Table of Content

30 August 2019, Volume 37 Issue 4

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INVUTED REPORTS

The success of parasitic disease control programs declares a substantial vitality of the socialistic system in China

Sen-hai YU

2019, 37(4):  381-382.  doi:10.12140/j.issn.1000-7423.2019.04.001

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On the 70th anniversary of the People’s Republic of China, the author looks back to the national programs for parasite control started in the early 1950s and summarizes their accomplishments. On the nationwide scale, the transmission of schistosomiasis has been controlled, no indigenous case of malaria reported in 2017, lymphatic filariasis eliminated since 2006, local prevalence or sporadic cases of visceral leishmaniasis reported only in the western region, and soil-transmitted nematode infections decreased to a fairly low level which poses no more threat in most previously endemic areas. Evidently, these achievements come from the socialistic system which endorses a firm government commitment, the dedicated technical teams, and the community involvement. Though an outstanding success among the developing countries, continued endeavors are needed to finally eliminate the above-mentioned parasites but also to control hydatidosis, clonorchiasis and other zoonotic parasitic infections.

The historical witness of the elimination of lymphatic filariasis in China

De-jian SUN

2019, 37(4):  383-387.  doi:10.12140/j.issn.1000-7423.2019.04.002

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China used to be one of the countries with the most serious epidemic of lymphatic filariasis in the world. Since the founding of New China, the Chinese Government has attached great importance to lymphatic filariasis control and included it into key disease prevention and control plans. Measures have been systematically and comprehensively adopted to the investigations, prevention and treatment, vector control and scientific research on filariasis that resulted in the eventual elimination of lymphatic filariasis nationwide by 2006. The author is one of the researchers and participants involved in the control of lymphatic filariasis in China. This paper retrospectively reviewed the several important time points that turned the filariasis under effective control.

Establishment and innovative practice of Integrative System of Prevention, Diagnosis and Management for echinococcosis in China

Aini ABUDUSALAMU, Ying-mei SHAO, Aji TUERGANAILI, Wen-bao ZHANG, Ren-yong LIN, Yan-yan HOU, Zhuang-zhi ZHANG, Hao WEN

2019, 37(4):  388-394.  doi:10.12140/j.issn.1000-7423.2019.04.003

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Echinococcosis (hydatid disease) is a neglected tropical zoonotic disease caused by the infection of Echinococcus larvae. It prevails globally and China has the highest disease burden of two main types of echinococcosis including cystic echinococcosis (CE) and alveolar echinococcosis (AE). The hydatid disease is a big threat of public health and a heavy burden of economic lose in China. The AE cases in western China account for over 90% of the total AE cases in the world. Great achievements have been made in the control and treatment of the diseases since the founding of People’s Republic of China, including remarkable improvement in clinical cure rate, significant reduction in morbidity and mortality in humans and infections in animals. China has made inspiring achievements in the research of the genomics, molecular epidemiology, immunology, diagnostic tools, treatment approaches, prevention strategies and vaccine development of echinococcosis especially since the beginning of the 21th century. This paper mainly described and discussed the establishment and innovative practice of Integrative System of Prevention, Diagnosis and Management for echinococcosis in China, which includes systems for scientific prevention and control, standardized diagnosis, innovative surgery and treatment, and relevant research and translational application.

China’s control process of soil-transmitted helminth infections from pandemic to low-endemic levels

Ying-dan CHEN, Ji-lei HUANG, Hui-hui ZHU, Ting-jun ZHU, Men-bao QIAN, Shi-zhu LI, Xiao-nong ZHOU, Mi-zhen ZHANG, Chang-hai ZHOU

2019, 37(4):  395-398.  doi:10.12140/j.issn.1000-7423.2019.04.004

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This paper retrospectively reviewed the great achievements in the control of soil-transmitted nematode (STN) infections nationwide since the founding of the People’s Republic of China. Based on the data of three national surveys including the 1st survey for the distribution of human parasitic diseases during 1988-1992, the 2nd survey for the status of human parasitic diseases during 2001-2004 and the 3rd survey of major human parasitic disease during 2014-2016, the infection rate of STNs has decreased dramatically from pandemic to the low-endemic levels after a few decades’ efforts. The role of the adopted control strategies and measures associated to the effective control of STN infections was discussed and the further measures to maintain the control was proposed.

ORIGINAL ARTICLES

Mutations and predicted structure change of G6PD isolated from a patient with primaquine-induced hemolysis in Yunnan Province

