Animals
The objective of the study was to establish an in vivo infection model to study the impact of genetic selection for mastitis susceptibility under defined conditions in dairy uniparous cows.
The experiment included 36 Holstein Friesian uniparous cows that were genetically selected for favorable (Q) and unfavorable (q) paternal chromosome-18-haplotypes for somatic cell count (SCC), as previously described [37-39]. The SCC served as a surrogate trait for mastitis susceptibility; therefore, low SCC was assumed to represent low mastitis susceptibility (Q), and high SCC was assumed to represent high mastitis susceptibility (q). The trial was conducted between January and September 2016 under the approval of the Lower Saxony Federal State Office for Consumer Protection and Food Safety (reference number 33.12-42502-04-15/2024; approval date: December 15th, 2015).
All uniparous cows were purchased from conventional private dairy farms across Germany and housed in individual pens at the Clinic for Cattle, University of Veterinary Medicine, Hannover, from at least four weeks before the calculated calving date until sacrifice after the intramammary challenge. The uniparous cows received constant veterinary care, including daily general examination, measurement of rectal temperature twice daily, calving management and treatments according to standard veterinary practice in case of diseases. After calving, detailed udder health monitoring was performed on a weekly basis, including udder palpation, macroscopic evaluation of milk secretion, cow-side California Mastitis Test (CMT) and quarter milk sampling for milk ingredients, SCC and microbiological examination.
Pens were cleaned twice daily and provided with fresh straw. The animals received a performance-adjusted component ration (dry-off, transition period, lactation period; concentrate adjusted daily according to milk yield). Water was given ad libitum. On days 1-6 postpartum (p.p.) all uniparous cows received 2,500 mg enrofloxacin (Enrotron®100) per day to create comparable conditions between the two groups with regard to antibiotic treatment. The overall aim of this systematic antibiotic treatment within the experimental setup was to strictly synchronize of the two haplotypes for maximal standardization of the experimental model including prevention of natural IMI before the start of the experiment. In case of disease, the treatment was prolonged.
Criteria for exclusion of animals from the experiment to create reliable and precise data and for ethical reasons were defined. Intramammary challenge was not conducted if (1) major pathogens have been detected in quarter milk samples at the last sampling before the start of the experiment, (2) treatment of occurring diseases had not been finished within one week before the start of the experiment, (3) withdrawal periods of applied pharmaceuticals had not expired at the start of the experiment or if (4) the respective animal showed clinical signs of a systemic disease before the start of the intramammary challenge. Furthermore, rectal temperature > 42.5°C, general depression and recumbency, as well as gangrenous mastitis, were criteria for discontinuing the experiment.
Intramammary infection model
The intramammary challenge experiment started on day 36 ± 3 p.p. The order in which the animals entered the experimental setup depended on the individual calving date. The uniparous cows were challenged intracisternally with either S. aureus (Q n = 12 vs. q n = 12) or E. coli (Q n = 6 vs. q n = 6). The time point before administration of the bacteria was defined as 0 h.
Intramammary S. aureus challenge was applied to the left and right hindquarters after cleaning and disinfection of the teats with 70 % ethanol. The inoculum stock was diluted with sterile pyrogen-free 0.9 % saline solution to a challenge dose of 10,000 CFU S. aureus1027 /2 ml. The inoculum was instilled into the teat canal with a sterile syringe and a teat cannula. Afterwards, the teat canal was kept close with two fingers, and the udder was massaged for 30 seconds to ensure distribution of the respective pathogen. The front left quarter received 2 ml sterile pyrogen-free 0.9 % saline solution, and the front right quarter remained untouched. Both front quarters served as control quarters. This protocol was followed for all uniparous cows within the S. aureus group with one exception: one cow had suffered from CM in the left hind quarter in the postpartum period, which had been treated and cured before the challenge, but to avoid an influence on the local intramammary reactivity, the pathogen was applied into the front right instead of the hind left quarter. The infection trial lasted 96 h after intramammary challenge with S. aureus.
Intramammary E. coli challenge (500 CFU E. coli1303 / 2 ml) was applied into the hind right quarter. The front right and front left quarter were left untreated, and the hind left quarter received 2 ml sterile pyrogen-free 0.9 % saline solution. The inoculum was instilled in the same manner as in the S. aureus group. The infection trial lasted 24 h after intramammary challenge with E. coli. This protocol was followed for all uniparous cows within the E. coli group with one exception: due to stenosis in the hind right and front left udder quarters, the pathogen was applied into the hind left quarter of this cow.
At the end of the experiment, the uniparous cows were killed with a captive bolt gun and exsanguination immediately followed by necropsy and tissue sampling for further investigations (Figure 1).
