Effect of LAG3 deficiency on natural killer cell function and hepatic fibrosis in mice infected with Echinococcus multilocularis / 中国血吸虫病防治杂志

Objective To investigate the effect of LAG-3 deficiency (LAG3-/-) on natural killer (NK) cell function and hepatic fibrosis in mice infected with Echinococcus multilocularis. Methods C57BL/6 mice, each weighing (20 ± 2) g, were divided into the LAG3-/- and wild type (WT) groups, and each mouse in both groups was inoculated with 3 000 E. multilocularis protoscoleces via the hepatic portal vein. Mouse liver and spleen specimens were collected 12 weeks post-infection, sectioned and stained with sirius red, and the hepatic lesions and fibrosis were observed. Mouse hepatic and splenic lymphocytes were isolated, and flow cytometry was performed to detect the proportions of hepatic and splenic NK cells, the expression of CD44, CD25 and CD69 molecules on NK cell surface, and the secretion of interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), interleukin (IL)-4, IL-10 and IL-17A. Results Sirius red staining showed widening of inflammatory cell bands and hyperplasia of fibrotic connective tissues around mouse hepatic lesions, as well as increased deposition of collagen fibers in the LAG3-/-group relative to the WT group. Flow cytometry revealed lower proportions of mouse hepatic (6.29% ± 1.06% vs. 11.91% ± 1.85%, P < 0.000 1) and splenic NK cells (4.44% ± 1.22% vs. 5.85% ± 1.10%, P > 0.05) in the LAG3-/- group than in the WT group, and the mean fluorescence intensity of CD44 was higher on the surface of mouse hepatic NK cells in the LAG3-/- group than in the WT group (t = −3.234, P < 0.01), while no significant differences were found in the mean fluorescence intensity of CD25 or CD69 on the surface of mouse hepaticNK cells between the LAG3-/- and WT groups (both P values > 0.05). There were significant differences between the LAG3-/- and WT groups in terms of the percentages of IFN-γ (t = −0.723, P > 0.05), TNF-α (t = −0.659, P > 0.05), IL-4 (t = −0.263, P > 0.05), IL-10 (t = −0.455, P > 0.05) or IL-17A secreted by mouse hepatic NK cells (t = 0.091, P > 0.05), and the percentage of IFN-γ secreted by mouse splenic NK cells was higher in the LAG3-/- group than in the WT group (58.40% ± 1.64% vs. 50.40% ± 4.13%; t = −4.042, P < 0.01); however, there were no significant differences between the two groups in terms of the proportions of TNF-α (t = −1.902, P > 0.05), IL-4 (t = −1.333, P > 0.05), IL-10 (t = −1.356, P > 0.05) or IL-17A secreted by mouse splenic NK cells (t = 0.529, P > 0.05). Conclusions During the course of E. multilocularis infections, LAG3-/- promotes high-level secretion of IFN-γ by splenic NK cells, which may participate in the reversal the immune function of NK cells, resulting in aggravation of hepatic fibrosis.

Dynamic observation on capillarization of liver sinusoidal endothelial cells induced by Echinococcus multilocularis infection / 中国血吸虫病防治杂志

Objective To investigate the capillarization of liver sinusoidal endothelial cells (LSECs) and its association with hepatic fibrosis during the development of alveolar echinococcosis, so as to provide the basis for unraveling the mechanisms underlying the role of LSEC in the development and prognosis of hepatic injuries and hepatic fibrosis caused by alveolar echinococcosis. Methods Forty C57BL/6 mice at ages of 6 to 8 weeks were randomly divided into a control group and 1-, 2- and 4-week infection groups, of 10 mice in each group. Each mouse in the infection groups was intraperitoneally injected with 2 000 Echinococcus multilocularis protoscoleces, while each mouse in the control group was given an equal volume of phosphate-buffered saline using the same method. All mice were sacrificed 1, 2 and 4 weeks post-infection and mouse livers were collected. The pathological changes of livers were observed using hematoxylin-eosin (HE) staining, and hepatic fibrosis was evaluated through semi-quantitative analysis of Masson’s trichrome staining-positive areas. The activation of hepatic stellate cells (HSCs) and extracellular matrix (ECM) deposition were examined using immunohistochemical staining of α-smooth muscle actin (α-SMA) and collagen type I alpha 1 (COL1A1), and the fenestrations on the surface of LSECs were observed using scanning electron microscopy. Primary LSECs were isolated from mouse livers, and the mRNA expression of LSEC marker genes Stabilin-1, Stabilin-2, Ehd3, CD209b, GATA4 and Maf was quantified using real-time fluorescence quantitative PCR (qPCR) assay. Results Destruction of local liver lobular structure was observed in mice 2 weeks post-infection with E. multilocularis protoscoleces, and hydatid cysts, which were surrounded by granulomatous tissues, were found in mouse livers 4 weeks post-infection. Semi-quantitative analysis of Masson’s trichrome staining showed a significant difference in the proportion of collagen fiber contents in mouse livers among the four groups (F = 26.060, P < 0.001), and a higher proportion of collagen fiber contents was detected in mouse livers in the 4-week infection group [(11.29 ± 2.58)%] than in the control group (P < 0.001). Immunohistochemical staining revealed activation of a few HSCs and ECM deposition in mouse livers 1 and 2 weeks post-infection, and abundant brown-yellow stained α-SMA and COL1A1 were deposited in the lesion areas in mouse livers 4 weeks post-infection, which spread to surrounding tissues. Semi-quantitative analysis revealed significant differences in α-SMA (F = 7.667, P < 0.05) and COL1A1 expression (F = 6.530, P < 0.05) in mouse levers among the four groups, with higher α-SMA [(7.13 ± 3.68)%] and COL1A1 expression [(13.18 ± 7.20)%] quantified in mouse livers in the 4-week infection group than in the control group (both P values < 0.05). Scanning electron microscopy revealed significant differences in the fenestration frequency (F = 37.730, P < 0.001) and porosity (F = 16.010, P < 0.001) on the surface of mouse LSECs among the four groups, and reduced fenestration frequency and porosity were observed in the 1-[(1.22 ± 0.48)/μm2 and [(3.05 ± 0.91)%] and 2-week infection groups [(3.47 ± 0.10)/μm2 and (7.57 ± 0.23)%] groups than in the control group (all P values < 0.001). There was a significant difference in the average fenestration diameter on the surface of mouse LSECs among the four groups (F = 15.330, P < 0.001), and larger average fenestration diameters were measured in the 1-[(180.80 ± 16.42) nm] and 2-week infection groups [(161.70 ± 3.85) nm] than in the control group (both P values < 0.05). In addition, there were significant differences among the four groups in terms of Stabilin-1 (F = 153.100, P < 0.001), Stabilin-2 (F = 57.010, P < 0.001), Ehd3 (F = 31.700, P < 0.001), CD209b (F = 177.400, P < 0.001), GATA4 (F = 17.740, P < 0.001), and Maf mRNA expression (F = 72.710, P < 0.001), and reduced mRNA expression of Stabilin-1, Stabilin-2, Ehd3, CD209b, GATA4 and Maf genes was quantified in three infection groups than in the control group (all P values < 0.001). Conclusions E. multilocularis infections may induce capillarization of LSECs in mice, and result in a reduction in the expression of functional and phenotypic marker genes of LSECs, and capillarization of LSECs occurs earlier than activation of HSC and development of hepatic fibrosis.

