Table of Content

30 June 2011, Volume 29 Issue 3

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Cloning, Expression and Stage-specific Analysis of Schistosoma japonicum P7 Antigen and Evaluation of its Value in Early Diagnosis

XU Bin, DUAN Xin-Wei, LU Yan, CHEN Shen-bo, FENG Zheng, HU Wei

2011, 29(3):  1-161-166. 

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  Objective   To clone and express Schistosoma japonicum P7 antigen（GenBank accession No. EU121231）, analyze stage-specific transcription and expression of the antigen, and evaluate its value in early diagnosis.  Methods  The positive clone （P7） screened from schistosomula cDNA library was amplified by PCR. The PCR product was subcloned into prokaryotic expression vector pET28a. The recombinant plasmids were identified by restrictive enzymes digestion. The positive recombinant plasmids were transformed into E. coli BL21（DE3）, induced by IPTG for expression and purified. The diagnostic value of P7 recombinant protein was evaluated by Western blotting analysis. RT-PCR and Western blotting were used to investigate the differential transcription and expression of P7 during the developmental stages. The specific antibodies against P7 recombinant protein in the sera of S. japonicum-infected rabbits at 14 d post-infection, sera of schistosomiasis （28 cases）, clonorchiasis （30 cases） and paragonimiasis （20 cases） patients, and sera of healthy people (30 cases) were detected by ELISA, respectively.  Results   The expression vector of p7/pET28a was established and the P7 recombinant protein （about M_r 20 100） was expressed in E. coli. Western blotting analysis showed that the recombinant protein was specifically recognized by immunized rabbit sera, and sera from mice on the 14th day post infection, but was not recognized by the sera of mice at 42 d post-infection. P7 mRNA was detected in cercariae, schistosomula and adult worms, while the protein was only found in schistosomula. The positive rate of rabbit sera collected at 14 d post-infection was 83.3%（15/18）. The sensitivity and specificity of ELISA for diagnosis of schisto-somiasis japonica were 75.0% （21/28） and 93.8% （75/80）, respectively. And the P7 protein showed cross reaction with sera of clonorchiasis and paragonimiasis patients with positive rates of 6.7%（2/30） and 5.0%（1/20）, respectively.  Conclusion   P7 antigen might be a potential candidate for early diagnosis of schistosomiasis.

Cloning, Expression and Immunodiagnostic Evaluation of Antigen EPC1 from Echinococcus granulosus

CAI Hui-Xia, SHEN Yu-Juan, HAN Xiu-Min, YUAN Zhong-Ying, WANG Hu, XU Yu-Xin, HU Yuan, LU Wei-Yuan, GUAN Ya-Yi, CAO Jian-Ping

2011, 29(3):  2-167-171. 

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Objective   To clone and express EPC1 gene of Echinococcus granulosus， and investigate its immunogenicity and diagnostic value.   Methods   Total RNA was extracted from hydatid cyst protoscoleces and EPC1 gene of Echinococcus granulosus was amplified by RT-PCR. The PCR product was cloned into pGEM-T vector, and then subcloned into the prokaryotic expression vector PET28a（+）. The positive recombinants were transformed into Escherichia coli BL21（DE3）, and followed by expression of the protein induced by IPTG. The recombinant protein was identified by SDS-PAGE and Western blotting, and used to establish ELISA. Serum samples from patients with cystic echinococcosis （60 cases）, alveolar echinococcosis （37 cases）, cysticercosis （16 cases）, clonorchiasis sinensis （7 cases）, schistosomiasis japonica （4 cases） and healthy persons （33 cases） were examined.   Results   The recombinant plasmid PET28a-EgEPC1 was identified by restriction enzyme digestion and sequencing. SDS-PAGE result showed that the recombinant containing recombinant plasmid PET28a-EgEPC1 expressed a soluble fusion protein of EgEPC1（about M_r 11 000）. The protein was recognized by pool sera of cystic echinococcosis patients. The overall sensitivity and specificity of diagnosis by ELISA for cystic echinococcosis were 78.3%（47/60） , and 98.3%（59/60）, respectively. The cross reaction with sera of alveolar echinococcosis was 40.5% （15/37）.   Conclusion   The recombinant EgEPC1 antigen has diagnostic value in cystic echinococcosis.

