| Mastitis is the most important diseases of dairy cows. Mastitis-causing Staphylococcus aureus is a contagious mastitis-causing pathogen which is difficult to eradicate. The objective of this thesis was to investigate the predominant strains of S. aureus recovered from mastitic cows on dairy farms distributed in 14 major dairy-farming provinces in China. The isolates were typed by pulsed-field gel electrophoresis (PFGE) using Sma I as the restriction enzyme. The relationship between PFGE types and patterns of superantigen genes as well as antibiotic resistance of S. aureus were also studied.1. Investigation on the major diseases in a commerical dairy herdTo investigate major diseases and incidence of dairy cows in large dairy farm, a survey had been conducted in a representative farm for one year by looking into case histories, interviewing with clinical veterinarian and on-site visits. Results showed that the most frequent diseases for lactating cows were bovine mastitis (20.9%), arthritis (20.7%), forestomach atony (7.7%), footrot (4.1%), placental retention (4.0%) and bovine endometritis (2.5%). The incidence of bovine (clinical) mastitis was 55.9%followed by arthritis (55.1%), forestomach atony (19.0%). The peak incidence of clinical mastitis was observed in August with a small peak in February during the period of low incidence which suggested that climate change is an important factor for the occurring of bovine clinical mastitis.The high incidences of arthritis and forestomach atony maybe result from lack of exercise and moldy forage.2. Investigation on the subclinical mastitis in the main dairy farming areas in Zhejiang ProvinceTo investigate the prevalence and major pathogens of subclinical mastitis for the main dairy farming area, Hangzhou, Jinhua, Ningbo and Taizhou in Zhejiang Province,30 representative farms were selected concerning size, location, and milking mode across the province. The incidence of subclinical mastitis was determined at cow and quarter levels based on Hangzhou mastitis test (HMT). Quarter milk samples with HMT score of 2 or 3 were collected aseptically for bacteriological assay. Results showed that the prevalence of subclinical mastitis was 54.3% on cow level and 28.0% on quarter level totally in this province. The highest prevalence in cow and quarter levels was 69.7% and 36.4%, respectively in Ningbo with the lowest prevalence 40.8% and 21.8% in cow and quarter levels in Taizhou. The prevalence of subclinical mastitis at cow and quarter levels in large farms (58.2%,30.0%), farm-scale larger than 300 cows, is a little higher than in small farms (50.0%,25.0%).Detection rate of Streptococcus agalactiae and Staphylococcus aureus were 27.0% and 12.2% which were the major pathogens for bovine mastitis in Zhejiang Province, especially for those large farms.Coagulase-negative staphylococci (CNS) were the most prevalence pathogens (34.0%). As a result, it is necessary to attach importance to its role in pathogenesis of bovine mastitis.3. Predominant strains of mastitis-causing Staphylococcus aureus in 14 provinces in ChinaTo investigate the predominant strains of bovine mastitis-causing S. aureus in China, S. aureus isolates were recovered from mastitic cows on dairy farms distributed in 14 provinces and typed by pulsed-field gel electrophoresis (PFGE) using Sma I as the restriction enzyme. Results showed that 191 isolates were assigned into 15 pulsed-field epidemic types, of which types A, B, C, D and E accounted for 91.1% of the all isolates with type A (56.5%) as the largest group observed in 13 provinces. Geographical distribution of the predominant types was obvious. Most provinces, mainly in southern, southeastern and central regions, belonged to type A which was highly similar to the reference strain S. aureus Newbould 305. Type D was only observed in Xinjiang while type C prevalent in Shandong and Inner Mongolia. These suggested that few widely distributed strains of S. aureus were responsible for the majority of bovine mastitic cases in China and variation existed for predominant types of S. aureus isolates in different provinces. Compared with other regions, Zhejiang Province had more PFGE types of S. aureus and rare types, only one isolate was observed for each, was just found in Zhejiang Province. The reason may be due to frequently imported cows from other provinces or even other countries for the expansion or replacement of the herds