Table of Content

30 June 2022, Volume 40 Issue 3

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EXPERT VIEWPOINTS

The strategy on One Health development in China based on SWOT analysis

LI Zhen, GUO Xiao-kui, WANG Yue-xiang, ZHENG Bin, ZHOU Xiao-nong

2022, 40(3):  271-277.  doi:10.12140/j.issn.1000-7423.2022.03.001

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The pandemic of Corona Virus Disease 2019 (COVID-19) worldwide has prompted the use One Health concept to solve health problems and improve the public health governance system. Using the Superiority Weakness Opportunity Threats (SWOT) analysis method to analyze the opportunities and challenges brought by the current development of One Health in China. The results show that the current advantage is that the Chinese government attaches great importance to One Health, and Chinese scholars are also actively involved in the development of One Health, but there are still disadvantages of weak foundation and low international influence. At the same time, with the opportunity for more recognition of the concept of One Health in the world, China is facing challenges such as insufficient talent competitiveness and unbalanced development in the development of One Health. In this regard, this paper puts forward the strategies and key research contents for developing One Health in China to provide ideas for promoting the development of One Health in China.

Molecular epidemiology and subtype distribution of Cryptosporidium parvum from dairy cattle in China

CHEN Yuan-cai, HUANG Jian-ying, LI Jun-qiang, ZHANG Long-xian

2022, 40(3):  278-284.  doi:10.12140/j.issn.1000-7423.2022.03.002

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Globally, most strains of Cryptosporidium parvum subtypes cause zoonotic infections. Dairy cattle are the important host for the natural infection of C. parvum and the important source of infection for human cryptosporidiosis. In this paper, the molecular epidemiology of C. parvum from dairy cattle was reviewed. The Ⅱd subtype distribution of C. parvum in dairy cattle in China was analyzed. The diversity distribution of Ⅱd subtype was summarized and the development trend of Ⅱd subtype was predicted. This review is of great significance for the prevention and control of C. parvum.

ORIGINAL ARTICLES

Identification and bioinformatics analysis of differentially expressed miRNAs in splenic lymphocytes in Echinococcus multilocularis-infected mice

ZHONG Shun-hu, SUN Yue, GUO Xiao-la, ZHENG Ya-dong, CHEN Yi-xia

2022, 40(3):  288-294.  doi:10.12140/j.issn.1000-7423.2022.03.003

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Objective To screen and identify the differentially expressed microRNAs (miRNAs) in spleen lymphocytes of mice infected with Echinococcus multilocularis, analyze the possible biological processes involved and signalling pathways, and provide experimental basis for further study of the role of miRNAs in parasite infection. Methods Twelve mice were randomly divided into two groups with 6 mice in each group. Each mouse in the experimental group was intraperitoneally injected with 600 protoscoleces, while the control group was injected with the same amount of PBS. On the 90th day after infection, the spleen lymphocytes of the mice in each group were separated by density gradient centrifugation, and the total RNA of the spleen lymphocytes was extracted by the TRIzol method. The differentially expressed miRNAs were identified and screened by high-throughput sequencing. Five differentially expressed miRNAs were randomly selected for real-time quantitative PCR (qRT-PCR) validation. MiRanda and RNAhybrid databases were used to predict the target genes of differentially expressed miRNAs, and gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis was performed. Cytoscape software was used to construct miRNA-mRNA interaction network diagrams. Results In the high-throughput sequencing, 12 104 631 and 10 856 249 clean reads were obtained in the control group and the experimental group, respectively. The sequences of clean reads were abundant in 20~24 nt in length. A total of 69 differentially expressed miRNAs (fold change> 2) were screened by high-throughput sequencing technology, of 40 were up-regulated, and 29 were down-regulated. The qRT-PCR results showed that the expression of miR-150-5p, miR-181a-5p, miR-467a-5p, and miR-467b-5p was down-regulated while the expression of miR-223-3p was up-regulated. GO enrichment results showed that the targets of 69 differentially expressed miRNAs were involved in mucosal immunity, stress response, and bacterial defence response. KEGG pathway analysis revealed that these genes were involved in the mitogen-activated protein kinase (MAPK) signalling pathway, phosphatidylinositol 3-kinase/protein kinase B (PI3K-Akt) signaling pathway, adenosine monophosphate-activated protein kinase (AMPK) signalling pathway, and other related pathways. The miR-150-5p, miR-181a-5p, miR-467a-5p, and miR-467b-5p and mRNA interaction network diagram shows that a miRNA interacted with multiple mRNAs, and a mRNA can also be regulated by multiple miRNAs. Conclusion The miRNA expression in mice spleen lymphocytes was significantly elevated during E. multilocularis infection, while the differentially expressed miRNAs were mainly enriched in some immune-related signalling pathways, which may play a role in the immune responses against E. multilocularis.

Effect of osteopontin expression level on the growth and development of Echinococcus multilocularis protoscoleces

ZHUO Yi-cheng, YANG Hai-cheng, LIU Cheng-hao, ZHANG Bao-cai, DUO Xiao-yong, ZHANG Shi-jie

2022, 40(3):  299-304.  doi:10.12140/j.issn.1000-7423.2022.03.004

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Objective To investigate the effect of osteopontin (OPN) expression level on the growth and development of Echinococcus multilocularis protoscoleces. Methods Protoscoleces were isolated from E. multilocularis-infected Meriones unguiculatus and cultured in vitro for 3 days. The E. multilocularis protoscoleces were assigned into four groups: LV-EmOPN-734 (knockdown EmOPN) group, LV3-NC (knockdown control) group, LV-EmOPN-0423 (overexpression EmOPN) group, and LV5-NC (overexpression control) group. Each group was tested in parallel triplicate wells, with 5 000 protoscoleces in each well. LV-EmOPN-734 lentivirus diluent (final concentration 1.4 × 10⁷ TU/ml) was added to the silenced EmOPN group, LV-EmOPN-0423 lentivirus diluent (final concentration 4.83 × 10⁸ copies/ml) was added to the EmOPN overexpression group, and the corresponding control group was added with the same amount of lentivirus diluent (blank plasmid). After 72 hours of lentivirus infection, the fluorescence intensity of each group of protoscoleces was observed under fluorescence microscope, the relative expression of OPN in protoscoleces was detected by Western blotting, Caspase-3 activity was detected by applying cysteine aspartate-3 (Caspase-3) assay kit, and the proliferation ability of protoscoleces was detected by applying EdU Imaging kit (Cy3). 72 hours after infection, the protosegment was co-cultured with rat hepatoma cells for 8-12 weeks, and the morphology of the germinal layer of E. multilocularis vesicles was observed by scanning electron microscopy. Statistical analysis of differences between groups using t-test. Results After 72 hours of lentivirus infection, it can be seen that the E. multilocularis protoscoleces were infected by lentivirus successfully, which is distributed in a circle or dots. Western blotting results showed that the relative expression of EmOPN in the LV-EmOPN-734 group was (0.43 ± 0.04), which was lower than that in LV3-NC group (0.80 ± 0.07) (t = 8.623, P < 0.01); the relative expression of EmOPN in LV-EmOPN-0423 group was (1.18 ± 0.21), which was higher than that in the LV5-NC group (0.73 ± 0.06) (t = 3.333, P < 0.05). Caspase-3 activity assay showed that the Caspase-3 activity was (61.55 ± 1.64) μmol/L in the LV-EmOPN-734 group, higher than (28.20 ± 2.16) μmol/L in the LV3-NC group (t = 24.57, P < 0.01); Caspase-3 activity in the LV-EmOPN-0423 group was (50.11 ± 6.45) μmol/L, which was lower than (78.22 ± 16.43) μmol/L in the LV5-NC group (t = 3.185, P < 0.01). The results of EdU Imaging Kits (Cy3) assay showed that the protoscoleces of EdU+ in the LV-EmOPN-734 group were(0.47 ± 0.06), which was lower than that of the LV3-NC group (0.72 ± 0.10) (t = 3.663, P < 0.05); the protoscoleces of EdU+ in the LV-EmOPN-0423 group had of EdU+ protoscolecesof (0.81 ± 0.09), which was higher than that of the LV5-NC group (0.54 ± 0.06) (t = 4.309, P < 0.05). Scanning electron microscopy observations showed that most of the germinal layer cells of the LV-EmOPN-734 group were collapsed, shrank, shed to a large extent, and lost their normal structure; while the germinal layer cells of the LV-EmOPN-0423 group had intact cell membranes and full morphology, forming germinal layer vesicles and growing small tips to connect with the germinal layer. Conclusion The OPN expression level upon rising-up or lowering-down may promote or inhibit the growth and development of the E. multilocularis protoscoleces.

