Table of Content

30 June 2019, Volume 37 Issue 3

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ORIGINAL ARTICLES

Epidemiological characteristics of malaria and the progress towards its elimination in China in 2018

Li ZHANG, Jun FENG, Shao-sen ZHANG, Zhi-gui XIA, Shui-sen ZHOU

2019, 37(3):  241-247.  doi:10.12140/j.issn.1000-7423.2019.03.001

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Objective To analyze the epidemiological characteristic of malaria and the updates of malaria elimination in China so as to provide evidence-based reference for facilitating the National Malaria Elimination Program. Methods The malaria epidemic data in 2018 were collected from 31 provinces/municipalities/autonomous regions (Taiwan, Hongkong and Macao regions not included) in China through Malaria Annual Reporting System, and analyzed using Microsoft Excel 2010 software. Results Total 2 678 malaria cases were reported from 796 institutions in China in 2018, which is decreased by 6.4% compared to the cases reported in 2017 (2 861 cases). The reported cases included 2 518 Chinese (94.0%) and 160 foreigners (6.0%). Most of the cases fell into the age range of 20-59 years (89.2%, 2 390/2 678) with a male-to-female ratio of 12.9 : 1. There were 397 cases infected with Plasmodium vivas (14.8%, 397/2 678), 1 765 infected with P. falciparum (65.9%, 1 765/2 678), 83 P. malariae(3.1%, 83/2 678), 376 P. ovale (14.0%, 376/2 678), 52 cases of mixed-infection (1.9%, 52/2 678) and 5 clinically diagnosed cases (0.2%, 5/2 678). No indigenous case was found while 2 671 (99.7%, 2 671/2 678) were identified as imported cases, 4 cases of introduced cases, 2 induced cases and 1 long-term dormant case. The top 5 provinces with highest number of malaria cases were Guangxi (254, 9.5%), Jiangsu (243, 9.1%), Shandong (233, 8.7%), Sichuan (221, 8.3%) and Yunnan (213, 8.0%). Total 117 cases with severe clinical symptoms were reported (4.4%, 117/2 678) from 15 provinces/municipalities/autonomous regions with 7 deaths (0.3%, 7/2 678) from 6 provinces/municipalities/autonomous regions. The 1-3-7 approach was implemented nationwide with good performance: all cases were reported within 24 hour (1 day) after diagnosis, 93.0% (2 491/2 678) of the cases were epidemiologically investigated within 3 days, and 2 250 sites with case identified were investigated and prevention measures were performed within 7 days. Conclusion Malaria was close towards elimination in China with no indigenous case reported for successive two years in the whole country from 2017-2018, however, more attention should be paid to the imported cases of malaria including the national surveillance and case management.

Study on the role of protein kinase 9 in the growth and development of Plasmodium yoelii

Xiang-yun CHENG, Tai-ping LIU, Sui-lin CHEN, Wen-yue XU

2019, 37(3):  248-253.  doi:10.12140/j.issn.1000-7423.2019.03.002

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Objective To explore the role of protein kinase 9 (PK9) in the growth and development of Plasmodium yoelii. Methods The PK9 sequence information of P. yoelii lethal strain 17XL (Py17XL) was obtained from PlasmoDB. The sequence homology of PK9 among P. yoelii (PyPK9), P. falciparum (PfPK9) and human protein kinase p78 was compared by BLAST. The primers were designed based on the PyPK9 DNA sequence and coding DNA was amplified from WT Py17XL genomic DNA. The homologous arm and sgRNA sequence were ligated into PYC-MCS plasmid by CRISPR-Cas9 technique to construct PK9 gene knockout (PK9KO) recombinant plasmid. The recombinant plasmid was transferred into Py17XL by electroporation, screened and cloned by pyrimidine. The recombinant plasmid was identified by PCR and DNA sequencing. To determine the virulence of erythrocytic stage of PK9KO P. yoelii, 5 female BALB/c mice were intraperitoneally infected with 1 × 10⁵ PK9KO P. yoelii erythrocytic stage. Another 5 mice were infected WT P. yoelii as control. Blood was taken every 24 h to detect parasitemia in mice, and the survival of the mice was observed(3 repeated experiments). To determine the development of PK9KO P. yoelii in mosquito, 200 Anopheles stephensi mosquitoes aged 3 to 5 days were randomly divided into two groups: WT group (n = 100) and PK9KO group (n = 100), each was infected with WT P. yoelii or PK9KO P. yoelii through feeding with infected mice. The mosquito stomach was dissected 8 days after being fed with blood, Each group was randomly selected from 15 Anopheles stephensi for necropsy, and the number of the developed oocysts in mosquito stomach was examined. To determine the infectivity of PK9KO P. yoelii sporozoites developed in infected mosquitoes, 10 female BALB/c mice were randomly divided into WT sporozoite infection group (n = 5) and PK9KO sporozoite infection group (n = 5). Each mouse was infected with 1 × 10³ sporozoites through intravenous injection(2 repeated experiments). Tail vein blood was taken every 12 hours after injection, and the occurrence time of parasitemia was observed by blood smear. Results The sequence homology of PyPK9 with PfPK9 was 82.5%, but only 39.4% with human protein kinase p78. The PCR product of PK9 of P. yoelii was 597 bp. The PK9KO recombinant plasmid was constructed and the PK9 gene was successfully knocked out in Py17XL strain conformed by PCR identification and DNA sequencing. All the mice died on the 5th to 6th day after being infected with WT P. yoelii, however, all mice infected with PK9KO P. yoelii were survived and the parasitemia disappeared on the 17 d after infection. However, there was no significant difference for the infection of mosquito (formation of oocyst) by feeding with mouse blood with WT P. yoelii (60.0%, 9/15) and with PK9KO P. yoelii (66.7%, 10/15) (P > 0.05). The infectivity of sporozoites developed from WT and PK9KO P. yoelii remained similar in infected mice. Parasitemia was found 72 h after intravenous infection of both sporozoites. Conclusion PK9 plays an important role in the virulence of erythrocytic stage of P. yoelii.

Endemic status of human important parasitic infection in Hainan Province, China in 2015

Ding-wei SUN, Ying LIU, Guang-ze WANG, Shao-xiong LIN, Chong-jin TONG, Dong-yan CHEN, Xi-min HU

2019, 37(3):  254-259.  doi:10.12140/j.issn.1000-7423.2019.03.003

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Objective To understand the current status and trends of human important parasitic diseases (HIPD) as a basis for their better control, a survey was carried out in Hainan Province in 2015. Methods According to the Protocol of the 3rd National Survey on the Status of Human Key Parasitic Infections. Total 18 survey sites (10 villages, 8 townships) were selected from 8 cities (counties) using a stratified random sampling method. The prevalence of soil-transmitted helminth and taenia infections was surveyed in all sites. The prevalence of intestinal protozoa infections was surveyed only in 10 village sites. In each survey site, no less than 250 permanent residents with age above 3 years old were recruited. Fecal sample was collected from each participant and the eggs of intestinal helminthes were examined in duplicate under microscope using Kato-Katz technique. Tube filter paper culture method was applied to culture the larvae and identify the species of hookworm. The Enterobius eggs were detected in children aged 3 to 6 years old by using adhesive tape. The cysts and trophozoites of intestinal protozoans were examined using saline direct smear and iodine staining. SPSS 21.0 software was used for statistical analysis. Results A total of 4 773 individuals were surveyed for HIPD from 18 selected sites with an overall infection rate of 13.3% (633/4 773) for HIPD infections. The prevalence of intestinal protozoa infections was 2.8% (76/2 698) with 5 species of protozoans identified. The prevalence of helminth infection was 12.1% (576/4 773) with 7 species of helminths including whipworm (5.6%) and hookworm (4.3%) as main species. Using adhesive tape method, 252 children of 3-6 years old were examined for the infection of E. vermicularis, and an infection rate of 31.4% (79/252) was found among the children examined. No Taenia infection was found. A total of 143 fecal samples with hookworm egg positive were cultured for identifying the species of hookworm, 120 of them were Necator ameicanus only, while other 23 were mixed with N. ameicanus and Ancylostoma duodenale. There was a significant difference (P < 0.01) in helminth prevalence between villages (18.1%, 488/2 698) and townships (4.2%, 88/2 075). A significant difference (P < 0.05) in helminth prevalence between male (10.9%, 247/2 273) and female (13.2%, 329/2 500) was observed. Among different age groups, people with age between 60-69 years old had the highest helminth prevalence (17.58%, 77/438), while group with age 15-19 years old had the lowest helminth prevalence (6.19%, 12/194) with statistical difference (P < 0.01). The Miao nationality had the highest helminth prevalence (19.1%, 97/508; P < 0.01) among different nationality people. Among different occupations, farmers had the highest helminth prevalence (14.1%, 384/2 717), while merchant had the lowest (3.5%, 8/228) with statistical difference (P < 0.01). Among different education levels, the illiterates had the highest helminth prevalence (20.8%, 33/159), while people with college and above education with the lowest helminth prevalence (5.8%, 34/591) with significant difference among different education levels (P < 0.01). The prevalence of intestinal protozoa infections was surveyed in Dongfang, Qionghai, Qiongzhong and Lingao counties only with 2.0% (16/790), 1.7% (14/829), 3.8% (20/527), and 4.7% (26/552), respectively. There was a significant difference in protozoa prevalence among different areas (P < 0.01). The protozoa prevalence in male and female were 2.8% (37/1 316) and 2.8 (39/1 382) respectively. The highest prevalence of intestinal protozoa infections was distributed in people with 20-29 years old (4.7%, 15/320) and Miao nationality (4.6%, 12/263). Among different occupations, preschoolers had the highest intestinal protozoa infections (4.6%, 12/260) with significant difference compared to other occupations (P < 0.01). Conclusion Although the overall infection rate of important human parasites has been showing a trend of significant reduction in Hainan province during past years, it still remains higher in context of nationwide level, particularly the infection rate of E. vermicularis in children appears notably high, thus it should be the prior target of intestinal parasitic disease control in Hainan in future.

