Jianping Tao

Apoptosis phenomenon in the schistosomulum and adult worm life cycle stages of Schistosoma japonicum

Apoptosis is an important aspect of a number of biological processes, from embryogenesis to the stress-injury response. It plays a central role in balancing cell proliferation and tissue remodeling activity in many organisms. In the present study, apoptosis in 14 days post infection schistosomula was evaluated using TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assays and DAPI staining. Additionally, flow cytometry using the Annexin V-FITC/propidium iodide (PI) (Annexin V/PI) assay confirmed the percentage of early apoptotic, late apoptotic, and necrotic cells in 14 and 23 days post infection worms. Conserved Domain Database (CDD) BLAST analysis and alignment analysis of known schistosome proteins demonstrated the feasibility of detecting the activity of caspase-3 and -7 using the caspase-3/7 Glo analysis assay. Analysis of caspase-3 and -7 activities in schistosome demonstrated that both caspases were active in each developmental stage of Schistosoma japonicum, but was highest in the 14 days post infection schistosomula. Additionally, the caspase peptide inhibitor (Z-VAD-FMK) inhibited the caspase-3/7 activity at all developmental stages examined. Therefore, we hypothesized that two main signaling pathways are involved in apoptosis in S. japonicum, the caspase cascade and the mitochondrial-initiated pathway. We have constructed a model of these two pathways, including how they may interact and their biological outcomes. qRT-PCR analyses of the gene expression profiles of apoptosis-related genes supported our hypothesis of the relationship between the apoptotic pathway and parasite development. The data presented here demonstrates that apoptosis is an important biological process for the survival and development of the schistosome, and identifies potential novel therapeutic targets.

Pathogenic effects of the coccidium Eimeria ninakohlyakimovae in goats.

Twenty-four coccidia-free goats were reared artificially in indoor cages and allocated to 6 groups of 4 animals each. At 20 days of age, goats in groups 1–3 received 104,105 and 106 sporulated oocysts of Eimeria ninakohlyakimovae per goat, respectively, each as a single dose. Goats in group 4 received daily doses increasing over a 3-week period, starting with 100/day for the first week, followed by 1000, and 10 000/day in weeks 2, 3, respectively. Goats in group 5 received 104 oocysts following a challenge dose of 106 oocysts on day 32. Goats in group 6 were kept as uninoculated controls. Infected animals showed diarrhoea and weight loss. Goats in group 4 showed longer periods of diarrhoea and patency than other infected goats. Goats in group 5 showed the same severe clinical signs as those in group 3 but produced very low oocyst output after a challenge dose. The diarrhoea was associated with a reduction in alkaline phosphatase activity and increases in packed cell volume and haemoglobin. No significant differences were found in serum aspartate aminotransferase, alanine aminotransferase, total protein, albumin, globulin, Na+, K+,Cl− between groups during 48 days after inoculation. There were no serum enzyme indications of damage to the liver. Histological examination performed 100 days after inoculation revealed that inoculated goats had mild subacute to chronic proliferative enteritis in the lower small intestine and the large intestine, and the mesenteric lymph nodes, gallbladders and livers also showed slight histological lesions. The results showed that E. ninakohlyakimovae was highly pathogenic.

MicroRNA expression profile in different tissues of BALB/c mice in the early phase of Schistosoma japonicum infection.

Schistosomiasis remains an important global public health problem that affects 200 million people in 76 countries. The molecular mechanisms of host-parasite interaction are complex, and in schistosome infection regulation of microRNA (miRNA) and the host micro-environment may be involved. In this study, an miRNA microarray was applied to investigate differences in miRNA expression in different tissues of mice before and 10 days post infection. In total, 220 miRNAs were detected in different tissues of the BALB/c mice before and after infection, including 8 miRNAs in liver, 8 in spleen and 28 in the lungs with up-regulated expression, and 3 miRNAs in liver, 5 in spleen and 28 in the lungs with down-regulated expression in mice 10 days post infection with schistosomes. The functions of these differentially expressed miRNAs are related mainly to the immune response, nutrient metabolism, cell differentiation, apoptosis, and signal pathways. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the differentially expressed miRNAs revealed that many important biological pathways are triggered by schistosome infection in BALB/c mice, such as the MAPK signaling pathway, insulin signaling pathway, Toll-like receptor signaling pathway and TGF-β signaling pathway.The results reveal that miRNAs may be an important regulator of schistosome-host interaction in the early phase of Schistosoma japonicum infection. The data presented here provide valuable information to increase understanding of the regulatory function of the miRNAs in the host micro-environment, as well as the mechanism of host-parasite interactions. This may be helpful in the search for potential new drugs, and for biomarkers of early S. japonicum infection applicable in the future control of schistosomiasis.

