Table of Content

28 April 2017, Volume 35 Issue 2

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ORIGINAL ARTICLES

Protective effects of adult-worm excretory-secretory protein of Trichinella spiralis against sepsis-induced acute liver injury and the mechanisms

Hui-hui LI, Wen-xin HE, Zhao-gen CAI, Da-peng QIU, Liang CHU, Xing-zhi CHEN, Qiang FANG, Hui XIA, Nan LI, Ning-ning CUI, Lan-song XU, Xiao-di YANG

2017, 35(2):  99-104. 

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Objective To investigate the protective effect of adult-worm excretory-secretory protein (AES) of Trichinella spiralis on sepsis-induced acute liver injury. Methods Twenty-four male BALB/c mice were randomly divided into sham operation group (Sham group), cecal ligation and puncture group (CLP group), AES treatment group (AES + CLP group), and AES control group (AES + Sham group) (n = 6 in each group). The mice in the CLP group and AES + CLP group received cecal ligation and puncture procedure to induce sepsis-associated acute liver injury. At 30 min after surgery, intraperitoneal injection of PBS or AES (25 μg) was performed. At 12 h after surgery, the serum levels of alanine transaminase (ALT) and aspartate transaminase (AST) were determined and mice were sacrificed to prepare paraffin sections of the liver for HE staining. Protein levels of Toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88) in liver were measured by Western blotting. Expression of nuclear factor-κB (NF-κB) p65 in hepatocytes was detected by immunohistochemistry, and the nuclear positive rate was analyzed. The levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-1β in liver were determined with ELISA. Results At 12 h after surgery, the serum levels of ALT and AST in the CLP group [(269.83 ± 77.60) and (712.00 ± 186.52) IU/L, respectively] were both significantly higher than those in the Sham group [(45.00 ± 15.01), (132.50 ± 36.95) IU/L] and the AES + CLP group [(136.67 ± 30.27), (419.00 ± 60.86) IU/L] (P < 0.05), and the serum levels of ALT and AST in the AES + Sham group [(50.83 ± 13.24), (134.67 ± 35.78) IU/L] had no significantly difference with those in the Sham group. HE staining revealed disordered hepatic cords, hepatocyte swelling, and infiltrations of inflammatory cells in the CLP group, but significantly ameliorated injury in the AES + CLP group. Results of Western blotting showed that the relative protein levels of TLR4 and MyD88 in the CLP group (0.66 ± 0.12, 0.69 ± 0.13, respectively) were significantly higher than those in the Sham group (0.31 ± 0.06, 0.22 ± 0.04) (P < 0.05). There was a significant difference between the AES + CLP and CLP groups in MyD88 level (0.47 ± 0.06 vs. 0.69 ± 0.13), but not for TLR4 (0.56 ± 0.09 vs. 0.66 ± 0.12). Immunohistochemistry revealed that the nuclear positive rate of NF-κB p65 was significantly higher in the CLP group [(18.93 ± 2.91)%] than in the Sham group [(0.5 ± 0.21)%] and the AES + CLP group [(9.6 ± 1.59)%] (P < 0.05). ELISA revealed that the levels of TNF-α, IL-6 and IL-1β in the CLP group [(26.21 ± 2.96), (80.80 ± 9.94) and (28.99 ± 3.70) pg/mg, respectively] were all significantly higher than those in the Sham group [(15.42 ± 1.21), (47.70 ± 4.77) and (16.86 ± 1.68) pg/mg] and the AES + CLP group [(18.93 ± 2.04), (61.82 ± 7.24) and (23.18 ± 2.21) pg/mg] (P < 0.05). Conclusion AES reduces the levels of TNF-α, IL-6 and IL-1β via decreasing MyD88 expression and the nuclear positive rate of NF-κB in liver, thereby relieving sepsis-induced acute liver injury in mice.

Immune response of mouse Th17 cells from mesenteric lymph node after treatment with soluble egg antigens and adult worm antigens of Schistosoma japonicum

Xue-ping LUO, Nan-gui YUAN, Jun HUANG

2017, 35(2):  105-109. 