Current status of intestinal parasite infections in rural areas of Shandong Province, China in 2015

Yan XU, Xiu-qin BU, Dian-bo ZHANG, Feng MIAO, Yong-bin WANG, Xiang-li KONG, Chang-lei ZHAO, Ben-guang ZHANG

2019, 37(3):  260-265.  doi:10.12140/j.issn.1000-7423.2019.03.004

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Objective To understand the endemic status of major human intestinal parasites in rural areas of Shandong Province, so as to provide scientific evidence for formulating prevention and control measures. Methods The survey was conducted based on the National Survey Plan and Implementation for Human Key Parasitic Diseases from 2014 to 2015. Total 52 pilot study sites were selected using a stratified cluster random sampling method based on different ecologic and geographic status, economic levels, each site with at least 250 residents enrolled. Fecal samples were collected from all participants and the intestinal helminth eggs in the feces were examined under microscope using Kato-Katz thick smear method. The direct smear method was used to detect intestinal protozoan trophozoites and cysts. The adhesive tape method was used to detect pinworm eggs around anal in children aged 3 to 6. The information for the natural factors, toilet, drinking water and diet and hygiene behaviors were collected for analyzing risks of infections. The difference in infection rates was statistically analyzed by chi-square or Fisher exact test using SPSS 18.0. Results A total of 13 620 people were investigated and the overall intestinal parasite infection rate was 1.06% (144/13 620), among them the intestinal helminth infection rate was 0.87% (118/13 620) and the intestinal protozoa infection rate was 0.19% (26/13 620). The pinworm infection rate in children with age of 3-6 was 1.71% (15/877). Total 5 species of intestinal helminthes and 3 species of intestinal protozoa were identified. The top three intestinal helminths were Trichuris trichiura (0.58%, 79/13 620), Ascaris lumbricoides (0.16%, 22/13 620) and Enterobius vermicularis (0.12%, 17/13 620). Among 118 helminth infecton cases, 4(3.39%) cases were mixed infection, and which were all mixed with Trichuris trichiura and Ascaris lumbricoides, while 26 protozoa infection cases were all single infection. Among 22 Ascaris lumbricoides infection cases, 20(90.91%) cases were mild infection, 1 (4.55%) case was moderate infection and 1 (4.55%) case was severe infection. 3 Ancylostoma sp. infection cases were all mild infection. Among 79 Trichuris trichiura infection cases, 70(88.61%) were mild infection, 8 (10.13%) were moderate infection and 1 (1.27%) case was severe infection. 1 Clonorchis sinensis infection case was mild infection. The top three counties with highest intestinal parasite infection rate were Rushan, Weihai (8.23%, 65/790); Mengyin, Linyi (1.51%, 16/1 063) and Yanggu, Liaocheng (1.26%, 10/793). The intestinal parasite infection rates in population between two ecologic zones were significantly different, with 0.61% in plain ecologic zone and 1.50% in hilly ecologic zone (P < 0.01). The pinworm infection rate was significantly higher in plain zone (0.21%) than in hilly zone (0.04%), while T. trichiura infection rate was significantly higher in hilly zone (1.13%) than in plain zone (0.01%) (P < 0.01). There was no significant difference in intestinal parasite infection between male (0.90%) and female (1.20%) (P > 0.05). The infection rate in old people with age of 70-79 (2.30%) was higher than other age groups with statistical difference (P < 0.01). There was no significant difference in infection rate in population with different occupations or different education even though the prevalence of intestinal parasites was high in preschool children (1.49%) (P > 0.05). Drinking water source was a risky factor, people drinking water from well had higher infection rate of intestinal parasites (1.88%) than people drinking tape water (0.82%) or spring water (0.37%) (P < 0.01). People eating raw vegetable had higher infection rate (1.25%) than people with cooked vegetable (0.74%) (P < 0.01). The population with regular massive anthelmintic drug administration had much lower intestinal parasite infection rate (0.06%) than those without implementation (1.19%) (P < 0.01). Conclusion The intestinal parasite infection rate had decreased significantly in recent years in rural area of Shandong Province and remained at low level, but whipworm infection remained high in some hilly areas and the pinworm infection kept endemic in children in plain area. People over 60 years old and preschool children are the key population who need special care for prevention and control of intestinal parasite infections in Shandong Province.

Current status of human intestinal protozoa infections in Jiangxi Province, China in 2014

Wei-ming LAN, Jun GE, Wei-sheng JIANG, Shu-ying XIE, Chun-qin HANG, Zhao-jun LI, Hong-yun LIU, Jing ZHANG, Cheng-jian HUANG, Xiao-jun ZENG

2019, 37(3):  266-271.  doi:10.12140/j.issn.1000-7423.2019.03.005

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Objective To investigate the current status of human intestinal protozoa infections in Jiangxi Province, so as to provide the evidence for the formulation of prevention and control measures. Methods A survey was performed in 2014 according to the Protocol of the 3rd National Survey on the Status of Human Key Parasitic Infections combining with the specific economic and geographic situation in Jiangxi Province. Total 84 survey sites were chosen from 28 counties (cities and districts) using stratified cluster sampling method based on the different geographic zones. No less than 250 permanent residents with no more than 1/3 of children with age less than 12 years old were recruited in the survey in each site. The fecal samples were collected from each participant. The infected protozoan cysts were examined using iodine staining and the trophozoites were examined using direct saline smear under microscope. Each fecal sample was examined once and the statistical analysis was performed using IBM SPSS Statistics 20 and chi-square test. Results A total of 21 569 people were participated in this survey. The overall prevalence of protozoa infections was 1.4% (307/21 569) with eight species of intestinal protozoa identified. Specifically the prevalence of Entamoeba histolytica was 0.2% (45/21 569), E. coli 0.5% (104/21 569), Gardia lamblia 0.1% (26/21 569), Blastocystis hominis 0.3% (68/21 569), E. hartmani 0.1% (28/21 569) and Endolimax nana 0.2% (46/21 569). Three cases of Iodamoeba butschlii and 1 case of Balantidium coli were identified. The prevalence of protozoa infections was highest in Nanling hilly areas (2.8%, 132/4 742) with B. hominis and E. nana as dominant infected species. The lowest prevalence of protozoa infections was identified in Zhejiang/Fujian hilly areas(0.3%, 18/5 887). There was significant difference in the prevalence of intestinal protozoa infections among different ecological areas(P < 0.01). The largest number of cases of E. histolytica infection was found in Jiujiang City, accounting for 93.3% (42/45) of the total cases in the province. E. coli and G. lamblia infections were common in Yichun City, accounting for 65.4% (68/104) and 46.2% (12/26) of the total cases in the province, respectively. The 1 case of B. coli infection was also found in Yichun City. The infections of B. hominis, E. hartmani and E. nana were mostly found in Ganzhou City, accounting for 69.1% (47/68), 82.1% (23/28) and 97.8% (45/46) of the total cases in the province, respectively. There were 12 cases of mixed protozoa infections identified province-wide. There was no significant difference in the prevalence of intestinal protozoa infections between male (1.4%, 148/10 582) and female (1.5%, 159/10 987) (P > 0.05). The highest prevalence of intestinal protozoa was observed in children with age 0-9 years old (2.0%, 64/3 243), and the lowest in the elders with age above 70 (0.9%, 16/1 710), however, there was no statistical significance among different age groups (P > 0.05). In different occupation groups, the highest prevalence of intestinal protozoa infections was found in workers and businessmen(1.8%, 6/329) and the lowest in teachers and retirees (0.8%, 2/248). There was no significant difference in the prevalence among different occupations (P > 0.05) though. In different level of education, the highest prevalence was identified in population with low education (illiterate, 1.5%, 56/3 635) and the lowest in people with high education (college and above, 0.2%, 1/219), but without statistical difference in people with different educational levels (P > 0.05). The prevalence of intestinal protozoa infections in the high income areas was 1.3% (102/7 837) with E. coli as a dominant infected species. The protozoa infection in people with medium income was 0.9% (50/5 456) mainly with E. histolytica and G. lamblia infected. The infection rate in people with low income was 1.9% (155/8 276), mainly with B. hominis and other protozoans. The prevalence of intestinal protozoa infections was significant difference between people with different economic level (P < 0.01). Conclusion The overall prevalence of human intestinal protozoa infections in Jiangxi Province is low, but more serious in the population in Nanling hilly areas with lower economic level. More control measures should be performed in this part of population.

