[Morphological and Molecular Identification of Acanthamoeba sp., A New Pathogen for Human Respiratory Tract Infection].

Objective: To identify the species of a morphologically Acanthamoeba-like pathogen in sputum from a patient with repeated cough. Methods: Protozoa were isolated from the sputum and cultured for morphological observation of the trophozoites and cysts. DNA was extracted from the cultivated sample, and PCR was performed using primers as follows: 18S universal primers for amoeba family（Ami6F1 and Ami9R） and for amoeba genus（JDP1 and JDP2）, and primers for 18S full-length sequence of S-7 ATCC reference strain of Acanthamoeba griffini （AacGF and AscGR）. The 18S rRNA was sequenced, followed by homology analysis. The maximum likelihood method was used to construct phylogenetic tree. Results: Microscopic examination showed that the trophozites had spine and irregular-shape pseudopodia bulge. The cysts were encapsulated by double membrane layer with the inner membrane having star-like processes. As expected, PCR amplification resulted in bands of 830, 479 and 1 957 bp, respectively, which were blasted to be 99%, 99% and 100% homologous to those of A. griffini（U07412.1）. Phylogenetic tree indicated that this acanthamobe in the patient's sample was 91.4%, 99.6%, 94.5% and 91.8% homologous to keratitis-associated A. castellanii, A. polyphage, A. cullbertsoni and A. rhysodes. Conclusion: The parasite in sputum of the patient with respiratory tract infection is Acanthamoeba griffini.

[Research Progress on Opportunistic Parasitic Infections].

The incidence of opportunistic parasitic infections is increasing as a result of the growing population with immune deficiency. Currently, studies on opportunistic parasitic infections are limited by the lack of animal models, due to the limited biological knowledge on the opportunistic parasitic pathogens as well as the small number of studies on species identification and typing, epidemiologic status, as well as the source and route of infection. The prevalence of HIV has promoted the research and understanding of opportunistic parasitic infections, which, in turn, has greatly reduced the morbidity and mortality of opportunistic infections. However, there still exists a bottleneck for the control and prevention of infectious diseases, i.e., the lack of sensitive and specific diagnostic methods and effective therapy, due to the complicated clinical manifestations and insufficient notification by clinical physicians. Supported by accumulating basic research, the discovery of diagnostic and therapeutic molecular targets is the key to overcome these problems.

[Diagnosis of an Imported Case of Plasmodium ovale Infection in Nanping City, Fujian].

An imported case previously misdiagnosed as vivax malaria was reviewed. The epidemiological data and blood sample were collected. The detection was conducted by microscopy, rapid diagnostic test (RDT) and nested PCR. The case was finally comfirmed as the first imported case of Plasmodium ovale infection in Nanping.

[Identification of three common sandfies in southern Xinjiang with multiplex PCR].

Based on the variable part of mtDNA CO I gene sequence, a multiplex PCR method was developed for the identification of the three common sandflies (Phlebotomus longiductus, Ph. wui, and Ph. alexandri) in southern Xinjiang. The results demonstrated that this multiplex PCR method was reliable, and could be used to identify the three Phlebotomus species. The PCR product of CO I gene from Ph. longiductus, Ph. wui and Ph. alexandri was 248, 632, and 395 bp, respectively.

[Cultivation of pathogenic free-living amoebae].

The isolation and culture of pathogenic free-living amoebae are useful in the diagnosis and research. This review focuses on the methods of isolation and cultivation of pathogenic free-living amoebae, including sample treatment, culture conditions, passage culture, pathogen detection, and maintenance.

[On revision of the tribe aedini mosquitoes taxa record in China, with a proposed new classification system (Diptera: Culicidae)].

This paper reports the rectification results of the tribe aedini mosquitoes formerly recorded in China, using the classification system proposed by Reinert during the recent years. Among all the 171 species of Chinese aedini mosquitoes examined, 160 species could be included in the new classification system. The other 11 species were listed in traditional taxonomic status for further study. The proposed new classification system of the Chinese aedini mosquitoes contained 29 genera, i.e. Aedes, Armigeres, Ayurakitia, Bothaella*, Bruceharrisonius*, Christophersiomyia*, Collessius*, Danielsia*, Downsiomyia*, Edwardsaedes*, Finlaya*, Fredwardsius*, Gilesius*, Heizmannia, Himalaius*, Hopkinsius*, Hulecoeteomyia*, Jihlienius*, Kenknightia*, Luius*, Mucidus*, Neomelaniconion*, Ochlerotatus, Phagomyia*, Scutomyia*, Stegomyia*, Tanakaius*, Udaya, and Verrallina. Among them, 22 genera (*) were new records in China. Besides, the authors made a significant revision to the following 4 species recorded formerly in "Fauna Sinica, Insecta Vol. 8, Diptera: Culicidae": Ae. (Edw.) antuensis as the synonym of Ed. pingpaensis, while Ae. (Sin.) occidentayunnanus, Ae. (Och.) flavidorsalis, and Ae. (Fin.) subsimilis should be rectified as Hz. (Mat.) occidentayunnana, Oc. albineus, and Ud. subsimilis, respectively.

