Clinical and subclinical endometritis are known to affect the fertility of dairy cows by inducing uterine inflammation. We hypothesized that clinical or subclinical endometritis could affect the fertility of cows by disturbing the molecular milieu of the uterine environment. Here we aimed to investigate the endometrial molecular signatures and pathways affected by clinical and subclinical endometritis. For this, Holstein Frisian cows at 42–60 days postpartum were classified as healthy (HE), subclinical endometritis (SE) or clinical endometritis (CE) based on veterinary clinical examination of the animals and histological evaluation the corresponding endometrial biopsies. Endometrial transcriptome and miRNome profile changes and associated molecular pathways induced by subclinical or clinical endometritis were then investigated using GeneChip® Bovine Genome Array and Exiqon microRNA PCR Human Panel arrays, respectively. The results were further validated in vitro using endometrial stromal and epithelial cells challenged with subclinical and clinical doses of lipopolysaccharide (LPS). Transcriptome profile analysis revealed altered expression level of 203 genes in CE compared to HE animals. Of these, 92 genes including PTHLH, INHBA, DAPL1 and SERPINA1 were significantly upregulated, whereas the expression level of 111 genes including MAOB, CXCR4, HSD11B and, BOLA, were significantly downregulated in CE compared to the HE animal group. However, in SE group, the expression patterns of only 28 genes were found to be significantly altered, of which 26 genes including PTHLH, INHBA, DAPL1, MAOB, CXCR4 and TGIF1 were common to the CE group. Gene annotation analysis indicated the immune system processes; G-protein coupled receptor signaling pathway and chemotaxis to be among the affected functions in endometritis animal groups. In addition, miRNA expression analysis indicated the dysregulation of 35 miRNAs including miR-608, miR-526b* and miR-1265 in CE animals and 102 miRNAs including let-7 family (let-7a, let-7c, let-7d, let-7d*, let-7e, let-7f, let-7i) in SE animals. Interestingly, 14 miRNAs including let-7e, miR-92b, miR-337-3p, let-7f and miR-145 were affected in both SE and CE animal groups. Further in vitro analysis of selected differentially expressed genes and miRNAs in endometrial stroma and epithelial cells challenged with SE and CE doses of LPS showed similar results to that of the array data generated using samples collected from SE and CE animals. The results of this study unraveled endometrial transcriptome and miRNome profile alterations in cows affected by subclinical or clinical endometritis which may have a significant effect on the uterine homeostasis and uterine receptivity.

Background: The equine periodontium provides tooth support and lifelong tooth eruption on a remarkable scale. These functions require continuous tissue remodeling. It is assumed that multipotent mesenchymal stromal cells (MSC) reside in the periodontal ligament (PDL) and play a crucial role in regulating physiological periodontal tissue regeneration. The aim of this study was to isolate and characterize equine periodontal MSC.Tissue samples were obtained from four healthy horses. Primary cell populations were har-vested and cultured from the gingiva, from three horizontal levels of the PDL (apical, midtooth and subgingival) and for comparison purposes from the subcutis (masseteric region). Colony-forming cells were grown on uncoated culture dishes and typicalin vitrocharacteristics of non-human MSC, i.e. self-renewal capacity, population doubling time, expression of stemness markers and trilineage differentiation were analyzed.Results: Colony-forming cell populations from all locations showed expression of the stemness markers CD90 and CD105. In vitro self-renewal capacity was demonstrated by colony-forming unit fibroblast (CFU-F) assays. CFU-efficiency was highest in cell populations from the apical and from the mid-tooth PDL. Population doubling time was highest in subcutaneous cells. All investigated cell populations possessed trilineage differentiation potential into osteogenic, adipogenic and chondrogenic lineages.Conclusions: Due to the demonstrated in vitro characteristics cells were referred to as equine subcutaneous MSC (eSc-MSC), equine gingival MSC (eG-MSC) and equine periodontal MSC (eP-MSC). According to different PDL levels, eP-MSC were further specified as eP-MSC from the apical PDL (eP-MSCap), eP-MSC from the mid-tooth PDL (eP-MSCm) and eP-MSC from the subgingival PDL (eP-MSCsg). Considering current concepts of cell-based regenerative therapies in horses, eP-MSC might be promising candidates for future clinical applications in equine orthopedic and periodontal diseases.