Effects of Fasciola gigantica infection and released excretory-secretory products on the mRNA expression of Toll-like receptors in mice

Ke-jing LU, Lin-jing HOU, Bin ZHU, Wei SHI, Yi-ying LIANG, Wei-yi HUANG, Wei-yu ZHANG

2019, 37(3):  272-279.  doi:10.12140/j.issn.1000-7423.2019.03.006

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Objective This work aims to investigate the effects of Fasciola gigantica infection and the released excretory-secretory products (FgESP) on the mRNA expression of mice Toll-like receptors (TLRs). Methods To determine the effect of F. gigantica infection on the TLR1-9 mRNA expression, 20 female BALB/c mice were orally infected with 20 metacercariae of F. gigantica, another 20 mice were given with PBS as non-infection control. Four mice from each group were sacrificed 1, 4, 7, 14 or 28 days post infection. The liver tissue of mice was obtained, total RNA was extracted, reverse transcribed into cDNA, and the relative expression of TLR1-9 mRNA was detected by qRT-PCR. To prepare the FgESP, the flukes from liver of buffalo infected with F. gigantica was collected from slaughterhouse and incubated in PBS for 6 hours, the incubation supernatant were collected as native FgESP(nFgESP). To determine the effect of FgESP on the TLR1-9 mRNA expression in mouse liver, 9 mice were treated with 1 mg/ml nFgESP by intraperitoneal injection. The same number of mice were given PBS only as control. Three mice from each group were sacrificed 1, 4 and 7 days post treatment. The liver was collected from each mouse for RNA extraction, the relative mRNA expression of TLR1-9 in the liver was detected by qRT-PCR. The nFgESP was inactivated by heating at 95 ℃ for 15 min to make the heat-inactivated ESP(hiFgESP). To determine the effect of nFgESP and hiFgESP on the TLR1-9 mRNA expression in macrophage cell line RAW264.7, 2 × 10⁶ cells were incubated with 25, 50, 100 or 200 μg/ml of nFgESP or hiFgESP, or PBS in duplicates for 24 h. The cell experiment was repeated three times. The cells were harvested for RNA extraction. The TLR1-9 mRNA expression in the cells were detected by qRT-PCR. Results In the F. gigantica infection experiment, qRT-PCR results demonstrated that the relative mRNA expression of TLR6 and TLR7 in the livers of mice infected for 7 days (2.9 ± 0.3 and 2.5 ± 0.5) were significantly higher than that in the control mice (1.0 ± 0.2 and 1.3 ± 0.5) (P < 0.01). The mRNA expression of TLR1 and TLR9 in the infected mice at 14 days (3.4 ± 0.8 and 2.8 ± 0.9) were significantly higher than that in the control mice (1.1 ± 0.2 and 1.1 ± 0.3) (P < 0.05, P < 0.01). After being infected for 28 days, the TLR1, TLR8 and TLR9 mRNA expression levels in the livers (3.8 ± 1.3, 4.2 ± 1.5 and 3.1 ± 0.9) were significantly higher than that in the control mice (1.0 ± 0.2, 1.2 ± 0.4 and 1.1 ± 0.3) (both P < 0.01, P < 0.05). In the FgESP treatment experiment, the mRNA levels of TLR1 and TLR2 in the livers of mice treated with nFgESP for 1 day (2.5 ± 0.4 and 1.9 ± 0.4) were significantly higher than that in the PBS-treated mice (1.0 ± 0.1) (P < 0.05, P < 0.01). For in vitro FgESP stimulation experiment on RAW264.7 cell line, the TLR1, TLR3, TLR7 and TLR8 mRNA expression levels in the cells stimulated with 100 μg/ml of nFgESP were significantly upregulated (1.6 ± 0.0, 1.4 ± 0.0, 1.6 ± 0.0 and 1.9 ± 0.0, respectively) than the cells treated with PBS（1.0 ± 0.0）（P < 0.05, all P < 0.01）. The TLR-8 mRNA expression level was significantly higher in cells stimulated with 200 μg/ml of nFgESP and hiFgESP (3.4 ± 0.0 and 4.0 ± 0.5) than that in cells treated with PBS（1.0 ± 0.0）（P < 0.01）. The TLR2 and TLR8 mRNA expression levels in cells stimulated with 100 μg/ml hiFgESP (1.5 ± 0.1 and 2.0 ± 0.0) were significantly higher than that in PBS treated cells (1.0 ± 0.0) （P < 0.01）. However, the TLR3 mRNA level was significantly lower in cells stimulated with 25 μg/ml hiFgESP (0.6 ± 0.0) than that in PBS treated cells（1.0 ± 0.0）（P < 0.01）. Conclusion F. gignatica may regulate host immune response by affecting the transcriptional expression of TLR1, TLR7 and TLR8 in mice through releasing ESP.

Cloning and expression of must mite allergen Der f 32 and its activation on mouse dendritic cells

Bei JIANG, Xiao-jun XIAO, Chun-yan OUYANG, Xin-ping LUO, Bao-qing SUN, Jing LI, Zhi-gang LIU

2019, 37(3):  279-285.  doi:10.12140/j.issn.1000-7423.2019.03.007

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Objective To clone and express must mite allergen Der f 32 protein and identify its antigenicity and its activation on dendritic cellline (DC2.4). Methods The DNA coding for allergen Der f 32 of dust mite was synthesized by genetic engineering technology, and cloned into expression vector pET-24a(+), and then transformed into Escherichia coli BL21 for expression. The expressed recombinant Der f 32 protein(rDer f 23)with His-tag was purified by nickel affinity chromatography and analyzed by SDS-PAGE and Western blotting with sera from patients allergic to dust mite. The rDer f 32 was used for skin prick test to detect its antigenicity in patients with allergic to dust mite. The rDer f 32 was co-cultured with mouse-derived DC2.4 for 24 h, flow cytometry was used to detect the expression of cell surface costimulatory molecules(CD40, CD83 and CD80)on the DC2.4. The DC2.4 cells were incubated with different amount of rDer f 32(10, 20, 30, 40, 50, 60 μg/ml)for 48 h and the expression of T cell immunoglobulin and mucin 4(TIM4)on DC2.4 was detected by Western blotting. Results The SDS-PAGE results showed that the rDer f 32 was successfully expressed in E. coli BL21 with a relative molecular mass of about 35 000. Clinical skin prick test showed that 6 of the 42 patients allergic to dust mite were positive with a positive rate of 14.2%. Western blotting showed that rDer f 32 was specifically recognized by IgE in sera of patients with positive in skin test. The results of flow cytometry showed that the expression levels of CD40, CD83 and CD80 were 24.5%, 9.6% and 24.6% on DC2.4 after being co-cultured with rDer f 32, which was significant higher than that of DC2.4 co-cultured with PBS(11.9%, 3.1% and 15.6%)(P < 0.01), however lower than that of the positive control group with stimulation of Der f 1(38.2%, 15.0%, and 39.1%)(P < 0.05). The results of Western blotting showed that the expression of TIM4 was increased in DC2.4 cells after being co-cultured with rDer f 32 at a dose-dependent pattern, with highest expression when Der f 32 was added at 50 μg/ml. The relative density of TIM4 expression was 1.112. Conclusion The dust mite allergen Der f 32 was successfully cloned and expressed as recombinant protein which could be recognized by the IgE in sera of patients with allergic to dust mite. rDer f 32 could stimulate DC maturation with increased expression of surface costimulatory molecules and TIM4.

