Table of Content

28 February 2011, Volume 29 Issue 1

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Malaria Situation in the People′s Republic of China in 2009

ZHOU Shui-Sen, WANG Yi, JIA Zhi-Gui

2011, 29(1):  1-1-3. 

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Therapeautic Effect on Murine Asthma with Sublingual Use of Dermatophagoides farinae/Chitosan Nanoparticle Vaccine

YU Hai-Qiong-1, 2 *, LIU Zhi-Gang-2, GUO Hua-2, ZHOU Yi-Beng-1

2011, 29(1):  2-4-9. 

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Objective   To prepare Dermatophagoides farinae （Der f）/chitosan nanoparticle vaccine （DCN）, and to investigate the effect of sublingual administration with DCN in asthma mice model.  Methods   DCN were prepared by ionotropic gelation. 30 BALB/c mice were randomly divided into 5 groups： normal control group（A）, PBS control group （B）, Chitosan group （C）, Der f group （D）, DCN group （E）. Group A were treated with normal saline （100 μl） all the time. Mice in other groups were sensitized intraperitoneally with 50 μg dust mite extracts plus 2 mg Al（OH）3, and on day 28 given a sublingual vaccination of PBS（group B）, or empty CS nanoparticles （group C）, or Der f （group D, 1 mg Der f） or DCN （group E, loaded with 1 mg Der f）. All the mice received 18 doses at 1-day intervals. One week after the last immunization, mice in group B, C, D， and E were intranasally challenged with 50 μg Der f extract daily for seven days. Twenty-four hours after the last challenge, airway hyper-responsiveness （AHR） was assessed by using whole-body plethymography. Two days post challenge, mice were sacrificed and bronchoalveolar lavage fluid （BALF） was collected. Number of the total cells and eosinophils was determined. Level of cytokines in the supernatant of splenocyte culture was assayed by ELISA. Level of Der f specific IgE, IgG2a and IgA in the sera was determined by ELISA. Airway inflammation was analyzed by HE staining. Spleen lymphocyte proliferation responses were analyzed by MTT colorimetry.   Results   Compared with group B， AHR and the lung inflammation in groups D and E were greatly reduced. Numbers of total cells and eosinophils in BALF of groups D （36.50×10⁴/ml， 3.72×10⁴/ml） and E （34.25×10⁴/ml， 2.25×10⁴/ml） were significantly lower than that of group B （61.67×10⁴/ml， 14.17×10⁴/ml） （P＜0.05）. The level of specific IgE was significantly lower in groups D （0.22） and E （0.22）， and that of IgA in groups D （0.88） and E （1.03） was significantly higher than that in group B （0.79）. The level of IL-4 in BALF （D: 28.49 pg/ml， E: 20.93 pg/ml） and cultured splenocytes （D: 27.82 pg/ml， E: 20.80 pg/ml） of groups D and E was significantly lower than that of group B （56.33 pg/ml， 45.84 pg/ml） （P＜0.05）. While IFN-γ （D: 18.80 pg/ml， E: 37.32 pg/ml） and IL-10 （D: 118.90 pg/ml， E: 129.15 pg/ml） in BALF in groups D and E were significantly higher than that of group B （13.60 pg/ml， 29.61 pg/ml） （P＜0.05）， and same with IFN-γ （D: 20.68 pg/ml， E: 42.42 pg/ml） and IL-10 （D: 36.31 pg/ml， E: 161.37 pg/ml） in spleen cultured supernatants of groups D and E （P＜0.05）. The allergen-specific splenocyte proliferation was inhibited in groups D （SI： 0.14） and E （SI： 0.13）， and there was no significant difference between group C （SI： 0.22） and group B （SI： 0.23）.   Conclusion   Dermatophagoides farinae （Der f） /chitosan nanoparticle vaccine has therapeautic effect on murine asthma.

In Vitro Effect of Seven Anthelmintic Agents against Adult Clonorchis sinensis

XU Chi-Chi, XUE Jian, ZHANG Yong-Nian, JIANG Hui-Qin, XIAO Shu-Hua-*

2011, 29(1):  3-10-15. 

