Animals and study design
Five multiparous, clinically healthy Holstein–Friesian cows in late-gestation without a history of clinical mastitis in the current lactation, and with a low SCC (≤ 250 000 cells/mL milk) and without (a) major pathogen infected quarter(s) (three to five weeks before dry off were selected from the research dairy farm of Ghent University (Biocentrum Agri-Vet, Melle, Belgium). Staphylococcus aureus, Streptococcus uberis, esculin-negative cocci, Trueperella pyogenes, Escherichia coli, Klebsiella spp., and other gram-negative bacteria were regarded as major pathogens.
Four of the selected cows were challenged in each quarter with S. chromogenes strain IM (see further) at dry off (D0), with the four quarters of an additional, unchallenged cow serving as negative controls. Around 8 a.m., cows were milked one last time before dry off and moved to a straw-bedded yard. Two hours later, immediately before inoculation, the last streams of milk were removed by hand and teats were pre-foamed with a lactic acid based foam product (Oxy-foam D, Ecolab, Northwich, UK). Subsequently, the teat ends were disinfected with 70% ethanol and the inocula were administered directly into the gland cistern using a polyvinyl chloride catheter of 18 cm (Vygon, Ecouen, France). Immediately after the inoculation, all teat ends were dipped using an iodine-based barrier dip (Io-Shield, Ecolab, Northwich, UK) and some quarters received an internal teat sealant (Orbeseal®, Zoetis, NJ, USA), administered as described by the manufacturer. Briefly, the teat end was first disinfected again and the teat was pinched at the base of the udder. Subsequently, one complete syringe was injected per teat while it remained secluded from the rest of the quarter. None of the quarters received (long-acting) antibiotics at dry-off.
In total, 16 quarters from 4 different cows were challenged, of which 4 quarters from 2 different cows received 100 CFU in 5 mL sterile phosphate buffered saline solution (PBS) (Thermo Scientific, Waltham, USA). Two of those quarters were sealed and 2 were not sealed during the dry period. Eight quarters from 4 different cows (3 sealed and 5 not sealed) were challenged with 100 000 CFU in 5 mL sterile PBS. From the 4 quarters of 2 different cows that received 10 000 000 CFU in 5 mL sterile PBS, 3 were sealed and 1 was not sealed. All quarters that served as negative controls, remained unsealed.
The dry periods lasted between 47 and 59 days. Immediately after calving, the cows were moved to a separate tie-stall barn and kept there until the end of the experiment (7 days after calving). The animals were clinically examined and their rectal temperature was measured every day during the entire dry period and the first week of lactation.
Staphylococcus chromogenes IM, an udder-adapted strain isolated from a cow with a persistent IMI lasting over 11 months , was used to challenge the dry quarters. This strain has been used in several other experiments [11,12,13, 34,35,36,37, 42]. A growth curve of the strain was grafted by aerobic incubation of the bacteria in sterile brain–heart infusion broth (BHI; Oxoid, Hampshire, UK) at 37 °C as was done by others with modifications . To prepare a stock solution, the bacteria were collected during the late logarithmic growth phase and a 30% (v/v) glycerol stock was stored at −80 °C. The concentration and viability of the bacterial stock was tested by plating serial dilutions on tryptic soy agar (TSA; Oxoid, Basingstoke, UK). To prepare the inoculum, the stock solution was thawed and bacteria were grown aerobically in sterile brain–heart infusion broth at 37 °C. The overnight culture was washed 2 times with sterile PBS by centrifugation for 10 min at 3220×g (4 °C) and the pellet was resuspended and diluted with sterile PBS to obtain the desired inoculum concentration. Serial dilutions of the inoculum were plated on TSA to confirm the inoculation dose. The inoculum was transported at 4 °C to the farm immediately before inoculation at dry off.