Progress in diagnosis and treatment of hepatic alveolar echinococcosis / 器官移植

Hepatic echinococcosis is a chronic parasitic disease, which is caused by the larvae of Echinococcus multilocularis. It has a high risk of disability and mortality, which is also known as "parasite cancer". In clinical practice, hepatic echinococcosis can be divided into hepatic alveolar echinococcosis and hepatic cystic echinococcosis. Hepatic echinococcosis is widely prevalent worldwide. It mainly occurs in the populations residing agricultural and pastoral areas in western China, posing significant threats to the quality of life of local residents. At present, surgery is the main treatment for hepatic echinococcosis in clinical settings. With rapid development of surgical diagnosis and treatment technology and deepening understanding of hepatic echinococcosis, diagnosis and treatment regimens have also been constantly improved. In this article, research progresses on the diagnosis and treatment of hepatic alveolar echinococcosis were reviewed, aiming to provide reference for clinicians, deliver early diagnosis and treatment, mitigate adverse effects of this disease upon patients and improve clinical prognosis.

Prevalence of Echinococcus infection in small mammals captured from Shiqu County, Sichuan Province from 2015 to 2020 / 中国血吸虫病防治杂志

Objective To investigate the prevalence of Echinococcus infection in small mammals in Shiqu County, Sichuan Province from 2015 to 2020, so as to provide insights into echinococcosis control in Shiqu County. Methods One setting with frequent activity of small mammals was sampled as the survey site from each of 9 townships where human alveolar echinococcosis was hyperendemic, in Shiqu County, Sichuan Province from 2015 to 2020. Two quadrats measuring 50 m × 50 m were assigned in each survey site during the period between July and August from 2015 to 2020 to capture all small mammals in quadrats, and the species of small mammals were identified by morphological characteristics. All captured small mammals were dissected in the field and Echinococcus infection was identified by visual examinations. The affected organs of Echinococcus-infected small mammals were collected, and Echinococcus infection was detected using PCR assay, with Echinococcus species characterized. The prevalence of Echinococcus infection was calculated in small mammals, and the trends in the prevalence of Echinococcus infection were analyzed during the period from 2015 to 2020. In addition, the prevalence of Echinococcus infection was compared in small mammals using visual examinations and PCR assay. Results A total of 2 692 small mammals were captured in the survey sites of Shiqu County from 2015 to 2020, and morphology characterized 1 360 Microtus fuscus (50.52%) and 1 332 Plateau pika (49.48%). The prevalence rates of Echinococcus infection were 35.63%, 19.16%, 21.41%, 8.40%, 7.68% and 4.44% by visual examinations and 18.96%, 5.36%, 5.61%, 4.58%, 3.30% and 0.37% by PCR assay in small mammals in Shiqu County from 2015 to 2020, both showing a tendency towards a decline year by year (χ2 = 215.024 and 117.045, both P values < 0.001). The prevalence of Echinococcus infection was significantly higher in small mammals by visual examinations than by PCR assay during the period from 2015 to 2020 except in 2018 (χ2= 33.597, 21.815, 51.373, 17.268 and 9.537, all P values < 0.01). PCR assay detected a reduction in the prevalence of E. multilocularis infection from 10.21% to 0.37% and a reduction in the prevalence of E. shiquicus infection from 8.75% to 0 in small mammals in Shiqu County from 2015 to 2020, both appearing a tendency towards a decline year by year (χ2 = 117.045 and 43.436, both P values < 0.001). In addition, the prevalence of E. multilocularis and E. shiquicus infections reduced from 15.19% to 0.45% and from 8.23% to 0 in M. fuscus, and the prevalence of E. multilocularis and E. shiquicus infections reduced from 7.76% to 0 and from 9.01% to 0 in P. pika in Shiqu County from 2015 to 2020. Conclusions M. fuscus and P. pika were dominant species of small mammals in Shiqu County, Sichuan Province from 2015 to 2020, and E. multilocularis infection was mainly found in M. fuscus and E. shiquicus infection mainly found in P. pika. The prevalence of Echinococcus infection appeared a tendency towards a decline in both M. fuscus and P. pika year by year during the period from 2015 to 2020.