Evaluation of Clonorchis sinensis PPMP Ⅰ Antigen Cs2 Recombinant Protein for Immunodiagnosis of Clonorchiasis

ZHOU Yan, XU Xua-Nian, YAO Kai-Ling, ZHANG Hong-Man, CHENG Na, BAO Yi-Fang, ZHANG Lu-Juan, XU Bin, JIANG He, LI Xua-Ming, Peter Chun, FENG Zheng

2011, 29(3):  3-172-176. 

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Objective  To develop and preliminarily evaluate two immunodiagnostic methods for clonorchiasis using Clonorchis sinensis PPMP Ⅰ antigen Cs2 recombinant protein （rCs2）.  Methods  Using the soluble rCs2， an indirect ELISA and a colloidal-gold immuno-chromatography assay （GICA） dynamic flow strip was developed for detecting specific antibodies in serum. Serum samples from 35 egg-positive clonorchiasis patients， 33 healthy individuals， 15 schistosomiasis patients， 15 paragonimiasis westermani patients and 13 cysticercosis patients were examined by ELISA and GICA strip test. To further evaluate the diagnostic value of these two methods， eight New Zealand rabbits were randomly divided into infected group and treatment group. Each rabbit was infected with 600 C. sinensis metacercaria. Rabbits in treatment group were treated with praziquantel [150 mg/（kg·d）×2 d] individually at day 56 post-infection. ELISA and GICA strip test were used to observe the dynamic changes of specific antibodies against rCs2 in the two parallel groups during the period of 0-44 weeks.  Results  The sensitivity， specificity and total coincidence rate determined by the ELISA method were 71.4%（25/35）， 93.4%（71/76）, and 86.5%（96/111）， respectively， and the cross reaction with schistosomiasis， paragonimiasis and cysticercosis patients were 1/15， 1/15, and 1/13， respectively. The sensitivity， specificity and coincidence rate in the GICA strip test were 85.7%（30/35）， 92.1%（70/76）, and 90.1%（100/111）， respectively. In C. sinensi-infected rabbits， antibodies level began to increase at 4 weeks after infection， peaked at the 6th week， and declined rapidly to a lower level in the 20th week， while the changing pattern of antibodies level in the treatment group was similar with that of infected group （P>0.05）. In the GICA strip test， antibodies in two groups could be detected in 4-16 weeks.  Conclusion   Indirect ELISA and the GICA dynamic flow strip developed in this study may be of value in the immunodiagnosis of clonorchiasis.

Cloning and Expression of Adenine Phosphoribosyltransferase （APRT） and its Differential Expression Analysis during the Developmental Stages of Schistosoma japonicum

KONG Juan, FENG Zheng, XU Bin, DENG Wang-Ping, YANG Zhong, HU Wei

2011, 29(3):  4-179-182. 

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Objective  To clone and express adenine phosphoribosyltransferase gene of Schistosoma japonicum， and analyze its stage-specific transcription and expression at different developmental stages of S. japonicum.  Methods  Specific primers were designed according to the reported EST sequence of APRT1 gene （GenBank Accession No. AAW24796）. RT-PCR was used to investigate the differential transcription of SjAPRT1 gene during the developmental stages. The gene was cloned into pET28a(+) plasmid. The recombinant plasmid rSjAPRT1/pET28a(+) was transformed into E. coli BL21(DE3) and induced with IPTG. The recombinant protein was purified with Ni-NTA resin and analyzed by SDS-PAGE. The purified protein was used to immune New Zealand white rabbits to obtain the antiserum. Western blotting was used to investigate the immunogenicity and the differential expression of rSjAPRT1 at different developmental stages.  Results  RT-PCR result showed that the specific bands were detected in eggs， cercariae， schistosomula, and adult worms （561 bp）. Western blotting analysis showed that the recombinant protein （rSjAPRT1, about M_r 25 000） existed in eggs， schistosomula and adult worms. The recombinant protein was recognized by pooled sera of infected rabbits.  Conclusion   The recombinant protein （rSjAPRT1） shows specific immunoreactivity, and is detected in the stage of eggs, schistosomula, and adult worms.

