| Background Given that milk is an necessity for infants and it gradually plays an important role in residents’ dietary with the development of economy in China, milk-related Public heath problems have attracted more and more attention.On one hand, mastitis is a disease of high incidence in the worldwide while milk is suitable for growth of microorgnisms. Milk-associated microbiological problems have long been the focus of animal husbandry and food hygiene. On the other hand,because of the widespread use of antibiotics in livestock, resistant strains and resistance genes have become topics of international interest in recent years.Objective 1 To investigate prevalence of subclinical mastitis in China and the microbial flora distribution of quarter milk.2 To investigate antibiotic use in dairy farming and then give an overall evaluation about impact of antibiotic use in dairy cows on the diversity of microorganisms isolated from quarter milk through multiple phenotyping and genotyping methods.Methods Choosing dairy cows in 15 dairy herds from 5 provinces of China on the principle of representativeness and feasibility. Informat ion about use of antibiotics in each herd was obtained by visiting the vet and by examining veterinary prescriptions and drug inventory records. The incidence of subclinical mastitis was determined based on the result of California Mastitis Test (CMT). Bacteriological assay was carried out by traditional culturing method. Identification of the isolates was done according to biochemical methods and molecular method. Phenotype and genotype analysis of the isolated strains was determined by broth microdilution method, serotyping, pulsed-field gel electrophoresis and PCR-based typing methods.Besides, swabs of extramammary sites (bovine teat skin and milking machine liners)were also collected and were subjected to correlation analysis with srains isolated from milk samples.Result The incidence of subclinical mastitis of investigated herds was 41%～90% at cow level and 14%～41% at quarter level. Most of the cows diagnosed with subclinical mastits had only one quarter infected [48.3% (256/530)].Mastitis was the most common disease treated with antibiotics. Antibiotics used to treat cows with mastitis included penicillin, streptomycin, eftiofur, clindamycin and ciprofloxacin. Cloxacillin Benzathine was the most commonly used antibiotics for dry cow therapy.A total of 748 milk samples were included in the study, among which 527 were from subclinical mastitis quarters, 142 were from normal quarters and 79 were from clinical mastitis quarters. 447 samples give positive results for microbiological testing.Positive rate of milk diagnosed with subclinical mastits, clinical mastitis and normal milk was 40.7%, 69.9% and 66.2%, respectively. 568 mastitis pathogens were separated from these samples, including 536 bacteria and 32 fungi. Corynebacterium bovis (21.4%), Coagulase-negative Staphylococci (CNS)(16.5%) , Staphylococcus aureus (11.4%) and Streptococcus agalactiae (11.1%) were predominant findings of bacteria. Staphylococcus epidermidis and Staphylococcus chromogenes were two major kinds of CNS with isolation rates of 7.8% and 3.4%. Isolation rate for Staphylococcus aureus of 150 swabs was 22.7%.The results of antimicrobial resistance tests showed as follows. (1) 92.8%(103/111) Staphylococcus aureus isolates were resistant to at least one antimicrobial.38.7% (43/111) strains were multi-drug resistant. Resistance of Staphylococcus aureus to penicillin (90.1%), erythromycin (48.6%), ciprofloxacin (36.9%), clindamycin(27.9%), gentamycin (18.9%), chloramphenicol (9.0%) and tetracycline (7.2%) of these strains were observed. All isolates were sensitive to vancomycin, selectrin and there was no MRSA isolated. The resistance rate of isolates in farms [100%, (48/48)]was higher than in artels [87%,(55/63 )] and the difference was statistically significant (χ2=4.80, P<0.05). The multi-drug resistance rate of isolates in farms[54%,(26/48)] was higher than in artels [27%, (17/63)] and the difference was statistically significant (χ2=8.48, P<0.05). (2) 85.3% (93/109) of CNS isolates were resistant to at least one antimicrobial. 