Treatment Of Bovine Clinical Mastitis With Nisin-based Formulation

Despite the fact that much research and effort has been dedicated to mastitis prevention and treatment for so many years, bovine mastitis is one of the most prevalent and costly diseases in dairy industries around the world. At present, intramammary administration of antimicrobial drugs is the main approach used for treatment of bovine mastitis. However, poor responses to antibiotic treatments have become very common in veterinary practice, because antimicrobial resistance of mastitis pathogens increased following so many kinds of antibiotics widely used. And the extensive use of antibiotics on dairy farm may cause antibiotic residues in milk which often brought great economic losses to dairy industry because of bad quality of milk products. Antibiotic residues in milk also have the potential threats to consumers'health. Therefore, it was necessary to develop a non-antibiotic preparation for the treatment of bovine mastitis, which was not harmful to public health. This study was designed to analyze the distribution of major mastitis-causing bacteria and common antimicrobial drugs in milk in vitro. And then therapeutic efficiency of nisin-based formulation in the treatment of clinical mastitis in lactating dairy cows was analyzed.1. Distribution of major mastitis-causing bacteria in milkTo investigate the distribution of major mastitis-causing bacteria in milk, the variations in major constituents of milk during milking in healthy dairy cows were analyzed. Quarter milk samples were collected from 4 Hostein dairy cows during the first, middle and last period of milking and their major constituents were analyzed. Results indicated that milk fat concentration gradually increased during the progress of milking, and was significantly higher in the milk collected during the last period than those collected during other periods of milking (P＜0.05). The concentration of milk protein, solids-not-fat and lactose had a rise trend at first, and then declined. There ware significant differences in the milk collected between the last period and other periods of milking (P＜0.05). After adding a certain amount of Staphylococcus aureus or Escherichia coli in milk and standing for 8,12,24 h at 4℃, Sta. aureus and E. coli were found largely in upper portion of milk, and the colony forming units (CFU)/mL of both isolates were significantly higher than those in middle and lower portions (P<0.001). However, there was no difference between the middle and lower portions (P>0.05). It is suggested that bacteria getting entrance into teat canal may float up through attaching to fat globes and invade the deep mammary tissues.2. Distribution of common antimicrobial drugs in milkIn order to analyzed the distribution of common antimicrobial drugs in mammary gland tissues after intramammary infusion, the distribution of common antibiotics and nisin Z in milk in vitro was investigated by measuring their inhibitory effect on indicator bacteria, after adding a certain concentration of common drugs and standing for 8,12,24 h at 4℃. The majority of all selected antimicrobial drugs were found to have higher inhibitory activities in lower portion of milk than in middle and upper portions. Four antibiotics including penicillin, gentamicin, lincomycin and ampicillin which had higher polarity had distinct differences among 3 parts. Nisin Z was distributed more evenly but a little higher concentration in the upper portion, suggesting that nisin Z have a good distribution in mammary tissues and be suitable to make an intramammary preparation.3. Inhibitory effects of nisin Z on Sta. aureus in vitroOf 37 Sta. aureus strains isolated from bovine clinical mastitis,83.8% were resistant to penicillin, and 16.2% to gentamicin (GM), but none to nisin Z.4. Efficacy of nisin-based formulation in treatment of clinical mastitis in lactating dairy cowsA total of 92 cows with 107 naturally occurring clinical mastitic quarters were randomly assigned to nisin-based formulation (48 cows with 51 quarters) and GM (44 cows with 56 quarters) treated groups. In nisin-based formulation treated group, cows received an intramammary infusion of nisin Z at a dose of 2,500,000 IU, in the GM-treated group, intramammary infusion of GM was administrated at a dose of 0.8 g. The treatment was performed twice daily (administrating after morning and afternoon milkings) until the inflammatory signs disappeared and mammary secretion became visibly normal. The treatments were repeated 4.1±0.2 and 6.1±0.3 times in groups of nisin-based formulation and GM, respectively. Nisin-based formulation offered a clinical cure rate similar to GM (90.2% vs.91.1%) and no difference in a higher bacteriological cure rate than GM-treated group (60.8% vs.44.6%, respectively). Proportions of the quarters with milk somatic cell counts lower than 500,000 cell/mL were no