Aizhen Guo

Comparative Geno-Plasticity Analysis of Mycoplasma bovis HB0801 (Chinese Isolate)

Mycoplasma bovis pneumonia in cattle has been epidemic in China since 2008. To investigate M. bovis pathogenesis, we completed genome sequencing of strain HB0801 isolated from a lesioned bovine lung from Hubei, China. The genomic plasticity was determined by comparing HB0801 with M. bovis strain ATCC® 25523™/PG45 from cow mastitis milk, Chinese strain Hubei-1 from lesioned lung tissue, and 16 other Mycoplasmas species. Compared to PG45, the genome size of HB0801 was reduced by 11.7 kb. Furthermore, a large chromosome inversion (580 kb) was confirmed in all Chinese isolates including HB0801, HB1007, a strain from cow mastitis milk, and Hubei-1. In addition, the variable surface lipoproteins (vsp) gene cluster existed in HB0801, but contained less than half of the genes, and had poor identity to that in PG45, but they had conserved structures. Further inter-strain comparisons revealed other mechanisms of gene acquisition and loss in HB0801 that primarily involved insertion sequence (IS) elements, integrative conjugative element, restriction and modification systems, and some lipoproteins and transmembrane proteins. Subsequently, PG45 and HB0801 virulence in cattle was compared. Results indicated that both strains were pathogenic to cattle. The scores of gross pathological assessment for the control group, and the PG45- and HB0801-infected groups were 3, 13 and 9, respectively. Meanwhile the scores of lung lesion for these three groups were 36, 70, and 69, respectively. In addition, immunohistochemistry detection demonstrated that both strains were similarly distributed in lungs and lymph nodes. Although PG45 showed slightly higher virulence in calves than HB0801, there was no statistical difference between the strains (P>0.05). Compared to Hubei-1, a total of 122 SNP loci were disclosed in HB0801. In conclusion, although genomic plasticity was thought to be an evolutionary advantage, it did not apparently affect virulence of M. bovis strains in cattle.

Characterization of Extensively Drug-Resistant Mycobacterium tuberculosis Clinical Isolates in China

ABSTRACT Thirteen extensively drug-resistant tuberculosis isolates which were highly resistant to a broad spectrum of antituberculosis drugs were identified from 1,926 clinical isolates in China. They had highly diverse mycobacterial interspersed repetitive-unit-variable-number tandem-repeat patterns. Most, but not all, of the drug target genes had mutations contributing to resistance to the corresponding drug.

Evaluation of a Novel Chimeric B Cell Epitope-Based Vaccine against Mastitis Induced by Either Streptococcus agalactiae or Staphylococcus aureus in Mice

ABSTRACT To construct a universal vaccine against mastitis induced by either Streptococcus agalactiae or Staphylococcus aureus, the B cell epitopes of the surface immunogenic protein (Sip) from S. agalactiae and clumping factor A (ClfA) from S. aureus were analyzed and predicted. sip-clfA, a novel chimeric B cell epitope-based gene, was obtained by overlap PCR, and then the recombinant Sip-ClfA (rSip-ClfA) was expressed and purified. rSip-ClfA and inactivated S. agalactiae and S. aureus were formulated into different vaccines with mineral oil as the adjuvant and evaluated in mouse models. The rSip-ClfA vaccination induced immunoglobulin G (IgG) titers higher than those seen in groups immunized with inactivated bacteria. Furthermore, the response to rSip-ClfA immunization was characterized as having a dominant IgG1 subtype, whereas both bacterial immunizations produced similar levels of IgG1 and IgG2a. The antiserum capacities for opsonizing adhesion and phagocytosis were significantly greater in the rSip-ClfA immunization group than in the killed-bacterium immunization groups (P < 0.05). The immunized lactating mice were challenged with either S. agalactiae or S. aureus via the intramammary route. At 24 h postinfection, the numbers of bacteria recovered from the mammary glands in the rSip-ClfA group were >5-fold lower than those in both inactivated-bacterium groups (P < 0.01). Histopathological examination of the mammary glands showed that rSip-ClfA immunization provided better protection of mammary gland tissue integrity against both S. agalactiae and S. aureus challenges. Thus, the recombinant protein rSip-ClfA would be a promising vaccine candidate against mastitis induced by either S. agalactiae or S. aureus.