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 Objective   To observe the in vitro effect of praziquantel, tribendimidine, levamisole, artemether, artesunate, albendazole and mebendazole against adult Clonorchis sinensis.  Methods   Seventy rats infected with 50-100 C. sinensis metacercariae for 5-7 weeks were euthanized, and adult C. sinensis were collected from the common bile duct. Three to four worms were placed in each well of a 24-well falcon plate, and treated by Hanks’ balanced salt solution-20% calf serum containing aforementioned drugs at various concentrations. The motor activity and morphology change of the worms were observed under an inverted microscope at 4，24，48 and 72 h post treatment.  Results   Praziquantel could reduce the motor activity of the worms rapidly which resulted in detachment of oral sucker from the well wall, curl of the worm body and emergence of vacuoles from the tegument. The minimal concentration of praziquanel to kill adult C. sinensis was 0.1 g/ml. After adult C. sinensis exposed to tribendimidine at concentrations of 0.5，1 and 10 g/ml, they revealed in paralysis, looseness and stretch of the worm body rapidly or immediately. The minimal concentration of tribendimidine to kill adult worms was 0.05 g/ml. When worms exposed to levamisole at 10 and 20 g/ml, there was a gradual decrease in the worm’s motor activity accompanied by looseness of the worm body. But 48 h post exposure, most worms showed apparently recovery of motor activity. In a higher levamisole concentration of 50 g/ml, all worms revealed in stretch and paralysis which was similar to that induced by tribendimidine. When adult C. sinensis were exposed to artemether or artesunate 10 and 50 g/ml, the motor activity of worm body and oral sucker reduced which accompanied by worm contraction, then followed by loosness of the worm body and emergence of vacuoles along the tegument. At 72 h post exposure, the worm mortalities induced by the two concentrations of the two drugs were about half, respectively. In adult C. sinensis exposed to albendazole and mebendazole at concentrations of 10 and 50 g/ml, only stimulation of motor activity of oral sucker was seen which revealed in vigorous contraction within 24 h post exposure. During 72 h observation period, no any other changes in worm activity and morphology were seen.  Conclusion   Praziquantel and tribendimidine exhibit strong in vitro killing effect on adult C. sinensis. The minimal concentration of levamisole used to kill adult worm is 50 times higher than that of tribendimidine. The higher concentrations of artemether and artesunate show slower action to reduce the worm activity and kill part of the worms. Higher concentrations of albendazole and mebendazole exhibit no killing effect on C. sinensis, besides stimulating the motor activity of worm oral sucker.

Recombinant Plasmid ZLW/pEGFP-C2 Transfection into Schistosomula of Schistosoma japonicum

LIU Pan-1, 2 , CENG Qiang-Ren-1 *, YANG Qing-Hui-1, 4 , WEI Qi-1, ZHOU Jun-3, LI Li-Xin-1, LAN Ling-Mei-1

2011, 29(1):  4-16-20. 

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Objective   To study the efficiency of ZLW/pEGFP-C2 plasmid transfection into Schistosoma japonicum schistosomula and observe its in vitro effect of anti-schistosomula.   Methods   Recombinant plasmid ZLW/pEGFP-C2 was transfected into mechanically transformed schistosomula by immersion in 0.75% DMSO and high concentration of plasmid. Enhanced green fluorescent protein （EGFP） transfected cells were observed under inverted fluorescence microscope. At 48 hours after culture, total RNA and proteins from transfected schistosomula were extracted, and the presence of the transgenes （ZLW and EGFP） in schistosomula were confirmed by RT-PCR and Western blotting. At 24, 48, 72， and 96 hours after transfection, the schistosomula were counted by light microscope with methylene blue staining. pEGFP-C2 empty plasmid group and TBS group served as controls.  Results   The transfection rate was about 10%. The fluor-escence of ZLW/EGFP protein was mainly localized in the tegument of the worms, especially abundant around oral sucker and ventral sucker. The expected size of 259 bp fragment was successfully amplified by RT-PCR and confirmed by DNA sequencing. Western blotting analysis showed that ZLW/EGFP was expressed in schistosomula. No statistically significant difference was established for schistosomula mortality among ZLW/pEGFP-C2 group （14.0%， 48.8%）, pEGFP-C2 group （15.9%， 45.7%） and TBS group （16.9%， 50.3%） at 24 and 48 hours after transfection （P＞0.05）. At 72 hours after trans-fection the mortality rate of ZLW/pEGFP-C2 group （92.7%） was significantly higher than that of pEGFP-C2 group （73.2%） （P＜0.01）, and after 96 h the mortality in ZLW/pEGFP-C2 group increased to 100%.  Conclusion   ZLW/pEGFP-C2 plasmid has been introduced into juvenile S. japonicum by immersion in 0.75% DMSO and high concentration of plasmid, and was expressed in the parasite.