Allergen-specific immunotherapeutic effect of recombinant Blo t 21 T protein on asthma in a mouse model

Qiang CHAI, Chao-pin LI

2019, 37(3):  286-290.  doi:10.12140/j.issn.1000-7423.2019.03.008

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Objective To investigate the effect of allergen-specific immunotherapy of recombinant protein Blo t 21 T on asthma in mice sensitized by the extracts of mite Blomia tropicalis. Methods Thirty mice were randomly divided into three groups: the asthma group, the immunotherapy group, and the control group (PBS group)(n = 10 in each group). Mice in the asthma and immunotherapy group were sensitized by intraperitoneal injection of 10 μg B. tropicalis crude extracts formulated with 2 mg Al(OH)₃ in total volume of 200 μl on day 0, 7 and 14. On the 21^th day, the mice were challenged with the same allergen extracts (0.5 μg/ml) through spray inhalation for 30 min for consecutive 7 days. In the immunotherapy group, mice were treated intraperitoneally with 10 μg recombinant Blo t 21 T protein 30 min before the challenge inhalation from 25^th to 27^th days. In the asthma group, mice were not treated. In the control group, mice were not sensitized and treated, only receiving PBS. All mice were sacrificed 24 h after the last inhalation, the sera, bronchoalveolar lavage fluid (BALF) and the lung tissue were collected. The number of eosinophils (EOS) was counted in BALF, the levels of cytokine IL-4, IL-13, IL-17 and IFN-γ in BALF, IgE and IgG_2a in sera were determined by ELISA. The lung histochemical sections were made to evaluate the pathological changes. Single factor F test was used for multiple group comparison. SNK test was used to compare the data from two groups. Results The count of EOS in immunotherapy group was (1.23 ± 0.35) × 10⁵/ml, which was significantly lower than that in asthma group [(5.32 ± 1.22) × 10⁵/ml] (P < 0.05). The levels of IFN-γ, IL-4, IL-13, IL-17 in the immunotherapy group [(316.80 ± 29.12), (70.96 ± 17.62), (159.20 ± 21.08) and (220.80 ± 25.95) pg/ml, respectively] was significantly lower than that in the asthma group [(72.84 ± 10.10), (111.96 ± 13.62), (321.40 ± 31.88) and (425.40 ± 34.83) pg/ml, respectively] (P < 0.05). The levels of IFN-γ, IL-4, IL-13 and IL-17 in asthma group was significantly different from the levels in control group [(125.60 ± 22.38), (12.57 ± 3.62), (109.62 ± 15.38) and (114.38 ± 18.43) pg/ml] (P < 0.05). The level of serological IgG_2a and IgE in the immunotherapy group [(49.73 ± 10.95) and (316.80 ± 29.12) μg/ml, respectively] was higher than that in the asthma group [(27.30 ± 4.83) and (57.40 ± 15.96) μg/ml, respectively] (P < 0.05), however, the levels of IgE in asthma group is higher than that in control group(P < 0.05). HE staining of lung tissue showed that lung tissue in immunotherapy group had reduced pathological changes characterized by the reduced thickened tracheal wall and less inflammatory cell infiltration around the vascular mucosa and lung tissue compared with the un-treated asthma group. Conclusion the Blomia tropicalis recombinant protein Blo t 21 T has allergen-specific immunotherapeutic effect on mite-induced asthma in a mice model.

Current endemic status of Clonorchis sinensis infection in population of Jilin Province in 2015

Xin WANG, Ben-he WANG, Shu-ya WANG, Qiang YIN, Chang-xi XU

2019, 37(3):  291-295.  doi:10.12140/j.issn.1000-7423.2019.03.009

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Objective To understand the current endemic status of Clonorchis sinensis infection in opulation of Jilin Province so as to provide a scientific basis for better prevention and control of clonorchiasis. Methods According to the National Survey Program and Implementation Rules for the status of human key parasites, total 78 pilot survey sites in rural areas and 21 sites in township from 35 counties (cities and districts) in Jilin Province were selected using stratified cluster sampling method in 2015. No less than 250 permanent residents over 3 years old were recruited from each site in the survey. Fecal samples were collected from each participant and the eggs of C. sinensis in the feces were examined using a modified Kato-Katz thick smear microscopy. The general demographic and personal information were collected by a written questionnaire. The infection status among residents was analyzed based on their different gender, age and occupation with statistical analysis using Chisquare test. Some residents from 50 pilot survey sites were randomly selected to conduct a questionnaire survey on clonorchiasis related knowledge to understand their awareness of parasites and infection risks. Results A total of 24 963 people were examined, of whom the total prevalence of C. sinensis infection was 3.0% (749/24 963), mainly with mild infection 88.5% (663/749). The medium and heavy infections only accounted for 11.0% (82/749) and 0.4% (3/749), respectively. The infection rate in rural areas (3.8%, 746/19 683) was significantly higher than that in urban areas (0.1%, 3/5 280) with statistical difference (P < 0.05). Male people had higher infection rate (3.6%, 459/12 843) than female (2.4%, 290/12 120）with statistical difference (P < 0.05). Among different age groups, it was shown that people with age between 41-50 had the highest infection rate (4.4%, 227/5 175) with significant difference compared to other age groups (P < 0.01). Among different occupations, the infection rate was highest in famers ( 4.1%, 712/17 455) (P < 0.01). In terms of educational levels, people educated with junior high school had the highest infection rate (4.4%, 546/12 322) (P < 0.01). With respect to different ethnic groups, Han population had the highest infection rate (3.1%, 746/23 794) (P < 0.01), possibly related to different food and cuisine habit. Totally 2 850 questionnaires were collected from 50 pilot survey sites. The awareness rate for clonorchiasis in people living in urban cites (58.5%, 350/598) was higher than that in people living in rural area（34.9%, 787/2 252）(P < 0.01), however, more people in urban areas ate raw freshwater fish and shrimp (19.0%, 113/598) than people living in rural areas (11.9%, 269/2 252) (P < 0.01), even though the former knew better about the parasite and the infection risks. Conclusion The prevalence of C. sinensis infection in Jilin Province still remains at a relatively high level. The awareness for the knowledge of infection and behavior-related infection risks among people are low and not much changed.

Association of raw-freshwater fish-eating practice with the infection of Clonorchis sinensis

Men-bao QIAN, Zhi-hua JIANG, Tao GE, Xin WANG, Zhuo-hui DENG, Chang-hai ZHOU, Hui-hui ZHU, Ying-dan CHEN, Xiao-nong ZHOU

2019, 37(3):  296-301.  doi:10.12140/j.issn.1000-7423.2019.03.010

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Objective To quantitatively evaluate the relationship between the raw-freshwater fish-eating practice and the infection of Clonorchis sinensis. Methods Total 16 counties were enrolled from 4 provinces highly endemic with clonorchiasis (Guangxi, Guangdong, Heilongjiang and Jilin), and 5 villages were selected from each investigated counties as survey sites. Total 200 residents from each site were recruited for the investigation. The fecal samples were collected from each participant and the eggs of C. sinensis in the feces were examined under microscope using Kato-Katz thick smear method, and the information on eating raw freshwater fish among the participants was collected. The linear relationship between the percentage of participants eating raw freshwater fish and the prevalence of C. sinensis infection was analyzed in people living in different counties, with different genders and age. The parameter of coefficient of determination (R²) was determined accordingly. The relative risk (RR) and 95% confidential intervals (95% CI) were calculated for those with raw-freshwater fish-eating practice compared to those without the practice. Subsequently, attributable risk (AR), attributable risk percent (ARP), population attributable risk (PAR) and population attributable risk percent (PARP) were analyzed. Results A total of 16 230 participants from 16 counties were enrolled in this survey. The percentage of participants with practice of eating raw freshwater fish was 26.5% (4 295/1 6230) and the overall prevalence of C. sinensis infection was 10.8% (1 747/1 6230) with a positive linear association between them in investigated counties (R² = 0.44, P < 0.01). The percentage of eating raw freshwater fish was 31.7% (2 687/8 478) in males and 20.7% (1 608/7 752) in females (P < 0.01), while the corresponding prevalence of C. sinensis infection was 13.7% (1 161/8 478) and 7.6% (586/7 752) (P < 0.01). The percentage of eating raw freshwater fish in males aged ≤14, 15-29, 30-44, 45-59 and ≥60 was 1.9%, 19.4%, 40.2%, 44.5% and 36.3%, respectively, positively correlating with the prevalence of C. sinensis infection of 1.5%, 7.5%, 15.1%, 20.8% and 15.4%, correspondingly (R² = 0.96, P < 0.01). The percentage of eating raw freshwater fish in females aged ≤14, 15-29, 30-44, 45-59 and ≥60 was 2.3%, 13.9%, 25.3%, 29.2% and 22.4%, correspondingly, with positive linear association with, the prevalence of C. sinensis infection of 1.2%, 6.1%, 9.0%, 10.4% and 8.0%, respectively(R² = 0.99, P < 0.01). Compared to those without the practice, RR of C. sinensis infection in those with the practice reached 12.9 (95% CI: 11.4-14.7). RR was 11.8 (95% CI: 10.0-14.0) in males and 13.4 (95% CI: 10.9-16.4) in females between eating raw freshwater fishes and the chance of getting infected. RR ranged from 8.2-16.1 in different age groups. Overall, AR for eating raw freshwater fishes and the infection of C. sinensis was 30.9%, ARP was 92.3%, PAR was 8.2%, and PARP was 75.9%. In males, AR was 33.5%, ARP was 91.5%, PAR was 10.6% and PARP was 77.4%, compared to those in female of 26.2%, 92.5%, 5.4% and 72.0%, respectively. AR ranged between 15.6% and 34.0%, ARP between 87.9% and 93.8%, PAR between 0.3% and 12.7%, and PARP between 24.1% and 79.7% in different age groups. Conclusion Raw-freshwater fish-eating practice is seriously associated with the infection of C. sinensis and the difference in raw-freshwater fish-eating practice determines the different distribution of C. sinensis infection in areas and population in these investigated endemic areas.