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Objective To observe the immune response of mouse Th17 cells from mesenteric lymph node (MLN) after treatment with soluble egg antigens (SEA) and soluble adult antigens (SWA) of Schistosoma japonicum. Methods Two New Zealand white rabbits were infected with S. japonicum cercariae through abdominal skin attachment for 20 min. After 45 days, SWA and SEA were collected. Twenty C57BL/6 mice were randomly divided into the infection group and the healthy group (n = 10 in each group). The mice in the infection group were infected with 40 ± 5 worms of S. japonicum through the abdominal skin. At 5 to 6 weeks, the serum levels of SEA- and SWA-specific IgG were determined by ELISA. The MLN lymphocytes were isolated and divided into four groups. Lymphocytes in group A received no stimulation, those in group B were treated with 100 μg/ml SEA + 1 μg/ml anti-CD28 mAb, those in group C were treated with 100 μg/ml SWA + 1 μg/ml anti-CD28 mAb, and in group D they were treated with 1 μg/ml anti-CD3 mAb + 1 μg/ml anti-CD28 mAb. The levels of IL-17 and IFN-γ in the supernatant were detected by ELISA after 72 h treatment, and Th17 cells were detected by flow cytometry after 9 h treatment. Results ELISA results showed that the A₄₅₀ values for serum SEA- and SWA-specific IgG in the infection group (2.66 ± 0.20, 1.68 ± 0.66) were both significantly higher than those of the healthy mice (0.19 ± 0.05, 0.25 ± 0.12, respectively) (P < 0.01). Flow cytometry showed that the proportions of CD4⁺IL-17⁺ cells and CD4⁺IFN-γ⁺ cells in group B lymphocytes from infected mice (0.43% and 0.56%, respectively) were significantly higher than the healthy group (0.05% and 0.20%) (P < 0.05). Similar results were found for group C (0.39% and 0.76% vs. 0.04% and 0.19%) (P < 0.05). The levels of IFN-γ and IL-17 were (49.13 ± 14.71) and (41.73 ± 2.42) pg/ml, respectively in group B lymphocytes from infected mice, which were significantly higher than the (3.27 ± 0.33) and (9.22 ± 0.58) pg/ml in healthy mice (P < 0.01). Similar results were found in group C [(46.92 ± 16.73) and (36.14 ± 4.82) pg/ml vs. (3.38 ± 0.34) and (8.78 ± 0.93) pg/ml] (P < 0.01). The IL-17 level in group B was (41.72 ± 2.42 ) pg/ml, significantly higher than the (36.14 ± 4.82) pg/ml in group C. Conclusion Both SEA and SWA can induce secretion of IL-17 and IFN-γ from MLN lymphocytes of C57BL/6 mice infected with S. japonicum, and the secreted IL-17 is significantly higher for SEA than SWA. SEA and SWA can also induce the production of CD4⁺IL-17⁺ cells and CD4⁺IFN-γ⁺ cells in the mesenteric lymph nodes of C57BL/6 mice infected with S. japonicum.

Effects of cystatins derived from two species of helminths on the release of nitric oxide and secretion of cytokines from murine peritoneal exudate cells

Xiao-di YANG, Hui-hui LI, Zhi-yong TAO, Qiang FANG, Yang CHENG, Lan-song XU, Ren-min XUE, Yong CHEN, Hui XIA, Hui ZHANG, Hui JIANG, Tao LIU, Kun PENG, Xing-zhi CHEN

2017, 35(2):  110-114. 

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Objective To investigate the effects of cysteine protease inhibitors(cystatins) from Trichinella spiralis (Tscystatin) and Angiostrongylus cantonensis (Accystatin) on the release of nitric oxide (NO) and secretion of cytokines from mouse peritoneal exudate cells (PECs). Methods Ten BALB/c mice were sacrificed under anesthesia to collect intraperitoneal fluid, from which PECs were prepared, consisting mostly of macrophages. The lipopolysaccharide(LPS)-stimulated PECs were incubated with Tscystatin or Accystatin. PECs were assigned into four groups, RPMI group, LPS group, Accystatin + LPS group and Tscystatin + LPS group. The cells in the latter three groups were stimulated with lipopolysaccharides (LPS 2 μg/ml) 2 hours prior to 2 μg/ml Accystatin or Tscystatin treatment for 24 hours. Each group had six replicates. The levels of tumor necrosis factor-α(TNF-α), interleukin IL-6(IL-6) and IL-10 in the culture supernatant were measured by ELISA. The concentration of nitric oxide(NO)was measured by nitrate reductase method. Statistic analysis was performed using SPSS11.5 software. Results The level of pro-inflammatory cytokines TNF-α and IL-6 was(507.50 ± 66.32) and (1 440.49 ± 77.25) pg/ml respectively in the Accystatin + LPS group, significantly higher than (454.15 ± 53.11) and (1 016.27 ± 115.10) pg/ml in LPS group (P < 0.05), but compared with LPS group and Accystatin + LPS group, the level of TNF-α [(296.35 ± 55.30) pg/ml] and IL-6 [(793.54 ± 86.61) pg/ml] in the Tscystatin + LPS group was significantly lower than those in the LPS group and Accystatin + LPS group (P < 0.05). The level of IL-10 was (38.80 ± 6.71) pg/ml in the RPMI group and (53.66 ± 7.72) pg/ml in the LPS group, with no significant difference(P > 0.05). The level of IL-10 in the Accystatin + LPS group [（149.74 ± 26.01) pg/ml] and the Tscystatin + LPS group [(158.76 ± 19.67) pg/ml] was significantly higher than that in the LPS group(P < 0.05). The level of NO in the Accystatin + LPS group and the Tscystatin + LPS group was(12.54 ± 2.12) μmol/L and (7.95 ± 1.40) μmol/L, respectively, significantly decreased compared to the LPS group (P < 0.05). In addition, the concentration NO in the of Tscystatin + LPS group was significantly lower than the Accystatin + LPS group (P < 0.05). Conclusion Cystatins derived from the two species of helminths can both inhibit the release of NO and up-regulate the level of IL-10. Accystatin remarkably promotes the secretion of TNF-α and IL-6, while Tscystatin has the opposite effects.

