Table of Content

30 December 2020, Volume 38 Issue 6

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ORIGINAL ARTICLES

Expression and angiogenic effect of VEGFA/VEGFR2 in mice hepatic metacestode tissue of Echinococcus multilocularis

JIANG Hui-jiao, GUI Xian-wei, GUO Li-jiao, YANG Xiong-feng, WANG Xiao-yi, CHEN Xue-ling, WU Xiang-wei

2020, 38(6):  673-681.  doi:10.12140/j.issn.1000-7423.2020.06.001

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Objective To detect the mRNA and protein levels of VEGFA and VEGFR2 in mouse liver tissue and liver cells of mouse infected with Echinococcus multilocularis(Em), in order to explore the roles of vascular endothelial growth factor A (VEGFA) and endothelial growth factor receptor 2 (VEGFR2) in tissue angiogenesis. Methods Twenty female C57BL/6 mice were randomly divided into two groups, the experimental group and the control group, 10 animals each. Each mouse in the experimental group was directly injected Em metacestodes into the liver, while the control group mice were injected with PBS in the same way. Sera were separated from the whole blood of mice on days 0, 16, 37, 58, 81, and 105 after infection. The mice were euthanized on day 120 to observe the growing status of metacestode and collect. Metacestode tissue, liver tissue around metacestode, and normal liver tissue of control group mice for examining the angiogenesis in metacestode tissue by HE staining. The expression and distribution of VEGFA and VEGFR2 in these tissue samples were detected by immunohistochemistry. The liver cells and protoscoleces were sampled for in vitro incubation in three groups in 12 Petri dishes each group, comprising the group of liver cells-protoscoleces co-incubation, liver cells and protoscoleces. The culture supernatant, liver cells and protoscoleces in each group were collected on incubation day 1, 2 and 3 to assay the levels of VEGFA, VEGFR2 mRNA and protein in normal liver tissue, metacestode tissue, liver tissues around metacestode within 4 mm, and liver cells after co-incubation with protoscoleces using qRT-PCR and Western blotting. ELISA was performed to detect the content of VEGFA in peripheral blood, as well as the protein levels of VEGFA in normal liver tissue, metacestode tissue, liver tissue around metacestode, as well as in the culture supernatant of the co-incubation group, the liver cell group and the protoscolex group. Results qRT-PCR showed that the relative transcription levels of VEGFA mRNA in healthy liver tissues, liver tissues around metacestode, and metacestode tissue were 1.033 ± 0.102, 1.222 ± 0.501, and 0.276 ± 0.092, respectively. The relative transcription level of VEGFA in metacestode tissue was lower than those in the normal liver tissue and the liver tissue around metacestode (P < 0.05); while the transcription of VEGFR2 mRNA was 1.042 ± 0.071, 1.836 ± 0.062, and 0.226 ± 0.077, respectively. In comparision, the relative transcription level of VEGFR2 in the liver tissue around metacestode was higher than that in normal liver tissue (P < 0.01), whereas the transcription in metacestode tissue was lower than those in the normal liver tissue and the liver tissue around metacestode (P < 0.01). Western blotting showed that the proteins of GAPDH, VEGFA and VEGFR2 were not detected in the protein extracts of metacestode tissue, while the relative expression of VEGFA protein in the liver tissue around metacestode and normal liver tissues were 0.920 ± 0.096 and 0.816 ± 0.129, and those of VEGFR2 protein in the two groups were 1.439 ± 0.160 and 0.515 ± 0.022, respectively (P < 0.05). ELISA indicated that the content of VEGFA in the normal liver tissue, liver tissue around metacestode, and metacestode tissue was (6.581 ± 0.722), (6.363 ± 0.638), and (0.670 ± 0.105) pg, respectively. The immunohistochemical assay for VEGFA by scoring showed the scores of normal liver tissue, liver tissue around metacetode, and metacestode tissue was 3.552 ± 0.683, 3.355 ± 0.807, and 9.450 ± 1.292, respectively, revealing the expression of VEGFA in metacestode tissue being higher than those in the other two liver tissue samples (P < 0.05); while the assay for VEGFR2 scored 0.361 ± 0.547, 4.093 ± 1.042, 5.275 ± 1.003, respectively, with the expressionlevel in metacestode tissue being the highest among the three tissue samples (P < 0.01). ELISA results presented that the VEGFA level in peripheral blood on days 0, 16, 37, 58, 81, and 105 of the experimental group was (73.233 ± 7.651), (156.925 ± 5.111), (176.571 ± 40.343), (204.212 ± 9.601), (201.335 ± 24.161) and (185.745 ± 37.902) pg/ml, respectively, which were significantly higher than those of the control group of (70.355 ± 24.751), (56.144 ± 14.736), (81.094 ± 13.753), (76.172 ± 5.689), (64.393 ± 19.060) and (70.871 ± 23.966) pg/ml, respectively(P < 0.05). qRT-PCR results showed that the relative transcription levels of VEGFA and VEGFR2 mRNA on day 2 of the co-incubation group were 9.380 ± 1.165 and 2.764 ± 0.871, respectively, indicating significantly higher than those of the liver cell group of 1.028 ± 0.252 and 1.062 ± 0.201, respectively(P < 0.05). Western blotting showed that the relative expression levels of VEGFA and VEGFR2 protein on day 2 in the co-incubation group were 1.500 ± 0.148 and 1.540 ± 0.079, respectively, while those in the liver cell group were 1.322 ± 0.050 and 0.303 ± 0.003, and the expression levels in the metacestode group were higher than those in the liver vell group (P < 0.01). ELISA demonstrated that the content of VEGFA in the culture supernatant liver cell group on the incubation day 1, 2, and 3 was (24.923 ± 1.427), (151.760 ± 4.282), and (223.033 ± 10.061) pg/ml, respectively, all significantly lower than those in the co-incubation group (P < 0.01), which were (95.218 ± 4.932), (240.295 ± 15.121) and (366.148 ± 4.822) pg/ml, respectively. Conclusion E. multilocularis infection induces host cells to continuously express VEGFA, which spreads to the surrounding tissue by secretion enters the blood circulation, and further inducing the peripheral cells to highly express VEGFR2 targeting to the infection site, promoting angiogenesis in metacestode tissue.

