Cysticercosis, caused by the larval stage of T. solium, remains a major neglected tropical disease with severe clinical and socioeconomic consequences in endemic regions. Although cyclic AMP-dependent protein kinase (PKA) signaling is fundamental to parasite development and survival, its therapeutic relevance in T. solium has not been comprehensively explored. This study provides an integrated molecular and functional characterization of the T. solium PKA catalytic subunit (TsPKA-c) and evaluates its potential as a novel therapeutic target for cysticercosis control. Recombinant TsPKA-c was successfully expressed in Pichia pastoris and purified in an active form, demonstrating robust kinase activity. Phylogenetic analysis revealed strong evolutionary conservation among cestode PKA homologues, underscoring its essential biological function. Immunohistochemical analysis confirmed stage-specific expression in both larval and adult parasite forms. Optimal enzymatic activity occurred at pH 7.5 and 35°C, with kinetic parameters showing a Km of 36.37 ± 2.64 µM and Vmax of 0.7648 ± 0.013 nmol/min/µg. Pharmacological inhibition experiments identified H89 as a highly potent inhibitor of TsPKA-c (IC50 = 14.55 ± 1.5 µM), exhibiting greater efficacy than PKI (14-22) (IC50 = 23.09 ± 0.5 µM). Both inhibitors induced significant dose- and time-dependent mortality in T. pisiformis cysticerci, with H89 causing rapid lethality. Metabolic analyses demonstrated a marked reduction in glucose uptake following TsPKA-c inhibition, accompanied by alterations in excretory-secretory protein profiles. Collectively, these findings establish TsPKA-c as a critical regulator of parasite metabolism and survival, supporting its strong candidacy as a promising molecular target for the development of novel anti-cysticercosis therapeutic strategies.

Taenia hydatigena is a globally distributed cestode of veterinary importance that infects canids (definitive hosts) and ungulate livestock (intermediate hosts). Although non-zoonotic, its transmission ecology overlaps with zoonotic taeniids, making surveillance relevant within a One Health framework. Its epidemiology and genetic diversity remain poorly understood in West Africa. Nigeria, with its large and mobile livestock populations, provides a critical setting for investigating parasite transmission and phylogeographic dynamics. We conducted a large-scale abattoir survey of small ruminants (goats and sheep) across four major Nigerian states to assess infection prevalence, organ tropism, and demographic correlates. T. hydatigena cysticerci were characterized using full-length mitochondrial nad1 and nad4 genes, as well as concatenated nad1-nad4 datasets. Sequence diversity, haplotype structure, and phylogenetic relationships were analyzed in comparison with global T. hydatigena isolates. Among 1726 small ruminants examined, 8.9% (153/1726) were infected, with infection biased toward adult animals and cysts predominantly localized in mesenteric and omental tissues. Mitochondrial analyses revealed high haplotype richness, with nad4 showing greater variability than nad1, and concatenated sequences providing superior phylogenetic resolution. Nigerian isolates clustered both locally and globally, indicating long-standing endemicity reinforced by porous livestock trade networks. Unique regional haplotypes highlighted West Africa's contribution to global T. hydatigena diversity. This study provides the most comprehensive molecular epidemiological assessment of T. hydatigena in Nigeria to date, underscoring an endemic, economically important burden and the need for integrated One Health interventions targeting the dog-livestock transmission cycle.