Milk samples, dry cow secretion and colostrum samples
Two milk samples before dry off (D-35/D-21 and D-1), three dry cow secretion samples (D14, D27 and D41), one colostrum sample on the day of parturition (C) and seven milk samples after calving until the end of the first week of lactation (L1—L7) were taken aseptically in duplicate from all quarters for bacteriological examination, for quarter milk somatic cell count (qSCC) (not on L7) and for cytokine measurements (not on L7) (Figure 1). Sealed quarters remained untouched during the entire dry period.
Quarter milk somatic cell counts (qSCC) and bacteriological examination
The qSCC was determined by a DeLaval Cell Counter (DeLaval, Tumba, Sweden) on all milk and colostrum samples that were taken both before and after the dry period. The qSCC was expressed as cells/µL.
Bacteriological culturing was performed on all milk, dry cow secretion and colostrum samples that were taken before, during and after the dry period. The bacteriological results from the duplicate sample were only used if the first sample turned out to be contaminated (yielding 3 or more phenotypically different colony types) to define a quarter as positive. Still, S. chromogenes was actively screened for in both duplicate samples to increase sensitivity and to increase the likelihood of defining quarters as colonized with S. chromogenes IM.
Ten microliter of the samples was plated on an aesculin-blood and MacConkey agar (Oxoid, Hampshire, United Kingdom) with a sterile loop according to the guidelines of the National Mastitis Council . The plates were aerobically incubated at 37 °C and evaluated after 24 h. If the first sample was contaminated, results of the bacteriological culture of the second, duplicate sample were used, as beforementioned, to determine the colonization status (see further).
All phenotypically different colonies were counted (colony-forming units (CFU)/mL) and Gram staining was performed. Based on an inspection of the colony morphology with light microscopy, Bacillus spp. and Corynebacterium spp. were differentiated from Gram-positive cocci, and by using a catalase test, the latter group was divided into catalase-negative and catalase-positive bacteria. A blood aesculin test was used to classify the catalase-negative bacteria into aesculin-negative or aesculin-positive. While aesculin-negative cocci could be further identified as Streptococcus agalactiae or Streptococcus dysgalactiae using the Christie, Atkins, and Munch-Petersen test, aesculin-positive cocci were divided into Streptococcus uberis or other aesculin-positive streptococci using a bile-aesculin agar (Oxoid). As mentioned before, we wanted to optimize the likelihood of finding S. chromogenes. Therefore, we kept one colony of each phenotypically different, Gram-positive, catalase-positive coccus that was found in either one of the duplicate samples taken between D-35/D-21 and L6. Those isolates were aerobically grown on an aesculin-blood agar for 24 h and stored in Microbank vials (Pro-Lab Diagnostics, Richmond Hill, Canada) at –80 °C until further analyses with MALDI-TOF MS.
MALDI-TOF mass spectrometry and multilocus sequence typing
The phenotypically different Gram-positive, catalase-positive cocci found between D-35/D-21 and L6 that were stored at –80 °C, were identified at the species level by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS (MALDI Biotyper, Bruker Daltonics, Bremen, Germany) . A score value ≥ 2.000 was considered reliable at species level for most species although this was lowered to 1.700 for the species-level identification of NAS isolates as previously described [39, 40], and a validated and updated bovine NAS library was used . If isolates could not be reliably identified by MALDI-TOF MS, 16S rRNA sequencing was carried out .
DNA was extracted from the first and last, when present, collected S. chromogenes isolate cultured from a quarter with a commercially available kit (DNeasy Blood and Tissue kit, Qiagen, Venlo, The Netherlands). The DNA was submitted to multilocus sequence typing (MLST)  to verify whether the recovered S. chromogenes isolates belonged to the same type as the inoculated S. chromogenes IM (ST 1). Four S. chromogenes isolates with a known sequence type (one ST1, two ST 44 and one ST 18) served as controls.
Milk, colostrum and dry cow secretion was centrifuged at 16 000×g (4 °C) for 30 min. After removing the fat layer, 1 tablet cOmplete™ Mini, EDTA-free Protease Inhibitor Cocktail (Sigma-Aldrich, St. Louis, MO, USA) was added to 10 mL whey. The whey was aliquoted (500 µL) and stored at –20 °C until ELISA analyses could be performed.
