Publikationsdatenbank

Studies of the development of endometritis in cattle on a cellular basis (2017)

Art

Hochschulschrift

Autor

Ibrahim, Mohammad Abdelwahab Ali (WE 3)

Quelle

Berlin: Mensch und Buch Verlag, 2017 — IV, 104 Seiten

Sprache

Englisch

Verweise

URL (Volltext): https://refubium.fu-berlin.de/handle/fub188/10123

Kontakt

Institut für Veterinär-Biochemie

Oertzenweg 19 b
14163 Berlin
+49 30 838 62225
biochemie@vetmed.fu-berlin.de

Abstract / Zusammenfassung

Nearly half of dairy cows develop uterine diseases in the first weeks after parturition, the most common manifestation being endometritis. Such diseases profoundly affect reproductive performance and reduce the profit potential of dairy farms, with an associated yearly cost of €1.4 billion within the European Union. Bacteria that enter into the uterus after calving trigger an innate immune response that results in the release of cytokines and antimicrobial peptides (AMP). There are different classes of AMP, which exert their antimicrobial action through different mechanisms. The beta-defensin (DEFB) family consists of cationic AMP that can permeabilize bacterial membranes. This family includes DEFB1, DEFB4A, and DEFB5, lingual AMP (LAP), and tracheal AMP (TAP). The bactericidal/permeability-increasing protein is also a cationic AMP that bind to bacterial lipopolysaccharides (LPS) that eventually results in the death of bacteria. Another AMP family is the S100 calcium-binding protein (S100A) family including the following members: S100A8, S100A9, S100A11, and S100A12. These AMP exert their antimicrobial action through chelation of several ions. The host immune response and release of AMP is essential for the removal of invading bacteria. A variety of bacterial strains have been discovered in the uterus so far. These include Escherichia coli and Trueperella pyogenes, which are the most important bacterial species associated with endometritis. The aim of this research project was to better understand the innate immune response in the context of postpartum uterine diseases. In the current project, the mRNA expression pattern of selected AMP (DEFB1, DEFB4A, DEFB5, LAP, TAP, BPI, S100A8, S100A9, S100A11, and S100A12) was evaluated in bovine endometrial cells collected (1) at different stages of the oestrous cycle; (2) during the puerperium depending on uterine health status (healthy, subclinical, or clinical endometritis) starting on Day 24 to 30 postpartum for 3 weeks on a weekly basis; and (3) in cell cultures in presence of Bacillus pumilus at three different multiplicities of infection (MOI 1, 5, and 10) up to 6 hours. Furthermore, to gain insights into bacterial factors contributing to the host-pathogen interactions, two strains of T. pyogenes were included in this study: one strain (TP2) was isolated from the uterus of a postpartum dairy cow developing CE and a second strain (TP5) was isolated from a uterus of a healthy cow. The two strains were compared in terms of their metabolic fingerprints, growth rate, virulence gene transcription, and effect on bovine endometrial epithelial cells in vitro. Finally, the effect of the presence of immune cells on the response of endometrial cells to T. pyogenes was evaluated. The endometrium samples were either collected at the abattoir (oestrous cycle) or from postpartum cows on a dairy farm using the cytobrush technique. For in vitro experiments, endometrial epithelial cells isolated from different animals were co-cultured with live B. pumilus at MOI of 1, 5, and 10, or T. pyogenes (TP2 or TP5) in form of live, heat-inactivated (HI) or bacteria-free filtrate (BFF) at a MOI of 1, or live TP2 and/or PBMC at a ratio of 1:1 to epithelial cells. Total RNA was isolated from endometrial cells and subjected to a reverse transcription-polymerase chain reaction for quantification of the mRNA expression of selected AMP or pro-inflammatory mediators. The results showed that the mRNA expression of all candidate AMP, except DEFB1, S100A8, and S100A9, was oestrous cycle-dependent. Higher mRNA expression was observed around ovulation compared with luteal phases. The mRNA expression of almost all candidate AMP was uterine health status-dependent, with higher mRNA expression in inflamed than in healthy endometrium, especially during the late stage of the puerperium (week 5–7). In cell culture experiments, the presence of B. pumilus had no significant effect on mRNA expression of the DEFB family but higher mRNA expression of S100A8 and S100A9 was observed in the presence of B. pumilus. TP2 grew at a faster rate, expressed more virulence factors such as collagen-binding protein (cbpA), neuraminidase H cell wall-bound protein (nanH), and fimbrial protein subunit E and G (fimE and fimG), and elicited a higher mRNA expression of pro-inflammatory factors such as prostaglandin-endoperoxide synthase 2 (PTGS2), chemokines (C-X-C motif) ligands 3 (CXCL3), and interleukin (IL) 8 in bovine endometrial epithelial cells compared with TP5. The presence of PBMC amplified the mRNA expression of pro-inflammatory factors (PTGS2, CXCL3, IL1A, IL6, and IL8) in bovine endometrial epithelial cells co-cultured with live TP2 compared with untreated cells, especially as early as after 4 h. In summary, it can be assumed that AMP are important components of the innate immune system in the uterus that create an environment that is free of pathogenic bacteria. Higher mRNA expression of the candidate AMP around ovulation or in inflamed endometrium during the puerperium suggests their crucial role in uterine innate immunity to defend against invading pathogenic bacteria. TP2 has distinct differences in the mRNA expression of certain virulence factors, a distinct metabolic profile, and different growth characteristics compared with TP5. Therefore, the strain of T. pyogenes could be a crucial factor for the development of endometritis in dairy cows after parturition. Moreover, the pro-inflammatory response of endometrial cells was amplified in the presence of immune cells. Therefore, communication between endometrial cells and immune cells might be important for bacterial clearance.