Abstract:
The pathogen Clostridium difficile (C. diff.) causes severe diarrhoea in humans. Almost one of three patients suffered from C. diff. infection undergoes recurrences due to failed antibiotic treatment. The supplementary administration of a bovine whey protein concentrate enriched with anti-C. diff. IgA may improve the treatment success of antibiosis by diminishing the rate of recurrences as proven previously by v. Dissel et al. (2005). The production of bovine milk containing anti-C. diff. IgA requires the immunization of dairy cows against the virulent factors of C. diff.. The object of this doctoral thesis is to investigate the bovine immune response to repeated vaccinations against C. diff..
Nine primiparous Brown Swiss cows were vaccinated several times against the pathogen within the 18-month treatment period. Different types of vaccines, containing inactivated C. diff. and the toxoids / toxins A and B, were administered nasally (MucoCD-N) and parenterally via injection (MucoCD-I batches A, B). The treated cows were divided into low responder (LR) and high responder (HR) cows measured by the level of anti-C. diff. milk IgA. The associated threshold was set at 8.00 μg / mL anti-C. diff. milk IgA. Untreated Brown Swiss cows were used as controls. The following investigations were performed to determine treatment effects: veterinary monitoring of the treated cows' health; milk yield recording by use of the TRU-Test milk meter; testing of the milk composition by means of the Fourier-transform infrared spectroscopy for milk fats and proteins plus fluorescence flow-cytometry for somatic cell counts (SCC); total cell counting of peripheral blood leukocytes (PBL); differential cell counting (DCC) of SCC and PBL by aid of light microscopy; IgA quantification in milk and blood with the enzyme-linked immunosorbent assay (ELISA); gene expression analysis of receptors and mediators associated with the immune system in SCC and PBL with the real-time quantitative polymerase chain reaction (RT-qPCR); correlation analysis of milk IgA and the main dairy production factors or rather DCC and associated gene expression data using the statistic software, SigmaPlot 11.0; immunohistochemically examination of different lymphocytes in mammary gland (MG) and supramammary lymph node (SLN) tissues.
The health of the treated cows was impaired after injecting MucoCD-I batch A, which contained an imbalanced mixture of C. diff. toxoids / toxins A and B. As a result, milk yield and quantities of milk fats and proteins (P < 0.05) were significantly diminished in early lactation of the treated cows compared to the control. The anti-C. diff. and total IgA concentrations in HR's milk were significantly higher up to 80% related to the "before treatment control" values (BTC) and the measurement values in LR's milk at any lactation stage. Only in the late lactation, the LR's total milk IgA differed significantly from the associated BTC by roughly 47%. Total and anti-C. diff. IgA contents were affected more in milk than in blood due to the vaccinations. The total SCC of milk were untouched by the treatment. In blood, a short-term leukopenia followed by a leukocytosis were induced by application of MucoCD-I batch A. Significant changes in DCC of SCC and PBL among LR and HR were only partially treatment-related. Generally, the treatment influenced stronger the gene regulations in PBL than in SCC. The significantly different gene regulations in PBL and SCC among the treated groups were primarily triggered in response to MucoCD-I batch A. Especially the gene expressions of the chemokines / cytokines like CXCL8, IL1β, IL12β and IFNγ in PBL as well as lactoferrin and also IFNγ in SCC were significantly different between LR and HR. During the immunization period with MucoCD-I batch B, the treated groups' gene expressions were predominantly synchronized. They differed significantly from the control in case of CXCL8, IL6, IL12β, IFNγ, IL2, TLR2, CD3δ, CD4 and CD126 in PBL and for lactoferrin, TNFα, IL6, IL12β, CCL20 and CXCL3 in SCC. At no time of the treatment, the gene expressions of the epithelial IgA cell receptor PIGR were altered. The correlations among total or anti-C. diff. milk IgA and the main dairy production factors were determined as weak (r < 0.5). A significant linear dependency existed between anti-C. diff. and total milk IgA (r = 0.69). The correlation analysis of DCC and associated gene expression data revealed strong interrelationships between the SCC percentages of neutrophilic granulocytes and the related cell surface determinants C5AR1 and CXCR2 (r > 0.8) and a middle interrelationship between the SCC percentages of lymphocytes and the plasma cell marker CD126 (r = 0.75). The median population sizes of CD4+ and CD8+ T-cells as well as IgA+ antibody secreting cells in MG and SLN tissues of HR were predominantly larger than in those of LR and control.
In conclusion, the persistent production of C. diff. specific IgA in milk depends on two important factors: a potent and well-tolerated C. diff. vaccine capable to induce a primarily mucosa-related immune response, and sensitive dairy cows as recipients. In advance, HR cows might be preselectable by in vitro testing of their PBL when challenged with C. diff. antigens and followed by gene expression analysis of CXCL8, IL1β, IL12β and IFNγ to check the induction of these genes. Overall, the following assumptions were deduced from the presented results and recommended as topics of subsequent investigations:
• In response to repeated immunizations against C. diff., HR cows activated the humoral response to antigen directed by TH2-lymphocytes, whereas LR cows emphasized rather the cellular immune response controlled by TH1-lymphocytes, as indicated by the gene expression profiles of the chemokines / cytokines.
Because the PIGR gene expressions were unaffected by the immunization, the epithelial transport capacity would not be a limiting factor for the secretion of milk IgA.
The intensive homing of lymphocytes to the MG and SLN - as determined in HR's tissues - is crucial prerequisite for the persistent production of anti-C. diff. milk IgA.