Table of Content

30 August 2023, Volume 41 Issue 4

Previous Issue    Next Issue

ORIGINAL ARTICLES

Function analysis of Anopheles stephensi peptidoglycan recognition protein S2 in regulating homeostasis of symbiotic microbiota

WANG Zhiqian, WANG Jingwen, SONG Xiumei

2023, 41(4):  397-403.  doi:10.12140/j.issn.1000-7423.2023.04.001

Asbtract ( )   HTML ( )   PDF (2686KB) ( )  

Figures and Tables | References | Related Articles | Metrics

Objective To study the function of Anopheles stephensi peptidoglycan recognition protein S2 (PGRP-S2) and its role in regulating the homeostasis of symbiotic microbiota. Methods The female An. stephensi mosquitoes aged 2-4 days after eclosion were divided into control group (by biting the 10% sucrose solution), infected blood group (by biting the Plasmodium berghei ANKA-infected mice with 4%-8% parasitemia) and healthy blood group (by biting healthy mice), 60 mosquitoes in each group, the midgut and carcass were isolated after 24 h of full blood meal. The RNA was extracted by TRIzol, and relative transcription level of the pgrp-s2 gene was detected by real-time fluorescence quantitative PCR (RT-qPCR). Female An. stephensi mosquitoes 0-1 day after eclosion were divided into control group (fed with 10% sucrose solution) and antibiotic treatment group (fed with 10% sucrose solution containing penicillin, streptomycin and gentamicin), 30 mosquitoes in each group, midgut and carcass were isolated after fed for 5 days to detect the relative transcription level of pgrp-s2 gene by RT-qPCR. Female An. stephensi mosquitoes were divided into pgrp-s2 knockdown group and green fluorescent protein (gfp) control group, 60 mosquitoes in each group, each group were injected with pgrp-s2 double-stranded RNA (dsRNA) or green fluorescent protein gene dsRNA (69 nl/mosquito) respectively. After 2 days, mosquito RNA was extracted and RT-qPCR was used to detect the relative transcription level of pgrp-s2, mosquito DNA was extracted and PCR was used to detect the total amount of symbiotic bacteria in the mosquitoes using 16S rRNA specific primers. RNA was extracted from 30 An. stephensi mosquitoes of the knockdown group and control group respectively, which were fed with cotton balls soaked with 10⁷/ml Morganella morganii for 5 days, and subsequently, the relative total amount of 16S rRNA specific symbiotic bacteria and the relative number of M. morganii were detected by RT-qPCR; the RNA extracted from the midgut tissues of the knockdown group and gfp control group mosquitoes was use for transcriptome sequencing, cluster analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotation and enrichment analysis. The PGRP-S2 amino acid sequence was predicted and analyzed using SMART website, and aligned using CLC Main Workbench software. The pgrp-s2 gene was cloned from An. stephensi mosquito cDNA, and the PGRP-S2 recombinant protein was expressed in SF9 cells using the insect baculovirus expression system (pFastbacI), and the protein expression was verified by Western blotting. The purified PGRP-S2 recombinant protein solution of 10, 20 and 40 μg/ml (protein buffer as control) was separately incubated with 40 μg of Lys-type peptidoglycan or DAP-type peptidoglycan, and the relative absorbance value (A₅₄₀) was detected every 12 h to check the degradation of peptidoglycan and verify the amidase activity of PGRP-S2. Results The RT-qPCR results showed that 24 h after the blood meal, the An. stephensi mosquitoes midgut pgrp-s2 relative transcription levels in the infected blood group, healthy blood group and control group were 1 590.0 ± 665.2, 126.8 ± 100.4 and 15.84 ± 6.92, respectively. The pgrp-s2 relative transcription levels of the infected blood group was higher than that of the control group (t = 2.38, P < 0.05); the pgrp-s2 of An. stephensi midgut relative transcription level of the control group was higher than that of the carcass (1.71 ± 0.51) (t = 2.04, P < 0.05). The pgrp-s2 of An. stephensi midgut relative transcription level of antibiotic treatment group was 0.33 ± 0.18, which was lower than that of the control group (117.9 ± 54.5) (t = 2.16, P < 0.05). The relative total amount of 16S rRNA of symbiotic bacteria in the pgrp-s2 knockdown group was 3 653 ± 2 023, which was lower than 14 982 ± 3 892 in the gfp control group (t = 2.58, P < 0.05); after feeding with M. morganii, the relative number of M. morganii in the An. stephensi of pgrp-s2 knockdown group was 571 517 ± 61 258, which was lower than 919 754 ± 123 397 of GFP control group (t = 2.53, P < 0.05). Transcriptome analysis results showed that pgrp-s2 knockdown could up-regulate An. stephensi Toll/Imd pathway, mTOR pathway, FoxO pathway and immune-related genes, while down-regulate metabolism-related genes such as fatty acid metabolism and tricarboxylic acid cycle. After incubation of PGRP-S2 recombinant protein at concentrations of 10, 20 and 40 μg/ml with DAP-type peptidoglycan for 48 h, the relative A₅₄₀ values were 0.49 ± 0.07, 0.40 ± 0.10 and 0.44 ± 0.07, respectively, which were lower than 0.90 ± 0.09 of control (t = 3.53, 3.65, 3.97; all P < 0.05); after incubation with Lys-type peptidoglycan for 48 h, the relative A₅₄₀ was 0.52 ± 0.03, 0.62 ± 0.03 and 0.65 ± 0.04, respectively, and there was no difference from 0.64 ± 0.05 of control (t = 1.95, 0.31, 0.11; all P > 0.05). Conclusion pgrp-s2 is mainly expressed in the midgut of An. stephensi, and the expression level is regulated by symbiotic bacteria. PGRP-S2 recombinant protein can degrade DAP-type peptidoglycan, having amidase activity, and may regulate the level of symbiotic bacteria in An. stephensi by negatively regulating immune responses and regulating metabolic responses.

Polymorphism analysis of multidrug resistance protein 1 gene in imported Plasmodium vivax in Yunnan Province

DING Hongyun, DONG Ying, XU Yanchun, DENG Yan, LIU Yan, WU Jing, CHEN Mengni, ZHANG Canglin

2023, 41(4):  404-411.  doi:10.12140/j.issn.1000-7423.2023.04.002

Asbtract ( )   HTML ( )   PDF (2202KB) ( )  

Figures and Tables | References | Related Articles | Metrics

Objective To analyze the polymorphism of Plasmodium vivax multidrug resistance protein 1 gene (Pvmdr1) from imported vivax malaria patients in Yunnan Province. Methods The whole Pvmdr1 gene was amplified and sequenced in the blood samples of patients who diagnosed with vivax malaria in 2020 and 2021 in Yunnan Province. The sequence of P. vivax SalⅠ isolate (GenBank accession number: NC_009915.1) was taken as the reference sequence for designing primers. MEGA 5.04 software was used to align the spliced sequences with the coding region reference sequence (GenBank accession number: XM_001613678.1). Haplotype and single nucleotide polymorphism (SNP) sites and their mutation types, nucleotide diversity π, expected heterozygosity (He), etc were analyzed by DnaSP 6.11.01. Haplotype medium network diagram was constructed using Network 10.0 Software. Results A total of 276 blood samples were collected from vivax malaria patients and were imported from overseas. The full gene sequences with 4 392 bp of Pvmdr1 were obtained from 259 blood samples, and the accession numbers were OP559204-OP559462 assigned by GenBank. The π of 259 DNA coding sequences was 0.000 76. A total of 22 SNPs were detected from 259 coding sequences, including 13 non-synonymous mutant and 9 synonymous mutant loci. Only the detection rate difference of c.4074C>T mutation between 2020 and 2021 [15.0% (21/140), 5.9% (7/119)] was statistically significant (χ² = 5.546, P < 0.05). The minor allele frequency was c.1587A>G (97.9%, 253/259), and the newly discovered SNPs included c.2499G>T (2.3%, 6/259), c.3358C>T (0.4%, 1/259), and c.3832C>T (0.4%, 1/259). All of c.3064C>T, c.4065A>G, c.3358C>T and c.3832C>T mutant sites were detected from the blood samples in 2021. The 259 complete coding region sequences of Pvmdr1 were defined as 26 haplotypes (Hap_2-Hap_27) by comparison with the reference sequence (haplotype Hap_1, all of which were mutant type haplotypes, and He was 0.890 7. Among them, the detection rate of Hap_8 was the highest (18.5%, 48/259). There were 15 and 22 haplotypes found from blood samples in 2020 and 2021, respectively. Among them, 4 haplotypes were only detected in blood samples from 2020, and 10 haplotype were only detected in blood samples from 2021. The difference of Hap_10 detection rate between 2020 and 2021 [15.0% (21/140), 3.4% (4/119)] was statistically significant (χ² = 22.264, P < 0.05). The proportions of quadruple, quintuple, six-fold, seven-fold, nine-fold and ten-fold multiple mutations in 26 haplotypes were 0.4% (1/26), 19.2% (5/26), 38.5% (10/26), 30.8% (8/26), 0.4% (1/26), and 0.4% (1/26), respectively. The haplotype medium network diagram displayed that the gradual evolution of 26 haplotypes started with Hap_1, and then passed through quadruple mutation (Hap_24), moved towards quintuple, six-fold, seven-fold, nine-fold, and ten-fold multiple mutations away from. Conclusion There is high polymorphism in the Pvmdr1 gene in the P. vivax from the imported vivax malaria cases in Yunnan Province.