Correlation between angiogenesis and disease progression of hepatic Echinococcus multilocularis in C57BL/6 mice

Qing ZHOU, Xiong-feng YANG, Huan-huan HAN, Li-jiao GUO, Hui-jiao JIANG, Xiao-yi WANG, Lin-lin LI, Zhen-yu LIAO, Xue-ling CHEN, Xiang-wei WU

2019, 37(3):  302-310.  doi:10.12140/j.issn.1000-7423.2019.03.011

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Objective To investigate the relationship between angiogenesis and disease progression in hepatic Echinococcus multilocularis (Em) in mice. Methods Total 60 C57BL/6 female mice with 6-8 weeks old were randomly divided into two groups: the experimental group and the control group with 30 for each. Each mouse in the experimental group was infected by direct injection into liver with 100 μl of suspension containing 400 Em protoscoleces under anesthesia of 5% chloral hydrate. In the control group mice were injected with the same volume of PBS. Some of mice were euthanized and the liver blood vessel was filled with silicone to show the distribution of blood vessels and angiogenesis 30 d, 60 d, 90 d and 120 d after infection. The vascular endothelial cell growth factor A (VEGFA) in the sera of mice were measured by ELISA. The mouse livers were collected and subjected to hematoxylin and eosin (HE) staining for observing pathological changes. The dynamic changes of VEGFA, CD31 and CD34 expression in infected liver was analyzed by immunohistochemical staining. Results The liver blood vessel filled with silicone showed that Em gradually grew in the livers of mice, surrounded by the increased blood vessels as the infection continued in the experimental group. The levels of VEGFA in the sera of infected mice were 269.00 pg/ml at 30 d, 420.62 pg/ml at 60 d, 539.00 pg/ml at 90 d and 271.73 pg/ml at 120 d after infection, which were significant higher than that in un-infected control mice (194.00 pg/ml at 30 d, 173.00 pg/ml at 60 d, 234.00 pg/ml at 90 d and 127.00 pg/ml at 120 d after infection) with statistical difference (P < 0.05). HE staining of infected liver tissue demonstrated that the growing Em tissue surrounded by the filtration of eosinophils, granuloma and fibrosis. The protoscoleces and calcification could be observed in the middle of the lesion. Immunohistochemical staining demonstrated different degrees of expression of VEGFA, CD34 and CD31 in the endothelial cells of Em tissue. The VEGFA immunohistochemical scores of Em tissues were 1.60 ± 0.52 at 30 d, 3.10 ± 0.87 at 60 d, 4.80 ± 1.32 at 90 d and 2.40 ± 1.07 at 120 d after infection, which were significant higher than that in the surrounding normal liver tissue of infected mice or livers of control mice (0) (P < 0.01). Among the infected mice, the VEGFA immunohistochemical scores at 60 d and 90 d were significant higher than that at 30 d after infection (P < 0.05, P < 0.01, respectively). The expression of CD34-MVD in the Em tissues were 38.70 ± 11.06 at 30 d, 65.50 ± 8.46 at 60 d, 109.90 ± 9.40 at 90 d, and 56.86 ± 6.64 at 120 d after infection, which were significant higher than those expressed in normal liver cells (P < 0.01). The expression of CD34-MVD at the different time of infection was also significantly different (P < 0.01). Expression of CD31-MVD in the Em tissues were 19.80 ± 3.12 at 30 d, 30.70 ± 2.50 at 60 d, 47.90 ± 4.77 at 90 d and 31.10 ± 3.84 at 120 d after infection with significant difference among different infection time points (P < 0.01), but significant lower than that expressed in normal liver tissues (P < 0.01). Conclusion E. multilocularis invasively grows in infected liver along with significant angiogenesis and expression of CD31 possibly due to the stimulated secretion of VEGFA. The secretion of VEGFA stimulated angiogenesis, neovascularization induced by Em infection in liver of mouse.

Preparation of polyclonal antibody against-triosephosphate isomerase of Toxoplasma gondii and its application to detect T. gondii infection

Zheng-hu ZHAO, Sheng-nan QIAN, Jing-yi WAN, Zi-ru TANG, Shuang SHEN

2019, 37(3):  311-316.  doi:10.12140/j.issn.1000-7423.2019.03.012

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Objective Triosephosphate isomerase (TPI) of Toxoplasma gondii is an immunodominant antigen secreted by T. gondii during infection and therefore an important diagnostic antigen. To explore its potential for diagnostic purpose, the polyclonal antibody anti-TPI was made in rabbit and its application to detect T. gondii infection was explored. Methods A New Zealand white rabbit was immunized with 200 μg recombinant TPI protein expressed in Escherichia coli and emulsified with Freund’s adjuvant for three times. The serum was collected from the immunized rabbit 2 weeks after the last immunization. The total IgG in immunized-rabbit serum was purified with Protein A affinity purification column. SDS-PAGE was used to detect the purity of the purified IgG. The anti-TPI antibody titers in the purified IgG and in the immune sera was measured by ELISA and its specific recognition of recombinant TPI and native TPI in T. gondii excretory/secretory (ES) products was detected by Western blotting. The anti-TPI IgG was conjugated on magnet beats to pull down the TPI in T. gondii-infected mouse sera and the pull-downed TPI was applied on NC membrane to be detected by the anti-TPI IgG, and the results were compared with that detected by ELISA kit. Results The recombinant TPI protein was expressed in E. coli and the purified TPI protein was used to immunize a rabbit. The rabbit anti-TPI serum was obtained and the total IgG in the serum was purified with Protein-A affinity column. SDS-PAGE analysis identified that the purified IgG contained typical IgG heavy chain and light chain. The anti-TPI specific antibody titers were 1 : 128 000 in immunized rabbit serum and in the purified IgG measured by ELISA. Western blotting showed that the recombinant TPI protein and native TPI in T. gondii ES products could be specifically recognized by the purified IgG. The purified rabbit IgG was able to pull down the TPI antigen in 10 of 11 sera of mice infected with T. gondii, however, one of the 11 normal mouse sera also showed positive. The results detected by ELISA kit showed that 6 of the 11 infected mice were positive and 5 were negative; 11 healthy mice were all negative. There was no significant difference in the results between Dot blot and ELISA detection. Conclusion The anti-TPI polyclonal antibody was prepared in immunized rabbit. An anti-TPI IgG-based immunological assay was potentially established to detect T. gondii infection. This TPI-specific IgG could be used to develop an immunodiagnostic assay to detect T. gondii infection.