Evaluation of Clumping Factor A Binding Region A in a Subunit Vaccine against Staphylococcus aureus-Induced Mastitis in Mice

ABSTRACT The present study evaluated the potential of recombinant binding region A of clumping factor A (rClfA-A) to be an effective component of a vaccine against mastitis induced by Staphylococcus aureus in the mouse. rClfA-A and inactivated S. aureus were each emulsified in Freunds adjuvant, mineral oil adjuvant, and Seppic adjuvant; phosphate-buffered saline was used as a control. Seven groups of 12 mice each were immunized intraperitoneally three times at 2-week intervals. The titers of IgG and subtypes thereof (IgG1 and IgG2a) in the rClfA-A-immunized group were more than 1,000-fold higher than those in the killed-bacteria-immunized group (P < 0.01). Of the three adjuvants used, mineral oil adjuvant induced the highest antibody levels for both antigens (P < 0.001). Furthermore, the anti-rClfA-A antibody capacities for bacterial adhesion and opsonizing phagocytosis were significantly greater in the rClfA-A-immunized group than in the killed-bacteria-immunized group (P < 0.05). Lactating mice immunized with either rClfA-A or inactivated vaccine were challenged with S. aureus via the intramammary route. The numbers of bacteria recovered from the murine mammary glands 24 h after inoculation were significantly lower in the rClfA-A group than in the killed-bacteria-immunized group (P < 0.001). Histologic examination of the mammary glands showed that rClfA-A immunization effectively preserved tissue integrity. Thus, rClfA-A emulsified in an oil adjuvant provides strong immune protection against S. aureus-induced mastitis in the mouse.

Protective effect of ligand-binding domain of fibronectin-binding protein on mastitis induced by Staphylococcus aureus in mice.

The immunoprotective effect of the ligand-binding domain of fibronectin-binding protein (lFnBP) from Staphylococcus aureus (S. aureus) was investigated in a mouse mastitis model. The recombinant lFnBP (rlFnBP) and inactivated S. aureus were emulsified in Freunds adjuvant, mineral oil adjuvant or Seppic adjuvant, respectively. Seven groups of mice (n=12 each) were immunized with these six vaccines or phosphate-buffered saline. The immunoglobulin G (IgG) titers of mice immunized with rlFnBP vaccine were higher than those in the inactivated vaccine group (P<0.01). Antiserum capacities to opsonize adhesion and phagocytosis were significantly greater in the rlFnBP immunization group than in the killed bacteria group (P<0.05). The immunized lactating mice were challenged with S. aureus. At 24h postinfection, the numbers of bacteria recovered in the rlFnBP group were significantly lower than those in the killed bacteria group (P<0.001). Levels of both interleukin-6 (IL-6) and interferon-gamma (IFN-gamma from the mammary glands in the rlFnBP group were higher than those in the inactivated group (P<0.05). Better protection of mammary gland tissue was shown in the rlFnBP group. Thus, the rlFnBP, emulsified in an oil adjuvant, provided strong immune protection against S. aureus mastitis in mice, and could therefore be a promising vaccine candidate against bovine mastitis induced by S. aureus.