The immunological response was measured by sandwich ELISA to determine IL-6, IL-10 and IFN-γ levels. All standard curve points and samples were run in duplicate and if the OD difference between the duplicates was more than 0.05 and more than 20% of the OD measurement, ELISA was repeated for this sample. The average of each duplicate measurement was used to determine the average optical density for each sample. The concentrations of cytokines in the samples were calculated by extrapolating from the respective standard curves, and the values expressed as the concentration of each cytokine in pg/mL.
The protocol for IL-10 was adapted from another study . Flat-bottom 96-well plates were coated with a capture antibody for bovine IL-10 (Bio-Rad Laboratories, Inc., California) diluted in bicarbonate coating buffer at a concentration of 5 µg/mL. The plates were sealed and incubated overnight at 4 °C before being washed three times with 200 µL wash buffer (0.05% Tween in PBS) by an autowasher (Hydroflex, Tecan, Männedorf, Switzerland). Subsequently, plates were blocked with 200 µL 3% casein from bovine milk (Sigma-Aldrich), sealed and incubated at room temperature for 1 h. A standard curve was prepared by making 1:2 dilutions of the bovine IL-10 recombinant protein (Bio-Rad Laboratories, Inc., California) with 9 ng/mL as the highest concentration. Plates were washed again and 100 µL of standards, undiluted samples and a negative control (wash buffer) were added in duplicate and sealed plates were incubated for 2 h at room temperature. The detection antibody for bovine IL-10 (Bio-Rad Laboratories, Inc., CA, USA) was diluted in wash buffer to get a concentration of 5 µg/mL. Plates were washed again as previously described and 100 µL of the detection antibody solution was added. The sealed plates were incubated in the dark for 1 h at room temperature. Plates were washed, 100 µL Streptavidin-HRP (DivBioScience, Breda, The Netherlands) was added and plates were sealed and incubated in the dark for 1 h at room temperature. After washing, 100 µL of TMB substrate (Sigma-Aldrich) was added to each well and left to incubate for 15–30 min at room temperature in the dark. Next, 100 µL stop solution (Sigma-Aldrich) was added to each well and OD was measured at 650 nm and 450 nm (background measurement) by use of an ELISA plate reader (Multiskan GO, Thermo Scientific, Waltham, MA, USA).
IFN-γ and IL-6
A commercially available kit was used to measure bovine IL-6 (DY8190, R&D Systems, Minneapolis, USA) and bovine IFN-γ (DY2300, R&D Systems, Minneapolis, USA) according to the manufacturer’s protocol with minor adaptations. Briefly, flat-bottom 96-well plates were coated with 100 µL capture antibody at a concentration of 2 µg/mL, and sealed and incubated overnight at room temperature. Plates were washed three times with 300 µL wash buffer by an autowasher and blocked with 200 µL 3% casein from bovine milk. The plates were sealed and incubated for 1 h at room temperature. A standard curve was prepared by making 1:2 dilutions of the recombinant proteins for IFN-γ and IL-6 with 5 ng/mL and 0.5 ng/mL, respectively as the highest concentration. Plates were washed and 100 µL of standards and undiluted samples was added in duplicate together with a negative control (reagent diluent). Sealed plates were left to incubate at room temperature for 2 h and washed again. One hundred microliter of detection antibody at a concentration of 0.4 µg/mL for IFN-γ and 2 µg/mL for IL-6 was added. Plates were sealed and left to incubate in the dark for 2 h at room temperature and then washed. One hundred microliter of Streptavidin-HRP was added and plates were sealed and incubated again for 30 min at room temperature in the dark. After the final wash step, 100 µL of substrate solution was added and the sealed plates were left to incubate for 15–30 min at room temperature in the dark. Finally, 50 µL stop solution was added and OD was measured at 450 nm and 540 nm (background measurement) by use of an ELISA plate reader.
