Global profiling of the proteome, phosphoproteome, and N-glycoproteome of protoscoleces and adult worms of Echinococcus granulosus.

Introduction: Cystic echinococcosis (CE) is a chronic zoonosis caused by infection with the metacestode of the Echinococcus granulosus. A unique characteristic of E. granulosus protoscolex (PSC) is their ability to develop bidirectionally into an adult worm in the definitive host or a secondary hydatid cyst in the intermediate host. Furthermore, cestodes have a complex life cycle involving different developmental stages; however, the mechanisms underlying this development remain unknown. Several studies have demonstrated that certain matrix proteins undergo posttranslational modifications (PTMs), including phosphorylation and glycosylation, which have important regulatory effects on their functional properties. Methods: Systematic analyses of the proteome, phosphorylated modified proteome, and glycosylated modified proteome of protoscoleces (PSCs) and adult worms were performed using a proteomic strategy. Data are available via ProteomeXchange with identifier PXD043166. Results: In total, 6,407 phosphorylation sites and 1757 proteins were quantified. Of these, 2032 phosphorylation sites and 770 proteins were upregulated, and 2,993 phosphorylation sites and 1,217 proteins were downregulated in adult worms compared to PSCs. A total of 612 N-glycosylation sites were identified in the 392 N-glycoproteins. Of these, 355 N-glycosylation sites and 212 N-glycoproteins were quantified. Of these, 90 N-glycosylation sites and 64 N-glycoproteins were upregulated, and 171 N-glycosylation sites and 126 N-glycoproteins were downregulated in adult worms compared to PSCs. GO enrichment analysis indicated that the differentially expressed phosphoproteins were mainly enriched in the regulation of oxidoreduction coenzyme metabolic processes, myelin sheath, and RNA helicase activity, whereas the differentially expressed N-glycoproteins were enriched in the cellular response to unfolded proteins, endoplasmic reticulum lumen, and nucleic acid binding. KEGG enrichment analysis indicated that the differently expressed phosphoproteins were mainly enriched in RNA transport, hypertrophic cardiomyopathy (HCM), glycolysis/gluconeogenesis, HIF-1 signaling pathway and pyruvate metabolism. Differentially expressed N-glycoproteins were enriched in the PI3K-Akt signaling pathway, ECM-receptor interactions, and protein processing in the endoplasmic reticulum. Discussion: To our knowledge, this study is the first global phosphoproteomic and N-glycoproteomic analysis of E. granulosus, which provides valuable information on the expression characteristics of E. granulosus and provides a new perspective to elucidate the role of protein phosphorylation and N-glycosylation in the development of E. granulosus.

Molecular evaluation of Cryptosporidium spp. in sheep in southern Xinjiang, China.

Cryptosporidium spp. are diarrheagenic intestinal parasites with multiple hosts worldwide. A total of 1252 fresh fecal samples of sheep were collected from 10 large-scale farms in southern Xinjiang. Based on the small subunit ribosomal (SSU rRNA) gene of Cryptosporidium, 100 Cryptosporidium-positive samples (8.0%, 100/1252) were detected by PCR. Nine out of 10 farms were positive for Cryptosporidium, with the highest infection rate being 18.4% (23/125) on farm 9 in Qira. The infection rates of Cryptosporidium in pre-weaned lambs, weaned lambs, fattening sheep, and adult sheep were 20.3% (61/301), 10.3% (34/329), 0.9% (3/327), and 0.7% (2/295), respectively. Three Cryptosporidium species were identified, namely, C. xiaoi (n = 61), C. parvum (n = 22), and C. ubiquitum (n = 17). Of them, C. xiaoi was detected on all positive farms and in different age groups of sheep. The subtypes of C. parvum and C. ubiquitum were identified by PCR at the 60 kDa glycoprotein (gp60) gene. Two C. parvum subtypes were identified: IIdA19G1 (n = 21) and IIdA15G1 (n = 1). One C. ubiquitum subtype was identified with XIIa (n = 17). These results indicated the common transmission and genetic diversity of Cryptosporidium in sheep in southern Xinjiang, and further investigations are needed on the zoonotic potential of C. parvum and C. ubiquitum in this region.

