Modification On Immune Responses And PBMC Transcription Profiles Of Calves Infected With Attenuated And Virulent Mycoplasma Bovis Strains

Mycoplasma bovis(M.bovis)is an important pathogen in beef and dairy industries.While M.bovis is primarily considered a respiratory pathogen,it may become systemic by causing several other diseases such as mastitis,arthritis,otitis,etc.Because of the inefficiency of antibiotics to M.bovis and the increasing of antibiotic resistance,it is difficult to eradicate the pathogen in cattle farms once it was obversed.Commercial inactivated vaccines on the market show poor efficiency in animal experiments.Therefore,it needs an effective attenuated mycoplasma vaccine for the control of M.bovis infection.The mechanism of immune protection and pathogenesis of M.bovis is not clear,which is an important factor hindering the development of vaccine.To reveal the underlying mechanism for pathogenesis of virulent P1 strain and immunity of attenuated P150 strain,transcriptome of peripheral blood mononuclear cells and humoral immunity and cellular immunity in calves induced by both M.bovis strains were comparative analysed.Twenty one-month-old male calves were divided into 4 groups.Twelve calves were infected with P1 strain(P1 group,n=6)and P150(P150-ND group,n=6)with nasal dripping,respectively.Four cavles were infected with P150 by hypodermic injection(P150 HI group,n=4),and negative control group(NC group,n=4)was set by nasal dripping with a corresponding volume of PPLO.The comparative study was carried out on the basis of comparison of the results of nasal inoculation with P1 strain and P150 strain,P150-HI group and NC group.The effects of different virulent mycoplasma strains on humoral and cellular immunity were compared,and then the expression levels of 20 cytokines in the blood were detected by applying the microarray technique.The second part was the research on the transcription spectrum of peripheral blood mononuclear cells(PBMC)from calves infected with the wild strain P1 and attenuated strain P150 of M.bovis.1.Animal infection model of M.bovis infection The results of clinical symptom observation showed that the virulence of P1 strain stimulated the calves to produce severe respiratory symptoms.The rectal temperature(Tm)was maintained for 4 days at a higher level(p < 0.05).There was no clinical signs and normal Tm in P150-ND group.Stress reaction was observed in P150-HI group,including one calf shaking in 8h post-infection,and higher Tm at 2-4 dpi.The histopathological observation and scoring test in the anatomy and the pathological HE and IHC staining and the results of M.bovis isolation from tissues showed that the virulent strain of P1 mainly invaded and colonized on the respiratory tract,and its pathological changes were mainly concentrated in the lung tissue,and the time of excretion of bacteria was longer than 22 days.There were no significant clinical symptoms observed on the calves incubated with P150 strain by nasal mucosal inoculation,no mycoplasma was isolated from the tissues.The calves in P150-HI group were observed to have greater stress after inoculation,including tremor and lying down after 8 h post-inoculation,they recovered from the stess in the second day.2.Analysis the difference on humoral immunity of calves induced by the attenuated and virlent stains of M.bovis The results showed that M.bovis strains could induce the production of serum Ig G,Ig G1,Ig G2,IgA and IgM,in which Ig G and IgA were main.The sera Ig G2 was increased in the P150-ND group calves at 28 dpi than that in the P1 group.The wild and attenuated strains of M.bovis induced the secretion of salivary saliva IgA and IgM In addition,the level of IgA in the nasal lavage fluid was increased significantly(p < 0.05)in P1 group.The antibodies of P150 HI group has similar trends but lower level than those in P150-ND group in serum,saliva and nasal mucosa.3.Analysis the difference and cellular immunity of calves induced by the attenuated and virlent stains of M.bovis The proliferation of PBMC were increased in the three infected groups on the 14 dpi(p < 0.05)compared to NC group.The early PBMC apoptosis in 14 dpi,the early and total PBMC apoptosis in 21 dpi of P1 group calves were higher than that in P150-ND group(p < 0.05),resulting in the lower live PBMC percent of P1 group in 21 dpi.The proportion of CD4+ cells and ratios of CD4+/CD8+ cells in PBMC of P150-ND group was increased significantly(p < 0.05),which indicated that P150 strain induced higher proliferation of CD4 cells.There was no significant change in the proportion of CD8+ cells of PBMC in the three groups of calves.The significantly lower levels of MIG,TNF-?,IFN-? and IL-13 in the blood of P1 group than those of the NC group on 7 dpi and 14 d dpi(p < 0.05)indicated that the virulent strain P1 induced suppression of secretion of some cytokines of calves.IL1F5 in the blood of P150-ND group was significantly higher than the NC group(p < 0.05),and the levels of IFN-?,IL1F5,TNF-?,MIG,IL-1? and IP10 in the blood of P150-ND group were higher than that in the P1 group on 7 dpi and 14 dpi(p < 0.05).The lower levels of IL-1? and GASP1 in the blood of the P150 HI group indicated that the immune response induced by P150 through subcutaneous injection was later than that through mucosal immunization.4.Transcriptome profiles of the different expression genes in PBMC of calves infected with the attenuated and virlent stains of M.bovis To reveal its virulence related factors,a virulent M.bovis HB0801 and its derived vaccine strain P150 were infected calve and the transcriptome profiles of peripheral blood mononuclear cells(PBMC)were compared by using of the microarray at 7 days after infection.Compared with NC group,874 and 519 genes of P1 group were unique up-regulated and down-regulated,and 540 and 274 genes of P150 group were unique up-regulated and down-regulated.The unique up-regulated genes in P1 group were mainly enriched in the GO terms as inflammatory