As for the cytokine ELISA, all samples were run in duplicate on the same plate and if the OD difference between the duplicates was more than 0.05 and more than 20% of the OD measurement, ELISA was repeated for this sample. A background correction reading at 450 nm was subtracted from the 650 nm absorbance reading. The average of each duplicate measurement was used to determine the average optical density for each sample. If a sample was diluted, the average optical density for that sample was corrected based on the dilution that was used. The total protein concentration was measured, and the values for IgG1 and IgG2 are expressed as the average OD divided by the total protein concentration.
Total protein concentration
The concentration of solubilized protein in each sample was determined by a commercially available protein assay (Bio-Rad Laboratories, Inc., CA, USA). Ten milligram bovine serum albumin (BSA; Sigma-Aldrich) was added to 10 mL filtered, demineralized water containing 1 tablet cOmplete™ Mini, EDTA-free Protease Inhibitor Cocktail to prepare a stock solution. A standard curve with a range of 100 µg/mL to 1000 µg/mL BSA was prepared, and filtered, demineralized water with the protease inhibitor cocktail served as a negative control. The standard curve and the negative control were tested in duplicate. Samples were diluted 1:90 with filtered, demineralized water, and if the OD of the sample was higher than the OD of the standard curve, the sample was diluted 1:150, 1:200 or 1:300. The protein assay dye reagent was diluted 1:5 with filtered, demineralized water and 200 µL of the solution was added to each well of a 96-well plate. Ten microliter of each standard curve point, the negative control or diluted sample was added and the plate was incubated for 5 min at room temperature. The OD was measured at 595 nm by use of an ELISA plate reader. The total protein concentration of each sample was calculated by extrapolating from the respective standard curves, and the values expressed as the total protein concentration in µg/µL.
IgG1 and IgG2
The antibodies IgG1 and IgG2 directed against Staphylococcus chromogenes IM were measured by modification of an ELISA protocol for IgG1  and IgG2 . First, S. chromogenes IM was grown under aerobic conditions in 5 mL BHI at 37 °C. After 5 h of incubation, 50 µL of the bacterial suspension was transferred into 5 mL fresh BHI and left to aerobically incubate overnight at 37 °C. Subsequently, the overnight culture was centrifuged for 10 min at 3220×g (4 °C) and supernatant was removed. The cell pellet was resuspended in 5 mL sterile wash buffer (0.05% Tween in PBS) and 1:10 dilutions were plated on TSA agar to determine the number of bacteria (CFU/mL). The bacteria were inactivated in a warm water bath for 1 h at 60 °C and frozen at –20 °C until further use. A serial dilution of the bacterial suspension was plated on TSA to confirm the heat inactivation.
The heat-inactivated S. chromogenes IM suspension was diluted in bicarbonate coating buffer to reach a concentration corresponding to 107 CFU/mL before inactivation to coat flat-bottom 96-well plates with 100 µL of bacterial suspension. The plates were sealed and left to incubate overnight at 4 °C before they were washed 3 times with 200 µL wash buffer with an autowasher. Subsequently, plates were blocked with 200 µL 3% casein from bovine milk, sealed and incubated for 2 h at room temperature. In the meantime, colostrum samples were diluted 1:10 for both IgG1 and IgG2 analyses and milk samples were used undiluted. Dry period secretion was used undiluted for IgG2 and diluted 1:100 for IgG1 ELISA. The plates were washed again and 100 µL of each (diluted) sample was added in duplicate. One hundred microliter fetal bovine serum (FBS; Life Technologies Corp., Rockville, MD, USA) served as a negative control. Sealed plates were incubated for 1 h at room temperature. Plates were washed again and 100 µL of detection antibody for IgG1 or IgG2, with a concentration of 40 ng/mL or 1 µg/mL respectively, was added to the plates. The plates were sealed and incubated for 1 h at room temperature in the dark and subsequently washed again. One hundred microliter of TMB substrate was added and the sealed plates were incubated for 15–30 min at room temperature in the dark. Next, 100 µL stop solution was added to each well and OD was measured at 650 nm and 450 nm (background measurement) by use of an ELISA plate reader.