Bioinformatics characteristics and immunoreactivity of protein kinase A of Echinococcus granulosus

FAN Jun-jie, HAN Xiu-min, Nur Fazleen Binti Idris, LI Kai, TAN Qing-qing, CAO Wen-qiao, LI Xiang, LIAO Peng, YE Bin

2020, 38(6):  682-687.  doi:10.12140/j.issn.1000-7423.2020.06.002

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Objective To investigate the bioinformatics characteristics of protein kinase A gene in Echinococcus granulosus (EgPKA), and analyze the immunoreactivity of its recombinant protein. Methods The coding region of EgPKA gene was amplified using primers designed based on the whole-genome sequencing data of Echinococcus granulosus, and the physicochemical properties, subcellular localization, and tertiary structure of EgPKA were comprehensively analyzed using bioinformatics methods. The recombinant plasmid of GS115/EgPKA-pPIC9K was constructed and induced by methanol in Pichia pastoris eukaryotic system. The recombinant EgPKA protein was purified by nickel-affinity chromatography with anti-His label, and the immunoreactivity of purified EgPKA was detected by Western blotting with serum of a patient with cystic echinococcosis and sera from mice 4, 8, 12 and 16 months after infected with E. granulosus protoscolex. Results The EgPKA gene had a full length of 1 053 bp and encoded 350 amino acid residues. A 48-bp deletion mutation (417-464 bp) occurred in the coding region. After mutation, the bases A⁴¹⁵, G⁴¹⁶ and T⁴⁶⁵ in the EgPKA coding region formed a new amino acid Ser¹⁵⁴. Bioinformatics analysis showed that EgPKA had theoretical isoelectric point of 8.63, instability coefficient of 29.04, fat coefficient of 82.49, and a total average hydrophilicity of -0.374, with no signal peptide and no transmembrane region. Cell-ploc 2.0 analysis showed that the EgPKA protein may be localized to the cell membrane, cytoplasm, mitochondria and nucleus. Western blotting analysis showed that the yield of recombinant protein EgPKA was highest after induction for 72 h in 1% methanol. The purified recombinant protein EgPKA could react specifically with sera of infected mice at 4, 8, 12 and 16 months post-infection, resulting in two protein bands at M_r 40 000 and 35 000, while no specific reaction occurred in sera from healthy mice and healthy humans. Conclusion The coding region of EgPKA has a gene deletion mutation, and the recombinant EgPKA protein has good immunoreactivity, which could be a potential diagnostic antigen for early diagnosis of echinococcosis.

Effect of Elsholtzia eriostachya in combination with albendazole in treatment of secondary Echinococcus multilocularis metacestode infection in rats

ZHU Ji-hai, CAO De-ping, QIE Yangrangzhong, LIU Jun, ZHAO Jun, LIU Yan

2020, 38(6):  688-694.  doi:10.12140/j.issn.1000-7423.2020.06.003

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Objective To investigate the efficacy of the Tibetan medicine Elsholtzia eriostachya and albendazole, either alone or in combination, in treatment of secondary Echinococcus multiulocularis metacestode in rats, and to explore a new approach for treatment of the metacestode infection with Tibetan medicine combined with western medicine. Methods The SD male rats were laparotomized to expose the liver for injection with 0.2 ml of protoscoleces suspension (containing 2 × 10³ protoscoleces) to establish the rat model of secondary E. multiulocularis metacestode infection. The 40 model rats were divided into the albendazole treatment group [50 mg/(kg·d)], Elsholtzia eriostachya treatment group [50 mg/(kg·d)], albendazole [25 mg/(kg·d)]+Elsholtzia eriostachya [25 mg/(kg·d)] group, and the infection control group. Another 10 healthy SD rats were used as the healthy control group. Rats in the 3 treatment groups received two courses of treatment by gavage with once daily for consecutive 4 weeks, at an interval of 5 days between courses. Rats in the infection control group and healthy control group were given an equal volume of normal saline. After two courses of treatment, the rats body were weighed, the liver lesion diameter was measured, and the spleen index was calculated. The liver and spleen tissues in each group were obtained and sectioned for HE staining. Histopathological changes were observed under optical microscope and the ultrastructure of liver lesions was further observed under a transmission electron microscope. Results The diameters of rat liver lesions in the albendazole group, Elsholtzia eriostachya group and the combination group were (0.72 ± 0.06) cm, (0.79 ± 0.10) cm, and (0.62 ± 0.05) cm, respectively, all of which were smaller than the (1.10 ± 0.11) cm of the control group (P < 0.01). The diameter of the lesion in the combination group was significantly smaller than that of the albendazole group and the Elsholtzia eriostachya group (P < 0.01). The spleen index of the combined medication group was 5.28 ± 0.59, which was significantly higher than that of the albendazole group (3.35 ± 0.43) and the Elsholtzia eriostachya group (3.56 ± 0.40) (P < 0.01). Furthermore, the HE staining revealed obvious proliferation of white marrow in rat spleen tissues in the infection group and the three treatment groups, with the presence of a large number of heteromorphic megakaryocytes. The liver lesions in three treatment groups all showed collapse of the cyst wall and significant reduction of protoscoleces. Obvious lymphocyte infiltration was seen in the Elsholtzia eriostachya group, and necrosis and hyaline degeneration were obvious in the combined treatment group. Ultrastructural observations of liver tissues showed that treatment with Elsholtzia eriostachya and albendazole alone or in combination lead to the destruction of the mitochondrial structure of the germinal membrane cells of the metacestode. In the combination treatment group, the metacestode showed loose wall structure, and the microvilli of the germinal cortex became thicker, shorter or even disappeared,. eosinophils and macrophages increased significantly in the Elsholtzia eriostachya group. Conclusion The treatment with combination of Elsholtzia eriostachya and albendazole presented a good therapeutic effect on secondary E. multiulocularis metacestode infection in rats.