[On a new checklist of the anopheline mosquitoes in China with rectification for some specific names].

This paper presents a new revised "Checklist of the Anopheline Mosquitoes in China" based on the development of the mosquito-taxonomic researches during the years of 1988-2007. The new checklist contained 61 species (subspecies) of anopheline mosquitoes all in China. Twelve species among the past records were omitted because of their invalid specific names which were allocated into following categories: (1) A doubtful record in China, with no typical specimen up to date since last century, e.g. Anopheles campestris reported in Yunnan; (2) Misidentification: An. atroparvus and An. indiensis; (3) Confirmed as synonyms by hybridizing experiments or molecular identification, including 9 species as follows: An. changes, An. dazhaius, An. kiangsuensis, An. anthropophagus, An. kunmingensis, An. xiaokuanus, An. junlianensis, An. yutsushiroensis (part) and An. fluviatilis. Meanwhile, the following rectified 4 anopheline mosquito species should be added to the new checklist: An. belenrae, An. lester, An. pulls, and An. baimaii.

[Maturation of dendritic cells and activation of B-lymphocytes in spleens of ICR mice infected with chloroquine-resistant Plasmodium berghei].

OBJECTIVE: To investigate the relation between activation of B-cells and maturation of dendritic cells (DC) in the spleens of ICR mice infected with chloroquine-resistant (RC) or chloroquine-sensitive (N) strain of Plasmodium berghei. METHODS: Spleens were taken after the mice were infected with N or RC strains of P. berghei and attained certain degree of parasitemia. Changes of B-cells and DCs were examined by pathological method, immunohistochemistry and immunofluorescence methods, transmission electron microscopy (TEM) and flow cytometry technology. RESULTS: Proliferation of white pulps in the spleen of mice infected with RC strain was found as compared to that with N strain. The percentage of cluster of differentiation (CD) 45R/B220, CD19 cells increased in the spleen cells, number of medium and small lymphocytes increased in the germinal centers, the immature and mature plasma cells also increased in the red pulps of spleen in RC strain-infected mice. On the contrary, in the N strain-infected mice spleen, the white pulps were reduced and the red pulps were filled with parasite-infected red blood cells; less small lymphocytes, immature and mature plasma cells were observed in red pulps. The number of CD11c DCs increased, especially in the periarteriolar lymphoid sheath, T cell area; the expression of major histocompatibility complex II (MHC II) on DC was up-regulated in RC strain-infected mice as compared to that in N strain-infected mice. TEM showed that the DCs in RC strain-infected mice spleens were more active than that in N strain-infected mice. CONCLUSION: Infection of RC strain P. berghei increases mature DCs in the spleen, which induces the proliferation of B cells and immune response.

[New channels and transporters as anti-malaria drug targets].

In order to get more nutrition from outside the erythrocyte, new channels were induced by malaria parasite. These channels play an important role in physiology of the parasitized cell. They are of interest both as potential targets in their own right and as potential drug targeting routes capable of mediating the entry of cytotoxic drugs into the appropriate compartment of the infected cell. It is hoped that this new anti-malarial strategy will help to create a sustainable anti-malaria-drug-development portfolio for the treatment of malaria.

[Investigation of mosquito abundance and composition around the Rare Birds National Nature Reserve of Yancheng, Jiangsu Province].

OBJECTIVE: To investigate the mosquito abundance and their relative species composition within and outside the Rare Birds National Nature Reserve of Yancheng, Jiangsu Province. METHODS: Sampling was carried out between May and Oct. 2004 at two weeks interval in two foci (the Reserve and nearby residential district) in Sheyang County. Mosquitoes were collected with the modified CDC light trap. Density was calculated, and species were identified. Environmental temperatures, rainfall and relative humidity were monitored during the study. RESULTS: A total of 40,912 mosquitoes were captured in the two foci. The sampled mosquitoes were identified as 4 species belonging to three genera (Anopheles sinensis, Culex pipiens pallens, Cx. tritaeniorhynchus, and Armigeres subalbatus). The most abundant mosquito species was An. sinensis and Cx pipiens pallens, which accounted for 97.7% of the whole number. 92% and 8% of the total amount of mosquitoes were collected from the nature reserve and residential district respectively. The most abundant species in the nature reserve and residential district was An. sinensis (60.6%) and Cx. pipiens pallens (76%), respectively. Within the nature reserve, there were two peaks occurred in adult abundance, in mid- and late July and mid-Sept. The abundance of mosquitoes in the area was positively correlated to the temperature (r = 0.765, P = 0.005). CONCLUSION: The wetland is an ideal breeding place for An. sinensis and Cx pipiens pallens. The peaks of mosquito abundance are in mid- and late July and mid-Sept. It is of importance to carry out surveillance on mosquito vectors with pathogen-transmitting potential.