Identification of miRNAs in the infectious third stage larvae and parasitic female adult of Strongyloides stercoralis

QIN Peixi, ZHOU Caixian, LU Zhigang, ZHANG Biying, ZHOU Taoxun, HU Min

2023, 41(4):  412-420.  doi:10.12140/j.issn.1000-7423.2023.04.003

Asbtract ( )   HTML ( )   PDF (2009KB) ( )  

Figures and Tables | References | Related Articles | Metrics

Objective To identify the microRNA (miRNA) of the Strongyloides stercoralis infective third-stage larvae (iL₃) and parasitic female adult (pF), and analyze the differential expression of miRNA and the function of their target genes. Methods The iL₃ was collected from the feces of dogs infected with S. stercoralis. Healthy dogs were subcutaneously injected with 4 000-5 000 iL₃ at the back of the neck. The pF was collected from the dog intestine 21 days post-infection. The total RNA of iL₃ and pF was extracted and sequenced. The sequencing data was aligned with the S. stercoralis genome and S. ratti miRNA to identify the conserved miRNA. The true miRNA sequences were screened out using miRDeep2 through loop structure analysis and mature sequence readings alignment. The miRNA expression profiles of iL₃ and pF were analyzed to screen the differentially expressed miRNA, and predict their target genes. Functional analysis was performed to those of unannotated miRNAs target genes with the values of log₂(Fold chang) > 1 and transcripts per million (TPM) > 1 000. GO enrichment analysis of differentially expressed miRNA target genes were analyzed using ClusterProfiler. Eleven unannotated or conserved differentially expressed miRNAs were selected for real-time fluorescent quantitative PCR (qRT-PCR) verification. The relative transcription level was calculated with the glyceraldehyde-3-phosphate dehydrogenase gene of S. stercoralis (GenBank accession number: BI773092.1) as reference gene. Results A total of 265 miRNA were identified, including 130 conserved miRNAs and 135 novel miRNAs, of them 134 were differentially expressed between iL₃ and pF (77 conserved and 57 unannotated miRNAs), 251 miRNAs were shared by iL₃ and pF, 10 were specific in iL₃ and 4 were specific in pF. The prediction results of differentially expressed miRNA target genes showed that of the 57 unannotated miRNA, 12 miRNA with fold chang > 2 and TPM > 1 000 have 248 target genes homologous with Caenorhabditis elegans. 35.08% (87/248) of the target genes were related to development, while the rest were related to stress, exercise and molting. Most of the highly expressed unannotated miRNA in pF target SSTP_0000114700. GO analysis of differentially expressed miRNA target genes showed that a total of 5 595 target genes were enriched, among which the top 5 enriched components were membrane components (1 821), nucleic acid binding (561), nucleus (450), proteolysis (365) and signal transduction (284). The qRT-PCR analysis showed that the log₂(fold change) of sst-miR-86-5p, sst-84-5p, sst-novel-104, sst-miR-92-3p, sst-miR-34a-3p, sst-miR-81a-5p, sst-miR-1-3p, sst-novel-108, sst-miR-124-5p, sst-miR-50-3p, sst-novel-51 were -2.12, 6.39, 4.46, -3.69, -3.69, 2.34, -2.48, -2.41, -2.30, 2.25, -3.32; and the log₂(fold change) obtained from transcriptome analysis were -3.05, 4.98, 4.07, -4.9, -3.66, 0.98, -3.79, -2.61, -0.99, 0.63, -1.55, respectively. The upregulation and downregulation trends obtained from qRT-PCR and transcriptome analysis are consistent. Conclusion The miRNA expression profiles and differentially expressed miRNAs of S. stercoralis infective third-stage larvae and parasitic female adult are identified. The differentially expressed miRNAs are associated with the growth, development, and reproductive function of S. stercoralis.

Establishment and application of PCR-CRISPR/Cas12a-based detection method for Clonorchis sinensis metacercaria

XU Yin, LIU Ting, XU Hui, ZENG Xiaojun, LAN Weiming, GONG Zhihong, DAI Kunjiao, QIU Tingting, HAO Xian, XIE Shuying

2023, 41(4):  421-426.  doi:10.12140/j.issn.1000-7423.2023.04.004

Asbtract ( )   HTML ( )   PDF (1948KB) ( )  

Figures and Tables | References | Related Articles | Metrics

Objective To develop a method for rapid detection of Clonorchis sinensis metacercaria based on the polymerase chain reaction (PCR) technology combined with clustered regularly interspaced short palindromic repeats-associated 12a protein (CRISPR/Cas12a). Methods PCR amplification was performed using internal transcribed spacer (ITS) primers with the C. sinensis genomic DNA as template. The CRISPR reaction system was prepared by mixing PCR amplification product, Cas12a protein, CRISPR-derived RNA (crRNA), and single stranded DNA (ssDNA) reporter molecule, subsequently, the mixture was observed under ultraviolet light to verify the feasibility of the detection system, in which the ssDNA reporting molecule concentration was optimized by designing 5 gradients (100, 200, 300, 400, 500 nmol/L), and the Cas12a/crRNA ratio was optimized by formulating 5 proportional gradients (1.0 : 1, 1.5 : 1, 2.0 : 1, 2.5 : 1, 3.0 : 1) while maintaining crRNA concentration at 50 nmol/L, while the fluorescence signals were detected by using a fluorescence quantitative PCR instrument. The sensitivity of the detection system was evaluated through PCR and CRISPR reaction using the recombinant plasmid diluted to 10 different concentrations (10^-2-10⁷ copies/μl) to examine the reacted fluorescence signal. In addition, the genomic DNA extracted from C. sinensis metacercaria, Metorchis orientalis metacercaria, Spirometra erinaceieuropaei plerocercoid, Anisakis pegreffii and Taenia asiatica used as the template to assess the specificity of the detection system through PCR and CRISPR reaction, judged by checking the fluorescence signal. Samples of small-sized freshwater fish flesh from the clonorchiasis endemic area was examined by microscopy on crushed smears, from them 8 types of mixed infection were separately selected: the samples infected with mixed metacercaria of C. sinensis, M. orientalis and unknown species, metacercaria of C. sinensis and M. orientalis, C. sinensis and unknown species, M. orientalis and unknown species, C. sinensis alone, M. orientalis alone, metacercaria of unknown species alone, and none cercaria infection. These selected sample were used to extract DNA respectively for performing PCR-CRISPR/Cas12a detection, of which the reaction results were observed under ultraviolet light. The data was analyzed by using software GraphPad Prism 9.0 to perform one-way ANOVA. The comparison between groups was analyzed using graph based multiple test. Results The complete PCR-CRISPR/Cas12a detection system was able to generate fluorescence signal under ultraviolet light. When the reported molecular concentration of ssDNA is 400 nmol/L, the average fluorescence value of the reaction system is (40 786 ± 1 758) AU, which is higher than (32 029 ± 2 651) AU at the level of 300 nmol/L (P < 0.05), but no statistically significant difference (P > 0.05) was found when compared with the vlaue of (42 698 ± 4 260) AU at the level of 500 nmol/L. Therefore, the reported molecular concentration of ssDNA was 400 nmol/L. When the Cas12a/crRNA ratio was 2.0 : 1, the average fluorescence value of the reaction system was (48 950 ± 3 723) AU which was higher than (37 700 ± 3 887) AU with the ratio of 1.5 : 1 (P < 0.05) and no statistically significant difference (P > 0.05) compared to (55 630 ± 3 110) AU with the ratio of 2.5 : 1. Thus, the Cas12a/crRNA ratio was selected as 2.0 : 1. When the concentration of the recombinant plasmid was 10^-1 copies/μl, the fluorescence value of the reaction system was (39 336 ± 7 231) AU, which showed a statistically significant difference compared to the negative control (2 216 ± 743) AU (P < 0.05); when the concentration decreased to 10^-2 copies/μl, the average fluorescence value was (5 451 ± 1 957) AU, which had no statistically significant difference with that of the negative control (P > 0.05). The results of PCR showed that ITS primers could simultaneously amplify 5 kinds of genomic DNA of C. sinensis, M. orientalis, S. erinaceieuropaei, A. pegreffii, and T. asiatica. The CRISPR detection results of the same amplification products showed that only the reaction system using the genomic DNA of C. sinensis as a template generated fluorescence signals under ultraviolet light. The results of PCR-CRISPR/Cas12a detection showed that fluorescence signals were observed in four tubes containing C. sinensis metacercariae in the reaction system of 8 types of fish sample infected with the different metacercaria. Conclusion This study established a PCR-CRISPR/Cas12a method for detection of C. sinensis metacercaria, and the method is highly sensitive, specific and modularized, with significant potential for field application.

