Neutrophil potential phagocytic capacity (NPPC), measured on 25 AI Canadian Holstein bulls, was investigated for evidence of association with production and type traits, SCC, and survival in dairy cows. Bulls were ranked based on different degrees of NPPC (Uptakes of 0, 1, 2, and 3 or more latex beads), using the solutions coming from an animal model. A total of 42,103 first lactation records, collected from 1985 through 1993 in 2,919 Quebec dairy herds, were used to obtain EBV's for SCC and for log SCC (LogSCC) for 697 sires. Correlations between NPPC measurements and somatic cell EBV's were null. Canadian official ETA's for type traits related with mammary system had a tendency of being positively correlated with higher NPPC and negatively with Uptake-0. Canadian official ETA's for production traits were negatively correlated with higher NPPC and positively with Uptake-0. A total of 17,202 first lactation records of daughters of the 25 AI bulls were used to study the effect of NPPC and log SCC on survival in dairy cows. Survival after first lactation was more closely related to sires' NPPC-EBV's than to LogSCC-EBV.

Diapedesis, phagocytosis and microbicidal activity are important parameters of neutrophil functionality and thus outcome of mastitis. An in vitro model of an "alveolar pavement" using the MAC-T3 bovine mammary epithelial cell line was developed to assess neutrophil diapedesis. Features of this biologically-meaningful barrier include: characteristic transepithelial resistance, tight junction complexes and polarity. Continuous transepithelial resistance measurements showed no significant changes throughout the assay period. Neither a Staphylococcus aureus challenge ($1 times10 sp7$ and $2 times10 sp9$ cfu/ml), or the presence of neutrophils, both resting and challenged had any deleterious effects on monolayer integrity over a short term (1-2 h) exposure. Neutrophils, both resting and challenged gave no indication of causing damage to the epithelium over the short term. Neutrophils isolated from proven sires and evaluated for phagocytic activity were found to differ significantly (p $<$ 0.05) in activity, rate and capacity to uptake particles. Correlations between phagocytic parameters and production traits were negative and small in magnitude. Microbicidal activity of neutrophils isolated from proven sires showed a highly significant variation between animals due to test day (p $<$ 0.001), however variation due to source of cells (i.e. animal) was not significant. in vitro analysis of diapedesis and phagocytosis is promising as a tool for the assessment of resistance or susceptibility to mastitis.

Bovine mastitis due to bacterial infection is one of the most costly diseases affecting the dairy industry. The polymorphonuclear neutrophils (PMNs) present in milk have a central protective role against invading pathogens, However, the manner by which PMNs traverse the secretory epithelia and the relationship between PMN diapedesis and the epithelial damage are unclear. This in vitro study investigated the process and rate of bovine PMN transepithelial migration. The bovine mammary epithelial cell line, MAC-T, formed a confluent monolayer with characteristic tight junctions, polarity and functional barrier to the dye trypan blue. In the first series of experiments, neutrophils were added into the upper compartment of the culture insert and stimulated to migrate across the epithelium in an apical-to-basal direction by the addition of Staphylococcus aureus to the lower compartment. Light and transmission electron microscopy revealed the following series of events for PMN transmigration: (1) adherence of PMNs to the surface of the epithelium; (2) projection of pseudopods toward the intercellular junction; (3) migration between adjacent epithelial cells; and (4) re-approximation of epithelial cell membranes and reformation.