Molecular Characterization of a Tetraspanin TSP11 Gene in Echinococcus granulosus and Evaluation Its Immunoprotection in Model Dogs.

Cystic echinococcosis (CE) is a cosmopolitan zoonosis caused by the larval stage of Echinococcus granulosus, which affects humans and a wide range of mammalian intermediate hosts. Parasite tetraspanin proteins are crucial for host-parasite interactions, and therefore they may be useful for vaccine development or disease diagnosis. In the present study, the major antigen coding sequence of tetraspanin 11 (Eg-TSP11) from E. granulosus was determined. The results of immunolocalization showed that Eg-TSP11 was mainly located in the tegument of adult worms and protoscoleces. Western blotting analysis showed that the serum from dogs injected with recombinant Eg-TSP11 (rEg-TSP11) could recognize Eg-TSP11 among natural protoscolex proteins. Moreover, the serum from dogs with E. granulosus infection also recognized rEg-TSP11. Serum indirect enzyme-linked immunosorbent assays demonstrated that IgG levels gradually increased after the first immunization with rEg-TSP11 compared with those in the control group. Furthermore, the serum levels of interleukin 4, interleukin 5, and interferon gamma were significantly altered in the rEg-TSP11 group. Importantly, we found that vaccination with rEg-TSP11 significantly decreased worm burden and inhibited segment development in a dog model of E. granulosus infection. Based on these findings, we speculated that rEg-TSP11 might be a potential candidate vaccine antigen against E. granulosus infection in dogs.

Molecular characterization and immune protection of the 3-hydroxyacyl-CoA dehydrogenase gene in Echinococcus granulosus.

BACKGROUND: Cystic echinococcosis (CE) is a serious parasitic zoonosis caused by the larvae of the tapeworm Echinococcus granulosus. The development of an effective vaccine is one of the most promising strategies for controlling CE. METHODS: The E. granulosus 3-hydroxyacyl-CoA dehydrogenase (EgHCDH) gene was cloned and expressed in Escherichia coli. The distribution of EgHCDH in protoscoleces (PSCs) and adult worms was analyzed using immunofluorescence. The transcript levels of EgHCDH in PSCs and adult worms were analyzed using quantitative real-time reverse transcription PCR (RT-qPCR). The immune protective effects of the rEgHCDH were evaluated. RESULTS: The 924-bp open reading frame sequence of EgHCDH, which encodes a protein of approximately 34 kDa, was obtained. RT-qPCR analysis revealed that EgHCDH was expressed in both the PSCs and adult worms of E. granulosus. Immunofluorescence analysis showed that EgHCDH was mainly localized in the tegument of PSCs and adult worms. Western blot analysis showed that the recombinant protein was recognized by E. granulosus-infected dog sera. Animal challenge experiments demonstrated that dogs immunized with recombinant (r)EgHCDH had significantly higher serum IgG, interferon gamma and interleukin-4 concentrations than the phosphate-buffered saline (PBS) control group. The rEgHCDH vaccine was able to significantly reduce the number of E. granulosus and inhibit the segmental development of E. granulosus compared to the PBS control group. CONCLUSIONS: The results suggest that rEgHCDH can induce partial immune protection against infection with E. granulosus and could be an effective candidate for the development of new vaccines.

De Novo Sequencing and High-Contiguity Genome Assembly of Moniezia expansa Reveals Its Specific Fatty Acid Metabolism and Reproductive Stem Cell Regulatory Network.

Moniezia expansa (M. expansa) parasitizes the small intestine of sheep and causes inhibited growth and development or even death. Being globally distributed, it causes considerable economic losses to the animal husbandry industry. Here, using Illumina, PacBio and BioNano techniques, we obtain a high-quality genome assembly of M. expansa, which has a total length of 142 Mb, a scaffold N50 length of 7.27 Mb and 8,104 coding genes. M. expansa has a very high body fat content and a specific type of fatty acid metabolism. It cannot synthesize any lipids due to the loss of some key genes involved in fatty acid synthesis, and it may can metabolize most lipids via the relatively complete fatty acid ß-oxidation pathway. The M. expansa genome encodes multiple lipid transporters and lipid binding proteins that enable the utilization of lipids in the host intestinal fluid. Although many of its systems are degraded (with the loss of homeobox genes), its reproductive system is well developed. PL10, AGO, Nanos and Pumilio compose a reproductive stem cell regulatory network. The results suggest that the high body lipid content of M. expansa provides an energy source supporting the high fecundity of this parasite. Our study provides insight into host interaction, adaptation, nutrient acquisition, strobilization, and reproduction in this parasite and this is also the first genome published in Anoplocephalidae.