Effect of recombinant protein P29 on the TGF-β/Smad signaling pathway in the liver of mice infected with Echinococcus granulosus

Rui MA, Cheng LIU, Shi-mei XU, Ming-xing ZHU, Jia-qing ZHAO, Qiao-feng WAN, Jia-jia ZHU, Wei ZHAO

2019, 37(3):  316-321.  doi:10.12140/j.issn.1000-7423.2019.03.013

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Objective To evaluate the effect of recombinant protein P29 (rP29), a 29 kDa antigen identified in hydatid cyst fluid which induces protective immunity, on the transforming growth factor-β (TGF-β)/Smad signaling pathway in the liver of mice infected with Echinococcus granulosus. Methods A group of 12 female BALB/c mice were subcutaneously immunized with 10 μg of rP29 emulsified with Freund’s adjuvant, then boosted twice with two weeks interval. Another two groups of mice were given with Freund’s adjuvant or PBS only as controls. The three groups of mice were intraperitoneally challenged with 1 500 protoscolex of E. granulosus per mouse two weeks after the last immunization. Twelve weeks after infection, the mice were euthanized and the liver tissues were collected. The TGF-β1 mRNA expression level was detected by RT-PCR in the liver of each infected mouse. The expression levels of TGF-β/Smad signaling pathway regulated downstream proteins such as TGF-RI, TGF-RII, p-Smad2,3, Smad4 and Smad7 were measured by Western blotting in the liver tissues. Results RT-PCR results showed that the relative expression level of TGF-β1 mRNA in the liver tissues of mice immunized with rP29 (1.24 ± 0.56) was reduced compared to PBS control mice (1.71 ± 0.29) with significant difference (P < 0.01), however, it was higher than that in adjuvant control mice (0.98 ± 0.42). The changes of the TGF-β1 mRNA expression level in the liver tissues were consistent with the TGF-β1 protein expression level detected by Western blotting. The expression density of TGF-β1 protein in the rP29 immunized group was 372.04 ± 0.01 (measured by densitometry) which was lower than that in the PBS group (673.02 ± 0.06), but higher than that in the adjuvant group (213.69 ± 0.31). The TGF-β/Smad signaling pathway-regulated downstream proteins also showed the similar changes except for Smad 7. The expression levels of TGF-β-RI, TGF-β-RII, p-Smad 2,3 and Smad 4 in the liver of rP29 immunized mice (357.59 ± 0.83, 289.07 ± 0.21, 204.06 ± 0.19, 396.11 ± 0.01, respectively) were reduced compared with the PBS group (496.89 ± 0.09, 378.41 ± 0.01, 428.11 ± 0.29, 566.95 ± 0.21, respectively), but higher than that of the adjuvant control group (171.99 ± 0.82, 185.77 ± 0.09, 128.09 ± 0.08, 205.87 ± 0.59, respectively). However, Smad 7 level expressed in rP29 immunized mice (278.89 ± 0.12) was up-regulated compared with PBS group (142.32 ± 0.07), but significantly lower than that in adjuvant group (384.17 ± 0.51). Conclusion Immunization of recombinant P29 protein significantly reduced the expression of TGF-β1 at both mRNA and protein levels in the liver of mice infected with E. granulosus. Most of the TGF-β/Smad signaling pathway-regulated downstream proteins (TGF-β-RI, TGF-β-RII, p-Smad 2,3 and Smad 4) were also reduced except for Smad 7 which was up-regulated.

Infection and genotype of Giardia lamblia among HIV/AIDS patients in Guangxi

Hua LIU, Ning XU, Yu-juan SHEN, Yuan HU, Jian-ping CAO

2019, 37(3):  321-325.  doi:10.12140/j.issn.1000-7423.2019.03.014

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Objective To investigate the co-infection rate and the genotype of Giardia lamblia among HIV/AIDS patients in Guangxi Autonomous Region. Methods Fecal specimens were collected from 285 HIV/AIDS patients and 303 HIV-negative people from Guangxi Autonomous Region. DNA were extracted from the feces and triosephosphate isomerase (TPI) gene of G. lamblia was amplified by nested PCR to determine the infection of G. lamblia. The amplified TPI PCR products were sequenced and the obtained sequences were aligned and analyzed by BLAST search. Results The overall co-infection rate of G. lamblia with HIV/AIDS was 2.8% (8/285). There was no G. lamblia infection detected in people without HIV-AIDS (P < 0.01). However, no significant risk factors were found to be related to the infection of G. lamblia among HIV-AIDS patients such as age, gender, sanitation, antiretroviral treatment or clinical manifestations (diarrhea, CD4⁺T lymphocyte level). Seven samples were identified to be zoonotic assemblage B and 6 had 100% sequence homology with the reported human-derived isolate. The other isolate was assemblage C and sequence identical to the reported human-derived isolate in our previous study. Conclusion HIV-AIDS patients have higher co-infection rate with G. lamblia than people without HIV infection. The major genotype of infected G. lamblia was assemblage B.

Effect of Angiostrongylus cantonensis infection on the expression of G-type lysozyme in Pomacea canaliculata

Zhi-yuan YUE, Yi ZHANG, Yun-hai GUO, Zhi-qiang QIN, Yun HUANG, Wei ZHANG, Guang-yao MAO

2019, 37(3):  326-331.  doi:10.12140/j.issn.1000-7423.2019.03.015

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Objective To evaluate the effect of Angiostrongylus cantonensis infection on the expression of G-type lysozyme in snail Pomacea canaliculate. Methods Primers were designed based on the DNA sequence coding for G-type lysozyme of P. canaliculate and the coding DNA was amplified by PCR with the primers from the snail tissue. The basic bioinformatics of P. canaliculate G-type lysozyme was analyzed using ExPASy, signalP 4.1, TMHMM2.0, Smart, MEGA7. P. canaliculate snails were infected with A. cantanensis by incubating with rat feces containing A. cantonensis first-stage larva for 24 h. The tissues of hepatopancrea, kidney, intestine, cephalopod and gill were collected from 3-5 P. canaliculate snails after being infected for 0, 1, 10, 20 and 30 days. The same number of uninfected snails were collected as negative control. Total RNA was extracted from these tissues and cDNA was synthesized by reverse transcription. Fluorescence quantitative PCR (qPCR) was used to analyze the tissue gene expression level of G-type lysozyme in P. canaliculata after being infected with A. cantonensis at different time points. Results The DNA coding for G-type lysozyme of P. canaliculata was amplified by PCR with the size of about 800 bp. The sequencing results showed that the coding DNA for G-type lysozyme of P. canaliculata was 828 bp encoding for an open reading frame of 275 amino acids. The P. canaliculate G-type lysozyme is a stable hydrophobic protein with a signal peptide at first 1-19 amino acids, mainly playing a role in the extracellular activity on the cell membrane. A SH3b domain is located between 18-85 amino acids. The phylogenetic analysis indicated that P. canaliculata G-type lysozyme is genetically close to the homologue in Physella acuta with 79% sequence identity. It clusters with homologues in other mollusks such as Argopecten irradians. qPCR results showed that the transcription level of G-type lysozyme in the hepatopancreatic tissues was significantly higher in female snail (2 238 ± 158) than in the male (685 ± 27) (P < 0.05), also significantly higher than that in other 4 tissues of the snail (P < 0.01). After being infected with A. cantonensis, the relative expression levels of G-type lysozyme in the tissues of hepatopanpancreas, kidney, intestine, gill and cephalopod all decreased to a certain extent compared with the tissues in un-infected snails. The relative expression levels of G-type lysozyme mRNA in gill 1, 10, 20 and 30 d after infection were 0.002 ± 0.002, 0.012 ± 0.050, 0.001 ± 0.000, and 0.004 ± 0.003, respectively, that were significantly lower than that in the un-infected snail (P < 0.01). The relative expression levels of G-type lysozyme in kidney were significantly lower than control only at 10 d and 20 d after infection (P < 0.05). The relative expression levels of G-type lysozyme was significantly lower than control in hepatopancreatic tissues at 1, 10 and 20 d after infection (P < 0.05). The relative expression levels of G-type lysozyme in the intestine at 1, 10, 20 and 30 d were 0.280 ± 0.040, 0.070 ± 0.030, 0.039 ± 0.002, and 0.120 ± 0.050, respectively, significantly lower than that in control group (P < 0.05). Similarly, the relative expression levels in cephalopod tissues were 0.280 ± 0.150, 0.150 ± 0.008, 0.031 ± 0.011, and 0.120 ± 0.030, significantly lower than that in control group (P < 0.05). Conclusion After being infected with A. cantonensis, the expression of G-type lysozyme gene in the tissues of gills, intestines and cephalopods of P. canaliculate was decreased for 1-30 d, decreased in hepatopancreas for 1-20 d and in kidneys for 10 d and 20 d after infection.

INFORMATION REPORTS

Analysis on the articles published in Chinese Journal of Parasitology and Parasitic Diseases from 2013 to 2018

Zheng-yan ZHANG, Feng-yun YI, Pin YANG, Qin CHEN, Jin CHEN, Fan YNAG, Hui-feng SHENG

2019, 37(3):  332-335.  doi:10.12140/j.issn.1000-7423.2019.03.016

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In order to understand the basic information for the articles published in the Chinese Journal of Parasitology and Parasitic Diseases from 2013 to 2018, all information related to the articles published in the journal during this period were collected and analyzed using method of bibliometrics. The statistic results showed that total 777 articles were published in the journal during the six years period with 51.2% (398/777) as original articles. Among the 428 original papers (including original articles, experimental studies, field studies, clinical studies, etc.), those related to the research of echinococcosis accounted for 18.7% (80/428), followed by the research of schistosomiasis (18.0%, 77/428). Among the research articles, experimental research accounted for 63.3% (271/428). Regarding author information, most of them came from disease prevention and control institutions (46.5%, 200/428) and universities (45.0%, 191/428), followed by the medical institutions (8.5%, 37/428). The authors from disease prevention and control institutions increased from 32.9% (25/76) in 2013 to 60.8% (45/74) in 2018, suggesting that researches in the field of disease prevention and control are the major source of authors who conduct the high level research work and contribute most to the publication of articles related to the parasitology and parasitic diseases.

