Supported by The Ministry of S&T, “Construction of the Platform of Natural Resourses”(No.2004DKA30480) LM Malaria remains one of the most devasting infectious diseases in the world. The annual case number of the...Supported by The Ministry of S&T, “Construction of the Platform of Natural Resourses”(No.2004DKA30480) LM Malaria remains one of the most devasting infectious diseases in the world. The annual case number of the disease is estimated to be 350～500 million, and it causes about 1～3 million deaths each year, most of them are children. Due to the emergence of the drug resistance of the parasite and the insecticide resistance of mosquitoes, it becomes more difficult to control malaria. Furthermore, Simian malaria parasites infecting human were reported many times,which might cause severe public health problem. Since prevention of malaria epidemics hasn’t achieved tremendous success, emergence of primate malaria infection in human makes the situation even more complicated. Recently when we examine retrospectively the smears of “P. vivax” collected from Yunnan Province(the patient had spent sometimes in China-Myanmar border,logging), we found the similarity in morphology between the smears of the Yunnan patient and the naturally acquired P. knowlesi infection of human reported in Thailand, which belongs to primate malaria. P. knowlesi usually infects monkeys in nature and is shown to be infective to human occasionally from the report of oversea. It hadn’t drawn much attention of scientists, because its clinic symptoms were neither specific nor severe. The morphology of P. knowlesi in human infection could not easily differentiate with other Plasmodium species, especially for atypical form of P. vivax and P.malariae. We observed all stages of the erythrocytic cycle of the parasite in Giemsa-stained blood films. Early trophozoites appeared as ring forms as early trophozoites of P. falciparum in multi-infection,except the rings were a little larger. Frequently, more than one ring forms were noted in erythrocytes and double or tri-chromatin dots were also seen. Late trophozoites were usually amoeboid, as that of P. vivax. Sometimes, the cytoplasm was compact, which occupied no more than two-third of the erythrocytes. Some late trophozoites had a trend to form “band” that are typical for P. malariae. Mature schizonts had a central cluster of 8 to 16 merozoites and did not fill the whole erythrocyte. Late trophozoites and schizonts were densely pigmented with dark brown/black malaria pigment. Gametocytes filled most part of the erythrocytes and malaria pigment was scattered. The structure of gametocytes was compatible with that of P. vivax,but smaller. From the observation above, we can’t conclude which species of Plasmodium the patient was infected with. A DNA-based diagnosis with the polymerase chain reaction (PCR) method targeting the small subunit ribosomal RNA (SSU rRNA) genes of two human malaria species and P. knowlesi was introduced. The blood films were scraped using a clean blade. All the films were incubated overnight at 37℃ and extracted with pheonl-chlorolform and DNA was precipitated with isopropanol. Using primers targeting SSU rRNA of different Plasmodium species, the extacted DNA was amplified by PCR with different primes for P.falciparum, P.vivax and P. knowlesi,respectively. The primer pair specific for P. knowlesi produced a single fragment of 150 bp. The amplified fragments were digested by endonuclease, cloned into T-vector, and then transformed into E. coli XL-10. Analysis of sequences showed that the amplified fragment was identical to the sequence of P. knowlesi. This result confirmed that the patient was infected with P. knowlesi, not P. vivax. A pair of primer was designed according to the reported msp-1 partial sequence of P. knowlesi. The partial msp-1 encoding gene was amplified by PCR from the extracted DNA. The amplified fragments were digested by endonuclease, cloned into pET-32a vector, and then transformed into E. coli XL-10. Analysis of sequences showed that the partial msp-1 gene was identical to the sequence of P. knowlesi, which was deposited in the GenBank previously. The recombinant plasmid pET-32a/msp-1 was transformed into E. coli BL21(DE3), and then the positive recombinant clone was expressed by induction with IPTG. The expressed E. coli BL21(DE3) was analyzed by SDS-PAGE gel electrophoresis. A 31 kDa fusion protein band could be discerned. E. coli BL21(DE3) containing recombinant msp-1 was disintegrated with supersonic, then centrifuged. SDS-PAGE gel electrophoresis showed that the msp-1 is expressed in E. coli as precipitation. The protein was purified by Ni-NTA affinity chromatography column. Western blot result showed that the purified protein could react with the blood serum from the patient infected with P.falciparum and its molecular weight accorded with the theoretical expectation. This is the first report of naturally human infection of P. knowlesi in China,and also established the basis for molecular diagnosis and differentiation of Plasmodium species. Further working idea for identifying the P. vivax multinucleatum, and the importance of human infection of simian malaria are discussed.展开更多