Anti-hepatofibrosis Effect of Fasudil Hydrochloride on Schistosoma japonicum-infected Mice

ZHENG Dan, LIANG Yue-Jin, MAO Wen-Qian, LI Ran, WANG Yong

2011, 29(3):  5-183-188. 

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Objective   To investigate the anti-fibrotic effect of fasudil hydrochloride on Schistosoma japonicum-infected mice， and the effect of fasudil hydrochloride on hepatic stellate cells （HSCs）.   Methods   Thirty female BALB/c mice were randomly divided into 3 groups viz. normal control group （NC group）, infection group, and experiment group. Mice in both infection group and experiment group were infected with （14±2） cercariae of S. japonicum. At 6 weeks post infection, mice in experiment group were intraperitoneally injected with fasudil hydrochloride （10 mg/kg） twice a day for 7 d, while mice in NC group and infection group received the same volume of physiological saline. All mice were sacrificed 12 h after the last injection. Livers from NC group and infection group were used to prepare tissue sections for hematoxylin and eosin （HE） staining, or sirius red staining, and observed under light microscope. Livers from all three groups were used to detect content of hydroxyproline （Hyp） and the mRNA expressions of α-smooth muscle actin （α-SMA）, type Ⅰ collagen α1 （Col1α1） and epithelial cell transforming sequence 2 （Ect2）. HSCs from mice in all three groups were isolated to detect the mRNA levels of α-SMA, Col1α1, and Ect2, respectively.  Results   Pathological sections showed that in livers from mice in infection group, inflammatory cells infiltrated and collagenous fibre proliferated around portal areas and egg granulomas. The content of Hyp in liver from mice of NC group, infection group, and experiment group was （279.7±21.2） μg, （528.0±15.0） μg, and （355.4±22.6） μg, respectively. The content of Hyp in livers from mice of experiment group was significantly reduced compared to infection group （P<0.01）. The mRNA expression of α-SMA, Col1α1 and Ect2 in livers and HSCs from mice in experiment group were significantly down-regulated compared to infection group （P<0.05）.  Conclusion   Fasudil hydrochloride can depress hepatofibrosis in Schistosoma japonicum-infected mice.

Cloning, Expression and Purification of Arginine Kinase from Blattella germanica and its Immune Activity

YAN Hao, XIA Li-Xin, CHEN Jia-Jie, LIU Jiao, DENG Zhi-Qiong, YI Hai-Tao, LIU Xiao-Ping

2011, 29(3):  6-191-194. 

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Objective   To clone and express the arginine kinase （AK） gene of Blattella germanica and analyze its immune activity.   Methods   The cDNA of AK was cloned using specific primers from the total RNA of Blattella germanica. The open reading frame （ORF） of AK was cloned into pET-28A vector, and expressed in Escherichia coli BL21（DE3） with IPTG induction. The recombinant protein was purified by Ni²⁺ chelating affinity chromatography. The recombinant protein was detected by SDS-PAGE， and its immune activity was analyzed by Western blotting.  Results   The cloned cDNA ORF sequence （GenBank accession No. FJ514482） contained 1 071 bp and encoded 356 amino acids. Its sequence homology with the published one （GenBank accession No. EU429466） was 97.2% at nucleotide level. The recombinant containing recombinant plasmid pET-28a-AK expressed a soluble protein of AK （M_r 45 000） after being induced with IPTG. The recombinant AK protein was recognized by sera of allergic patients, indicating that the recombinant AK protein has an adequete response activity.  Conclusion   The AK gene of Blattella germanica has been cloned and the recombinant AK protein has been confirmed with immune activity.

