Comparative proteomic analysis of Leptospira interrogans serogroup Icterohaemorrhagiae human vaccine strain and epidemic isolate from China.

Leptospira interrogans serogroup Icterohaemorrhagiae is the predominant pathogen causing leptospirosis in China and is still used as the vaccine strain for the current human inactivated vaccine. Unlike the clade ST17, which is distributed worldwide, ST1 is the most prevalent in serogroup Icterohaemorrhagiae in China. To further characterize leptospiral pathogens, isobaric tags for relative and absolute quantitation and parallel reaction monitoring were used to analyze differences at the proteomic level between serogroup Icterohaemorrhagiae vaccine strain 56001 (ST1) and circulating isolate 200502 (ST17) from different periods. Two hundred and eighty-one proteins were differentially expressed between the circulating isolate and vaccine strain, of which 166 were upregulated (> 1.2-fold change, P < 0.05) and 115 (< 0.8-fold change, P < 0.05) were downregulated. Function prediction revealed that nine upregulated proteins were outer membrane proteins, including several known immunogenic and/or virulence-related proteins, such as ompL1, LipL71, and LipL41. Furthermore, important expression differences in carbohydrate, amino acid, and energy metabolism and transport proteins were identified between both strains from different clusters, suggesting that these differences may reflect metabolic diversity and the potential of the pathogens to adapt to different environments. In summary, our findings provide insights into a better understanding of the component strains of the Chinese human leptospirosis vaccine at the proteomic level. Additionally, these data facilitate evaluating the mechanisms by which pathogenic Leptospira species adapt to the host environment.

Disruption of Plasmodium falciparum histidine-rich protein 2 may affect haem metabolism in the blood stage.

BACKGROUND: Haem is a key metabolic factor in the life cycle of the malaria parasite. In the blood stage, the parasite acquires host haemoglobin to generate amino acids for protein synthesis and the by-product haem for metabolic use. The malaria parasite can also synthesize haem de novo on its own. Plasmodium falciparum-specific histidine-rich protein 2 (PfHRP2) has a haem-binding site to mediate the formation of haemozoin, a biocrystallized form of haem aggregates. Notably, the gene regulates the mechanism of haemoglobin-derived haem metabolism and the de novo haem biosynthetic pathway in the Pfhrp2-disrupted parasite line during the intraerythrocytic stages. METHODS: The CRISPR/Cas9 system was used to disrupt the gene locus of Pfhrp2. DNA was extracted from the transgenic parasite, and PCR, Southern blotting and Western blotting were used to confirm the establishment of transgenic parasites. RNA-sequencing and comparative transcriptome analysis were performed to identify differences in gene expression between 3D7 and Pfhrp2--3D7 parasites. RESULTS: Pfhrp2- transgenic parasites were successfully established by the CRISPR/Cas9 system. A total of 964, 1261, 3138, 1064, 2512 and 1778 differentially expressed genes (DEGs) were identified in the six comparison groups, respectively, with 373, 520, 1499, 353, 1253 and 742 of these DEGs upregulated and 591, 741, 1639, 711, 1259 and 1036 of them downregulated, respectively. Five DEGs related to haem metabolism and synthesis were identified in the comparison groups at six time points (0, 8, 16, 24, 32, and 40 h after merozoite invasion). The genes encoding delta-aminolevulinic acid synthetase and ferrochelatase, both related to haem biosynthesis, were found to be significantly upregulated in the comparison groups, and those encoding haem oxygenase, stromal-processing peptidase and porphobilinogen deaminase were found to be significantly downregulated. No GO terms were significantly enriched in haem-related processes (Q value = 1). CONCLUSION: Our data revealed changes in the transcriptome expression profile of the Pfhrp2--3D7 parasite during the intraerythrocytic stages. The findings provide insight at the gene transcript level that will facilitate further research on and development of anti-malaria drugs.

Genetic diversity of Leptospira interrogans circulating isolates and vaccine strains in China from 1954-2014.