[Human natural infection of Plasmodium knowlesi].

A blood film slide taken from a patient previously diagnosed as vivax malaria in Mojiang County, Yunnan Province, showing atypical forms. The ring forms had multinuclei, and the late trophozoites trended to form band. The schizonts and gametocytes were somewhat alike to Plasmodium vivax. PCR amplification confirmed that the patient was infected by P. knowlesi.

[Molecular identification of naturally acquired Plasmodium knowlesi infection in a human case].

OBJECTIVE: To confirm the diagnosis of a human case with atypical vivax-malaria from Yunnan Province by molecular technique. METHODS: DNA was extracted from blood films of unidentified sample, and of four known Plasmodium species (P. vivax, P. falciparum, P. knowlesi, and P. cynomolgi). A DNA-based diagnosis with the polymerase chain reaction (PCR) method targeting the small subunit ribosomal RNA (SSU rRNA) genes of genus- and species-specific (two human malaria species and P. knowlesi) was introduced. RESULTS: The PCR amplification with primer pair specific for P. knowlesi produced a single fragment of 150 bp. Sequence analysis showed that the amplified fragment was identical to the sequence of P. knowlesi. CONCLUSION: The patient was naturally infected with P. knowlesi.

[Identification of recombinant aldolase of Plasmodium falciparum and its monoclonal antibody preparation].

OBJECTIVE: To identify the recombinant aldolase (ALD) of Plasmodium falciparum, and to develop monoclonal antibodies (McAbs) against the recombinant ALD. METHODS: ALD gene was amplified by PCR from genomic DNA of FCC1/HN strain, and expressed in E. coli DH5 alpha. BALB/c mice were immunized with the recombinant ALD of P. falciparum via celiac injection for 3 times with 2 weeks interval. Three days after a booster injection, spleen cells of the immunized mice were used for producing McAbs. The immune serum was tested by IFAT and Western blotting. RESULTS: BALB/c mice immunized with purified aldolase protein developed strong immune response to the antigen, and the titer of specific antibody reached 1:10(5) in all immune sera after the third immunization. Moreover, immune sera specifically recognized the cultured P. falciparum. Western blotting showed that the immune sera recognized specifically a Mr 41000 band of crude malaria antigen. No cross-reaction with human red cells was detected. Seven positive hybridoma cell lines were obtained after 3 rows of selection. All the McAbs' subclasses belong to IgG1. IFAT showed that only 4 McAbs could recognize the cultured P. falciparum. CONCLUSION: Plasmodial aldolase has been successfully expressed and purified, and the established hybridoma cell lines can secrete McAbs specific to the aldolase of P. falciparum.

[Advances in researches on ß-carbonic anhydrases as anti-parasitic drug targets].

ß-carbonic anhydrases (ß-CAs) are ubiquitous metalloenzymes which active site contains a zinc ion (Zn²âº), and they could catalyze the hydration of carbon dioxide to bicarbonate and protons efficiently and are involved in many biological processes, such as respiration, pH and CO2 homeostasis, biosynthetic reactions, virulence regulation and so on, and may play a critical role in the life activity of many organisms which contain these enzymes. ß-CAs are widely distributed in fungi, bacteria, algae, plants and a small number of protozoan and metazoan except vertebrates. Therefore, as potential drug targets for designing and developing antibacterial and anti-parasitic drugs, ß-CAs promise a broad application prospect. This paper focuses on the distribution, physiological function and the progress of researches on ß-CAs in parasites and their vectors.

[Preparation and identification of monoclonal antibodies against the Region II+ motif in circumsporozoite protein of Plasmodium falciparum].