Effect of Echinococcus multilocularis infection on Tim3 expression in spleen natural killer cells of mice / 中国血吸虫病防治杂志

Objective To investigate the effect of Echinococcus multilocularis infection on Tim3 expression and its co-expression with immune checkpoint molecules 2B4 and LAG3 in spleen natural killer (NK) cells of mice. Methods C57BL/6 mice, each weighing (20 ± 2) g, were randomly divided into a high-dose infection group (15 mice), a low-dose infection group (13 mice), and a control group (11 mice). Mice in the high- and low-dose infection groups were inoculated with 2 000 and 50 Echinococcus multilocularis protoscolices via the hepatic portal vein, while animals in the control group was injected with an equivalent amount of physiological saline via the hepatic portal vein. Mouse spleen cells were harvested 12 and 24 weeks post-infection, and Tim3 expression and its co-expression with 2B4 and LAG3 in NK cells were detected using flow cytometry. Results There were significant differences in the proportions of Tim3 expression (F = 13.559, P < 0.001) and Tim3 and 2B4 co-expression (F = 12.465, P < 0.001) in mouse spleen NK cells among groups 12 weeks post-infection with E. multilocularis, and the proportion of Tim3 expression was significantly higher in mouse spleen NK cells in the low-dose infection group [(23.84 ± 2.28)%] than in the high-dose infection group [(15.72 ± 3.67)%] and the control group [(16.14 ± 3.83)%] (both P values < 0.01), while the proportion of Tim3 and 2B4 co-expression was significantly higher in mouse spleen NK cells in the low-dose infection group [(22.20 ± 2.13)%] than in the high-dose infection group [(14.17 ± 3.81)%] and the control group [(15.20 ± 3.77)%] (both P values < 0.01). There were significant differences in the proportions of Tim3 expression (F = 5.243, P < 0.05) and Tim3 and 2B4 co-expression (F = 4.659, P < 0.05) in mouse spleen NK cells among groups 24 weeks post-infection with E. multilocularis infection, and the proportions of Tim3 expression and Tim3 and 2B4 co-expression were significantly lower in mouse spleen NK cells in the high-dose infection group [(20.55 ± 7.04)% and (20.98 ± 7.12)%] than in the control group [(31.38 ± 3.19)% and (31.25 ± 3.06)%] (both P values < 0.05), and there were no significantly difference between the proportions of Tim3 expression and Tim3 and 2B4 co-expression in splenic NK cells in the low-dose infection group [(26.80 ± 6.47)% and (26.48 ± 6.48)%] and the control group (both P > 0.05). There were no significant differences in the proportions of Tim3 and LAG3 co-expression in mouse spleen NK cells among groups 12 (F = 2.283, P > 0.05) and 24 weeks post-infection (F = 0.375, P > 0.05). In the low-dose infection group, there were no significant differences in the proportions of Tim3 expression or Tim3 and 2B4 co-expression in mouse spleen NK cells 12 (t = −1.137, P > 0.05) or 24 weeks post-infection (t = −1.658, P > 0.05), and the proportion of Tim3 and LAG3 co-expression increased in mouse spleen NK cells 24 weeks post-infection relative to 12 weeks post-infection (t = −5.261, P < 0.01). In the highdose infection group, there was no significant difference in the proportion of Tim3 expression in mouse spleen NK cells 12 and 24 weeks post-infection (t = −1.546, P > 0.05); however, the proportions of Tim3 co-expression with 2B4 and LAG3 increased in mouse splenic NK cells 24 weeks post-infection relative to 12 weeks post-infection (t = −2.425 and −4.745, both P values < 0.05). Conclusions The Tim3 expression and Tim3 co-expression with LAG3 and 2B4 on spleen NK cells is affected by doses of E. multilocularis infection and disease stages, and present different phenotypes during the course of alveolar echinococcosis. NK cells tend to form an immunosuppressive phenotype with the progression of E. multilocularis infection, which facilitates immune escape and chronic parasitism of E. multilocularis.

Research progress in microRNA as a novel diagnostic marker for bone echinococcosis / 国际生物医学工程杂志

Bone echinococcosis is usually caused by Echinococcus granulosus and E. multilocularis. People become infected when they eat food or water contaminated with the eggs. Treatment of bone echinococcosis usually includes surgery and medication, but the lengthy and costly treatment imposes a heavy burden on patients. MicroRNAs (miRNA) are known to be involved in a variety of biological processes and host-host interactions, including development, cell growth and death, lifespan-related target regulation, transcription, signal transduction, and cell motility, which will help us find new strategies and targets for the treatment and control of osteonechoconiosis. For further understanding of bone echinococcosis, it is important to understand the molecular basis of E. multilocularis development in both final and intermediate hosts. The miRNA found in E. granulosus and E. multilocularis have gene and developmental stage specificity in their respective host expression regulation. In this review paper, the progress of research on miRNA as a novel diagnostic marker for osteoblastic echinococcosis is reviewed.

