| Bovine mastitis is a common disease of dairy cows and constitutes a source of huge economic losses for the dairy industry.The main causes of bovine mastitis are the intramammary infection(IMI)of mastitis bacterial pathogens.Until now,over 150 pathogens have been reported as mastitis pathogens.A.viridans is well known as an environmental microorganism and opportunistic pathogen;however,it has been associated with clinical and subclinical mastitis in several countries recently.It is still not clear that whether A.viridans can cause bovine mastitis and how the immune system of bovine udder will react to the infection of this pathogen.Therefore,the aims of this thesis are to investigate.1)the phenotypic and genotypic characteristics of A.viridans isolated from the subclinical mastitis cases in the dairy farms of China.2)the effect of A.viridans on udder health in vitro and in vivo.3)the immune response of bovine mammary epithelial cells(bMECs)and mice mastitis model induced by A.viridans.One thousand and eight subclinical mastitis milk samples were collected for bacteriology analysis from 19 different farms located in Beijing,Tianjin and Hebei provinces of China.All the suspected strains would be identifed for A.viridans.The identified isolates of A.viridans were tested for biochemical test and antibiotic susceptibilty test.Subsequently,the genotypic diversity of strains was analysed by 16S rRNA gene sequenceing,pulsed field gel electrophoresis(PFGE)and random amplified polymorphic DNA(RAPD).In total,60 strains were isolated and identified from 10 dairy farms in China,and the overall prevalence of A.viridans in subclinical cases was 5.9%.The results of biochemical test showed a great phenotypic diversity of A.viridans.Especially the catalase test and VP test exhibited a different results compared to the strains from other countries.The minimum inhibitory concentration of different drugs showed 95%of strains were susceptible to(3-lactam antibiotics,and over 50%of strains were resistant to streptomycin,sulfamethoxazole trimethoprim,tetracycline and clindamycin.As compared to the sequence of 16S rRNA gene,both PFGE and RAPD showed their capacities to discriminate the intra-species diversity of A.viridans.Furthermore,most of the isolates obtained from the same herd or region belonged to the same major RAPD group,which indicated that RAPD is an appropriate assay for tracking the origins of isolates and epidemiological studies of A.viridans.The data suggest that A.viridans associated with subclinical mastitis has a considerable phenotypic and genotypic diversity.Nine strains were selected randomly(one strain from each RAPD subtype)and challeged to the mice for survival analysis.The virulent and avirulent strain were selected based on the survival rate.bMECs were challenged with both virulent and avirulent strain for the cytotoxity,adherence rate and invading rate.The scanning electron microscope and transmission electron microscope were used to observe the pathological changes of bMECs caused by the challenge of A.viridans.The results of mice survival anlyasis showed that A.V.40 had a strong lethal effect on mice,hence selceted as virulent strain,while A.V.36 had the least effect which was selected as avirulent strain.The virulent strain has proved its strong cytotoxity to bMECs,while avirulent strain had much less cytotopathic ability compared to the virulent strain.Besides,the strong adherence and invading capablity to bMECs has been found in virulent strain,while it was not in avirulent strain.The image presented by TEM and STM confirmed the adherence and invading of virulent strain into bMECs.It was also noticed that the disruption of cell microvilli and membrane,the swelling of mitochondria and endoplasmic reticulum,vacuolar degeneration of nucleus,and finally the apoptosis of bMECs.The expression of mRNA of Toll-like receptors and several important cytokines in bMECs were detected by real-time PCR after challenged by A.viridans.The mRNA expressions of TLR2 and IL-6 increased rapidly within 3 h after injection,while that of TLR-4,IL-1?,IL-8 and TNF-a increased drastically between 3 to 6 h.The bMECs challenged by avirulent strain showed slight increase of mRNA of receptors and cytokines.The treatment of ceftiofur greatly attenuated the expression of mRNA level,protecting the cell from the bacterial challenge.The mice mastitis model challenged by A.viridans was built,investigating the pathogenic effect of this bacteria on mice udder health in vivo.The results of bacerial burdan showed that the virulent strain maintained a high bacterial level(103 CFU/gland),causing a severe inflammatory reaction with the infiltration of innumerous lymphocyte and neutrophil.The level of TNF-a increased in the udder tissure after the challenge of virulent strain.However,the avirulen strain failed to induce a proper inflammatory reaction,without any diruption of mammary gland structure.Ceftiofur and ciprofloxacin(30 mg/kg)showed a great effect on the reduction of baterial level and protection of mammary gland.In summary,A.vridans had a moderate prevalence(5.9%)in the subclinical mastitis cases from the dairy farms located in Beijing,Tianjin and Hebei province of China,having great phenotypic and genotypic diversity.The virulent strain adhered to,invaded and disrupted bMECs,inducing a strong immune reaction of bMECs.This virulent strain caused a severe inflammatory reaction in mice mammary gland,damaging mice mammary gland tissue,while the antibiotic treament achieved great curative effect.The avirulent strain was much less harmful to the bMECs and mice mammary gland.The results in this thesis demonstrated that some virulent strains of A.viridans are highly pathogenic to bMECs and mice mammary gland,suggesting that these strains can also cause bovine mastitis and influence the milk production,which needs to be confirmed in the cow model challenged by A.viridans.Overall,we have systematically studied the genotype and phenotype of A.viridans from the subclinical mastitis cases.Furthermore,the cell damage and immune reaction caused by A.viridans has been investigated in bMECs(in vitro)and mice model(in vivo).The present thesis for the first time explored the effect of A.viridans on bovine udder health,laying the foundation of further research and providing scientific theory for the prevention and treatment of mastitis caused by A.viridans. |
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