epithelium in Drosophila. Rapamycin can also significantly affect the FOXO associated genes in intestine and up-regulate the negative regulators of IMD/Rel pathway, consequently delaying the microbial expansion in the aging guts. Collectively, these findings reveal that rapamycin can delay the intestinal aging by inhibiting mTOR and thus keeping stem cell proliferation in check. These results will further explain the mechanism of healthspan and lifespan extension by rapamycin in Drosophila.

Clinical efficacy of botanical extracts from Eupatorium adenophorum against the Sarcoptes scabiei (Sarcoptidae: Sarcoptes) in rabbits

The aims of present study were to evaluate the therapeutic efficacy of extracts from Eupatorium adenophorum against Sarcoptes scabiei. A 30-day experiment was performed using New Zealand rabbits that were naturally infested with S. scabiei in the toes (n=30) or artificially infected in the external ear margin with S. scabiei (n=30). Rabbits were randomly divided into five groups (6 animals per group, A-E groups for rabbits of naturally infested and F-J groups for artificially infected rabbits), respectively. All 60 rabbits were treated twice on days 0 and 7 successively. Animals in groups A/F, B/G, and C/H were treated on each toe/external ear margin with topical E. adenophorum ethanol extract at 1.0, 0.5 and 0.25 g/ml (w/v), respectively. Animals in groups D/I and E/J were treated with ivermectin by injections (positive controls) or by glycerol with water only rubbed onto the affected area (negative controls). After two treatments with extracts of E. adenophorum with relatively high concentrations of 0.5 and 1g/ml, the S. scabiei was completely eliminated in rabbits between days 14 and 30. Our results showed that rabbits treated with ivermectin (positive controls) and those treated with the extracts of concentrations of 1.0 or 0.5 g/ml achieved remarkable therapeutic efficacy; no mites were present in toes of rabbits in these groups on day 14, which confirmed a 100% therapeutic efficacy rate up to day 30 of the end of the trial. The clinical effects of treatment with 1.0 and 0.5 g/ml E. adenophorum extracts (groups A and B) were similar to ivermectin treatment. However, the therapeutic efficacy in group C and E rabbits only reached 43.25% and 7.13% by day 14. Furthermore, the therapeutic efficacy improved slightly by the end of the experiment on day 30, and rabbits in groups F, G and I also achieved good efficacy according to the recovery scoring criteria. These results indicate that E. adenophorum contains potent compounds for the effective control of sarcoptidosis.

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.