Ying DONG, Shu-ping LIU, Yan-chun XU, Yan LIU, Yan DENG, Meng-ni CHEN

2019, 37(4):  399-405.  doi:10.12140/j.issn.1000-7423.2019.04.005

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Objective Glucose-6-phosphate dehydrogenase (G6PD) deficiency is related to primaquine-induced hemolysis. This study aims to determine the mutations of G6PD gene isolated from a patient with primaquine-induced hemolysis and the possible structure change related to the pathology. Methods Blood sample was collected from a primaquine-induced hemolysis patient infected with Plasmodium vivax. The G6PD enzyme activity was measured to reduce up to 75% in this patient. The genomic DNA was extracted from the blood sample, and G6PD gene fragments containing exon2, exon3-7, exon8-9 and exon10-13 were amplified by PCR using primers designed based on the G6PD coding sequence. The amplified PCR products were sequenced to obtain the full-length coding sequence of G6PD and then aligned with the wildtype (GenBank: NC_000023.11) and mutant (GenBank: X55448.1) G6PD sequences to identify the mutations using MEGA5.04 software. The 3D structure of G6PD was predicted based on the deduced amino acid sequences by SWSS-MODEL(www.swissmodel.expasy.org/interactive) and modified by PyMOL 2.2.0 software. Results The DNA fragments containing G6PD exon2, exon3-7, exon8-9, exon10-13 were amplified from genomic DNA isolated from the blood sample of a P. vivax infected patient with primaquine-induced hemolysis, with the length of 336 bp, 2 277 bp, 976 bp and 1 421 bp, respectively. The full-length DNA of 1 545 bp coding for G6PD of the patient was obtained by aligning with reference wild-type and mutant G6PDs deposited in GenBank. Alignment of patient’s G6PD sequence with wild-type and mutant reference sequences identified two mutations with c.1 311T > C and c.1 376G > T. The first mutation(c.1 311T > C) was synonymous without changing the coding amino acid(Y), the second mutation(c.1 376G > T) leads to the coding amino acid changed from Arg(R) to Leu(L) at position 459. However, the mutation at position 459 was far away from the binding site of G6PD with NADP⁺ and glycolic acid ligands. The structure of patient G6PD could be the dimer form or tetramer form with the dimer more stable according to the GMQE and QMEAN modeling systems. Compared with the wild-type model of the dimer G6PD (6e07.1.A), the patient G6PD mutant amino acid at 459 was on the surface of the structure, and the binding region with NADP⁺ ligands contained 16 residues including 238 and 357 positon amino acids, similar to the wild-type form. However, the 3D structure of patient’s G6PD tetramer form was not as stable as wild-type G6PD. The ligand binding regions of glycolic acid contained only 6 residues including 47 position amino acid which is less than 11 residues of wild-type model. Conclusion The sequence of G6PD isolated from a P. vivax infected patient with primaquine-induced hemolysis contained a synonymous mutation of c.1 311T > C and a nonsynonymous mutation of c.1 376G > T compared to the wild-type G6PD. The nonsynonymous mutation at c.1 376G > T leads to the coding amino acid change from Arg(R) to Leu(L) at postion 459 which may not change the dimer structure, but interfere with the formation of tetramer structure of G6PD.

Investigation on malaria vectors in Jinghong, a border area in Yunnan Province

Rui YANG, Yu-ting ZHENG, Xiao-yu YANG, Li-min DONG, Zu-rui LIN, Yao-wu ZHOU, Xu-can ZENG, Hong-bin LI, Jin-yong JIANG

2019, 37(4):  406-410.  doi:10.12140/j.issn.1000-7423.2019.04.006

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Objective To understand the species, ecology of mosquitoes involved in the transmission of malaria in Jinghong, a border area in Yunnan Province. Methods A village called Manghuilong with historically high incidence of malaria along the border area in Jinghong City, Yunnan Province, was selected as a pilot site for the investigation of mosquitoes involved in the transmission of malaria. Mosquitoes were captured with light in 5 living houses and livestock houses evenly located in the village three days a month from June to October in 2015. The mosquito density was recorded in each house. The host species of blood in the stomach of captured mosquitoes were identified by multiple PCR to determine their bloodthirsty habit. The nocturnal activities of mosquitoes were observed by tent trap in and out of the village. The Plasmodium-specific primer-based nest PCR was used to detect sporozoite infection in captured mosquitoes. Results A total of 1 286 mosquitoes belonged to 13 species of Anopheles were captured, including 936 An. sinensis (72.8%) and 188 An. minor (14.6%) as dominant species. The main source of Anopheles was from livestock houses, accounting for 85.6% (1 101). PCR detection on blood from 125 An. sinensis showed that all blood was from pig, no one from human source. The biting incidence of An. sinensis on human in the village was 0.6 per person per hour, and outside village was 0.2 per person per hour. The peak of nocturnal activity was from 8 : 00 pm to 10 : 00 pm. Results of nested PCR on 101 An. minimus, 517 An. sinensis and 40 An. multiflora showed that no Plasmodium sporozoite infection was detected in all captured mosquitoes. Conclusion An. sinensis was the dominant species of mosquito in Jinghong, Yunnan Province, followed by An. minimus. However, An. sinensis mainly feeds on blood of domestic livestock, not on humans. There was no Plasmodium infected mosquito detected in this area.

Investigation on the status of major human parasitic infections in Sichuan Province in 2015

Jing-wen LUO, Hong-chun TIAN, Hong XIE, Yang LIU, Bo ZHONG, Zi-song WU, Lin CHEN, Ji ZHANG, Yi-lan CHEN, Li LI, Xi GONG, Li-ping ZHANG