Preliminary study on the effect of Echinococcus multilocaris on phenotypic transformations of glucose metabolism and polarization types in macrophages / 中国血吸虫病防治杂志

Objective To investigate the effects of Echinococcus multilocularis on the phenotypic transformations of glucose metabolism, polarization types and inflammatory responses in macrophages, so as to provide insights into elucidation of echinococcosis pathogenesis. Methods Bone marrow cells were isolated from C57BL/6J mice at ages of 6 to 8 weeks, and induced into bone marrow-derived macrophages (BMDMs) with mouse macrophage colony-stimulating factor (M-CSF), which served as controls (BMDMs-M0). BMDMs-M0 induced M2 macrophages by interleukin-4 for 24 hours served as the IL-4 induction group, and BMDMs-M0 co-cultured with 2.4 ng/mL E. multilocularis cystic fluid (CF) served as the BMDM-CF co-culture group, while BMDMs-M0 co-cultured with E. multilocularis protoscolex (PSC) at a ratio of 500:1 served as the BMDM-PSC co-culture group. The types of polarization of BMDMs co-cultured with E. multilocularis CF and PSC were analyzed using flow cytometry, and the expression of macrophage markers, inflammatory factors, and glucose metabolism-related enzymes was quantified using fluorescent quantitative real-time PCR (qPCR) and Western blotting assays. Results There were significant differences among the four groups in terms of Arginase-1 (Arg1) (F = 1 457.00, P < 0.000 1), macrophages-derived C-C motif chemokine 22 (Ccl22) (F = 22 203.00, P < 0.000 1), resistin-like α (Retnla) (F = 151.90, P < 0.000 1), inducible nitric oxide synthase (iNOS) (F = 107.80, P < 0.001), hexokinase (HK) (F = 9 389.00, P < 0.000 1), pyruvate kinase (PK) (F = 641.40, P < 0.001), phosphofructokinase 1 (PFK1) (F = 43.97, P < 0.01), glucokinase (GK) (F = 432.50, P < 0.000 1), pyruvate dehydrogenase kinases1 (PDK1) (F = 737.30, P < 0.000 1), lactic dehydrogenase (LDH) (F = 3 632.00, P < 0.000 1), glucose transporter 1 (GLUT1) (F = 532.40, P < 0.000 1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (F = 460.00, P < 0.000 1), citrate synthase (CS) (F = 5 642.00, P < 0.01), glycogen synthase1 (GYS1) (F = 273.30, P < 0.000 1), IL-6 (F = 1 823.00, P < 0.000 1), IL-10 (F = 291.70, P < 0.000 1), IL-1β (F = 986.60, P < 0.000 1), and tumor necrosis factor (TNF)-α (F = 334.80, P < 0.000 1) and transforming growth factor (TGF)-β mRNA expression (F = 163.30, P < 0.001). The proportion of M2 macrophages was significantly higher than that of M1 macrophages in the BMDM-PSC co-culture group [(22.87% ±1.48%) vs. (1.70% ±0.17%); t = 24.61, P < 0.001], and the proportion of M2 macrophages was significantly higher than that of M1 macrophages in the BMDM-CF co-culture group [(20.07% ±0.64%) vs. (1.93% ±0.25%); t = 45.73, P < 0.001]. The mRNA expression of M2 macrophages markers Arg1, Ccl22 and Retnla was significantly higher in the BMDM-CF and BMDM-PSC co-culture groups than in the control group (all P values < 0.01), and no significant difference was seen in the mRNA expression of the M1 macrophage marker iNOS among the three groups (P > 0.05), while qPCR assay quantified higher mRNA expression of key glycolytic enzymes HK, PK and PFK, as well as inflammatory factors IL-10, IL-1β, TNF-α and TGF-β in the BMDM-CF and BMDM-PSC co-culture groups than in the control group (all P values < 0.01). Western blotting assay determined higher HK, PK and PFK protein expression in the BMDM-PSC co-culture group than in the control group (all P values < 0.05), and qPCR quantified higher GLUT1, GAPDH and IL-6 mRNA expression in the BMDM-CF co-culture group than in the control group (all P values < 0.05), while higher HK, PK and PFK protein and mRNA expression (all P values < 0.01), as well as lower IL-6 and TNF-α and higher TGF-β mRNA expression (both P values < 0.05) was detected in the IL-4 induction group than in the control group. Glycolytic stress test showed no significant difference in the extracellular acidification rate (ECAR) of mouse BMDM among the control group, IL-4 induction group and BMDM-PSC co-culture group (F = 124.4, P < 0.05), and a higher ECAR was seen in the BMDM-PSC co-culture group and a lower ECAR was found in the IL-4 induction group than in the control group (both P values < 0.05). Conclusions Treatment of E. multilocularis CF or PSC mainly causes polarization of BMDM into M2 macrophages, and phenotypic transformation of glucose metabolism into high-energy and high-glycolytic metabolism, and affects inflammatory responses in BMDM.

A case of echinococcosis in Hunan Province / 中南大学学报(医学版)

Echinococcosis is mainly prevalent in the agricultural and pastoral areas in the northwest of China, but it is relatively rare in Hunan Province. Here, we reported the clinical data of a case of echinococcosis in Hunan Province. The patient was an 11-year-old male, who sought treatment at the Second Xiangya Hospital of Central South University due to abdominal mass. According to the symptoms, signs, and laboratory examinations, he was initially diagnosed as "intra-abdominal mass" and "spleen cyst". Subsequently, he underwent abdominal massive occupying resection and splenectomy. Postoperative pathological examination revealed the cuticle and germinal layer of hydatid and protoscolex, which was consistent with characteristics of echinococcosis. In addition, the serological examination showed that the specific anti-hydatid IgG antibody was positive. Combined with the patient's condition, he was given praziquantel treatment. After a month of follow-up, the patient was asymptomatic.

Progress of researches on infection with two species of Echinococcus causing human diseases in animal hosts and influencing factors / 中国血吸虫病防治杂志

Echinococcosis is a serious zoonotic parasitic disease caused by infections with larval Echinococcus. The life cycle of Echinococcus involves a variety of animal hosts, including hoofed animals and rodents as intermediate hosts and carnivores as definitive hosts. The transmission of human echinococcosis is closely associated with the life cycle of E. granulosus and E. multilocularis among animal hosts in nature. This review summarizes the recent advances in the prevalence and influencing factors of E. granulosus and E. multilocularis infections in animal hosts, so as to provide insights into precision control of echinococcosis.