Leptospirosis is one of the most important but neglected, infectious tropical diseases worldwide. Leptospira interrogans is now recognized as a leading cause of the disease. Little is known of the genetic diversity and phylogenetic characteristics of L. interrogans within China. To better understand the transmission and genetic diversity of L. interrogans populations, we characterized 271 isolates and seven vaccine strains from China during 1954-2014 using multilocus variable-number tandem repeat analysis (MLVA). 110 different L. interrogans MLVA profiles (MTs) were identified, of which five were predominant, reflecting a high level of genetic diversity in L. interrogans population in China. Different from that of circulating isolates, seven vaccine strains have different MT, of which some are phylogenetically away from the circulating isolates. The results showed that Icterohaemorrhagiae, Hebdomadis, and Canicola ranked as the top three serogroups among L. interrogans strains tested. The cluster analysis demonstrate the clonal links between rodent and human isolates, suggesting the rodent species played a key role in the transmission of leptospirosis to humans, and contributed to the circulation of the pathogen in humans. Taken together, these findings should provide insight into a better knowledge of the epidemiology and molecular evolution of L. interrogans in China. Furthermore, the results should facilitate the selection of candidate vaccine strains in the future.

Genomic Analysis of a New Serovar of Leptospira weilii Serogroup Manhao.

Leptospirosis, caused by pathogenic Leptospira spp., is recognized as an important emerging zoonotic disease throughout the world. In this study, multiple approaches were used to characterize the recently discovered serovar Heyan strain L231. This strain can infect guinea pigs and belonged to the pathogenic species L. weilii. Genome sequencing analysis revealed the draft genome of 4.2 M bp with a G+C content of 40.67% for strain L231, and a total of 4,794 ORFs were identified. The strain L231 genome was found to have a larger LPS biosynthesis locus than that of strains L. interrogans serovar Lai and L. borgpetersenii serovar Hardjobovis. Phylogenomic reconstructions showed that the evolutionary position of L. weilii serovar Heyan was different from that of other serovars from serogroup Manhao. These findings may lead us to a better understanding of Leptospira pathogenesis and evolution.

Whole genome sequencing revealed host adaptation-focused genomic plasticity of pathogenic Leptospira.

Leptospirosis, caused by pathogenic Leptospira spp., has recently been recognized as an emerging infectious disease worldwide. Despite its severity and global importance, knowledge about the molecular pathogenesis and virulence evolution of Leptospira spp. remains limited. Here we sequenced and analyzed 102 isolates representing global sources. A high genomic variability were observed among different Leptospira species, which was attributed to massive gene gain and loss events allowing for adaptation to specific niche conditions and changing host environments. Horizontal gene transfer and gene duplication allowed the stepwise acquisition of virulence factors in pathogenic Leptospira evolved from a recent common ancestor. More importantly, the abundant expansion of specific virulence-related protein families, such as metalloproteases-associated paralogs, were exclusively identified in pathogenic species, reflecting the importance of these protein families in the pathogenesis of leptospirosis. Our observations also indicated that positive selection played a crucial role on this bacteria adaptation to hosts. These novel findings may lead to greater understanding of the global diversity and virulence evolution of Leptospira spp.

Genetic stability of vaccine strains by multilocus sequence typing and pulsed-field gel electrophoresis analysis: Implications for quality control of the leptospiral vaccine.

Quality control of vaccine strains is directly associated with the safety and efficacy of inactivated whole bacterial vaccines. The assessment of genetic stability is one of the essential elements to guarantee the quality of vaccine strains. The multiple-valence inactivated leptospiral vaccine, comprising the main circulating serogroups, has played an important role in the control of Leptospira infection in China. In the present study, to assess the genetic stability of vaccine strains and develop novel quality control tests that enhance and extend the existing procedures, 7 Chinese leptospiral vaccine strains were characterized during in vivo and in vitro passages by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) analysis. The seven vaccine strains were found to have distinct sequence types (STs) and PFGE profiles. Further analysis showed that the ST and PFGE pattern of each vaccine strain, after in vivo or serial in vitro passages (up to 20 passages), were identical to those of the initial strain, demonstrating that these strains were genetically stable and homogeneous. Taken together, PFGE and MLST provide a reproducible and reliable means for confirming the identity and genetic stability of vaccine seeds, suggesting that these approaches can be used to evaluate the quality of leptospiral vaccine strains.

[Changes of IFN-gamma, IL-4 and T cells in Schistosoma japonicum-infected mice after praziquantel treatment].