Time-series anaylsis of malaria incidence in China during 2004-2013

XU Jun-fang, XIA Zhi-gui, ZHOU Xiao-nong, LI Shi-zhu, GUO Xiao-peng, QIN Si

2017, 35(2):  114-119. 

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Objective To estimate the trend of malaria incidence in China from 2004 to 2013 and clarify the pattern of malaria prevalence. Methods The malaria incidence trend during 2004-2013 was analyzed with the Joinpoint software to calculate the annual percent change (APC). Grey relational analysis was also performed. Results The joinpoint for the overall prevalence of malaria was found in 2006, with APC of 34.8% (P > 0.05) and -37.9%(P < 0.05), respectively. The joinpoint for vivax malaria was found in 2007, with APC of 14.7% (P < 0.05) and -51.5% (P < 0.05), respectively. The joinpoint for falciparum malaria was in 2009, with APC of -39.6% (P < 0.05) and -25.9% (P > 0.05), respectively. The joinpoint for unclassified malaria was in 2006, with APC of 31.7% (P > 0.05) and -40.7% (P < 0.05), respectively. The comprehensive correlation degrees of vivax malaria, falciparum malaria and unclassified malaria incidence with the overall prevalence of 0.887 7, 0.625 4, and 0.844 5, respectively. The joinpoint for indigenous malaria incidence was in 2008, with APC of 2.43% （P > 0.05) and -72.89% (P < 0.05), respectively. The comprehensive correlation degree between indigenous malaria incidence and the overall prevalence was 0.969 3. Conclusion There is a trend of decrease for overall prevalence, as well as for the incidences of indigenous malaria, vivax malaria, and unclassified malaria. The incidence of falciparum malaria was a trend of decrease from 2004 to 2009, but was steady from 2009 to 2013.

Isolation and identification of cultivable symbiotic bacteria from the intestinal tract of Musca domestica during development

Jing LIU, Dan CHEN, Gui-fen ZHUANG, Zhen-dong HUANG, Zhi-jing XUE, Rui-ling ZHANG, Zhong ZHANG

2017, 35(2):  120-124. 

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Objective To identify the intestinal cultivable bacteria in Musca domestica at different development stages. Methods Five eggs or 5 larvae at day 1 after egg hatch were collected under aseptic conditions, and were homogenized in 1 ml sterile water. At day 2 or above, 5 larvae. 5 pupae, and 5 adult worms were also collected, and their intestinal tract was isolated and homogenized in 1 ml sterile water. The intestinal symbiotic bacteria were cultured from the homogenate. Single colonies with different morphology were picked up and cultured in LB medium for small-scale production. DNA was extracted to amplify 16S rDNA by PCR. The amplification products were sequenced and blasted in GenBank to determine the bacteria genus. The numbers of genera at different developmental stages were recorded. Results A total of 26 bacterial genera were obtained, comprising 11 in larvae stage, 19 in pupae stage, and 14 in adult worms. None cultivable symbiotic bacterium was isolated from eggs. There were genus overlaps between stages. The larvae and pupae stages shared 6 genera, consisting of Proteus, Kurthia, Bordetella, Microbacterium, Alcaligenes and Leuconostoc. The pupae and adult stages shared 3 genera, consisting of Sphingobacterium, Enterococcus and Ochrobactrum. The larvae and adult stages shared 1 genus, the Klebsiella. The three stages shared 4 genera, consisting of Providencia, Staphylococcus, Myroides and Pseudomonas. The proportions of gram-positive bacteria were 4/11, 6/19 and 3/14 in the larvae, pupae and adult stages, respectively. Conclusion The genus number of cultivable bacteria from Musca domestica shows a trend of increase followed by a decrease during the development from larvae to pupae stage, accompanied with a gradual decrease of proportions of gram-positive bacteria.

Investigation of the source regions of Babesia spp. infection in the central and south areas of Zhejiang Province

Wei RUAN, Ling-ling ZHANG, Hua-liang CHEN, Qiao-yi LU, Xuan ZHANG, Yan FENG, Li-nong YAO

2017, 35(2):  125-130. 