Role of CD155 in hepatocyte apoptosis and liver fibrosis induced by Echinococcus multilocularis infection in mice / 国际生物医学工程杂志

Objective:To investigate the role of CD155 in hepatocyte apoptosis and liver fibrosis in mice infected with Echinococcus multilocularis. Methods:Thirty-six female C57BL/6 mice were randomly divided into a sham surgery group and a model group, with 18 mice in each group. Mice in the model group were injected with protoscolex via the portal vein to create an animal model of E. multilocularis infection. Mice in the sham surgery group were injected with the same amount of saline. The mice were sacrificed at 1 month, 3 months, and 6 months after modeling, and liver samples were collected. Hepatic pathological changes were observed by hematoxylin-eosin staining. Liver fibrosis was detected by Sirius red staining, and expression of Caspase-3 and CD155 in hepatocytes was detected by immunohistochemical staining. The correlation between CD155 expression in hepatocytes and Caspase-3 and liver fibrosis levels were analyzed by Person. Results:There were obvious lesions in the liver of the model group accompanied by severe liver fibrosis. Compared with the sham surgery group, the expression of CD155 and Caspase-3 in mouse hepatocytes at different stages in the model group was significantly increased, and the differences were statistically significant ( P<0.05). The model group's liver fibrosis level was significantly higher at different stages than the sham surgery group, with statistical significance ( P<0.05). In addition, correlation analysis showed that expression of CD155 in hepatocytes was positively correlated with the expression of Caspase-3 ( r=0.956 8; P<0.001; 95% CI: 0.885 5-0.984 1) and that expression of CD155 in hepatocytes was positively correlated with the degree of liver fibrosis( r=0.853 9; P<0.001; 95% CI: 0.643 7-0.944 3). Conclusions:CD155 expression was significantly up-regulated in mouse hepatocytes infected with E. multilocularis at different stages, which was positively correlated with the degree of hepatocyte apoptosis and liver fibrosis, suggesting that CD155 may be involved in the process of hepatocyte apoptosis and liver fibrosis caused by E. multilocularis infection.

Establishment and preliminary application of a recombinase-aided isothermal amplification assay-based multiplex nucleic acid assay for detection of three Echinococcus species / 中国血吸虫病防治杂志

Objective To establish a multiplex nucleic acid assay for rapid detection of Echinococcus multilocularis, E. granulosus and E. shiquicus based on the recombinase-aided isothermal amplification assay (RAA) and to preliminarily assess its diagnostic efficiency. Methods The mitochondrial genomic sequences of E. multilocularis (GenBank accession number: NC_000928), E. granulosus (GenBank accession number: NC_044548) and E. shiquicus (GenBank accession number: NC_009460) were used as target sequences, and three pairs of primers were designed based on the RAA primer design principle and synthesized for the subsequent multiple RAA amplification. The genomic DNA of E. multilocularis, E. granulosus and E. shiquicus at different concentrations and the recombinant plasmids containing the target gene at various concentrations were amplified to evaluate the diagnostic sensitivity of the multiplex RAA assay, and the genomic DNA of E. multilocularis, E. granulosus, E. shiquicus, Taenia multiceps, T. saginata, T. asiatica, Dipylidium caninum, T. hydatigena, Toxocara canis, Fasciola hepatica, T. pisiformis, Mesocestoides lineatus and Cryptosporidiumn canis was detected using the multiplex RAA assay to evaluate its specificity. In addition, the reaction condition of the multiplex RAA assay was optimized, and was then employed to detect the tissues with echinococcosis lesions, simulated canine fecal samples and field captured fox fecal samples to examine its application values. Results The multiplex RAA assay was effective to specifically amplify the mitochondrial gene fragments of E. multilocularis, E. granulosus and E. shiquicus within 40 min at 39 °C, with sequence lengths of 540, 430 bp and 200 bp, respectively. This multiplex RAA assay showed the minimum detection limits of 2.0, 2.5 pg/μL and 3.1 pg/μL for detection of the genomic DNA of E. multilocularis, E. granulosus and E. shiquicus, and presented the minimum detection limit of 200 copies/μL for detection of the recombinant plasmids containing E. multilocularis, E. granulosus and E. shiquicus target genes. This multiplex RAA assay was effective to simultaneously detect single and multiple infections with E. multilocularis, E. granulosus and E. shiquicus, but failed to amplify the genomic DNA of T. multiceps, T. saginata, T. asiatica, D. caninum, T. hydatigena, T. canis, F. hepatica, T. pisiformis, M. lineatus and C. canis. In addition, the optimized multiplex RAA assay was effective to detect all positive samples from the tissue samples with echinococcosis lesions, simulated canine fecal samples and field captured fox fecal samples, which was fully consistent with the detection of the single PCR assay. Conclusion A sensitive and specific multiplex nucleic acid assay for rapid detection of E. multilocularis, E. granulosus and E. shiquicus has been successfully established.