Cystic echinococcosis (CE), caused by Echinococcus granulosus sensu lato, is a major zoonotic disease with significant public health and economic impacts. Despite its endemicity in Africa, molecular data on Echinococcus granulosus s.l. genotypes in Nigeria remain limited, with existing knowledge primarily derived from sparse epidemiological studies. This study presents the first nationwide molecular assessment of CE in Nigerian cattle, evaluating prevalence, genetic diversity, and risk factors. We conducted a cross-sectional study across four abattoirs to investigate the zoonotic risk of cystic echinococcosis (CE) by examining the fertility, organ distribution and molecular characterization of the hydatid cysts in cattle, genotyping by polymerase chain reaction (PCR) and sequencing of the cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1) genes. Results revealed an overall CE prevalence of 2.05 %, with regional variations (highest in Abubakar Saraki [5.79 %] and Akinyele [2.59 %]). Cysts were primarily found in lungs (1.62 %) and liver (0.43 %). Molecular analysis identified E. granulosus sensu stricto (G1 genotype) as the dominant genotype, with 3 cox1 and 6 nad1 haplotypes demonstrating substantial genetic diversity. Regional differentiation suggested localized transmission linked to livestock movement. This study provides crucial epidemiological and genetic data on E. granulosus sensu stricto in Nigeria, confirming the presence of the globally dominant G1 genotype and highlighting significant genetic diversity. The findings emphasize the need for a region-specific, One Health-based approach integrating veterinary, medical, and environmental interventions to control CE. Future research should focus on expanding surveillance to other intermediate hosts and evaluating control strategies to mitigate disease transmission.

Alveolar echinococcosis (AE) is a severe and life-threatening parasitic disease caused by Echinococcus multilocularis. Liver fibrosis is a significant pathological feature of advanced AE, characterized by the excessive production and accumulation of extracellular matrix (ECM). However, the precise underlying mechanism remains largely unknown. In this study, we show that the long noncoding RNA gm40262, predominantly expressed in hepatic stellate cells (HSCs), is upregulated in AE. Interestingly, its knockdown leads to liver fibrosis resolution, accompanied by a substantial suppression of parasite growth. Gm40262 functions by targeting miR-193b-5p to activate HSCs and stimulate their proliferation in a TGF-β-dependent manner, thereby promoting ECM production by upregulating Col1α1. Moreover, gm40262 is also involved in inflammation through the gm40262-miR-193b-5p-TLR4 axis. Our findings suggest that gm40262 plays a pivotal role in parasite-induced liver fibrosis through multiple mechanisms, highlighting its potential as a therapeutic target for hepatic fibrosis. IMPORTANCE:Echinococcus multilocularis is a tiny parasite with significant medical implications. The chronic parasitism of E. multilocularis in the liver generally leads to liver fibrosis, but the underlying mechanisms are poorly understood. We herein show that gm40262, a long noncoding RNA predominantly expressed in hepatic stellate cells (HSCs), is involved in hepatic fibrogenesis during infection by activating HSCs and promoting extracellular matrix production. The gm40262-orchestrating fibrogenesis occurs through the gm40262-miR-193b-5p-TLR4 and gm40262-miR-193b-5p-Col1α1 axes. The knockdown of gm40262 remarkably alleviates liver fibrosis, with decreased parasite growth. Our findings reveal a key role of gm40262 in liver fibrosis during E. multilocularis infection, rendering it a therapeutic target for hepatic fibrosis.

Regulatory T cells (Treg) play a crucial role in creating an immunosuppressive microenvironment surrounding the metacestode during chronic alveolar echinococcosis (AE). However, the mechanisms by which E. multilocularis induces Treg differentiation, particularly the role of parasite-derived microRNAs (miRNAs), remain largely unexplored. Here, we demonstrate that E. multilocularis can significantly induce the differentiation of Treg in mice. Emu-let-7-5p is upregulated in peripheral blood lymphocyte cells (PBLC) and splenic lymphocytes of E. multilocularis-infected mice. Exosomes enriched with emu-let-7-5p were found to upregulate the expressions of Treg markers. Conversely, exosomes collected following the knockdown of worm-derived emu-let-7-5p via RNA interference resulted in a reversal of Treg marker expression in PBLC. Mechanistically, emu-let-7-5p regulates Treg differentiation by targeting NFκB2. Knockdown of emu-let-7-5p in E. multilocularis-infected mice resulted in diminished Treg differentiation, leading to a significant reduction in worm load. These findings reveal that emu-let-7-5p drives Treg differentiation by suppressing NFκB2, representing a novel immune evasion strategy of E. multilocularis. Sustained inhibition of parasite-derived emu-let-7-5p may provide a therapeutic avenue for controlling AE progression.