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Objective To investigate the infectious status of Babesia spp. in hosts and vectors in the central and south areas of Zhejiang Province, and analyze the 18S rRNA gene sequence from a patient confirmed to have human babesiosis in Zhejiang Province and align it with that in GenBank, in order to understand the genetic relationship and evolutionary characteristics of Babesia. Methods Anticogulated blood was collected from animal hosts and Babesia spp. vectors were collected. DNA was extracted from these samples and PCR was performed to amplify the 18S rRNA gene of Babesia. The PCR products were further sequenced and sequence alignment was performed to identify the species of Babesia. The family- and species-specific 18S rRNA sequences of Babesia spp., as well as those of B. microti from different animal hosts and from different geographical source regions were searched in the NCBI database and analyzed by BLAST. Phylogenetic trees were constructed by the MEGA5 software. Results Thirty-seven samples from a total of 261 produced positive bands. The positive rate of anticogulated blood from animal hosts was 14.2% (32/225), and that of vectors was 13.9%(5/36). The positive bands were sequenced, and 20 underwent successful sequencing. Results of sequence alignment revealed five samples with B. microti infection from rodents, four samples with Sarcocystis sp. infection, two with Hepatozoon sp. infection, one with Coccidia sp. infection, five with Theileria orientalis infection, two with T. buffeli infection and one B. caballi infection in rodent and cattle hosts/vetors. The 18S rRNA gene sequence of the confirmed human babesiosis in the Province had 98.1%-99.8% homology with the sequences of B. microti from different hosts and different geographical regions. And it was in the same branch as the human-source B. microti from Yunnan and Japan, and the rodent-source B. microti from Fujian, and Hangzhou, Tiantai and Xianju of Zhejiang. The Chinese CNMM-2 strain and Xinjiang 1647 strain of B. microti had a closer genetic distance with American GI strain and German Jena strain. Conclusion The B. microti was commonly found in rodents in the central and south areas of Zhejiang Province. Studies should be extended to other small mammals and domestic livestock, as well as to the biological vectors to see if they are carrying Babesia species pathogenic to humans.

Genetic diversity of Angiostrongylus cantonensis in Nan’ao Island of Guangdong Province

Qiu-an HU, Shan LV, Yun-hai GUO, He-xiang LIU, Yi ZHANG

2017, 35(2):  130-135. 

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Objective To investigate the genetic diversity of Angiostrongylus cantonensis in Nan’ao Island of Guangdong Province, and analyze the phylogenetic relationship among A. cantonensis isolates in different regions of China. Methods Three villages (Gongqian Village, Jinshan Village and Liudu Village) were selected in Nan’ao Island using the stratified random sampling method during 2015-2016. Rodents were captured by the night feeding and trapping method and were dissected to collect adult worms. Pomacea canaliculata and Achatina fulica collected from fields were examined for the stage-Ⅲ larvae, which were then used to infect SD rats to collect adult worms. Genomic DNA was extracted from the adult worms, and PCR was performed to amplify internal transcribed spacer 2 (ITS2) and cytochrome c oxidase subunitⅠ (COⅠ). The PCR products were sequenced and aligned by Clustal X1.83. The genetic diversity was analyzed with DnaSP5.10. The ITS2 sequences of A. cantonensis from different areas in the country, such as Wenzhou in Zhejiang (Accession number HQ540551.1), Shenzhen in Guangdong (Accession number HQ540546.1), Hualian in the Region of Taiwan (Accession number KF591125.1), were obtained from GenBank or published reports. The genetic distances between these strains were determined with Mega6.06. The phylogenetic trees were constructed by the neighbor-joining (NJ) and maximum parsimony (MP) methods to analyze the phylogenetic relationship. Results A total of 110 rodents were captured and 32 were positive for A. cantonensis (29.1%). And 1 190 P. canaliculata and 24 A. fulica were examined, with a larva infection rate of 6.1% (72/1 190) and 83.3% (20/24), respectively. The amplification products of ITS2 and CO I of A. cantonensis had a length of 693 bp and 1 174 bp, with a site mutation rate of 4.7% and 1.0%, respectively. The haplotype diversity index, nucleotide diversity index, and the average nucleotide difference index were 0.927, 0.007 and 4.549 respectively in ITS2, and 0.440, 0.004 and 3.743 in CO I. The genetic distance of A. cantonensis in Nan’ao Island with isolates from other sources was 0.181-0.775. Phylogenetic tree results showed that most isolates of A. cantonensis in Nan’ao Island were clustered into one branch with isolates from Shenzhen, Fuqing, Puer and Nanning, while a small portion of isolates from Nan’ao Island were clustered into an independent branch. Conclusions There exist some degrees of genetic differentiation and genetic diversity within the population of A. cantonensis in Nan’ao Island. They might have multiple types and sources, but more studies are needed to clarify this.

Investigation of Crytosporidium infection in primates in a zoo of Shanghai

Shang-rui ZHANG, Yu-juan SHEN, Bin WANG, Hua LIU, Sheng-kui CAO, Zhong-ying YUAN, Jian-ping CAO, Hui LIU

2017, 35(2):  136-139. 