Prevalence and distribution of Echinococcus spp. in wild and domestic animals across Africa: A systematic review and meta-analysis.

Cystic echinococcosis (CE), a worldwide zoonosis, is highly prevalent in Africa particularly in northern and eastern Africa where data are more abundant than other regions. However, harmonization of available data through systematic review and meta-analysis may foster improved transboundary cooperation for the control of CE in Africa. Using the Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines, research articles (from 2000 to 2019) were retrieved from ScienceDirect, PubMed, African Journals OnLine and Google Scholar databases. A total of 98 studies of 806,624 animals from 13 countries comprising 264,016 goats, 247,326 sheep, 251,106 cattle, 28,314 camels, 4,764 buffaloes, 2,920 equids, 1,966 pigs, 408 wild boars and 50 Norway rats were available for systematic review and meta-analysis of pooled prevalence including 5,048 dogs, 345 lions, 220 hyenas, 94 wolves and 47 jackals/foxes analysed for Echinococcus infection. In total, 46,869 animals were infected and pooled prevalence of CE in intermediate hosts was highest in camels (17.1%; 95% CI: 12.1-22.8) and lowest in pigs (0.3%; 95% CI: 0.1-0.6). Results also showed uneven species/genotype distribution across the continent such that Echinococcus granulosus sensu stricto (G1, G3) constituted 74.45% of the total isolates from East Africa, E. canadensis (G6/7) accounted for 60.3% and 97.4% in North and West Africa, respectively, while 81.3% of E. ortleppi (G5) were recorded for southern Africa. The comparatively higher prevalence estimates for eastern and northern Africa than other regions indicate where efforts on CE management should now be given greater attention in Africa. Additionally, this study also advocates for better cooperation between countries within the same sub-region and the establishment of joint CE control programmes.

Evaluation of dynamic developmental processes and the molecular basis of the high body fat percentage of different proglottid types of Moniezia expansa.

BACKGROUND: Moniezia expansa (Cyclophyllidea: Anoplocephalidae) is a large species of tapeworm that occurs in sheep and cattle and inhabits the small intestine, causing diarrhea and weight declines, leading to stockbreeding losses. Interestingly, the body fat percentage of M. expansa, which lacks the ability to synthesize fatty acids, is as high as 78% (dry weight) and all of the proglottids of M. expansa exhibit a dynamic developmental process from top to bottom. The aim of this paper is to identify the molecular basis of this high body fat percentage, the dynamic expression of developmental genes and their expression regulation patterns. RESULTS: From 12 different proglottids (four sections: scolex and neck, immature, mature and gravid with three replicates), 13,874 transcripts and 680 differentially expressed genes (DEGs) were obtained. The gene expression patterns of the scolex and neck and immature proglottids were very similar, while those of the mature and gravid proglottids differed greatly. In addition, 13 lipid transport-related proteins were found in the DEGs, and the expression levels showed an increasing trend in the four proglottid types. Furthermore, it was shown that 33 homeobox genes, 9 of which were DEGs, had the highest expression in the scolex and neck section. The functional enrichment results of the DEGs were predominantly indicative of development-related processes, and there were also some signal transduction and metabolism results. The most striking result was the finding of Wnt signaling pathways, which appeared multiple times. Furthermore, the weighted gene co-expression networks were divided into 12 modules, of which the brown module was enriched with many development-related genes. CONCLUSIONS: We hypothesize that M. expansa uses lipid transport-associated proteins to transport lipids from the host gut to obtain energy to facilitate its high fecundity. In addition, homeobox genes and Wnt signaling pathways play a core role in development and regeneration. The results promote research on the cell differentiation involved in the continuous growth and extension of body structures.