Trematode egg internal transcribed space-2 sequence based analysis for diagnosis of Opisthorchis viverrin and Opisthorchis sinensis infections

CAI Zhanghuang, ZHANG Zhiping, ZHUO Mingying, GUO Liping, FU Zhihui, LIU Yiruo

2023, 41(4):  427-433.  doi:10.12140/j.issn.1000-7423.2023.04.005

Asbtract ( )   HTML ( )   PDF (5453KB) ( )  

Figures and Tables | References | Related Articles | Metrics

Objective To explore the diagnosis and differentiation between Opisthorchis viverrin and Opisthorchis sinensis infection through morphological examination of small trematode eggs, egg internal transcribed space 2 (ITS-2) sequence amplification and epidemiological investigation. Methods The epidemiological data of 6 trematode egg positive cases diagnosed by the modified Kato-Katz thick smear method were collected, and positive fecal samples were collected for 3 consecutive days for collection of eggs by washing with water and sedimentation prior to microscopy. The eggs DNA was extracted, amplified and sequenced using universal primers for the rDNA ITS-2 region. The product sequences were aligned in the NCBI database to identify the species, and a phylogenetic tree based on ITS-2 sequences was constructed using the neighbor-joining method. The diagnosis was reached based on data analysis of the egg morphology, gene sequence alignment and epidemiological findings. Results Microscopically, the eggs in the fecal samples of cases 1 to 3 were similar to those of cases 4 to 6 in the shell, morphology, color, egg cover, acromion, hair and small wart at the bottom of the eggs. The egg length, width and aspect ratio in the fecal samples from cases 1 to 3 were (26.94 ± 2.28) μm, (14.43 ± 1.22) μm, 1.88 ± 0.18. Compared with (29.70 ± 1.21) μm, (14.36 ± 0.70) μm and 2.07 ± 0.14 in cases 4 to 6. The difference between length and aspect ratio was significant (t = 4.318, 4.816; P < 0.01). The former is shorter and wider than the latter, The Forde-Meleni index (FMI) of the two eggs was 5 676.69 ± 1 317.97 and 6 134.25 ± 626.27 (t = 0.428, P > 0.05). In cases 1 to 3, PCR and sequencing results showed that the ITS-2 fragments were 355, 362 and 359 bp, respectively, with the sequence identity of 99.14%, 98.86% and 99.39% to C. sinensis (GenBank No. MK886663). In cases 4 to 6, ITS-2 gene fragments were 394, 395 and 396 bp, respectively, with the sequence identity of 100%, 99.49% and 100% to O. viverrin (GenBank No. OK103575.1). The phylogenetic tree showed that the ITS-2 sequence of the eggs in cases 1 to 3 clustered on a branch with C. muscidiode and the ITS-2 sequence of the eggs in cases 4 to 6 clustered on a branch with C. sinensis. The epidemiological investigation results showed that cases 1 to 3 were all foreign migrant women from Cambodia who had the habit of eating pickled freshwater fish since childhood; cases 4 to 6 were all local farmers. Combining the morphological, molecular biology and epidemiological findings, cases 1 to 3 were diagnosed as imported testicular fluke infection, and cases 4 to 6 were C. sinensis infection. Conclusion Combining morphological detection as well as epidemiological investigation and analysis, trematode egg ITS-2 amplification and sequencing could effectively differentiate C. sinensis and O. viverrin in infection.

Applied research of serum IgG sialylation modification in the diagnosis of hepatic echinococcosis

FENG Xiaoxiao, BAIMA Yangjin, ZHANG Ting, LU Haojie, WEI Liming

2023, 41(4):  434-439.  doi:10.12140/j.issn.1000-7423.2023.04.006

Asbtract ( )   HTML ( )   PDF (1706KB) ( )  

Figures and Tables | References | Related Articles | Metrics

Objective To assess the application value of serum IgG sialylation in diagnosis and type-differention of echinococcosis. Methods Blood samples of 98 hepatic cystic echinococcosis patients and 29 hepatic alveolar echinococcosis patients were collected from Tibet Autonomous Region, Gansu Province, Sichuan Province and Qinghai Province from 2014 to 2016, in addition, 30 blood samples were from hepatocellular carcinoma patients hospitalized in the Affiliated Hospital of Guangxi Medical University during January to December 2017, and 22 from examinees of the Physical Examination Department of Guangxi Medical University collected. With the serum samples collected, sialylation modification of IgG was quantitatively analyzed using fluorescence chromatography-based glycomic technology. IgG was isolated and purified from 50 μl serum using protein G agarose purification kit. Reductive alkylation reaction was performed for 50 μg purified IgG in 25 mmol/L dithiothreitol solution and 25 mmol/L dithiothreitol solution, respectively, followed by adding 1 μl N-glycosidase buffer for enzymolysis at 37 ℃ for 18 h. Following enrichment with graphitized carbon column the glycans were freeze-dried, and then dissolved in a mixed solution of dimethyl sulfoxide and glacial acetic acid at a proportion ratio 7 : 3 (v/v), in which 50 μl each of 5 mmol/L 2-aminobenzamide solution and 10 mmol/L sodium cyanoborohydride was quickly added, respectively, to carry on the labelling reaction at 65 ℃ for 2 h, and purification of the reactants afterwards with graphitized carbon column. Upon the final step, the labelled N-glycans were analyzed by fluorescence chromatography to define monosialylation, bisialylation and total sialylation modification using a Waters Acquity UPLC（H-Class）system for hydrophilic chromatographic column separation, and the resultants were detected by fluorescence detector with the setting of excitation and emission wavelengths at 330 and 420 nm, respectively. Empower 3 software was used for data collection and processing, while SPSS 25 for statistical analysis. Inter-group data were compared by D'Agostino & Pearson omnibus normal distribution test (P₁ value) and one-way ANOVA test (P₂ value). Pairwise comparison was performed using Tukey's multiple comparisons test (P₃ value). Results A total of 21 peaks were detected in the chromatogram of IgG glycans, including five kinds of mono-sialylation modified glycans: FA2BG1S1 (17.79 min), A2G2S1 (19.02 min), A2BG2S1 (20.02 min), FA2G2S1 (20.27 min) and FA2BG2S1 (21.13 min), and four kinds of di-sialylation modified glycans, namely A2G2S2 (22.70 min), A2BG2S2 (23.29 min), FA2G2S2 (23.78 min) and FA2BG2S2 (24.26 min). All kinds of sialylation modifications in serum samples of each group had Gaussian distribution characteristics (P₁ > 0.1). The content of mono-sialylation was (14.15 ± 2.89)% and (10.24 ± 2.97)%, and the content of di-sialylation was (3.02 ± 0.98)% and (1.92 ± 0.65)%, respectively, in hepatic cystic echinococcosis and hepatic alveolar echinococcosis groups. The content of total sialylation in hepatic cystic echinococcosis and hepatic alveolar echinococcosis groups was (17.25 ± 3.69)% and (12.27 ± 3.65)%, respectively. The above sialylation of IgG glycan from hepatic cystic echinococcosis and hepatic alveolar echinococcosis group were lower than those of the healthy control group with (16.55 ± 2.95)%, (3.71 ± 1.04)% and (20.26 ± 3.79)% (F = 23.70, 11.14, 22.98, P₂ < 0.000 1). There was a statistically significant difference in the content of the mono-sialylation, di-sialylation and total sialylation between the hepatic cystic echinococcosis group and the hepatic alveolar echinococcosis group (t = 6.352, 4.701, 6.392, P₃ < 0.000 1). The potential diagnostic performance of the sialylation of IgG glycans was also evaluated using ROC curves. Between hepatic cystic echinococcosis and hepatic alveolar echinococcosis groups, it demonstrated an AUC value of 0.829 (95%CI: 0.738 to 0.921) with a sensitivity of 82.7% and a specificity of 87.7%. The mono-sialylation, di-sialylation and total sialylation of IgG glycans in hepatocellular carcinoma group is (15.09 ± 2.68)%, (3.28 ± 1.30)% and (18.37 ± 3.69)%, respectively, which shown statistically significant difference with hepatocellular carcinoma group (t = 6.587, 4.318, 6.372, P₃ < 0.000 1). ROC analysis showed the model has a differential diagnosis of hepatic alveolar echinococcosis group and hepatocellular carcinoma group response (AUC = 0.840) with a sensitivity of 64.00% and a specificity of 100%. Conclusion The present study found that the serum IgG sialylation modification may be related to the pathogenesis of echinococcosis, and possess certain application value in diagnosis of liver echinococcosis, and in the study on differentiation of hepatic cystic echinococcosis from hepatic alveolar echinococcosis, and liver tumor from hepatic alveolar echinococcosis.