Analysis of the influencing factors of lesion activity in hepatic cystic echinococcosis patients

HOU Jiao, WEN Hao, WANG Ming-kun, JIANG Tie-min, FANG Bin-bin, LI Jing, ZHANG Chuan-shan, WANG Hui

2022, 40(3):  309-314.  doi:10.12140/j.issn.1000-7423.2022.03.005

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Objective To identify the main factors affecting the active state of lesions in patients with hepatic cystic echinococcosis and provide the basis for decision-making to improve the diagnosis and treatment of cystic echinococcosis. Methods A total of 251 patients with hepatic cystic echinococcosis receiving surgical treatment at the First Affiliated Hospital of Xinjiang Medical University from between January 2018 and June 2020 were enrolled, and their demographic information and clinical data were collected, Based on the imaging and biological characteristics, the cysts classified as CE1, CE2 and CE3 were grouped as active group, and CE4 and CE5 inactive group. Using the cyst activity as the dependent variable, univariate analysis was performed, based on which unconditinal logistic regression model was constructed to analyze the independent factors influencing the cyst activity status. Results In this study, clinical data of 251 patients with hepatic cystic echinococcosis meeting the inclusion criteria were collected, with 57.0% (143 cases) female and 43.0% (108 cases) male. The age of the patients ranged from 4 to 85 years old, and the average age was (41.31 ± 15.05) years old. Most of the patients had a low educational level, with high school or below accounting for 79.3% (199 cases); the rural resident accounted for 66.9% (168 cases), which is higher than that of the urban resident 33.1% (83 cases). The percentage of CE in Han nationality was 42.2% (106 cases), while that in other minority nationalities was 57.8% (145 cases). The percentage of patients with their first infection was 78.1% (196 cases), which is higher than that of the recurrent infection (21.9%, 55 cases). The active lesions accounted for 76.9% (193 cases), and the inactive lesions accounted for 23.1% (58 cases). The results of the univariate analysis showed that there were significant differences among patients with different age groups, ethnic groups, the active state of lesions between patients with hepatic cystic echinococcosis and those without recurrence, the number of lesions, the size of lesions, red blood cell count, prothrombin time, direct bilirubin and alkaline phosphatase (χ² = 17.110, 7.797, 2.906, 4.702, 16.520, Z = -1.989, 2.446, 2.003, 1.914, P < 0.1). Unconditional Logistic regression results showed that the main factors affecting the active status of lesions in patients with hepatic cystic echinococcosis were age and lesion size. Larger lesions tend to occur in younger patients(P < 0.05). Conclusion The analysis on the clinical data of 251 hepatic cystic echinococcosis patients receiving surgical treatment demonstrated that younger age and larger cyst size were the independent factors affecting the activity status of cysts.

Bioinformatics analysis of the sensitization mechanisms and molecular targets of Echinococcus granulosus

XILIZATI Kulaixi, WANG Chun-sheng, WANG Jia-ling, LI Meng, FANG Zhi-yuan, WANG Si-jia, ZHOU Jing-ru, XIANYIDAN Abulajiang, WU Er-ge-li, YE Jian-rong

2022, 40(3):  319-323.  doi:10.12140/j.issn.1000-7423.2022.03.006

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Objective To analyze the sensitization mechanisms and molecular targets of Echinococcus granulosus using bioinformatics approaches. Methods Eighteen BALB/c mice were randomly grouped as infection- unsensitized (CE), infection-sensitized (ANPC), and uninfected controls (CTRL). Mice in the infected unsensitized group were injected with protoscoleces suspension (0.2 ml/mouse), followed with saline (0.2 ml/mice) after 90 days. Mice in the infected sensitized group were injected with protoscoleces suspension (0.2 ml/mice), followed with crude cyst fluid (0.2 ml/mice) 90 days after infection. Mice in the uninfected control group were injected with an equal amount of saline. Splenic tissue from three mice of each group were collected 1 h after injection for RNA extraction and transcriptome sequencing. Genes of differential expression were identified by bioinformatics analysis. Significance analysis of differential expression differences was performed using edgeR (quantified with RNA-Seq by expectation-Maximization RSEM) software. Network interaction analysis was performed using the String database and Cytoscape software with the score set as > 500. The network analysis of the genes was then performed using the Cytoscape software and use cytoHubba to obtain the degree of nodes (degree) and the nodes with degrees over 3 were filtered for display. The analysis was performed using the DAVID online enrichment analysis tool, and the results were visualized using the R language ggplot2. Results A total of 743 genes were differentially expressed in the ANPC group compared to the CTRL group, including 568 upregulated genes and 175 downregulated genes. A total of 554 genes were differentially expressed in the CE group compared to the CTRL group, including 393 upregulated genes and 161 downregulated genes. There are 458 genes specifically expressed in the ANPC group compared to the CTRL group, 269 genes specifically expressed in the CE group compared to the CTRL group and 285 genes co-expressed. GO enrichment of 458 genes, which are unique to the ANPC group, and the CTRL group showed that 147 genes are involved in the biological process, 10 genes are involved in cellular components, and 43 genes are involved in molecular functions. The GO enrichment of shared genes showed that 56 were involved in biological processes, 23 in cellular components, and 25 in molecular functions. The GO enrichment of genes that are unique to the CE group and CTRL group revealed that 51 genes are involved in biological processes, 13 genes are involved in cellular components, and 18 genes are involved in molecular functions. The functions of the ANPC group-specific genes and the CTRL group-specific genes mainly focus on inflammatory response and immune response. The functions of genes expressed in both the ANPC and CTRL groups mainly focus on the neuropeptide signaling pathway and cellular response to interleukin-1. The function of CE group-specific genes and the CTRL group-specific genes mainly focuses on the nucleosome assembly and the positive regulation of ERK1 and ERK2 cascades. The genes expressed explicitly in the ANPC group, and the CTRL group was enriched in 32 pathways, including cytokine-cytokine receptor interaction and the JAK-STAT signalling pathway. The genes commonly expressed in all groups were enriched in 9 pathways, including the phosphatidylinositol 3-kinase/protein kinase B (PI3K-Akt) signaling pathway and complement and coagulation cascades. The CE specific genes and the CTRL group-specific genes are enriched to 12 pathways, including Tyrosine metabolism and Linoleic acid metabolism, and identified key genes through the PPI network. Conclusion The inflammatory response plays an important role in metacestode sensitizing process; high expression of IL-6 promotes the occurrance of inflammation; IL-10 functions as an anti-inflammatory factor confronting the host’s inflammation response; IL-13 may be the key gene in developing immune tolerance after metacestode infection. ERK signaling pathway may play an immunomodulatory role during the growth of metacestodes. Cytokine-cytokine receptor interaction pathway and PI3K-Akt signaling pathway were significantly correlated with the sensitization induced by Einococcus infection.