Cystic echinococcosis (CE) and Taenia spp. infections are major zoonotic helminthiases with substantial public health and economic burdens, particularly in endemic regions. Despite their classification as neglected tropical diseases (NTDs), these tapeworm infections remain understudied in Nigeria. Accurate epidemiological data are essential for evidence-based control strategies, yet comprehensive estimates of their prevalence and geographical distribution in Nigeria are lacking. This systematic review and meta-analysis provide the first nationwide synthesis of CE and Taenia spp. infections in both human and animal populations, elucidating high-risk areas and guiding targeted interventions. A comprehensive systematic search was conducted across four major databases (PubMed, Google Scholar, Africa Journal Online, and ScienceDirect) to identify relevant studies published between 1979 and October 2024. Eligible studies were selected based on predefined inclusion criteria and subjected to meta-analysis using RStudio (RStudio Team, 2024). Heterogeneity was evaluated using Cochran's Q test and the I² statistic, while publication bias was assessed through the Luis Furuya-Kanamori (LFK) index, funnel plot asymmetry, and Egger's regression test to ensure robustness of the findings. The study compiled 156 datasets from 80 publications, primarily abattoir surveys. Of 86 reports on cystic echinococcosis (CE) in animals, half were from the Northwest. The South-South and North-Central regions had the most reports on Taenia infection (21.9% each). Post Mortem Examination and Microscopy (PME/MCS) were used in 76.5% of CE studies, while microscopy alone detected Taenia in 84.4% of cases. Among 5,542,336 hosts examined, 14,216 infections were identified, yielding an overall pooled prevalence of 0.26% (95% CI: 0.174-0.346%) likely an underestimate due to diagnostic limitations. Meta-analysis of CE in animals and human Taenia infections showed high variability (Q= 83,102.51; I²= 99.9%, P= 0.000), with individual study prevalence ranging from 0.00 to 27.91%. Human studies had weights of 2-3.5% (Q= 1003.05; I²= 96.9%, P<0.0001). This study reveals a high prevalence of cestode infections, particularly in Northern Nigeria, emphasizing the persistent burden of these neglected diseases. Findings highlight critical gaps in surveillance, diagnostics, and species data, underscoring the urgent need for integrated One Health interventions and targeted public health strategies. Enhanced research and regular monitoring are essential for effective control.

Alveolar echinococcosis is a zoonotic disease that poses serious threats to public health. We observed subcutaneous cysts (SCs) of E. multilocularis had fewer protoscoleces (PSCs) compared to intraperitoneal cysts (ICs) at 60 days post-infection. However, the mechanisms underlying the development of E. multilocularis cysts in different tissues remain unclear. In this study, we compared the proteomic profiles of E. multilocularis cysts derived from mice intraperitoneally and subcutaneously infected with PSCs at 30 days post-infection, prior to the development of mature PSCs. Proteomic analysis identified 284 differentially expressed proteins (DEPs) in SCs compared to ICs, with 147 upregulated DEPs and 137 downregulated DEPs. Enzymatic proteins involved in carbohydrate and amino acid metabolism were predominantly upregulated in SCs compared to ICs, whereas proteins associated with protein folding, sorting, a degradation were downregulated. Western blotting analysis confirmed that phosphoenolpyruvate carboxykinase (PEPCK) and fructose-bisphosphate aldolase (FBA) were upregulated, whereas transitional endoplasmic reticulum ATPase (TER ATPase) was downregulated in SCs compared to ICs. The identified DEPs may play crucial roles in shaping the unique characteristics of E. multilocularis cysts. This study offers valuable insights into exploring the mechanisms underlying the occurrence and development of metacestodes.