42.2% (46/109) strains were multi-drug resistant. Resistance of CNS to penicillin (76.1%), erythromycin (49.5%), selectrin(35.8%), oxacillin (33.9%), tetracycline (22%), ciprofloxacin (12.8%), clindamycin(8.3%) , chloramphenicol (6.4%) and gentamycin (4.6%) were observed. (3) 90%(81/90) of streptococcus isolates were resistant to at least one antimicrobial. 33.3%(27/90) of the strains were multi-drug resistant. Resistance to tetracycline (85.6%),clindamycin (43.3%), erythromycin(33.3%) and ofloxacin(10%) were observed.All isolates were sensitive to penicillin, cefotaxime , vancomycin and chloramphenicol.The results of ge no typing and detecting for virulence determine nts of Staphylococcus aureus showed as follows. 77 Staphylococcus aureus strains were clustered into 7 PFGE types according to the similarity of 75.2%. Each herd included at least 1 type and at most 4 types. Each PFGE type prevailed in at least 1 herd and at most 8 herds. Genotyping results based on PCR amplification of coa gene, auxiliary gene regulatory factor and capsular polysaccharide were in consistency with PFGE result. (2) Among 34 swab isolates, there were 21 strains clustered into the same type as milk isolates isolated from the same herd and 10 swab isolates cannot be digested by Sma I enzyme. (3)Among 77 milk isolates, all of the strains harboured the genes nuc、coa、hla、hlb、clfA、clfB、ebp、eno、fib、map、spa(x-region) and spa(IgG-binding region). Detection rate offnbB、can、pvl and SAgs was 51.9%, 42.9%, 39.0% and 54.5%, respectively. Sei was the kind of SAgs with the highest detection rate. All the isolates lacked amplification of the gene bbp and fnbA.The results of serotyping, genotyping, detecting for resistance genes and virulence determinents of Streptococcus agalactiae showed as follows. (1)Among 68 Streptococcus agalactiae, 66.2% of the strains can be typed by serotyping with 66.2%of the strains belonged to serotype I a and 10.3% of the strains belonged to serotypeⅡ. 68 Streptococcus agalactiae strains were clustered into 14 PFGE types according to the similarity of 76.8%. For each herd, there was one type predominant and different herds may share the same type. (2)All 21 erythromycin resistant isolates were simultaneously positive for genes ermA、ermB and mefA. Amog 62 isolates resistant to tetracycline, tetM was the most prevalent gene associated with tetracycline resistance with detection in 41 isolates. 21 stains left harboured tetO and these strains were resisitant to erythromycin as well. Strains with these five kinds of resistance genes distributed in multiple PFGE types. (3) Almost all of the strains harboured virulence genes hylB、glnA、cfb、scaA、cyl and all of the stains were negative for virulence genes bac、bca、scpB、lmb.Conclusion The prevalence of subclinical mastitis was high and the condition should be taken seriously. Corynebacterium bovis, CNS, Staphylococcus aureus and Streptococcus agalactiae were 4 kinds of main pathogens. Management and use of antibiotics on farms were in better situation than in alters.Staphylococcus aureus isolats showed very high drug resistance rate and multi-drug resistance rate while most stains harboured multiple kinds of virulence genes, which could be major obstacles for the treatment of mastitis. All of the Streptococcus agalactiae strains were susceptible to penicillin which was commonly used for treatment of mastitis. However, there was high resistance rate to erythromycin and clindamycin for which potential hazard of transfer of resistance genes should be watched out. Mechanisms for resistance to erythromycin were ribosomal modification by methylase and drug efflux by membrane-bound proteins.Tetracycline resistant strains may play an important role in the spread of erythromycin-resistant strains.A few specialized Staphylococcus aureus clones were responsible for most of the cases of bovine mastitis in a single herd and some clones might have a broad geographic distribution. Teat skin may act as origins and reservoirs of Staphylococcus aureus. Isolats from milk and skin may transmitted via the milking machines. For Streptococcus agalactiae, serotype I a was the predominant type. Certain PFGE type prevailed in each herd and there was certain relevance between PFGE types, serotypes and antimicrobial profiles. |
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