response,regulation of apoptotic process,positive regulation of gene expression,RNA processing,etc.These genes were found to be involved in the bacterial invasion of epithelial cells and FC gamma R mediated phagocytosis pathway;The genes unique down-regulated by virulent strains were mainly enriched in the oxidation-reduction process,viral process,cell-cell adhesion,I-kappa B kinase/NF-kappa B signaling,regulation of m RNA stability,etc.The kegg pathway involved were mainly the pathway of ABC transporters and NFKB signaling pathways.Furtherly,SYK,TP53,IL-6,STAT3,ATP5 B,GSK3B,ACTG1,UBC,INS,TNF,TLR4,HSPA8,NFKBIA,INSR,NOS3 and BIRC3.SYK(Spleen tyrosine kinase)and UBC(Ubiquitin C)were the key genes of the virulent strain of P1 to regulate the suppression of responses on calves.The unique up-regulated genes in P150 group were mainly enriched in the GO terms as GTPase activity,cell proliferation,translational initiation,cell division,protein sumoylation and protein ubiquitination,global genome nucleotide-excision repair,etc.These genes were found to be involved in the pathways of nucleotide excision repair and ubiquitin mediated proteolysis;only genes in the unique down-regulated by attenuated strain were mainly enriched in go terms such as the cell adhesion,mitochondrial ATP synthesis coupled proton transport,ATP biosynthetic process,protein K48-linked deubiquitination oxidation-reduction process,cell-cell adhesion,etc.The kegg pathway involved were mainly the pathway of ABC transporters and NFKB signaling pathways.Furtherly,the most important DEGs were UBE2 I,RAD23B,GTF2H4,SOCS1,MDM2,TCEB1,CDK7,ITCH,UBE2D3,UBE2E3 and RCHY1.Most of these genes are enzymes associated with protein ubiquitination,and the other two are involved in nucleotide cleavage repair.17 genes were expressed as P1>P150>control group,and 171 graduate increased genes were found as the screening criteria were further reduced by change > 1.5 times,and p < 0.05.These genes are mainly involved in the process regulation of uclear-transcribed m RNA catabolic process,nonsense-mediated decay,SRP-dependent cotranslational protein targeting to membrane,platelet activation,RNA transcription GO terms and the pathways of the inflammatory mediator regulation of TRP channels and bacterial invasion of epithelial cells may be involved in the regulation of immune response induced by M.bovis with the gradually up-regulated genes of TRPV4,ITPR1 and CALM.TRP channel involved by TRPV4,CALM and ITPR1 were related with the lung injury induced by P1 strain.5.Verification of the important differential expression genes in hosts induced by the wild and attenuated strains of M.bovis The correlations of the key DEGs with the average daily gain and pathological score of calves were analyzed.It was found that 18 genes and 9 genes were positively and negatively correlated with gross score and lung score respectively(p < 0.05),another one and two genes were positively and negatively correlated with gross score(p < 0.05),and one gene and four genes were positively and negatively with average daily gain.There was a significant negative correlation between IFNG and pathological change score(p < 0.05),a significant negative and positive correlation between IL-17 D and average daily gain and pathological score(p < 0.05),respectively.The expression levels of key molecules involved in Th17 cell differentiation pathway in PBMC and lung tissues post-infection were verified.As a results,q PCR confirmed the up-regulation of IL-23 R and IL-21 R in PBMC,as well as the up-regulation of IL-6,IL-23 R,and IL-21 R in the lung tissues of calves in P1 group relative to the other two groups.Meanwhile,IL-6 and IL-21 R were confirmed to increase in the lung tissues by western blot assay,which is also in agreement with the microarray results.Further,the level of IL-17 A in whole blood of calves after different days post infection was also found to be significantly higher than that in NC and P150 groups on 14 dpi(p < 0.05).With comprehensive analysis of these data,it was speculated that Teff17 over-responsiveness may have occurred in P1 group due to both the up-regulation of Teff17 cells and the down-regulation of Treg17 cells,and it was related to the pathological damage induced by M.bovis.The Th1 response cytokine,IFN-?,was verified at the protein level to be significantly higher in the blood samples from the P150 than P1 group,which was in agreement with the results of microarray analysis.So,it might be a universe rule that IFN-? production representing enhanced Th1 response might play a protective role against Mycoplasma infection.In conclusion,the differences of responses induced by the attenuated and virlent stains of M.bovis were mainly on the cellular immunity.P1 induced early apoptosis of PBMC,inhibited the secretion of inflammatory factors such as IFN-? and TNF-? and the expression of many genes such as TNF and TLR,which made the escaping and clearing of the specific immune response of hosts.The virulent M.bovis enhanced pathogenic Teff17 response and suppressed Treg17 response of the host and increased the secretion of IL-17 A,which related with the lung lesion induced by M.bovis.SYK and UBC were the key genes in the unique regulated genes of P1.Meanwhile,the lung injury induced by P1 was related to the TRP channel in which TRPV4 was the dominant gene.P150 induced the differation of CD4 cells in PBMC,increased Th1 response and the levels of cytokines such as IFN-?,and the regulation of these responses was related to the proteins degradation by ubiquialization and the repair of nucleotides.Additionally,both of the two strains could induce humoral immunity,mainly on the increasing of Ig G and IgA in the serum.P150 induced higher serum Ig G2 in 28 dpi than that of P1 induced,while P1 induced higher IgA in nasal lavage fluid.This research improves the understanding of M.bovis-induced pathogenesis and immunity that could be used in the development of control measures against M.bovis associated diseases.