2019, 37(4):  411-416.  doi:10.12140/j.issn.1000-7423.2019.04.007

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Objective To understand the endemic status of major human parasitic infections in Sichuan Province. Methods According to the National Survey Plan and Implementation Rules for the Status of Human Major Parasitic Infections, total 45 survey sites were selected from 15 counties (cities and districts) within 5 ecological zones of Sichuan Province using stratified cluster and randomly sampling method for investigation of major human parasitic infections from April to June in 2015. No less than 250 rural permanent villagers (above 0 year old) were enrolled from each site in the survey and the fecal samples were collected from each participant. The intestinal helminth eggs were examined under microscope using modified Kato-Katz thick smear method (duplicate for each sample). The intestinal protozoan cysts and trophozoites were examined using Lugol’s iodine staining method and saline smear method, respectively. The Enterobius vermicularis eggs were identified using transparent tape anal swab method for children with 3-6 years old. SPSS 23.0 software was used for statistical analysis. The infection difference among different population and areas was compared by χ² test. Unconditional logistic multivariate regression analysis was used to analyze the risk factors of common parasitic infections. Results Total 11 403 participants were involved in the survey with overall prevalence of 15.5% (1 762/11 403) for all intestinal parasite infections. Five intestinal nematodes were identified in infected people including Ascaris lumbricoides (4.7%, 540/11 403), hookworm (7.1%, 811/11 403), Trichuris trichiura (3.2%, 360/11 403), Enterobius vermicularis (0.1%, 10/11 403) and Strongyloides stercoralis (0.1%, 6/11 403). Three tapeworms were identified with 55 cases of Taenia infections, 3 cases infected with Hymenolepis and 1 case with Spirometra mansoni. Total 9 protozoan species were identified in infected people including Entamoeba histolytica, E. coli, Gardia lamblia, Blastocystis hominis, E. hartmani, Endolimax nana, Balantidium coli, Iodamoeba butschlii and Embadomonas intestinalis with overall prevalence of 2.0% (223/11 403). The highest prevalence of intestinal parasite infections of 24.9% (563/2 257) was determined in the Southwest Sichuan-North Yunnan mountainous ecological zone and lowest (8.2%, 186/2 273) in the Eastern Tibet-Western Sichuan ecological zone, with statistical significance for the different prevalence among different ecological zones (P < 0.01). In terms of the distribution of parasitic infections, Leibo County had the highest prevalence of 53.9% (407/755), and Ruoergai County had the lowest of 1.9% (10/522), with significant difference among different counties (cities and districts) (P < 0.01). The prevalence of intestinal parasites in females (16.2%, 994/6 156) was significant higher than that in males (14.6%, 768/5 247) (P < 0.05). Regarding the age, the highest prevalence was identified in people aged 70-79 and over 80 years (21.4%, 221/1 034, 46/215) and the lowest prevalence in people aged 20-29 (12.3%, 100/813) and 40-49 (12.3%, 273/2 217), with significant difference of prevalence among different age groups (P < 0.01). Regarding the education distribution, the highest prevalence of intestinal parasites was found in people with low education of primary school (17.4%, 931/538) and the lowest in people with high school and secondary school (5.5%, 19/346), with significant difference among people with different educational levels (P < 0.01). Regarding the occupation distribution, the highest prevalence of 16.5% (1 397/8 475) was found in farmers, followed by school students (12.7%, 190/1 491), with significant difference among different occupations (P < 0.01). Regarding the ethnic distribution, the highest prevalence was found in Yi people (65.0%, 253/389), followed by Han (16.0%, 1 255/7 857), with significant difference among different ethnic groups (P < 0.01). Unconditional logistic multivariate regression analysis demonstrated that female, elders, Yi nationality and people with low education level were susceptible populations of intestinal parasite infections. Conclusion The soil-transmitted nematodes A. lumbricoides, hookworm and T. trichiura are the main species infecting people in Sichuan Province. The prevention and control measures should be focused on female, elders, Yi nationality and people with low education level, and those living in Southwest Sichuan-North Yunnan mountainous and Sichuan basin ecological zones who have the highest prevalence of intestinal parasites.

Infection and species identification of Anisakis larvae in sea fishes along Fujian coast

Chen-xin LIN, Shu-ling HUANG, Shi-han LIN, Dian-wei JIANG, Han-guo XIE

2019, 37(4):  417-421.  doi:10.12140/j.issn.1000-7423.2019.04.008

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Objective To understand the infection status and species of the larvae of Anisakis nematodes in sea fishes along Fujian coast. Methods The samples of marine fishes were randomly collected from three major fisheries along Fujian coast (Eastern, Middle and Southern) during January 2016 to December 2018. The fish species were identified and the Anisakis larvae were isolated from the visceral and muscle tissues of the infected fishes. The species of infected Anisakis spp. was examined and identified under dissecting microscope. The data of fish species, Anisakis species and infection rate were collected and analyzed. Results A total of 810 marine fishes classified as 32 species were examined, and the overall infection rate of Anisakis nematodes was 34.2% with average 11.2 worms in each infected fish. The fish species with highest infection rates of the nematode were Trichiurus lepturus (85.6%, 95/111), Scomberomorus niphonius (63.5%, 47/74), Ditrema temmincki Bleeker (36.7%, 67/162) and Plectorhynchus nitens (36.7%, 18/49), with average 23.1, 7.6, 4.5 and 4.2 larvae for each infected fish, respectively. The distribution of Anisakis larvae differed in different fish species, commonly seen in the mesenteric and intestinal wall, body cavity and gastric wall of those infected fishes followed by livers, pancreas and genitals, rarely seen in the muscle. Total four species of Anisakis nematodes were identified in the larvae collected from the fishes: Anisakis type Ⅰ, Anisakis type Ⅱ, Hysterothylacium amoyense and other Hysterothylacium. Conclusion The infection rate of Anisakis larvae in sea fishes along Fujian coast is high, and more prevention and control measures should be adopted and strengthened.

Prevalence and molecular characteristics of Cryptosporidium infection in pre-weaned dairy calves in Sichuan Province

Ding JIA, Jia-ming DAN, Yan SUN, Jing-yi LUO, Zi-qi ZHU, Xue-ping ZHANG, Hai-feng LIU, Zi-yao ZHOU, Xiao-ping MA, Guang-neng PENG, Zhi-jun ZHONG