Preliminary Study on the Comprehensive Evaluation Method of Schistosomiasis Health Education ——A Field Study in a Heavy Endemic Area in Lake Region

TANG Qi-Qiang, ZHAO An, ZHANG Jing-Wei

2011, 29(3):  7-195-199. 

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Objective   To explore a comprehensive evaluation method of knowledge, attitude and behavior （KAP） about schistosomiasis control among students of susceptibility zone in lake region.  Methods   A comprehensive KAP evaluation indicator system was developed by using analytic hierarchy process （AHP）. The system consists of three layers. First-layer indices include knowledge, altitude and behavior of schistosomiasis control. 17 specific parts compose second-layer indices. Third-layer indices have 30 specific aspects. The experts were invited to provide comments for these indicators, and indicator weights were formulated by using AHP. A questionnaire survey was conducted among students of grade 3 and 4 from Central Primary School， Liyuzhou Primary School and Meichi Primary School in Wuxing Farm. Status of health education for schistosomiasis was evaluated by this method.   Results   In the indicator system， the weights of behavior， altitude and knowledge were 0.54, 0.30, and 0.16, respectively. The average score of KAP indicator was 8.45. The score for behavior, altitude and knowledge indicators was 8.86, 8.90, and 6.25, respectively. The scores of some indicators such as fishing outside and inside levee, washing outside levee and grazing in marshland were 8.17-9.29.  Conclusion   This comprehensive evaluation method can be used to evaluate basic situation and effect of health education in schistosomiasis intervention. The health education should be strengthened in the survey site.

Resistance Assay of Malaria Vectors to Four Kinds of Common Insecticides in Some Endemic Areas of Hainan Province

ZENG Lin-Hai, WANG Shan-Qing, SUN Ding-Wei, ZHAO Wei, LI Shan-GAn, YANG Xia

2011, 29(3):  8-200-203. 

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Objective   To determine the resistance of malaria vectors to four kinds of common insecticides in some endemic areas of Hainan Province.  Methods   Anopheline mosquitoes were collected between 2008 and 2010 from malaria endemic areas where insecticides were used for years. Anopheles dirus were collected from human-baited trap in Wangxia Town of Changjiang County. An. minimus and An. sinensis were collected by cow-baited trap in Jiangbian Town of Dongfang City. F₀ generation female An. sinensis, F₁ generation of female An. dirus and An. minimus were selected and exposed to insecticide impregnated papers with discriminating concentrations of DDT （4%）, deltamethrin （0.05%）, cyfluthrin （0.15%）, and malathion （5%） using WHO standard assays. Knockdown rate was recorded at 10, 15, 20, 30, 40, 50, and 60 min, and KT₅₀ values were calculated. Mortality was recorded after 24 hours of exposure.  Results   Mortality in An. dirus was 100% to DDT, deltamethrin and malathion. Knockdown rate of An. dirus exposed to DDT and deltamethrin was 82.0% and 100%, with a KT₅₀ value of 46.9 and 18.4 min, respectively. Mortality of An. minimus to DDT, deltamethrin, cyfluthrin and malathion was 98.1%, 99.0%, 100%, and 100%, respectively. The knockdown rate of An. minimus to DDT, deltamethrin, and cyfluthrin was 96.3%, 99.0%, and 100%, respectively， and the KT₅₀ value was 31.3, 16.8, and 7.4 min, respectively. Mortality of An. sisensis to DDT, deltamethrin, and malathion was 19.8%, 22.9%, and 43.8%, respectively. Knockdown rate of An. sisensis to DDT and deltamethrin was 2.0%, the KT₅₀ can not be calculated.   Conclusion   An. dirus and An. minimus, the main malaria vectors in the survey sites of Hainan Province, are susceptible to the four insecticides, while secondary malaria vector An. sinensis showed resistance to DDT, deltamethrin, and malathion.

