Mastitis is inflammation of the mammary gland and is one of the most common and harmful diseases of dairy ruminants worldwide, causing economic losses owing to low milk quality and quantity [1]. Mastitis is commonly caused by bacterial infections of the teat canal [2]. Bacterial infection of the mammary gland induces the production of pro-inflammatory cytokines, such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), and chemokines, such as IL-8 [3]. The activation of these cytokines induces an acute-phase response to inflammation and recruits leukocytes to the infection site [4].

Immune responses that protect epithelial surfaces present specialized characteristics that define mucosal immunity [5]. In mucosal immunity, epithelial cells within specialized cells (M cells) take up antigens from the lumen or the environment and deliver them to the second layer, which includes macrophages and dendritic cells, to process the antigen [5]. B cells are then activated in lymphoid tissues [6]. B cells are recruited to the mucosa and express mucosal IgA on the mucosal surfaces. IgA binds to the immunoglobulin receptor (pIgR) on mucosal epithelial cells, and subsequently to transcytoses at the surface, and is secreted into the mucus [7]. Immune responses have been induced locally, not only at the delivery site of antigens, but also at distant mucosal surfaces [8].

Usually, live pathogenic bacteria enter the mammary gland through the teat canal and cause inflammation, although bacterial components such as lipopolysaccharides (LPSs), a cell wall component of gram-negative bacteria, also induce mammary gland inflammation [9]. Furthermore, LPSs can be translocated to different organs. Jin et al. [10] reported that feeding cows a long-term high-concentrate diet causes ruminal acidosis, resulting in the release of LPS from gram-negative bacteria. These LPSs are then translocated from the rumen to the mammary gland through the blood vessels, resulting in an inflammatory response.

LPSs consist of three regions: O-specific polysaccharide (O-antigen), core polysaccharide, and lipid A. Over 180 serogroups have been defined according to different O-antigens from O1 to O185. A variety of serotypes is important for host-specific antibody responses [11,12]. LPS obtained from Escherichia coli O111:B4 and O55:B5 is commonly used for the experimental induction of inflammation. However, the differences in the virulence of these two strains in the mammary glands are not fully understood.

Endometritis is an infectious uterine disease caused by gram-negative bacteria such as E. coli, Fusobacterium necrophorum, Prevotella, and Bacteroides, and gram-positive bacteria such as Trueperella pyogenes [13]. LPS is released when gram-negative bacteria are killed by an immune response. Purba et al. [14] found that cows with endometritis had higher somatic cell counts (SCC) than healthy cows, suggesting the translocation of bacterial components between organs. To confirm that LPSs can be translocated from the uterus to the mammary glands, Purba et al. [15] infused LPS into the uterus of healthy goats and measured the cytokine levels in milk. The results showed that IL-1β and IL-6 levels increased after intrauterine LPS infusion, and LPS was detected in the mammary gland, indicating their translocation from the uterus to the mammary gland. Additionally, Purba et al. [16] used immunosuppressive goats to examine the same experiment and showed that the SCC, IL-1β, and lactoferrin levels in their milk increased, which indicated heavier inflammation compared with that in healthy goats. They infused LPSs into the uterus only once, whereas naturally occurring endometritis continued for several days, not just a single day. LPS should be infused repeatedly into the uterus to mimic natural endometritis. However, the inflammatory status of the udder after repeated intrauterine LPS infusions has not been elucidated. Therefore, in this study, we aimed to determine the effect of repeated infusions of LPSs into the uterus on the inflammation status of the mammary gland.