Attenuated Mycoplasma bovis strains provide protection against virulent infection in calves

Mycoplasma bovis is a major etiological agent of pneumonia and arthritis in feedlot beef cattle. To develop a novel live vaccine against M. bovis, two attenuated M. bovis strains, P150 and P180, were tested in calves for protection against challenge with the virulent M. bovis parental strain. Twenty calves were divided into four groups of five calves each that were designated as the P150, P180, positive control (PC), and negative control (NC) groups. Each calf in the P150 and P180 groups was immunized with 10(9)CFU of P150 or P180, respectively, via the nasal cavity, and the PC and NC groups received the mock inoculation. Baseline data were collected for 46 days post-immunization. The clinical signs were scored, and rectal temperatures and daily weight gain were recorded. The blood leukocyte count, the neutrophil ratio, and the serum levels of IgG, IgA, IFN-β, and TNF-α were quantified using laboratory tests, and the nasal shedding was evaluated using microbiological methods. The P150, P180, and PC calves were challenged with a dose of 10(10)CFU of virulent M. bovis by intratracheal injection on 3 consecutive days. The calves were monitored for 25 days post-challenge to observe changes in the baseline parameters. On day 25 post-challenge the calves were euthanized for necropsy and analysis of tissue samples. The P150 and P180 immunizations caused no clinical abnormality, and did not affect daily weight gain. The post-inoculation neutrophil ratio and serum levels of IgG and IFN-β significantly increased in the P150, P180, and PC calves, whereas the serum levels of IgA and TNF-α did not. After challenge, the PC group developed the typical clinical signs and pathology associated with M. Bovis infection, whereas immunization with P150 or P180 provided efficacious protection. Based on the scores for gross pathology and lung pathology, the protection rates of the P150 and P180 immunizations were 87.7% and 70.8%, respectively. The P150 attenuated strain is a promising candidate for a live vaccine against M. bovis infection in cattle.

Characterization of the Mechanism of Inhibin α-Subunit Gene in Mouse Anterior Pituitary Cells by RNA Interference

Inhibin, a member of the transforming growth factor-β [TGF-β] superfamily, is a suppressor of follicle-stimulating hormone [FSH] release through pituitary–gonadal negative feedback loop to regulate follicular development. In this study, Inhibin α-subunit [Inha] gene was knocked down successfully in mice primary anterior pituitary cells at both transcriptional and translational levels by RNAi-Ready pSIREN-RetroQ-ZsGreen Vector mediated recombinant pshRNA vectors. The results indicated that inhibin silencing significantly promoted apoptosis by up-regulating Caspase-3, Bax and Bcl-2 genes without affecting p53 both at transcriptional and translational levels. Furthermore, it markedly impaired the progression of G1 phase of cell cycle and decreased the amount of cells in S phase [as detected by flow cytometry]. Inhibin silencing resulted in significant up-regulation of mRNA and protein expressions of Gondotropin releasing hormone receptors [GnRHR] and down-regulated mRNA levels of β-glycans with parellel change in the amount of its protein expression. Silencing of inhibin-a significantly increased [P<0.05] activin-β concentration without affecting FSH and LH levels in anterior pituitary cells. These findings revealed that up regulation of GnRH receptors by silencing inhibin a-subunit gene might increase the concentration of activin-β in the culture medium. Inhibin a silencing resulted in increased mRNA and protein expressions of inhibinβ which may demonstrate that both inhibin subunits co-participate in the regulation of reproductive events in anterior pituitary cells. This study concludes that inhibin is a broad regulatory marker in anterior pituitary cells by regulating apoptosis, cellular progression and simultaneously by vital fluctuations in the hormonal signaling.

Mycobacterium bovis and BCG induce different patterns of cytokine and chemokine production in dendritic cells and differentiation patterns in CD4+ T cells.