Effects of persistent Echinococcus multilocularis infections on hepatic fibrosis in mice / 中国血吸虫病防治杂志

Objective To investigate the effects of persistent Echinococcus multilocularis infections on hepatic fibrosis in mice, so as to provide insights into the understanding of liver fibrogenesis induced by E. multilocularis infections and the treatment of alveolar echinococcosis. Methods Hepatic stellate HSC-T6 and LX-2 cells were exposed to the sera (25, 50 and 100 μL) from Meriones unguiculatus infected with E. multilocularis, and E. multilocularis, germinal layer cells (GCs) and protoscoleces (PSCs) for 48 hours, respectively. The cell proliferation was measured using a CCK-8 assay, and the levels of collagen 1 (Col1) and α-smooth muscle actin (α-SMA) were measured in the culture supernatant of HSC-T6 cells using ELISA. In addition, the serum and liver samples were collected 1, 2, 4, 6, 8 months post-infection with E. multilocularis, respectively. The serum Col1 and α-SMA concentrations were measured using enzyme-linked immunosorbent assay (ELISA), and the deposition of collagen fibers was examined in mice livers using Sirius red staining. Results The sera of E. multilocularis-infected gerbils promoted the proliferation of HSC-T6 and LX-2 cells in vitro, and there were significant differences seen in the proliferative rate of HSC-T6 (FHSC-T6 = 126.50, P < 0.05) and LX-2 cells (FLX-2 = 201.50, P < 0.05) among different serum groups, with the highest proliferative rate of HSC-T6 (573.36% ± 206.34%) and LX-2 cells (940.38% ± 61.65%) found following exposure to 100 μL mouse sera. Exposure to serum from E. multilocularis-infected gerbils resulted in an increase in the Col1 and α-SMA levels in the culture supernatant of HSC-T6 cells, with the greatest Col1 (20.99 ng/mL ± 2.01 ng/mL) and α-SMA levels (305.52 pg/mL ± 16.67 pg/mL) measured following exposure to 100 μL sera. The metacestodes (142.65% ± 9.17% and 189.99% ± 7.75%), GCs (118.55% ± 8.96% and 122.54% ± 0.21%) and PSCs of E. multilocularis (156.34% ± 17.45% and 160.59% ± 31.41%) all promoted the proliferation of HSC-T6 and LX-2 cells in vitro, and there were significant differences in the proliferative rates of HSC-T6 (FHSC-T6 = 11.24, P < 0.05) and LX-2 cells among groups (FLX-2 = 47.72, P < 0.05). Exposure to E. multilocularis resulted in an increase in Col1 and α-SMA levels in the culture supernatant of HSC-T6 cells, and the highest Col1 (4.43 ng/mL ± 2.23 ng/mL) and α-SMA levels (285.20 pg/mL ± 90.67 pg/mL) were detected following treatment with E. multilocularis metacestodes. In addition, a persistent increase was seen in the deposition of collagen fibers in mice livers 1 to 8 months post-infection with E. multilocularis, with the greatest Col1 level (280.26 ng/mL ± 23.04 ng/mL) seen 6 months post-infection and the highest α-SMA level (33.68 ng/mL ± 4.45 ng/mL) detected 8 months post-infection, respectively. Conclusions Persistent E. multilocularis infections promote hepatic stellate cell proliferation, induce an increase in mouse serum Col1 and α-SMA levels, and cause elevated deposition of collagen fibers in mice livers. The infective stage of E. multilocularis is a critical period for inducing hepatic fibrosis of alveolar echinococcosis.

Effect of Echinococcus multilocularis infections on mitochondrial functions of macrophages / 中国血吸虫病防治杂志

Objective To investigate the changes of mitochondrial metabolic functions of macrophages following Echinococcus multilocularis infections, so as to provide insights into the pathogenesis of alveolar echinococcosis. Methods Two groups were assigned according to different treatment methods. In the culture group, mouse leukemic monocyte macrophage RAW264.7 cells were cultured with 2 000 E. multilocularis at a ratio of 500∶1, while RAW264.7 cells in the control group were given no treatment. Then, both the culture and control groups were further divided into the 24 h and 72 h subgroups. Mitochondria were stained with MitoTracker® Deep Red FM and the mean fluorescence intensity of macrophage mitochondria was measured with the Cytation 5 Cell Imaging Multi-Mode Reader. The mitochondrial DNA copy number was quantified using the quantitative real-time PCR (qPCR) assay, and the mitochondrial energy metabolism was monitored using the Seahorse XF assay. In addition, the mitochondrial reactive oxygen species and mitochondrial membrane potential were detected using flow cytometry. Results The mean fluorescence intensities of macrophage mitochondria were significantly lower in the 24 h (15 341 ± 2 532 vs. 17 823 ± 3 429; t = 6.379, P < 0.01) and 72 h (18 102 ± 3 505 vs. 21 511 ± 5 144; t = 17.680, P < 0.01) culture subgroups than in the corresponding control subgroups, and lower mitochondrial DNA copy numbers were measured in the 72 h culture subgroup than in the 72 h control group [(3.23 × 109 ± 1.78 × 107) vs. (4.39 × 109 ± 3.70 × 107); t = 8.85, P < 0.001]. The oxygen consumption rates were significantly greater in the 24 h [(241.70 ± 73.13) pmol/min vs. (69.05 ± 52.30) pmol/min; t = 7.89, P < 0.01] and 48 h culture groups [(249.50 ± 42.06) pmol/min vs. (60.28 ± 40.66) pmol/min; t = 8.64, P < 0.01] than in the corresponding control groups, and a higher extracellular acidification rate was seen in the 48 h culture group than in the 48 h control group ([ 111.6 ± 17.49) mpH/min vs. (35.05 ± 7.57) mpH/min; t = 16.90, P < 0.01]. In addition, flow cytometry detected higher mean fluorescence intensity of mitochondrial reactive oxygen species (58 264 ± 10 087 vs. 4 307 ± 97; t = 12.930, P < 0.01) and lower mitochondrial membrane potential (9.833% ± 2.285% vs. 2.667% ± 0.208%; t = 6.645, P < 0.01) in the 72 h culture group than in the control group. Conclusions E. multilocularis infection may impair mitochondrial functions and inhibit oxidative phosphorylation of macrophages, resulting in increased macrophage glycolysis. It is speculated that the alteration of macrophage metabolic states may contribute to the mechanisms underlying the development and progression of alveolar echinococcosis.