in Zhejiang Province.Different dairy farming regions also had different predominant strains in Zhejiang Province. At the farm level, most of dairy farm tended to have a unique type during a period of time.4. Comparison of Xingjiang's and Zhejiang'isolates of Staphylococcus aureus on the antibiotic resistanceAntibiotic resistance of bovine mastitis-causing S. aureus was evaluated by using 75 S. aureus strains isolated from 23 representative dairy farms distributed in Zhejiang Province. Results showed that 90.7% of all strains were resistant to at least one antimicrobial class. S. aureus isolates were observed mostly resistant to penicillin and ampicillin (77.3%) with different resistance patterns in different regions.No completely susceptibility strains of S. aureus were found from Xinjiang Province.93.0% penicillin-resistant strains were observed while strains of resistant to tetracycline, clindamycin, gentamicin was 23.3%,9.3%, and 16.3%, respectively which was better than in Zhejiang Province.8.0% ciprofloxacin-resistant strains from Zhejiang Province were found, but all of the strains from Xinjiang Province are susceptible to ciprofloxacin. These results suggested that different geographical environments, bacterial strain and veterinarian's experience could give impact to S. aureus resistance.The emergence of multi antimicrobial-resistance is usually observed among bovine isolates. The present study indicated that most of S. aureus strains were resistant to more than one antimicrobial agent tested, and the predominant pattern was simultaneously resistant to penicillin, ampicillin, tetracycline, and erythromycin. It was also noted that therapy of other infectious diseases in dairy farm might have influence on antimicrobial-resistance of mastitis-causing S. aureus.No association was observed between antibiotic resistance patterns and particular S. aureus PFGE types in present study. 5. Superantigen genes in different PFGE types of Staphylococcus aureus isolatesA total of 191 bovine mastitis-causing S. aureus isolates collected from 14 provinces in China were tested for sequences of genes encoding superantigen by conventional PCR. It was found that 72 (37.7%) isolates harbored some type of genes which were arranged in 8 superantigen genotypes. The most frequent genes of the S. aureus strains harboring were sei (53,27.7%), seg (25,13.1%) and sea (15,7.9%). Furthermore, sei (28,14.7%), seg-sei (18,9.4%) and sea (9,4.7%) were the most frequent genotypes.Our study showed an association between the superantigen genotypes and PFGE types among the S. aureus strains. Type A, the predominant strain in China, had a very low frequency of superantigen genes (3.7%) while the frequency of type B (76.2%), type C (92.3%), type D (54.5%) and type D (50.0%) were much higher. These results suggest that S. aureus strain with different PFGE type play a different role in the pathogenesis of bovine mastitis.For all of the 191 mastitis-causing S. aureus isolates, only one isolate was positive for pvl which implied that pvl was not important for the infection of the mammary gland.6. Cloning and prokaryotic expression of clumping factor A (ClfA) gene in Staphylococcus aureusThe fibrinogen-binding adhesin clumping factor A (ClfA) is important for the pathogenesis of S. aureus. With specificity primer designed according to GenBank, sequences of A domains of clumping factor A (ClfA) was amplified from a predominant strain of mastitis-causing S. aureus in China by conventional PCR technique, and the PCR product was about 1560 bp in length. The PCR product was cloned into pMD18-T vector successfully and constructed plasmid pMD18-ClfA. After digested by double enzymes with BamH I and EcoR I, the purfied ClfA gene was subcloned into pET-His, the expression vector, and the recombinant plasmid pET-His-ClfA was constructed successfully which was transformed into the host cell E. coli BL21 (DE3). After induced by IPTG (1 mmol/L), approximately 57 KD target protein was observed on the SDS-PAGE. The expressed protein was purified with HisTrapFF chromatography. Polyclonal antibody was obtained by immunized ICR mice and analyzed by Western blotting. Result showed that the protein was the antibody of ClfA. Our study was expected to be useful for S. aureus mastitis adjuvant therapy and development of engineering vaccine for preventing S. aureus matitis of dairy cows.
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