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Objective To determine Cryptospordium infection in primates in a zoo of Shanghai. Methods Fresh fecal samples of primates were collected in a zoo in Shanghai from March 2013 to October 2014. The samples underwent Auramine phenol and modified acid-fast staining to detect oocysts of Cryptosporidium. The 18S rRNA gene was amplified by nested PCR. The PCR products were sequenced and phylogenetic trees were constructed with the neighbor-joining method using Mega 7 software. Results A total of 83 fecal samples were collected from primates from 8 families, 37 species. None was positive for oocysts of Cryptospordium. Nested PCR of 18S rRNA gene generated positive products from 3 samples from No. 3 orangutan, No. 42 and No. 44 mandrills, respectively, with a positive rate of 3.61%(3/83). The sequences of the three products all shared 99% identity with Cryptosporidium andersoni（GenBank Accession No. KT175424, KF271479 and KF271467). The phylogenetic tree showed that the three samples were in the same branch as other species of Cryptosporidium andersoni(GenBank Accession No. KT175424, KF271479, KF271467, KF826314 and KJ917578). This implied that the three Cryptosporidium isolates were C. andersoni. Conclusion Cryptospordium infection exists in primates in the zoo in Shanghai, indicating a potential risk of zoonosis.

An epidemiological survey on hydatid disease in Tibetan autonomous areas of Gansu Province

Dong WANG, Yu FENG, Fan LI, Peng-fei GE, Ting ZHANG, Wei HU, Hong LIANG, Guo-bing YANG, Da-wei YU, Cheng-ming YANG, Jun-ke YANG

2017, 35(2):  140-144. 

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Objective To investigate the prevalence of echinococcosis in 10 Tibetan autonomous prefectures(counties) of Gansu Province. Methods Stratified random sampling was carried out from September 2011 to June 2012, 16 villages for each. The subjects were examined by B ultrasonography. Statistics were performed using the SPSS 20.0 software. Data of prevalence were analyzed with χ² test. Results A total of 37 815 residents were examined, and echinococcosis was found in all the surveyed sites with an average county-level prevalence rate of 0.59%(224/37 815) (0.05%-1.59%). The counties with prevalence of over 1% were Xiahe(1.59%, 64/4 019), Maqu(1.37%, 44/3 206) and Sunan(1.20%, 60/5 000). In the overall populations, the prevalence in males and females was 0.53%(108/2 0276) and 0.66%(116/17 539) respectively(χ² = 2.647, P > 0.05), while in the Tibetan ethnic population, the prevalence showed a considerable difference(0.71% in females versus 0.46% in males, P < 0.05). There was also a significant difference by age(χ² = 109.346, P < 0.05), with the lowest prevalence in the group of 20-30 years old(0.28%, 19/6 687), and highest in the group of > 80 years old(2.41%, 4/166). Overall, the prevalence showed a trend of increase with ageing. There was no significant difference in prevalence among ethnicities(χ² = 1.710, P > 0.05) though the Hui ethnicity seemed higher(0.92%, 5/546). The prevalence varied significantly among residents with different occupations(χ² = 33.345, P < 0.05), with businessmen ranking first(3.32%, 2/62). In this study, we identified 223 cystic echinococcosis cases and a case of alveolar echinococcosis. No mixed infection was found. Single-organ infection accounted for 98.21%, with a liver dominance(94.76%, 217/229). The single cyst type accounted for 81.75% (183/224). The proportion of multi-cyst type was higher in residents > 60 years old(28.57%, 22/77) than in those < 60 years old(12.93%, 19/147) (χ² = 8.273, P< 0.05). Conclusions Cystic echinococcosis is present in all the counties surveyed, particularly in counties of Xiahe, Maqu and Sunan. Target populations are mainly females of Tibetan ethnicity, residents aged over 60 years, and merchants.

A survey on Echinococcus infections in animals in Xinjiang Uygur Autonomous Region

WUMAIER Maimaitijiang, OSMAN Yisilayin, SIMAYI Adili, Yan-yan HOU, Ning XIAO

2017, 35(2):  145-149. 

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Objective To make a systematic survey on Echinococcus infection among domesticated (shepherd) dogs, rodents and livestock in Xinjiang Uygur Autonomous Region. Method According to the Xinjiang Protocol of Epidemiological Survey on Echinococcosis, 366 administrative villages were selected as the survey spots from 26 counties in districts with echinococcosis multilocularis prevalence, including Altay Mountain, Taer Basin, Yili River Valley, the Northern Tianshan Mountains and the Central Tianshan Mountains, from August 2012 to September 2013. Dog feces were collected for examination of echinococcus antigen with ELISA. Hydatid cyst was examined through touching and visual observation of rodents and livestock at the slaughterhouses including sheep, horses, yaks, cattle and goats. The positive rate of echinococcus antigen in feces and the infection rate of hydatid cysts were analyzed with the chi-square test. Results Of the 8 493 totally collected fecal samples, ELISA showed a positive rate of 2.63% (223/8 493) for echinococcus antigen, and the Taer Basin (4.69%, 74/1 600) had the highest positive rate, followed by Ili River Valley (2.52%, 66/2 615), Altay Mountain (2.14%, 50/2 341), the Central Tianshan Mountains (2.08%, 27/1 298) and the Northern Tianshan Mountains (0.94%, 6/639) (χ² = 13.65-18.01, P < 0.05). In addition, the detection rate of hydatid cysts was 6.18% (1 616/26 170). All species except yak and horses had different degrees of infection. The detection rate was higher in sheep (6.47%, 1 550/23 943) than in cattle (3.18%, 30/943) and goats (2.89%, 36/1 247) (χ² = 25.85-16.54, P < 0.05). The livestock infection rate was highest in the Central Tianshan Mountains (18.04%, 545/3 021), followed by the Ili River Valley (6.44%, 305/7 312), Northern Tianshan Mountains (4.17%, 65/2 010), Taer Basin (3.59%, 239/6 652) and Altay Mountain (3.23%, 462/7 175) (χ² = 321.46-248.35, P < 0.05). The ratio of hydatid cysts found in the liver, lung, lung + liver, and other organs was 116 ∶ 12 ∶ 5 ∶ 1. Regarding the rodent infection, 25 400 of 46 species underwent necropsy, and 374 of 20 species had a positive result (1.47%, 374/25 400). Hydatid infection was found in all the survey spots except for the Northern Tianshan Mountains, with a highest infection rate in the Ili River Valley (3.94%, 322/8 120) followed by the Central Tianshan Mountains (0.55%, 18/3 260), the Taer Basin (0.55%, 28/5 076) and the Altay Mountain (0.09%, 6/6 942) (χ² = 93.50-264.36, P < 0.05). Conclusions The positive rate of fecal antigen in dogs is 2.63%. The hydatid cysts are found in 6.18% of livestock and 1.47% of wild rodents in Xinjiang Uygur Autonomouse Region.

