Treatment Of Bovine Subclinical Mastitis With Nisin

Mastitis, one of widespread disease for dairy cows, made a great loss to dairy production with high incidence. Subclinical mastitis can result in an elevated milk SCC and decrease milk yield, change the composition of milk to influnce milk quality. If not treated in time, it may lead to clinical mastitis, even teat blind or culling. In general, subclinical mastitis was not treated because of high incidence and antibiotic residues in milk. When bulk milk somatic cell count was obviously increased to influence milk sale, it was still treated with antibiotics. However, this could increase the resistance of mastitis pathogens, residues in milk and accumulation in vivo, which did harm to human health. As a result, it was necessary to develop a non-antibiotic preparation for the treatment of bovine subclinical mastitis, which was more highly-efficient, low toxicity and residues free. This study was designed to identify the pathogens with the sensitive test for routine antibiotics, and use nisin to treat bovine subclinical mastits, especially Staphylococcus aureus subclinical mastitis.1. The patogens of bovine subclinical mastitis was investigated in Jin Jiang Dairy FarmTo investigate the main pathogen of bovine subclnical mastitis, a survey had been conducted for 232 cows with HMT in a dairy farm of Hangzhou suburb.89 milk samples with HMT "+++" were collected to detect main pathogens, SCC and milk samples. Results showed the main pathogens were Staphylococcus aureus (25.0%), CNS (31.7%) and Streptococcus agalactiae (34.9%) respectively. S. aureus and CNS had been resistant to Penicillin G, Ampicillin and Oxacillin, and the resistant rates were (88.8%,86.4%), (83.3%,77.3%) and (72.2%,68.2%), while Streptococcus agalactiae was sensitive to these routine antibiotics. This showed the main pathogens in the dairy farm were Staphylococcus aureus, CNS and Streptococcus agalactiae, which had been resitant to Penicillin Q Ampicillin and Oxacillin except Streptococcus agalactiae.2. The irritancy test of Nisin for bovine quartersIn order to select the optimum dose to treat bovine subclinical mastitis, Nisin based formulation was evaluated for the dairy quarters before treatment. One heathy Holstein cow was selected to make an irritant test with three doses of nisin-based formulation (5.0×106IU,2.5×106IU,1.25×106IU), saline (250mL) as a control. Results indicated that the visible symptoms were observed in the quarter after treatment at the dose of 5.0×106IU alone. The acticity of NAGase in milk quarters was increased after 24h with infusion of Nisin for three groups, it was returned to the normal level after one week. All the results indicated that two doses (2.5×106IU, 1.25×106IU) had little irritative for dairy mammary glands. According to the efficacy of nisin-based formulation for clinica mastitis, the dose of 2.5×106IU was suitable to treat bovine subclinical mastitis.3. Efficacy of nisin-based formulation in treatment of subclinical mastitis in lactating dairy cowsA total of 90 cows with 90 subclinical mastitic quarters were randomly assigned to Nisin treated group and control group. In nisin-based formulation treated group, the cows were received an intramammary infusion of Nisin Z at the dose of 2.5×106IU, the control group without treatment. The treatment was performed once daily for 3 days in the nisin treatded group. The main pathogens in all cases were Staphylococcus aureus (27.78%), CNS (31.11%) and Streptococcus agalactiae (33.33%) respectively. After treatment, the bacteriological cure rate of nisin-based group and control group were 65.2%and 15.9%in the 2nd week with significant difference (P<0.01) in the 2nd wk. And the bacteriological cure rates of the main pathogens in the nisin-treated group and control group were (50.0%,18.2%), (90.9%,1.40%) and (58.8%,27.3%) respectively including Staphylococcus aureus, CNS and Streptococcus agalactiae. No significant differences were observed for S.aureus and CNS between the two groups, while significant difference for S.agalactia. The NAGase acticvity in the nisin-treated group were decreased significantly (P<0.01), while no difference in the control group (P>0.05). And the percentage of SCC≤5.0×105/mL in the two groups were (41.3%, 18.2%) and (32.9%,9.1%) in the 1st and 2nd week after treatment, the differences at the same time point were both P<0.01. All data indicated that nisin-based formulation had a better therapeutic effect for bovine subclinical mastitis.4. Efficacy of nisin-based formulation to decrease bulk tank somatic cell count after treatment of bovine subclinical mastitis in lactating dairy cows.Two cowsheds were selected to evaluat nisin-based formulation to decrease BTMCC, total 85 and 90 cows in nisin and control group. Before the treatment, a survey was conductecd to detect BTSCC, bulk bacteria and bulk milk composition from March to July. The high BTSCC herd was selected as the nisin-based group, and the low BTSCC herd was the control group. From August to December, nisin-based formulation was used to treat the subclinial cases once daily for 3 days in the treated group, while the control group without treatment. The efficacy of nisin-based formulation to treat subclinical mastitis was evaluated with BTSCC, bulk bacteria, bulk milk composition and Nisin Z residues detected by ECLIPSE50.Results indicated that BTSCC, bulk bacteria in the treated group were significantly lower than the control group (P<0.05), while bulk milk composition including