Exhaustion of CD8 T cell immune functions in spleen of mice with different doses of Echinococcus multilocularis infections / 中国血吸虫病防治杂志

ObjectiveTo examine the changes in the immune functions of CD8+ T cells in the spleen of mice following Echinococcus multilocularis infections at various doses and at different time points. MethodsThe E. multilocularis protoscoleces were collected, and E. multilocularis infection was modeled in mice via the hepatic portal vein at doses of 50 (low-dose), 500 (medium-dose) and 2 000 protoscoleces (high-dose), while physiological saline served as controls. Mouse spleen was isolated 2 (earlystage), 12 (middle-stage) and 24 weeks post-infection (late-stage), and spleen lymphocytes were harvested. The phenotype of memory CD8+ T cells and 2B4 expression were quantified in the mouse spleen, and the secretion of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-17A and IL-10 was measured. Results A central-memory phenotype was predominant in the CD8+ T cells in the spleen of mice at the early stage of high-dose protoscolece infections, and the proportion of central-memory CD8+ T cells was significantly greater in the high-dose group than in the control group (35.50% ± 2.00% vs. 25.90% ± 2.46%, P < 0.01), while a effector- memory phenotype was predominant in the CD8+ T cells in the spleen of mice at the late stage of medium- and high-dose protoscolece infections, and the proportions of effector-memory CD8+ T cells were significantly greater in the medium- (25.70% ± 4.12%) and high-dose group (28.40% ± 4.12%) than in the control group (10.50% ± 6.45%) (P < 0.05). The proportions of the central-memory CD8+ T cells were significantly higher in the high-dose group than at middle and late stages than at the early stage (P < 0.01), and the proportion of effector-memory CD8+ T cells was significantly greater in the high-dose group at the late stage than at early and middle stages (P < 0.05). The secretion of IFN-γ and IL-17A by spleen CD8+ T cells was elevated in the low- and medium-dose groups at the early stage of infection, and high-dose protoscolece infection promoted the secretion of IFN-γ and TNF-α by spleen CD8+ T cells; however, the levels of IFN-γ and TNF-α were significantly lower at the late stage than at the early and middle stages (P < 0.05). In addition, high 2B4 expression was detected in spleen CD8+ T cells in the middle- and high-dose groups at the late stage of infection, and the 2B4 expression was significantly higher in the medium(4.73% ± 1.56%) and high-dose groups (4.94% ± 1.90%) than in the low-dose group (2.49% ± 0.58%) and the control group (2.92% ± 0.60%) (P < 0.05). Conclusions E. multilocularis may be killed and eliminated through the host immune responses at the middle and late stages of low- and medium-dose protoscolece infections, while high-dose protoscolece infections may trigger the upregulation of 2B4 expression in mouse spleen CD8+ T cells at the late stage, which leads to immune exhaustion and the resultant chronic infections.

Establishment of a recombinase-aided isothermal amplification assay for nucleic acid detection of Echinococcus multilocularis and its preliminary application / 中国血吸虫病防治杂志

Objective To establish a rapid nucleic acid detection technique for identification of Echinococcus multilocularis based on the recombinase aided isothermal amplification assay (RAA) and assess its diagnostic efficiency. Methods The mitochondrial gene sequence of E. multilocularis (GenBank accession number: AB018440) was used as a target sequence. The primers were designed according to the RAA reaction principle and synthesized, and RAA was performed using the generated primers. E. multilocularis genomic DNA at various concentrations and the pMD19-T (Simple) vector containing various copies of the target gene fragment were amplified using RAA to evaluate its sensitivity for detection of E. multilocularis, and RAA was em- ployed to detect the genomic DNA of E. granulosus G1 genotype, Taenia saginata, T. asiatica, T. multiceps, Dipylidium caninum, Toxocara canis, Trichuris trichiura, Giardia lamblia, Fasciola hepatica, Paragonimus westermani, Fasciola gigantica and Clonorchis sinensis to evaluate its specificity. In addition, the optimized RAA was employed to detect nine tissue specimens of E. granulosus-infected animals, 3 fecal samples from E. granulosus-infected dogs and 2 fecal samples from field infected dogs to examine its reliability and feasibility. Results The established RAA was able to detect the specific target gene fragment of E. multilocularis within 40 min. The lowest detect limit of RAA was 10 pg if E. multilocularis genomic DNA served as a template. If the re- combinant plasmid was used as a template, the minimally detectable copy number of RAA was 104. In addition, RAA was nega- tive for the genomic DNA of E. granulosus G1 genotype, T. saginata, T. asiatica, T. multiceps, D. caninum, T. canis, T. trichiura, G. lamblia, F. hepatica, P. westermani, F. gigantica and C. sinensis. The established RAA was positive for detection of the tissue specimens of infected animals, and simulated and field dog stool samples. Conclusion A rapid, sensitive and specific RAA is established, which shows promising values in identification of E. multilocularis and gene diagnosis of alveolar echinococcosis.

Study on the molecular phylogeny of Echinococcus multilocularis in Qinghai Province / 中国血吸虫病防治杂志

Objective To amplify and sequence Coxl and Nadl genes in Echinococcus multilocularis isolates from Qinghai Province, and to create phylogenetic trees and molecular clocks, so as to provide evidence for estimating the evolutionary relationships and origins of E. multilocularis in Qinghai Province. Methods Twenty-two post-surgical specimens of patients with hepatic alveolar echinococcosis were sampled from Qinghai Provincial People’s Hospital in 2017. The Coxl and Nadl genes were amplified from E. multilocularis samples and sequenced. Then, the gene sequences were aligned to the Coxl and Nadl genes of Echinococcus species in GenBank database. The intra-species variation was observed, and the phylogenetic tree and molecular clock were created. Results All E. multilocularis samples shared more than 99% genetic homology with the sequences of Coxl and Nadl genes from the E. multilocularis Asian strain in the GenBank database. A total of 6 genotypes were identified, including 2 isolates that had no 100% homology with the sequences of known genes in the GenBank database. Phylogenetic tree analysis revealed remarkable clustering of the E. multilocularis samples with the E. multilocularis Asian strain, and the E. multilocularis isolates from Qinghai Province were estimated to date back to 94 000 years ago by the molecular clock. Conclusions The present study characterizes 6 E. multilocularis genotypes in Qinghai Province, including 2 novel genotypes. Asian strain is the predominant strain of E. multilocularis in Qinghai Province, and the E. multilocularis isolates from Qinghai Province date back to 94 000 years ago.

