# Retained Placenta as a Potential Source of Mastitis Pathogens in Dairy Cows
## 1 Introduction  
Bovine uterine infections and mastitisti are two of the most important causes of economic losses in dairy farms [1, 2]. Environmental bacteria play an important role in the contamination of uterine lumen from the vagina through to the cervical canal in the immediate postpartum period, and can lead to uterine infections [3]. Bacteria causing uterine infections after calving come from the feces and from a contaminated environment, with the establishment of an infection depending on the animal's immune status, bacterial load and the pathogenicity of the invading microorganisms [4, 5, 6].  
Retained placenta (RP) is a reproductive-metabolic disease [7], defined as a failure to expel fetal membranes within 12-24 h after calving. The persistence of placental membranes and lochia is a source of nutrients for bacterial growth. The economic consequence of RP, in addition to direct veterinary costs and impaired milk production, is the increased risk for further metabolic diseases and enhanced incidence of metritis and endometritis [8]. RPenhances the severity of infection, as it is the perfect medium for bacterial growth [6]. The interactions among pathogenic, opportunist and microbiome species form a complex and highly dynamic process [2; 9; 10; 11].  
Uterine microbiota is indeed more diverse than once was thought, and due to the significant impact of the related reproductive disorders, its characterization has long been a focus of research. The techniques have included traditional culture-based techniques and more advanced molecular ones, such as real-time polymerase chain reaction (RT-PCR), denaturing gradient gel electrophoresis (DGGE) or ribosomal RNA clone libraries [12; 13; 14]. More recently, next-generation sequencing (NGS) technologies (sequencing of the rrs gene encoding 16S rRNA), allowing researchers to conduct in-depth sequencing and facilitating the analysis of thousands of sequences per sample, permit the description of previous unculturable bacteria of the complex uterine bacterial community [15; 16; 17; 18].  
The establishment of uterine microbiota is a normal and dynamic postpartum process. Less uterine microbial diversity and microbial dysbiosis with an increased abundance of several bacterial species such as _Bacteroides_, _Fusobacterium_, _Trueperella_ and _Porphytromonas_ contributes to uterine infection [19]. _Trueperella pyogenes_, _Escherichia coli_, _Bacillus_ spp. and _Streptococcus uberis_ are the bacteria most frequently isolated from the uterine content in cows presenting clinical endometritis [2]. Most of these bacteria are also mastitis pathogens. The etiological agents of mastitis include a variety of Gram-positive and Gram-negative bacteria, and can be environmental (e.g., _E. coli_, _Enterococcus_ spp., _coagalase-negative Staphylococcus_ (CNS), _Strep. uberis_, _Serratia marcescens_) or contagious (e.g., _Staphylococcus aureus_, _Streptococcus agalacliae_, _Mycoplasma_ spp.) [1]. In a previous study, _Strep. uberis_ was simultaneously observed in both the genital tract (vaginal and uterine secretions) and udder, in 82.6% of cases [20]. The carriage of _Mycoplasma boyeinitialium_ from the lochia of postpartum cows to their udder was also reported [21].  
Nonetheless, bacterial data from uterine samples of primiparous and multiparous dairy cows presenting RP are limited (e.g., [4; 22; 23]). There is also scarce information on the relationship among bacterial species present in the uterine lumen during RP and the clinical signs observed, such as fever. We hypothesized that dairy cattle with RP may be a source of mastitis pathogens. Therefore, we aimed at evaluating the presence of mastitis pathogens in dairy cows with RP, using RT-PCR, and determining if the severity of the disease in terms of the presence of fever related to certain species. The agreement between RT-PCR and bacterial culture was also assessed.