Detection of specific IgG in the Sera of Patients with Chronic Schistosomiasis Japonica by Dot-ELISA with the Recombinant Sj26-Sj32 Fusion Protein

CA Shi-Fei, LI Wen-Gui-*, WANG Min

2011, 29(1):  5-21-24. 

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Objective   To study the diagnostic value of the Dot ELISA with rSj26-Sj32 fusion protein for chronic schistosomiasis japonica.   Methods   rSj26-Sj32 fusion protein and SjAWA were used to establish the HRP-IgG-Dot-ELISA. Serum samples from patients with chronic schistosomiasis japonica（40 cases）, clonorchiasis sinensis（21 cases）, paragonimiasis westermani（13 cases）, alveolar echinococcosis（10 cases）, cystic echinococcosis（9 cases）, hepatitis B（20 cases）, pulmonary tuberculosis（20 cases） and healthy persons（43 cases） were examined.   Results   Sensitivity and specificity were respectively 92.5%（37/40）and 95.4%（41/43）for rSj26-Sj32-Dot-ELISA and 95.0%（38/40）and 93.0%（40/43）for SjAWA-Dot-ELISA, and there was no significant difference between two antigens（P>0.05). There were different cross reactions to the sera of patients with clonorchiasis sinensis, paragonimiasis westermani or alveolar echinococcosis, but no cross reaction to the sera of patients with cystic echinococcosis, hepatitis B or pulmonary tuberculosis. The positive and negative predictive value and efficiency of diagnosis of rSj26-Sj32-Dot-ELISA for chronic schistosomiasis japonica were 84.1%（37/44）, 97.7%（129/132）, and 94.3%（166/176）, respectively, and those of SjAWA-Dot-ELISA were 77.6%（38/49）, 98.4%（125/127）, and 92.6%（163/176）, respectively. There was no significant difference between the two methods（P>0.05).   Conclusion   rSj26-Sj32 fusion protein can be applied to immunodiagnosis for chronic schistosomiasis japonica.

Function of TEP1 Gene during Plasmodium yoelii Infection in Anopheles dirus

WANG Yan-Yan, WANG Yang, ZHANG Jian, DUAN Jian-Hua, HUANG Fu-Sheng-*

2011, 29(1):  6-25-28. 

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Objective   To study the role of TEP1 gene from Anopheles dirus during Plasmodium yoelii infection by RNA interference.  Methods   TEP1 primers with T7 promoter were designed based on the sequence of An. dirus TEP1 gene from GenBank database. PCR amplification of TEP1 gene was completed with An. dirus cDNA as template. The AdTEP1 double-stranded RNA was synthesized by using in vitro transcription kit with purified PCR products. Female An. dirus emerged for 1-2 days were divided into three groups each with 200 mosquitoes: TEP1 interference group，EGFP interference group and control. Mosquitoes in TEP1 and EGFP interference groups were microinjected in chest with 147 ng of AdTEP1 and EGFP double-stranded RNA, respectively, while those of control group were untreated. Effect of TEP1 interference on P. yoelii in An. dirus was estimated through semi-quantitative PCR with internal reference AdS7 at 3 d after injection. On 4 d after injection, mosquitoes were infected by EGFP-expressing P. yoelii BY265. The infection rate and infectiosity of mosquitoes were observed through anatomizing 25 midguts from each group at 9 d post-infection.  Results   The AdTEP1 double-stranded RNA did well in the interference of TEP1 expression in An. dirus. The infection rate in the groups of control, EGFP and TEP1 interference was （24±2.83）%, （24±0.71）%, and （80±3.54）%, respectively；and the infectiosity of the three groups was 0.32±0.7, 0.44 ± 0.85, and 5.52 ± 4.84, respectively.   Conclusion  AdTEP1 interference increases the infection rate and infectiosity of An. dirus by P. yoelii, and raises the susceptibility of An. dirus to P. yoelii significantly. TEP1 plays a critical role in the process of P. yoelii infection.

Prokaryotic Expression and Identification of S-dsRNA Gene from Cryptosporidium parvum Virus

DIAO Yu-Mei, GONG Feng-Chao, LI Wei, SU Li-Bei, HUANG Xiang-Cheng, LI Jian-Hua, ZHANG Xi-Chen-*

2011, 29(1):  7-29-32. 