Experimental observation on the changes of hepatic stellate cells stimulated in vitro with tissue protein of Echinococcus multilocularis

CAO Deping, LI Jiajing, SONG Mengwei, MO Gang

2023, 41(4):  440-445.  doi:10.12140/j.issn.1000-7423.2023.04.007

Asbtract ( )   HTML ( )   PDF (5483KB) ( )  

Figures and Tables | References | Related Articles | Metrics

Objective To observe the changes in morphology and intracellular lipid droplets and ɑ-smooth muscle actin (α-SMA) expressed in the hepatic stellate cell (HSC)-LX2 stimulated with Echinococcus multilocularis tissue protein in vitro. Methods The E. multilocularis tissue was collected from infected mice under aseptic conditions, with which the tissue protein was extracted by ultrasonic-splitting. The protein extractant was added into HSC-LX2 culture in vitro at a final concentration of 0.125 mg/ml to stimulate for 24, 48, 72, 96 and 120 h, then the cells were stained with oil red O to observe the distribution of intracellular lipid droplets under light and fluorescence microscopes. The total protein of HSC-LX2 cells was extracted after stimulation for 48, 72, 96 and 120 h to detect the changes of relative expression of α-SMA using Western blotting. Results Under the light microscope, the morphology of HSC-LX2 changed gradually from polygonal to long spindle shape, and with prolonged stimulation with the E. multilocularis tissue protein, the number of intracellular lipid droplets increased, and lipid droplets distributed around the nuclei. The color of lipid droplets gradually changed from light yellow to bright red. After 120 h, almost all HSC-LX2 became long spindle-shaped. The number and volume of lipid droplets decreased, and the color changed from red to yellow. Under fluorescence microscopy, red fluorescent lipid droplets could be seen surrounding the nucleus, and the fluorescence signal did not decrease with the increase of irradiation times. Western blotting showed that the relative expression α-SMA in HSC-LX2 gradually increased with the prolongation of stimulation time, which peaked at 4.846 ± 0.741 after 96 hours and decreased to 2.420 ± 0.349 after 120 hours, but both were higher than that of the control group (t = 8.988, 7.045, both P < 0.01). Conclusion The E. multilocularis tissue protein can promote the activation of HSC-LX2 in vitro, inducing changes in cell morphology, the number and size of intracellular lipid droplets being raised first and then reduced, and the expression of intracellular α-SMA protein showing a similar changing trend as well.

Study on the inhibitory effect of artemether liposome on Toxoplasma gondii proliferation in vitro

ZHAO Ziqi, LV Fangli

2023, 41(4):  446-451.  doi:10.12140/j.issn.1000-7423.2023.04.008

Asbtract ( )   HTML ( )   PDF (2454KB) ( )  

Figures and Tables | References | Related Articles | Metrics

Objective To investigate the inhibitory effect of artemether liposome (ARM-Lip) on Toxoplasma gondii tachyzoite proliferation in vitro. Methods Human cervical cancer cells (Hela cells) were cultured in 96-well flat bottom cell culture plate, with 100 μl cell culture medium in each well containing 1 × 10⁴ Hela cells. Setting ARM-Lip groups with different concentrations (3.125, 6.250, 12.500, 25.000, 50.000, 100.000, 200.000, 400.000 and 800.000 μmol/L), a control group (only containing 1 × 10⁴ human cervical cancer cells), and a blank group (only containing culture medium). A total of 100 μl corresponding drugs were added to each well in the ARM-Lip groups after 24 h, and 10 μl CCK-8 solution was added to each well after 48 h. A microplate reader was used to detect the absorbance at 450 nm wavelength after 1 h (A-value) to calculate the cell survival rate and 50% inhibitory concentration (IC₅₀) of ARM-Lip. Hela cells were infected with T. gondii green fluorescence-expressing RH (RH-GFP) straintachyzoites, and co-cultivated with different concentrations of ARM-Lip to screen safe concentrations of ARM-Lip those rendered higher than 50% cell survival rate. Through setting of 400 μmol/L sulfadiazine (SDZ) group, and untreated group, the proliferations of tachyzoites were observed under fluorescence microscope after cultured for 24, 48, and 72 h. The average fluorescence gray scale values of each group at 48 h were measured and the group having optimal concentration of ARM-Lip was selected. After Giemsa staining, the parasitophorous vacuoles in Hela cells and the growth of T. gondii tachyzoites were observed and counted. One-way ANOVA was used for inter-group comparison, and the least significant difference (LSD) test was used for pairwise comparison. Results ARM-Lip at a concentration of 25 μmol/L or lower had little toxic effect on Hela cells, and its IC₅₀ was 285.1 μmol/L. Treated with 100.000, 200.000, 400.000, and 800.000 μmol/L ARM-Lip, the cell survival rate was 89.03%, 76.30%, 42.12% and 27.85%, respectively. ARM-Lip concentrations of 6.250, 12.500, 25.000, 50.000, and 100.000 μmol/L were selected to conduct subsequent experiments. Under a fluorescence microscope, a large amount of T. gondii RH-GFP strain tachyzoites were observed in the untreated group, with normal morphology and high intensity of green fluorescence. As the concentration of ARM-Lip was increased, the intensity of green fluorescence was gradually decreased. The optimal concentration for ARM-Lip to inhibit T. gondii growth in vitro was 100.000 μmol/L and the optimal action time was 48 h. After cultured for 48 h, the average gray scale values of the untreated group, ARM-Lip group (100.000 μmol/L) and SDZ group were 89.92 ± 1.12, 35.74 ± 1.55 and 7.34 ± 0.60 (F = 7 916.301, P < 0.01). Compared with the untreated control group, the average grey scale values of the ARM-Lip group and SDZ group were significantly decreased (t = 81.247, 123.831, both P < 0.01); compared with the ARM-Lip group, the average gray scale value of the SDZ group was significantly decreased (t = -42.584, P < 0.01). After Giemsa staining, the numbers of parasitophorous vacuoles of untreated group, ARM-Lip group, and SDZ group were 28.667 ± 2.658, 16.667 ± 0.817 and 12.667 ± 1.211 (F = 135.652, P < 0.01); the numbers T. gondii tachyzoites in each group were 176.167 ± 13.273, 105.333 ± 7.789 and 70.167 ± 5.947 (F = 192.763, P < 0.01). Compared with the untreated control group, the numbers of parasitophorous vacuoles of ARM-Lip group and SDZ group were significantly decreased (t = 0.083, 0.063, both P < 0.01), and the numbers of T. gondii tachyzoites of ARM-Lip group and SDZ group were significantly decreased (t = 0.014, 0.009, both P < 0.01). The numbers of parasitophorous vacuoles and T. gondii tachyzoites in the SDZ group were significantly fewer than in the ARM-Lip group (t = -0.500, -0.057, both P < 0.01). Conclusion ARM-Lip have relatively good safety and can inhibit the proliferation of T. gondii tachyzoites in vitro, whereas its effectiveness remains insufficient when compared with SDZ.

Myotis fimbriatus ectoparasite infection and the morphological and phylogenetic analysis of Nycteribiidae in Dali, Yunnan Province

YANG Jinting, HUANG Xiaobin, WANG Yujuan, GUO Xianguo, ZHANG Xianzheng, YANG Huijuan, ZHENG Xiaoyan

2023, 41(4):  452-458.  doi:10.12140/j.issn.1000-7423.2023.04.009

Asbtract ( )   HTML ( )   PDF (1974KB) ( )  