Proteomic analysis of human proteins in extracellular vesicles secreted by protoscoleces of Echinococcus granulosus

SHI Chun-li, YANG Hui, PAN Wen, ZHANG Xin, ZHU Xiao-ting, ZHAO Jia-qing

2020, 38(6):  695-701.  doi:10.12140/j.issn.1000-7423.2020.06.004

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Objective To identify the protein of human source in extracellular vesicles (EVs) secreted by protoscoleces of Echinococcus granulosus by proteomic analysis. Methods Liver intact cysts were collected from cystic echinococcosis patients, and the protoscoleces were separated under aseptic conditions and then cultured in 30% exosome-free fetal bovine serum in vitro, with medium replacement every three days. The supernatant was collected, from which EVs were isolated by a simple and rapid extracellular vesicle separation kit. A transmission electron microscope was employed to observe and identify the morphology of EVs, and the concentration and size of EVs were evaluated by nanoparticle tracking analysis. The EVs underwent Liquid chromatography-mass spectrometry analysis for identification and characterization of their protein content. The secondary mass spectrometry data were used to retrieve and annotated with Maxquant (v1.5.2.8). The enrichment analysis of tissues, disease and pathway for the human source proteins in EVs were were carried out using online tool David. Results The EVs secreted by protoscoleces in vitro were cup-shaped bilayer membranous vesicles with a diameter less than 200 nm. The proteomic analysis found a total of 20 317 peptides, and identified 1 461 proteins, including 328 human source proteins. Exosome marker proteins, including filament protein, 14-3-3 protein, heat shock protein 90, heat shock protein 70 and pyruvate kinase, are highly enriched in EVs secreted by protoscolex. The top 3 tissues in enrichment extent were plasma, Cajal-Retzius cells and liver. The disease enrichment found involving infection, immunity and cancer. The Bio Carta pathways analysis indicated top 3 enriched pathways, which were the alternative complement pathway, lectin complement pathway and complement pathway; the top 3 of KEGG pathways enrichment were adhesion plaque, extracellular matrix receptor-related action, and proteasome. Conclusion The proteins of human source in EVs secreted by protoscoleces cultured in vitro are related to human plasma and liver, indicating that the protoscoleces exert substance exchange with human via seretion of EVs.

Epidemic status of imported malaria at different stages of malaria elimination in Fujian Province

CHEN Zhu-yun, OUYANG Rong, XIE Han-guo, LIN Yao-ying, XIAO Li-zhen, ZHANG Shan-ying

2020, 38(6):  702-708.  doi:10.12140/j.issn.1000-7423.2020.06.005

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Objective To analyze the epidemic status of imported malaria at different stages of malaria elimination program in Fujian Province, in order to provide scientific basis for the formulation of malaria prevention and control strategy and implementation of control measures in later stage of elimination efforts. Methods The process of malaria elimination in Fujian Province was divided into three stages: the pre-elimination stage from 2005 to 2009, the elimination stage from 2010 to 2015, and the post-elimination stage from 2016 to 2019. Data of malaria cases and epidemiological investigations at different stages were collected to analyze the epidemic situation of imported malaria cases using SPSS 17.0 software. Results A total of 1 105 malaria cases were reported in Fujian Province from 2005 to 2019, and all were imported cases except for 3 locally transmitted cases. The annual average number of imported cases reported in the post-elimination stage (116.3 cases per year) was higher than those in the elimination (74.2 cases) and the pre-elimination (38.4 cases) stages. The proportion of falciparum malaria cases increased with staging, from 21.9% (42/192) in the pre-elimination stage, to 60.9% (271/445) in the elimination stage, and to 76.8% (357/465) in the post-elimination stage (P < 0.01). With regard to the infection source, there were cases with a source of local transmission in the pre-elimination and elimination stages, while in the post-elimination stage all cases were imported from abroad, among the imported, the case proportion from Africa increased from 27.1% (52/192) in the pre-elimination stage to 75.7% (337/445) in the elimination stage and to 92.3% (429/465) in the post-elimination stage (P < 0.01). The proportion of cases reported from medical institutions increased, which was significantly higher in the post-elimination stage (95.1%, 442/465) than in the elimination (65.6%, 292/445) and pre-elimination stages (65.1%, 125/192) (P < 0.01). There was an obvious seasonal distribution of reported cases in the pre-elimination stage (peak occurred in summer), but not in the elimination stage, while in the post-elimination stage the reporting peak occurred in the beginning of the year (January-February) and the middle of the year (June-July). The regional distribution of reported cases in three stages found that the reporting was mainly concentrated in Fuzhou, but the proportion increased, showing the proportion in the pre-elimination, elimination and post-elimination stages was 35.9% (69/192), 72.8% (324/445) and 78.1% (363/465) respectively, with statistically significant differences (P < 0.01). The age distribution revealed that the cases were largely in young and middle-aged males (20-59 years old), accounting the proportions in the elimination and post-elimination stages were 96.2% (428/445) and 96.1% (447/465), both higher than 83.3% (160/192) in the pre-elimination stage(P < 0.01). Conclusion During the malaria post-elimination stage, there is a more severe challenge of imported malaria in Fujian Province, especially falciparum malaria imported from Africa, thus public health education to key population and training for medical institutions should be strengthened.