Global health cooperation strategy for National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention: a SWOT analysis

Wei DING, Lu-lu HUANG, Xue-jiao MA, Hong-mei LI, Lei DUAN, Ying-jun QIAN, Duo-quan WANG, Ya-yi GUAN, Ning XIAO, Xiao-nong ZHOU

2019, 37(3):  336-342.  doi:10.12140/j.issn.1000-7423.2019.03.017

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To identify the current situation and the future direction for National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention (NIPD, China CDC) engaging in global tropical disease prevention and control, a SWOT analysis (strengths, weaknesses, opportunities, threats) was conducted among NIPD’s senior-level staff, directors and global health center staff, through a questionnaire survey on their perceptions of the internal advantages/disadvantages, external opportunities/threats faced by NIPD in global health cooperation. Four aspects of strategies were identified by analyzing the strengths-opportunities, weaknesses-opportunities, strengths-threats and weaknesses-threats combining with the survey results. The identified strategies suggested NIPD to adapt China’s experience into local settings and consider the demand of local partners during cooperation in global health, while improving its management mechanism of human resource and international cooperation, defining strategic work plan, preparing fundamental reserves such as human resource, technology and products to enhance overall capacities engaging in the global health.

Current status of toxoplasmosis misdiagnosis in clinic

Hai-ying SU, Shu-jun YANG, Hong-juan PENG, Chun-mei WANG

2019, 37(3):  342-345.  doi:10.12140/j.issn.1000-7423.2019.03.018

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To understand the current status of Toxoplasmosis misdiagnosis in clinic, information for the misdiagnosed Toxoplasmosis were collected by searching through PubMed and Tsinghua Tongfang CNKI database from January 2000 to August 2018 and analyzed regarding the type of diseases to which the Toxoplasmosis was misdiagnosed, the location of the infection and lesion, the method of definitive diagnosis, co-infection with HIV and organ transplantation. The possible causes for the misdiagnosis were further discussed. Total 70 related literatures were obtained and a total of 251 misdiagnosed cases were collected. The analytic results showed that Toxoplasmosis was misdiagnosed to 61 different types of diseases. The top two misdiagnosed diseases were iridocyclitis (11.6%, 29/251) and retinitis (8.8%, 22/251) in eyes. The most common locations of the infection and lesion were in brain (33.5%, 84/251), eye (25.5%, 64/251) and liver (12.4%, 31/251). 132 cases were positive for Toxoplasma antibody by serological test; 68 cases (including 25 death cases) were observed with Toxoplasma gondii trophozoites or cysts in tissues by histopathological examination; 13 cases were diagnosed by identification of T. gondii in blood, body fluid and puncture fluid under microscope. Out of the total 251 misdiagnosed Toxoplasmosis, 49 had co-infection with HIV with symptoms of AIDS, 13 had received bone marrow transplantation and 2 received kidney transplantation.

REVIEWS

Research progress on detection technology of Echinococcus and echinococcosis

Hong-rang ZHOU, Mu-xin CHEN, Ying WANG, Qing YU, Ning XIAO

2019, 37(3):  346-352.  doi:10.12140/j.issn.1000-7423.2019.03.019

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Echinococcosis is a chronic parasitic disease caused by the infection of Echinococcus larvae. Due to the complex life cycle involved with a large number of hosts, it is very challenging to control the spread of infection and eliminate echinococcosis. Early and accurate detection of Echinococcus parasite, real-time monitoring of various types of host infection are very critical for blocking the epidemic and the transmission of the disease. This paper reviews the recent advances in the detection technology for Echinococcus parasite and the related research on the diagnosis of echinococcosis, including morphological identification, imaging diagnosis, immunological and molecular biology diagnostic techniques, in order to provide useful information for the advanced detection of Echinococcus infections and diagnosis of echinococcosis.

Review on epidemiological characterization of asymptomatic malaria infection and its risk factors

Xiao-xiao WANG, Shui-sen ZHOU, Fang HUANG, Zhi-gui XIA, Shao-sen ZHANG

2019, 37(3):  352-359.  doi:10.12140/j.issn.1000-7423.2019.03.020

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Asymptomatic Plasmodium infection plays an important role in malaria transmission. The research on the epidemiological characterization of asymptomatic malaria infection poses high public health significance for malaria elimination. Prevalence of asymptomatic infections was documented to be highly diversified, depending on different host, Plasmodium, environment and vector breeding. This review updates definition, epidemiology and risk factors of asymptomatic malaria infections, in order to provide data for malaria elimination.

SHORT COMMUNICATIONS

Study on the optimal intermediate host of Fasciola gigantica in Dali

Wen FANG, Tian-mei LI, Ting LI, Jian-ping ZHANG, Ming-ming HAO, Yu-hua LIU, Shao-rong CHEN

2019, 37(3):  360-363.  doi:10.12140/j.issn.1000-7423.2019.03.021

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To identify the optimal intermediate host for Fasciola gigantica in Dali so as to provide information for developing control measures, the eggs of F. gigantica were collected and incubated in water at 28 ℃until being hatched to miracidia. The hatched miracidia were used to infect three different snails: Physa acuta, Radix swinhoei, and Galba previa collected from Dali area in a 24-well plate with a ratio of 1 : 3 for snail and miracidia. Three kinds of snails were randomly divided into the infected group and the un-infected group. The number of snail in the infected group was 141 (P. acuta), 240 (R. swinhoei) and 251 (G. previa). The number of un-infected snails was 124(P. acuta), 136 (R. swinhoei) and 87 (G. previa). Four hours after infection, the snails were placed in incubation pots with 48 to 60 snails for each pot at room temperature. The un-infected group was placed directly into a snail basin, which was fed in the same way as the infected group. The dead snails were picked out daily and the development of the larvae in the snail was observed under a microscope. The room temperature and water temperature were recorded daily. When the larvae in the snail developed to rediae stage, a plastic membrane was placed on the surface of water in the pot to collect metacercaria. The results showed that the miracidia were hatched from eggs on day 11. In infected groups, snail P. acuta and R. swinhoei could survive till 72 days and G. previa survived till 53 days. The culture room temperature maintained at 21 ℃ to 27 ℃ (average 24 ℃), and the water temperature was maintained at 21 ℃ to 25 ℃ (average 23 ℃). All 141 P. acuta snails were negative for the infection, 12 kept survived and the rest died on day 72. For infection in 240 R. swinhoei, only one was infected and died on the day 28, 36 were survived till 72 days and the rest died. Total 112 out of 251 infected G. previa snails were positive with an infection rate of 44.6%, but all died 53 days after infection. In un-infection groups, snail P. acuta and R. swinhoei could survive till 77 days and G. previa survived till 57 days. In the un-infection groups, all 124 P. acuta snails were negative and 8 were survived till 77 days, all 136 R. swinhoei snails were negative and 30 survived till 77 days, all 87 G. previa snails were negative and died before 57 days. No sporocyst was observed in all positive snails. In the only one infected P. acuta, mother rediae was observed. The stages of rediae and mother rediae were observed on day 11, 23 respectively. The daughter rediae containing the immature and mature cercaria were observed on day 26, 30 respectively. The cercariae were observed in the body of dead G. previa 40 days after infection. The metacercaria were obtained on the day 40 after infection and collection lasted 11 days. Total 511 metacercaria were obtained. Most of the metacercaria were collected at 8:00 in the morning (97.7%, 499/511) and some were collected at 2:30-4:30 in the afternoon (2.3%, 12/511). During this collection period, 29 G. previa kept alive and 24 of them were infected (21.4%, 24/112). The average of 21 metacercaria were formed from each infected snail.