Cloning, Expression and Purification of Silent Information  Regulator 2 from Giardia lamblia

ZHANG Xia, JU Hong-Mei, WANG Yun-Hua, LI Ya-Jie

2011, 29(3):  9-204-207. 

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Objective  To clone and express silent information regulator 2 （Sir2） gene from Giardia lamblia.  Methods   The GlSir2 gene was amplified by PCR from genomic DNA of Giardia lamblia（Chinese strain C2 clone）. PCR product was cloned into pMD-19T vector and transformed into E. coli JM109. The recombinant plasmid was sequenced and then cloned into the pET28b vector. The pET28b-GllSir2 recombinant plasmid was transformed into E. coli BL21（DE3）， followed by expression of the protein induced by IPTG. The recombinant protein was analyzed by SDS-PAGE. Inclusion bodies were dissolved with 8 mol/L urea, and the supernatant was collected and applied to Ni²⁺ affinity chromatography. The purified recombinant protein was renatured by dialysis and verified by Western blotting using anti-His tag antibody.  Results  GlSir2 gene sequence was cloned. The GlSir2 open reading frame （1 680 bp） encoded a 559-amino acid protein with M_r 62 800. The recombinant plasmid pET28b-GlSir2 expressed an inclusion body protein of GlSir2 after being induced with IPTG. The protein purity reached above 80% after purification. The purified protein was renatured by dialysis. The recombinant GlSir2 was recognized by anti-His tag antibody.  Conclusion  The coding sequence of GlSir2 gene was cloned and expressed in vitro. The recombinant protein was identified by anti-His tag antibody.

X-ray Irradiation against Echinococcus multilocularis Protoscoleces in vitro

BAO Yong-Xing, MAO Rui, QI Hong-Zhi, ZHANG Yue-Fen, NI Ya-Qiong, XIE Zeng-Ru, Aziguli Tursun, WEN Hao

2011, 29(3):  10-208-211. 

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Objective   To explore the effect of X-ray irradiation on Echinococcus multilocularis protoscoleces in vitro.  Methods   Echinococcus multilocularis protoscoleces were collected from cysts of infected Meriones meridianus and then cultured in RPMI 1640 medium. Protoscoleces were subpackaged into culture flasks at a density of about 104 per flask after culture for 3 days. Each group has 10 culture flasks. There were seven groups named as blank control group, low dose group （15 Gy and 30 Gy）, medium dose group （45 Gy and 60 Gy）, high dose group（75 Gy and 90 Gy）, albendazole group （2 500 ng/ml）, 45 Gy X-ray+2 500 ng/ml albendazole group, and 75 Gy X-ray +2 500 ng/ml albendazole group. Protoscoleces received three radiations on every other day with a source-skin distance of 100 cm and at a dose rate of 200 cGy/min after 3 days in culture. At each day after irradiation, protoscoleces were counted by light microscope with 0.1% eosin staining, and calculated mortality rate （per 100 protoscoleces） until all the parasites in experimental groups died. At the same time, the morphological changes of protoscoleces were observed.  Results   There were significant differences in protoscolex mortality between X-ray groups and blank control group（P＜0.05）， between X-ray+albendazole groups and albendazole group（P＜0.05）. Protoscolex mortality in albendazole group were higher than that of blank control group（P＜0.05）. Significant difference were also found in protoscolex mortality between albendazole combined with radiation and radiation only（P＜0.05）. Before radiation, protoscoleces was normal with complete structure. After radiation, the parasites were mostly valgus type protoscoleces with disordered rostellar hooks and deformed acetabulum， and finally died.  Conclusion   X-ray can kill Echinococcus multilocularis protoscoleces in vitro.

Research Progress on the Molecular Pathogenesis of Trichomonas vaginalis

TANG Zi-Hao, XU Jing-Bo, MEI Jun, GAO Xing-Zheng

2011, 29(3):  11-215-218,223. 