Effect of albendazole-loaded vesicles on the vitality of protoscoleces of Echinococcus granulosus

QIAO Shi-yuan, ZHOU Xue, LIU Cheng-hao, JIANG Hui-jiao, BU Yuan-yuan, CHEN Xue-ling, WU Xiang-wei

2022, 40(3):  324-329.  doi:10.12140/j.issn.1000-7423.2022.03.007

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Objective To investigate the effect of albendazole-loaded extracellular vesicles (drug-loaded vesicles) on the vitality of Echinococcus granulosus protoscolex in vitro. Methods The mouse hepatoma cells H22 culture suspension was assigned into low, medium and high concentration groups, to each of which albendazole was added at final concentrations of 200, 400 and 600 μmol/L, respectively. UV irradiation (UVB, 300 J/m²) was performed for 1 hour, followed by 18-20 hours of incubation. The drug-loaded vesicles were produced by ultra-high speed differential centrifugation. Vesicles without albendazole were prepared using the same method. The shape of the drug-loaded vesicles was observed with a transmission electron microscope, the particle diameter was measured with a laser particle sizer, and the optimal dose determined by liquid chromatography. The E. granulosus protoscolex was divided into 4 groups and was cultured in vitro with pure medium (blank control group), no-loading vesicles (no-loading vesicle group), drug-loaded vesicles (drug-loaded vesicle group) and albendazole (albendazole positive control group), respectively. The final concentration of albendazole in the drug-loaded vesicle group and albendazole positive control group was 13 μmol/L. The protoscolex was stained with Eosin to observe the morphology and activity at 1, 3, 5 and 7 days post-treatment. The survival rate of protoscolex was calculated. The caspase-3 expression level of E. granulosus protoscolex was detected by using caspase-3 detection kit 3, 5, 7 days post-treatment to identify the apoptosis of protoscolex. One-way ANOVA was used for comparisons between groups. Results The transmission electron microscopy showed variations in vesicle of different size with double membrane structure. The particle diameter sizer showed that the particle diameter was 200-400 nm. The liquid chromatography showed that the effective drug concentrations in low, medium and high concentration groups were (36.3 ± 2.85), (79.0 ± 2.30), (99.5 ± 4.20) μmol/L. The increment of albendazole concentration from the low concentration group to the medium concentration group was higher than that from the medium concentration group to the high concentration group. The difference between the groups was statistically significant (F = 21.43, P < 0.05). The optimal concentration of the final drug-loaded vesicles was 400 μmol/L. Eosin staining showed that on day 7 post-treatment, the protoscolex remained active, and the morphology and structure of the protoscolex were clear in the blank control group and the no-loading vesicle group. In the albendazole positive control group, the protoscolex was inactive, and unsharpness and its volume decreased. In the drug-loaded vesicle group, the structure of protoscolex was disordered, shrunken and partially died. On day 7 post-treatment, the survival rate of the protoscolex in the blank control group, no-loading vesicle group, albendazole positive control group and drug-loaded vesicle group were (91.2 ± 1.07)%, (88.9 ± 1.43)%, (64.5 ± 1.19)% and (45.3 ± 0.98)%. The survival rate curves in the drug-loaded vesicle group were lower than that of the the other groups (F = 1 021.17, P < 0.05). The apoptosis of the protoscolex results showed, on day 3 post-treatment, the caspase-3 of E. granulosus protoscolex in blank control group, no-loading vesicle group, albendazole positive control group and the drug-loaded group were (41.80 ± 3.02), (40.26 ± 2.78), (55.20 ± 3.09) and (68.15 ± 3.60) μmol/L, respectively; on day 5 post-treatment, were (43.18 ± 2.43), (43.02 ± 3.13), (52.17 ± 4.13) and (62.74 ± 3.16) μmol/L, respectively; while on day 7 post-treatment, were (52.93 ± 1.46), (53.08 ± 1.60), (57.32 ± 1.81) and (61.99 ± 1.14) μmol/L, respectively. The differences were significant between the groups on 3, 5, 7 days post-treatment (F = 51.97, 24.53, 23.82; P < 0.05) and the caspase-3 in the drug-loaded vesicle group were all higher than that in the albendazole positive control group (F = 22.36, 12.43, 14.33; P < 0.05). Conclusion Albendazole-loaded vesicle could improve the solubility of albendazole, enhancing the killing effect on E. granulosus protoscolex.

Preliminary study on the changes of plymorphonucler myeloid-derived suppressor cells in the spleen of mice infected with Schistosoma japonicum

ZHANG Xiao-cheng, GAO Yuan, HU Yuan, CAO Jian-ping

2022, 40(3):  330-336.  doi:10.12140/j.issn.1000-7423.2022.03.008

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Objective To explore the dynamic changes of the proportion, function and spleen histopathology of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) in the spleen of mice infected with Schistosoma japonicum. Methods Thirty-six BALB/c mice aged 6-8 weeks were randomly assigned into the infected group and control group, with 18 mice in each group. Mice in the infection group were infected with S. japonicum cercariae (20 ± 1)/mouse. At 4, 6 and 8 weeks post-infection, the spleen from 6 mice, which were randomly selected from each group, were collected, followed by calculating the spleen coefficients after weighting. The spleen tissues were fixed, sliced and stained with hematoxylin-eosin (HE) to observe the pathological changes microscopically. Spleen single cell suspension was prepared for examining the dynamic changes of PMN-MDSCs proportion in splenic lymphocytes by flow cytometry. Fluorescent quantitative PCR was used to determine the mRNA relative expression expression level of PMN-MDSC related inflammation factors, including interleukin-6 (IL-6), S100 calcium binding protein A8 (S100A8）, S100A9, and the cell function factor arginase 1 (Arg1), nitric oxide synthase (iNOS), heme-binding membrane glycoprotein 91(gp91), transforming growth factor-β(TGF-β), and IL-10 in the spleen tissues. Results At 4, 6 and 8 weeks after infection, the spleen weight and spleen coefficient in infection group were(179 ± 10.19)mg, (350.3 ± 16.84)mg, (414.3 ± 18.98)mg, and (0.93 ± 0.03)%, (1.97 ± 0.10)%, (2.31 ± 0.08)%, respectively, which were all significantly higher than that in the control group[(108.2 ± 9.93)mg and (0.51 ± 0.04)%] (F = 101.3, 143.7, P < 0.01). HE staining showed that at 4-6 weeks after infection, the inflammatory cell band gradually increased, while the lymphoid follicles of the spleen decreased, and the germinal center decreased or even disappeared compared with the control group. At 6-8 weeks after infection, the inflammatory cell band decreased gradually, and the lymphoid follicles and germinal centre structure of the spleen proliferated gradually compared with the control group. Splenic PMN-MDSCs proportion in infection group were (1.53 ± 0.16)%, (28.40 ± 2.35)%, (38.67 ± 1.94)%, which were all significantly higher than that in the control group (0.80 ± 0.10)% (F = 326.5, P < 0.01). Real-time quantitative PCR showed that at 6 weeks post-infection, the relative level of mRNA expression of IL-6, S100A8, S100A9, gp91, Arg1, iNOS, IL-10 and TGF-β in splenic tissue were 2.74 ± 0.25, 51.4 ± 1.25, 39.20 ± 2.83, 2.15 ± 0.08, 2.33 ± 0.39, 1.57 ± 0.08, 2.20 ± 0.39 and 1.44 ± 0.05, respectively, which were all significantly higher than that in the control group [1.05 ± 0.10, 1.01 ± 0.11, 1.02 ± 0.07, 1.04 ± 0.09, 1.01 ± 0.06, 1.00 ± 0.05, 0.98 ± 0.20 and 1.00 ± 0.04(t = 6.367, 40.07, 13.50, 9.311, 3.315, 5.642, 2.764, 6.914, P < 0.05, P < 0.01)]. The relative iNOS mRNA expression in splenic PMN-MDSCs was higher than that in the control group (t = 0.6134, P < 0.01) and the relative of IL-10 mRNA expression level was no statistically significantly different between the infection group and the control group(t = 1.176, P > 0.05). At 8 weeks after infection, the relative IL-6, S100A8, S100A9, Arg1, iNOS and IL-10 mRNA expression levels in splenic tissue were all significantly higher than those in the control group (t = 2.496, 5.145, 9.518, 3.938, 4.819, 2.251, P < 0.05, P < 0.01). In the splenic of PMN-MDSCs, the relative iNOS mRNA expression level and IL-10 were 32.12 ± 2.30, and 2.64 ± 0.37, respectively, which were higher than that at 4 weeks after infection (1.08 ± 0.01, 1.14 ± 0.35), and also higher than that at 6 weeks after infection (12.06 ± 1.80, 1.50 ± 0.36), as well as the control group (1.02 ± 0.13, 1.06 ± 0.12) (F = 100.6, 5.471, P < 0.01). Conclusion At 4~6 weeks post S. japonicum infection, the spleen tissue presented strong inflammatory response, at 6~8 weeks after infection, the secretion of the inhibitory factor IL-10 of PMN-MDSC was significantly increased, and the spleen tissue structure was gradually proliferated.