Pathogens for intramammary challenge
The applied strains of S. aureus1027 and E. coli1303 are field isolates from cases of subclinical and clinical mastitis, respectively. Genomic and proteomic characteristics of S. aureus1027, including common virulence markers and virulence gene expression, have been examined [47], and the genome sequence of E. coli1303 has been published [48]. The strains were stocked in a cryobank system at -80°C. To create a stock solution for comparable intramammary challenge doses, E. coli was cultured on Violet-Red-Bile-Agar (VRB), and S. aureus was cultured on Columbia-Sheep-Blood-Agar (CSB) and incubated (24 h, 37°C). Afterwards, one colony per bacterial strain was applied into a tube with 10 ml brain heart infusion broth (BHI) and subsequently incubated (6 h, 37°C). Of this solution, 100 µl was applied into 9.9 ml tryptic soy broth (TSB). After 18 h of incubation, the inoculum was prepared to perform serial dilutions. These serial dilutions were plated on VRB (E. coli) or CSB (S. aureus) and incubated for 24 h to determine the counts of colony forming units per ml (CFU/ml) in the inoculum. The prepared inoculum was aliquoted and stored at ‑80°C.
Monitoring and sampling
To monitor local and systemic signs of mastitis, all uniparous cows were examined immediately before the challenge (0 h) and every 12 h after challenge with regards to their general health and signs of inflammation in the udder and milk secretion. To evaluate the general health status, such parameters as heart rate, respiratory frequency, rectal body temperature, feed intake, rumen activity and content were recorded. Additionally, the inner body temperature was measured over the entire experimental challenge every three minutes via a temperature logger (HOBO U12 Stainless Temperature Data Logger, Onset Computer Corporation, Bourne; USA) attached to an intravaginal plastic device (EAZI-BEED CIDR-blank, Zoetis, USA) containing no progesterone. Due to data loss because of rejection of the intravaginal device in one animal, statistical analysis of the inner body temperature within the E. coli group could only be performed for n = 11 uniparous cows (Q: n = 6, q n = 5).
For assessing udder health before and during the challenge, all udder quarters were examined for signs of inflammation such as swelling, redness, pain or increased udder surface temperature as well as for the evaluation of milk secretion according to Table 1 and Table 2. Sterile quarter milk sampling was performed for bacterial examination (including colony counting) every 12 h before milking of the uniparous cows. These samples were stored on ice until further processing in the laboratory (see below). The milking procedure was conducted with a special quarter milker (WestafliaSurge, Bönen, Germany) to determine the exact amount of milk per quarter and to take quarter milk samples for the determination of SCC, pH and milk contents. Respective milk samples were preserved with bronopol for further analysis at Milchwirtschaftlicher Kontrollverband Mittelweser e.V. (Rehburg-Loccum, Germany) using the MilkoScan FT Plus (FOSS, Hilleroed, Denmark).
Bacterial recovery from milk
Bacterial recovery was assessed via sterile sampling of quarter milk samples. Each sample was streaked onto three different agar plates (CSB, VRB, Edwards-Agar) and incubated at 38°C. The plates were checked for bacterial growth after 24 h and 48 h. Growing bacteria were identified via colony morphology and growth patterns. Additionally, these quarter milk samples were stored at -20°C until the end of the experiment. For analysis, the samples were defrosted and prepared to perform serial dilutions according to Petzl et al. (2016) [49]. Therefore, the quarter milk samples were diluted with 0.9 % sterile saline solution. Three degrees of dilution were plated on CSB agar in the case of S. aureus challenge and on VRB agar in the case of E. coli -challenge. For each degree of dilution, one triplicate was used. The agar plates were incubated for 24 h at 38°C, and the CFUs were determined by manual colony counting. Evaluable plates contained a minimum of two and a maximum of 300 colonies. Calculation of the colony forming units (CFU/ml) was performed according to Farmiloe et al. (1954) [50]. The value obtained after calculation was multiplied by the factor 10, as only 100 µl were incubated in each degree of dilution.
Statistical Analysis
Data were managed using Microsoft Excel and Access (Microsoft, Redmond, WA, USA). Statistical analyses were performed with GraphPad PRISM 5.04. Power calculation for sample size determination was performed based on results from previously performed IMI models which had indicated higher variability of target parameters during IMI [40, 45]. Two-sample t test power calculation with the target variable SCC in quarter milk samples resulted in the necessity of n=12 per S. aureus-group and n=6 per E. coli-group. Data were tested for normal distribution via the Shapiro-Wilk test. In the case of normally distributed data, the results are presented as the mean ± standard error of the mean (SEM), in the case of non-normally distributed data or if individuals per subgroup were less than n = 7, the results are presented as the median ± interquartile range (IQR). Normally distributed data were compared via unpaired t-tests. If data were not normally distributed or if individuals per subgroup were less than n = 7, the nonparametric Mann-Whitney test was applied. Accordingly, all analyses concerning data originating from the E. coli group were performed using the nonparametric Mann-Whitney test, as maximum n = 6 individuals were counted in each group. As described above, uniparous cows in the S. aureus group received the pathogen in two udder quarters. Concerning udder secretion and palpatory score as well as SCC and CFU, a mean value was calculated per uniparous cow and used for the statistical analysis and data illustration. Calculated differences with P < 0.05 were regarded as significant.