Expression of fusion protein of Sj28GST epitopes and cholera toxin B subunit in baculovirus/insect cells

Yu-mei XU, Shi-de CAO, Chuan-gang ZHU, Shi-qing ZHANG

2019, 37(3):  364-367.  doi:10.12140/j.issn.1000-7423.2019.03.022

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To induce mucosal protective immunity for schistosomiasis, 4 major epitopes from a leading vaccine antigen of Schistosoma japonicum 28 000 glutathione-S-transferase (Sj28GST) was fused with cholera toxin B subunit (CTB) as a mucosal adjuvant and the fusion protein was expressed as recombinant protein in insect cell Sf9 using Bac-to-Bac baculovirus expression system. The coding DNA for fusion CTB-Sj28GST was PCR amplified from previous constructed plasmid and then subcloned into transposed plasmid pFastBac using SalⅠand SphⅠsites. The recombinant pFastBac-CTB-Sj28GST plasmid DNA was transformed into DH10Bac for homologous recombination. The successfully obtained recombinant viruses were identified by PCR using M13 universal primers and CTB-Sj28GST specific primer and then transfected into Sf9 insect cells. The recombinant viruses were amplified by repetitively infecting Sf9 cells. The expressed recombinant fusion CTB-Sj28GST in infected insect cells was identified by indirect immunofluorescent assay (IFA), and the expressed fusion protein in the cell lysates identified by Western blotting with polyclonal antibody against Sj28GST. Results demonstrated that a 519 bp DNA fragment containing 4 epitopes of Sj28GST (189 bp) fused with CTB coding DNA was successfully amplified and cloned into pFastBac plasmid to construct pFastBac-CTB-Sj28GST. The obtained recombinant viruses from infected Sf9 cells contained the same size of fusion DNA identified by PCR. The transfected Sf9 insect cells enabled to express fusion CTB-Sj28GST identified by IFA with anti-Sj28GST specific antibody. Western blotting showed a specific band at the M_r 22 000, the same size as the expected molecular weight of fusion CTB-Sj28GST, which could be recognized by the polyclonal antibody against-Sj28GST.

Mitochondrial cox3 based PCR detection of the first imported case of Plasmodium ovale in Guizhou province

Yu-ting HUANG, Tian-yi HUANG, Li-dan LU, Dan-ya SHE, Shi-jun LI

2019, 37(3):  368-371.  doi:10.12140/j.issn.1000-7423.2019.03.023

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To develop a PCR-based detection of Plasmodium ovale infection, DNA was extracted from blood of a patient diagnosed as the first imported case of P. ovale infection in Guizhou province, two sets of PCR based on the Plasmodium ribosomal RNA small subunit (SSU rRNA) and mitochondrial cytochrome oxidase subunit 3 gene(cox3) were established. The genus, species-specific primers were designed accordingly, the specificity of Plasmodium species detection was performed using the designed primers and the amplified cox3 PCR products were sequenced. The obtained sequences were aligned with reference sequence of the P. ovale wallikeri subspecies(GenBank accession number: HQ712053.1)and the curtisi subspecies(GenBank accession number: HQ712052.1)using DNAMAN V6. A phylogenetic tree was constructed based on the Plasmodium cox3 gene using the neighbor-joining method and Mega 7.0.26 software. The PCR results showed that only a genus-specific band with 1 200 bp was obtained based on Plasmodium SSU rRNA and no species-specific bands were amplified. However, a PCR product with 880 bp was amplified from the patient sample based on Plasmodium cox3 gene. The Blast alignment showed that the obtained cox3 sequence shared 99.4% identity with P. ovale wallikeri subspecies and 97.4% with curtisi subspecies.

A survey of Clonorchis sinensis and soil-transmitted nematode infections in Qingxin District of Qingyuan City, Guangdong Province

Wei-qiang LUO, Xue-liang ZHANG, Zhi-shan ZHOU, Xiu-hong FAN, Xing-ru LI, jie LI

2019, 37(3):  372-375.  doi:10.12140/j.issn.1000-7423.2019.03.024

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To understand the infection status and infection risks of Clonorchiasis sinensis and soil-transmitted nematodes (STNs) in population in Qingxin district of Qingyuan city, Guangdong Province, a survey was performed in this area from October to December, 2018. Fecal samples were collected from people living in this area and the eggs of C. sinensis, hookworm, roundworm and whipworm in the collected feces were examined under microscope in duplicate using modified Kato-Katz thick smear method. The larvae were cultured from hookworm egg positive feces using filter tube method to determine hookworm species. A transparent adhesive tape test around anus was used to examine pinworm eggs for children with 3-9 years old. Total 1 254 residents were participated in this survey and 30 of them were infected with C. sinensis (2.3%), 36 infected with STNs (2.7%). Among them, the infection rate for hookworm was 0.3%, whipworm 0.2%, roundworm 0.5% and pinworm of 1.9%. The infection rate of C. sinensis in male people (3.0%, 22/747) was higher than that in female (1.6%, 8/507), however, the difference was not statistically different (P > 0.05). With respect to the different occupations, age and education related to the infection of C. sinensis, people with farmer as occupation (7.9%, 10/126), age over 60 (4.2%, 11/260) (P < 0.01) and education lower than elementary school (3.6%, 14/386）(P < 0.01) had the highest infection rates. The risk for infection of C. sinensis was positively correlated with the eating habit with raw fish or fish porridge (P < 0.01). The STN infections in male was 3.2%（24/747), which was higher than that in female (2.4%, 12/507), but without significance (P > 0.05). The highest STN infection rates happened in pre-school children (11.4%, 23/201), children with age 3-9 years old (9.5%, 24/252) and people with education lower than elementary school (8.6%, 33/386). The risk for STN infections was positively correlated to the lack of washing hands before eating and after pooping (P < 0.01). This survey results suggested that prevention and control of C. sinensis infection should focus on the intervention of the people with low education level and elder population. More intervention measures of prevention and control of STN infection should focus on the children and people with low education.

Effects of Cu²⁺ stress on the expression of metallothionein mRNA in Pomacea canaliculata

Guang-yao MAO, Yun-hai GUO, Yi ZHANG, Ning XIAO

2019, 37(3):  375-378.  doi:10.12140/j.issn.1000-7423.2019.03.025

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Fifteen Pomacea canaliculata were exposed to 100 μg/L Cu²⁺ for 0, 1, 7, 14 and 21 days. The total RNA was isolated from livers of three P. canaliculate at each time points and the expression level of P. canaliculate metallothionein (PcMT) mRNA was detected by real-time fluorescence quantitative PCR. The DNA coding for the full-length PcMT was amplified from P. canaliculata total cDNA, and then cloned into the bacterial expression vector pET28a. The recombinant plasmid DNA was sequenced to confirm the correct insert, then transformed into E. coil BL21 (DE3). The recombinant PcMT protein was expressed in BL21 under induction of IPTG and analyzed by SDS-PAGE. The results showed that the expression of PcMT mRNA in the liver of P. canaliculate was continuously upregulated upto 14 days under stress of 100 μg/L Cu²⁺ and reached to a peak value of 2.1 ± 0.2, then decreased to 1.1 ± 0.1 on Day 21. The full length DNA of PcMT gene was about 300 bp. The correct insert in the constructed recombinant pET28a-PcMT plasmid was confirmed by PCR, double digestion and DNA sequencing. The recombinant PcMT protein was successfully expressed in BL21 under induction of IPTG and conformed by SDS-PAGE with relative molecular weight (M_r) of 17 000. These results provide information for further study on the dynamic expression of PcMT in P. canaliculata under stress of heavy metals and its role in the viability of the snail.

Surveillance on the prevalence of soil-transmitted nematode infections in Ningxia during 2016-2017

Hong-ju DUAN, Rong MA, Yi-ren FU, Xiang-lin WU, Tian-bo MA, Rong-ting QI

2019, 37(3):  379-381.  doi:10.12140/j.issn.1000-7423.2019.03.026

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To understand the current prevalence of soil-transmitted nematode (STN) infections in Ningxia, total 3 counties (Helan County, Haiyuan County and Pingluo County) were chosen and 5 villages were selected from each county, based on the evenly geographical distribution, as survey sites. Each site included 200 permanent residents with age of over 3 years old. The stool samples were collected from each enrolled villager and the eggs of STNs were examined under microscope in duplicate using modified Kato-Katz thick smear method. Questionnaire was used to collect the information of the respondents. Results showed that an overall prevalence of STN infections in Ningxia from 2016-2017 was 1.4% (83/6 047). The STN prevalence in Haiyuan County (2.2%, 45/2 059) was higher than that in other counties with significant difference (P < 0.05), however, there was no significant difference in prevalence between females (1.6%, 50/3 099) and males (1.1%, 33/2 948) (P > 0.05). Participants with age over 60 or between 40-59 had the highest prevalence (1.7%, 26/1 519 and 1.8%, 33/1 852, respectively) with statistical significance compared to that in other age groups (P < 0.05). Farmers had higher infection rate (1.6%, 71/4 528) than people with other occupations, and participants with education of high school and above had higher infection rate (1.6%, 17/1 034）（P > 0.05) than people with other education levels, but these difference had no statistical difference. The infection of soil-transmitted nematodes keeps a decreasing trend in Ningxia, and it is necessary to adopt different measures and guidance in different areas to reduce the infection.