2019, 37(4):  422-427.  doi:10.12140/j.issn.1000-7423.2019.04.009

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Objective To understand the infection and molecular characteristics of Cryptosporidium spp. in pre-weaned dairy calves in Sichuan Province. Methods The fecal samples were collected from 278 pre-weaned dairy calves with age less than one month in 11 dairy farms(7 in-house farming and 4 free-range farming) located in 10 different regions of Sichuan Province from June 2016 to March 2017. The parasites in the collected fecal samples were concentrated using a modified floatation method with saturated sucrose solution and the fecal parasite DNA was extracted. The small subunit ribosomal RNA(SSU rRNA) of Cryptosporidium was amplified from the fecal DNAs by nested-PCR. The PCR positive fecal samples were used to calculate Cryptosporidium infection rate in these examined caves. Chi-square test was used to compare the difference of infection rates between in-house farming and free-range farming calves. The amplified SSU rRNA PCR products were sequenced and the obtained sequences were BLAST searched against Cryptosporidium sequences deposited in GenBank to identify the infected species. The phylogenetic tree was constructed based on the SSU rRNA sequences by MEGA 7 adjacency method. The M_r 60 000 glycoprotein(gp60) gene was further amplified from the samples identified as C. parvum by SSU rRNA sequencing to further determine their genotypes. Results A total of 278 calves were investigated and the fecal samples were collected from each of them. The SSU rRNA amplification results identified the total infection rate of Cryptosporidium was 14.4% (40/278) in the examined calves. Cryptosporidium infection was found in 10 of the 11 examined farms, among which the highest infection rate was 35.7% (10/28) in Mianyang followed by Aba(35%, 7/20) and Ziyang (30.8%, 8/26). No Cryptosporidium infection was found in Meishan. The infection rate was statistically different among the 10 positive farms(P < 0.05). The Cryptosporidium infection rate in calves in in-house farms was 17.4% (34/195) which is significantly higher than that in free-range farms (7.2%, 6/83) (P < 0.05). Among the 40 Cryptosporidium-positive samples, 28 were identified as C. bovis, 7 as C. parvum and 5 as C. ryanae. After removing the identical sequences, total 10 unique sequences were obtained (ABC881, AYC6953, AYC6969, CDC16111, CDC16117, DYC014, MYC116, MYC117, MYC126, ZYC6874, GenBank login number: MF671870-MF671879). Among them AYC6953, AYC6969, CDC16117 and ZYC6874 were 99% identical to C. bovis (JX515546), DYC014 is 99% identical to C. ryanae (HQ179574), MYC126 is 99% identical to C. bovis (JX416366). ABC881, CDC16111, MYC116 and MYC117 is 100% identical to C. parvum (AH006572), C. ryanae (HQ179574), C. bovis (HQ179573) and C. bovis (MH166335), respectively. The 7 C. parvum-positive samples were further determined as IIdA15G1 subtype which is 100% identical to the Ningxia isolate of KM067092, based on gp60 gene sequencing results. Conclusion The Cryptosporidium infection was prevalent in pre-weaned dairy calves in Sichuan Province and the major infected species were C. bovis, C. parvum and C. ryanae.

Risk factors of echinococcosis in Shiqu County, Sichuan Province

Wei HE, Qian WANG, Yan HUANG, Wen-jie YU, Guang-jia ZHANG, Sha LIAO, Qi WANG, Liu YANG, Fan CHEN, Rui-rui LI, Bo ZHONG

2019, 37(4):  428-432.  doi:10.12140/j.issn.1000-7423.2019.04.010

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Objective To analyze the main influencing and risk factors related to the epidemic of human echinococcosis and their impacts on the prevalence of echinococcosis as a basis for providing a strategy for the better control of echinococcosis in the region. Methods The data of an epidemiological survey on echinococcosis in Shiqu, Sichuan, from November 2015 to June 2017 were collected to identify the factors related to the endemic of echinococcosis as independent variables, and the population prevalence of echinococcosis as dependent variables. Linear regression analysis was conducted to identify the factors with statistical significance related to the endemic of echinococcosis. A multiple linear regression model was established to analyze the factors that significantly effect the prevalence of echinococcosis in the region. Results The results of single factor linear regression analysis showed that the average number of dog per household, the infection rate of dogs, the average number of sheep per household, the average number of cattle per household, the prevalence rate of cattle, the average slaughtered number of cattle and sheep per household, the density of small mammals, and people’s awareness for the knowledge to prevent and control echinococcosis were all the influencing and risk factors (with b = 5.278, 0.818, 0.183, 0.274, 0.234, 3.035, 0.038, -0.539, respectively, P < 0.05). The average number of dog per household has the greatest impact. The prevalence rate of the population could increases by 5.278 times if one dog would be increased in each household. The results of multiple linear regression analysis showed that the knowledge awareness(X9), the average number of dog per household (X1), the infection rate of dogs (X2) and the density of small mammals (X7) had significant effect on the prevalence of echinococcosis in local population(P < 0.05). The equation of multiple linear regression model was Y = 11.213 - 0.146X9 + 2.184X1 + 0.231X2 + 0.009X7. Among them, the average number of dogs per household has the greatest impact. Conclusion The effective and risk factors such as the number of dog per household, the rate of canine infection, the density of small mammals, and the awareness rate of prevention and treatment of echinococcosis are related to the prevalence of echinococcosis in the endemic area.

Analysis of surveillance results of echinococcosis in Ningxia in 2017

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

2019, 37(4):  433-436.  doi:10.12140/j.issn.1000-7423.2019.04.011

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Objective To understand the current prevalence and epidemiological characteristics of echinococcosis in Ningxia, so as to provide the references for its better prevention and control. Methods One village was chosen from each of eight endemic countries including Helan, Pingluo, Tongxin, Xiji, Yanchi, Yuanzhou, Zhongning and Haiyuan in Ningxia as monitoring site and all villagers with age over 3 years old from these eight monitoring sites were examined for hydatid cysts by abdominal ultrasound imaging. The dog fecal samples were collected from each site for detecting Echinococcus antigens by ELISA. The sheep’s visceral organs were collected for examining the hydatid cysts by necropsy. Results Among the 16 058 villagers receiving B ultrasound examination, 53 were detected to have hydatid cysts (0.33%). Most of the hydatid cysts were identified in livers (94.3%, 50/53). Among the eight monitoring sites, the villagers in Zhongning had the highest detection rate of hydatid cysts(1.40%, 28/2 002), there was no cyst detected in Pingluo and Yanchi, with statistical difference in the distribution of hydatidosis in different countries (P < 0.05). Females had the higher detection rate of hydatid cysts (0.34%, 29/8 434) than male villagers(0.31%, 24/7 624) (P < 0.05). The old villagers with age ≥ 60 years had the highest detection rate (0.81%, 29/3 574) compared to other age groups with statistical difference (P < 0.05). The Echinococcus coproantigen was detected in 12 dog fecal samples from total 1 134 collected dog feces with Echinococcus infection rate of 1.06%. Out of 6 226 sheep visceral organs examined, the hydatid cysts were identified in 87 organs with Echinococcus infection rates of 1.40% in sheep. There was significant difference in the detection of dog Echinococcus coproantigen and the sheep Echinococcus infection rate among the different monitoring sites(P < 0.05). Conclusion The prevalence rate of Echinococcus infection in human, dogs and livestock still remains high in some endemic areas in Ningxia and the further efforts and measures are needed for preventing and controlling of echinococcosis in Ningxia.