Figures and Tables | References | Related Articles | Metrics

Objective To analyze the prevalence of ectoparasites on the surface of Myotis fimbriatus in Dali, Yunnan Province, and describe the morphological characteristics of two Nycteribiidae, exploring their phylogenetic relationship. Methods In July 2022, M. fimbriatus were captured in Jianchuan County, Dali Prefecture, Yunnan Province. The ectoparasites were collected and identified. The constituent ratio, infection rate, mean abundance, and mean intensity were calculated for each ectoparasite. Dominant species were determined using the dominance index (Y). Their spatial distribution types were determined using the dispersion coefficient (C), I index, patchiness index (m*/m), and K index. The association coefficient (V) was used to analyze the interaction between dominant species. The morphological characteristics of the two Nycteribiidae were described. Genomic DNA was extracted from Nycteribiidae, and the mitochondrial cytochrome C oxidase subunit 1 (cox1), cytochrome B (cytb) and 16S ribosomal RNA (16S rRNA) was amplified by PCR and sequenced, then the sequences in tandem were used to construct the phylogenetic tree using the maximum likelihood method in MEGA 11. Results A total of 72 M. fimbriatus (24 males, 48 females) were captured, and the ectoparasites infection rate was 97.3% (70/72). A total of 1 745 ectoparasites were collected, including 1 463 gamasid mites belonging to 7 species in 5 genera and 2 families, namely Spinturnix myoti, Spinturnix kolenatii, Steatonyssus periblepharus, Macronyssus zhijinensis, Choronyssus scutatus, Eyndhovenia euryalis, and Macronyssus coreanus. Additionally, 282 Nycteribiidae were collected, which belongs to 2 species in 2 genera, namely Nycteribia formosana and Penicillidia dufourii. No other ectoparasites were detected. The infection rate, mean abundance, and mean intensity of gamasid mites are 94.4% (68/72), 20.32 and 21.53, respectively, all of which are higher than those of the Nycteribiidae [91.7% (66/72), 3.92, 4.27] (Fisher's exact test, P < 0.05; U = 700, 424; both P < 0.01). S. myoti, S. periblepharus and M. zhijinensis are the dominant species (Y = 0.30, 0.23, 0.22). The dominant species, including S. myoti (C = 1.98, I = 0.98, m*/m = 1.18, K = 5.55), M. zhijinensis (C = 9.43, I = 8.43, m*/m = 2.15, K = 0.87) and S. periblepharus (C = 6.50, I = 5.50, m*/m = 2.02, K = 0.98), exhibit an aggregated distribution among different individuals of M. fimbriatus. There is a positive correlation relationship between S. myoti and S. periblepharus (V = 0.33, P < 0.05), between S. myoti and M. zhijinensis (V = 0.34, P < 0.05), and between S. periblepharus and M. zhijinensis (V = 0.72, P < 0.01). N. formosana has a body length of about 3 mm. Its dorsal plate Ⅰ is wide and round, with about 25 long bristles at the tip. There are many long hairs between the dorsal plates Ⅱ and Ⅵ, and the dorsal plate Ⅵ has 6 long bristles and 10 long spikes. P. dufourii has a body length of 3.5-4.0 mm. The dorsal plate Ⅰ is elongated and wide, with a row of short bristles along the edges. The dorsal plate Ⅱ is approximately three times longer than it is width. At the back of the dorsal plate Ⅱ, there are two protrusions with concave surfaces. The posterior edge of the protrusions is sharp and has pigmented deposits. The cox1, cytb and 16S rRNA sequences of N. formosana were 875, 338 and 455 bp, respectively. The GenBank accession numbers are OQ127648, OQ134944, and OQ127330. The cox1, cytb and 16S rRNA sequences of P. dufourii were 832, 332 and 451 bp, respectively. The GenBank accession numbers are OQ119629, OQ129602, and OQ119632. The results of the phylogenetic tree analysis show that N. formosana and the N. parvula are clustered together in the same branch, and then they are clustered with P. dufourii. Conclusion The infection rate of ectoparasites on M. fimbriatus in Yunnan Dali is high, mainly with the presence of gamasid mites and Nycteribiidae, among them, N. formosana shows the closest genetic relationship to its congeneric N. parvula, while P. dufourii is related closer to N. formosana and N. parvula.

Clinical characteristics of patients with intestinal Diphyllobothrium tapeworm infection

LI Xiaoli, LI Shaogang, WU Zhaoyong

2023, 41(4):  459-463.  doi:10.12140/j.issn.1000-7423.2023.04.010

Asbtract ( )   HTML ( )   PDF (1522KB) ( )  

Figures and Tables | References | Related Articles | Metrics

Objective To summarize and analyze the characteristics of clinical manifestation and laboratory indicators of patients with intestinal infection of Diphyllobothrium tapeworm. Methods The medical record, including gender, age, epidemiological history (residence, history of overseas traveling and dietary habits), clinical manifestation, laboratory examinations when admitted (blood routing test, liver function, fecal examination for parasite eggs, and parasite species identification), treatment record and prognosis were collected from 18 diphyllobothriasis cases diagnosed at the Beijing Friendship Hospital, Capital Medical University from January 2009 to July 2022. Information on patients' clinical features and the changes in laboratory findings were summarized and analyzed. Results The 18 cases included 9 male and 9 female respectively, of them the age ranged from 8 to 47 years with an average of (28.88 ± 11.57) years. Epidemiological inquiry indicated that 12 cases were locals from Beijing, while 6 patients were from other provinces, among them 7 patients had overseas traveling history, and all patients had history of consuming raw/undercooked fish meat and beef. The clinical manifestation of the 18 cases were abdominal pain and distension in 6 cases, and dizziness in 2 cases, whereas anal pruritus and body weight loss were not seen, and 10 were asymptomatic. The inflammatory indicator of leukocyte count, C-reactive protein, erythrocyte sedimentation rate, anemia indicator of erythrocyte and hemoglobin, eosinophils count, and liver function showed no abnormal changes. Fecal examination found Diphyllobothrium tapeworm eggs in the samples from 8 cases, and proglottids from all cases. Deworming regime with Chinese medicine areca-pumpkin seed cured all patients. Conclusion Most of the cases infected with Diphyllobothrium tapeworm are asymptomatic or showed only mild gastrointestinal symptoms, with no significant abnormal changes in blood routine test and liver function examination. Early diagnosis could be made by fecal examination etiologically and epidemiological inquiry.

Survey on Angiostrongylus cantonensis infection in common snails in Yunnan Province from 2017 to 2022

ZHANG Juan, TAO Hong, LI Yanzhong, WANG Tingting, YANG Jingjing, XIANG Yibin, CHEN Yishan, ZHOU Xiaomei

2023, 41(4):  464-469.  doi:10.12140/j.issn.1000-7423.2023.04.011

Asbtract ( )   HTML ( )   PDF (1310KB) ( )  

Figures and Tables | References | Related Articles | Metrics

Objective To investigate the prevalence and characteristics of Angiostrongylus cantonensis infection in snails in parts of Yunnan Province. Methods Fresh snails were collected in the counties (districts) of 16 prefectures (cities) in Yunnan Province from public places, wild environment, farmers' markets, and the food material sold in restaurants, during April to October every year in 2017—2022. Each snail sample weights approximately 1 kg. Suspected larvae were observed and identified for species under optical microscope by enzyme digestion method, and the third-stage larvae of A. cantonensis were detected. Results A total of 2 402 samples of snails were collected and examined from 2017 to 2022, among them, 217 samples were found positive, with a total positive rate of 9.03%. The positive rate of snails collected in different years showed a decreasing trend year by year. The highest positive rate was seen in 2018 (15.05%) and the lowest rate was seen in 2022(3.36%) (χ² = 55.427, P < 0.01). Among different snails, the Achatina folia infection rate was the highest, reaching 47.06% (88/187), followed by Pomacea canaliculata (7.89%, 50/634), the infection rates in Bellamya aeruginosa and Cipangopaludina cahayensis were 4.57% (54/1 181) and 6.25% (25/400), respectively (χ² = 362.406, P < 0.01). A. cantonensis was detected in snail samples from 15 cities in the province except for Nujiang prefecture, and the positive rate of A. cantonensis in samples collected from Honghe prefecture was the highest, with 19.75% (63/319), followed by Xishuangbanna (19.69%, 51/259), Pu'er (9.52%, 12/126), Yuxi (9.09%, 16/176), etc (χ² = 123.135, P < 0.01). In different sampling environments, the snail-positive rate was the highest in public places such as parks, which was 33.33% (28/84), in field environments such as fields and rivers, which was 17.61% (50/284), in farmers' markets, which was 6.94% (130/1 872), and in restaurants, which was 5.56 (χ² = 98.076, P < 0.01). Among the snail samples collected in different months, the A. cantonensis positive rate was the highest in June, accounting for 50.00% (14/28). The lowest was 4.36% (18/413) in May (χ² = 117.157, P < 0.01). Conclusion A. cantonensis infection was found in fresh snails collected from wild environment and commercial markets in Yunnan Province among which A. fulica and P. canaliculata showed higher infection rate.

