Isolation And Characterization Of Nocardia Cyriacigeorgica From Bovine Mastitis And Its Related Pathogenic Mechanism

Nocardia cyriacigeorgica,a gram-positive environmental pathogen,is reported to cause multidrug resistant infections in humans and dairy cattle.Nocardial mastitis is the mostly important nocardiosis in dairy cattle,which can result in a serious financial loss to dairy farming industry.Mastitis caused by various species of Nocardia has been reported in several countries.However,there is paucity of reports which characterized N.cyriacigeorgica from bovine mastitis.Moreover,the mechanisms by which this pathogen causes the bovine mastitis is not yet studied.Therefore,herein we report colonial morphology,biochemical characteristics,antimicrobial susceptibility patterns and molecular identification of N.cyriacigeorgica isolated from an outbreak of clinical mastitis in a dairy herd in northern China.Furthermore,in this study,histopathology,electron microscope,flow cytometry,western blot and real-time PCR are used to evaluate the histopathological changes in mice mammary gland challenged with N.cyriacigeorgica in vivo;to analyse the ultrastructural changes,apoptotic effects and the innate immune response elicited by Nocardia in the bovine mammary epithelial cells line MAC-T in vitro;finally to demonstrate pathogenicity and mechanism underlying bovine mastitis caused by N.cyriacigeorgica.Results showed that the 12 Nocardia isolated from were 87 mastitic milk samples were N.cyriacigeorgica,which were all able to grow at 37℃ and 45℃,and were positive for catalase,acetamide and nitrate reduction tests.Moreover,using standard minimum inhibitory concentrationmethod,all isolates were found to be multidrug resistant to ampicillin,amoxicillin-clavulanic acid,ciprofloxacin,minocycline,rifampicin and aminoglycosides;susceptible to amikacin,sulfamethoxazol,cefotaxime and impenem.The drug sensitive test result played an essential role in the clinical therapy of noacrdial mastitis.In vivo,mice intramemmary inoculated with N.cyriacigeorgica had remarkable signs of messy fur,anorexia and were sluggish.The histopathology of mice mammary gland revealed chronic inflammatory changes with hyperaemia,edema and infiltration of lymphocytes and neutrophils as well as hyperplasia of lymph nodules in mammary glands.In vitro,the results showed that N.cyriacigeorgica possessed the abilities of adhesion and invasion to MAC-T cells and growth inside cells.Nocardia infection induced cell demage and necrosis manifested specific features under electron microscope,such as rupturing of cell membrane,cristae degeneration and swelling of mitochondria,clumping of chromatin and karyolysis.N.cyriacigeorgica was also found to collapse mitochondrial transmembrane potential,decrease Bcl-2 mRNA level,increase Bax mRNA level,release mitochondrial cytochrome c and ultimately induce cell apoptosis.Additionally,it promoted casepase-9 and casepase-3 levels,increased the release of LDH and promoted DNA fragmentation which further confirmed the apoptosis.These results indicated that the clinical N.cyriacigeorgica induced both necrosis and apoptosis in MAC-T cells;meanwhile mitochondrial-caspase dependent apoptotic pathway involved in the Nocardia-induced apotosis process.The analysis of inflammatory cytokines in MAC-T cells showed that the mRNA levels of various proinflammatory factors(including TNF-a,IL-1β,IL-8,IL-12 and IL-18)were increased with the enhancing of TLR2,TLR4,NLRP3,ASC and caspase-1 mRNA levels.The results of western blot analysis revealed a degradation trend of IκB-α and an increase of phosphorylate NF-KB-p65,NLRP3 and caspase-1.These results suggested that MAC-T cells may recognize N.cyriacigeorgica through TLR2 and TLR4,then may initiate the inflammatory response by targeting NF-κB signal pathway.Besides,N.cyriacigeorgica might also activate caspase-1 though NLRP3 inflammasome,inducing the production of IL-1β andIL-18 and pyroptotic cell death.In conclusion,we draw the conclusion that N.cyriacigeorgica can induce bovine mastitis through the following pathogenic mechanisms that(ⅰ)N.cyriacigeorgica can quickly adhere and invade into bovine mammary epithelial cells and grow inside cells,inducing cell damage and necrosis;(ⅱ)N.cyrziacigeorgica induced cell apoptosis through mitochondrial-caspase dependent apoptotic pathway;(ⅲ)N.cyriacigeorgica elicited bovine mammary epithelial cells to secrete various proinflammatory factors.