Objective:To study the genetic diversity at the msp—1,msp—2,and glurp genes of Plasmodium falciparum(P.falciparum) isolates from 3 endemic areas in Thailand:Tak.Kanchanaburi and Ranong provinces.Methods:A total of 1...Objective:To study the genetic diversity at the msp—1,msp—2,and glurp genes of Plasmodium falciparum(P.falciparum) isolates from 3 endemic areas in Thailand:Tak.Kanchanaburi and Ranong provinces.Methods:A total of 144 P.falciparum isolates collected prior to Irealmenl during January.2012 to June,2013 were genotyped.DNA was extracted:allele frequency and diversity of msp-1.msp-2,and glurp genes were investigated by nested polymerase chain reaction.Results:P.falciparum isolates in this study had high rate of multiple genotypes infection(96.5%)with an overall mean multiplicity of infection of 3.21.The distribution of allelic families of msp-1was significantly different among isolales from Tak.kanchanahnri.and Ranong but not for the msp-2.K1 and MAD20 were the predominant allelic families at the msp-1 gene,whereas alleles belonging to 3D7 were more frequent at the msp-2 gene.The glurp gene had the least diverse alleles.Population structure of P.falciparum isolates from Tak and Ranong was quite similar as revealed by the presence of similar proportions of MAD20 and K1 alleles at msp-1 loci.3D7 and FC27 alleles at msp-2 loci as well as comparable mean MOI.Isolates from Kanchanaburi had different structures;the most prevalent alleles were K1 and RO33.Conclusions:The present study shows that P.falciparum isolates from Tak and Ranong provinces had similar allelic pattern of msp—1 and msp—2 and diversity but different from Kanchanaburi isolates.These allelic variant profiles are valuable baseline data for future epidemiological study of malaria transmission and for continued monitoring of polymorphisms associated with antimalarial drug resistance in these areas.展开更多
Objective: To determine the effects of toll-like receptor 2(TLR-2) agonist, Pam3 CSK4, on cellular and humoral immune response against recombinant Mycobacterium bovis bacille Calmette-Guérin(rBCG) expressing the ...Objective: To determine the effects of toll-like receptor 2(TLR-2) agonist, Pam3 CSK4, on cellular and humoral immune response against recombinant Mycobacterium bovis bacille Calmette-Guérin(rBCG) expressing the C-terminus of merozoite surface protein-1 of Plasmodium falciparum.Methods: Six groups of mice(n=6 per group) received intraperitoneal phosphate buffered saline T80(PBS-T80), BCG or rBCG in the presence or absence of Pam3 CSK4. Enzymelinked immunosorbent assay was carried out to measure serum total IgG, IgG1, IgG2 a, and Ig G2 b production. Spleens were also harvested and splenocytes were co-cultured with rBCG antigen for in vitro determination of IL-4 and IFN-γ via enzyme-linked immunosorbent assay.Results: The production of total IgG and the isotype IgG1, IgG2 a and IgG2 b was significantly higher in rBCG-immunised mice than in the BCG and PBS-T80-immunised mice, and Pam3 CSK4 further enhanced their productions. A similar pattern was also observed in IFN-γ production. Moreover, there was no significant difference in IL-4 production in all groups either in the presence or absence of Pam3 CSK4.Conclusions: We present evidence of the adjuvant effects of TLR-2 agonist in enhancing the production of total IgG, IgG1, IgG2 a, IgG2 b, as well as IFN-γ in response to rBCG. However, the presence or absence of Pam3 CSK4 had no effect on IL-4 production.展开更多
Objective: To determine the role of toll-like receptor 4(TLR-4) in eliciting cellular and humoral immune responses against recombinant Mycobacterium bovis bacille Calmette-Guérin(rB CG) expressing the C-terminus ...Objective: To determine the role of toll-like receptor 4(TLR-4) in eliciting cellular and humoral immune responses against recombinant Mycobacterium bovis bacille Calmette-Guérin(rB CG) expressing the C-terminus of merozoite surface protein-1 of Plasmodium falciparum. Methods: Six groups of mice(n=6 per group) were injected with phosphate buffered saline T80, BCG or r BCG intraperitoneally, in the presence or absence of a TLR-4 inhibitor; TAK-242. Enzyme-linked immunosorbent assay was carried out for serum total IgG, IgG 1, Ig G2 a and Ig G2 b determination. Spleens were also harvested and splenocytes cultured for determination of intracellular cytokines; IL-4 and IFN-毭 via enzyme-linked immunosorbent assay. Results: The production of total Ig G, and the subclasses Ig G1, Ig G2 a and Ig G2 b was significantly higher in rB CG-immunised mice than BCG and phosphate buffered saline immunised mice in the absence of TAK-242. A significant rise in total IgG occurred with more booster immunisations. The level of IgG 2 a was highest, followed by IgG 2 b, then IgG 1. The production of both IL-4 and IFN-were inhibited in t毭 was also highest in the rB CG immunised groups. These significant riseshe presence of TAK-242. Conclusions: We present evidence of the role of TLR-4 in the increased production of total IgG, IgG 1, IgG 2 a and IgG 2 b, as well as IL-4 and IFN-毭 in response to our rB CG construct.展开更多