Study on the molecular phylogeny of Echinococcus multilocularis in Qinghai Province / 中国血吸虫病防治杂志

Objective To amplify and sequence Coxl and Nadl genes in Echinococcus multilocularis isolates from Qinghai Province, and to create phylogenetic trees and molecular clocks, so as to provide evidence for estimating the evolutionary relationships and origins of E. multilocularis in Qinghai Province. Methods Twenty-two post-surgical specimens of patients with hepatic alveolar echinococcosis were sampled from Qinghai Provincial People’s Hospital in 2017. The Coxl and Nadl genes were amplified from E. multilocularis samples and sequenced. Then, the gene sequences were aligned to the Coxl and Nadl genes of Echinococcus species in GenBank database. The intra-species variation was observed, and the phylogenetic tree and molecular clock were created. Results All E. multilocularis samples shared more than 99% genetic homology with the sequences of Coxl and Nadl genes from the E. multilocularis Asian strain in the GenBank database. A total of 6 genotypes were identified, including 2 isolates that had no 100% homology with the sequences of known genes in the GenBank database. Phylogenetic tree analysis revealed remarkable clustering of the E. multilocularis samples with the E. multilocularis Asian strain, and the E. multilocularis isolates from Qinghai Province were estimated to date back to 94 000 years ago by the molecular clock. Conclusions The present study characterizes 6 E. multilocularis genotypes in Qinghai Province, including 2 novel genotypes. Asian strain is the predominant strain of E. multilocularis in Qinghai Province, and the E. multilocularis isolates from Qinghai Province date back to 94 000 years ago.

Death pattern and cause analysis in mice after establishment of the liver alveolar hydatid model / 中国人兽共患病学报

We studied the death pattern and the main causes of death after artificial liver inoculation with alveolar hydatid model,and explored the risk factors and prevention of major complications resulting from death after modeling.The 50 healthy Kunming mice were inoculated with open-staring liver puncture and the physiological changes of the mice were observed dy-namically within 1 5 days after operation.The time and amount of death were recorded,and the causes of death and related fac-tors were analyzed emphatically.On the 1 5 day after surgery,1 9 died;1 died in the ninth day,and the remaining 1 8 died with-in 7 days of the operation.The postoperative death rate began to rise in the second day,and gradually decreased to 1 after the peak of the 4 th and 5 th days.The main causes of death of the mice after inoculation were closely related to the complications of surgical methods,anaesthesia,infection,antigenicity of vesicle,and liver injury.Effective prevention of these complications is the key to improve the survival rate.

Analysis of Lethality in Echinococcal Disease

The information on mortality from echinococcosis is important not only for a better understanding of the severity of the disease, but also for evaluating the effectiveness of public health interventions. The aim of this research was to study the causes of mortality from echinococcosis. We have collected and analyzed the materials of 1,470 patients in 10 age - groups in the Republic of Armenia (from 2000 to 2016). To find out the causes of mortality from echinococcosis, we have analyzed the medical histories and protocols of postmortem examinations of 19 deaths from echinococcosis and 17 deaths due to other indirect causes not associated with the parasite. The average annual death rate from echinococcosis is 0.007 per 10,000 population, and the mortality is 1.29 (per 100 patients). The highest mortality occurs in people aged 70–79. Mortality from echinococcosis is also recorded among the unoperated children. The rupture of the parasitic cyst and hepatic insufficiency are major among the direct causes of mortality. Sometimes the hydatid cysts unrecognized during the life were first diagnosed at autopsy. Insufficient qualification of doctors in the field of helminthology, as well as the latent course of the disease or manifestation of minor symptoms in echinococcosis over a long period often led to medical errors. Further decline in mortality can be achieved by early diagnosis, timely hospitalization and treatment before the development of severe complications worsening the prognosis and outcomes of surgical intervention.

Emerging alveolar echinococcosis and gene fragments of Nad 2 and Cob in Nileke County, Yili area of Xinjiang, China / 中国人兽共患病学报

We investigated the endemic situation of alveolar echinococcosis (AE) in Nileke County,Yili Prefecture,Xinjiang and its genetic diversity of Echinococcus multilocularis.Human AE cases in the county were retrospectively investigated including their location and surrounding ecological conditions.Sequence detection and analysis of nad2,cob gene fragments of AE mtDNA from patients was used to identify genotype variation.Results showed that a total of 48 AE cases were diagnosed and the first AE patient was identified in 1989 in the county.The 45.8％ of AE cases were found in the recent 5 years (2011-2015) and annual prevalence was 1.7 per 100 000.The patients were distributed along the Kashgar River,particularly intensive in Wulasitai Township.The 38.6％ of the patients were aged arranged 35-44 years old,64.6％ were male and 95.8％ were farmers and herdsmen.AE cases were confirmed further and there was only one haplotype by sequencing analysis from 11 AE clinical patients in the county.It suggests that Nileke County is AE foci,and alveolar echinococcosis with sequences conserved is an emerging disease in the county.