OBJECTIVE: To develop and identify the monoclonal antibodies (McAbs) against Region II+ motif in circumsporozoite protein of Plasmodium falciparum. METHODS: BALB/c mice were immunized with 12 peptides within Region II+ in circumsporozoite protein of P. falciparum. Spleen cells isolated from the immunized mice were fused with myeloma cell. After three times screening with ELISA, 3 positive hybridoma cell lines were obtained. RESULTS: ELISA test indicated that the McAbs reacted with recombinant circumsporozoite protein fragment containing tandemly repeat region and conserved Region II+. IFA test showed that the McAbs recognized not only the sporozoites of P. falciparum, but also the sporozoifes of P. yoelii. CONCLUSION: McAbs obtained can probe the Region II+ motif in circumsporozoite protein of P. falciparum, which might also recognize that of other Plasmodium species.

[Cloning and expression of aldolase encoding gene of Plasmodium falciparum FCC1/HN strain].

OBJECTIVE: To clone, sequence, and express the aldolase (ALD) encoding gene of Plasmodium falciparum FCC1/HN strain. METHODS: The ALD encoding gene was amplified by PCR from genomic DNA of FCC1/HN strain. The positive clones were screened and identified by agarose gel electrophoresis and endonuclease. The recombinant plasmid was transformed into E. coli M15. The fusion protein was expressed by IPTG induction and purified by Ni-NTA affinity chromatography and anion exchange column. RESULTS: The ALD gene of P. falciparum was amplified. Analysis of sequencing showed that the ALD gene of P. falciparum was identical with the sequence of other reported isolates. A Mr 41,000 fusion protein was induced by IPTG and was purified by chromatography. CONCLUSION: The ALD gene of P. falciparum FCC1/HN strain was identical to the other reported isolates. ALD fusion protein of P. falciparum was expressed and purified.

In vitro molecular evolution of AL NEIBMs improved immunoglobulin (Ig) binding and antibody detection.

AL (SpA A domain-PpL B3 domain), LD5 (PpL B3 domain-SpA D domain-PpL B3 domain-SpA D domain-PpL B3 domain, L-D-L-D-L) and LD3 (PpL B3 domain-SpA D domain-PpL B3 domain, L-D-L) are novel evolved Ig binding molecules (NEIBMs) derived from the in vitro molecular evolution of combinatorial phage libraries displaying randomly rearranged Ig-binding domains of protein A and protein L. These molecules all showed novel Ig-binding properties of double-site binding to the VH3 and Vκ regions of human Ig Fab and high affinity for human IgM, which enhanced IgM detection in the anti-HCV ELISA assay. In this double-site binding, the A domain binds to the VH3 chain with low affinity. Whether the appropriate mutations in the A domain could improve this binding remains unknown. In this study, four combinatorial phage libraries displaying AL mutants with random mutations at different amino acid positions in the A domain were constructed. Seven AL mutant phages with significantly improved Ig binding activity were obtained from the phage library displaying AL mutants randomly mutated at positions 27 and 34 through human IgM-directed in vitro evolution. Two of the seven prokaryotically expressed AL mutants, AL (VV) and AL (KA), exhibited IgM and IgG binding activities equivalent to those of wild-type AL, whereas other mutants showed attenuated binding. However, after labeling with HRP, AL (VV) and AL (KA) showed improved IgM and IgG binding activity, which significantly improved the detection in the anti-HCV assay. Thus, the present study demonstrates that the binding properties of AL were successfully improved through phage-based molecular evolution, which could substantially contribute to the use of AL in antibody detection, and provides an example of successful protein engineering through in vitro molecular evolution.

Three Gorges Dam and its impact on the potential transmission of schistosomiasis in regions along the Yangtze River.

Large-scale hydroprojects have a propensity for incurring schistosomiasis epidemics by altering the environments of their vicinities. As the construction of the Three Gorges Dam, one of the world's largest hydroprojects to date, draws near its conclusion, an assessment of the dam's capacity in causing schistosomiasis becomes more urgent and pressing. This article reviews recent investigations into the possible effects of the dam on schistosomiasis in the Three Gorges region and areas along the Yangtze downstream from the dam. Data used in this article were extracted from peer-reviewed papers found in PubMed, Chinese Journal of Parasitology and Parasitic Diseases, and Chinese Journal of Schistosomiasis Control. Results indicate that the Three Gorges Dam is capable of inducing a wide variety of environmental and ecological changes both within the Three Gorges region and in downstream areas. These changes, however, carry ambivalent implications for the reproduction of Oncomelania snails and the spreading of schistosome infections. Furthermore, major changes in the demographics and agricultural practices of the Three Gorges and downstream Yangtze areas caused by the dam could also exert significant influence on the transmission of schistosomiasis in these regions. Major conclusions of this review include the need for further ecological simulations of the Three Gorges Dam and the need for deploying monitoring and intervention systems to provide successful prophylaxis of the Three Gorges Dam-associated schistosomiasis emergence.