文摘Supported by The Ministry of S&T, “Construction of the Platform of Natural Resourses”(No.2004DKA30480) LM Malaria remains one of the most devasting infectious diseases in the world. The annual case number of the disease is estimated to be 350～500 million, and it causes about 1～3 million deaths each year, most of them are children. Due to the emergence of the drug resistance of the parasite and the insecticide resistance of mosquitoes, it becomes more difficult to control malaria. Furthermore, Simian malaria parasites infecting human were reported many times,which might cause severe public health problem. Since prevention of malaria epidemics hasn’t achieved tremendous success, emergence of primate malaria infection in human makes the situation even more complicated. Recently when we examine retrospectively the smears of “P. vivax” collected from Yunnan Province(the patient had spent sometimes in China-Myanmar border,logging), we found the similarity in morphology between the smears of the Yunnan patient and the naturally acquired P. knowlesi infection of human reported in Thailand, which belongs to primate malaria. P. knowlesi usually infects monkeys in nature and is shown to be infective to human occasionally from the report of oversea. It hadn’t drawn much attention of scientists, because its clinic symptoms were neither specific nor severe. The morphology of P. knowlesi in human infection could not easily differentiate with other Plasmodium species, especially for atypical form of P. vivax and P.malariae. We observed all stages of the erythrocytic cycle of the parasite in Giemsa-stained blood films. Early trophozoites appeared as ring forms as early trophozoites of P. falciparum in multi-infection,except the rings were a little larger. Frequently, more than one ring forms were noted in erythrocytes and double or tri-chromatin dots were also seen. Late trophozoites were usually amoeboid, as that of P. vivax. Sometimes, the cytoplasm was compact, which occupied no more than two-third of the erythrocytes. Some late trophozoites had a trend to form “band” that are typical for P. malariae. Mature schizonts had a central cluster of 8 to 16 merozoites and did not fill the whole erythrocyte. Late trophozoites and schizonts were densely pigmented with dark brown/black malaria pigment. Gametocytes filled most part of the erythrocytes and malaria pigment was scattered. The structure of gametocytes was compatible with that of P. vivax,but smaller. From the observation above, we can’t conclude which species of Plasmodium the patient was infected with. A DNA-based diagnosis with the polymerase chain reaction (PCR) method targeting the small subunit ribosomal RNA (SSU rRNA) genes of two human malaria species and P. knowlesi was introduced. The blood films were scraped using a clean blade. All the films were incubated overnight at 37℃ and extracted with pheonl-chlorolform and DNA was precipitated with isopropanol. Using primers targeting SSU rRNA of different Plasmodium species, the extacted DNA was amplified by PCR with different primes for P.falciparum, P.vivax and P. knowlesi,respectively. The primer pair specific for P. knowlesi produced a single fragment of 150 bp. The amplified fragments were digested by endonuclease, cloned into T-vector, and then transformed into E. coli XL-10. Analysis of sequences showed that the amplified fragment was identical to the sequence of P. knowlesi. This result confirmed that the patient was infected with P. knowlesi, not P. vivax. A pair of primer was designed according to the reported msp-1 partial sequence of P. knowlesi. The partial msp-1 encoding gene was amplified by PCR from the extracted DNA. The amplified fragments were digested by endonuclease, cloned into pET-32a vector, and then transformed into E. coli XL-10. Analysis of sequences showed that the partial msp-1 gene was identical to the sequence of P. knowlesi, which was deposited in the GenBank previously. The recombinant plasmid pET-32a/msp-1 was transformed into E. coli BL21(DE3), and then the positive recombinant clone was expressed by induction with IPTG. The expressed E. coli BL21(DE3) was analyzed by SDS-PAGE gel electrophoresis. A 31 kDa fusion protein band could be discerned. E. coli BL21(DE3) containing recombinant msp-1 was disintegrated with supersonic, then centrifuged. SDS-PAGE gel electrophoresis showed that the msp-1 is expressed in E. coli as precipitation. The protein was purified by Ni-NTA affinity chromatography column. Western blot result showed that the purified protein could react with the blood serum from the patient infected with P.falciparum and its molecular weight accorded with the theoretical expectation. This is the first report of naturally human infection of P. knowlesi in China,and also established the basis for molecular diagnosis and differentiation of Plasmodium species. Further working idea for identifying the P. vivax multinucleatum, and the importance of human infection of simian malaria are discussed.
