Bovine postpartum uterine disease, metritis, affects about 40% of animals and is widely considered to have a bacterial aetiology. Although the γ-herpesvirus bovine herpesvirus 4 (BoHV-4) has been isolated from several outbreaks of metritis or abortion, the role of viruses in endometrial pathology and the mechanisms of viral infection of uterine cells are often ignored. The objectives of the present study were to explore the interaction, tropism and outcomes of BoHV-4 challenge of endometrial stromal and epithelial cells. Endometrial stromal and epithelial cells were purified and infected with a recombinant BoHV-4 carrying an enhanced green fluorescent protein (EGFP) expression cassette to monitor the establishment of infection. BoHV-4 efficiently infected both stromal and epithelial cells, causing a strong non-apoptotic cytopathic effect, associated with robust viral replication. The crucial step for the BoHV-4 endometriotropism appeared to be after viral entry as there was enhanced transactivation of the BoHV-4 immediate early 2 gene promoter following transient transfection into the endometrial cells. Infection with BoHV-4 increased cyclooxygenase 2 protein expression and prostaglandin estradiol secretion in endometrial stromal cells, but not epithelial cells. Bovine macrophages are persistently infected with BoHV-4, and co-culture with endometrial stromal cells reactivated BoHV-4 replication in the persistently infected macrophages, suggesting a symbiotic relationship between the cells and virus. In conclusion, the present study provides evidence of cellular and molecular mechanisms, supporting the concept that BoHV-4 is a pathogen associated with uterine disease. Abstract Bovine postpartum uterine disease, metritis, affects about 40% of animals and is widely considered to have a bacterial aetiology. Although the γ-herpesvirus bovine herpesvirus 4 (BoHV-4) has been isolated from several outbreaks of metritis or abortion, the role of viruses in endometrial pathology and the mechanisms of viral infection of uterine cells are often ignored. The objectives of the present study were to explore the interaction, tropism and outcomes of BoHV-4 challenge of endometrial stromal and epithelial cells. Endometrial stromal and epithelial cells were purified and infected with a recombinant BoHV-4 carrying an enhanced green fluorescent protein (EGFP) expression cassette to monitor the establishment of infection. BoHV-4 efficiently infected both stromal and epithelial cells, causing a strong non-apoptotic cytopathic effect, associated with robust viral replication. The crucial step for the BoHV-4 endometriotropism appeared to be after viral entry as there was enhanced transactivation of the BoHV-4 immediate early 2 gene promoter following transient transfection into the endometrial cells. Infection with BoHV-4 increased cyclooxygenase 2 protein expression and prostaglandin estradiol secretion in endometrial stromal cells, but not epithelial cells. Bovine macrophages are persistently infected with BoHV-4, and co-culture with endometrial stromal cells reactivated BoHV-4 replication in the persistently infected macrophages, suggesting a symbiotic relationship between the cells and virus. In conclusion, the present study provides evidence of cellular and molecular mechanisms, supporting the concept that BoHV-4 is a pathogen associated with uterine disease.

Experimental infection with the γ-herpesvirus bovine herpesvirus 4 (BoHV-4) rarely establishes disease, yet BoHV-4 is commonly associated with uterine disease in cattle. Uterine disease involves co-infection with bacteria such as Escherichia coli, which stimulate the production of prostaglandin E₂ (PGE₂) by endometrial cells. BoHV-4 replication depends on immediate early 2 (IE2) gene transactivation and, in the present study, PGE₂, E. coli or its lipopolysaccharide upregulated the IE2 gene promoter in uterine cells. Bacterial co-infection is important for BoHV-4 uterine disease. Abstract Experimental infection with the γ-herpesvirus bovine herpesvirus 4 (BoHV-4) rarely establishes disease, yet BoHV-4 is commonly associated with uterine disease in cattle. Uterine disease involves co-infection with bacteria such as Escherichia coli, which stimulate the production of prostaglandin E₂ (PGE₂) by endometrial cells. BoHV-4 replication depends on immediate early 2 (IE2) gene transactivation and, in the present study, PGE₂, E. coli or its lipopolysaccharide upregulated the IE2 gene promoter in uterine cells. Bacterial co-infection is important for BoHV-4 uterine disease.