Inhibitory effect of anti-profilin antibody on the proliferation of Toxoplasma gondii in mouse embryonic fibroblasts

Rui YANG, Yi-ding XIONG, Shan-shan GUO, Ye LIAO, Qiang XIA, Jin-jin GUO, Lian-feng DU

2019, 37(4):  437-443.  doi:10.12140/j.issn.1000-7423.2019.04.012

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Objective A polyclonal antibody against Toxoplasma gondii profilin (TgPRF) was prepared in two rabbits and its inhibitory effect on the proliferation of T. gondii in mouse embryonic fibroblasts was observed. Methods The total RNA was extracted from T. gondii RH strain and total cDNA was reversely transcribed. The cDNA encoding for TgPRF gene was amplified from the total cDNA and then cloned into prokaryotic expression vector pET-30a(+). The recombinant pET-30a(+)-TgPRF plasmid was transformed into Escherichia coli BL21 (DE3) and the recombinant TgPRF protein was expressed under IPTG induction. The expressed recombinant protein with His-tag was purified by affinity chromatography with AKTA system and the purified recombinant TgPRF was analyzed by SDS-PAGE and Western blotting. Two New Zealand rabbits were each immunized with 1 mg recombinant TgPRF emulsified with the same volume of Freund’s complete adjuvant by multiple subcutaneous injections on the back, then boosted three times with the same amount of protein formulated with incomplete adjuvant with two weeks interval. Blood was collected from rabbit heart 10 days after the last immunization. The anti-TgPRF specific antibody in the post-immune serum was identified by Western blotting and titrated by ELISA. Mouse embryonic fibroblasts were seeded in 96-well plates (4 × 10³ cells/well) and then infected with 10⁴ T. gondii tachyzoites (RH-GFP). The inhibition of T. gondii tachyzoites in infected embryonic fibroblasts by anti-TgPRF serum was observed when anti-TgPRF serum was added at 1 : 20 (B1), 1 : 80 （B2） and 1 : 200 （B3） dilutions. The pre-immune serum at 1 : 20 was added as negative control (C) and blank medium was used as blank control(A). Each sample was added in triplicate and cultured for 72 h, then observed under fluorescence microscope. Proliferation of tachyzoites was analyzed by Image pro 6.0 software. Results The cDNA encoding for TgPRF was amplified from T. gondii total cDNA by PCR with size of 510 bp, and then subcloned into pET-30a(+) for expression in E. coli. The cloned recombinant pET-30a-TgPRF plasmid contained the correct target sequence confirmed by DNA sequencing. The recombinant TgPRF protein was successfully expressed in E. coli BL21 as size of 25 000 mainly in soluble supernatant fraction. After being purified and concentrated, the concentration of recombinant TgPRF protein was determined by BCA as 4 mg/ml. The expressed recombinant TgPRF was used to immunize two rabbits to obtain antiserum and the recombinant TgPRF was recognized by the rabbit anti-TgPRF serum determined by Western blotting analysis. The rabbit anti-TgPRF serum had no toxic effect on embryonic fibroblasts determined by CCK-8 assay. The anti-TgPRF antibody titer was > 1 : 10⁵ determined by ELISA. After being incubated with anti-TgPRF for 24 h, the T. gondii tachyzoites proliferation was significantly inhibited in group incubated with 1 : 20 antiserum (the relative parasite amount 0.35 ± 0.16) compared with blank control (0.62 ± 0.23) and pre-immune serum control (0.74 ± 0.25) (P < 0.05), however, there was no significant difference among groups incubated with antiserum of 1 : 80, 1 : 200 and blank control and pre-immune serum control (P > 0.05). The tachyzoite proliferation inhibitory rates in group B1-B3 were 43.5%, 11.4%, 3.6% respectively. After being incubated with antiserum for 48 h, the relative tachyzoites amount in group incubated with antiserum of 1 : 20, 1 : 80 and 1 : 200 were 1.09 ± 0.58, 1.92 ± 0.73 and 2.47 ± 0.84, respectively, that were significant lower than blank control group (3.61 ± 0.66) and pre-immune serum group (3.38 ± 0.78) (P < 0.01) with inhibitory rates of 69.9%, 46.9%, and 31.7%, respectively. After being incubated with antiserum for 72 h, the tachyzoites proliferation was significantly inhibited in groups with antiserum at 1 : 20 (5.58 ± 2.43), 1 : 80 (8.06 ± 2.66) (P < 0.01) and 1 : 200 (16.02 ± 6.46) compared with blank control (19.90 ± 3.92) and pre-immune serum control (20.61 ± 4.07), with inhibitory rates of 72.0%, 59.5%, and 19.5%, respectively. Conclusion The E. coli expressed recombinant TgPRF protein was immunogenic and induced strong antibody response in immunized rabbits. The rabbit anti-TgPRF serum could effectively inhibit the proliferation of T. gondii tachyzoites in mouse embryonic fibroblast cells at a dose-dependent pattern in vitro, but could not completely eliminate the parasite.