Investigation on visceral leishmaniasis in domestic dogs and sandfly density in epidemic area in Shanxi Province from 2021 to 2022

ZHENG Yuhua, TIE Ping, BAI Yongfei, YAN Changfu, WANG Ting, WANG Jingying, TIAN Xiaodong, DAI Peifang

2023, 41(4):  470-475.  doi:10.12140/j.issn.1000-7423.2023.04.012

Asbtract ( )   HTML ( )   PDF (1381KB) ( )  

Figures and Tables | References | Related Articles | Metrics

Objective To investigate the population density of domestic dogs, the prevalence of Leishmania infection in domestic dogs, and the density of sandfy in the visceral leishmaniasis (VL) endemic areas in Shanxi Province. Methods The investigation was carried out in 2 to 6 villages randomly selected from suburbs of Yangquan City, Wuxiang County of Changzhi City, and Xiangning County of Linfen City in Shanxi Province from 2021 to 2022. The domestic dog population density was obtained through household survey in the survey areas. The sample size of domestic dogs was estimated based on the previous survey results. Venous blood sampls were collected from the selected dogs and their detailed information were recorded. The canine rk39 immunochromatographic test strip was used to detect serum anti-Leishmania antibodies in the sampled dogs. From each of 3 survey areas (county), 2 natural villages were selected, which had local case reports or no case reports over the last five years. The sandfy density was investigated by light trap method in 2 farmer's households and livestock barns, respectively, selected from each village. For calculation of mean sandfy density, the survey was conducted once at early and latter part of every month during May to September yearly. The VL case report data from Shanxi endemic areas were collected through the Population-based Healthcare System. Statistical analysis was performed using SPSS 25.0, and the chi-square test was used to compare the positive rate of anti-Leishmania antibody in domestic dogs in different years in 3 survey areas (counties). Results The average domestic dogs density in the 3 survey areas (counties) in Shanxi Province from 2021 to 2022 was 0.09 dogs per household. The mean domestic dog density in the outer suburbs of Yangquan City, Wuxiang County of Changzhi City and Xiangning County of Linfen City were 0.07, 0.16, and 0.13 dogs per household, respectively. A total of 1 149 blood samples were collected, of which 73 were positive for anti-Leishmania antibodies, resulting in a positivity rate of 6.4% (73/1 149). The positivity rate of anti-Leishmania antibodies in male dogs was 7.6% (41/542), and in female dogs the positivity rate was 5.1% (31/607), with no significant difference in the positivity rate of anti-Leishmania antibodies between sexes (χ² = 2.94, P > 0.05). Among the 1 093 dogs whose age was known, 75.6% (826/1 093) were aged four or younger. The positivity rate of anti-Leishmania antibodies in dogs aged five years was the highest (8.2%, 7/85), and there was no significant difference in the positivity rate of anti-Leishmania antibodies among different age groups (P > 0.05). A total of 25 537 sandflies were captured, of which 23 012 were females (accounting for 90.1%), and 2 525 were males (accounting for 9.9%). The mean density of sandflies was 26.1 flies per lamp-night, and the mean density of sandflies in the outer suburbs of Yangquan City, Wuxiang County of Changzhi City and Xiangning County of Linfen City were 3.9, 36.6 and 45.9 flies per lamp-night, respectively. A total of 57 cases of visceral leishmaniasis were reported in 3 districts (counties) of Shanxi Province from 2021 to 2022, with 45, 3 and 9 cases reported from the outer suburbs of Yangquan City, Wuxiang County of Changzhi City and Xiangning County of Linfen City, respectively. Conclusion In three survey areas (county) of Shanxi Province, the sandfly density and the infection rate of Leishmania in domestic dogs are relatively high.

Enterocytozoon bieneusi infection in dairy cows and its genotype identification in Yinchuan area of Ningxia Province

LI Yuqiong, YU Youli, GAO Junrong, LIU Yunyun, LI Hongbing, NIU Xiaohao

2023, 41(4):  476-479.  doi:10.12140/j.issn.1000-7423.2023.04.013

Asbtract ( )   HTML ( )   PDF (1616KB) ( )  

Figures and Tables | References | Related Articles | Metrics

Objective To clarify the prevalence and main genotypes of Enterocytozoon bieneusi infection in dairy cows in Yinchuan area of Ningxia Province. Methods From August 2021 to August 2022, cow fresh fecal samples (including diarrheal and non-diarrheal samples) were collected from 6 scaled dairy farms in Yinchuan City and the surrounding counties. The genomic DNA was extracted from the fecal samples, and nested PCR was performed using E. bieneusi internal transcriptional spacer (ITS) primers. The PCR amplicaons were seqeuced, and sequence alignment was conducted using BLAST to identify the genotype of E. bieneusi. SPSS 25.0 software was used for statistical analysis, and χ² test for comparing the differences in positive rates between the cows of different months of age. Results A total of 772 fresh cow fecal samples were collected. The E. bieneusi positive rate from nested PCR amplification was 8.7% (67/772). The target band size was approximately 400 bp. The E. bieneusi positive rate found in different farms ranged from 4.8% (6/124) to 11.5% (18/156), with statistically significant differences (χ² = 30.828, P < 0.05). The highest positive rate was found in cows aged 5-7 months (15.6%, 23/147) and the lowest was found in cows aged above 12 months (3.4%, 5/149). There was a statistically significant difference in the positive rate of E. bieneusi among cows of different months of age (χ² = 59.529, P < 0.05). The E. bieneusi positive rate in diarrheal samples was 10.6% (42/398), which was higher than that in non-diarrheal samples (6.7%, 25/374) (χ² = 3.419, P < 0.05). The 66 E. bieneusi sequences could be classified into 4 genotypes, including CHN4, BEB4, J and I. The accession obtained by GenBank were OR352930, OR352929, OR352932 and OR352931 respectively. 3 sequences were CHN4 (account for 4.5%), which showed a 98.4% identity to the sequence of CHN4 (HM992511) in G1. 18 sequences were BEB4 (account for 27.3%), which showed a 96.3% identity to the sequence of BEB4 (AY331008) in G2. 22 sequences were J (account for 33.3%), which showed a 93.1% identity to the sequence of J (AF135837) in G2. 23 sequences were I (account for 34.9%), which showed a 91.1% identity to the sequence of I (AF135836) in G2. The dominant E. bieneusi genotypes in cows varied with age, but BEB4, J and I presented in all age groups. The BEB4, J and I accounted for 88.1% (37/42) and 60.0% (15/25) of the total positive diarrheal samples and positive normal samples respectively, with statistically significant differences (χ² = 3.419, P < 0.05). Conclusion E. bieneusi infection was found in dariy cows in Yinchuan area of Ningxia Province, with the main genotypes BEB4, J and I.

REVIEWS

Research advances on the taxonomy of Anisakis, Anisakidae

CHEN Yao, ZHANG Benguang

2023, 41(4):  480-485.  doi:10.12140/j.issn.1000-7423.2023.04.014

Asbtract ( )   HTML ( )   PDF (1310KB) ( )  

References | Related Articles | Metrics

Anisakis is a zoonotic parasite that is widely distributed in marine areas around the world. The adult nematodes are mainly parasitic in dolphins, seals, whales and other marine mammals, and the larvae are mainly parasitic in fish, cephalopods and other small marineorganisms. If the larvae of Anisakis are eaten by humans, they can cause anisakiasis and harm health. The traditional morphological analysis method is unable to accurately identify anisakis, which hinders the in-depth study of Anisakis population. With the development and application of molecular biotechnology, many important achievements have been made in the study of Anisakis. In this paper, research progress on the life history, host preference, morphology of adults and larvae, molecular biology classification, global distribution of Anisakis nematodes was reviewed.

Research progress of antimalarial drugs

WEI Luanting, LI Runze, GUAN Liangchao, ZHANG Qianyu, LI Cheng, CAO Yaming, ZHAO Yan

2023, 41(4):  486-491.  doi:10.12140/j.issn.1000-7423.2023.04.015

Asbtract ( )   HTML ( )   PDF (1376KB) ( )  

Figures and Tables | References | Related Articles | Metrics

Antimalarial drug resistance presents the biggest challenge for treatment against malaria. Plasmodium parasites have developed different degrees of resistance to common traditional antimalarial drugs including artemisinin. Therefore, the improvement of traditional drugs and the research and development of new drugs are urgently needed. This paper discusses the malaria control strategies based on a systematic review of the resistance mechanisms against traditional antimalarial drugs, the improvement strategies and optimisation achievements based on traditional drugs, and the research advances of new antimalarial drugs.

Research progress on the immune evasion mechanism in Trichinella spiralis infection

ZHANG Xu, SUN Ximeng

2023, 41(4):  492-496.  doi:10.12140/j.issn.1000-7423.2023.04.016

Asbtract ( )   HTML ( )   PDF (1297KB) ( )  

References | Related Articles | Metrics

Trichinella spiralis infection can cause zoonotic trichinellosis that seriously endangers human health. During the entire T. spiralis life cycle stages, the parasite can manipulate the host's immune responses. The immune evasion mechanism, as a common natural selection mechanism, is an adaptation of pathogens to the immune system under natural selection. It enables the pathogens to relieve or alleviate the host's immune responses, avoid attacking from the immune system, and ensure that they can survive and reproduce. This paper reviewed the research advance of T. spiralis immune evasion mechanism in disturbing complement response, innate immune response, humoral immune response, and T cell immune response. Further research of the immune evasion mechanism of T. spiralis is of great significance for the prevention and control of trichinosis, the treatment of human autoimmune diseases, and allergic diseases.

