Experiment animals and treatment
The Chinese Holstein cows were obtained commercially from Sichuan Ninggang Animal Husbandry Co., Ltd. Twelve healthy cows and 36 cows with clinical mastitis having similar date of parturition and milk production were selected form 50 healthy and 50 cows with mastitis with weight 612 ± 47 kg, 3-4 years of age, and 2-3 parity in a semi-closed unified dairy farm. Twelve healthy cows were placed into control group (group A), the other 36 cows with clinical mastitis were divided into 3 groups (group B, C, and D) randomly by simple rando mizaton. Intramuscular injection of 15, 30, and 60 mL of Pulsatilla saponin B4 QD in the brachiocephalicus muscle of the cows in group B, C and D, respectively was administered, and recorded the first day of the experiment as day 1, continued 4-6 days treatment until the recovery of cows. No administration for the control group. All the groups were under observation for 12 days.All experimental cows had the same feeding and management procedure, and no other diseases occurred, nor antibiotic treatment administration during the experimental period. We selected a small sample size because the Pulsatilla saponin B4 was evaluated for the first time in the present study, and therefore, the initial intention was to gather basic evidence regarding the use of this drug in more complex experimental designs.
Sample collection
10 mL tail venous blood of experimental cows were collected before morning feeding at 8 am. The sampling was done on the days 1, 3, 5, and 7 in group B, C, and D, also on same day during the experiment in group A. Collected venous blood was placed in a centrifuge tube without anticoagulant and centrifuged at 3 000 rpm for 10 min to separate serum after 1 hour rest at room temperature (20-25 ℃), and upper serum was transferred to EP tube, stored at -70 ℃. Washed the udder by warm water, and sterilized by 75% ethanol. Discarded the first three streaks of milk, then measured the SCC of the milk in the fourth streak on the days 1, 3, 5, 7, 9, and 11 in group A and group D. And averaged the SCC of group A during the experiment. All sampling process were administrated before dosing.
Animals treatment after experimentation
Breeding management of cows in group A and cured cows in group B, C, and D returned to normal after the study, and the rest were treated by other medicine until cured. All cows will be fed until death.
Equipment and reagents
The equipment shown below: Centrifuge (Sigma, Germany); Ultra-low temperature freezer (Haier, China); Clean bench (Sujing Co., Ltd, China); Full wavelength microplate reader (Thermo Scientific, U.S.A), UV-visible spectrophotometer (Lambda 45, U.S.A), Microsampler (Thermo Scientific, U.S.A); Electronic thermostatic water bath (Zhongxing Instrument, China), Stratagene Mx3005P (Agilent Technologies, U.S.A).
The reagents shown below: Pulsatilla saponin B4 (100 mL/bottle, concentration 66%, ethanol solution), donated by Sichuan Innovate Medical Technology Co., Ltd, Chengdu, China, identified by the National Engineering Research Center of Traditional Chinese Medicine Solid Preparation Manufacturing Technology of Jiangxi University of Traditional Chinese Medicine; ELISA kit of IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, TNF-α, LTB4, PGE2, SAA, and HP, all were purchased from Shanghai Enzyme Biotechnology Co., Ltd, Shanghai, China. Milk pathogenic bacteria DNA extraction kit, bioeasy Co., Ltd, Shenzhen, China. Eight combined bovine mastitis pathogen nucleic acid detection reagents kit, bioinfee Biotechnology Co., Ltd, Shenzhen, China.
Criteria of clinical mastitis
All cows with clinical mastitis were diagnosed by the same branch veterinarian of the dairy farm. Detailed criteria included red and swollen mammary gland with sensitive tenderness, higher surface temperature of the incidence quarter, significantly decreased milk production, milk with yellow or red color, SCC higher than 500 000/mL, and other abnormal traits [21].
Clinical cure and bacteriological cure
A cow was regarded as clinically cured if both milk and mammary gland had a normal appearance in the clinical examination performed approximately 24 hours after the last infusion.
A cow was considered bacteriologically cured if a microorganism was identifiedin the milk sample collected on d 1, and the same species was not isolated in any of milk samples collected posttreatment (d 1 or 11). If the same pathogen isolated on d 1 was identified in either of the posttreatment samples, the quarter was considered noncured. Only cows with culture results for posttreatment milk samples were included in this evaluation. In addition, samples with negative culture (no growth) on d 1 were not included in the analysis of bacteriological cure.
PCR tests and indices measurement
Bacterial incubation and isolation were operated on the milk sample of d1 and d 11 of all test groups, then use qPCR (40 loops, threshold fluorescence 500 dR) to verify the bacteria (Pseudomonas aeruginosa, Streptococcus agalactiae, Streptococcus dysgalactiae, Klebsiella species, Escherichia coli, and Staphylococcus aureus) and β-lactam resistance.
Serum concentrations of IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, TNF-α, LTB4, PGE2, SAA, and HP in experimental cows were measured by the ELISA kit.
SCC measurement and cure rate calculation
Detected the SCC of milk by De Laval milk somatic cell detector and observed the clinical symptoms of the cows with mastitis to judge the treatment condition every day during the experiment, and then calculated the cure time, effective rate, and cure rate.
Data Analysis
Used SPSS 19.0 to verify the data distribution, distinguished the differences and correlations between groups by independent sample t-test and Person relation analysis, respectively. All data was performed by `X ± SD, compared data with P-value lower than 0.05 was considered to be significant, lower than 0.01 was considered to be very significant.
For each cow, three different investigators were involved as follows: a first investigator administered the treatment and collected sample based on the randomization table. This investigator was the only person aware of the treatment group allocation. A second investigator was responsible for testing the cure rate, bacterial infection rate, somatic cell count, inflammatory factors, and qPCR. Finally, a third investigator (also unaware of treatment) analyzed the above test results.