Expression and function of a potential antigen Pb280 as transmission-blocking vaccine in Plasmodium berghei

WU Yu-di, LIU Fei, YANG Fan, CAO Ya-ming

2020, 38(6):  710-717.  doi:10.12140/j.issn.1000-7423.2020.06.006

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Objective To investigate the expression and function of a transmission-blocking vaccine candidate Pb280 (PBANKA_041710) in Plasmodium berghei. Methods Homologous sequences of Plasmodium species were downloaded from NCBI and compared using MEGA7. The domains of Pb280 were predicted by SMART. Ten female Kunming (KM) mice aged 6-8 weeks were randomly selected, injected with 1 × 10⁶ P. berghei-infected red blood cells (iRBC) through tail vein. When the parasitemia in the mice reached 3%-5%, orbital blood was obtained and cultured in vitro for isolation of schizonts. The hemagglutinin (HA) tagged plasmid (Pb280HA) and Pb280 knockout plasmid (Pb280KO) were linearized and transfected to mature schizonts, which were then injected in the mice (1 × 10⁷ schizonts/mouse) through tail vein; transgenic parasites in the infected mice was identified by PCR, and underwent monoclonal screening. As a result, one strain of Pb280HA and two strains of Pb280KO (Pb280KO-C1 and Pb280KO-C2) were obtained. The expression of Pb280 in P. berghei was analyzed by Western blotting and indirect immunofluorescence assay (IFA). Another nine female KM mice were randomly divided into 3 groups (n = 3): Pb280KO-C1 group (C1 group), Pb280KO-C2 group (C2 group) and control group. Each mouse was injected with 5 × 10⁶ Pb280KO-C1, Pb280KO-C2 or wild-type (WT) P. berghei through tail vein. From day 3 to day 10 post-infection, mice tail vein blood was sampled for monitoring daily parasitemia by examining blood smears. On day 3 after infection, gametocytemia and female/male gametes ratio were calculated; mice tail vein blood was mixed with ookinete culture medium for 15 min to count the number of exflagellation centers, number of male-female integrating and number of macrogametes followed by further culture for 24 h to count the number of ookinetes. The comparisons of parasitemia, gametocytemia and female/male gamete ratio were performed by Chi-square test, and other data were analyzed with one-way ANOVA. Results Phylogenetic tree constructed by MEGA7 showed that Pb280 was genetically most close to the PY04819 of P. yoelii. SMART prediction showed that Pb280 contained an N-terminal signal peptide, 10 transmembrane domains and 1 growth factor receptor domain. Western blotting demonstrated that Pb280 protein expression occurred in P. berghei, with a relative molecular weight of 280 000. IFA results showed that Pb280 was secreted from cytoplasm to plasma membrane during the development from gametocyte to ookinete. Gene function analysis revealed that on day 3 after infection, the three groups had parasitemia of about 15%, which reached over 60% on day 10 post-infection. The parasitemia rate in the group C1 and the C2 did not differ significantly from that in the control group (P > 0.05). On day 3 post-infection, the gametocytemia rate, gamete sex ratio, number of exflagellation centers per 10 fields of view, number of male-female integrating and number of macrogametes in group C1 were 39.50‰, 1.65, 21.63 ± 4.03, 12.50 ± 8.02, and 930.00 ± 79.20, respetively; those in the group C2 were 34.50‰, 1.71, 18.25 ± 5.85, 13.75 ± 9.54, and 885.00 ± 130.11, respetively; and those in the control group were 41.50‰, 1.74, 21.44 ± 4.73, 15.31 ± 8.06, and 1 018.50 ± 58.69. There were no statistically significant differences among the three groups (P > 0.05). The numbers of ookinetes in the C1, C2 and control groups were 410.00 ± 67.88, 557.50 ± 2.12 and 782.00 ± 41.01, respectively. The numbers of ookinetes in the C1 and C2 groups did not differ significantly, but both were lower than that in the control group (P < 0.05). Conclusion Pb280 is genetically conserved in Plasmodium spp. and expressed in schizont, gametocyte and ookinete stages of P. berghei. Knockout Pb280 may result in the decrease of ookinete formation.

Molecular characteristics of Cryptosporidium sp. isolated from imported breeding dairy cows in a dairy farm in Shenzhen, China

JIANG Yan-yan, YUAN Zhong-ying, SHEN Yu-juan, XU Ning, ZHANG Ren-li, HUANG Da-na, CAO Jian-ping