Comparison of the differential expression miRNAs in Wistar rats before and 10 days after S.japonicum infection

BackgroundWhen compared to the murine permissive host of Schistosoma japonicum, Wistar rats are less susceptible to Schistosoma japonicum infection, and are considered to provide a less suitable microenvironment for parasite growth and development. MicroRNAs (miRNAs), are a class of endogenous, non-coding small RNAs, that impose an additional, highly significant, level of gene regulation within eukaryotes.MethodsTo investigate the regulatory mechanisms provided by miRNA in the schistosome-infected rat model, we utilized a miRNA microarray to compare the progression of miRNA expression within different host tissues both before and 10 days after cercarial infection, in order to identify potential miRNAs with roles in responding to a schistosome infection.ResultsAmong the analysed miRNAs, 16 within the liver, 61 within the spleen and 10 within the lung, were differentially expressed in infected Wistar rats. Further analysis of the differentially expressed miRNAs revealed that many important signal pathways are triggered after infection with S. japonicum in Wistar rats. These include the signal transduction mechanisms associated with the Wnt and MAPK signaling pathways, cellular differentiation, with a particular emphasis on adipocyte and erythroid differentiation.ConclusionsThe results presented here include the identification of specific differentially expressed miRNAs within the liver, lungs and spleen of Wistar rats. These results highlighted the function of host miRNA regulation during an active schistosome infection. Our study provides a better understanding of the regulatory role of miRNA in schistosome infection, and host–parasite interactions in a non-permissive host environment.

Cloning and characterization of an Eimeria necatrix gene encoding a gametocyte protein and associated with oocyst wall formation

BackgroundGametocyte proteins of Eimeria (E.) spp. are important components of the oocyst wall and some have been used to develop transmission-blocking vaccines against avian coccidiosis.MethodsTotal RNA isolated from E. necatrix gametocytes was utilized as templates for RT-PCR amplification and sequencing of cDNA encoding a gametocyte protein using gene-specific primers. The cDNA was cloned into the bacterial expression vector pET28a(+) and expressed in E. coli BL21 cells. The antigenicity of the recombinant gametocyte protein and its localization in different E. necatrix life-cycle stages were determined by western blot and indirect immunofluorescence analyses, respectively.ResultsA 731-nucleotide sequence of cDNA [GenBank: KF649255] of E. necatrix had 97.7% identity to that of Etgam22 of E. tenella. The cDNA ORF encoded a 186-amino acid protein containing a histidine-proline-rich region. The recombinant gametocyte protein (rEnGAM22) was predominately expressed in the insoluble inclusion body and recognized by antiserum from chickens immunized with oocysts of E. necatrix, E. maxima and E. tenella. A specific antibody to the rEnGAM22 protein recognized the wall-forming bodies in macrogametocytes and the walls of oocysts and sporocysts.ConclusionsThe gene cloned from E. necatrix gametocytes is an ortholog to Etgam22 of E. tenella and presents a potential target for future recombinant subunit vaccines against coccidiosis.

Loop-mediated isothermal amplification assay for detection of Histomonas meleagridis infection in chickens targeting the 18S rRNA sequences

Histomonas meleagridis is the causative agent of histomonosis, a disease of gallinaceous fowl characterized by necrotic typhlitis, hepatitis, and high mortality. To develop a rapid and sensitive method for specific detection of H. meleagridis, an assay based on loop-mediated isothermal amplification (LAMP) targeting the 18S rRNA gene was established. The detection limit of the LAMP assay was 10 copies for standard plasmids containing an 18S rRNA gene fragment, which was superior to that of a classical PCR method. Specificity tests revealed that there was no cross-reaction with other protozoa such as Trichomonas gallinae, Blastocytis sp, Tetratrichomonas gallinarum, Plasmodium gallinaceum, Toxoplasma gondii, Eimeria tenella, Leucocytozoon caulleryi and Leucocytozoon sabrazesi. The assay was evaluated for its diagnostic utility using liver and caeca samples collected from suspected field cases, the detection rate was 100 and 97.92%, respectively. These results indicate that the LAMP assay may be a useful tool for rapid detection and identification of H. meleagridis in poultry.

Differential expression of microRNAs in the non-permissive schistosome host Microtus fortis under schistosome infection.

The reed vole Microtus fortis is the only mammal known in China in which the growth, development and maturation of schistosomes (Schistosoma japonicum) is prevented. It might be that the anti-schistosomiasis mechanisms of M. fortis associate with microRNA-mediated gene expression, given that the latter has been found to be involved in gene regulation in eukaryotes. In the present study, the difference between pathological changes in tissues of M. fortis and of mice (Mus musculus) post-schistosome infection were observed by using hematoxylin-eosin staining. In addition, microarray technique was applied to identify differentially expressed miRNAs in the same tissues before and post-infection to analyze the potential roles of miRNAs in schistosome infection in these two different types of host. Histological analyses showed that S. japonicum infection in M. fortis resulted in a more intensive inflammatory response and pathological change than in mice. The microarray analysis revealed that 162 miRNAs were expressed in both species, with 12 in liver, 32 in spleen and 34 in lung being differentially expressed in M. fortis. The functions of the differentially expressed miRNAs were mainly revolved in nutrient metabolism, immune regulation, etc. Further analysis revealed that important signaling pathways were triggered after infection by S. japonicum in M. fortis but not in the mice. These results provide new insights into the general mechanisms of regulation in the non-permissive schistosome host M. fortis that exploits potential miRNA regulatory networks. Such information will help improve current understanding of schistosome development and host–parasite interactions.