OBJECTIVE: To investigate the serum levels of IFN-gamma and IL-4, and the dynamic changes of IFN-gamma-specific and IL-4-specific lymphocytes in mice with Schistosoma japonicum infection after treatment by praziquantel. METHODS: Ninety BALB/c mice were randomly divided into three groups (n = 30) named as infection group, treatment group and control group. The mice in treatment group and infection group were infected with (25 +/- 2) S. japonicum cercariae through the abdominal skin. At 6 weeks post-infection, the mice in treatment group were administered orally with praziquantel [300 mg/(kg x d)] for 3 d. At 4, 6, 8 and 12 weeks post-treatment, the mice were weighed, and serum samples were collected. Serum levels of IFN-gamma and IL-4 were measured by ELISA. At the same time, the spleens were aseptically removed to prepare cell suspension, and the counts of IFN-gamma and IL-4 specific lymphocytes were examined by ELISPOT after stimulation of Schistosoma japonicum soluble egg antigen (SEA). RESULTS: From 4 to 12 weeks after praziquantel treatment, the body weight of mice in treatment group were significantly heavier than that of infection group (P < 0.05), but no significant difference was found between treatment group and control group (P < 0.05). At 4 weeks posttreatment, there was no significant difference in serum levels of IFN-gamma and IL-4 between treatment group and infection group (P > 0.05). At 6, 8, and 12 weeks after treatment, the serum levels of IFN-gamma (0.038 +/- 0.013, 0.028 +/- 0.001, and 0.027 +/- 0.007) and IL-4(0.051 +/- 0.020, 0.045 +/- 0.019, and 0.043 +/- 0.016) in treatment group were significantly lower than that of infection group (IFN-gamma: 0.057 +/- 0.004, 0.060 +/- 0.023, and 0.052 +/- 0.017; IL-4: 0.150 +/- 0.014, 0.148 +/- 0.014, and 0.123 +/- 0.017) (P < 0.05). Serum IFN-gamma and IL-4 levels in treatment group and infection group were significantly higher than that of control group (P < 0.05). ELISPOT results showed that at 4, 6 weeks post-treatment, there was no significant difference in the number of IFN-gamma-specific lymphocytes between treatment group and infection group (P > 0.05). While at 8 and 12 weeks after treatment, the IFN-gamma-specific lymphocytes in treatment group (39.9 +/- 22.8 and 38.5 +/- 6.2) were significantly less than that of infection group (141.9 +/- 39.3 and 106.8 +/- 28.6) (P < 0.05). At 4-week post-treatment, the IL-4-specific lymphocytes in treatment group were much more than that of infection group (175.6 +/- 62.3) (P < 0.05), and then began to decline. At 8 and 12 weeks after treatment, the IL-4-specific lymphocytes (111.3 +/- 14.3 and 113.0 +/- 44.2) in treatment group were significantly less than that of infection group (220.3 +/- 107.1 and 208.1 +/- 17.2) (P < 0.05). The IFN-gamma-specific and IL-4-specific lymphocytes in treatment group and infection group were significantly more than that of control group (P < 0.05). CONCLUSION: After praziquantel treatment, the serum levels of IFN-gamma and IL-4 in mice with S. japonicum infection decrease, and the number of IFN-gamma and IL-4 specific lymphocytes reduces.

[Polymorphism analysis of Plasmodium falciparum histidine-rich protein II and III].

OBJECTIVE: To analyze the polymorphism of Plasmodium falciparum histidine-rich protein (PfHRP) II and III. METHODS: Genomic DNA was isolated from blood samples of 20 patients infected with Plasmodium falciparum in Yunnan Province. Blood samples were tested by microscopy and RDTs. The Pfhrp2 and Pfhrp3 gene fragments were amplified by PCR and sequenced. The sequencing results were analyzed and compared using the bioinformatics software. RESULTS: 20 patients infected with Plasmodium falciparum tested by microscopy and RDTs. PCR showed that the Pfhrp2 gene was with 389~986 bp, and Pfhrp3 gene with 329-640 bp. All PfH-IRP II sequences started with type 1 repeat (AHHAHHVAD) and ended with the type 12 repeat (AHHAAAHHEAATH). The number of type 7 (AHHAAD), type 2 (AHHAHHAAD) and type 6 (AHHATD) within PfHRP II was more than the other types of repeats, as well as type 16 (AHHAAN) and type 17 (AHHDG) for PfHRP III. Type 11 repeat (AHN) was not found from the PfHRP II and PfHRP III sequences. CONCLUSION: There is an extensive diversity in Pfhrp2 and Pfhrp3 fragments in the individuals infected with P. falciparum in Yunnan. Some types of repeats are shared by PfHRP II and PfHRP III.