fat, protein, and lactose was increased in the nisin-based formulation group. Nisin Z could not be detected by ECLIPSE50 in bulk tank milk when the number of quarters infused with nisin-based formulation was less 10. All results showed that nisin-based formulation could decrease bulk tank somatic cell counts effectively.5. The residues of nisin-based formulation in mik samples after treatment for bovine subclinical mastitis3 Holstein cows with subclinial mastitis were selected to evaluate nisin residues in treated quarters after treatment. The treatment was performed once daily for 3 days in the nisin treatded quarters at the dose of 2.50×106IU, while the control quarters without treatment. The results showed that Nisin Z in milk(75.81±36.50 IU/mL) was detected only in the treated group at 24h following intramammary infusion, which was much lower than the upper limit (0.5g/kg, equivalent to 500IU/mL) allowed as preservative in milk products by the China authority, while no residues of Nisin Z in milk samples from the control quarters. Reduced fermentation with low acidity from treated quarters was found in milk samples at 24h, where no inhibition was detected in fermentation from control quarters throughout the experiment. All the results showed that nisin-based formulation was safe and litte fermentation was found in a short time after treatment.6. Degradation of Nisin Z in normal, subclinically and clinically mastitic milk samples in vitro9 Holstein cows were selected and divided into 3 normal cases (SCC 0-2.0×105cells/mL),3 subclinical cases (SCC 1.0-2.0×106cells/mL) and 3 clinical cases. At first, the NAGase activities in different milk samples were detected after freezing and thawing, then Nisin Z (104IU) was added into lOmL milk samples to detect the residues at different time points (Oh,2h,4h,6h and 8h) after culture at 37℃in vitro. The results indicated that the NAGase activity in milk samples were increased from normal, subclinical and clinical mastitis cases in turn. Nisin residues were detected by National Standards to be decreased gradually after cultivation at 37℃in vitro from normal, subclinical and clinical mastitis cases.7. Efficacy of vaccination in combination with Nisin Z treatment against Staphylococcus aureus mastitis in lactating dairy cowsAlthough nisin-based formulation could have been proved effective on bovine sublinical mastitis, how to improve the efficacy of Nisin Z had been a new concern in the following research. At first, Staphylococcus aureus vaccine was attempted to improve the effect of nisin-based formulation to treat bovine subclinical mastitis.75 Holstein cows with subclinical mastitis were divided into 3 groups, nisin combination with Staphylococcus aureus vaccine group (25), Nisin treated group (25) and control group (25). Staphylococcus aureus vaccine (2mL) was injected into lymph mammary gland, after one week, Nisin-based formulation (2.50×106IU) was infused to the mastitic quarters once daily for 3 days, then one week later,2mL Staphylococcus aureus vaccine for lymph mammary gland again; In the Nisin-treated group, Nisin-based formulation was performed once daily for 3 days with the dose of 2.50×106IU. The indicators of milk samples were HMT, SCC, NAGase, bacteriological exzamination and milk composition after treatment in the 2nd,4th and 6th week.The percentage of SCC≤5.0×105cells/mL was (56.0%,48.0%,100%), (64.0%, 52.0%,100%) and (60.0%,48.0%,100%) among three groups at the same time point. And the NAGase activity in two treated groups were (34.2±9.8U/L,35.5±12.5U/L) (30.3±12.0U/L,31.7±13.5U/L)and(29.9±14.2U/L,32.2±15.4U/L)in the 2nd,4th and 6th wk after infusion. No significant differences were observed in the two treated groups after HMT, SCC and NAGase test at the same time point (P>0.05), while significant differences (P<0.01) compared with pretreatment and control group among three groups.The bacteriological cure rates of three groups were (68.0%,44.0%,0) (72.0%,40.0%,0) and (72.0%,40.0%,0) at the same time point after treatment. No significant difference was observed between two treated groups in the 2nd week after infusion, while significant differences (P<0.05) in the 4th and 6th week after treatment. The percentage of milk fat, protein, lactose and NSF were all increased in the same time point after treatment. Compared with pre-treatment and the control group, significant differences (P<0.01) were observed, while no significant differences (P>0.05) between two treated groups. All the results indicated that Nisin combination with vaccine had a better therapeutic effect than nisin-based formulation alone for subclinical mastitis with Staphylococcus aureus treated.In summary, the main pathogens of the dairy farm were Staphylococcus aureus, Streptococcus agalactiae and CNS, and the Staphylococcus was resistant to Penicillin G, Ampicillin and Oxacillin. After the irritant test of nisin-based formulation for mammary gland, the dose of 2.50×106IU was suitable to treat bovine subclinical mastitis. Nisin-based formulation had a better therapeutic effect for bovine subclinical mastitis, and the residues of Nisin Z in milk after treatment was much lower than the upper limitallowed as preservative in milk products by the China authority. Nisin-based formulation could decrease BTSCC, bulk bacteria, and improve bulk milk compositions. Combination with Staphylococcus aureus vaccine, nisin-based formulation had a better therapeutic effect for subclinical mastitis, especially for those subclinical cases caused by Staphylococcus aureus.