Max Paape

Escherichia coli and Staphylococcus aureus Elicit Differential Innate Immune Responses following Intramammary Infection

ABSTRACT 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 1β (IL-1β), 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.

Genetically enhanced cows resist intramammary Staphylococcus aureus infection

Mastitis, the most consequential disease in dairy cattle, costs the US dairy industry billions of dollars annually. To test the feasibility of protecting animals through genetic engineering, transgenic cows secreting lysostaphin at concentrations ranging from 0.9 to 14 mg/ml in their milk were produced. In vitro assays demonstrated the milks ability to kill Staphylococcus aureus. Intramammary infusions of S. aureus were administered to three transgenic and ten nontransgenic cows. Increases in milk somatic cells, elevated body temperatures and induced acute phase proteins, each indicative of infection, were observed in all of the nontransgenic cows but in none of the transgenic animals. Protection against S. aureus mastitis appears to be achievable with as little as 3 mg/ml of lysostaphin in milk. Our results indicate that genetic engineering can provide a viable tool for enhancing resistance to disease and improve the well-being of livestock.

Defense of the Bovine Mammary Gland by Polymorphonuclear Neutrophil Leukocytes

The primary phagocytic cells of the bovine mammary gland, polymorphonuclear neutrophil leukocytes (PMN), and macrophages, comprise the first line of defense against invading pathogens. In the normal healthy mammary gland, macrophages predominate and act as sentinels to invading mastitis-causing pathogens. Once invaders are detected, macrophages, and possibly mammary epithelial cells, release chemoattractants that direct migration of PMN into the area. In the mammary gland, protection is only effective if rapid influx of PMN from the circulation and subsequent phagocytosis and killing of bacteria occur. The second line of defense against infection consists of a network of memory cells and immunoglobulins that interact with the first line of defense. To minimize mammary tissue damage caused by bacterial toxins and oxidative products released by PMN, elimination of invading bacteria must proceed quickly. Therefore, the inflammatory response needs to be regulated. Hormones, metabolites, and acute phase proteins act to influence the outcome of mastitis, especially around parturition. The number of circulating PMN in cows during early lactation is highly heritable and closely related to susceptibility to clinical mastitis at this time. Advances in molecular biology are making available the tools, techniques, and products to study and modulate host–pathogen interactions. For example, the cloning and expression of proteins such as recombinant bovine soluble (rbos) CD (cluster of differentiation) 14 antigens, may provide ways of minimizing damaging effects of endotoxin during acute coliform mastitis. Soluble CD14 binds and neutralizes lipopolysacharide (LPS) and causes local recruitment of PMN after binding of CD14-LPS complexes to mammary epithelial cells. Development of transgenic animals that express rbosCD14 in their milk could prevent infection by Gram-negative pathogens.

Role of the neutrophil leucocyte in the local and systemic reactions during experimentally induced e.coli mastitis in cows immediately after calving

Mammary leucocytes are the major contributors to natural defence against mastitis after a microorganism has entered the gland. This paper reviews the role of the neutrophil granulocyte during acute coliform mastitis in cows in the periparturient period. Qualitative and quantitative aspects of several neutrophil cell functions before and during experimentally induced infections are briefly discussed.

Immune surveillance of mammary tissue by phagocytic cells

The leukocytes in milk consist of lymphocytes, neutrophil polymorphonuclear leukocytes (PMN) and macrophages. Lymphocytes together with antigen-presenting cells function in the generation of an effective immune response. Lymphocytes can be divided into two distinct subsets, T- and B-lymphocytes, that differ in function and protein products. The professional phagocytic cells of the bovine mammary gland are PMN and macrophages. In the normal mammary gland macrophages are the predominate cells which act as sentinels to invading mastitis causing pathogens. Once the invaders are detected, macrophages release chemical messengers called chemoattractants that cause the directed migration of PMN into the infection. Migration of neutrophils into mammary tissue provides the first immunological line of defense against bacteria that penetrate the physical barrier of the teat canal. However, their presence is like a double-edged sword. While the PMN are phagocytosing and destroying the invading pathogens, they inadvertently release chemicals which induces swelling of secretory epithelium cytoplasm, sloughing of secretory cells, and decreased secretory activity. Permanent scarring will result in a loss of milk production. Resident and newly migrated macrophages help reduce the damage to the epithelium by phagocytosing PMN that undergo programmed cell death through a process called apoptosis. Specific ligands on the neutrophil surface are required for directed migration and phagocytosis. In response to infection, freshly migrated leukocytes express greater numbers of cell surface receptors for immunoglobulins and complement and are more phagocytic than their counterparts in blood. However, phagocytic activity rapidly decreases with continued exposure to inhibitory factors such as milk fat globules and casein in mammary secretions. Compensatory hypertrophy in non-mastitic quarters partially compensates for lost milk production in diseased quarters. Advances in molecular biology are making available the tools, techniques, and products to study and modulate host-parasite interactions. For example the cloning and expression of proteins that bind endotoxin may provide ways of reducing damaging effects of endotoxin during acute coliform mastitis. The successful formation of bifunctional monoclonal antibodies for the targeted lysis of mastitis causing bacteria represents a new line of therapeutics for the control of mastitis in dairy cows.