2020, 38(6):  718-722.  doi:10.12140/j.issn.1000-7423.2020.06.007

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Objective To investigate the status of Cryptosporidium infection in imported breeding dairy cows in a dairy farm in Shenzhen, and analyze the molecular characteristics of Cryptosporidium isolates. Methods Fresh cow fecal samples were collected from a dairy farm in Shenzhen. Genomic DNA was extracted from the samples, and the small subunit ribosomal ribonucleic acid gene (SSU rDNA) of Cryptosporidium was amplified by nested PCR. The PCR products were sequenced and blasted to determine the genotype, and construct phylogenetic tree. Data were analyzed with Chi-square test using Graphad Prism 7.0. Results A total of 20 cow fecal samples were collected, including 5 from adult cows, 10 from weaned calves and 5 from pre-weaning calves. The PCR results showed that 8 samples were positive of Cryptosporidium, with positive rate of 40%, including 1 adult cow (1/5), 5 weaned calves (5/10), and 2 pre-weaning calves (2/5). Sequencing results showed that 2 Cryptosporidium genotypes were detected, including C. ryanae (n = 5) and C. bovis (n = 3). The C. ryanae infection was found in all age groups. After blasting with the SSU rDNA gene of C. ryanae, four of the five isolates of C. ryanae were 100% similar to the SSU rDNA gene (KT922233) of bovine-borne C. ryanae from Ethiopia, and the other one was similar to that of the isolate of C. ryanae (HQ179574, 99.9%) from Northeast China. The three isolates of C. bovis were 100% similar with the SSU rDNA gene (KT922231) of bovine-borne C. bovis from Ethiopia. After constructing the phylogenetic tree of C. ryanae based on the sequence of SSU rDNA gene, the sequences of C. ryanae and C. bovis were in two branches, which were consistent with the same isolated Cryptosporidium species in different regions. Conclusion Cryptosporidium infection is common in this dairy farm in Shengzhen, with C. ryanae as a dominant species in infection.

Cloning, expression, purification and immunogenicity analysis of the tenth-class allergen of Dermatophagoides pteronyssinus

GUO Chu-tong, OUYANG Chun-yan, HE Jun-xian, JI Shu-yu, YANG Li-teng, LIU Xiao-yu

2020, 38(6):  723-729.  doi:10.12140/j.issn.1000-7423.2020.06.008

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Objective To clone the gene of the tenth-class allergen Der p 10 (tropomyosin) of Dermatophagoides pteronyssinus, express and purify recombinant protein Der p 10, and analyze the immunogenicity and biological information of the protein. Methods Cultured mites were picked for total RNA extraction, then cDNA was generated by reverse transcription and underwent RT-PCR to amplify the Der p 10 gene. The pET-24a vector was ligated with the Der p 10 gene and transformed into the Top10 cloned bacteria. After BamH I and Xho I double enzyme digestion and sequencing, the construct was transformed into Escherichia coli BL21 (DE3), induced by 1 mmol/L IPTG, and examined in 12% SDS-PAGE to analyze the expression of recombinant protein. After purification by Ni⁺ ion affinity chromatography, the immunogenicity of Der p 10 recombinant protein was analyzed by Western blotting using serum of patient with dust mite allergy as the primary antibody. The ProtParam Tools, HNN, SWISS MODEL, DNAStar were used to predict the physiochemical properties of Der p 10, the secondary and tertiary structure of tropomyosin, and B cell epitope; Blastpand MEGA tool were used to construct Der p 10 phylogenetic tree. Results The target gene Der p 10 was obtained by RT-PCR, with an open reading frame of 855 bp, encoding 284 amino acids. The results of double enzyme digestion and sequencing showed that Der p 10 was linked to the vector. SDS-PAGE analysis showed that the relative molecular mass of Der p 10 recombinant protein was 38 000, which was a soluble protein. Western blotting analysis showed that the Der p 10 recombinant protein reacted with the serum of dust mite allergy patient. The bioinformatics analysis indicated that the Der p 10 protein was unstable, and its secondary and tertiary structures were mainly by α-helices. DNAStar predicted the presence of one B cell epitope peptide sequence in Der p 10. Blastp alignment and MEGA analysis showed that it was closely related to Der f 10, with sequence similarity reaching 94.4%. Conclusion The Der p 10 recombinant plasmid was constructed, and the expressed soluble recombinant protein of Der p 10 can specifically bind to the IgE antibody in patient serum with dust mite allergy, demonstrating its apparent immunogenicity.

INFORMATION EXCHANGE

Analysis of the results of parasitic disease control skills contest in Jiangsu Province in 2018

CAO Yuan-yuan, ZHU Guo-ding, LIU Yao-bao, DAI Yang, LI Wei, WANG Jie, ZHOU Hua-yun, WANG Wei-ming, WANG Yi, YANG Kun, CAO Jun, YANG Hai-tao

2020, 38(6):  731-736.  doi:10.12140/j.issn.1000-7423.2020.06.009

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Jiangsu province organized a technical contest on parasitic disease control skills was late September, 2018. Fifty-six professionals from 13 reprentative city-level and provincial teams participated in the contest. The scores of theoretical examination and technical operation were recorded and analyzed to understand the professional capability of the participants specialized in parasitic disease control in the province. The average age of the 56 participants was 32.5 ± 5.7 years. The average score of written examination of theoretical knowledge was 83.27 ± 8.31 with a passing rate of 98.21%; among them, the examination in theoretical knowledge of malaria (59.01%) and control execution (65.29%) showed lower scoring rate (χ² = 128.78, 246.43, P < 0.01). The average score of technical skill was 74.80 ± 14.41 with a passing rate of 83.93%. More specifically, the score of plasmodium blood smear preparation was 8.97 ± 0.59 with a passing rate of 83.93%; the score of Kato thick smear preparation was 8.87 ± 0.83 with a passing rate of 98.21%; the score of microscopy for Plasmodium spp. was 19.03 ± 5.91 with a passing rate of 62.50%; and the score of microscopy for common parasitic eggs was 37.93 ± 10.67 with a passing rate of 83.93%. The participants lost more scores in thick blood smear appearance, thick blood smear volume, thin blood smear diameter and thin blood smear appearance, with the scoring rate of 7.14%, 33.93%, 26.79% and 19.64%, respetively, all less than 50%. The scoring rate in the pressing process of Kato’s smear preparation was only 17.86%. These results suggest that despite the overall good results in the contest, the technical skills remain to be improved.