Comparative transcriptome analysis of second- and third-generation merozoites of Eimeria necatrix

BackgroundEimeria is a common genus of apicomplexan parasites that infect diverse vertebrates, most notably poultry, causing serious disease and economic losses. Eimeria species have complex life-cycles consisting of three developmental stages. However, the molecular basis of the Eimeria reproductive mode switch remains an enigma.MethodsTotal RNA extracted from second- (MZ-2) and third-generation merozoites (MZ-3) of Eimeria necatrix was subjected to transcriptome analysis using RNA sequencing (RNA-seq) followed by qRT-PCR validation.ResultsA total of 6977 and 6901 unigenes were obtained from MZ-2 and MZ-3, respectively. Approximately 2053 genes were differentially expressed genes (DEGs) between MZ-2 and MZ-3. Compared with MZ-2, 837 genes were upregulated and 1216 genes were downregulated in MZ-3. Approximately 95 genes in MZ-2 and 48 genes in MZ-3 were further identified to have stage-specific expression. Gene ontology category and KEGG analysis suggested that 216 upregulated genes in MZ-2 were annotated by 70 GO assignments, 242 upregulated genes were associated with 188 signal pathways, while 321 upregulated genes in MZ-3 were annotated by 56 GO assignments, 322 upregulated genes were associated with 168 signal pathways. The molecular functions of upregulated genes in MZ-2 were mainly enriched for protein degradation and amino acid metabolism. The molecular functions of upregulated genes in MZ-3 were mainly enriched for transcriptional activity, cell proliferation and cell differentiation.ConclusionsTo the best of our knowledge, this is the first RNA-seq data study of the MZ-2 and MZ-3 stages of E. necatrix; it demonstrates a high number of differentially expressed genes between the MZ-2 and MZ-3 of E. necatrix. This study forms a basis for deciphering the molecular mechanisms underlying the shift from the second to third generation schizogony in Eimeria. It also provides valuable resources for future studies on Eimeria, and provides insight into the understanding of reproductive mode plasticity in different Eimeria species.

Comparative transcriptome analysis of Eimeria necatrix third-generation merozoites and gametocytes reveals genes involved in sexual differentiation and gametocyte development

Eimeria necatrix is one of the most pathogenic parasites causing high mortality in chicken older than 8 weeks. Eimeria spp. possess a coccidian lifecycle including both sexual and asexual stages. Sexual differentiation and development occupies a central place in the life cycle of the Eimeria parasite. However, our knowledge of the sexual differentiation and gametocyte development of Eimeria is very limited. Here using RNA sequencing, we conducted a comparative transcriptome analysis between third-generation merozoites (MZ-3) and gametocytes (GAM) of E. necatrix to identify genes with functions related to sexual differentiation and gametocyte development. Approximately 4267 genes were differentially expressed between MZ-3 and GAM. Compared with MZ-3, 2789 genes were upregulated and 1478 genes were downregulated in GAM. Approximately 329 genes in MZ-3 and 1289 genes in GAM were further analyzed in the evaluation of stage-specific genes. Gene Ontology (GO) classification and KEGG analysis revealed that 953 upregulated gametocyte genes were annotated with 170 GO assignments, and 405 upregulated genes were associated with 231 signaling pathways. We also predicted a further 83 upregulated gametocyte genes, of which 53 were involved in the biosynthesis of the oocyst wall, and 30 were involved in microgametocyte development. This information offers insights into the mechanisms governing the sexual development of E. necatrix and may potentially allow the identification of targets for blocking parasite transmission.

A case report of Apatemon gracilis (Szidat, 1928) infection in domestic geese in mainland China

This is the first report of the diagnosis and treatment of Apatemon gracilis infection in domestic geese in mainland China confirmed at the molecular level. The mortality of geese reached 10% (150/1500). Necropsy examinations confirmed hemorrhagic inflammation of the small intestine and blood and catarrhal mucus in the enteric cavity. A large number of flukes were found in the first third of the small intestine, with one end buried in the intestinal mucosa, forming lesions at the absorption sites. The ITS-1 sequence of the parasites was amplified by polymerase chain reaction and sequence analysis confirmed that the isolated worms were Apatemon gracilis (Szidat, 1928). Expulsion of the parasites with praziquantel effectively controlled the disease on the goose farm.