Hormonal patterns during heat stress following PGF2α-tham salt induced luteal regression in heifers

Abstract Ten, normally cycling, Holstein heifers were assigned to one of two environmental treatment groups (21.3 C, 59% RH or 32.0 C, 67% RH). PGF2α was used to induce luteal regression and synchronize estrus in order to evaluate temperature effects on various hormonal and physiological responses during the proestrous through metestrous periods. Environmental temperature (32.0 C) evoked a 1.4 C increase in rectal temperature and a 3.6 C increase in skin temperatures. Length of estrus was shorter (P vs 21 hr.). Average plasma progestin concentration between treatments was not different (P>.10). Mean estradiol concentrations were significantly (P .10) were detected in mean concentrations of LH between heifers at 21.3 C and 32.0 C. Preovulatory peak LH concentrations were 32.2 and 33.2 ng/ml plasma, respectively. All animals had a preovulatory surge of LH, suggesting that hyperthermia did not alter factors which regulate hypothalamic control of LH release. Mean basal concentrations of prolactin and corticoids were not different between temperature treatments (P>.10). However, mean corticoid response following ACTH was of lower magnitude, earlier to peak, and of shorter duration in heat stressed heifers. Heat stress did not appear to affect the hormonal milieu in peripheral plasma associated with corpus luteum regression (decrease in progestin) and ovulation (LH surge). However, duration of estrus, concentrations of estradiol at proestrus and corticoid response to injection of ACTH were reduced.

Escherichia coli induces apoptosis and proliferation of mammary cells.

Mammary cell apoptosis and proliferation were assessed after injection of Escherichia coli into the left mammary quarters of six cows. Bacteriological analysis of foremilk samples revealed coliform infection in the injected quarters of four cows. Milk somatic cell counts increased in these quarters and peaked at 24 h after bacterial injection. Body temperature also increased, peaking at 12 h postinjection. The number of apoptotic cells was significantly higher in the mastitic tissue than in the uninfected control. Expression of Bax and interleukin-1β converting enzyme increased in the mastitic tissue at 24 h and 72 h postinfection, whereas Bcl-2 expression decreased at 24 h but did not differ significantly from the control at 72 h postinfection. Induction of matrix metalloproteinase-9, stromelysin-1 and urokinase-type plasminogen activator was also observed in the mastitic tissue. Moreover, cell proliferation increased in the infected tissue. These results demonstrate that Escherichia coli-induced mastitis promotes apoptosis and cell proliferation. Cell Death and Differentiation (2001) 8, 808–816

Differentiation and enumeration of somatic cells in goat milk

Non-leukocytic cell-like particles commonly observed in goat milk were examined ultrastructurally and cytochemically. Transmission electron microscopy indicated that these particles were generally membrane-bound and anucleate. They contained granular material in the dilated cisternae of the endoplasmic reticulum and homogeneous electron translucent inclusions that resembled lipid. Histochemical and fluorescent staining indicated that the particles contained large amounts of protein, some lipid, but no deoxyribonucleic acid. Several methods routinely used for estimating somatic cell counts in cow milk were compared to determine which one would give accurate estimates of somatic cell counts in goat milk. No significant difference was found (P> .05) among methods which specifically measure deoxyribonucleic acid. These included Membrane Filter-DNA, direct microscopic somatic cell counts using Pyron in Y-methyl green stain, and Fossomatic cell counts. Results of the Wisconsin Mastitis Test did not differ significantly from Fossomatic cell counts. Because Coulter electronic counts and direct microscopic somatic cell counts using Levowitz-Weber stain could not differentiate between the cell-like particles and the actual leukocytes, these methods resulted in elevated cell counts that were highly variable. Results indicate that only those counting methods that are specific for deoxyribonucleic acid can distinguish cell-like particles from somatic cells, and thereby give reliable estimates of somatic cell numbers in goat milk.

Effect of Fat and Casein on Intracellular Killing of Staphylococcus aureus by Milk Leukocytes

Summary Suspensions of PMN isolated from milk and S. aureus were incubated together at different ratios and with different types and concentrations of mammary fluids. Results showed that greater numbers of S. aureus were left unphagocytosed and alive within PMN at the higher S. au-reus: PMN ratios (2:1) than at the lower ratios. This effect was more noticeable in whole milk than in skimmed milk. Phagocytosis and destruction of S. aureus by PMN (ratio 1:10) were reduced (p < 0.01) to a greater extent in whole milk or in skimmed milk containing 10% cream than in either skimmed milk or whey. There was no difference between skimmed milk or whey. Increasing the concentration of either whole milk or skimmed milk in the incubation mixture from 10 to 40% resulted in fewer (p < 0.01) S. aureus phagocytosed but had no affect on intracellular kill. The data show that the milk fat globule is a major deterrent to the phagocytosis and destruction of S. aureus by PMN. Casein inhibited phagocytosis, but only when the concentration of skimmed milk in the incubation medium was increased to 40%. Casein did not affect intracellular kill. We thank Jane Hawes for technical assistance.

Recombinant Soluble CD14 Reduces Severity of Intramammary Infection by Escherichia coli

ABSTRACT 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.