Neospora caninum, one of the main causes of abortion in cattle, is very effective at crossing the placental barrier and placental damage is crucial in the pathogenesis of abortion. Bovine trophoblast and caruncular cell layers are key cellular components in the maternal-foetal interface in placentomes, playing a fundamental role in placental functionality. We studied tachyzoite adhesion, invasion, proliferation and egress of high- (Nc-Spain7) and low- (Nc-Spain1H) virulence N. caninum isolates in established cultures of bovine caruncular epithelial (BCEC-1) and trophoblast (F3) cells. The parasite invasion rate (pInvR) and the cell infection rate (cInfR) were determined by immunostaining plaque assay at different time points and multiplicities of infection (MOIs), respectively. In addition, tachyzoite growth kinetics were investigated using real-time PCR (qPCR) analysis and immunostaining plaque assay at different times. Neospora caninum invaded and proliferated in both cell lines. The pInvR was higher in F3 compared to BCEC-1 cells for the Nc-Spain7 isolate (P < 0.05), and higher for the Nc-Spain7 than the Nc-Spain1H in F3 cells (P < 0.01). The cInfR was also higher in F3 cells than in BCEC-1 cells for both isolates (P < 0.0001), and the cInfR for the Nc-Spain7 isolate was higher than for the Nc-Spain1H isolate in both cell lines (P < 0.05). Tachyzoite growth kinetics showed tachyzoite exponential growth until egress at 58 hpi for both isolates in F3, whereas Nc-Spain1H showed a non-exponential growth pattern in BCEC-1. Asynchronous egress of both isolates was observed from 22 h post-infection onwards in BCEC-1. In addition, the tachyzoite yield (TY58h) was higher in F3 than in BCEC-1 infected by both isolates (P < 0.0001), highlighting better replication abilities of both parasites in F3. Nc-Spain7 showed shorter doubling times and higher TY58h compared to Nc-Spain1H in F3 cells; adhesion, invasion and proliferation mechanisms were very similar for both isolates in BCEC-1. Our results indicate a highly similar behavior of high- and low-virulence isolates in their interactions with maternal caruncular cells and suggest an important role of foetal trophoblasts in the pathogenesis of N. caninum infection.

Bovine neosporosis, one of the main causes of reproductive failure in cattle worldwide, poses a challenge for the immune system of pregnant cows. Changes in the Th-1/Th-2 balance in the placenta during gestation have been associated with abortion. Cotyledon and caruncle cell layers form the maternal-foetal interface in the placenta and are able to recognize and induce immune responses against Neospora caninum among other pathogens. The objective of the present work was to elucidate the immunomodulation produced by high- (Nc-Spain7) and low-virulence (Nc-Spain1H) isolates of N. caninum in bovine trophoblast (F3) and caruncular cells (BCEC-1) at early and late points after infection. Variations in the mRNA expression levels of toll-like receptor-2 (TLR-2), Th1 and Th2 cytokines (IL-4, IL-10, IL-8, IL-6, IL-12p40, IL-17, IFN-γ, TGF-β1, TNF-α), and endothelial adhesion molecules (ICAM-1 and VCAM-1) were investigated by RT-qPCR, and protein variations in culture supernatants were investigated by ELISA. A similar pattern of modulation was found in both cell lines. The most upregulated cytokines in infected cells were pro-inflammatory TNF-α (P < 0.05–0.0001) and IL-8 (P < 0.05–0.001) whereas regulatory IL-6 (P < 0.05–0.001) and TGF-β1 (P < 0.05–0.001) were downregulated in both cell lines. The measurement of secreted IL-6, IL-8 and TNF-α confirmed the mRNA expression level results. Differences between isolates were found in the mRNA expression levels of TLR-2 (P < 0.05) in both cell lines and in the mRNA expression levels (P < 0.05) and protein secretion of TNF-α (P < 0.05), which were higher in the trophoblast cell line (F3) infected with the low-virulence isolate Nc-Spain1H. Neospora caninum infection is shown to favor a pro-inflammatory response in placental target cells in vitro. In addition, significant immunomodulation differences were observed between high- and low-virulence isolates, which would partially explain the differences in virulence.