Staphylococcus aureus and Escherichia coli are among the most prevalent species of gram-positive and gram-negative bacteria, respectively, that induce clinical mastitis. The innate immune system comprises the immediate host defense mechanisms to protect against infection and contributes to the initial detection of and proinflammatory response to infectious pathogens. The objective of the present study was to characterize the different innate immune responses to experimental intramammary infection with E. coli and S. aureus during clinical mastitis. The cytokine response and changes in the levels of soluble CD14 (sCD14) and lipopolysaccharide-binding protein (LBP), two proteins that contribute to host recognition of bacterial cell wall products, were studied. Intramammary infection with either E. coli or S. aureus elicited systemic changes, including decreased milk output, a febrile response, and induction of the acute-phase synthesis of LBP. Infection with either bacterium resulted in increased levels of interleukin 1␤ (IL-1␤), gamma interferon, IL-12, sCD14, and LBP in milk. High levels of the complement cleavage product C5a and the anti-inflammatory cytokine IL-10 were detected at several time points following E. coli infection, whereas S. aureus infection elicited a slight but detectable increase in these mediators at a single time point. Increases in IL-8 and tumor necrosis factor alpha were observed only in quarters infected with E. coli. Together, these data demonstrate the variability of the host innate immune response to E. coli and S. aureus and suggest that the limited cytokine response to S. aureus may contribute to the well-known ability of the bacterium to establish chronic intramammary infection.

Gastrointestinal disease caused by the apicomplexan parasite Cryptosporidium parvum is one of the most important diseases of young ruminant livestock, particularly neonatal calves. Infected animals may suffer from profuse watery diarrhoea, dehydration and in severe cases death can occur. At present, effective therapeutic and preventative measures are not available and a better understanding of the host–pathogen interactions is required. Cryptosporidium parvum is also an important zoonotic pathogen causing severe disease in people, with young children being particularly vulnerable. Our knowledge of the immune responses induced by Cryptosporidium parasites in clinically relevant hosts is very limited. This review discusses the impact of bovine cryptosporidiosis and describes how a thorough understanding of the host–pathogen interactions may help to identify novel prevention and control strategies.

The uptake of respiratory syncytial virus (RSV) antigen by cattle dendritic cells was investigated. Pathways of antigen uptake were monitored by flow cytometry using specific tracers and by proliferation assays, which were used to measure the presentation of RSV antigen and ovalbumin. Inhibitors that differentially affected pathways were used to distinguish them. Presentation of RSV antigen, but not ovalbumin, was inhibited by phorbol myristate acetate and filipin, which have been reported to inhibit caveolae, but not by cytochalasin D, amiloride, or mannose. These inhibitors have been reported to block macropinocytosis and other actin-dependent uptake mechanisms, endocytic pathways involving clathrin-coated pits, and the mannose receptor. Furthermore, co-localization of RSV antigen and caveolae was observed by confocal microscopy. Thus, the major route for uptake of RSV antigen by cattle dendritic cells is one mediated by caveolae, adding a pathway of antigen uptake by dendritic cells to those established. J. Leukoc. Biol. 66: 50-58; 1999.

Natural killer (NK) cells have not previously been precisely identified or characterized in cattle or any other ruminant species. We have generated a monoclonal antibody against bovine NKp46, which is expressed exclusively by NK cells in man. NKp46 + cells comprised 1-10% of blood mononuclear cells in cattle, and did not stain with antibodies against CD3, CD4, TCR1, B cell or granulocyte markers. The majority of the NKp46 + cells expressed CD2, and a variable fraction also expressed CD8. The tissue distribution of NKp46 + cells in cattle was compatible with the tissue distribution of NK cells in other species. Bovine NKp46 + cells had typical, large granular lymphocyte morphology, and proliferated vigorously in response to bovine IL-2 for a limited number of cell divisions. IL-2-activated NKp46 + cells killed the bovine kidney cell line MDBK. This cytotoxicity was inhibited by preincubation with antibody against NKp46. In a redirected lysis assay, IL-2-activated NKp46 + cells killed the Fc + R + target cell line P815 after preincubation with antibody against NKp46. Together, these data indicate that bovine NKp46 is an activating receptor and demonstrate the existence of a subset of leukocytes in cattle that, in terms of surface markers, morphology and function, represent NK cells.

-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 proinflammatory 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 wellstudied, 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-1β, IL-8, IL-10, IL-12, IFN-γ, TNF-α, 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-1β, IL-10, IL-12, IFN-γ, and C5a, relative to S. marcescens infection. Together, these data demonstrate the variability of the innate immune response to two distinct mastitis pathogens. cytokines / innate immunity / mastitis / Serratia / Streptococcus

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Ten multiparous Holstein cows were used to determine the effects of negative energy balance (NEB) on the immune response to a Streptococcus uberis (strain O140J) mastitis challenge during midlactation. Before the study, milk from all quarters of each cow was bacteriologically negative, with a composite somatic cell count of <200,000 cells/mL. Cows were paired based on parity, days in milk, and milk yield. At approximately 77 d in milk, half the cows (n = 5) were feed-restricted to 60% of calculated net energy for lactation requirements to induce NEB. Feed restriction lasted 7 d. Control cows (n = 5) were fed the same diet ad libitum (i.e., positive energy balance; PEB). After 5 d, one rear quarter in all cows was inoculated with 5,000 cfu of Strep. uberis. Jugular blood and aseptic quarter milk samples were collected daily until inoculation and every 6 h postinoculation for 36 h. Blood was analyzed for nonesterified fatty acids, beta-hydroxybutyrate, insulin, cortisol, albumin, serum amyloid A (SAA), and haptoglobin (Hp). Periodically throughout the trial period, blood neutrophils were isolated for determination of cell morphology, chemotaxis, and phagocytosis capability in vitro. Quarter milk samples were analyzed for concentrations of SAA, Hp, cytokines (tumor necrosis factor-alpha, IL-10 and IL-1beta), and activity of respiratory burst enzymes (superoxide dismutase and glutathione peroxidase). All cows developed local and systemic signs of mastitis and calculated NEB was similar to that of cows experiencing postpartal NEB. Serum glucose and insulin concentrations increased in both groups after challenge, most likely because of enhanced glycogenolysis and gluconeogenesis; results indicate that immune cell function may be glucose dependent. Serum cortisol concentration was higher in NEB than PEB cows during feed restriction only (before inoculation), and serum albumin concentration was higher in NEB than PEB cows during the infection period. Compared with PEB, cows in NEB had lower SAA concentrations in serum after 5 d of feed restriction but higher SAA concentrations in milk after Strep. uberis challenge. Serum Hp concentration was higher by 36 h postchallenge in NEB than in PEB cows. Phagocytic capability of neutrophils was lower in NEB than in PEB cows at 0 h of infection but decreased in both PEB and NEB cows through 36 h postinfection. Our results indicate that cows subjected to dietary-induced NEB during midlactation had relatively minimal alterations in immune function.