Tick distribution in border regions of Northwestern China.

Ticks are important vectors of emerging and re-emerging pathogens. The aim of this study was to determine tick species occurring in Xinjiang Uygur Autonomous Region (XUAR), especially on border regions. A total of 22,994 ticks (including 22,629 adults, 365 larvae and nymphs), belonging to six tick genera (i.e. Dermacentor, Hyalomma, Rhipicephalus, Haemaphysalis, Ixodes and Argas) and fourteen tick species, were collected from ten animal hosts in thirty-five counties (cities) in XUAR during 2011 - 2017. Rhipicephalus turanicus, Dermacentor niveus, Hyalomma asiaticum and Dermacentor marginatus were dominantly sampled from domestic animals while Dermacentor nuttalli, Haemaphysalis punctata, Haemaphysalis concinna, Rhipicephalus sanguineus sensu lato, Dermacentor silvarum, Hyalomma scupense and Argas persicus were sporadically found. Based on 16S rDNA, phylogenetic analyses showed that: i) R. turanicus genotypes in XUAR showed geographical separation, and belonged to clade I (major distribution in the Central Asian) rather than clade II (major distribution in the Mediterranean Basin); ii) Ixodes kaiseri, firstly sampled from Asian badgers (Meles leucurus), was in ancestral position compared to European tick species when combining COI haplotypes; and iii) Haemaphysalis erinacei from marbled polecats in China was a separate genotype compared with that in Mediterranean and Europe. Our findings suggest that geographical range plays a more important role than host-association in tick phylogeny, especially for R. turanicus, I. kaiseri and H. erinacei.

Systematic Analysis of Long Non-Coding RNAs and mRNAs in the Ovaries of Duroc Pigs During Different Follicular Stages Using RNA Sequencing.

The dynamic process involving the selection and maturation of follicles is regulated and controlled by a highly synchronized and exquisitely timed cascade of gene expression. Studies have shown that long non-coding RNA (lncRNA) is essential for the normal maintenance of animal reproductive function and has an important regulatory function in ovarian development and hormone secretion. In this study, a total of 2076 lncRNAs (1362 known lncRNAs and 714 new lncRNAs) and 25,491 mRNAs were identified in libraries constructed from Duroc ovaries on days 0, 2 and 4 of follicle development. lncRNAs were shorter, had fewer exons, exhibited a shorter ORF (Open Reading Frame) length and lower expression levels, and were less conserved than mRNAs. Furthermore, 1694 transcripts (140 lncRNAs and 1554 mRNAs) were found to be differentially expressed in pairwise comparisons. A total of 6945 co-localized mRNAs were detected in cis in 2076 lncRNAs. The most enriched GO (Gene Ontology) terms were related to developmental processes. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis revealed that the differentially expressed lncRNAs targeted mRNAs, and the differentially expressed mRNAs were related to the TGF-ß signaling pathway, the PI3K-Akt signaling pathway, the Retinol metabolic pathway and the Wnt signaling pathway. This study deepened our understanding of the genetic basis and molecular mechanisms of follicular development in pigs.

Identification of emu-TegP11, an EF-hand domain-containing tegumental protein of Echinococcus multilocularis.

Tegumental proteins (TegPs) are a group of proteins that coat on the surface of worms, mainly being involved in ion uptake and immune evasion. Echinococcus species have many TegPs, but none of them have been characterized and their role remains unclear. The genome-wide analysis revealed that there were at least 14 tegp genes (tegp1 - 14) in Echinococcus species, the majority of which were found to contain an EF-hand domain or a dynein light chain-like domain or both. Despite low identity, all TegP11 proteins from 25 flatworms were conserved in structure. Echinococcus multilocularis TegP11 (emu-TegP11) was verified to be secreted by extracellular vesicles and to be localized in different spatiotemporal patterns in protoscoleces. Moreover, emu-TegP11 was also shown to have weak or no Ca2+-binding capacity. In treated macrophages, emu-TegP11 interfered with the small RNA-induced silencing pathway via inducing ectopic expression of some key component genes. Additionally, emu-TegP11 remarkably promoted NO secretion possibly by upregulation of inos gene expression (p < 0.05). It was further shown that emu-TegP11 acted as a suppressor of inflammation, with il-12B and il-1ß being significantly down-regulated (p < 0.01), and il-10 and il-4 being significantly upregulated (p < 0.05). The study demonstrates a regulatory role of emu-TegP11, likely acting as a immunomodulator to be involved in regulation of host immune system during Echinococcus infection.