Taenia pisiformis (T. pisiformis) cysticerci, belonging to Taeniidae, attaches to the wall of the mesentery and omentum of rabbits, causing cysticercosis pisiformis that can seriously affect the healthy development of the rabbit breeding industry. Helminths can produce exosomes containing proteins and RNAs. In our previous study, tpi-miR-10a-5p was found to be highly enriched in the exosomes from T. pisiformis cysticerci. In this study, we report that tpi-miR-10a-5p is significantly up-regulated in the blood and peripheral blood lymphocytes (PBLCs) of T. pisiformis-infected rabbits. Furthermore, tpi-miR-10a-5p targets mitogen-activated protein kinase kinase kinase 7 (MAP3K7), the key gene involved in the c-Jun N-terminal kinase (JNK) signaling pathway. Knockdown of MAP3K7 inhibited the JNK signaling pathway, suppressing the production of inflammatory cytokines such as IFN-γ and TNFα, whereas overexpression of MAP3K7 activated the JNK signaling pathway in PBLCs. The same trend was observed with knockdown of MAP3K7 when PBLCs were treated with exosomes from T. pisiformis cysticerci. In vivo experiment further demonstrated that the expression of MAP3K7, JNK, p-JNK, IFN-γ, and TNFα was significantly decreased in PBLCs during T. pisiformis infection. Therefore, tpi-miR-10a-5p can suppress the JNK signaling pathway and inflammatory response by targeting MAP3K7 in host PBLCs. These findings may imply a mechanism used by the parasites releasing exosomes to sense and adapt to the host environment by regulating the immune reaction.

Metacestodiasis is an infectious disease caused by the larval stage of cestode parasites. This disease poses a serious health hazard to wildlife, livestock, and humans, and it incurs substantial economic losses by impacting the safety of the livestock industry, the quality of meat production, and public health security. Unfortunately, there is currently no available molecular diagnostic method capable of distinguishing cysticercus- and Echinococcus-derived microRNAs (miRNAs) from other helminthes and hosts in the plasma of metacestode-infected animals. This study aims to develop a specific, sensitive, and cost-efficient molecular diagnostic method for cysticercosis and echinococcosis, particularly for early detection. The study developed a rolling circular amplification (RCA)-assisted CRISPR/Cas9 detection method based on parasite-derived miRNA let-7-5p. Using a series of dilutions of the let-7 standard, the limit of detection (LOD) of the qPCR, RCA, and RCA-assisted CRISPR/Cas9 methods was compared. The specificity of qPCR and CRISPR/Cas9 was evaluated using four artificially synthesized let-7 standards from different species. A total of 151 plasma samples were used to evaluate the diagnostic performance. Additionally, the study also assessed the correlation between plasma levels of let-7-5p, the number of Taenia pisiformis cysticerci, and the weight of Echinococcus multilocularis cysts. The results demonstrated that the RCA-assisted CRISPR/Cas9 assay could significantly distinguish let-7 from cestodes and other species, achieving a LOD of 10 aM; the diagnostic sensitivity and specificity for rabbit cysticercosis and mouse E. multilocularis were 100% and 97.67%, and 100% and 100%, respectively. Notably, let-7-5p gradually increased in the plasma of T. pisiformis-infected rabbits from 15 days post infection (dpi), peaked at 60 dpi, and persisted until 120 dpi. In E. multilocularis-infected mice, let-7-5p gradually increased from 15 dpi and persisted until 90 dpi. Furthermore, the expression of let-7-5p positively correlated with the number of cysticerci and cyst weight. These results indicated that the let-7-5p-based RCA-assisted CRISPR/Cas9 assay is a sensitive and specific detection method that can be used as a universal diagnostic method for metacestodiasis, particularly for early diagnosis (15 dpi).