Mycobacterium tuberculosis can infect dendritic cells (DCs), but the molecular mechanism by which these cells contribute to tuberculosis pathogenesis is largely unclear. Using Mycobacterium bovis and the attenuated strain M. bovis BCG as model strains, we analysed cytokine and chemokine secretion in murine DCs infected with M. bovis and BCG at 6, 12 and 24 h post-infection. BCG enhanced production of MCP-1, RANTES, IL-12, TNF-α and IL-6 in DCs, while M. bovis promoted secretion of IL-1β, IL-10 and IL-23. Heat-killed BCG and M. bovis both stimulated cytokine production, but at significantly lower concentrations than corresponding live bacteria. Quantitative RT-PCR and Western blotting indicated that NF-κB regulates production of most cytokines and chemokines. After DCs were infected for 24 h, the culture was used to activate naïve CD4(+) T cells. A combination of the supernatant and activated DCs infected with M. bovis gave high expression of foxp3 and IL-10, directing differentiation of naïve CD4(+) T cells into regulatory T cells (CD4(+)CD25(+)Foxp3(+)) more effectively than BCG. Furthermore, M. bovis-infected DC cultures induced CD4(+) T cells to express significantly higher levels of IL-17, a Th17-type cytokine, while BCG-infected DC cultures stimulated an apparently higher production of IFN-γ, a Th1-type cytokine. In addition, the mycobacteria did not exert a direct effect on the differentiation of CD4(+) T cells. These differential cytokine profiles in DCs and CD4(+) T cells, and the resultant development of CD4(+) T subsets, may be related to the pathogenesis of tuberculosis.

Efficient production and characterization of Bacillus anthracis lethal factor and a novel inactive mutant rLFm-Y236F

Lethal factor (LF) is a 90kDa zinc metalloprotease that plays an important role in the virulence of anthrax. Recombinant LF (rLF) is an effective tool to study anthrax pathogenesis and treatment. In this study, the LF gene was cloned into the Escherichia coli expression vector pGEX-6P-1 and expressed as a GST fusion protein (GST-rLF) in E. coli BL21-codonPlus (DE3)-RIL cells with 0.2mM IPTG induction at 28 degrees C. The GST-rLF protein was purified and the GST-tag was then cleaved in a single step by combining both GST-affinity column and treatment with 3C protease. This procedure yielded 5mg of rLF protein per liter of culture. The purified rLF was functional as confirmed by cytotoxicity assay in RAW264.7 cells and Western blot assay. Furthermore, the rLF could induce strong immune response in BALB/c mice and the presence of a specific antiserum could neutralize the cytotoxicity of rLF in vitro. In addition, a novel inactive mutant (rLFm-Y236F) was obtained. Compared to the wild-type rLF, an increase by 3700 folds of the purified rLFm-Y236F was needed to achieve a similar level of cytotoxicity of the wild-type rLF. This mutant might be of significance in the study of anthrax pathogenesis and treatment.

Mycoplasma bovis MBOV_RS02825 Encodes a Secretory Nuclease Associated with Cytotoxicity

This study aimed to determine the activity of one Mycoplasma bovis nuclease encoded by MBOV_RS02825 and its association with cytotoxicity. The bioinformatics analysis predicted that it encodes a Ca2+-dependent nuclease based on existence of enzymatic sites in a TNASE_3 domain derived from a Staphylococcus aureus thermonuclease (SNc). We cloned and purified the recombinant MbovNase (rMbovNase), and demonstrated its nuclease activity by digesting bovine macrophage linear DNA and RNA, and closed circular plasmid DNA in the presence of 10 mM Ca2+ at 22–65 °C. In addition, this MbovNase was localized in membrane and rMbovNase able to degrade DNA matrix of neutrophil extracellular traps (NETs). When incubated with macrophages, rMbovNase bound to and invaded the cells localizing to both the cytoplasm and nuclei. These cells experienced apoptosis and the viability was significantly reduced. The apoptosis was confirmed by activated expression of phosphorylated NF-κB p65 and Bax, and inhibition of Iκβα and Bcl-2. In contrast, rMbovNaseΔ181–342 without TNASE_3 domain exhibited deficiency in all the biological functions. Furthermore, rMbovNase was also demonstrated to be secreted. In conclusion, it is a first report that MbovNase is an active nuclease, both secretory and membrane protein with ability to degrade NETs and induce apoptosis.