[Differential Expression of Six wnt Gene Family Members in Echinococcus granulosus Protoscoleces and Adult Worms].

Objective: To investigate the differential mRNA expression and tissue distribution of wnt [wingless-type mouse mammary tumor virus (MMTV) integration site family, wnt] gene members wnt1, wnt2, wnt4, wnt5, wnt11A and wnt11B in protoscoleces and adult worms of Echinococcus granulosus. Methods: The mRNA expression of wnt1, wnt2, wnt4, wnt5, wnt11A and wnt11B was determined by qRT-PCR. Tissue distribution of wnt1, wnt2, wnt4, wnt5, wnt11A and wnt11B in Echinococcus granulosus protoscoleces was determined by the whole-mount in situ hybridization. Results: The qRT-PCR results showed that the mRNA expression levels of wnt1 and wnt2 in the adult worms were 1.49 （P>0.05） and 2.53 folds（P<0.05） of those in the protoscoleces, respectively. The mRNA expression levels of wnt4, wnt5, wnt11A and wnt11B in the protoscoleces were 25.00（P<0.01）, 33.33（P<0.01）, 14.29（P<0.01） and 1.03 folds（P>0.05） of those in the adult worms, respectively. In brief, there was no significant difference of mRNA expression in wnt2 and wnt11B between protoscoleces and adult, but there was a significant difference of mRNA expression in wnt1, wnt4, wnt5 and wnt11A between protoscoleces and adults. Results of the whole-mount in situ hybridization showed that in protoscoleces wnt1 was mainly localized in the epidermal tissue, wnt2 in suckers, wnt4 in suckers and rostellum, wnt5 and wnt11B in suckers and epidermal tissue, and wnt11A in rostellum and hooks. Conclusion: The mRNA expression of wnt2 in adult E. granulosus was higher than that in protoscoleces, and the mRNA expression ofwnt4, wnt5, wnt11A and wnt11B in protoscoleces was higher than that in the adult worms. The six wnt gene family members were all distributed in the forward region of protoscoleces.

[mRNA Expression Analysis of 10 Reproduction-related Genes in Different Developmental Proglottids of Moniezia expansa].

According to the results of transcriptome sequencing in Moniezia expansa, 10 functional genes which represent different expression patterns in different developmental proglottids were selected, including KIFC1, Kif17, tgf-beta, SmadD, tgf-beta receptor, HSD5, aminopeptidase puromycin, Methionine aminopeptidase 2, transcription factor fork head and Sox transcription factor. A real-time fluorescent quantitative PCR assay was developed for detection of mRNA expression level of these taget genes with its beta-tubulin as an internal control. The results showed that compared to scolex-neck proglottids, the mRNA levels of 2 genes (KIFC1, Kif17) in immature proglottides, 4 genes (KIFC1, Kif17, tgf-beta receptor, and amniopeptidase puromycin) in mature proglottides, and 3 genes (HSD5, tgf-beta receptor, and Methionine aminopeptidase 2) in gravid proglottides were up-regulated (P < 0.01).

Purification and characterization of a novel antimicrobial peptide from sheep reproductive tract.

A novel antimicrobial peptide, SRTAP-40 has been purified and characterized from sheep reproductive tract. The isolation procedure entailed acetic acid extraction, gel filtration chromatography, and HPLC. SRTAP-40 is composed of 40 amino acid residues with a MW of 4,820.47 Da from MALDI-TOF-MS. Its N-terminal sequence was AYVLDEPKP. SRTAP-40 cDNA was cloned by 3'-RACE. SRTAP-40 showed activity against E. coli Staphylococcus aureus, Streptococcus sp. and, Candida albicans with MIC values of 12, 12, 24, 6 µg/ml, respectively. By BLAST search, SRTAP-40 had no significant similarity to any known peptide.