TEACHING RESEARCH

Establishment and improvement of a case base for multi-mode problem-based learning teaching of human parasitology

YANG Xiao-di, WANG Yu-hang, CHENG Yang, YUAN Yuan, CHU Liang, ZHANG He, CHANG Xue-lian, TAO Zhi-yong, LI Hui-hui, LI Kun-long, JIANG Hui, SUN Si-ying, ZHENG Dong-xue, XIA Hui, FANG Qiang, CHEN Xing-zhi

2020, 38(6):  737-741.  doi:10.12140/j.issn.1000-7423.2020.06.010

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Cases are essential to teach based on the problem-based learning (PBL), and are the basis of students’ independent or teamed learning. The design and quality of PBL curriculum determines the teaching efficacy. However, to compose PBL cases is not a easy matter. Based on our practice in establishment of case base of multi-mode PBL teaching for human parasitology, this paper summarizes setting up objectives for PBL curriculum, recomposing the clinical cases, verifying and finalizing the teaching cases, in order to provide reference for teachers specialized in teaching human parasitology in colleges and universities.

REVIEWS

Functions and roles of miRNA during the infection of Anopheles by Plasmodium

ZHU Ling-qian, FENG Xin-yu, HU Wei, LI Shi-zhu

2020, 38(6):  742-748.  doi:10.12140/j.issn.1000-7423.2020.06.011

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miRNA is a type of single-stranded non-coding RNA with a length of 21-24 bases. miRNA may lead to mRNA degradation or translation inhibition by targeting the 3′-end untranslated region of mRNA, and plays an important role in the defense response of Anopheles against pathogen invasion among many other biological processes. This article reviews the possible functions, and target genes and signaling pathways involved of miRNAs, during Anopheles infection with Plasmodium.

Research progress on economic burden of malaria

XU Qiu-li, ZHOU Hong-rang, QIAN Men-bao, WANG Duo-quan, XIAO Ning

2020, 38(6):  749-752.  doi:10.12140/j.issn.1000-7423.2020.06.012

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Malaria is an old mosquito-borne parasitic disease, imposing great threat to human health, impacting social stability and economic development, and placing heavy burden to the global medical and health resources. In this paper, we systematically review the economic burden caused through malaria by searching literature from China and other countries, with the aim to provide reference for credible estimation of disease burden for effective allocation of health resources on nationwide and local base, and for appropriate management of the challenges of disease burden.

Research progress on odor binding proteins and odor receptors of mosquitoes

GU Zhen-yu, ZHAO Teng, LI Chun-xiao

2020, 38(6):  753-757.  doi:10.12140/j.issn.1000-7423.2020.06.013

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Prevention and control of mosquitoes is the main measure to control mosquito-borne diseases, and chemical control remains the primary approach for mosquito control. However, with the increasing occurrences of resistance to insecticides in mosquitoes, there is an imperative need for a novel, efficient and environmentally friendly method for mosquito control. Olfaction plays an important role in the life activities of mosquitoes. Many physiological behaviors of mosquitoes depend on olfaction, such as mating, blood-sucking and oviposition. This paper summarizes research progress on the olfaction-related odor-binding proteins and odor receptors of mosquitoes and discusses the related mechanisms and targets in mosquito repellence and attraction behaviors, in order to provide theoretical references for the application and improvement of mosquito vector control measures.

Prevalence and challenges in elimination of schistosomiasis mekongi

WANG Li-ping, QIN Zhi-qiang, LV Shan, LI Shi-zhu, ZHOU Xiao-nong, XU Jing

2020, 38(6):  758-763.  doi:10.12140/j.issn.1000-7423.2020.06.014

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Schistosomiasis mekongi has been a major public health problem in Southeast Asia since its discovery. Although Laos and Cambodia have achieved steady control of schistosomiasis mekongi through continuous prevention and control measures, there are still many challenges and difficulties that hinder the march towards elimination. In addition, with the gradual progression of “the Belt and Road” initiative, there has been an increased risk of schistosomiasis mekongi infection in China, either by case import or via contracting infection among Chinese visitors in those countries. This review summarizes the prevention and control measures and current problems in controlling schistosomiasis mekongi, and discusses the possible negative impact of the disease to China and “the Belt and Road” initiative, in order to provide referential information for future south-south cooperation and elimination of schistosomiasis.

Research advances on the innate immunity mechanisms against Toxoplasma gondii

DU Kai-ge, ZHUO Xun-hui, LU Shao-hong

2020, 38(6):  764-770.  doi:10.12140/j.issn.1000-7423.2020.06.015

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The innate immunity is the first defense line against invasion of pathogenic microorganisms. Toxoplasma gondii infection induces effective innate immune responses in the host, which play an important role in restricting Toxoplasma gondii infection and promoting the adaptive immunity. In the innate immunity, the pattern recognition receptors, immune cells and cytokines hold their respective roles while on the other hand interacting with each other, constituting a complex but ordered network. Some regulatory mediators and transcription factors regulate signaling transduction and gene expression, and participate in the defense response, autophagy and other biological processes. In this paper, we review the research progress of host innate immunity against T. gondii, to provide scientific basis for control of the parasite.