Alveolar echinococcosis (AE) is an important infectious disease caused by the metacestode larvae of Echinococcus multilocularis, seriously threatening global public health security. Kupffer cells (KCs) play important roles in liver inflammatory response. However, their role in hepatic alveolar echinococcosis has not yet been fully elucidated. In this study, qRT-PCR was used to detect the expression level of miR-374b-5p in KCs. The target gene of miR-374b-5p was identified through luciferase reporter assays and loss of function and gains. Critical genes involved in NFκB signaling pathway were analyzed by qRT-PCR and western blot. This study reported that miR-374b-5p was significantly upregulated in KCs during E. multilocularis infection and further showed that miR-374b-5p was able to bind to the 3'-UTR of the C/EBP β gene and suppressed its expression. The expression levels of NF-κBp65, p-NF-κBp65 and pro-inflammatory factors including iNOS, TNFα and IL6 were attenuated after overexpression of miR-374b-5p while enhanced after suppression of miR-374b-5p. However, the Arg1 expression level was promoted after overexpression of miR-374b-5p while suppressed after downregulation of miR-374b-5p. Additionally, increased protein levels of NF-κBp65 and p-NF-κBp65 were found in the C/EBP β-overexpressed KCs. These results demonstrated that miR-374b-5p probably regulated the expression of inflammatory factors via C/EBP β/NF-κB signaling. This finding is helpful to explore the mechanism of inflammation regulation during E. multilocularis infection.

Cysticercosis pisiformis, a highly prevalent parasitic disease worldwide, causes significant economic losses in the rabbit breeding industry. Previous investigations have identified a novel microRNA, designated as novel-miR1, within the serum of rabbit infected with Cysticercus pisiformis. In the present study, we found that C. pisiformis-derived novel-miR1 was released into the rabbit serum via exosomes. Through computational analysis using TargetScan, miRanda, and PITA, a total of 634 target genes of novel-miR1 were predicted. To elucidate the functional role of novel-miR1, a dual-luciferase reporter assay was utilized and demonstrated that novel-miR1 targets rabbit Toll-like receptor 2 (TLR2). Rabbit peripheral blood lymphocytes (PBLCs) were transfected with novel-miR1 mimic and mimic NC, and the in vitro experiments confirmed that novel-miR1 suppressed the expression of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 through the nuclear factor kappa B (NF-κB) pathway. In vivo experiments demonstrated that novel-miR1 was significantly upregulated during the 1–3 months following infection with C. pisiformis in rabbits. Notably, this upregulation coincided with a downregulation of TLR2, P65, pP65, TNF-α, IL-1β, and IL-6 in PBLCs. Collectively, these results indicate that the novel-miR1 derived from C. pisiformis inhibited the rabbits’ immune response by suppressing the NF-κB-mediated immune response. This immune modulation facilitates parasite invasion, survival, and establishment of a persistent infection.

旨在探讨多房棘球蚴感染早期对小鼠肝脂质代谢的影响.选取60只清洁级BALB/c小鼠(6周龄,雄性)随机分为试验组和对照组,每组30只.试验组小鼠每只腹腔接种600个原头蚴,对照组小鼠腹腔注射等体积的PBS缓冲液.采用原位两步循环灌流及多步低速差速离心分离纯化肝实质细胞.利用高通量测序分析感染3月小鼠肝实质细胞差异表达的mRNA,并对其进行KEGG、GO分析和qRT-PCR验证.制备多房棘球蚴感染2、3和6月小鼠的肝组织切片,分别进行油红O染色,并通过qRT-PCR检测多房棘球蚴感染不同感染时期肝实质细胞脂质代谢相关基因的表达水平.结果表明,与未感染对照组相比,小鼠感染多房棘球蚴后3、6月肝脂肪沉积明显增多;感染2月的肝实质细胞脂质合成相关基因Scd1、Fasn和Srebf1,以及脂质氧化分解相关基因Acox1和Cpt1α均明显上调;感染3月的肝实质细胞Scd1、Fasn、Srebf1及Cd36的表达水平均明显升高,而Acox1、Cpt1α、Pparγ和Pparα的表达水平明显降低.上述结果表明,多房棘球蚴感染早期可能通过促进肝实质细胞的脂质合成,抑制脂质氧化分解,从而导致肝脂质沉积.