[Observation on dynamic changes of SEA specific antibody in sera of BALB/c mice infected with Schistosoma japonicum].

OBJECTIVE: To investigate the dynamic changes of SEA-induced specific IgG, IgM in sera of BALB/c mice infected with Schistosoma japonicum in 18 weeks. METHODS: After mice were infected with S. japonicum cercarial for 2 weeks, the sera were collected from 2 to 18 weeks post-infection. The serum levels of SEA-specific IgG and IgM antibodies were measured respectively by ELISA, and the different fractions of IgG and IgM antibodies were identified by the Western blotting method. RESULTS: The ELISA results showed that the serum levels of SEA-specific IgG increased 5, 6, 9, 11 week, after the infection, and SEA-specific IgM increased obviously 5, 9 weeks after the infection. The Western blotting results showed that 140, 180 kDa molecules were recognized by IgG antibodies in the mouse sera 4 weeks after the infection. The specific IgG antibodies of 43, 50 kDa antigens appeared 5 weeks after the infection. 60-130 kDa fractions were recognized by IgG in the sera 6 weeks post-infection, and 38, 73 kDa proteins were recognized by IgG in the sera 9 weeks post-infection. The IgG antibodies of 26, 32, 35, 80 kDa molecules appeared 11 weeks post-infection and reacted strongly 12 weeks post-infection. The IgM antibodies of 100, 140, 180 kDa molecules appeared 3 weeks after the infection, and 73 kDa protein was recognized by sera 6 weeks after the infection, but the reaction became strong 9 weeks after the infection. The 38, 43, 50 kDa proteins induced IgM antibodies in 9-week-infection sera and the reaction became stronger 9 weeks after the infection. CONCLUSIONS: There is a dynamic change in the levels of specific IgG and IgM antibodies induced by S. japonica SEA and the appearance of the antibodies is related to different infection stages. The 43, 50, 10, 140, 180 kDa antigens might have the potential value of early immunodiagnosis. The 73 kDa antigen shows high diagnostic value in both acute and chronic schistosomiasis. The 28, 32, 35, 38, 80 kDa antigens are not only the diagnostic molecules for chronic schistosomiasis, but they may also have therapeutic effects, and in addition, they may be the candidate vaccines of the disease.

Development of O-antigen gene cluster-specific PCRs for rapid typing six epidemic serogroups of Leptospira in China.

BACKGROUND: Leptospira is the causative agent of leptospirosis. The O-antigen is the distal part of the lipopolysaccharide, which is a key component of outer membrane of Gram-negative bacteria and confers serological specificity. The epidemiology and clinical characteristics of leptospirosis are relative to the serology based taxonomic unit. Identification of Leptospira strains by serotyping is laborious and has several drawbacks. RESULTS: In this study, the O-antigen gene clusters of four epidemic Leptospira serogroups (serogroup Canicola, Autumnalis, Grippotyphosa and Hebdomadis) in China were sequenced and all genes were predicted in silico. Adding published sequences of two serogroups, Icterohaemorrhagiae (strain Lai and Fiocruz L1-130) and Sejroe (strain JB197 and L550), we identified six O-antigen-specific genes for six epidemic serogroups in China. PCR assays using these genes were developed and tested on 75 reference strains and 40 clinical isolates. CONCLUSION: The results show that the PCR-based assays can be reliable and alternative means for rapid typing of these six serogroups of Leptospira.

The synthesis of GoldMag nano-particles and their application for antibody immobilization.

Fe3O4/Au (GoldMag) particles with core/shell structure were synthesized by reduction of Au3+ with hydroxylamine in the presence of Fe3O4. The synthesized particles have an average size smaller than 100 nm in diameter with of superparemagnetic properties due to their Fe oxide cores. The particles show optical features with a plasmon resonance peak from 550, 570 to 590 nm correlating with increasing diameters from 50 nm, 70 nm to 100 nm. The GoldMag particles need only a single step for antibody immobilization and have high binding capacity for antibodies. These advantages permit improved methods of isolating and detecting biomolecules.