Research progress on microRNAs of ticks

PEI Ting-wei, YU Zhi-jun, LIU Jing-ze

2020, 38(6):  771-776.  doi:10.12140/j.issn.1000-7423.2020.06.016

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MicroRNAs (miRNAs) are a class of non-coding small RNA molecules involved in the regulation of development, stress, cell differentiation, cell apoptosis and other physiological and cellular processes. Ticks, as obligate blood-sucking ectoparasites, serve as a vector of a great diversity of pathogens. Research on the miRNAs of ticks has mainly focused on their regulation of growth and development, as well as their roles in functional regulation during the tick-pathogen interactions. This paper reviews the recent progress on the identification of miRNAs in ticks and their roles in functional regulation, which may provide a theoretical basis for subsequent design of prevention and control strategies for ticks and tick-borne diseases.

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Sexing Schistosoma japonicum cercariae by sequence characterized amplified region-PCR

MO Xiao-jin, WU Qun-feng, FENG Zheng, XU Bin, ZHANG Ting, CHEN Shen-bo, HU Wei

2020, 38(6):  777-780.  doi:10.12140/j.issn.1000-7423.2020.06.017

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To identify the sex of Schistosoma japonicum cercaria, single sex cercariae were prepared. Monosexual miracidia were used to infect single parasite-negative snail for breeding positive snails being able to shed single sex cercaria. The single-sex cercariae released from each positive snail were collected for sex identification using the sequence characterized amplified region-PCR (SCAR-PCR) method. To confirm the sex of cercariae obtained, single-sex cercariae from the same snail were used to infect mice, and the adult worms were harvested on 21 d post-infection for verifying the worm gender morphologically, and examining the accuracy of the PCR method for detecting DNA of one single cercaria. The PCR results indicated that the female cercariae presented a specific band at 153 bp, showing a concordance with the findings of infected mouse experiment. The minimum DNA amount detectable in the PCR assay was 16.5 ng, equivalent to the DNA amount of one cercaria. To verify the accuracy of the PCR method, 40 female and 40 male sex-known cercariae verified morphologically on their derived adult worms were examined using the PCR method, and found 37 females and 40 males respectively from the sex-known samples, indicative of a sex-discriminating accuracy of 92.5% (37/40) for female and 100% (40/40) for male cercariae of S. japonicum. The results demonstrated that the cercaria sex could be differentiated using the SCAR-PCR method.

Survey on current status of human hookworm infection in Yunnan Province in 2015

WU Fang-wei, WANG Li-bo, LI Ben-fu, YAN Xin-liu, ZI Jin-rong, PENG Jia, CAI Xuan, BAO Xue-ying, YANG Ya-ming

2020, 38(6):  781-784.  doi:10.12140/j.issn.1000-7423.2020.06.018

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To understand the prevalence of human hookworm infection in Yunnan Province, a total of 20 survey sites in 10 counties (cities) in Yunnan Province were selected by the stratified cluster random sampling method in 2015, according to the National Survey Plan for the Status of Key Human Parasitic Diseases and the implementation guideline. In each site, no less than 250 permanent residents aged 1 year and above were surveyed. Fecal samples were collected from the participants for examining, hookworm eggs using modified Kato-Katz method. The hookworm species was identified by the in vitro filter culture method. A total of 5 067 residents were examined, with an infection rate of 9.0% (457/5 067), among them, 94.5% (432/457) were severe, 2.8% (13/457) moderate and 2.6% (12/457) mild infection. Species identification was performed for 160 cases, of them, 139 was infected with Ancylostoma americanum, 16 with A. duodenale, while 5 with mixed infection. The infection rate was 8.4% (204/2 422) in males and 9.6% (253/2 645) in females. Highest infection rate was detected in the age group of 60 years and above (12.93%), in Dulong ethnic group (39.55%), in farmers (9.4%), and in the group of education level of primary school level (9.5%). The human hookworm infection rate was highest in the ecological area of southern part of Yunnan, Guangxi and Guangdong (17.14%), with Hekou County in particular having infection rate up to 30.6%. Human hookworm infection rate was 0.8% (6/759), 13.6% (311/2 287) and 6.9% (140/2 021) in regions with high, medium and low economic levels, respectively, with significant differences between the regions(P < 0.01). In conclusion, the human hookworm infection rate remains high in part of Yunnan Province, efforts should be made to continuously strengthen prevention and control measures in farmers, elders, and strait through ethnic minorities.

Relationship between the infectivity of encysted larvae and the developmental stage of Trichinella spiralis

WANG Guo-ying, CHEN Dan-dan, ZHENG Xue-li, LI Xiang-hui, ZHANG Hao, ZHANG Jun, TENG Tie-shan

2020, 38(6):  785-788.  doi:10.12140/j.issn.1000-7423.2020.06.019

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To investigate the relationship between the infectivity of encysted larvae and the developmental stage of Trichinella spiralis. Eighteen female Kunming mice were orally infected with 20 encycted larvae each mouse. On days 60, 100, 200, 300, 400 and 500 after infection, three mice in each group were sacrificed by euthanatized, and 10 flesh samples (20 cysts/flesh sample) were used to orally infect 10 female Kunming mice. On day 30 after infection, intact diaphragms were obtained from the mice, weighted, and observed under a microscope to count the cysts. The worm load per gram of diaphragmatic muscle was calculated. In the day 60 group, the walls of cysts were intact, the edge of the deep staining layer was transparent, and larvae were seen. In the day 100 group, the cyst walls were intact, transparency appeared outside the edge of the deep staining layer, and the larvae could be seen; two cysts had no cyst wall or internal structure, with faintly visible larvae. In the day 200 group, the cyst walls were intact, light traces appeared outside the edge of the deep staining layer, and the larvae could be seen; four cysts had no cyst wall or internal structure, with barely visible larvae. In the day 300 group, the cyst walls were intact, the transparent zone and trace seen, and the larvae were visible; four cysts had no cyst wall or internal structure, with faintly visible or invisible larvae. In the day 400 group, most of the cysts had intact walls, the transparent zone and trace were visible, and the larvae were visible or faintly visible; in twelve cysts devoid of wall and cystic structure, the larvae in the cysts were visible, indistinct or invisible. In the day 500 group, the cyst walls were inapparent or even disappeared, with a transparent zone at the edge of the deep staining layer, and the larvae were visible or faintly visible. In the day 60-500 infection group, the average length of diaphragm muscle worm was (1 026.6 ± 64.8), (1 041.1 ± 62.8), (1 031.5 ± 75.6), (1 047.9 ± 56.0), (1 030.5 ± 72.2), and (1 011.2 ± 95.0) μm, respectively. In the day 60-400 infection group, the mean cyst length was (371.4 ± 69.1), (309.4 ± 40.3), (311.9 ± 48.1), (299.5 ± 43.8) and (294.3 ± 23.4) μm respectively, and the difference was statistically significant between day 60 and day 100 groups (P < 0.01). In microscopic examination of all 6 experimental groups, larval cysts were found in diaphragm muscle. This suggested that with prolonged parasitism, increased number and degree of aging of larval cysts would lead to decreasing infectivity.