Therapeutic effect of NK cells stimulated by Toxoplasma gondii excretory/secretory antigens on B16F10 melanoma in mice

Yu-meng JIAO, Hui XIA, Xue-mei WANG, Zhi-yong TAO, Lin ZHU, Qiang FANG

2019, 37(4):  444-447.  doi:10.12140/j.issn.1000-7423.2019.04.013

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Objective To observe the therapeutic effect of natural killer (NK) cells stimulated by Toxoplasma gondii excretory/secretory antigens (TgESA) on the growth of B16F10 melanoma in mice. Methods TgESA was obtained from the culture of T. gondii tachyzoites in vitro for 12 hours. The tumor-bearing animal models were established by direct injection of melanoma B16F10 cells into the right axillary of ten mice (2 × 10⁵/mouse). Five of the melanoma-inoculated mice were intraperitoneally treated with TgESA(100 μl/mouse) 7 days after inoculation of B16F10 cells, the rest 5 mice were treated with PBS only as control. One week after the treatment, all mice were euthanized, and the NK cells were isolated from the splenocytes of two groups (named NK_B16F10 and NK_ESA). Total 30 naïve C57BL/6 mice were randomly divided into 3 groups with 10 mice each group. Each mouse was inoculated with 2 × 10⁵ B16F10 cells subcutaneously into axillary. One week after melanoma inoculation, one group of mice were passive transferred with 2 × 10⁵ of NK_B16F10 cells through tail vein injection (group of NK_B16F10). Another group of mice were transferred with the same number of NK_ESA cells (group of NK_ESA). The third group of mice were given with PBS only as control. After passive transfer, the tumor growth, mortality and survival rate in the transferred mice were observed and recorded for 35 days. Results The visible time of tumor in mice of NK_ESA group [(14.70 ± 0.95) d] was significantly longer than that in NK_B16F10 group [(12.60 ± 0.70) d] and in control group [(8.50 ± 0.85) d] (P < 0.05). The tumor size in NK_ESA group [(577.79 ± 49.70) mm²] was also significantly smaller than that in NK_B16F10 group [(686.53 ± 17.84) mm²] and in control group [(787.84 ± 19.94) mm²] at D35. The delayed appearance of tumor and smaller tumor size in group of NK_B16F10 was also significant compared to control group. Mice began to die at D24 for control group, D27 for NK_B16F10 group and D30 for NK_ESA group after tumor inoculation, and the number of mice remain alive was 3, 4 and 6 respectively at terminal time D35 with significant difference between NK_ESA group and control group.Conclusion Passive transfer of NK cells stimulated by TgESA conferred therapeutic effect on B16F10 melanoma in recipient mice.

Polymorphism of mitochondrial pnad1 gene of Spirometra erinaceieuropaei sparganum isolated from different species of snake in Hunan Province

Lei TAN, Ai-bing WANG, Xiao-xian KONG, Xuan LIANG, Jun-lin HE, Jie LI, Dan HU, wei LIU

2019, 37(4):  448-452.  doi:10.12140/j.issn.1000-7423.2019.04.014

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Objective Spirometra erinaceieuropaei sparganum larvae were isolated from different species of snake and identified by morphology and molecular biology. The polymorphism of partial mitochondrial NADH dehydrogenase subunits 1 gene(pnad1) amplified from these isolated spargana was analyzed for its potential as a species marker. Methods Eleven sparganum larvae of S. erinaceieuropaei were isolated from different species of wild snake [Elaphe carinata (3), Zaocys dhumnade (2), Naja (1), Ptyas korros (2), Ptyas mucosus (2) and Bungarus multicinctus(1)] in Yongzhou, Chenzhou, Yueyang of Hunan Province and identified by morphology and DNA sequence. The pnad1 gene was amplified from these spargana isolated from different snakes and sequenced. The obtained sequences were aligned to determine their nucleotide variations and compared with their homologues from other tapeworms, and then a phylogenetic tree was constructed accordingly using MEGA7.0. Results The 11 larvae were identified as Spirometra erinaceieuropaei spargana by their morphology under microscopy and DNA sequence. The pnad1 gene was amplified from these collected spargana with size of 648 bp. The sequence results showed that the amplified pnad1 sequences were almost identical with 0-1.4% variation, but with 97.8%-99.3% sequence identity with different isolations of Spirometra erinaceieuropaei deposited in GenBank, and with less than 74.3% identity with pnad1 sequence of other tapeworms. Constracted phylogenetic tree based on the pnad1 gene sequences showed that the Hunan isolates were clustered in the same clade of Spirometra erinaceieuropaei, genetically closer to Dibothriocephalus latus and Hymenolepis nana, but further to other tapeworms. Conclusion The sparganum larvae isolated from different species of snakes in Hunan in the present study were identified as Spirometra erinaceieuropaei, and the mitochondrial pnad1 gene can be used as a genetic marker to identify tapeworm species.

Comparative analysis of antennal olfactory gene expression between Culex pipiens pallens and Culex pipiens molestus

Jie ZHOU, Chun-xiao LI, Ce-jie LAN, Jian GAO, Qin-mei LIU, Ai-juan SUN, Tong-yan ZHAO

2019, 37(4):  453-457.  doi:10.12140/j.issn.1000-7423.2019.04.015

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Objective To analyze the difference in antennal olfactory gene expression between Culex pipiens pallens and Cx. pipiens molistus to provide a reference for revealing the characteristics of their genetic differentiation. Methods Total RNAs were extracted from antennas of Cx. pipiens pallens and Cx. pipiens molistus before and after blood feeding, and the RNA libraries were constructed using NEBNext Ultra^TM RNA ^® RNA Library Prep Kit. The transcriptome were obtained by RNAseq and their difference in different species of Cx. was analyzed by FPKM. Those olfactory genes with significant difference before and after blood feeding were identified by qRT-PCR. Results RNA-Seq results identified 50 odorant binding protein (OBP) genes and 31 odor receptor(OR) genes expressed in Cx. pipiens pallens, 36 OBPs and 12 ORs in Cx. pipiens molestus. The regulation trend of OBP44 was, in consistence, the same with the RNAseq results while OBP31 was contrary to that. The expression of OBP17 was significantly higher in Cx. pipiens molestus than in Cx. pipiens pallens before blood-sucking, and the expressions of OBP31 and OBP44 displayed the same trend after blood-sucking while the expression of OBP99a was significantly lower than Cx. pipiens pallens(P < 0.05). Expression of OR31, OR65 and CPIJ001069 were significantly up-regulated in Cx. pipiens molestus post blood feeding, but not in Cx. pipiens pallens, consistent with RNAseq results. Expression of OR65 and CPIJ001069 was significantly higher in Cx. pipiens molestus than that in Cx. pipiens pallens post blood feeding(P < 0.05). There was no significant change of OR gene expression in Cx. pipens pallents post blood feeding. Conclusion OBPs and ORs of Cx. pipiens pallens and Cx. pipiens molestus showed genetic differentiation. OBP17 and OBP44 is possibly involved in the regulation of behavior post blood feeding of Cx. pipiens molestus. OR31, OR65, and CPIJ001069 could be involved in regulating the behavior of Cx. pipiens molestus post blood feeding.