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Objective   To clone and express S-dsRNA gene of Cryptosporidium parvum virus， and investigate the reactionogenicity of the recombinant.  Methods   Total RNA was extracted from Cryptosporidium parvum and S-dsRNA gene was amplified by RT-PCR. The PCR product was cloned into pET-28a（+） expression vector. The recombinant plasmid pET-28a（+）-S was transformed into E. coli BL21 （DE3） and induced with IPTG. The expression situation of recombinant protein was analyzed by SDS-PAGE. Its reactionogenicity was examined by Western blotting analysis.   Results   pET-28a（+）-S was identified by PCR and double endonuclease digestion. SDS-PAGE result showed that the recombinant protein （M_r 37 000） was expressed in the form of inclusion body. High level expression of recombinant protein was found at 1 mmol/L IPTG condition after incubation at 37 ℃ for 4 h and reached up to 72.6 % of the total protein. The protein was recognized by the antisera from mice immunized with antigens from Cryptosporidium parvum oocysts.  Conclusion   The S-dsRNA gene of Cryptosporidium parvum virus has been expressed with adequate reactionogenicity.

The Correlation between Osteopontin and Metastasis of Hepatic Echinococcus multilocularis Infection

ZHANG Long-1, ZHANG Qi-Jie-1 *, CAO Yu-Wen-2, TUN Xiang-Wei-1, BANG Xin-Yu-1, YANG Hong-Jiang-1, SUN Gong-1

2011, 29(1):  8-33-36. 

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Objective   To investigate the expression and distribution of osteopontin （OPN） in Echinococcus multilocularis cyst, and explore the role of OPN in the metastasis of hepatic E. multilocularis infection.  Methods   Forty gerbils were infected with 20% E. multilocularis suspension （0.1 ml per gerbil） through abdominal opening injection in liver. Gerbils were sacrificed at 100 days postinfection for observing the growth and metastasis of hepatic echinococcus cyst. The liver, hepatic echinococcus cyst and metastasis tissue were observed on HE stain; the expression of OPN were measured by immunohistochemistry staining （SP method）.   Results   E. multilocularis were spread over the liver and abdominal cavity. Expression of OPN was found at different degree in echinococcus cysts. The positive expression rate of OPN in echinococcus cysts was 70% （28/40）. OPN was mainly distributed in the germinal layer, inflammatory cells and some liver cells. 60%（24/40） occurred thoracic lymph node metastases. The OPN expression rate in hepatic echinococcus cysts with thoracic lymph node metastases （83%， 20/24） was significantly higher than that of hepatic echinococcus cysts without thoracic lymph node metastases（50%， 8/16） （P＜0.05）. The positive expression of OPN in lymph node metastases （92%， 22/24） was higher than that of hepatic echinococcus cyst （70%, 28/40） （P＜0.05）.   Conclusion   Osteopontin mainly distributes in the germinal layer of hepatic echinococcus cyst and inflammatory cells, which might be involved in metastasis of hepatic E. multilocularis infection.

Cloning and Expression of var2csa DBL Domains from Plasmodium falciparum Hainan Isolate and Functional Analysis of the Recombinant Protein

KANG Wei, CHANG Zhi-An, LIU Hui-Jun, JIANG Ning, YIN Ji-Gang, CHEN Qi-Jun-*

2011, 29(1):  9-37-41. 

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Objective   To clone and express three VAR2CSA duffy antigen-binding ligand （DBL） domains （DBL4/5/6） encoded by var2csa gene of a Hainan isolate of Plasmdium falciparum, and study the difference of chondroitin sulfate A （CSA）-binding activity among them.  Methods   Three DBL domains was amplified by PCR and cloned into the vector pMD18-T. The recombinant plasmids were identified by enzyme digestion and sequencing, and then subcloned into the prokaryotic expression vector pET-22b. The recombinant plasmid was transformed into E. coli BL21 （DE3） and followed by expression of the protein induced by IPTG. The recombinant protein was purified with His GraciTrap kit and identified by SDS-PAGE and Western blotting. CSA-binding activity of the three recombinant DBL domains was assayed by ELISA.   Results   The target genes were amplified with the length of 996 bp, 859 bp and 894 bp. The constructed recombinant plasmids were identified by enzyme digestion and DNA sequencing. The recombinant proteins（DBL4/5/6） were purified, the relative molecular mass of DBL4, DBL5 and DBL6 was M_r  439 800, M_r  34 500 and M_r  36 000, respectively. The purified protein has been confirmed with immunogenicity by Western blotting. The results of adhesion experiment indicated that A₄₀₅ values of DBL5 domain with different concentration were significantly higher than that of DBL4 and DBL6.   Conclusion   The three recombinant proteins （DBL4/5/6） of VAR2CSA DBL domains were expressed, and DBL5 domain has high binding affinity with CSA.