differences in nisin-based formulation treated group (50.0% and 47.8%) compared with GM treated group (33.3% and 37.3%) 1 and 2 wk after treatment. Of the bacteria isolated from all the clinical mastitis cases, Streptococcus agalactiae (Str. agalactiae) and Sta. aureus were the predominant causative pathogens, being isolated from 37.4% and 15.9% of 107 cases, respectively. Coagulase-negative staphylococci seemed less important and accounted for 9.3% of all cases. Nisin-based formulation therapy eliminated 54.5% (6 of 11) of Sta. aureus intramammary infection (IMI), whereas GM eliminated 33.3% (2 of 6). For Str. agalactiae mastitis, 83.3% (15 of 18) and 50.0%(11 of 22) became bacteriologically negative in the cases treated by intramammary infusion of nisin-base formulation and GM, respectively. Nisin Z in milk (4.5±0.8 IU/mL) was detected only in the treated quarters at 12 h following intramammary infusion, which was much lower than the upper limit (0.5 g/kg, equivalent to 500 IU/mL) allowed as preservative in milk products by the China authority. Reduced fermentation with low acidity from nisin-based formulation treated cows with clinical mastitis was found in milk collected from the treated quarters until 36 h following the last intramammary infusion, whereas no inhibition was detected in fermentation of the milk from untreated quarters throughout the experiment. But for GM treated cows, the inhibited fermentation was found in milk collected from treated and untreated quarters until 72 h following the last intramammary infusion, with more inhibitory activity detected in the milk from the treated quarters. All data indicated that nisin-based formulation has a better therapeutic effect for bovine mastitis and reduced fermentation was found in a short time after treatment.5. ECLIPSE 50 test for the detection of nisin Z in milk after nisin-based formulation treatmentECLIPSE 50 is a test for the detection of inhibitors and antibiotics in milk. To analyze the feasibility of this kit for detecting nisin Z residue in milk, detection limit of this kit for standard nisin in milk was established. Then ECLIPSE 50 test was used to detect nisin Z in milk samples collected from nisin-based formulation treated cows. Results indicated that the detectable limit of ECLIPSE 50 test for nisin in cow's milk was 5×10-4-5×10-3 mg/mL (equal to 0.5-5 IU/mL). The maximum residue limit (MLR) of nisin in milk was 5 IU/mL. Milk samples collected from all the treated quarters were positive at 8 h after receiving an intramammary infusion of nisin-based formulation at a dose of 2,500,000 IU. After diluted 1:5000 with non-antibiotic milk, ECLIPSE 50 test became negative. For the milk collected from untreated quarters, ECLIPSE 50 test was negative throughout the experiment. Therefore, ECLIPSE 50 can be used for the detection of nisin Z in milk.6. Effect of nisin-based formulation treatment on lactating performance of clinical mastitic cowsNine dairy cows with clinical mastitis were selected and intramammary administrated 6 times with nisin-based formulation. Daily milk production and major milk constituents were analyzed to evaluate the effect of nisin-based formulation treatment on lactating performance. Daily milk productions of all the mastitic cases gradually recovered, but the milk production at 8 d after treatment decreased by an average of 2.5 kg compared with 6 d before infection. Milk fat, protein, lactose and solids-non-fat contents of milk collected from treated quarters increased after intramammary infusion of nisin-based formulation, but they were lower than those from untreated quarters (composite milk samples). The concentration of milk lactose was above 4.7% in all treated quarters except for 2 d after intramammary infusion, suggesting that mammary tissues damage may recover quickly and the synthesis of epithelia cells increase in large extent.In summary, major mastitis-causing bacteria and common antibacterial drugs may not contact each other sufficiently due to their contra-direct movements in mammary gland, resulting in poor responses of mastitic cases to antibacterial therapy. Nisin Z which was more efficient against Sta. aureus isolated from clinical mastitis in vitro was distributed more evenly in milk, suggesting that it be suitable to make an intramammary preparation. Nisin-based formulation had better therapeutic effects on bovine clinical mastitis in vivo with the bacteriological cure rate of 60.8% (Gentamicin 44.6%). Quarter milk SCC gradually declined and lactating performance including daily milk production and major constituents recovered more quickly after nisin-based formulation administrated. Small amounts of nisin Z residue and reduced fermentation were found in milk collected from treated quarters in short time after treatment. Nisin-based formulation will be widely used as a new medicine to treat bovine mastitis, as well as its safety in humans.