Changmin Hu

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.

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.

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.

Immunoproteomic identification of MbovP579, a promising diagnostic biomarker for serological detection of Mycoplasma bovis infection

A lack of knowledge regarding the antigenic properties of Mycoplasma bovis proteins prevents the effective control of bovine infections using immunological approaches. In this study, we detected and characterized a specific and sensitive M. bovis diagnostic biomarker. After M. bovis total proteins and membrane fractions were separated with two dimensional gel electrophoresis, proteins reacting with antiserawere detected using MALDI-TOF MS. Thirty-nine proteins were identified, 32 of which were previously unreported. Among them, immunoinformatics predicted eight antigens, encoded by Mbov_0106, 0116, 0126, 0212, 0275, 0579, 0739, and 0789, to have high immunological value. These genes were expressed in E. coli after mutagenesis of UGA to UGG using overlap extension PCR. A lipoprotein, MbovP579, encoded by a functionally unknown gene, was a sensitive and specific antigen for detection of antibodies in sera from both M. bovis-infected and vaccinated cattle. The specificity of MbovP579 was confirmed by its lack of cross-reactivity with other mycoplasmas, including Mycoplasma agalactiae. An iELISA based on rMbovP579 detected seroconversion 7 days post-infection (dpi). The ELISA had sensitivity of 90.2% (95% CI: 83.7%, 94.3%) and a specificity of 97.8% (95% CI: 88.7%, 99.6%) with clinical samples. Additional comparative studies showed that both diagnostic and analytic sensitivities of the ELISA were higher than those of a commercially available kit (p<0.01). We have thus detected and characterized the novel antigen, MbovP579, and established an rMbovP579-based ELISA as a highly sensitive and specific method for the early diagnosis of M. bovis infection.

Mycoplasma bovis NADH oxidase functions as both a NADH oxidizing and O2 reducing enzyme and an adhesin

Mycoplasma bovis causes considerable economic losses in the cattle industry worldwide. In mycoplasmal infections, adhesion to the host cell is of the utmost importance. In this study, the amino acid sequence of NOX was predicted to have enzymatic domains. The nox gene was then cloned and expressed in Escherichia coli. The enzymatic activity of recombinant NOX (rNOX) was confirmed based on its capacity to oxidize NADH to NAD+ and reduce O2 to H2O2. The adherence of rNOX to embryonic bovine lung (EBL) cells was confirmed with confocal laser scanning microscopy, enzyme-linked immunosorbent assay, and flow cytometry. Both preblocking EBL cells with purified rNOX and preneutralizing M. bovis with polyclonal antiserum to rNOX significantly reduced the adherence of M. bovis to EBL cells. Mycoplasma bovisNOX–expressed a truncated NOX protein at a level 10-fold less than that of the wild type. The capacities of M. bovisNOX– for cell adhesion and H2O2 production were also significantly reduced. The rNOX was further used to pan phage displaying lung cDNA library and fibronectin was determined to be potential ligand. In conclusion, M. bovis NOX functions as both an active NADH oxidase and adhesin, and is therefore a potential virulence factor.

1H-NMR Spectroscopy Revealed Mycobacterium tuberculosis Caused Abnormal Serum Metabolic Profile of Cattle

To re-evaluate virulence of Mycobacterium tuberculosis (M. tb) in cattle, we experimentally infected calves with M. tb andMycobacterium bovisvia intratracheal injection at a dose of 2.0×107 CFU and observed the animals for 33 weeks. The intradermal tuberculin test and IFN-γin vitro release assay showed that both M. tb and M. bovis induced similar responses. Immunohistochemical staining of pulmonary lymph nodes indicated that the antigen MPB83 of both M. tb and M. bovis were similarly distributed in the tissue samples. Histological examinations showed all of the infected groups exhibited neutrophil infiltration to similar extents. Although the infected cattle did not develop granulomatous inflammation, the metabolic profiles changed significantly, which were characterized by a change in energy production pathways and increased concentrations of N-acetyl glycoproteins. Glycolysis was induced in the infected cattle by decreased glucose and increased lactate content, and enhanced fatty acid β-oxidation was induced by decreased TG content, and decreased gluconeogenesis indicated by the decreased concentration of glucogenic and ketogenic amino acids promoted utilization of substances other than glucose as energy sources. In addition, an increase in acute phase reactive serum glycoproteins, together with neutrophil infiltration and increased of IL-1β production indicated an early inflammatory response before granuloma formation. In conclusion, this study indicated that both M. tb and M.bovis were virulent to cattle. Therefore, it is likely that cattle with M. tb infections would be critical to tuberculosis transmission from cattle to humans. Nuclear magnetic resonance was demonstrated to be an efficient method to systematically evaluate M. tb and M. bovi sinfection in cattle.

Attenuation of bovine herpesvirus type 1 by deletion of its glycoprotein G and tk genes and protection against virulent viral challenge

To develop a novel vaccine against infectious bovine rhinotracheitis (IBR), a bovine herpesvirus-1 (BoHV-1) mutant was constructed by deleting the genes for glycoprotein G (gG) and thymidine kinase (tk) through homologous recombination. The resulting sequences for both genes were shown to be correct and a gG expression defect was also confirmed. A parallel study of the BoHV-1 gG(-)/tk(-), gE(-)/tk(-) mutants and wild type (wt) in 31 calves was performed at three different doses, 4×10(5)PFU, 4×10(6)PFU and 4×10(7)PFU. Compared to wt BoHV-1, inoculation of BoHV-1 gG(-)/tk(-) and gE(-)/tk(-) produced no clinical signs and the virus was not reactivated by dexamethasone (dex). Inoculation of BoHV-1 gG(-)/tk(-) at the doses of 4×10(6) and 4×10(7)PFU provided full clinical protection for the cattle against wt BoHV-1 challenge at 4×10(7)PFU/calf. Although the mutants were associated with significantly lower levels of serum neutralizing antibody, interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) than wt BoHV-1 on days 3, 5 and 7 after immunization, stimulation of IFN-β by BoHV-1 gG(-)/tk(-) was significantly higher than that of wt BoHV-1 and gE(-)/tk(-) on days 3 and 5. We conclude that BoHV-1 gG(-)/tk(-) was attenuated adequately and that it maintains the ability to stimulate immune protection. Therefore, it may be a promising candidate for a marker vaccine against IBR.