Liver fibrosis is one of the histopathological characters during Echinococcus multilocularis infection. The activation of hepatic stellate cells (HSCs) is a key event in the development of liver fibrosis. However, the molecular mechanism of HSC activation in the E. multilocularis infection-induced liver fibrosis remains largely unclear. Here, we reported that mmu-miR-342-3p was most dominantly expressed in HSCs and was upregulated in the HSCs in response to E. multilocularis infection. We further showed that mmu-miR-342-3p was able to bind to the 3' UTR of the Zbtb7a gene and regulated its expression. Moreover, mmu-miR-342-3p expression was negatively correlated with its target gene Zbtb7a in HSCs during E. multilocularis infection. Knockdown of mmu-miR-342-3p promoted the expression of Gfap in the activated HSCs in vitro. In the E. multilocularis-infected mice, knockdown of mmu-miR-342-3p suppressed the expression of α-Sma, Col1α1, and TGF-β but promoted the expression of Gfap. Therefore, mmu-miR-342-3p is a key regulator for activation of HSCs, and inhibiting mmu-miR-342-3p to suppressed Zbtb7a-mediated TGF-β signaling in activated HSCs could be a novel strategy to treat liver fibrosis induced by E. multilocularis.

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 IL1β and NFκB2 respectively, which imply that novel334-IL1β/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.

目的 建立检测家兔豆状囊尾蚴感染的滚环扩增(RCA)方法.方法 以家兔血清中豆状囊尾蚴来源的novel-miR1为诊断靶标,设计连接序列和锁式探针,并对连接序列和锁式探针的比例、反应时间、酶用量、dNTP用量以及扩增反应时间等5个重要条件进行优化,建立检测豆状囊尾蚴感染的RCA方法.将novel-miR1标准品倍比稀释为1 fmol/L至100 nmol/L的9个不同浓度的样品,评价RCA方法的灵敏度.用优化后的RCA方法分别检测健康家兔和豆状囊尾蚴感染家兔血清miRNA各20份(实验室保存样品),并通过受试者工作特征(ROC)曲线分析其敏感性和特异性.20只雌性家兔每只经口感染1 000个豆状带绦虫虫卵,采集感染前和感染后每个月的家兔血样制备血清miRNA,用优化后的RCA方法进行检测,评价其应用效果.结果 电泳结果显示,RCA扩增产物的DNA停留在加样孔中,形成一条明亮条带.反应条件优化试验结果显示,连接序列浓度为2μmol/L、锁式探针浓度为1μmol/L(连接序列和锁式探针的比为2:1)、T4 DNA连接酶为350 U、连接180 min时连接效率最高.在100μl的扩增体系中,dNTP浓度为0.5 μmol/L,扩增240 min时,RCA扩增效果最佳.优化后RCA的最低检测浓度为10 pmoI/L.RCA检测健康家兔和豆状囊尾蚴感染家兔血清miRNA样品的平均荧光值分别为53.298±1.707和97.498±5.892,差异有统计学意义(t=7.206,P＜0.01).ROC分析结果显示,当阳性临界值为61.69时,RCA方法的敏感性和特异性均为95％,AUC为0.955 0,似然比为19.00.RCA检测豆状囊尾蚴感染后不同时间的家兔血清miRNA结果显示,20份感染前的血清中19份检测结果为阴性,1份为阳性;感染后1个月的血清中,17份为阳性,2份为疑似,1份为阴性;感染后2个月的血清,1份为阴性,其余为阳性;感染后3个月的血清,3份为阴性,其余为阳性.结论 建立了检测家兔豆状囊尾蚴感染的RCA方法,该方法在豆状囊尾蚴感染后3个月内的家兔血清中检出novel-miR1,具有良好的应用潜力..

