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Measurement of secreted interferon (IFN)-γ has proven to be a valuable tool for the detection of animals infected with mycobacterial pathogens, including Mycobacterium aviumsubsp. paratuberculosis.Previous reports have suggested that tuberculin skin testing can influence the performance of the IFN-γ assay. In the present study, healthy noninfected cows, and cows subclinically and clinically infected with M. paratuberculosiswere administered an intradermal injection of johnin purified protein derivative (JPPD) and effects on secreted and intracellular IFN-γ were observed. Intradermal injection resulted in significant increases in secreted IFN-γ for subclinically infected cows after stimulation of peripheral blood mononuclear cells (PBMC) with concanavalin A or M. paratuberculosisantigen preparations (whole-cell sonicate and JPPD) on days 7 and 10 postinjection. Intracellular IFN-γ was increased after intradermal injection in total PBMC for all treatment groups and was higher within CD4+ and CD8+ subpopulations for infected cows compared to healthy controls throughout the study. When T-cell populations were further defined by CD45RO expression, intracellular IFN-γ was higher within CD8+/CD45RO+ lymphocytes compared to CD4+/CD45RO+ cells for subclinically and clinically infected cows but similar within these subpopulations for healthy controls. These results indicate that intradermal sensitization of cows in the subclinical stage of infection will upregulate expression of IFN-γ, enhancing the sensitivity of this assay. In addition, CD8+ lymphocytes appear to play an important role as a mediator of M. paratuberculosisinfection in naturally exposed cattle.

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.

There is an increased incidence of infectious disease in periparturient dairy cows. During the periparturient period there is a decline in T-lymphocyte cell subsets, which parallels a reduction in functional capacities of blood lymphocytes and neutrophils. Mechanisms responsible for these changes in immune function during the periparturient period are poorly characterized. Ten mastectomized and eight intact multiparous Jersey cows were used to determine whether the periparturient changes in peripheral blood mononuclear cell populations are the result of the physiological demands associated with the onset of lactation or whether they are a result of the act of parturition. Blood mononuclear cells were phenotyped with monoclonal antibodies against T-cell subsets, B-cells, and monocytes. Blood samples were taken frequently from before 4 to 4 wk after parturition. In intact cows, all T-cell subset populations (i.e., CD3-, CD4-, CD8-, and gamma-delta positive cells) decreased at the time of parturition, while the percentage of monocytes increased. Mastectomy eliminated the changes in leukocyte subset populations (CD3-, CD4-, and gamma-delta positive cells, and monocytes) observed in intact cows around parturition. These results indicate that the mammary gland and metabolic stresses associated with lactation influence the composition of peripheral blood mononuclear cell populations in dairy cows during the periparturient period.

The objective of this study was to compare blood profiles of intact and mastectomized periparturient cows to discriminate those metabolic changes associated with the act of parturition from the metabolic changes caused by lactation. Mastectomized and intact cows had similar increases in plasma estrogens and cortisol concentrations around the time of calving. Mastectomy eliminated hypocalcemia and the rise in 9,13-di-cisretinoic acid observed in intact cows. Mastectomy reduced but did not eliminate decreases in plasma phosphorus, α-tocopherol, and β-carotene associated with parturition in intact cows, suggesting the mammary gland is not the sole factor affecting plasma concentrations of these compounds. Dry matter intake was similar in both groups before calving. The day of calving, dry matter intake was lower in intact cows than in mastectomized cows, but after calving the mastectomized cows exhibited a pronounced decline in feed intake. Plasmanonesterified fatty acid (NEFA) concentrations rose rapidly in intact cows at calving and did not return to baseline level for 10 d. In contrast, NEFA concentrations in mastectomized cow plasma rose moderately at calving and returned to baseline level 1 to 2 d after calving. This study provides evidence that hypocalcemia in the cow is solely a result of the calcium drain of lactation. The act of parturition affects blood phosphorus,dry matter intake, and NEFA concentration independent of the effect of lactation.

It is unclear why some cows fail to expel the placenta following calving. One theory suggests the fetal placenta must be recognized as “foreign” tissue and rejected by the immune system after parturition to cause expulsion of the placenta. We hypothesized that impaired neutrophil function causes retained placenta (RP). We examined the ability of neutrophils to recognize fetal cotyledon tissue as assessed by a chemotaxis assay, which utilized a placental homogenate obtained from a spontaneously expelled placenta as the chemoattractant. Neutrophil killing ability was also estimated by determining myeloperoxidase activity in isolated neutrophils. Blood samples were obtained from 142 periparturient dairy cattle in two herds. Twenty cattle developed RP (14.1%). Neutrophils isolated from blood of cows with RP had significantly lower neutrophil function in both assays before calving, and this impaired function lasted for 1 to 2 wk after parturition. The addition of antibody directed against interleukin-8 (IL-8) to the cotyledon preparation used as a chemoattractant inhibited chemotaxis by 41%, suggesting that one of the chemoattractants present in the cotyledon at parturition is IL-8. At calving, plasma IL-8 concentration was lower in RP cows (51±12 pg/ml) than in cows expelling the placenta normally (134±11 pg/ml). From these data, we suggest that neutrophil function is a determining factor for the development of RP in dairy cattle. Also, depressed production of IL-8 may be a factor affecting neutrophil function in cows developing RP.

Neutrophil function is reduced in periparturient dairy cows. Possible factors that reduce neutrophil function include endocrine changes associated with parturition and metabolic stresses associated with lactogenesis. In this study, mastectomized and intact cows were studied to specifically examine the effects of lactogenesis on neutrophil function in periparturient cows. Expression of adhesion molecules on neutrophils (L-selectin, mediating capture and rolling adhesion, and β2- integrin, mediating tight adhesion vital to egress) and neutrophil myeloperoxidase activity (an index of bactericidal activity) were assessed in mastectomized and intact cows. Expression of L-selectin decreased at parturition followed by rapid recovery to prepartum values in both intact and mastectomized cows. Expression of β2-integrins increased in intact cows at parturition but not in mastectomized cows. Expression of β2-integrins was greater in intact cows than in mastectomized cows throughout the study. Neutrophil myeloperoxidase activity decreased from baseline prepartum values as parturition approached in both intact and mastectomized cows, which suggests the endocrine changes associated with the act of parturition are predominant factors causing loss of neutrophil function. Myeloperoxidase activity recovered to prepartum values within a week of parturition in mastectomized cows; however, myeloperoxidase activity remained depressed in neutrophils obtained from intact cows throughout the first 20 d of lactation. The presence of the mammary gland and its attendant metabolic stresses slowed recovery of neutrophil function, which suggests that the metabolic stress of lactation exacerbated periparturient immunosuppression.