SHORT COMMUNICATIONS

Cloning and prokaryotic expression of Theileria equi rhoptry neck protein 5 gene

LU Xing, WANG Shuiyi, CHEN Linjun, LIU Mingming, LIU Yutong, ZHU Huiru, JIANG Bingbing, DU Shaolei, BAYIN Chahan, LIU Dandan, ZHANG Wei

2023, 41(4):  497-501.  doi:10.12140/j.issn.1000-7423.2023.04.017

Asbtract ( )   HTML ( )   PDF (1774KB) ( )  

Figures and Tables | References | Related Articles | Metrics

The rhoptry-neck protein 5 (ron5) gene was PCR amplified from the Xinjiang isolates of Theileria equi. The prokaryotic expression vector of pGEX-4T-ron5 was constructed and transformed into Escherichia coli BL21 (DE3) for prokaryotic expression. The expression products was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Western blotting analysis was performed using glutathione S-transferase labeled rabbit monoclonal antibody (1:2 000) and horse serum infected with T. equi (1:100) as primary antibodies, and horseradish peroxidase labeled sheep anti-rabbit IgG and rabbit anti-horse IgG (1:5 000) as secondary antibodies. The ron5 of the Xinjiang isolate of T. equi was 1 434 bp, and the entry number obtained by GenBank was OP777492. Sequence comparison results showed that the nucleotide and amino acid sequences of ron5 of the Xinjiang isolate were the most consistent with those of T. equi WA strain (GenBank accession number XM 004833657), which were 99.6% and 99.2%, respectively. Phylogenetic tree analysis showed that the Xinjiang isolate and the T. equi WA strain were clustered on the same branch, with the most closely relation. SDS-PAGE analysis showed that the protein expression was the highest at 3 h induced by isopropyl thiogalactoside. The relative molecular mass (M_r) of RON5 is about 79 000, which is mainly expressed in the form of inclusion bodies. Western blotting results showed that RON5 could be recognized by glutathione S-transferase labeled antibody and horse serum infected with T. equi, with an obvious band at M_r 79 000. In this study, ron5 was successfully cloned, and the RON5 expressed had good antigenicity.

Study on early warning of high risk environment of Schistosoma japonicum infection by quantitative real-time PCR

LAN Weiming, XU Hui, XU Yin, QIU Tingting, XIE Shuying, DENG Fenglin, HU Shaoliang, LIU Huan, GUO Jiagang, ZENG Xiaojun

2023, 41(4):  502-505.  doi:10.12140/j.issn.1000-7423.2023.04.018

Asbtract ( )   HTML ( )   PDF (1274KB) ( )  

Figures and Tables | References | Related Articles | Metrics

To rapidly monitor and warn high-risk environments with Schistosoma japonicum, detecting S. japonicum DNA in the visceral tissues of sentinel rats at the early stage of infection by quantitative real-time PCR (qRT-PCR). 20 infected Oncomelania hupensis were placed in 25 ℃ warm water to release cercariae. The rats (KM species) were divided into three groups randomly, i.e. Group A with natural cercarial infection, Group B with quantitative cercarial infection and Group C without cercarial infection as a negative control group. In Group A, 6 rats were transferred into the cage with the water with cercariae for 1 h, which imitate the natural infection field. 36 rats in Group B were infected with cercariae (40 ± 5 per mouse) via the abdominal skin route. 9 rats in Group C were not infected with cercariae. The rats in Group A were anatomized randomly on day 1, 2 and 5 after infection. The rats in Group B were anatomized randomly on day 1, 2, 3, 5, 7 and 14 after infection, respectively. The rats in Group C were randomly selected to anatomized synchronously with the Group A and B. The tissues of abdominal skin, heart, lung and liver of each rat were collected after the dissection. Then the genomic DNA of the total collected tissues was extracted and tested for schistosome DNA through qRT-PCR. The Ct values were recorded to determine the infection rates in each group, especially for Group B, in which each rat's DNA Ct value of the lung tissue needed to record at different time period. As the qRT-PCR results showed, schistosome DNA in Group A was detected in lung on day 1, 2 and 5 after infection, and in liver on day 5 after infection. While in group B, the schistosome DNA was detected in the lung tissue on day 1, 2, 3, 5, 7 and 14 after infection, and later in the liver tissue on day 2, 3, 5, 7 and 14. It showed that the schistosome DNA in lung in group B can be detected from the 2nd to the 5th day after infection. The results suggested that the detection of schistosome DNA in the lung tissue by qRT-PCR at the early stage of sentinel rat infection has the value for early monitoring and warning of the high-risk environment with schistosome infection.

Clinical analysis of imported COVID-19 cases complicated with severe falciparum malaria

ZHANG Le, XIA Jiawei, LI Xiang, MA Zhongxu, JIANG Jianjie, TANG Yalin, LIU Shu, ZHANG Kaiyi

2023, 41(4):  506-509.  doi:10.12140/j.issn.1000-7423.2023.04.019

Asbtract ( )   HTML ( )   PDF (1757KB) ( )  

Figures and Tables | References | Related Articles | Metrics

The medical records of 4 cases of imported coronavirus disease 2019 (COVID-19) complicated with severe falciparum malaria diagnosed in Kunming Third People's Hospital in August 2022 were collected. The epidemiology, clinical manifestations, laboratory and imaging examinations, course of treatment and prognosis of 4 cases were analysed retrospectively. All the 4 cases were male, aged 40-54 years, and had a history of work and life in Africa. All cases had acute onset of symptoms, including fever (4 cases), chills (3 cases), shivering (3 cases), nausea and vomiting (3 cases), diarrhea (4 cases), and poor appetite (4 cases) as the main clinical manifestations. Headache, dizziness, unclear consciousness were reported in 2 cases, urinary incontinence in 1 case, muscle soreness in 2 cases, cough in 2 cases, phlegm in 1 case, and pharyngalgia in 1 case. All cases were positive for coronavirus. Nucleic acid test by throat swabs, and Plasmodium falciparum was found by microscopy examination of peripheral blood smear. All cases met the diagnostic criteria for severe malaria and were accompanied by abnormal liver function and severe hypoproteinemia, 2 cases of hyperbilirubinemia, 3 cases of dyslipidemia, 3 cases of abnormal blood picture involving the third system, increased calcitonin to varying degrees, 2 cases of lactic acid acidosis, and 1 case of hypoglycemia. Chest CT showed changes of viral pneumonia in 1 case. Antiviral drug and artesunate (intravenous injection 120 mg/time, once at 0, 12 and 24 hours, and once a day later for 7 consecutive days) were given, and different schemes of precise treatment were given based on the condition of the patient. All the patients were cured and discharged, and the follow-up was stable. It is suggested that the awareness of COVID-19 and malaria co-prevention should be established among the entry personnel in the high malaria epidemic areas abroad, to avoid serious cases and deaths.

Mechanism of a Chinese patent medicine in the treatment of liver fibrosis caused by infection of Clonorchis sinensis based on network pharmacology

LI Tianxing, ZHANG Jiaming, XU Chenxi, WANG Zige, GUO Jingjie, LI Shan

2023, 41(4):  510-515.  doi:10.12140/j.issn.1000-7423.2023.04.020

Asbtract ( )   HTML ( )   PDF (3613KB) ( )  

Figures and Tables | References | Related Articles | Metrics

To explore the pharmacological efficacy and molecular mechanism of FufangBiejiaRuanganPian (FBRP, a Chinese patent medicine) in the treatment of liver fibrosis caused by Clonorchis sinensis infection, we obtained the active compounds from the 11 Chinese ingredients of FBRP and their potential targets as well as clonorchiasis and liver fibrosis related targets through the TCMSP, BATMAN-TCM, Swiss and GeneCards databases. The intersection targets were screened for the construction of protein-protein interaction and drug-component-target-disease networks as well as analysis of GO and KEGG enrichments. The core targets were used for molecular docking with their corresponding compounds. The results showed 17 targets from the intersection of FBRP active components, clonorchiasis and liver fibrosis. The top 5 targets from degree ranking were cysteinyl aspartate-specific proteinase-3 (CASP3), cysteine x chemokine ligand 8 (CXCL8), tumor necrosis factor (TNF), proto-oncogene tyrosine-protein kinase src (SRC), and interleukin 10 (IL-10). The compound that showed the lowest binding energy for molecular docking with these 5 targets were respectively baicalein, wogonin, 24-ethylcholest-4-en-3-one, 24-ethylcholest-4-en-3-one, and 24-ethylcholest-4-en-3-one. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment and Gene Ontology (GO) function analysis suggested that FBRP could regulate cell apoptosis and proliferation, reduce the inflammatory response and oxidative stress injury, and inhibit the expression of pro-fibrotic factors. It could play a role in the treatment of liver fibrosis caused by infection of Clonorchis sinensis through the phosphatidylinositol 3 kinase-protein kinase B (PI3K-Akt), mitogen-activated protein kinase (MAPK) and janus kinase-signal transducer and activator of transcription (JAK-STAT) pathways.

