Cow/ herd Selection and Sampling
Two commercial free-stall Holstein dairy herds with high bulk milk count of S. aureus (ranged from 50 to 60 CFU/mL), milking 800 (herd 1) and 300 (herd 2) cows were selected for this study. Daily milk yield were 38 and 40 L respectively. Cows eligible for study had to be in fine health with functional quarters free of clinical mastitis and notable teat lesions without receiving antimicrobial or anti-inflammatory agents within the last two weeks. The cows were dried off by gradual cessation of milking at 60±3 days prior to expected date of calving. Three foremilk samples from each quarter aseptically collected one week before and at the last milking of drying off and immediately transferred to the laboratory in a styrofoam box containing ice. Following the last milking, all quarters were disinfected and infused with Kanaclox DC® (Pars Dopharma, Iran); Kanamycin acid sulfate 100000 IU, Cloxacillin Benzathine 500 mg, Penicillin G Procaine 300000 IU and Oilbase: 10 gr). Post-calving milk samples were taken at 3 and 7 DIM.
Study Design
Following DCT cows were randomly allocated to one of the three groups (TYLO, MARB and CONT) via block randomization. Cows in TYLO received once daily SC injection of 10 mg/kg mg tylosin (Tyloject® 20%, Razak Laboratories Co, Iran) for three days, at 21±3 days prior to calving and cows in MARB received single SC injection of 8 mg/kg SC marbofloxacin (Marbox®, Ceva Santé Animale BV, Netherlands) at 21±3 days prior to calving while cows assigned to CONT remained untreated (Figure1).
Milk Sample Analysis
Somatic Cell Counts. Composite milk samples from last month before dry off were counted by electric counter (Fossomatic Milk Analysis, Foss Electric, Hillerød, Denmark) and linear score (LS) was calculated subsequently.
Laboratory Bacteriological Culture. Culture techniques and identification of bacteria were performed according to instructions from National Mastitis Council (45). Diagnosis of various pathogens was made based on morphology, growth on selective and differential media, Gram staining, hemolysis, catalase, MSA, hippurate and CAMP. A sample was considered contaminated if more than 3 bacterial species were present on a plate.
Identification of S.aureus Isolates. The isolates were submitted to VP (Voges Proskauer) and coagulase tests. The quarters considered to be infected if >100 CFU/ml of S. aureus isolated from each cultures of any one milk sample taken at dry off and post-calving. To confirm the identification of isolates as S. aureus, the gene was amplified by nucA via conventional PCR method.
Antimicrobial Susceptibility Test. The Agar disk diffusion method (ADD) was used to determine the susceptibility of S. aureus isolates. Procedures were based on the Clinical and Laboratory Standards Institute (CLSI) guidelines (46). S. aureus ATCC 29213 was used as the reference strain for quality control.
nucA PCR. DNA extraction was performed by rapid boiling according to method by Abdelhai et al.(47). Primers were selected on the basis of the published nucleotide sequence of the 613-bp nuc gene (F- CTG GCA TAT GTA TGG CAA TTG TT, R- TAT TGA CCT GAA TCA GCG TTG TCT).
RAPD-PCR. In quarters from which S. aureus was isolated from both dry off and post calving, the genomic variability of the S. aureus isolations was analyzed by RAPD-PCR method based on two primer AP4(5´ TCACGCTGCA 3´) and T3 (5´ATTAACCCTCACTAAAGG3´). The amplification conditions for AP4 primer performed based on study by Morandi et al. (48). Amplification cycle of the T3 primer has been described by Pinto et al. (49). Thermal cycler (BIO RAD®, USA).
Amplification products were electrophoresed on a 1.8% agarose gel (Bio-Rad, California, USA) in TAE buffer. Gels were stained with ethidium bromide and photographed under UV light. PCR fingerprint images were analyzed estimated band sizes using Gel Compar®II (Applied Maths, Inc, Austin, USA) software. Strains were clustered and displayed in dendrogram form with consideration of 60% genetic similarity.
Biochemical Analysis
Total Antioxidant Capacity (TAC). Antioxidant capacity of skim milk samples was assessed by the method of FRAP(50). The reduction of Fe3+-TPTZ to Fe2+-TPTZ was followed by the absorbance increase at 595 nm via spectrophometer (2150-UV, UNICO®, USA).
Total Oxidant Capacity (TOC). Oxidant capacity of skim milk samples was analyzed by measuring DTNB based on the method applied by (43). The decrease in absorbance at 412 nm was used to calculate the rate of oxidation of TNB to DTNB. The parameters have measured in each sample three times and coefficient of variation (CV) was calculated by the formula (SD/mean×100). Within-run CV were <1.0% at all values tested. Between-series imprecision and bias have been evaluated as well.
The investigator of the tests was blinded to group allocation.
Statistical Analysis
In general, the statistical analysis was performed using SPSS software (version 21.0; SPSS Inc., Chicago, USA). The sample size calculated based on confidence level of 95% and power of 90%. Chi-square analysis was used to investigate the differences in cure, new infection and clinical mastitis rates among treatment groups. Logistic regression was carried out to determine the effect of treatments on the probability of cure, new infection and clinical mastitis controlling for parity, milk yield, position of quarter and oxidant/antioxidant balance at dry period. Kaplan-Meier survival curves were plotted to show survival distribution function of quarters with clinical mastitis for each level of treatments. Mann-Whitney U test was used for comparison of DTNB and FRAP between cured and uncured and new infected and healthy quarters. All statistical differences were assessed at the two-sided 0.05 level of significance.
Definition of Dependent Variables
Developing New Infection Between Dry off and 7±3 DIM. A new IMI was defined as the presence of pathogen at 3 or 7 DIM in a quarter that was not previously infected at dry off, or the presence of a different pathogen from those present at dry off (51). In cases of mixed infections, a quarter was counted only once as having a new IMI, even if 2 new bacterial pathogens were cultured. In the cases which RAPD-PCR revealed an S. aureus isolate at calving belonged to different strain than S. aureus isolate at dry off, they were considered to new IMI.
Developing Clinical Mastitis. A clinical mastitis occurrence was defined as the appearance of obvious abnormal milk (with/without abnormal mammary gland or systemic signs of illness). A quarter developing one or more episodes of clinical mastitis between dry off and 30±3 DIM was classified as a positive clinical mastitis case.
Experiencing Cure. Cure was defined as the elimination of one or more pathogens at both 3 and 7 DIM that previously had been existed at dry off. The RAPD-PCR results were considered for the calculation of cure rate regarding the quarters which S. aureus isolated from both dry off and post-calving.