Establishment and preliminary evaluation of a visualized detection technique for Schistosoma mansoni nucleic acid based on recombinase polymerase amplification

WANG Li-ping, LV Chao, QIN Zhi-qiang, XU Jing, DENG Wang-ping

2022, 40(3):  337-343.  doi:10.12140/j.issn.1000-7423.2022.03.009

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Objective To establish and preliminarily evaluate a fast, convenient and visualized method for detection of Schistosoma mansoni nucleic acid by combining the recombinase polymerase amplification (RPA) and lateral flow dipstick (LFD). Methods Selecting the cytochrome c oxidase subunit 1 (COX1) gene of S. mansoni as the target sequence, specific primers and probes were designed using Primer Primer5 with manual assistance and screened to establish a LFD-RPA method for detection of S. mansoni. The sensitivity of the established method was evaluated by detecting the genomic DNA of adult S. mansoni at different concentrations (1 ng/μl, 100 pg/μl, 10 pg/μl, 1 pg/μl, 100 fg/μl, 10 fg/μl, 1 fg/μl, 0.1 fg/μl) and the recombinant SmCox1 plasmids with different copies (10⁵, 10⁴, 10³, 10², 10¹, 10⁰, 10^-1 copies/μl). The specificity of LFD-RPA was evaluated by detecting the genomic DNA of S. japonicum adult worms, S. haematobium eggs, S. mansoni infected and non-infected Biomphalaria spp., S. japonicum infected and non-infected Oncomelania hupensis and other trematodes. Thirty female BALB/c mice were randomly divided into low infection group (40 cercariae per mouse), high infection group (80 cercariae per mouse) and control group (no infection) to establish the mouse infection model. The DNA was extracted from the feces and serum samples from the mice at different time points post infection (1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks) and detected by LFD-RPA to evaluate its effectiveness in early detection of S. mansoni infection. Results The specific band of S. mansoni could be detected by the established SmCox1-LFD-RPA method after 20 min of reaction at 39 ℃. The detectable limits of LFD-RPA method for S. mansoni were 10 fg/μl for the genomic DNA of adult worms and 10 copies/μl for SmCox1 recombinant plasmid, respectively. The established SmCox1-LFD-RPA method was specific to the DNA of S. mansoni and S. mansoni infected Biomphalaria spp., no cross-reaction with other flukes were observed. When detecting DNA samples extracted from mouse blood and feces from the 40 cercariae group 1 week to 8 weeks post infection, SmCox1-LFD-RPA presented positive reaction since 3 weeks post infection. While for the mice of the 80 cercariae group, positive band began to appear from 1-week post infection. The color of the band was gradually deepened with the infection time. Conclusion A LFD-RPA based visualized rapid detection method for S. mansoni nucleic acid was developed, with higher sensitivity, specificity, rapidity and easy-to-use.

A reverse transcriptase aid-enzymatic recombinase isothermal amplification-based method for detection of West Nile virus

ZHANG Yi-long, YE Run, LE Bin, CHEN Wen-zhu, PAN Wei-qing, ZHANG Dong-mei

2022, 40(3):  344-348.  doi:10.12140/j.issn.1000-7423.2022.03.010

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Objective To establish a reverse transcription-enzymatic recombinase isothermal amplification (RT-ERA) assay for detection of specific gene segment of West Nile virus. Methods The highly conserved region of NS5 gene (120 bp) was selected as the target gene fragment to be detected. The primers and fluorescence probes were designed and synthesized based on the isothermal amplification principle to establish a fluorescence RT-ERA assay system. The fluorescence RT-ERA assay was performed to detect serial diluted recombinant plasmids (10⁴, 10³, 10², 10, 1 copy/μl) containing target gene fragments and West Nile virus RNA at different concentrations (10, 1, 10^-1, 10^-2, 10^-3 ng/μl) to determine the sensitivity. Further, this assay was applied to detect the genomic RNA of Tick-Borne Encephalitis virus, Chikungunya virus, Yellow fever virus, Dengue virus type I, Japanese Encephalitis virus and Influenza A virus (H2N1) to evaluate the specificity. Results A fluorescence RT-ERA assay was successfully established, which was effective in amplifying the specific gene fragments of West Nile virus within 20 min at 39 ℃. The minimum detectable limit of the fluorescence RT-ERA assay was 1 copy/μl using recombinant plasmids as templates and 10^-3 ng/μl using West Nile virus RNA samples as templates. The results of the fluorescent RT-ERA assays were all negative for detecting the genomic RNA from tick-borne encephalitis virus, chikungunya virus, yellow fever virus, dengue virus type I, Japanese encephalitis virus, and Influenza A virus (H2N1). Conclusion A fluorescence RT-ERA assay for detection of West Nile virus RNA is successfully established, which is easy to use, sensitive and specific.

Construction and identification of macrophage migration inhibitory factor gene knockout strain of Toxoplasma gondii