Application of Nested PCR in Diagnosis of Imported Malaria

ZHOU Shui-Mao, WANG Chong-Xin, TUN Kai, MAO Chong-Chi, YANG Yan

2011, 29(1):  10-43-45. 

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Objective   To evaluate the usefulness of nested PCR method in the diagnosis of imported malaria.  Methods   A total of 210 blood smears and blood samples on filter paper were taken from persons returned from highly malaria endemic countries. The results of both nested PCR and microscopy for 210 samples were compared.  Results  Among the 210 persons, 43 were hospitalized due to malaria, and positive by nested PCR test. Among the rest 157 people at high risk of getting malaria, 3 were found plasmodium-positive by microscope （1.91%）, and 5 were positive by nested PCR （3.18%）. In four samples with discrepancy between the two methods, 1 was microscopy positive and PCR negative, and 3 were microscopy negative and PCR positive. Positive and negative coincidence rate between the two tests was 66.7% and 98.1%, respectively. The coincidence between the two methods was 97.5%.   Conclusion   Nested PCR is useful for monitoring, identification and diagnosis of imported malaria.

Non-surgical Treatment for Nonresectable Advanced Hepatic Alveolar Echinococcosis

TANG Qun-Ke, ZHANG Ying, LI Yong-Shou, YUAN Chun-Ping, ZHANG Dong-Tian

2011, 29(1):  11-46-48. 

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Objective   To investigate the therapeutic methods of nonresectable advanced hepatic alveolar echinococcosis.   Methods   A retrospective study was carried out to analyze 25 cases of nonresectable advanced hepatic alveolar echinococcosis in the Fourth Hospital of PLA from 2006 to 2009.  Results   There were 18 male and 7 female patients with a mean age of 41 years. Twelve cases were treated with albendazole alone. Eleven patients were treated with albendazole combined with percutaneous puncture. Two cases were treated with albendazole combined with other intervention. A course of albendazole administration lasted 2 weeks with a dose of 15-20  mg/（kg·d） for 3 course in general. Eighteen patients were followed up for 1-4 years. In albendazole group, 2 cases were effective and 7 cases were symptom-improved. Five partients got improved and 2 cases showed effective in albendazole combined with percutaneous puncture group. Two cases of albendazole combined with intervention group showed no efficacy.   Conclusions   Longterm use of albendazole is the main treatment for nonresectable advanced hepatic alveolar echinococcosis.

Application of Artificial Neural Networks in Infectious Diseases

XU Dun-Fang, ZHOU Xiao-Nong-*

2011, 29(1):  12-49-54. 

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With the development of information technology， artificial neural networks has been applied to many research fields. Due to the special features such as nonlinearity， self-adaptation， and parallel processing， artificial neural networks are applied in medicine and biology. This review summarizes the application of artificial neural networks in the relative factors， prediction and diagnosis of infectious diseases in recent years.

Progress on Functional Genomics of Some Important Zoonotic Parasites

AI Lin-1, 2 , CHEN Shao-Gong-1, CHEN Jia-Xu-1 *

2011, 29(1):  13-58-63. 

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With the development of molecular biology， genomics research has been expanded from structural genomics to functional genomics, and from single gene to massive batch. This paper summarizes the progress of structural genomics of some zoonotic parasites and major technical methods.

Immunopathological Mechanism of Cerebral Malaria

LIU Tai-ping, FU Yong, XU Wen-Yue-*

2011, 29(1):  14-64-67. 

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Cerebral malaria is a severe complication of malaria. Early studies suggest that cerebral malaria is related to cytoadherence of parasitized red blood cells to the microvessel endothelium of brain. However， more and more evidence supported that the cause of cerebral malaria is uncontrolled inflammatory cytokines and infiltration of lymphocytes in brain microvessel. The article summarizes the research progress on immunological mechanism of cerebral malaria.