Clinical epidemiological analysis of imported malaria in Shanghai

Xu-hua JIANG, Yu-xian HUANG, Yun LING, Ai-hong ZHU, Shan-ke YE, Qin HUANG

2017, 35(2):  150-155. 

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Objective To evaluate the epidemiological characteristics of imported malaria and analyze the risk factors for severe malaria in Shanghai, China. Methods A retrospective review was made on imported malaria cases treated in Shanghai Public Health Clinical Center during 2013-2015, focusing on the demographic information, epidemiological data, and laboratory data. Comparisons between groups were made with Mann-Whitney test and Fisher’s exact test, and risk factors were analyzed with multivariate logistic regression. Results Eighty-seven imported malaria cases (82 males and 5 females) were reviewed, with an average onset age of 36.4, comprising 79 Chinese (90.8%), 3 South Sudanese (3.4%), 2 Indians (2.3%), and one from each of Cameroon, Nigeria, and Burundi (1.1%). Seventy-five cases had a defined region of malaria source, with a dominance in African countries (63, 84.0%) and then in Asia (11, 14.7%). Seventy-eight (89.7%) were confirmed by laboratory tests while 9(10.3%) were clinically diagnosed. Among the laboratory confirmed cases, 89.7% were infected with Plasmodium falciparum and 10.3% with P. vivax. Twenty cases(23.0%) had a history of malaria infection. The median of duration from symptom onset to admission was 5 days and 30 (34.5%) patients received anti-malaria drug treatment within 48 hours after onset. Eighteen patients met the diagnostic criteria of severe malaria, of whom 1 showed cerebral malaria, 17 had serum total bilirubin elevating to > 43 μmol/L and 3 had a high serum creatinine level above 265 μmol/L. Multivariate logistic regression showed that medication within 48 h after onset was an independent risk factor for severe malaria (OR = 0.05, 95%CI 0.01-0.43, P < 0.05). Conclusion Plasmodium falciparum remains a major pathogen for imported malaria in Shanghai. Africa is the major region of malaria source. Timely medication after disease onset reduces the risk for sever malaria.

Epidemiological analysis of imported malaria in Gansu Province during 2013-2015

Da-wei YU, Yu FENG, Fan LI, Cheng-ming YANG, Jun-ke YANG, Guo-bing YANG

2017, 35(2):  156-159. 

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Objective To analyze the imported malaria situation in Gansu Province during 2013-2015, in order to provide reference for improving elimination strategies and measures. Methods Data on malaria control and reported malaria cases during 2013-2015 were collected, and analysis was made on prevalence and regional distribution of malaria cases, species of plasmodium, diagnosis of patients, and sources of infection. Results Ninety-seven cases were reported in Gansu Province during 2013-2015, consisting of 52 cases of vivax malaria (53.6%). 42 falciparum malaria (43.3%, 42/97), and one for each of quartan malaria, ovale malaria and mixed infection (3.1%). All were imported, with a source dominance in southeast Asia (60.8%, 59/97), then the Africa (39.2%, 38/97). Most of the cases were young and middle-aged adults of 21-50 years who working overseas (93.8%, 91/97), and most of the cases occurred from April to August (51.6%, 50/97). The cases mainly distributed in Wenxian (50.5%, 49/97), and Lanzhou (15.5%, 15/97). The median of interval from onset to diagnosis was 4 days. The cases were mainly reported by clinical medical institutions (91.8%, 89/97). Only 8.2% (8/97) were reported by disease control institutions. No indigenous cases were reported in the period. Conclusions The reported malaria cases are predominated by vivax malaria and falciparum malaria imported from the southeast Asia and Africa.