WANG Jie, WEN Hong-yang, CHEN Ying, AN Ran, LUO Qing-li, SHEN Ji-long, DU Jian

2022, 40(3):  349-354.  doi:10.12140/j.issn.1000-7423.2022.03.011

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Objective To construct and validate a macrophage migration inhibitory factor (mif) gene knockout strain of Toxoplasma gondii RH strain. Methods Single guide RNA (sgRNA) and site-directed mutation primers the RH strain of T. gondii Tgmif gene were designed. The pSAG1::Cas9-U6::sgUPRT plasmid was mutated into the pSAG1::Cas9-U6::sgTgmif plasmid targeting the Tgmif gene. A Tgmif donor plasmid containing 3 fragments of 1 001 bp upstream of Tgmif (Tgmif-up), dihydrofolate reductase-thymidylate synthase gene (dhfr-ts) and 1 011 bp downstream of Tgmif (Tgmif-down) was constructed. The Tgmif donor DNA was amplified from the Tgmif donor plasmid by PCR. The pSAG1::Cas9-U6::sgTgmif plasmid and Tgmif donor DNA were mixed and electroporated into the tachyzoites of wild-type T. gondii RH strain (RH_WT) and cultured with pyrimethamine for 7 days prior to screening to obtain the stable expression of pyrimethamine-resistant tachyzoites. The upper and lower homology arms of dhfr-ts and Tgmif were amplified by PCR to identify Tgmif knockout monoclonal strains (RH_ΔTgmif). The protein of RH_ΔTgmif and RH_WT tachyzoites was extracted and the expression of TgMIF protein was analyzed by Western blotting. RH_ΔTgmif was cultured in human foreskin fibroblasts (HFF) cells for 10 passages, and the number of tachyzoites per 100 HFF cells was counted by Giemsa staining to evaluate the proliferation of RH_ΔTgmif in HFF cells in vitro. RH_WT was used as the control. Twenty BABL/c mice were randomly divided into RH_WT group and RH_ΔTgmif group (10 mice/group). The mice were injected intraperitoneally with tachyzoites of RH_WT or RH_ΔTgmif strains (1 000/mouse) for each group respectively. The survival of mice was recorded. Independent sample t-test was used to compare the proliferation of T. gondii, and Log Rank test was used to compare the survival rate of mice. Results PCR results showed that the Tgmif donor DNA fragment containing 3 fragments, including Tgmif-up, dhfr-ts and Tgmif-down was 5 049 bp long, which was as expected; RH_ΔTgmif strain amplified dhfr-ts upstream and downstream homology arm bands of 1 387 bp and 1 524 bp, respectively, while RH_WT strain had no corresponding bands; a 1 837 bp Tgmif band was amplified in RH_WT strain, while no corresponding band in RH_ΔTgmif strain. The results of Western blotting showed that the RH_WT strain had a protein band at a relative molecular mass (M_r) of 12 500, while the RH_ΔTgmif strain had no corresponding protein band. The results of Giemsa staining showed that the tachyzoites of RH_ΔTgmif strain were (2 986 ± 69.20) per 100 HFF cells, which was higher than the RH_WT strain (2 067 ± 51.08) (t = 18.50, P < 0.01). The results of the in vivo virulence test showed that the mice in the RH_WT group started to die on the 7th day and all died on the 9th day; the mice in the RH_ΔTgmif group started to die on the 5th day and all died on the 7th day, and the difference between the two groups was statistically significant (χ² = 17.45, P < 0.01). Conclusion The Tgmif gene knockout strain RH_ΔTgmif was successfully constructed, and the RH_ΔTgmif strain of Toxoplasma was more virulent than the RH_WT strain.

Isolation, identification and the activity of producing digestive enzymes of culturable bacteria from the larval intestine of laboratory line housefly

ZHANG Ke-xin, LIU Wen-juan, ZHANG Xin-yu, ZHANG Qian, ZHANG Rui-ling, ZHANG Zhong

2022, 40(3):  355-361.  doi:10.12140/j.issn.1000-7423.2022.03.012

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Objective To isolate cultivable bacteria from the intestine of housefly larvae, investigate the activity of digestive enzymes produced by these bacteria, and explore the effects of intestinal bacteria on the digestion of food and the growth and development of housefly larvae. Methods The bacteria in the intestine of housefly larvae were isolated and purified by traditional bacterial isolation and culture method, and 16S rRNA gene sequence was analyzed for molecular identification of identify species. Using the bacteria culture medium of different property to screen the bacteria producing amylase, cellulase, protease, or lipase, and the diameter ratio of hydrolytic circle (D) to bacterial colony (d) was measured to compare the activities of digestive enzymes produced by different bacteria. SPSS 20.0 software was used for statistical analysis. A one-way analysis of variance was used for comparison between groups. Results A total of 9 species of facultative anaerobic bacteria, which belongs to 6 genera, were isolated from the intestines of housefly larvae cultured in an anaerobic environment, among which there were 3 species in Providence (Providencia sneebia DSM 19967, P. rettgeri and P. stuartii), 2 species in Enterococcus (E. casseliflavus and E. faecalis), and 1 specie each in Pseudocitrobacter (P. faecalis), Morganella (M. morganii), Enterobacter (E. hormaechei) and Klebsiella (K. pneumoniae). A total of 10 species of bacteria belonging to 8 genera were isolated from the intestines of housefly larvae cultured in an aerobic environment, among which there were 2 species in Enterobacter (E. hormaechei and E. cloacae), 2 species in Providence (P. stuartii and P. vermicola), and 1 specie each in Klebsiella (K. pneumoniae), Pseudomonas (P. aeruginosa), Acinetobacter (A. bereziniae), Lactococcus (L. lactis), Lysinibacillus (L. fusiformis) and Bacillus (B. safensis). Among these facultative anaerobes were isolated from the intestines of housefly larvae cultured in an anaerobic environment, 9 produced amylase, and 8 produced cellulase and 1 produced lipase; no bacteria produced protease under anaerobic conditions. Among these facultative anaerobes, 9 produced amylase and 8 produced cellulase; no bacteria produced protease and lipase detected in aerobic condition. Under anaerobic conditions, there was no difference in amylase activity among 9 facultative anaerobic culture bacteria (F = 1.953, P > 0.05); among the cellulase activities of 8 facultative anaerobes, E. casseliflavus lead had the strongest cellulase activity, with a D/d value of 1.36 ± 0.06 (F = 3.367, P < 0.05); and only P. rettgeri produced lipase, with a D/d value of 2.28 ± 0.16. In the aerobic state, among the amylase activities of 9 facultative anaerobic bacteria, E. faecalis had the strongest amylase-producing activity, and the D/d value of 1.42 ± 0.06 (F = 3.881, P < 0.05). Among the cellulase activities of 8 facultative anaerobes, E. casseliflavus had the strongest cellulase activity, with a D/d value of 1.29 ± 0.01（F = 6.633, P < 0.05）. Among aerobic bacteria, P. aeruginosa and B. safensis produced protease, with D/d values of 3.67 ± 0.25 and 3.58 ± 0.31 (F = 0.087, P > 0.05); and only K. pneumoniae produced amylase, with a D/d value of 4.83 ± 0.12; no bacteria produced cellulase and lipase. Conclusion The composition of cultivable bacteria in the intestine of housefly larvae is relatively unitary, but most anaerobic bacteria and aerobic bacteria have the function of secreting digestive enzymes.

Prokaryotic expression of Theileria annulata recombinant TA04380 protein and establishment of ELISA

CAO Tian-xing, LIU Jun-long, ZHANG Zhi-gang, SHI Kang-yan, SHI Miao, GUAN Gui-quan, LI You-quan, YIN Hong, LUO Jian-xun