The endometrium is central to mammalian fertility. The endometrial stromal cells are very dynamic, growing and differentiating throughout the estrous cycle and pregnancy. In humans, stromal cells appear to have progenitor or stem cell capabilities and the cells can even differentiate into bone. It is not clear whether bovine endometrial stromal cells exhibit a similar phenotypic plasticity. So, the present study tested the hypothesis that bovine endometrial stromal cells could be differentiated along an osteogenic lineage. Pure populations of bovine stromal cells were isolated from the endometrium. The endometrial stromal cell phenotype was confirmed by morphology, prostaglandin secretion, and susceptibility to viral infection. However, cultivation of the cells in standard endometrial cell culture medium lead to a mesenchymal phenotype similar to that of bovine bone marrow cells. Furthermore, the endometrial stromal cells developed signs of osteogenesis, such as alizarin positive nodules. When the stromal cells were cultured in a specific osteogenic medium the cells rapidly developed the characteristics of mineralized bone. In conclusion, the present study has identified that stromal cells from the bovine endometrium show a capability for phenotype plasticity similar to mesenchymal progenitor cells. These observations pave the way for further investigation of the mechanisms of stroma cell differentiation in the bovine reproductive tract.

In captive breed turtles and tortoises conjunctival disease is common. Our aim was to investigate the bacterial and fungal flora present in the eyes of healthy and pathological chelonians and to compare findings in turtles with those in tortoises. Samples were taken from the conjunctival sacs of 34, diseased and healthy, chelonians (18 tortoises and 16 turtles) and submitted to bacterial and fungal investigation. All samples showed bacterial growth. Thirteen animals (38%), harboured a single bacterial species as sole isolate and twenty-one animals (62%) harboured more than one species. Detection of multiple bacterial infection was clearly greater in tortoises compared to turtles. Most frequently isolated bacterial species were Bacillus spp. (13 isolates), Staphylococcus xylosus (10 isolates), Sphingomonas paucimobilis (6 isolates), Staphylococcus sciuri and Aeromonas hydrophila/caviae (each 5 isolates), Ochrobactrum anthropi (3 isolates), Citrobacter freundii, Enterobacter cloacae and Pseudomonas luteola (each 2 isolates). Only one isolate of Kocuria varians/rosea, Staphylococcus aureus, Staphylococcus auricularis, Staphylococcus haemolyticus, Staphylococcus lentus, Morganella morganii, Pasteurella multocida, Pasteurella pneumotropica/haemolytica, Proteus spp., Pseudomonas putida, Salmonella enterica ssp. arizonae, Stenotrophomonas maltophilia and Vibrio parahaemolyticus was evidenced. The presence in 8 animals of Mycoplasma spp. and in 1 animal with severe conjunctivitis of Chlamydia spp. was detected by PCR. Candida spp. was also isolated from two healthy animals. A clear predominance of Gram positive isolates in tortoises and Gram negative isolates in turtles was found. However, we cannot ascribe the observed difference to the diversity of animal species, as other factors, including especially different characteristics of the living environments, may play a role. Almost all bacterial species isolated may have clinical significance, mostly as opportunistic pathogens, both for humans and animals. That chelonians are often carrier of bacteria with zoonotic potential is a well-known fact, in particular with regard to Salmonella spp. Therefore, it is not surprising the detection of a strain of Salmonella enterica ssp. arizonae in the eye of one of the animals tested. Worthy of note is the finding of Chlamydia spp. in a severe case of conjunctivitis, though we cannot epidemiologically assess a cause-effect relationship between presence of chlamydia and disease.