Differential diagnosis of Moniezia benedeni and M. expansa (Anoplocephalidae) by PCR using markers in small ribosomal DNA (18S rDNA).

Moniezia benedeni and M. expansa are common ruminant tapeworms of worldwide distribution, causing gastrointestinal disorders and even death in sheep and goats. In this study, a polymerase chain reaction- (PCR-) based approach for precise species identification was developed and also applied to faecal DNA diagnosis of the tapeworm infection. Since nuclear ribosomal DNA (rDNA) appears to be a useful target for species and/or strain markers, the 18S regions of the rDNA of M. benedeni and M. expansa were amplified and sequenced. The lengths and GC contents of the regions sequenced were 2476-2487 bp and 51.9-52.1% for M. benedeni and 2473 bp and 51.9-52.0% for M. expansa, respectively. Alignment and comparison of the 18S sequences of the two species showed 92.5-93.3% homology. No matches for the 18S regions of M. benedeni and M. expansa were found with other species by BLAST search, which made the 18S sequences appropriate markers for the design of distinctive primers for the two Moniezia species. Our 18S-based PCR could detect as low levels as 0.5 pg genomic DNA or the DNA extracted from 0.2 g faecal sample collected from the rectum of an M. expansa-infected goat. The results indicate that this PCR approach is a reliable alternative for the differential diagnosis of Moniezia species in faecal samples.

[mRNA transcription of translation elongation factor and primase in different segments of Moniezia expansa].

A SYBR green real-time fluorescent quantitative PCR assay was developed for detection of Moniezia expansa mRNA with its beta-tubulin as an internal control. The results showed a good linear relationship (>0.99) between the Ct value and the concentration of positive plasmid for each gene from scolex and various proglottids. Real-time PCR showed that the expression abundance of translation elongation factor and primase was different. In conclusion, the transcription level of translation elongation factor and primase was high in both scolex and immature segment, suggesting that they may play a role in the development of scolex and immature segment.

[Analysis of microbial diversity in rectum of calf in large-scale cattle farm].

OBJECTIVE: To analyze the diversity of bacterial community in rectum of diarrheic calves, and differences with health calves. METHODS: 16S rRNA clone libraries were constructed, positive clones were digested by Msp I and Hha I for restriction fragment length polymorphism (RFLP), and then a phylogenetic tree was depicted based on the 16S rRNA sequencing, to confirm the compose of microbe in the diarrheic calf rectum. RESULTS: The positive rate of clone was 98.75% (474/480) in diarrheic calves, the dominant bacteria included Lactobacillus (14%), Enterococcus (10%) and Escherichia (8%). The positive rate of clone was 96.45% (488/506) in health samples, the dominant bacteria included Clostridium (13%), Bifidobacterium (8%), Megasphaera (5%). CONCLUSION: Complexity and diversity of bacterial community in rectum in 2 weeks old calves had their own features, and significant increase of Lactobacillus, Enterococcus and Escherichia was found in diarrhea calves.

Gene expression profiling of in Moniezia expansa at different developmental proglottids using cDNA microarray.

Gene expression profiles of Moniezia expansa proglottids at varying developmental stages were analysed using cDNA microarray. A total of 4,056 spots, including full length and partial complementary DNAs that represent novel, known, and control genes, were studied. Results indicated an up-regulation of 55 genes in immature proglottids, 134 genes in mature proglottids and 103 genes in gravid proglottids were up-regulated, and a down-regulation of 7 genes in immature proglottids, 68 genes in mature proglottids and 78 genes in gravid proglottids compared to controls (scolex-neck proglottids). Many of these genes were identified as transcription factors and were involved in functions such as metabolism, transport, protein biosynthesis, apoptosis, cell differentiation, cell communication and nucleic acid binding. Expression level alterations in UBE2A, Cavß, RAD51, DAZ, PKAc and 2 unknown genes were confirmed by real-time quantitative polymerase chain reaction (RT-PCR). The complete microarray data set has been deposited in the NCBI Gene Expression Omnibus, GEO Series accession number GSE13982. Results provide a gene expression profile at various development stages of M. expansa proglottids, which prove invaluable in understanding the pathogenesis of the tapeworm and studying the genes concerned with reproductive organ development.