2022, 40(3):  362-368.  doi:10.12140/j.issn.1000-7423.2022.03.013

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Objective To express recombinant TA04380 protein (rTA04380) of Theileria annulata, establish an enzyme-linked immunosorbent assay (ELISA)-based detection method and evaluate its value of application. Methods Bioinformatics analysis of TA04380 protein was performed with TMHMM and SignalP, primers were designed according to the nucleotide sequence of T. annulata TA04380 published in GenBank, and the truncated fragment (1-636 bp) of TA04380 gene was amplified by PCR to construct pET30a(+)-TA04380 plasmid and was transformed into Rosetta (DE3) competent cells, from which the rTA04380 protein was expressed by induction and then purified. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to identify the expression of the recombinant protein. The reactivity of the rTA04380 protein and its cross-reaction with other bovine piroplasma were analyzed by Western blotting. The rTA04380 protein was used as the target antigen to establish an ELISA method for the detection of serum antibodies against T. annulata; the optimal antigen coating concentration, serum and secondary antibody concentrations and the time for optimal blocking were determined by screening tests. The established ELISA method was used to detect 56 standard T. annulata negative bovine sera and 76 standard T. annulata positive bovine sera to determine the threshold value. The reproducibility and robustness of the ELISA method in the first, second and third weeks after the rTA04380 protein was coated. A total of 125 bovine serum samples were detected with the established ELISA, and compared with the reported ELISA method based on sporozoite surface antigen (SPAG) protein, the coincidence rate was calculated, and 571 bovine serum samples from 9 provinces were detected by the established ELISA method to evaluate its application value. Results The bioinformatics analysis showed that the T. annulata TA04380 protein has no signal peptide sequence, there are four transmembrane regions in the 212-413 aa region, and the amino acid sequence identity with T. orientalis homologous protein is 55%. The PCR amplification fragment length was 636 bp. The SDS-PAGE showed that the rTA04380 protein mainly existed in the inclusion bodies, and the purified TA04380 protein band was located at a relative molecular weight (M_r) of 31 940; Western blotting analysis showed that the rTA04380 protein could react both with the anti-His monoclonal antibody and T. annulata positive serum, but no cross-reaction was observed with T. orientalis, T. sinensis, B. bovis, B. bigemina reactive sera and healthy bovine serum. The optimal conditions of the ELISA method based on rTA04380 protein were: antigen is coated at a concentration of 10 μg/ml, and blocked with 1% bovine serum albumin for 1 h followed by addition of 1 ∶ 100 diluted serum and incubate for 1 h. The rabbit anti-bovine horseradish peroxidase (HRP)-IgG was added as a secondary antibody at a concentration of 1 ∶ 6 000 and incubated for 1 h. The detection threshold was 16.9%, the sensitivity and specificity were 93.4% and 96.4%, respectively, and the false positive rate was 3.6%. The positive rates of the established ELISA method and the ELISA method based on SPAG protein were 54.4% (68/125) and 49.6% (62/125), respectively, and the coincidence rate between the two methods was 85.6%. a The positive rate of 571 bovine serum samples from 9 provinces was 43.1% (246/571) using the established ELISA method. The positive rate from high to low is Jilin (85.7%, 24/28), Ningxia (54.2%, 13/24), Guizhou (50.0%, 13/26), Henan (8/16), Gansu (43.8%, 57/130), Inner Mongolia (42.2%, 35/83), Liaoning (41.1%, 30/73), Yunnan (38.2%, 42/110) and Qinghai (29.6%, 24/81). Conclusion The rTA04380 protein was successfully expressed, and the established ELISA based method presents high sensitivity and specificity, good reproducibility and coincidence, impliying potential value for wide application in the field.

Comparative analysis of biological characteristics of Hyalomma asiaticum and H. anatolicum Xinjiang isolates at different developmental stages

SONG Rui-qi, ZHAI Xue-jie, LI Cai-shan, GE Ting, GAN Lu, ZHANG Meng-yuan, FAN Xin-li, LI Yong-chang, ZHANG Yang, BAYIN Cha-han

2022, 40(3):  369-378.  doi:10.12140/j.issn.1000-7423.2022.03.014

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Objective To analyze and compare the biological characteristics and morphologies at different developmental stages beween Hyalomma asiaticum and H. anatolicum isolates from Xinjiang geographic regions. Methods Hard ticks were captured from Qitai and Turpan, the tick endemikc areas of Xinjiang, in April to May, 2019. Species of the ticks were identified based on morphology. The tick genomic DNA was extracted and the conserved fragment of the mitochondrial cytochrome oxidase subunit 1(CO1) gene was amplified by PCR. The target gene was cloned using pEASY-T1 vector system, single bacteria colony was selected for sterile expansion culture and the cultured fluid was undergone sequencing. The homology of CO1 gene sequence with other sequences was aligned using NCBI Blast online analysis software, and phylogenetic tree was generated. Twelve New Zealand white rabbits were divided into 2 groups with 6 rabbits in each group, H. asiaticum and H. anatolicum at different developmental stages (200 larvae, 150 nymphs, or 50 adults) were fed with blood from two rabbits. H. asiaticum and H. anatolicum ticks were placed in equipment enclosing rabbit ears to feed on blood. Ticks were checked daily until repletion. The recovered ticks were collected and stored under controlled experimental conditions. The biological characteristics of H. asiaticum and H. anatolicum was observed systematically at various developmental stages of their life cycles. Results A total of 317 H. asiaticum and 291 H. anatolicum ticks were collected in Qitai and Turpan, respectively. The results of phylogenetic analysis based on the CO1 gene show that H. asiaticum from Qitai, Xinjiang (MW217459), Inner Mongolia (JX051135), Kazakhstan (KU364324), Wuwei, Gansu (MK292004) and Yuli, Xinjiang (KF527441) belong to the same cluster; the sequence of H. anatolicum from Turpan, Xinjiang (MW221948), Kazakhstan (MN853167), Gansu (JQ737067) and other Xinjiang strains (MN268557, KF583576) were categorized into another cluster. There was significant difference in the genetic relationship between H. asiaticum-Qitai strain and H. anatolicum-Turpan strain. The average life cycles of H. asiaticum and H. anatolicum were 131.5 and 112.5 days, respectively. At the egg stage, the oviposition and egg’s incubation of H. asiaticum (27.5 days and 31.5 days) were longer than H. anatolicum (12.0 days and 20.5 days). At the larva stage, H. asiaticum and H. anatolicum need 5.5/18.5 days and 9/18.5 days to feed/molt, respectively. At the nymph stage, H. asiaticum and H. anatolicum need 10.5/19 days and 4.5/30 days to feed/molt, respectively. H. asiaticum and H. anatolicum needed 8.5/10.5 days and 13.0/5.0 days to feed/preoviposition. The morphological comparison showed that the appear time of “cicatricle” (later development to anus) in H. asiaticum (day 19) is later than H. anatolicum (day 14) in the process of egg development, but there were no significant difference between H. asiaticum and H. anatolicum at larvae and nymphs stages; the differences among adult ticks were mainly reflected in neck furrow (the male H. asiaticum has long and deep, extending backward beyond the middle of scutum, while that of the male H. anatolicum was short and shallow), paraproct and hypopygium (male H. asiaticum paraproct is narrow and long, and the anterior tip is narrow and the posterior tip is blunt and round, and the inner edge convex is wide and triangular, the hypopygium is slightly smaller and papillary; in male H. anatolicum, the paraproct is slightly wide in the middle, narrow in the front, slightly round and blunt in the back, and short in the inner edge, hypopygium is vague), spiracular plate (in male H. asiaticum, it is in the shape like a curved-neck-bottle, the front end is oval, the dorsum process is slightly narrow and long; in male H. anatolicum: it is in the shape like a spoon, dorsal process slightly wider and longer, end at the edge of the backplane. In female H. asiaticum: it is long-elliptic, slightly wider and longer dorsum process, at right angles to the back, terminal to the edge of the backplane. In female H. anatolicum: it is long rectangular, dorsum slightly wider and longer, bent forward nearly at right angles), coxa (in female H. asiaticum, the outer spur of the coxaⅠ is slightly longer than the inner spur. The outer spur is short and coarse, tapering in sequence. In female H. anatolicum: the outer spur of coxaⅠis larger than the inner spur. The outer spur of coxa Ⅱ~Ⅳ are short without the inner spur), and the colour banding of podomere (in male H. asiaticum it is clearly visible; in male H. anatolicum it is vague), among which the key identification character for male ticks were paraproct, hypopygium, and spiracular plate; the main identification character for female ticks were podomere and spiracular plate. Conclusion The biological characteristics of Xinjiang tick isolates H. asiaticum and H. anatolicum vary in life cycle, morphology, and molecular biology.