The small ubiquitin-like modifier (SUMO) plays important roles, with the SUMOylation pathway as one of its core components. In the present work, a single SUMO gene was initially identified from Taenia pisiformis and designated as TpSUMO. Bioinformatic analysis showed that the TpSUMO gene contained a 309 bp open reading frame (ORF), encoding 102 amino acids, and had a predicted molecular weight of ∼12 kDa. The amino acid sequence of TpSUMO was deduced and it shared 44.00% identity with human SUMO2 (HsSUMO2) and exhibited more than 97.78% identity with SUMOs from Taenia and Echinococcus. TpSUMO possessed a putative non-consensus site (FK11MG) within its N-terminus and a typical di-glycine (GG) motif at the C-terminus. Basic local alignment search tool (BLAST) analysis showed that only a single SUMO-related ortholog was present in each set of known genome data for fourteen tapeworm species. The precursor His-TpSUMO-FL, mature His-TpSUMO-GG and mutant His-TpSUMO-GGK11R proteins (∼18 kDa) were expressed in Escherichia coli Rosseta (DE3), and rabbit polyclonal anti-TpSUMO was generated with a high titer of 1.28 × 105. In vitro SUMOylation assay results showed that TpSUMO multimer formation in the His-TpSUMO-GG reaction could be catalyzed by the human SAE1/SAE2 and UBC9 conjugation system, but K11R mutation disrupted TpSUMO chain synthesis. Quantitative real-time PCR (qRT-PCR) further revealed that TpSUMO was ubiquitously expressed in different stages of T. pisiformis and in higher levels during an early development phase (day 14) of adult worms. Immunofluorescence localization showed that TpSUMO was detected in the bladder wall of cysticerci, in the testis in immature segment, and within eggs in the gravid proglottids. These findings indicated that TpSUMO is a new member of the SUMO protein family and may play a vital role in regulation of functions within proteins involved in worm growth and development.

China is the country with the largest number of domestic small ruminants in the world. Recently, the intensive and large-scale sheep/goat raising industry has developed rapidly, especially in nonpastoral regions. Frequent trading, allocation, and transportation result in the introduction and prevalence of new pathogens. Several new viral pathogens (peste des petits ruminants virus, caprine parainfluenza virus type 3, border disease virus, enzootic nasal tumor virus, caprine herpesvirus 1, enterovirus) have been circulating and identified in China, which has attracted extensive attention from both farmers and researchers. During the last decade, studies examining the etiology, epidemiology, pathogenesis, diagnostic methods, and vaccines for these emerging viruses have been conducted. In this review, we focus on the latest findings and research progress related to these newly identified viral pathogens in China, discuss the current situation and problems, and propose research directions and prevention strategies for different diseases in the future. Our aim is to provide comprehensive and valuable information for the prevention and control of these emerging viruses and highlight the importance of surveillance of emerging or re-emerging viruses.