Preparation and identification of monoclonal antibodies targeting Giardia lamblia

Le-sheng ZHANG, Lei SUN, Yuan HU, Wen-ci GONG, Yan-juan WANG, yu-juan SHEN, Yu-xin XU, Tian-ping WANG, Jian-ping CAO

2017, 35(2):  160-163. 

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Objective To prepare and identify the monoclonal antibodies (mAb) against Giardia lamblia.Methods Two BALB/c mice of 8 weeks were immunized with antigens extracted from G. lamblia trophozoites, based on which mAb was prepared with traditional hybridoma technology. mAb isotyping was performed using enzyme-linked immunosorbent assay (ELISA) with coatings by soluble trophozoites antigen (STA) and excretory-secretory protein (ESP) prepared from G. lamblia trophozoites. mAb recognition of STA and ESP was identified by Western blotting. ELISA was performed to detect mAb reactions with G. lamblia trophozoite antigen as well as its cross-reactions with antigens from other parasite species. Results Twelve mAb-secreting hybridoma cell lines were obtained, comprising BB3, CE5, CC10, EG4, GC7, CC1, EF6, DB8, BG10, GH7, HC7 and EB2, all producing the IgG1-subtype mAb. The EB2 mAb specifically recognized proteins with M_r of 175 000 and 191 000 from STA and ESP, respectively, with no cross-reactions to the antigen extracts from Escherichia coli, Ascaris suum, Blastocystis hominis, eggs and adult worms of Schistosoma japonicum, and adult worms of Paragonimus westermani. Conclusion The specific IgG1-type mAb for G. lamblia trophozoites has been prepared.

Establishment of ITS-based PCR-RFLP for identifying three species of nematodes in a white yak

Liang HAN, Dong-hui ZHOU, Qing LIU, Wen-bin ZHEN, Xing-quan ZHU, Xiao-lin SUN

2017, 35(2):  164-168. 

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Objective To develop the polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method based on internal transcribed spacers (ITS) of ribosomal DNA to identity three nematode species that infected a white yak. Methods Twelve nematodes were collected from the small intestine of a white yak in Tianzhu Tibetan Autonomous County, Gansu Province, and DNA was extracted. Then the ITS region was amplified by PCR and sequenced. The sequences were analysed by DNAstar7.1 software and the phylogenetic tree was constructed by the neighbor-joining (NJ) method. Specific sites for restriction endonucleases were identified, based on which the PCR products were digested, and the PCR-RFLP method was established. Results PCR resulted in specific ITS bands of 900 bp for all the 12 nematodes. Sequencing results showed that each was approximately 900 bp in length. The sequences of 4 of the 12 nematodes had sequence similarity of > 99% with Cooperia oncophora (GenBank accession number AB534601), Teladorsagia circumcincta (GenBank accession number JF680984), and Ostertagia sp. (GenBank accession number HQ844228). However, the interspecies difference in ITS sequence among the three species was 4.3%-13.9%, while the intraspecific difference was < 1%. The interspecies difference in ITS sequence was most significant between C. Oncophora and T. circumcincta (12.9%-13.1%), medium between C. oncophora and Ostertagia sp. (13.6%-13.9%), and the least between T. circumcincta and Ostertagia sp. (4.3%-4.8%). The phylogenetic tree grouped the three nematode species into three branches, and revealed a closer ralationship between T. circumcincta and Ostertagia sp. PCR-RFLP analyses showed that the ITS sequences of C. Oncophora and T. circumcinctais were cut into fragments of 650 bp and 250 bp with endonuclease NdeⅠ, while only the ITS sequence of C. oncophora was cut into fragments of 700 bp and 200 bp with EcoR Ⅴ. The two endonucleases were ineffective for the ITS sequences of Ostertagia sp. Conclusions The three species of nematodes are successfully identified with ITS-based PCR-RFLP.

Preliminary exploration of loop-mediated isothermal amplification based on cox2 gene of Echinococcus granulosus

Yan-yan ZHANG, Qian YE, Zheng-rong WANG, Xin-wen BO

2017, 35(2):  169-172. 

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Objective To establish a loop-mediated isothermal amplification(LAMP) assay for rapid detection of Echinococcus granulosus cytochrome c oxidase subunit 2(cox2) gene in dog feces. Methods Four primers were designed based on the cox2 gene of Echinococcus granulosus and were used for the LAMP assay. LAMP assay and conventional PCR were performed to detect E. granulosus, Cysticercus tenuicollis, Moniezia expansa, Thysaniezia ovilla, Avitellina centripunctata, flagella nematode, Fasciola hepatica, E. multilocularis and Haemonchus contortus, and the specificity of LAMP was evaluated. The sensitivity of LAMP was compared with that of conventional PCR using gradient dilutions of recombinant plasmid carrying cox2 fragment of E. granulosus. In addition, LAMP, conventional PCR and ELISA were performed to detect E. granulosus in 50 dog fecal samples. The positive rate of E. granulosus was calculated and the efficacy of LAMP was evaluated. Results The LAMP assay only produced specific bands for E. granulosus among various species. The sensitivity of LAMP for E. granulosus was 16.09 ag/μl, which was 1 000 times higher than that of conventional PCR(16.9 fg/μl). Regarding the fecal samples, the three methods revealed same positive rate of 8.0%(4/50). Conclusion The LAMP assay is a convenient method to detect E. granulosus based on the cox2 gene with a high specificity and sensitivity, revealing a suitable technique for rapid detection of E. granulosus in dogs.