Characterization of a Moniezia expansa ubiquitin-conjugating enzyme E2 cDNA.

Ubiquitin and other ubiquitin-like proteins play important roles in post-translational modification. They are phylogenetically well-conserved in eukaryotes. Here we report a new ubiquitin-conjugating enzyme E2 cDNA, which containing a ubiquitin-conjugating enzyme UBCc-domain named UBE2AM. Its cDNA is 899 base pairs in length and contains an open reading frame from nucleotide 171 to 632 encoding 153 amino acids. The result of real time RT-PCR showed that UBEA2 M is expressed in most of M. expansa proglottides and over-expressed in the mature proglottides. Comparison of predicted UBE2AM with UBCc (protein) homologues/orthologous from other species revealed identities between species varying from 97.5 to 99.4% at the amino acid level. Phylogenetic analysis showed the UBE2AM is a member of the eukaryotic UBCc superfamily, which have diverged from a common ancestor and the gene is clustered in the same group with the ubiquitin-conjugating enzyme E2A-like protein from Taenia asiatica.

Generation and analysis of expressed sequence tags from a cDNA library of Moniezia expansa.

The Moniezia expansa is a parasite of sheep that causes diarrhea and fleshless leading to stockbreeding losses. A genomic resource for large-scale molecular studies in this parasite is lacking. To study the gene expression including development, digestion and reproduction organs of M. expansa, a cDNA library had been constructed and expressed sequence tags (ESTs) had been analyzed, which were helpful for the development of a powerful microarray platform which are used to analyze gene expression in this species. cDNAs are useful resources in annotating genes and providing functional analysis of genes. In this study, a cDNA library from adult cestode of M. expansa was created and 2642 ESTs from 5'-ends of the cDNA clones representing 1081 unigenes were obtained. Cluster analysis of these ESTs allowed identification of 1081 unique sequences containing 351 contigs and 730 singletons. BLASTX searches identified 780 significant (E-value<10(-5)) hits and further Gene Ontology (GO) analysis was used to annotate these genes. All the EST sequences were deposited under dbEST in GenBank (GenBank: FE905224-FE905315, FE942104-FE942773, FE969189-FE969190, FF677548-FF677734, FF848124-FF848253). Although we only obtained 1081 unigenes, the set of ESTs identified represents a significant proportion of the M. expansa and provides molecular resource for the development of microarrays for gene expression studies concerning development, metabolism and reproduction.

[Detecting hematolysis of Enterococcus from sheep].

OBJECTIVE: To characterize hematolysis of Enterococcus from sheep. METHODS: Using plate assay (PA), contact hemolysis (CH), supernatant assay (SA), culture hemolysis (CLH) and PCR, we studied hemolysis characteristics of 11 Enterococcus clinical isolates, 30 isolates from healthy sheep, a standard G-Streptococcus and a standard Enterococcus. RESULTS: Rabbit blood and sheep blood were not hemolysising in the 11 clinical isolates analyzed by SA and CH. Of the clinical isolates 63.6% had beta-hemolysis with rabbit blood and 36.4% had ALPHA-hemolysis with sheep blood analyzed by PA and CLH assay. Of the cylA gene 63.6% was detected in clinical isolates, the sequence of cylA gene was 99.3% homologous with cylA of plasmid pAD1 (GenBank accession number: L37110). J-hemolysis had 53.3% in rabbit blood, alpha-hemolysis and beta-hemolysis in sheep blood had 53.3% and 43.3% respectively in 30 healthy sheep initial isolates with PA. Only 6% had hemolytic capacity in rabbit blood after second generations. The cylA gene was not detected in 30 healthy sheep isolates by PCR. Standard Enterococcus strain of alpha-hemolysis of sheep blood had no hemolysis of rabbit blood. CONCLUSION: The red blood cells could induce enterococci hemolysis secreting in the bacteria growth. The result was different with the Phenotype and the Genotype.