基金Supported by a grant from Research Institute,Bansomdejchaopraya Rajabhat University(Grant no.2555)
文摘Objective:To study the genetic diversity at the msp—1,msp—2,and glurp genes of Plasmodium falciparum(P.falciparum) isolates from 3 endemic areas in Thailand:Tak.Kanchanaburi and Ranong provinces.Methods:A total of 144 P.falciparum isolates collected prior to Irealmenl during January.2012 to June,2013 were genotyped.DNA was extracted:allele frequency and diversity of msp-1.msp-2,and glurp genes were investigated by nested polymerase chain reaction.Results:P.falciparum isolates in this study had high rate of multiple genotypes infection(96.5%)with an overall mean multiplicity of infection of 3.21.The distribution of allelic families of msp-1was significantly different among isolales from Tak.kanchanahnri.and Ranong but not for the msp-2.K1 and MAD20 were the predominant allelic families at the msp-1 gene,whereas alleles belonging to 3D7 were more frequent at the msp-2 gene.The glurp gene had the least diverse alleles.Population structure of P.falciparum isolates from Tak and Ranong was quite similar as revealed by the presence of similar proportions of MAD20 and K1 alleles at msp-1 loci.3D7 and FC27 alleles at msp-2 loci as well as comparable mean MOI.Isolates from Kanchanaburi had different structures;the most prevalent alleles were K1 and RO33.Conclusions:The present study shows that P.falciparum isolates from Tak and Ranong provinces had similar allelic pattern of msp—1 and msp—2 and diversity but different from Kanchanaburi isolates.These allelic variant profiles are valuable baseline data for future epidemiological study of malaria transmission and for continued monitoring of polymorphisms associated with antimalarial drug resistance in these areas.
基金supported by the Universiti Sains Malaysia(USM)Research University(RU)Grants(No.1001/PPSK/8011100)
文摘Objective: To determine the effects of toll-like receptor 2(TLR-2) agonist, Pam3 CSK4, on cellular and humoral immune response against recombinant Mycobacterium bovis bacille Calmette-Guérin(rBCG) expressing the C-terminus of merozoite surface protein-1 of Plasmodium falciparum.Methods: Six groups of mice(n=6 per group) received intraperitoneal phosphate buffered saline T80(PBS-T80), BCG or rBCG in the presence or absence of Pam3 CSK4. Enzymelinked immunosorbent assay was carried out to measure serum total IgG, IgG1, IgG2 a, and Ig G2 b production. Spleens were also harvested and splenocytes were co-cultured with rBCG antigen for in vitro determination of IL-4 and IFN-γ via enzyme-linked immunosorbent assay.Results: The production of total IgG and the isotype IgG1, IgG2 a and IgG2 b was significantly higher in rBCG-immunised mice than in the BCG and PBS-T80-immunised mice, and Pam3 CSK4 further enhanced their productions. A similar pattern was also observed in IFN-γ production. Moreover, there was no significant difference in IL-4 production in all groups either in the presence or absence of Pam3 CSK4.Conclusions: We present evidence of the adjuvant effects of TLR-2 agonist in enhancing the production of total IgG, IgG1, IgG2 a, IgG2 b, as well as IFN-γ in response to rBCG. However, the presence or absence of Pam3 CSK4 had no effect on IL-4 production.
基金supported by the Universiti Sains Malaysia(USM) Fundamental Research Grant Scheme(FRGS)(No.203/PPSK/6171158)
文摘Objective: To determine the role of toll-like receptor 4(TLR-4) in eliciting cellular and humoral immune responses against recombinant Mycobacterium bovis bacille Calmette-Guérin(rB CG) expressing the C-terminus of merozoite surface protein-1 of Plasmodium falciparum. Methods: Six groups of mice(n=6 per group) were injected with phosphate buffered saline T80, BCG or r BCG intraperitoneally, in the presence or absence of a TLR-4 inhibitor; TAK-242. Enzyme-linked immunosorbent assay was carried out for serum total IgG, IgG 1, Ig G2 a and Ig G2 b determination. Spleens were also harvested and splenocytes cultured for determination of intracellular cytokines; IL-4 and IFN-毭 via enzyme-linked immunosorbent assay. Results: The production of total Ig G, and the subclasses Ig G1, Ig G2 a and Ig G2 b was significantly higher in rB CG-immunised mice than BCG and phosphate buffered saline immunised mice in the absence of TAK-242. A significant rise in total IgG occurred with more booster immunisations. The level of IgG 2 a was highest, followed by IgG 2 b, then IgG 1. The production of both IL-4 and IFN-were inhibited in t毭 was also highest in the rB CG immunised groups. These significant riseshe presence of TAK-242. Conclusions: We present evidence of the role of TLR-4 in the increased production of total IgG, IgG 1, IgG 2 a and IgG 2 b, as well as IL-4 and IFN-毭 in response to our rB CG construct.