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Trichomonas vaginalis is one of the most common human sexually transmitted pathogens that colonize the urogenital mucosa. This paper reviews those factors in the molecular pathogenesis of the parasite，including cell adhesin, interaction with fibronectin and laminin，G-proteins，pore-forming ptotein and proteinases.

Alternatively Activated Macrophages in Helminth Infections

BAI Xue, YU Jian-Li, WANG Feng, ZHAO Ying, LIU Meng-Yuan, WANG Guang-Ming

2011, 29(3):  12-219-223. 

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Macrophages not only initiate and modulate immune responses, but also are the final effector cells. Recent studies suggested that macrophages conventionally associated with IFN-γ dominant Th1-type responses and also playing an essential role in the Th2-type inflammatory response, exhibit a quite different activation from the classically activated macrophages （CAMΦ） stimulated during Th1-type responses, therefore named as alternatively activated macrophages （AAMΦ）. AAMΦ have multiple effects during helminth infection, including control of inflammatory reaction, contribution to fibrosis and repair at the site of injury, and anti-helminth effect. This article reviews recent findings regarding the role of AAMΦ in the development of disease and host protection following helminth infection.

Application and Progress of Fluorescence in situ Hybridization in Schistosome Biology

MO Xiao-Jin, FENG Zheng, HU Wei

2011, 29(3):  13-224-228,232. 

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Schistosomiasis is one of the most serious parasitic diseases. Schistosome genes research provides the basis for study of schistosomiasis diagnosis, vaccine and drug targets. Fluorescence in situ hybridization （FISH） in schistosome focuses on researches of location of functional genes on chromosomes, genome physical mapping and chromosome identification. This article reviews the application of FISH in schistosome biology and its potential development.

Progress on Classification and Identification of Acanthamoeba

PIAO Jie, XUAN Ying-Hua, ZHENG Shan-Zi

2011, 29(3):  14-229-232. 

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As a pathogenic free-living amoeba， Acanthamoeba is easy to be recognized at the genus level， but difficult to identify at species level on the morphological basis. This review summarizes the methods for Acanthamoeba species classification and identification.

Design and Trial of Computer Test System for Experiment Courses of Human Parasitology

LIAO Hua, LING Jin, SU Shui-Lian, ZENG Jie, XIE Qiong-Jun

2011, 29(3):  15-233-235. 

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Based on the traditional experimental test of human parasitology, a reform was conducted to avoid the shortage of specimens and a disclosure of test questions. An experimental test system of human parasitology based on client/server （C/S） structure was therefore developed. This practicable system can increase the efficiency and fairness of examination and reduce cost.

Strengthening the Research on Clonorchiasis in China

QIAN Men-Bao, ZHOU Xiao-Nong, FANG Yue-Yi, LIANG Song, CHEN Ying-Dan

2011, 29(3):  16-211-214. 

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Liver flukes mainly include Clonorchis sinensis, Opisthorchis viverrini, and Opisthorchis felineus. The international congress of liver flukes was held in Khon Kaen， Thailand， during 7-8th March， 2011. The congress assembled a wide array of studies and reflected the current status of research， control and prevention of liver flukes in the world. This paper summarizes basic information from the meeting. Meanwhile， based on the research status and needs for control and prevention， priorities of research on clonorchiasis in China are discussed.

Laboratory Diagnosis of HIV/AIDS Patients Complicated with Pneumocystis jirovecii Poneumonia

CHEN Jing-Jie, LI Yong, HE Han, SU Ling-Song, QIN Song-Shu, HU Hong-Bo, TANG Liu-Sheng, LI Shi-Li

2011, 29(3):  17-176-178. 