Investigation of Anisakis larvae infection in marine fish in markets of Anshan City, Liaoning Province

WEI Zhuo-chao, ZHANG Wei, DENG Xiao-li

2020, 38(6):  789-791.  doi:10.12140/j.issn.1000-7423.2020.06.020

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From 2015 to 2018, marine fish selling in market of four districts and three counties of Anshan City were sampled according to the National Food Safety Risk Monitoring Plan. Anisakis was examined by direct dissection, and isolated for species identification. A total of 303 fish from 60 samples of 12 species of marine fish were collected. Among them, Anisakis stage-3 larvae were detected in 158 fish from 28 samples of 6 species. The detection rate from the samples was 46.7% (28/60), the total positive rate among all the fish species was 52.2% (158/303), and the average infection intensity was 5.99 larvae/fish (947/158). Among the 6 species of fish infected, the infection rate was highest in Miichthys miiuy, 100% infection in all 5 fishes, followed by Pneumatophorus japonicus (3/4), Larimichthys polyactis(64.4%, 114/177), Trichiurus lepturus (50.8%, 32/63), Scomberomorus niphonius (3/18), and Cololabis saira (1/16). The infection intensity from the highest to the lowest larvae/fish was in following order: Miichthys miiuy (8.20), Larimichthys polyactis (6.28), Trichiurus lepturus (5.67), Scomberomorus niphonius (4.84), Pneumatophorus japonicus (3.67), and Cololabis saira (1). From 2015 to 2018, 22, 10, 14 and 14 marine fish samples were respectively collected, among them, the detection rate in 2015 was highest at 72.7% (16/22). In different sampling sites, the fish with highest detection rate were found from county supermarkets, showing 5 out of 6 samples being detected positive, followed by urban farm markets, with the detection rate of 46.7% (14/30). These findings indicate a considerably high infection rate of Anisakis found in marine fish from market of Anshan City, thus food health education should be strengthened.

Evaluation of the performance of three immunochromatographic strips in detecting Leishmania infection in dogs in Gansu Province

YU Da-wei, LI Fan, FENG Yu, YANG Cheng-ming, YANG Jun-ke, LIU Lin-lin, ZHANG Yong-fu

2020, 38(6):  792-795.  doi:10.12140/j.issn.1000-7423.2020.06.021

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Blood samples were collected from 537 domestic dogs in Wenxian County and Diebu County of Gansu Province, where canine visceral leishmaniasis were endemic, and from 37 domestic dogs from Tianzhu County of Gansu Province, the non-endemic area. Blood examination was carried out using three immunochromatographic test strips including human rk39 strip, canine rk39 strip and circulating antigen (CA) strip in parallel to assess the sensitivity and specificity of the methods, and using PCR assay as the standard to compare with. The results showed that among the total 574 dog blood samples, 221 samples were tested Leishmania positive by PCR with the positive rate of 38.5% (221/574). Examining the 221 positive samples with CA strip, the human rk39 strip and canine rk39 strip, the sensitivity was found of 90.0% (199/221), 65.2% (144/221) and 64.3% (142/221), respectively, all of which significantly differed from that of PCR method (χ² = 141.76, 20.31, and 17.96, P < 0.05). The specificity of CA test strip and the two rk39 test strips in testing the blood samples of 316 dogs without Leishmania infection from the endemic areas was 95.3% (301/316), 99.7% (315/316) and 99.4% (314/316), respectively, all of which significantly differed from that of the standard (χ² = 258.85, 312.01, and 308.05, P < 0.05). The positive-result coincidence rate of the CA strip and the canine and human rk39 strips with PCR in dogs with symptoms from the endemic areas was 88.5% (23/26), 88.5% (23/26) and 84.6% (22/26), respectively, all of which significantly differed from that of the PCR method (χ² = 23.22, 23.22, and 20.50, P < 0.05). The positive-result coincidence rate of CA test strip and the canine and human rk39 test strips with PCR in dogs without symptoms in the endemic areas was 91.8% (179/195), 62.1% (121/195) and 61.5% (120/195), respectively, all of which significantly differed from that of the PCR method (χ² = 136.25, 11.33, and 10.39, P < 0.05). Area under the curve (AUC) analysis was used to assess the method accuracy, showing the AUC value of all the 3 methods was above 0.8. The kappa consistency coefficient was 0.671-0.857, indicating that all of the three test strips are applicable for preliminary screening of Leishmania-infected dogs. The CA test strip is superior to the two rk39 test strips and could be a good method for screening Leishmania-infected dogs in endemic areas.