Innate immune response to intramammary infection with Serratia marcescens and Streptococcus uberis

Streptococcus uberis and Serratia marcescens are Gram-positive and Gram-negative bacteria, respectively, that induce clinical mastitis. Once initial host barrier systems have been breached by these pathogens, the innate immune system provides the next level of defense against these infectious agents. The innate immune response is characterized by the induction of pro-inflammatory cytokines, as well as increases in other accessory proteins that facilitate host recognition and elimination of the pathogens. The objective of the current study was to characterize the innate immune response during clinical mastitis elicited by these two important, yet less well-studied, Gram-positive and Gram-negative organisms. The pro-inflammatory cytokine response and changes in the levels of the innate immune accessory recognition proteins, soluble CD14 (sCD14) and lipopolysaccharide (LPS)-binding protein (LBP), were studied. Decreased milk output, induction of a febrile response, and increased acute phase synthesis of LBP were all characteristic of the systemic response to intramammary infection with either organism. Infection with either bacteria similarly resulted in increased milk levels of IL-1$\beta$, IL-8, IL-10, IL-12, IFN-$\gamma$, TNF-$\alpha$, sCD14, LBP, and the complement component, C5a. However, the duration of and/or maximal changes in the increased levels of these inflammatory markers were significantly different for several of the inflammatory parameters assayed. In particular, S. uberis infection was characterized by the sustained elevation of higher milk levels of IL-1$\beta$, IL-10, IL-12, IFN-$\gamma$, and C5a, relative to S. marcescens infection. Together, these data demonstrate the variability of the innate immune response to two distinct mastitis pathogens

Recombinant bovine soluble CD14 reduces severity of experimental Escherichia coli mastitis in mice

Endotoxin, or lipopolysaccharide (LPS), is responsible for pathogenesis of infections induced by Gram-negative bacteria, such as E. coli. The cellular response to LPS is modulated by interactions among LPS, LPS-binding protein (LBP) and CD14. Accumulated evidence shows that the soluble form of CD14 (sCD14) competes with membrane-bound CD14 (mCD14) for LPS and plays a pivotal role in regulating bacterial infection and septic shock caused by Gram-negative bacteria. Recombinant bovine sCD14 (rbosCD14) was produced by transfected insect sf/9 cells and its biological function was evaluated in mice. Eighty-one 8-week old BALB/cj female mice were randomly assigned to two groups, and injected intraperitoneally with either LPS (8 $\mu$g/g of body weight, $n = 41$) or LPS plus rbosCD14 (6.8 $\mu$g/g of body weight, $n = 40$). Survival rate at 24 h after injection for mice injected with either LPS or LPS plus rbosCD14 was 30 and 72%, respectively ($P < 0.01$). At 48 h survival rate was 7 and 37%, respectively ($P < 0.01$). To investigate the protective effect of rbosCD14 on experimentally induced mastitis in mice, two abdominal contralateral mammary glands of 7 lactating BALB/cj mice were injected through the teat canal with 10–20 colony-forming units (CFU) of Escherichia coli. One gland simultaneously received rbosCD14 (6 $\mu$g) and the other saline. At 24 h after challenge, glands that received rbosCD14 had less swelling and hemorrhaging, significantly lower bacterial counts ($P < 0.05$) and lower concentrations of TNF-$\alpha$ ($P < 0.05$). Results indicate that rbosCD14 is biologically functional and reduces mortality in mice from endotoxin shock and severity of intramammary infection by E. coli

Purification and characterization of bovine complement component C3 and its cleavage products