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Pneumocystis jirovecii was detected in sputum samples and bronchoalveolar lavage fluid （BALF） obtained from HIV/AIDS patients complicated with Pneumocystis jirovecii poneumonia by Giemsa staining. CD₄⁺ T lymphocytes of 500 patients were counted by flow cytometer. P. jirovecii positive rate in sputum samples （46.8%， 845/1 806） was significantly lower than that of BALF （55.8%， 106/190）（P<0.05）. The proportion of patients developing clinical symptoms in P. jirovecii positive cases （96.6%， 816/845） was higher than that of P. jirovecii negative cases （64.0%， 615/961）（P<0.05）. P. jirovecii positive rate increased with the decrease of CD₄⁺ T lymphocyte number. P. jirovecii positive rates in cases with CD₄⁺ ＞200×10⁶/L， CD₄⁺ 200×10⁶/L-100×10⁶/L， and CD₄⁺＜100×10⁶/L were 12.0%（6/50）, 39.0%（39/100）, and 54.6% （191/350）, respectively （P＜0.05）. Giemsa staining is an efficient, simple and feasible method for P. jirovecii detection, relying on the experience and skill of the operator.

Total Protein Analysis by Two-dimensional Electrophoresis in Cysticerci of Taenia solium and Taenia asiatica

FANG Wen, XIAO Liang-Liang, BAO Huai-En, MU Rong

2011, 29(3):  18-188-190. 

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Two 20-day-old three-way crossed hybrid pigs were infected with 80 000 Taenia solium or T. asiatica eggs, respectively. Immature cysticerci of the two species in liver were collected at 40 days after infection. The total proteins were separated by two-dimensional electrophoresis, and differentially expressed proteins were analyzed by ImageMaster 2D Plantinum 6.0 software. The results showed that there were （236±12） and （231±14） protein spots in 2D electrophoresis gel images of T. solium and T. asiatica, respectively, with 3 proteins up-regulated and 7 proteins down-regulated in T. solium cysticercus by 2-fold or more compared with those in T. asiatica cysticercus.

An Efficient and Accurate Method for Counting Target Molecules in Phage-Display Peptide Library

WANG Ke-Geng, ZENG Qing-Ren, YU Zheng-Yang, ZENG Tie-Bing, LIU Yan

2011, 29(3):  19-236-238. 

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The phage titer of samples representing the low, intermediate and high phage number was respectively determined by the double-layer agar plate （DLAP） method and real-time PCR assay. The two methods accurately measured the titer of samples. The plaques from about 1/3 double-agar layer plates could be used to determine the phage titer. The DLAP experiment should repeat 10 times with 10 μl sample each time, while the within-assay coefficient variation （CV） was 4.93%-30.38%. At the same time, the real-time PCR assay only repeated 3 times with 1 μl phage each time, while CV for within-assay ranged from 0.02% to 0.25%. Results indicated that real-time PCR is a simple and quick method for determining bacteriophage titer.

Serological Detection of Cryptosporidium spp. Infection in Outpatients in Changchun

SONG Jun-Peng, ZHAO Ji-Xue, GAO Hong, LIU Yan, YUE Hong-Xia, ZHANG Jing, SU Yan, YIN Ji-Gang

2011, 29(3):  20-239-封三. 

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CP23 gene of Cryptosporidium parvum was expressed in Escherichia coli， and the recombinant protein was purified. Its immunoreactivity was analyzed by Western blotting. Serum samples were collected from outpatients of different ages from August to November, 2010 in Changchun. Indirect ELISA was established to detect the anti-CP23 IgG in sera. Western blotting analysis indicated that the recombinant CP23 protein was recognized by sera from Cryptosporidium parvum-infected calves and positive human sera, but not recognized by sera of mice infected with Schistosoma japanicum, sera from falciparum malaria patients and negetive human sera. The overall anti-CP23 IgG positive rate was 3.2% （65/2 046）. The seropositive rate was 2.7% （28/1 036） in men and 3.7% （37/1 010） in women （P>0.05）. The seropositive rates were significantly different among age groups （P<0.05）, and the age group of 71-80 had the highest positive rate （8.6%, 13/152）.

One imported case of falciparum malaria in Harbin

LIU Yang, ZHANG Ying, LU Fu-Rong

2011, 29(3):  21-封二. 

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