目的 建立豆状囊尾蚴体外长期培养方法,并对其培养分泌产物进行鉴定.方法 将豆状带绦虫虫卵经口感染新西兰白兔(2 000枚/只),感染后3个月,从感染兔腹腔中收集豆状囊尾蚴.将虫体随机分为PBS组、RPMI1640组、胆汁组(RPMI1640+10％兔胆汁)和血清组(RPMI1640+10％无外泌体胎牛血清),10个/组,37℃、5％CO2培养后5min、1h、2h、12h、24 h、36 h、48 h及其后每天进行观察,记录豆状囊尾蚴头节翻出情况、虫体活力、体外存活时间及形态改变等,筛选适宜的培养条件.取50个幼虫在细胞瓶(25 cm2)中按筛选出的培养条件培养,每隔48小时收集培养液离心浓缩,取上清,蛋白质免疫印迹(Western blotting)检测豆状带绦虫烯醇化酶(Tpeno)和Tp14-3-3蛋白的表达情况.收集豆状囊尾蚴培养上清,提取外泌体,分别与阴性兔血清、CD63抗体、抗Tpeno单克隆抗体1D7和抗Tp14-3-3多克隆抗体孵育45 min,再加入胶体金标记的IgG,透射电镜观察外泌体形态,免疫电镜检测其标记蛋白CD63、Tpeno和Tp14-3-3表达情况.采用SPSS 20.0统计学软件进行数据分析,组间比较采用单因素方差分析.结果 培养前的豆状囊尾蚴呈椭圆形,有内凹的点状乳白色头节;PBS组可存活7d;RPMI 1640组可存活2个月;胆汁组存活时间最短,仅48h;血清组幼虫存活时间最长,可达3个月以上,高于其他3个组(P＜0.05).存活期内,PBS组、RPMI 1640组、胆汁组和血清组幼虫囊泡长度分别为(1.38±0.39)、(1.30±0.12)、(1.18±0.59)和(1.83±0.10)cm;宽度分别为(0.45±0.10)、(0.68±0.05)、(0.25±0.06)和(0.85±0.05)cm;血清组虫体囊泡长度和宽度与其余3组相比差异均有统计学意义(F长 度=7.58、65.93、7.11,F宽度=73.85、29.41、308.57,P＜0.05 或P＜0.01).培养 12 h 时,PBS组、RPMI 1640组、胆汁组和血清组幼虫头节翻出率分别为(33.3±5.8)％、(20.0±0.0)％、(100.0±0.0)％、(13.3±5.8)％,其中血清组与PBS和胆汁组头节翻出率差异有统计学意义(F=18.00、676.00,P＜0.01).对各组培养效果比较显示,血清组的培养液(RPMI 1640+10％无外泌体胎牛血清)为最适培养液.SDS-PAGE电泳分析结果显示,利用RPMI 1640+10％无外泌体胎牛血清培养豆状囊尾蚴,可成功提取到虫体的代谢_分泌产物和外泌体蛋白.Western blotting分析结果显示,用特异性抗体检测可见Tpeno和Tp14-3-3阳性条带,相对分子质量(Mr)分别约47 000和28 000,与预期相符.透射电镜下豆状囊尾蚴外泌体大小不一,直径为50～150 nm,为圆形或椭圆形的囊泡.免疫电镜分析显示,阴性兔血清不能识别外泌体蛋白,未见胶体金颗粒标记;外泌体标志蛋白CD63、虫体特异性蛋白Tpeno和Tp14-3-3均为阳性表达,胶体金颗粒标记明显.结论 初步建立了豆状囊尾蚴体外培养体系,可连续培养豆状囊尾蚴至少3个月以上.采用该体外培养方法培养可在培养上清中成功提取豆状囊尾蚴的外泌体.

Caused by Echinococcus multilocularis ( E. multilocularis ), alveolar echinococcosis is reported every year around the world and severely threatens the safety of human beings and animals. However, the molecular interaction relationships between host and E. multilocularis still remains unclear. With multiple functions, circRNA plays a crucial role in regulating the development of a parasitic disease. With that in mind, the main purpose of this study was to reveal the circRNA expression profiles and circRNA–miRNA–mRNA network relationships in hepatocytes (HCs), hepatic stellate cells (HSCs), and Kupffer cells (KCs) of murine liver after E. multilocularis infection. After sequencing, 6,290 circRNAs were identified from 12 hepatic cell samples. Based on the subsequent analysis, 426 and 372 circRNAs were significantly different in HC expression at 2 and 3 months after E. multilocularis infection, and similar results were also demonstrated in HSCs (426 and 372 circRNAs) and KCs (429 and 331 circRNAs), respectively. Eight candidate circRNAs were randomly selected to identify the accuracy of the sequencing results by using qRT-PCR. Additionally, three circRNAs–miRNA–mRNA networks in HCs, HSCs, and KCs were constructed. Taken together, our study provided a systematic presentation of circRNAs in murine liver cells after E. multilocularis infection, and these networks are essential for research in circRNAs associated with E. multilocularis infection.