INFORMATION REPORTS

Investigation on parasite density and treatment measures in malaria patients

LI Mei, ZHOU He-jun, YIN Jian-hai, ZHANG Li, TU Hong

2022, 40(3):  379-383.  doi:10.12140/j.issn.1000-7423.2022.03.015

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The blood smears and basic information of 272 malaria patients in 28 Provinces (Municipalities and Autonomous regions) in 2019 were randomly selected. According to the rough estimation of the parasite density, patients with parasite density higher than 4 000/μl and/or with complications were defined as in severe conditions. Following the definition and information recorded in the National Information System for Parasitic Disease Prevention and Control, the consistency between conditions of malaria patients and their therapeutic strategies (hospitalization or outpatient treatment) and medication regimens (injection or oral medication). One hundred sixty-three patients were categorized as severe cases, including 160 with high parasite density and 3 with complications but low parasite density. The patients tended to have complications (P < 0.05) with higher parasite density. First visits and treatment of most patients were in County, Prefecture and provincial medical agencies (83.82%, 228/272; 96.32%, 262/272). Patients visited and treated in Prefecture medical agencies were the most (40.81%, 111/272; 48.90%, 133/272) among medical facilities of different levels. The proportion of hospitalized patients was 77.94% (212/272), and the proportion of patients treated with injections was 57.93% (157/271). The general trend was to hospitalize those with severe conditions (92.02%, 150/163), while patients with mild conditions were treated as outpatients (7.98%, 13/163) (χ² = 46.92, P < 0.05). The trend of treatment methods for malaria patients in the county, prefecture, and provincial tertiary medical institutions was consistent with the general trend (P < 0.05). The overall trend of the patient’s medication regimen was that patients with severe disease were administrated with therapies (66.67%, 108/162), and those with mild disease were administrated with oral antimalarial therapies (55.05%, 60/109) (χ² = 12.61, P < 0.05). Only the drug use in prefecture-level medical institutions was consistent with the overall trend (χ² = 7.17, P < 0.05), while the trend in the county and provincial medical institutions was not significant (χ² = 0.62, 1.36, P > 0.05). The proportion of patients whose treatment and drug regimens were inconsistent with the condition was 27.53% (75/272) and 37.83% (103/272), respectively. The treatment measures implemented by the primary medical facilities for the treatment of malaria are generally good.

The logic and characteristics of the formulation of the Parasitic Disease Standard from the perspective of Healthy China

YU Cheng-hang, ZHENG Bin, CHEN Jia-xu, WEN Li-yong, ZHOU Xiao-nong

2022, 40(3):  384-389.  doi:10.12140/j.issn.1000-7423.2022.03.016

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Standards have gained increasing significance in supporting the national economic and social development. With the promulgation and implementation of the 《standardization law》 in China, we have reviewed the establishment, publicizing, implementation and research of the standards related to parasitic disease. The national standard system for parasitic diseases has been summarized, and the challenges while constructing the standard system have been analyzed in anticipation of providing scientific evidence for the construction and formulation of systemic parasitic disease standards under the recently revised standardization law. This review will eventually provide policy guidance for parasitic disease prevention and control.

REVIEWS

Research advances on exosome and its functions to parasitic helminths

PAN Xiao-wen, WU Yin-juan, HE Qing, YIN Ying-xuan, LI Xue-rong

2022, 40(3):  390-395.  doi:10.12140/j.issn.1000-7423.2022.03.017

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Exosomes are bilayer vesicular bodies secreted by a variety of variable cells, containing many important bioactive substances, including proteins, lipids and nucleic acids, which are involved in important physiological processes such as cell-to-cell communication and immune regulation. Exosomes from parasites play a critical role in parasite-host interaction, which can regulate the host immune response through various mechanisms and manipulate host cell function. It is an important medium for the interaction between parasite and parasite, as well as between parasite and host. This review has summarized the recent advances on the pathogenicity, immune regulation and immune evasion of exosomes in trematodes, nematodes and cestodes to help the study of exosomes in parasitic helminths.

Research progress on reticulocyte binding proteins associated with Plasmodium vivax invasion of reticulocytes

SHI Tian-qi, CHEN Jun-hu

2022, 40(3):  396-401.  doi:10.12140/j.issn.1000-7423.2022.03.018

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Plasmodium vivax is the most geographically widespread malaria parasite that causes most malaria infection cases outside the most malarious continent, sub-Saharan Africa. P. vivax preferentially invades reticulocytes, with a high species specificity. P. vivax reticulocyte binding protein (PvRBP) family has been implicated in roles in reticulocyte invasion and severity of P. vivax infections. The members of PvRBP family act as invasion ligands that mediate new pathways for the parasites to invade reticulocytes, and therefore, are considered important immune targets. Importantly, PvRBP2a-CD98 and PvRBP2b-TfR1 have been identified as two major ligand-receptor pairs implicated in the invasion of reticulocytes by P. vivax. The Pvrbp family is highly polymorphic and can generate immune evasion, which can increase the efficiency of vivax malaria invasion and the severity of the disease. With the advances in research on the molecular mechanisms of P. vivax invasion, insights have been provided on members of this protein family as promising antimalarial vaccine candidates, able to generate high titer antibodies for effective prevention and control of vivax malaria. This review summarizes the role of reticulocytes in P. vivax infection and the function of the PvRBP proteins family as immune targets in the human population.

Advances in research on the vacuolar membrane function and the associated proteins of plasmodium parasitophorous vacuole

GE Jie-yun, LIU Lei, SUN Yi-fan, CHENG Yang

2022, 40(3):  402-410.  doi:10.12140/j.issn.1000-7423.2022.03.019

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Malaria is one of the most important infectious diseases in the world and seriously threatens human health. It remains a critical problem to interrupt malaria transmission. Plasmodium parasites invade and multiply inside the host cells causing severe pathological damage to the host. As the interface between the host cells and Plasmodium parasites, PVM is of crucial importance for the survival of Plasmodium parasites. This manuscript briefly introduces the formation of PVM and its relationship with PPM and TVN. The PVM-associated proteins and their functions in blood-stage, liver-stage and sexual-stage parasites are summarized. This review also presents perspectives on malaria control strategies.

Classification system adjustment of Pseudophyllidea and the identification characteristics of the main taxa

LI Hai-yun

2022, 40(3):  411-419.  doi:10.12140/j.issn.1000-7423.2022.03.020

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Pseudophyllidea was officially categorized into two orders in 2008 internationally: Bothriocephalidea and Diphyllobothriidea. Due to the limited advances in local tapeworm research, the relevant systematic classification has not been updated. This review summarized the adjustment history and alternate status of the classification system and lists the differentiation and identification characteristics of the major taxa in the diagram and table form. It would provide a reference for the local tapeworm research and the update of the tapeworm classification system.