INFORMATION REPORTS

Application and funding status of parasite-related research projects funded by National Natural Science Foundation of China from 2009 to 2018

Zheng-gang YANG, Ai-xia ZHAI, Dou DOU, Jian-ye ZHANG, Zhi QI, Jing JI, Zhang-cai YAN

2019, 37(4):  458-463.  doi:10.12140/j.issn.1000-7423.2019.04.016

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This paper reviews the application and funding status of research projects related to parasitology within the category of of Medical Pathogen and Infection funded by National Natural Science Foundation of China (NSFC) from 2009 to 2018. The granted projects are categorized and analyzed based on the parasite classification, scientific aims and research contents. The characteristics and trends of these granted applications were deeply discussed to facilitate the future application in the field of parasitology. We found that there was still a big gap in the research of parasitology between China scientist and international top researchers even though some high-level scientific research achievements have been observed.

REVIEWS

Analysis of inconsistence of Plasmodium detection in some malaria cases

Mei LI, Zhi-gui XIA, Shui-sen ZHOU

2019, 37(4):  464-471.  doi:10.12140/j.issn.1000-7423.2019.04.017

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Microscopic examination, PCR and rapid diagnostic test (RDT) are three major methods commonly used for detecting Plasmodium and diagnosing malaria. During the re-check and confirmation of diagnosis of malaria cases in national and provincial malaria diagnosis laboratories, it is common to observe the inconsistence in the results of three detection methods for the same blood sample, even reversing the final diagnosis at the end in some cases. For this reason, the literature related to the sensitivity and specificity of the three detection methods was collected and analyzed combining with our practical experience in order to provide reference for better understanding and encountering the inconsistence for those who meet the similar situation.

Recent progress in immune checkpoint molecules in Plasmodium infection and immunity

Jun WANG, Yan SHEN, Yue LI, Ya ZHAO

2019, 37(4):  472-480.  doi:10.12140/j.issn.1000-7423.2019.04.018

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Malaria is still a life-threatening infectious disease transmitted by mosquito worldwide. It is necessary for host to maintain a delicate balance between pro-and anti-inflammatory immune responses to efficiently eliminate pathogens without inducing immunopathology. In recent years, breakthroughs in immune checkpoint molecules involved in anti-tumor and anti-autoimmune diseases have brought new enlightenment to the anti-malaria immunotherapy in chronic and severe infections. Studies have shown that blocking the immune checkpoint signaling in the chronic malaria infections could restore host immune response and accelerate the elimination of Plasmodium infection. In severe malaria caused by the acute infection of Plasmodium, moderately enhancing the signal of immune checkpoint molecules could reduce the immune response and alleviate the immunopathology caused by Plasmodium infection. This paper provides a brief review of the current progress in immune checkpoint molecules in Plasmodium infection and immunity.

Advances in the genetic polymorphism of Echinococcus granulosus

Yu-huan WEI, Yuan HU, Jian-ping CAO

2019, 37(4):  481-485.  doi:10.12140/j.issn.1000-7423.2019.04.019

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The larvae of Echinococcus granulosus parasitize in the liver and lungs of the intermediate host (human, cattle, sheep, etc.) and develop into cysts that damage the organs and cause hydatidosis. The disease is still a seriously public health problem that threats the health of people and hurdles the animal husbandry in the endemic areas. It is estimated that more than 1 million people are suffering from hydatidosis, listed as one of serious tropical diseases by the World Health Organization. Based on the genetic variation E. granulosus has been classified into 9 genotypes (G1, G3-G8, G10 and E. felidis) that differ in the regional distribution, host species and pathogenicity. This review focuses on the research progress on the genetic polymorphism of E. granulosus and research direction, providing supporting data for better understanding and control of hydatidosis.

SHORT COMMUNUCATIONS

Morphological observations of Toxocara canis egg development in vitro

Yi-lin ZHANG, Sen-zhao ZHANG, Xiao-bin GU, Guang-you YANG, Yue XIE

2019, 37(4):  486-489.  doi:10.12140/j.issn.1000-7423.2019.04.020

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To observe the morphological changes of Toxocara canis egg development, an automatic microscopic photographing system was setup to track and record the whole process of T. canis egg development during continuous 21-day egg culture in vitro in this study. The results demonstrated that the whole development of T. canis egg in vitro was divided into 12 developmental stages including one-cell, two-cell, three-cell, four-cell, early-morula, late-morula, blastula, gastrula, pre-larva, L1 (the first stage larva), pre-L2 and L2 stages. The cell started to divide to form two-cell egg in 24 hours culture, appeared as 3-4 cells egg, morula, blastula and gastrula stages in 3-6 days. Total 22.5% of eggs developed to embryonated L1 with phototactic movements in 7 days. Total 10.67% eggs developed to infective embryonated L2 in 8 days. At 17-day post culture, the infective larva have been hatched and the whole eggshell collapsed. These data provide more information for the developmental biology of T. canis egg in vitro.

