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Evaluation of the immunological status in clinically healthy and subclinically infected bovine mammary glands

dc.contributor.advisorCzerny, Claus-Peter Prof. Dr.
dc.contributor.authorSchwarz, Danielde
dc.titleEvaluation of the immunological status in clinically healthy and subclinically infected bovine mammary glandsde
dc.contributor.refereeCzerny, Claus-Peter Prof. Dr.
dc.description.abstractengMastitis is a major disease affecting dairy cattle worldwide. Especially the subclinical form of the disease causes high economic losses. The evaluation of udder health is based on somatic cell count (SCC) and bacteriological examinations. A SCC threshold of 100,000 cells/mL in quarter foremilk samples is used to differentiate between healthy and diseased mammary glands. However, the overall goal of this thesis was the analysis of the immunological status of clinically healthy and subclinically infected bovine mammary glands to contribute to a more detailed understanding of subclinical mastitis.Initially, the results of 615,187 quarter foremilk samples taken between 2000 and 2008 in Hesse, Germany, were analyzed. The data indicated SCC >100,000 cells/mL in 38% and ≤100,000 cells/mL in 62% of all samples; 31% revealed SCC ≤25,000 cells/mL. Mastitis pathogens were detected in 83% of samples with SCC >100,000 cells/mL and in 8.5% of samples in the SCC range from 1,000 to 100,000 cells/mL. Based on these results, inflammatory processes were already suspected in udder quarters with SCC ≤100,000 cells/mL. We argued that such inflammation can be detected by examination of the relationship of immune cells in milk. Hence, in a second and a third study 20 cows, respectively, were selected and differential cell count (DCC) patterns of apparently healthy (SCC ≤100,000 cells/mL) and diseased quarters (>100,000 cells/mL) were compared. While in the second study 100 milk cells of each quarter were differentiated into lymphocytes, macrophages and polymorphonuclear neutrophilic leukocytes (PMNL) using light microscopy, a flow cytometric method was applied for differentiation of 5,000 cells in the third study. In both studies almost all DCC patterns of quarter foremilk samples taken from apparently healthy mammary glands were dominated by lymphocytes. Interestingly, microscopic DCC patterns of three milk samples with 43,000 to 45,000 cells/mL and flow cytometric DCC patterns of six samples with SCC values from 9,000 to 46,000 cells/mL indicated already inflammatory reactions based on the predominance of PMNL. Both studies revealed PMNL as dominant cell population with proportions of ≥49% in milk samples drawn from diseased quarters. In both studies, almost all samples tested revealed macrophages as second predominant cell population in relationship to lymphocytes and PMNL. Further analysis of the flow cytometric data demonstrated significant differences of the cellular components between quarters infected by major or minor pathogens and culture-negative quarters. In the second and third study, inflammatory reactions in milk of udder quarters classified as healthy according to SCC ≤100,000 cells/mL were detected based on DCC.The aim of a fourth study was to identify cytological parameters for differentiation between healthy and diseased udder quarters. Quarter foremilk samples were collected from 48 cows and bacteriological status, SCC, and microscopic DCC were determined. Mean lymphocyte percentage was significantly higher in group N (normal secretion, n = 96 samples) than in the three groups of diseased quarters (LM, latent mastitis, n = 15 samples; UM, unspecific mastitis, n = 47 samples; M, mastitis, n = 30 samples). Mean PMNL percentage was significantly lower in group N than in groups UM and M, but not LM. Macrophages did not vary significantly between by the four groups. The mean value of phagocytes (macrophages and PMNL) was significantly lower in group N than in the other groups. Both logarithmic (log) PMNL:Lym and log phagocyte:lymphocyte ratios were significantly lower in group N than in groups LM, UM, and M. However, calculating Fisher values for all variables, the log PMNL:lymphocyte ratio revealed the highest value. A subsequent study concentrated on establishment and verification of cutoff values differentiating between healthy and diseased quarters applying the log PMNL:lymphocyte ratio. Initially, quarter milk and blood samples were taken from eight cows for five consecutive days to investigate short-term repeatability of DCC in milk of healthy mammary glands. While SCC and bacteriological status were determined only for milk samples, DCC was analyzed in all blood and milk samples using flow cytometry. Neither milk nor blood DCC patterns were significantly influenced by sampling day, parity, lactation stage, or quarter position suggesting that DCC can be reliably applied to evaluate udder health. A cutoff value of 0.495 for log PMNL:lymphocyte was established. For the second part of the study 16 animals were randomly selected in a different herd and quarter milk samples were taken on three consecutive days. When the cutoff value was applied to the data along with SCC, high sensitivity and good specificity of 97.3% and 92.3%, respectively, were found under field conditions.Since all DCC studies revealed lymphocytes as predominant cell population in milk of healthy udder quarters, in a sixth study quantitative relationships of CD2+ T and CD21+ B lymphocytes were investigated using flow cytometry. Percentages of CD2+ T cells were significantly higher in apparently healthy quarters (SCC ≤100,000 cells/mL, n = 65) than in diseased quarters (n = 15), whereas the behaviour of CD21+ B cells was the contrary. As a result of this antidromic effect, a new variable – the CD2/CD21 index – was defined. While diseased quarters indicated values <10, the CD2/CD21 index was >10 in apparently healthy quarters. To test whether CD2/CD21 indices <10 are primarily related to major pathogens, in a second part of this study quarters with SCC ≤100,000 cells/mL (n = 31) and >100,000 cells/mL (n = 32) – either culture-negative or containing minor or major pathogens – were selectively examined. Interestingly, CD2/CD21 indices <10 were found in quarters showing SCC ≤100,000 cells/mL and minor or major pathogens at the time of the current or previous sampling. The results of our examinations indicated a clear connection between the CD2/CD21 index and the bacteriological status. A CD2/CD21 index of 10 may be suitable to aid differentiation between unsuspicious and suspicious or diseased udder quarters.The aim of a seventh study was to explore DCC patterns of host-microbial interactions for improvement of disease diagnosis. Data collected from six bovine, two human, and one avian study with viral, parasite, or bacterial agents were analyzed. In all studies, no classic data structure (e.g., percentage of an individual cell population) discriminated disease-positive (D+) from disease-negative (D–) samples without overlapping. In contrast, advanced data analysis, like the 3D approach, distinguished three (steady, positive, and negative) feedback phases, in which D– data characterized the steady phase, and D+ data were found in the positive and negative phases. Due to the advanced data analysis methods, in the seventh study host-microbial interactions could be assessed, which might be helpful for earlier diagnosis, differentiation of D+ classes, and lower rates of false-negatives.In conclusion, this thesis indicated immunological processes and inflam-matory reactions appearing already in the SCC range of apparently healthy mammary glands. In addition, new concepts for data analysis and potential new tools for the diagnosis of subclinical mastitis were
dc.contributor.coRefereeKönig, Sven Prof.
dc.contributor.thirdRefereeHessel, Engel Prof.
dc.contributor.thirdRefereeUsleber, Ewald Prof. Dr.
dc.subject.engsubclinical mastitisde
dc.subject.engudder healthde
dc.subject.engflow cytometryde
dc.subject.engsomatic cell countde
dc.subject.engdifferential cell countde
dc.subject.engmilk productionde
dc.affiliation.instituteFakultät für Agrarwissenschaftende
dc.subject.gokfullLand- und Forstwirtschaft (PPN621302791)de

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