Experiment animals and treatment
The Chinese Holstein cows were obtained from Sichuan Ninggang Animal Husbandry Co., Ltd. Twelve healthy cows and 36 cows with first-class clinical mastitis with 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) equally and randomly by simple randomizaton. In group B, C and D, the cows were applied with Pulsatilla saponin B4 Injaction through brachiocephalicus intramuscular injection with 15, 30, and 60 mL respectively, and the first day of the experiment was recorded as day 1, then administrated the same doses continuously for 4-6 days until the recovery of cows. The control group didn’t receive any administration. Our research was a field trial. All cows were diagnosed as clinical mastitis for the first time and didn’t receive antibiotic treatment for 14 days before diagnosis. All the groups were under observation for 12 days. All experimental cows had the same feeding and management procedure without other diseases occurred and antibiotic treatment 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, the initial intention was to gather basic evidence regarding the usage of this drug in the further reseaches.
10 mL of tail venous blood of experimental cows were collected before feeding at 8 am. The sampling was done on day 1, 3, 5, and 7 in group A, B, C, and D. The blood was loaded in a centrifuge tube without anticoagulant and centrifuged with a centrifuge (Sigma, Germany) at 3 000 rpm for 10 min to separate serum after 1 hour deposition at room temperature (20-25 ℃), and the upper serum was transferred to EP tube, stored at -70 ℃ in refrigerator (Haier, China). Washed the udder by warm water, and sterilized by 75% ethanol. Discarded several streams of milk, then collected 5 mL milk and measured the SCC on day 1, 3, 5, 7, 9, and 11 in group A and group C. All sampling process were administrated before treatment.
Animals treatment after experimentation
Cows in group A and cured cows in group B, C, and D were under the normal breeding management again after the study, and the rest were treated by other medicine until cured. All cows are raised according to animal welfare principles.
Pulsatilla saponin B4 injection (100 mL/bottle, 66% Pulsatilla saponin B4 , ethanol solution), PubChem CID: 11636713 (https://pubchem.ncbi.nlm.nih.gov/compound/71307558), donated by Sichuan Innovate Medical Technology Co., Ltd, Chengdu, China, identified by National Pharmaceutical Engineering Center for Solid Preparation in Chinese Herbal Medicine, Jiangxi University of Traditional Chinese Medicine .
Criteria of clinical mastitis
All cows with clinical mastitis were diagnosed by the same veterinarian of the dairy farm. Detailed criteria included red and swollen mammary gland with sensitive tenderness, infected quarters heated, decreased milk production, milk with yellow or red color, SCC higher than 500 000/mL, and other abnormal traits .
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 approximately 24 hours after the last injection. A cow was considered bacteriologically cured if a microorganism was identifiedin the milk sample on d 1, and the same species was not isolated in any of milk samples collected post-treatment (d 1 or 11). If the same pathogen isolated on d 1 was identified in any of the post-treatment samples, the infected quarter was considered non-cured. Only milk samles with positive results 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
A commercial eight combined bovine mastitis pathogen nucleic acid detection reagents kit (bioinfee Biotechnology Co., Ltd, Shenzhen, China) was employed to measure pathogenic bacteria on the milk sample on day 1 and day 11 in the optimal dose group according to the manufacturer’s instructions with the help of real-time fluorescent quantitative PCR instrument (Stratagene Mx3005P, U.S.A). Kit item number: YRMBP7045-2; lot number: 20021001.
Serum levels of IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, TNF-α, LTB4, PGE2, SAA and HP in group A and the optimal dose group was measured by bovine ELISA kits (Shanghai Enzyme Biotechnology Co., Ltd, Shanghai, China) according to the manufacturer’s instructions with the help of full wavelength microplate reader (Thermo Scientific, U.S.A). The intra and inter-assay coefficient of variance was less than 10% and 15%, respectively. The Detection range and minimum detection dose of ELISA Kits was shown in table 1..
SCC measurement and cure rate calculation
Detected the SCC of milk in group A and the optimal dose group by milk somatic cell detector (De Laval, Sweden), observed the clinical symptoms of the cows with mastitis every day during the experiment period, and then calculated the cure time, effective rate, and cure rate.
Criteria of PCR results
The criteria of PCR results were shown in table 2.
Regarded the average of the data in group A as the control and compared it with the optimal dose group. The data was normally distributed and distinguished the differences and correlations between groups by independent sample t-test and Person relation analysis by SPSS 19.0 (IBM SPSS statistics for Windows, version 21.0). All data was recorded as`X ± SD.
For each cow, three different investigators were involved as follows: a first investigator administered the treatment and collected samples based on the randomization table. This investigator was the only person aware of the treatment group allocation. A second investigator was responsible for the measurement of somatic cell count, inflammatory factors, and qPCR and the calculation of cure rate. Finally, a third investigator (also unaware of treatment) analyzed the above results.