Malaria Epidemic Trend and Characteristics at Monitoring Sites in Yunnan Province in 2008

CHEN Guo-Wei-1, WEI Chun-1, LI Hua-Xian-1, YANG Li-Xiang-2, HUANG Jiang-3, YAN Han-Zhang-4, TIAN Guang-Jiang-5, BAI Zhi-Rong-6

2011, 29(1):  15-54-57. 

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Malaria situation in 5 monitoring sites of Yunnan showed a decline trend from 2005 to 2008. The average malaria incidence in 2008 was 11.84/10 000 with a decrease of 66.1% in comparison to 2005. The seropositive rate with immuno-fluorescence assay （IFA） was 4.61% for pupils. 82% of the cases chose town or township hospitals as the first place of seeking diagnosis and treatment. 83.6% cases were diagnosed over 3 days of symptom appearing. The main clinical manifestation was fever every other day attack （occupied 72.7%）. 98.4% of the cases were with light symptoms. The proportion of primary attacks and relapses among malaria patients were 95.3% and 4.7%， respectively. Plasmodium vivax was the main malaria parasite, occupying 81.2%. 97.2% of the local infected cases were found in the bordering areas of the country. The mosquito net utilization rate was 51.4%. Results showed that malaria has been effectively controlled in the monitoring sites of Yunnan.

Cloning and Sequence Analysis of Thioredoxin Peroxidase Gene from Taenia multiceps

LI Yong-Guang-1, 2 , LI Wen-Hui-1, GAI Wen-Yan-1, TAO Ju-Xia-1, QU Zi-Gang-1, GU Mo-Zhong-1, Radu Blaga3, FU Bao-Quan-1 *

2011, 29(1):  16-67-70. 

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Protoscoleces of Taenia multiceps were collected from the naturally infected sheep and total RNA was extracted. Specific primers were designed according to TaHc2-D11 mRNA sequence and T. multiceps thioredoxin peroxidase gene （TmTPx） was amplified by RT-PCR. PCR products were ligated into pMD18-T vector and transformed to E. coli DH5α. The recombinant plasmids were identified by restriction digestion and sequencing. A 614 bp cDNA was amplified. The TmTPx open reading frame （591 bp） encoded a 196-amino acid protein with M_r 21 690, pI 7.61. Bioinformatics analysis indicated that TmTPx had a typical 2-Cys Prx conserved domain. Phylogenetic tree revealed that T. multiceps had the closest relationship to T. asiatica， followed by T. solium and T. crassiceps， E. granulosus and E. multilocularis.

Localization of Acetylcholinesterase in the Nervous System of Cotylophoron indicum

ZHANG Hao-1, 2 , ZHANG Wei-2, ZHU Ran-2, ZHU Ge-Hong-Xiang-1 *

2011, 29(1):  17-71-73. 

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The nervous system of Cotylophoron indicum was studied by using acetylcholine esterase histochemical staining techniques. Cranial ganglia and transverse commissure situate at dorso-lateral body between oral sucker and genital sucker. From the cranial ganglia four pairs of nerves proceed cephalad and connect with nerve network of the oral sucker. The posterior nerve cords from the cranial ganglia consist of 3 pairs and the ventral ones are the stoutest and longest nerves. A few branches from the 3 pairs of nerve cords connect to ventral sucker. There is a developed nerve network distributed in its genital sucker. The nerve fibers on body surface in pairs and parallel are diagonal and cross to form a nerve network on body surface. Three kinds of neurocytes distribute at the prosomal region. Results show that the nervous system structure of C. indicum is consistent with the essential features of Digenea， but more special and complicated around genital sucker.

Investigation on Serology， Risk Factor and Awareness of Angiostrongylus cantonensis in Hainan Province

LI Yu-Chun-1, 2 , HU Ti-Min-1, 2 , TONG JIn-Jin-1, 2 , LIU Jian-1, LI Mei-Tong-3, WANG Shan-Qing-1, 2 *

2011, 29(1):  18-74-75. 

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1 Hainan Center for Disease Control and Prevention, Haikou 570203, China; 2 Hainan Provincial Key  Discipline of Medical Parasites, Haikou 570203, China; 3 Center for Disease Control and Prevention of Dingan County, Dingan 571200, China