Objective-To purify complement component C3 from bovine serum, characterize and analyze NH2-terminal amino acid sequences from its various cleavage products, and do cross-species homology comparisons. Animals-2 healthy lactating Holstein cows, and 2 healthy adult female New Zealand White rabbits. Procedure-Bovine C3 was isolated from serum, and was cleaved to C3b. The resulting protein was analyzed to determine apparent molecular mass of resulting protein segments, Bands were electroblotted onto a membrane and excised, then NH2-terminal amino acid sequences were determined. Results-The C3 preparation consisted of 6 segments, with molecular mass of 30, 40 (2 bands, a and b), 70, 75, and 115 kd. Via sequence comparisons, the 115-kd band was identified as the alpha chain; the 75-kd segment was determined to be the NH2-terminal portion of alpha chain; the 70-kd piece was identified as the intact beta chain; and the two 40-kd bands are believed to be located at the C-terminal portion of the alpha chain, at the cleavage site that yields C3f. The 30-kd band is the NH2-terminal portion of the alpha chain (minus the C3a segment). Sequence analysis of each band revealed a high degree of homology with human, rat, mouse, and horse C3, Polyclonal antibodies raised in rabbits yielded sera that reacted to the purified sample in manner similar to that of commercially available antibodies. Conclusions-The purified preparation contained intact C3, C3b, and the degradation products iC3b and C3c, which had high sequence homology with those of other species, The C3a and C3d, and C3g segments of the protein were not detected and may have been lost during the purification, lyophilization, or transfer steps. Structure and cleavage characteristics of bovine C3 can be used to better understand immune responses to bacterial pathogens in the mammary gland

The bovine neutrophil: Structure and function in blood and milk

Migration of polymorphonuclear neutrophil leukocytes (PMN) into the mammary gland provide the first line of defense against invading mastitis pathogens. Bacteria release potent toxins that activate white blood cells and epithelial cells in the mammary gland to secrete cytokines that recruit PMN that function as phagocytes at the site of infection. While freshly migrated PMN are active phagocytes, continued exposure of PMN to inhibitory factors in milk such as fat globules and casein, leads to altered PMN morphology and reduced phagocytosis. In the course of phagocytosing and destroying invading pathogens, PMN release chemicals that not only kill the pathogens but that also cause injury to the delicate lining of the mammary gland. This will result in permanent scarring and reduced numbers of milk secretory cells. The life span of PMN is limited by the onset of apoptosis. To minimize damage to mammary tissue, PMN undergo a specialized process of programmed cell death known as apoptosis. Macrophages quickly engulf and phagocytose apoptotic PMN, thereby minimizing the release of PMN granular contents that are damaging to tissue. The PMN possess an array of cell surface receptors that allow them to adhere and migrate through endothelium and to recognize and phagocytose bacteria. One receptor found on phagocytes that is receiving considerable attention in the control of infections by Gram-negative bacteria is CD14. Binding of lipopolysaccharide (LPS) to membrane bound CD14 causes release of tumor necrosis factor-$\alpha$ and sepsis. Binding of LPS to soluble CD14 shed from CD14-bearing cells results in neutralization of LPS and rapid recruitment of PMN to the site of infection. Recent advances in the fields of genomics and proteomics should greatly enhance our understanding of the PMN role in controlling intramammary infections in ruminants. Further, manipulation of PMN, through either recombinant proteins such as soluble CD14 that enhance PMN response or agents that mediate PMN apoptosis, may serve as novel therapeutics for the treatment of mastitis

Recombinant Soluble CD14 Reduces Severity of Intramammary Infection by Escherichia coli

The interaction among gram-negative bacteria, the innate immune system, and soluble CD14 (sCD14) has not been well documented. The effect of recombinant bovine sCD14 (rbosCD14) on milk somatic cell count (SCC), bacterial clearance, and cytokine production was investigated by using a bovine intramammary Escherichia coli infection model. We first determined whether rbosCD14 would increase the SCC during a lipopolysaccharide (LPS) challenge. Three quarters of each of six healthy lactating cows were injected with either 0.3 μg of LPS, 0.3 μg of LPS plus 100 μg of rbosCD14, or saline. In comparison with quarters injected with LPS alone, the SCC was twofold higher (P < 0.05) in quarters injected with LPS plus rbosCD14 after the challenge. We therefore hypothesized that when E. coli bacteria invade the mammary gland, sCD14 in milk would interact with LPS and rapidly recruit neutrophils from the blood to eliminate the bacteria before establishment of infection. To test this hypothesis, two quarters of each of nine healthy cows were challenged with either 50 CFU of E. coli plus saline or 50 CFU of E. coli plus 100 μg of rbosCD14. Quarters challenged with E. coli plus rbosCD14 had a more rapid recruitment of neutrophils, which was accompanied by a faster clearance of bacteria, lower concentrations of tumor necrosis factor alpha and interleukin-8 in milk, and milder clinical symptoms, than challenged quarters injected with saline. Results indicate that increasing the concentration of sCD14 in milk may be a potential strategy with which to prevent or reduce the severity of infection by coliform bacteria