SHORT COMMUNICATION

Expression of the high mobility group box 1 homologous protein of Clonorchis sinensis and its effect on nuclear transcription factor-κB activation in mouse macrophages

WANG Ting, YANG Qing-li, LENG Jing, LI Bao-ying, SHEN Ji-qing, DAI Yue

2022, 40(3):  305-308.  doi:10.12140/j.issn.1000-7423.2022.03.021

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The PET-28a(+)expression plasmids were constructed according to the identified HMGB1 homologous gene sequences (CDSs)of Clonorchis sinensis and cloned into Rosetta (DE3) competent cells. Isopropyl-β-D-thiogalactoside (IPTG) was used to induce the expression of CsHMGB1-His fusion proteins. After purification by Ni-TED agarose resin, the expression of His-tagged fusion proteins was confirmed by Western blotting analysis. The mouse macrophage RAW264.7 cells were cultured with mediums supplemented with CsHMGB1-159/127 at a concentration of 1 μg/ml and 10 μg/ml, for 24 h and 48 h, respectively. The cells cultured without stimulants were used as the blank control. The SEAP activity, which indicates NF-κB activation, was detected by using pNiFty2-SEAP plasmid and HEK-Blue^TM culture medium. The A₆₂₀ value of culture supernatant was detected by microplate a reader, and the intracellular NF-κB activation was also observed under a microscope. Our results showed that the CsHMGB1-159/127 proteins encoding 159 and 127 amino acids were expressed, and the relative molecular weights (M_r) were approximately 23 500 and 20 000, respectively. The A₆₂₀ values of supernatant from mouse macrophage stimulated with CsHMGB1-159 protein (1 and 10 μg/ml)for 24 hours were 0.66 ± 0.08 and 0.65 ± 0.03, respectively, which were higher than those in the control group (0.29 ± 0.02)(t = 11.1, 28.5; P < 0.01), and blue staining was observed in the cytoplasm. The A₆₂₀ values after 48 h reached 1.02 ± 0.08 and 1.07 ± 0.08, respectively, which were higher than those in the control group (0.62 ± 0.035)(t = 11.2, 12.9; P < 0.01), and the blue staining in the cytoplasm was stronger. Similarly, the A₆₂₀ values after 24 h stimulation with CsHMGB1-127 protein (1 and 10 μg/ml) were 0.52 ± 0.08 and 0.56 ± 0.08, respectively, which were higher than those in the control group(t =7.39, 8.37; P < 0.01), and the blue staining was observed in the cytoplasm. The A₆₂₀ values after 48 h reached 1.10 ± 0.05 and 0.90 ± 0.10, respectively, which were also higher than those in the control group (t = 20.30, 6.26; P < 0.01), and stronger blue staining was also observed in the cytoplasm. The A₆₂₀ values of CsHMGB1-159 protein (1 and 10 μg/ml) after 24 hours of stimulation were significantly higher than that of CsHMGB1-127 protein (t = 2.98, 2.75; P < 0.05). There were also significant differences in A₆₂₀ value after 48 h stimulation (t = 3.40, P < 0.01). The HMGB1 homologous proteins of Clonorchis sinensis encoding 159 and 127 amino acids can stimulate mouse macrophage NF-κB activation in vitro. The CsHMGB1-159 has stronger stimulatory effect than CsHMGB1-127 protein.

CASE REPORTS

Diagnosis and identification of a case with Taenia saginata infection

QIAO Fei, LIU Ao, YU Fu-chang, QI Meng

2022, 40(3):  285-287.  doi:10.12140/j.issn.1000-7423.2022.03.022

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A male patient with taeniasis was admitted Tarim University hospital in June 2020. The patient complained that there were milky white peristaltic worm fragments in the feces and the patient reported a history of taeniasis. After administration of areca nuts and pumpkin seeds, one milky tapeworm was expelled. Morphological observation of the expelled worm was carried out under stereo microscope. Combined with epidemiological data and patient’s dietary habits, the sample was preliminarily identified as Taenia saginata infection. The gene amplification results based on the nad1 and rrnS loci of the tapeworm mitochondrion showed that fragments of 529 bp and 357 bp were successfully amplified by PCR. BLAST analysis showed that the gene sequences of the nad1 and rrnS loci had 100% and 99.7% homology to T. saginata collected from human in Kenya (GenBank access number AM503345) and Japan (GenBank access number AB031355). Therefore this tapeworm was identified as T. saginata. The patients were followed up after 3 and 6 months, respectively. The patients were in good health and no tapeworm was expelled.

One case of Diphyllobothrium latum infection caused by raw salmon

OUYANG Bing, LI Ren-xi

2022, 40(3):  295-298.  doi:10.12140/j.issn.1000-7423.2022.03.023

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A 58-year-old male patient was admitted to the Affiliated Hospital of Xiangnan University in September 2021. He complained of a right neck mass accompanied by pain for 1 week and had a long history of having raw salmon and raw pickled shrimp diet. Physical examination showed a soft neck, midline trachea, and no palpable thyroid mass on both sides. The palpable right sternocleidomastoid muscle had a size of approximately 5 cm × 5 cm × 3 cm, medium texture, regular shape, unclear boundary, poor mobility, no movement with swallowing, and multiple enlarged lymph nodes were palpable in the right neck and supraclavicular fossa, the largest was about 2 cm × 1 cm, with medium texture and good mobility. Blood cell count showed eosinophil count 2.1 × 10⁹/L (0.02 × 10⁹/L-0.52 × 10⁹/L), eosinophil percentage 10.4% (0.4%-8%). Routine stool examination showed no obvious abnormality. Serum IgG antibody was positive. Colour ultrasound of the neck showed multiple cystic, solid mixed echo areas in the right sternocleidomastoid muscle, with a range of about 60 mm × 25 mm × 53 mm, irregular shape, unclear boundary and uneven internal echo. The capsule of the tumour is intact, there seems to be a coiled cord inside, and the blood supply around the capsule is rich. Contrast-enhanced CT of the neck showed that the lower end of the right sternocleidomastoid muscle was significantly enlarged with uneven density, with multiple low-density areas within it, and annular enhancement was observed in the parenchyma during enhancement. Intraoperative exploration revealed that the right sternocleidomastoid muscle mass had a complete capsule. After the incision, clear fluid flowed out of the capsule accompanied by 3 white parasites slowly peristalsis. The parasite body is milky white, long band, the head segment is small, spoon-shaped, the parasite body is relatively large, its dorsal and ventral surface each has a narrow and deep concave suction groove, the front end of the body is depressed and slightly larger, the body does not segment but has transverse folds, the tail is thin. Combined with the morphological identification and epidemiological history of the patients, the infection was considered as Diphyllobothrium latum infection. After the operation, praziquantel (total dose 120 mg/kg, divided into 5 days, three times a day) was treated with deworming, and no abnormality was found in follow-up 3 months later.

HIV co-infection with Blastocystis hominis and Giardia lamblia in a patient

ZHU Ming-chao, ZHU Ya

2022, 40(3):  315-318.  doi:10.12140/j.issn.1000-7423.2022.03.024

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A 64-year-old female patient, who is a farmer, was admitted to the hospital for intermittent pain and discomfort in the lower back. The patient was positive for human immunodeficiency virus (HIV) antigen/antibody test. Blastocystis hominis and Giardia lamblia flagellate cysts were found by microscopic examination for the normal saline smear of the fecal sample. The B. hominis cysts were round, oval, and varying in size, with 1 to 2 granular nuclei. Vacuoles were seen in the middle of the iodine-stained parasites with irregular, shiny, crescent-like edges between the vacuoles and the cell membrane. Many small bright spots were seen in the vacuoles. The G. lamblia flagellate cysts are oval in shape, with thick cyst walls. There is a clear gap between the cyst and the parasite. The iodine stains the inner axis of the cyst with 2 to 4 nuclei. The nuclei are located on both sides of the axis. Four purplish red nuclei were evident in the cyst after hematoxylin staining. Nested PCR amplified a 511 bp band from patient fecal DNA, which was 99% identical to the sequence TSA417 of G. lamblia (GenBank accession number AF065606). The ribosomal small subunit gene was amplified by PCR with the specific primers of BhRDr and RD5 of B. hominis, and a band of approximately 600 bp was amplified. The sequencing suggested it was B. hominis ST1 type. Combined with the patient’s epidemiological history of drinking unboiled water, using dry-toilet, breading cats and dogs, etc., the diagnosis of HIV co-infection with B. hominis and G. lamblia was made. After 4 days of treatment with metronidazole (0.2 g/time, 3 times/d), the patient’s symptoms were relieved, and the patient was discharged.