REVIEWS

Advances on the infection of parasites in treating inflammatory bowel diseases

Hui-wen XU, Jian-ping CAO, Kui-yang ZHENG, Wei PAN

2017, 35(2):  173-179. 

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Epidemiological evidence has shown that the morbidity of inflammatory bowel disease in human is negatively correlated with the infection rate of parasites, indicating a therapeutic potential of parasite infection for inflammatory bowel disease. There have been explorations on a switch from parasite elimination to using their excretory-secretory products to develop safe and effective inflammatory bowel disease therapies. This review summarizes the therapeutic effectiveness of parasite infection models in the treatment of inflammatory bowel disease and the underlying mechanisms.

Progress on parasite tubulin

Jia-qing YAO, Cong-shan LIU, Hao-bing ZHANG

2017, 35(2):  180-184. 

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Parasites threaten the health of humans and animals, so there is an imperative need for pharmaceutical control and treatment of parasitic infection. The anti-parasite drugs targeting the microtubules of parasites have attracted much attention. In this article, we summarize the properties of microtubules, their interactions with drugs, resistance to drugs, as well as the experimental methods, in order to provide theoretical basis for exploring new anti-parasite drugs.

The role of innate immunity in defending against infection with Cryptosporidium parvum

Xiao-jie LIU, Tie-bo MAO, Rui ZHOU

2017, 35(2):  185-192. 

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Cryptosporidium parvum is an important pathogen that causes diarrhea in humans, especially in children < 2 years. Because of the“minimally invasive” nature of Cryptosporidium parasites, the innate immune responses mediated by epithelial cells are critical for the host to defend against infection, particularly infection with Cryptosporidium. In this review, we focus on the effector molecules secreted by epithelial cells, pattern recognition receptor activation, miRNA regulation, exosome release, and the complement system, in order to elucidate the mechanisms underlying the role of innate immunity in defending against infection with Cryptosporidium parvum.

SHORT COMMUNICATIONS

Facial infection with Demodex spp. among Indian foreign students and native students in Jiamusi University

Yue DAI, Ju-xiang SU, Yan-jie LI, Sheng BI, Hui-ming ZHANG, Lian-shun CAI, Li-ping CHEN, Guang CHEN

2017, 35(2):  192-194. 

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This study aimed to investigate the prevalence of facial infection with Demodex spp. among 291 Indian students and 314 native students of class 2013 in Jiamusi University, China. Samples were collected with the method of skin squeeze/scraping smear and subjected to microscopic examination within a drop of glycerol. Statistics were performed with SPSS 19.0 software. The results showed that in foreign students the positive rate of infection in males was 16.4% (34/207), and that in females was 9.5% (8/84), while in native students they were 49.0% (71/145) and 18.9% (32/169), respectively (χ² = 43.14, P < 0.05 for males). There was a dominance of Demodex folliculorum infection followed by Demodex brevis infection and mixed infection in all the foreign students and the male natives, and a dominance of mixed infection in the female natives. In addition, in native students the infection rate in those with facial symptoms(68.2%) was significantly higher than those showing healthy facial skin (19.0%) （χ² = 69.43, P < 0.05）.

Surveillance of sand flies at desert-like area in Jiashi County of Xinjiang: potential vectors of visceral leishmaniasis

OSMAN Yisilayin, Duo-zhong WANG, Yan-yan HOU, KEYUMU Kaisuer, Xin-ping ZUO, Zi-chao MA, Duan-ming WANG, ABLIMIT Maimaitiaili, GHUPPUR Aibibula

2017, 35(2):  194-196. 

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To understand the changes of sand flies in endemic regions of visceral leishmaniasis in Xinjiang, sand flies were captured using arrest tubes on outer walls of human houses, light traps in field, and viscous oil-papers, in the sampling site of Gholtoghrak desert agricultural area in Jiashi County from June 15 to June 30, 2016. The density and species of sand flies were determined and identified, respectively. The infection status with leishmania promastigote in the digestive tract of female sand flies under natural conditions was examined. Totally 497 sand flies were collected, all identified to be Phlebotomus wui with a female-to-male ratio of 3.04 ∶ 1 (374 versus 123). A total of 182 sand flies were captured using arrest tubes, with a density of 30.3/work hour; 259 sand flies were captured using light traps in field, with a density of 43.2/work hour; and 56 using viscous oil-papers in courtyards, with a density of 5.6/piece. One hundred and nine female sand flies were dissected, and two showed infection with leishmania promastigote in the digestive tract, resulting in an infection rate of 1.83%. These findings suggest a high density of sand flies and the presence of Leishmania infection in Gholtoghrak area.