Epidemiological analysis of malaria in Zibo City of Shandong Province during 2014-2018

Jin XU, Chao LIU, Hai-ning ZHU, Yan-dong WANG, Qin WANG, Ben-guang ZHANG

2019, 37(4):  490-493.  doi:10.12140/j.issn.1000-7423.2019.04.021

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To analyze the epidemiological characteristics of malaria in Zibo City, Shandong Province, as a scientific basis for consolidating the elimination of malaria, the epidemiological data on malaria cases identified in this region from 2014 to 2018 were collected. The epidemic situation, infection source and species, the distribution of cases in different regions, gender, age, occupations and season, malaria diagnosis and treatment were analyzed using Microsoft Excel 2007 software. The results showed that total 30 cases of malaria were reported in Zibo, Shandong Province from 2014-2018, and the average annual incidence rate was 0.6/10 million. All cases were imported from Africa, mainly from 11 African countries including Nigeria(12 cases, 40.0%) and Mozambique (6, 20.0%). Most of the cases were falciparum malaria(26 cases, 86.7%). All cases were adult males, aged 23-60 years with the average age of 38 years old. Most of the cases were labor exporters to Africa including project workers and farmers(28 cases, 93.3%). Regarding the regional distribution of the cases, 8 counties(districts)in Zibo administrative region had reported malaria cases with highest case number in Zhangdian District(12 cases, 40.0%). Only one case was reported in each Gaoqing County and Wuyuan County(1 case, 3.3%). Regarding the case monthly distribution, the most cases were reported in October(7 cases)followed by January(5 cases), September(5 cases) and November(3 cases). There were no case reported in May. The time from disease onset to visiting doctor varied from 0 day to 16 days with average time of 3.1 days. All cases were diagnosed as malaria within 24 hours of doctor visit except for one case that was diagnosed as malaria 7 days after the first doctor visit (3.3%). The local hospital was the preferred selection for patients to visit after onset of the disease(22 cases, 73.3%), followed by the local Chinese Center for Disease Control and Prevention (8 cases, 26.7%). Total 25 out of 30 cases received confirmed diagnosis of malaria during the first visit and these medication facilities owned good experience and capacity to diagnose malaria with 83.3% accuracy of diagnosing malaria.

Infection of Plasmodium knowlesi and Babesia microti in farmed monkeys in Guangxi

Zi-yue WANG, Yi-chao YANG, Zhi-pin CHEN, Yun-liang SHI

2019, 37(4):  494-496.  doi:10.12140/j.issn.1000-7423.2019.04.022

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This study aims to understand the infection status of Plasmodium knowlesi and Babesia microti in farmed monkeys in Guangxi to assess its risk as reservoir host for human infections. Total 600 blood samples were collected from monkeys raised in a farm in Guangxi including 330 Macaca fascicularis and 270 M. mulatta. The infections of B. microti and P. knowlesi were detected by amplifying 18S rRNA of each species using nested PCR. The PCR products of positive samples were DNA sequenced, and then BLAST searched to confirm the species of infected parasite. The nested PCR results showed that 16 monkeys were infected with B. microti with overall positive rate of 2.7% （16/600）. These infected monkeys included 13 Macaca fascicularis and 3 Macaca mulatta with infection rates of 3.9% (13/330) and 1.1% (3/270), respectively. The difference of B. microti infection between these two monkey species was statistically significant (P < 0.05). Four PCR products were sequenced and the obtained sequences possessed 99.9% sequence identity to B. microti (GenBank accession number: KC904078.1). No infection of P. knowlesi was detected in the examined 600 monkey blood samples. The results suggested that infection of B. microti was detectable in the farmed moneys which could be the potential reservoir host for human Babesia infection in Guangxi, while infection of P. knowlesi was not identified in these farmed monkeys.

Epidemic situation of imported malaria in Fushun from 2009 to 2018

Xiao-qiang DENG, Jing-hua DING

2019, 37(4):  497-500.  doi:10.12140/j.issn.1000-7423.2019.04.023

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To analyze the epidemiological characteristics of imported malaria in Fushun City so as to provide scientific basis for the development of prevention and control strategies and measures, the data of imported malaria cases in Fushun were collected from 2009 to 2018, the epidemiological information including the source of infection, the distribution of cases in different regions, gender, age, occupations and seasons, diagnosis and transmission vector was analyzed using SPSS 17.0 software. The results showed that total 33 cases of imported malaria were reported in Fushun from 2009-2018. All of them were laboratory confirmed cases including 29 cases of falciparum malaria(87.9%), 2 cases of vivax malaria(6.1%), and 2 undetermined malaria(6.1%). All cases were imported from overseas, 31 cases of them(93.9%) were from Africa, mainly from Angola(12 cases). Two cases (6.1%) were imported from Asia, in particular 1 case from Pakistan and another one from Malaysia. The imported malaria cases have been identified in 7 counties or districts of Fushun City except for Fushun County. Xinbin County owned the highest number of the cases(14 cases). More male patients(87.9%, 29/33) than females(12.1%, 4/33) have been identified with significant difference(P < 0.05). The youngest age is 24 years old, the oldest age is 58 years old. Most of the cases happened within the age between 40 to 49(45.5%, 15/33). Among the occupational distribution, most of the cases were farmers(36.4%, 12/33). There was no significant seasonal distribution for the case onset including 8 cases in spring, summer and autumn(each 24.2%), and 9 cases in winter(27.3%). The rate of correct diagnosis at the first medication at county, city and provincial level facilities was 1/6, 12/14 and 13/13, respectively, and the difference was statistically significant (P < 0.01). The longest time from disease onset to the first seeking medication was 51 days, the shortest was 1 day and the median time was 5 days. The time from the first medication to the confirmed diagnosis varied from 1 day to 31 days with median time of 2 days. The time from the disease onset to the confirmed diagnosis varied from 1-57 days with the median time of 8 days. There was no malaria vector mosquito Anopheles sinnensis found in Fushun City urban area, but found in suburban counties, mainly distributing in livestock sheds. The density of An. sinnensiswas significantly different from residential areas to parks, hospitals and farmer houses (P < 0.01). The imported malaria is still a public health problem in Fushun City. The malaria diagnostic rate in county health medical institutions was low and more training for malaria diagnosis and treatment is needed.