All data were entered in an electronic spreadsheet program (Excel 2016, Microsoft Corp., Redmond, WA, USA) and checked for unlikely values.
Bacterial shedding of S. chromogenes IM (CFU/mL), IL-6 (pg/mL), IFN-γ (pg/mL), IL-10 (pg/mL), IgG1 [OD/total protein (µg/µL)], IgG2 [OD/total protein (µg/µL)] and IgG2/IgG1 were available as outcome variables from the day before inoculation, the dry period and the first week of lactation, complemented with qSCC (cells/mL) in early lactation.
In order to obtain a normal distribution, a natural logarithmic transformation was performed for the qSCC (Ln qSCC) and a log10-transformation was performed for the shedding of S. chromogenes IM (log10 CFU/mL; all values were increased by + 1 before a log10-transformation as S. chromogenes was not present in all samples) and IFN-γ (log10 IFN-γ). Because the conventional ways of transformation (e.g. log10, ln, inverse, square root, quadratic) were not sufficient to obtain normally distributed variables for IL-6, IL-10, IgG1, IgG2, IgG2/IgG1, a 2-step approach was applied . The first step involved transforming the variables into a percentile rank, which resulted in uniformly distributed probabilities. The second step applied the inverse-normal transformation to the results of the first step to form a variable consisting of normally distributed z-scores. In this 2-step approach, the mean and standard deviation of the original variables were retained, facilitating the interpretation of the results.
All linear mixed models contained cow as random effect to correct for the clustering of quarters within a cow and quarter as repeated effect to account for the clustering of the repeated measurements within quarters (Dry period: 4 sampling times; Lactation: 7 sampling times) and were fitted using PROC MIXED in SAS 9.4 (SAS Institute Inc., Cary, NC, USA). In all linear mixed regression models, a first-order autoregressive correlation structure was used to account for the clustering of the repeated sampling times within a quarter. The goodness-of-fit measures included −2 × log-likelihood, Akaike information criterion, and Bayesian information criterion. The conditional Pearson residuals were evaluated graphically and plotted against the normal values and predicted values to check whether the assumptions of normality and homogeneity had been fulfilled, respectively. Also, plots of standardized residuals versus the dependent variables were generated to check whether the assumption of linearity had been fulfilled. No problems were detected. Significance was assessed at P ≤ 0.05.
Effect of inoculation status
A quarter was considered to be colonized with S. chromogenes IM if this strain was recovered from that particular quarter at least once during the entire dry period or the first week of lactation. The association between the S. chromogenes IM inoculation status (categorical predictor variable of main interest with 3 levels: non-inoculated, inoculated and non-colonized, inoculated and colonized) and the transformed outcome variables were determined fitting 15 separate linear mixed regression models, using the values for the dry period (4 sampling times; before dry off: D-1; dry period secretion: D14, D28, and D41; no Ln qSCC values) and early lactation (7 sampling times; colostrum: C; milk samples: L1, L2, L3, L4, L5, and L6) separately. Beside inoculation status as categorical predictor variable of main interest, the models also included time of sampling (4 and 7 sampling times, respectively for the dry period and early lactation). In all models, the interaction term between time of sampling and inoculation status was included.
Effect of inoculation dose
The association between the inoculation dose (categorical variable of main interest with 4 levels; 0 CFU (control), 100 CFU, 100 000 CFU, and 10 000 000 CFU) and S. chromogenes IM shedding during the dry period (4 samplings, D-1, D14, D27, and D41) and early lactation (7 sampling times, C, L1, L2, L3, L4, L5, L6) separately, was determined fitting two linear mixed regression models with log10 CFU/mL S. chromogenes IM during dry period and log10 CFU/mL S. chromogenes IM in early lactation, respectively, as continuous outcome variables. In both models, the interaction term between time of sampling and inoculation dose was included.