Prevalence of parasitic infections in wild mice in Wuhu City, Anhui Province

WANG Feng, WU Fan, LI Linlin, HUANG Qingqing

2023, 41(4):  516-519.  doi:10.12140/j.issn.1000-7423.2023.04.021

Asbtract ( )   HTML ( )   PDF (1648KB) ( )  

Figures and Tables | References | Related Articles | Metrics

To understand the distribution of zoonotic parasite infections in the wild mice, the wild environment of 13 administrative villages in 9 counties (districts) of Wuhu City, Anhui Province was selected to capture wild mice in October, 2022. The mice species and gender were identified and age was determine according to the weight. Fecal samples were collected and eggs were detected by the modified Kato thick smear method (Kato-Katz method). The liver, mesentery and other tissues of the wild mice were collected, and parasite infection was detected by tissue biopsy or tablet microscopy. SPSS 26.0 software was used for statistical analysis. The χ² test was used to compare between groups. The results showed that the total infection rate of wild mice parasites in the 13 surveyed villages in 9 counties (districts) was 78.1% (125/160). Except for Sanshan District (2/2), the infection rate of wild mice in Jiujiang District (90.3%, 28/31) was the highest, and the difference was statistically significant compared with that in Economic Development District (52.2%, 12/23) and Nanling County (3/6) (χ² = 8.253, 6.016, both P < 0.05). Eggs of six parasite species were detected by Kato-Katz method, with detection rates of hookworm 72.5% (116/160), Ascaris sp. 2.5% (4/160), Capillaria hepatica 4.4% (7/160), Syphacia spp. 1.9% (3/160), and Hymenolepis diminuta 5.6% (9/160) and Hymenolepis nana 3.1% (5/160). Two parasite species, C. hepatica and hookworm, were detected by tissue biopsy or microscopic examination, with detection rates of 1.3% (2/160) and 3.1% (5/160), respectively. The proportion of wild mice infected with a single parasite was 84.8% (106/125) and the proportion of wild mice infected with two or more parasites was 15.2% (19/125). Among the 160 wild mice, Rattus tanezumi and Apodemus agrarius had parasite infection rates of 78.6% (99/126) and 76.5% (26/34), respectively (χ² = 0.403, P > 0.05). The parasites infection rates between male and female mice were 79.7% (59/74) and 76.7% (66/86), respectively (χ² = 0.207, P > 0.05). The parasite infection rates of juvenile mice, subadult mice and adult mice were 68.8% (33/48), 81.7% (89/109) and 3/3, respectively (χ² = 3.659, P > 0.05). Parasitic infection in wild mice was widely present and diverse in Wuhu City, Anhui Province. The local population is at risk of infection with zoonotic parasites.

CASE REPORTS

A child case of alveolar echinococcosis in Guizhou Province

ZHU Aiya, WANG Xu, WANG Jiangyou, WANG Ying, LI Yang, SONG Shan, GENG Yan, LAN Ziyao, DAI Jiarui

2023, 41(4):  520-523.  doi:10.12140/j.issn.1000-7423.2023.04.022

Asbtract ( )   HTML ( )   PDF (2857KB) ( )  

Figures and Tables | References | Related Articles | Metrics

The patient is a 9-year-old boy, a student from Ziyun County, Anshun City, Guizhou Province. He has experienced constant pain and discomfort in his upper right abdomen since August 2021. He was admitted to the 302 Hospital of the China Guihang Group on September 10, 2022 with a "liver occupying lesion: suspected liver abscess with calcification or tumor". The epidemiological investigation indicated that the boy has no history of travelling to epidemic areas. His parents have been engaged in livestock (dogs) slaughter for a long time, and some dogs were purchased from Echinococcosis epidemic areas such as Tibet Autonomous Region and Sichuan Province. The patient often came into contact with those dogs. The admission examination showed no yellowing of the skin and sclera throughout the body, slight tenderness in the right upper abdomen, and no palpable liver mass. Blood cell count showed that the percentage of eosinophils was 20.1%. The liver function testing results were showed γ Glutamic transferase was 36 U/L. The enhanced CT scan of the upper abdomen showed a patchy mixed-density shadow in the right posterior lobe of the liver, which is lobulated. Multiple patchy and tortuous earthworm-like high-density calcification shadows distributed in clusters. Multiple grid-like septal shadows were observed in the enhanced scan at the lesion, with mild continuous enhancement at the edges and septal areas. The abdominal color ultrasound showed an elliptical-shaped, uneven, strong echogenic mass in the right posterior lobe of the liver. Laparoscopic resection of the right posterior lobe of the liver was performed on September 14th, and compensatory enlargement of the right posterior lobe was observed. A milky white mass was seen in segments Ⅵ and Ⅶ, approximately 6 cm × 7 cm × 5 cm, with uneven surface and hard texture. The postoperative pathological section results showed inflammatory necrosis of the liver tissue. Postoperative blood cell count showed a normal of eosinophil percentage. Postoperative CT reexamination of the upper abdomen revealed postoperative changes, and no other obvious abnormalities were found. Therefore, the child was discharged. On November 10th, the patient went to the Affiliated Hospital of Guizhou Medical University for review, and the serum test indicated that Echinococcus antibody was positive. The cytochrome c oxidase subunit 1 (cox1) and nicotinamide adenine dinucleotide dehydrogenase subunit 1 (nad1) gene fragments of Echinococcus were amplified by PCR from the lesion tissue DNA by National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention (Chinese Center for Tropical Diseases Research) on November 29th. The amplified cox1 and nad1 gene fragments have high sequence similarity of 99.42% and 99.59% with the cox1 gene (reference sequence registration number AB477010) and nad1 gene (reference sequence registration number AJ132907) of E. multilocularis, respectively. The diagnosis of echinococcosis multilocularis was confirmed. The patient was administered with 15 mg/(kg·d) orally albendazole, which was taken separately after breakfast and dinner for a course of 6 months and followed-up the patient regularly.

A case of acute primary amoebic meningoencephalitis

ZHU Canmin, PENG Weijian, WANG Dili, ZHOU Huajing, JIN Qiangjian, CHANG Chang

2023, 41(4):  524-526.  doi:10.12140/j.issn.1000-7423.2023.04.023

Asbtract ( )   HTML ( )   PDF (1229KB) ( )  

Figures and Tables | References | Related Articles | Metrics

The 50-year-old male patient is a construction worker from Wuhan, Hubei. On August 3rd, 2022, he was administrated with oral antibiotics and antipyretic treatment at the fever clinic of the First People's Hospital/Union Medical College Jiangnan Hospital in Jiangxia District, Wuhan due to fever. However, his condition did not improve. The next day, the family discovered that the patient was mentally ill, restless, and talking nonsense, so the patient was transferred to the neurology department. The family reported that the patient swam in a lake near the construction site on July 29th, 2022. On August 3rd, he developed a fever with a maximum body temperature of 39.0 ℃, accompanied by pain in the neck and vomiting. Admission examination showed clear consciousness, mild delirium, unable to cooperate with the examination, positive bilateral Pap sign, stiffed neck with a gap of 4 fingers, and positive bilateral K sign. Blood routine examination shows that white blood cell count is 17.5 × 10⁹/L with 86.5% neutrophils and procalcitonin 0.16 ng/ml. Lumbar puncture cerebrospinal fluid examination on August 5th showed a normal cerebrospinal pressure (145 mmH₂O, 1 mmH₂O = 9.779 Pa), an increase in white blood cell count (4 265.00 × 10⁶/L), multinucleated cells were increased (88.00%), Pan's protein test was positive, the total protein increased (2 394.2 mg/L), glucose was decreased (0.15 mmol/L), chloride was normal (114.66 mmol/L), potassium wad decreased (2.75 mmol/L). The head CT scan showed lacunar infarction in the left basal ganglia area. The high-throughput genetic examination of pathogenic microorganisms in the cerebrospinal fluid revealed Naegleria fowleri, with a relative abundance of 98.76%. Based on the epidemiological history, clinical symptoms and laboratory examination results, the diagnosis of primary amoebic meningoencephalitis was made. Dehydration, intracranial pressure reduction, anti-infection, anti-epilepsy, and maintenance of water-electrolyte balance were administered with mannitol (125 ml/6 h), ceftriaxone sodium (2.0 g/12 h), amphotericin B (1 mg on August 6th, 2 mg on August 7th, 3 mg on August 8th, and 4 mg on August 9th) were administered. Although the patient managed to control the seizure after treatment, the condition worsened gradually and on August 9th, he developed a cerebral hernia and died.