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Bovine mammary glands respond to infection by foreign pathogens such as Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) through changes in gene expression. Monitoring the gene expression profiles will contribute to better understanding of the pathology of mastitis, and provide important selective markers for future animal breeding programs. Using cultured bovine mammary duct epithelial cells and somatic cells from infected bovine mammary glands, this study first examined the existence of Toll Like Receptors in these two systems. In cultured duct epithelial cells stimulated with E. coli LPS, both TLR 4 and 2 mRNA up regulation was detected at 2h-72h and 12h-48h respectively. For S. aureus LTA TLR 2 mRNA was up regulated at 48 and 72h whereas for TLR 4 mRNA expression up regulation was detected at 24, 48, and 72h in comparison to the Oh (p&lt;0.05). In the case of PGN, an abundant structural component of S. aureus, the expression of TLR 2 mRNA was significant (p&lt;0.05) at 72h whereas TLR 4 mRNA expression increased at 24, 48, and 72h. The expression of these receptors was also monitored in milk cells from cows infected with either E. coli or S. aureus. However, results obtained from the milk cells were inconclusive due to the high individual variability. Afterwards, differential gene expression profiles were monitored by the Differential Display Polymerase Chain Reaction technique in the cultured duct epithelial cells in response to E. coli and S. aureus structural components. A total of 6 candidate fragments were identified for E. coli LPS induction, whereas only one fragment was identified for S. aureus LTA induction. After LTA induction, a specific band was found to be up regulated and confirmed to be GCP-2, a chemokine involved in neutrophil recruitment. In contrast, PGN induction resulted in no change in GCP-2 levels. In different preparations of cultured duct epithelial cells both GCP-2 and IL-8 were confirmed by real time PCR to be up regulated by LTA with a significance of (p&lt;0.01) when compared to the control cells. In the case of the E. coli identified bands, a different approach is necessary to potentially confirm the origin of these fragments. Further large scale screening of the GCP-2 and IL-8 genes in dairy cattle is necessary to test for their potential use as targets to differentiate the mastitis resistant from the mastitis prone cows.

The purpose of this study was to investigate which type(s) of somatic cells release nitric oxide (NO) in response to Escherichia coli lipopolysaccharide (LPS) and cytokines in vitro and how NO affects superoxide anion (O2-) production by bovine neutrophils and blood monocytes. Mammary epithelial cell line (FbE) released NO after stimulation with recombinant bovine interleukin-1beta (rBoIL-1beta). Moreover, monocytes produced NO in response to recombinant bovine interferon gamma (rBoIFN-gamma) alone or in combination with LPS in a dose- and time-dependent manner. Nitric oxide production was diminished by addition of inducible nitric oxide synthase (iNOS) inhibitors L-N 6-(1-Iminiethyl)lysine or aminoguanidine. However, NO release could not be induced in freshly isolated bovine neutrophils under the experimental conditions used, even after 96 h of incubation. Interestingly, when reverse transcriptase polymerase chain reaction (RT-PCR) with specific primers for iNOS was performed to study mRNA expression, iNOS expression was observed in both monocytes and neutrophils in response to LPS and rBoIFN-gamma. / Unlike neutrophils, monocytes were poor producers of superoxide anion under the experimental conditions. A neutrophil-monocyte co-culture system was set up to study the effect of monocyte derived-NO and iNOS inhibitors on superoxide anion production by neutrophils. Neither NO derived from activated monocytes nor iNOS inhibitors seemed to have an effect on bovine neutrophil ability to release O2-. These results suggest that mammary epithelial cells and mononuclear phagocytes are among the cell types responsible for the important quantities of NO released by somatic cells recovered from LPS-infused mammary quarters during endotoxin-induced bovine mastitis. In addition, NO or iNOS inhibitors have no effect on the ability of activated bovine neutrophils to produce superoxide anions.

The objective of this study was to investigate the effect of recombinant human interleukin-1beta (rHIL-1beta) and interleukin-8 (rHIL-8) on bovine neutrophil migration and degranulation. An in vitro co-culture system was used to study bovine neutrophil migration. This simulative system allowed studying neutrophil migration across endothelium (bovine aorta endothelial cells), extracellular matrix (ECM), and epithelium (MAC-T) in the correct sequences and directions. Quantification of neutrophil migration was carried out by assaying the activity of myeloperoxidase, a major enzyme of neutrophils. Degranulation of azurophilic, specific, and tertiary granules was studied by measuring releases of myeloperoxidase, lactoferrin, and gelatinase, respectively. The results showed that bovine neutrophils were able to migrate across the simulative co-culture system in response to zymosan activated serum. Recombinant HIL-8 was demonstrated to have a dose-dependent effect on bovine neutrophil migration. Furthermore, rHIL-8 had a dose-dependent effect directly on degranulation of azurophilic and specific granules, but not on tertiary granules. On the other hand, rHIL-1beta only had a significant effect on degranulation of azurophilic granules when the concentration of 100 ng/ml was used. The dose effect of rHIL-1beta on specific degranulation was much stronger. Moreover, the effect of 100 ng/ml rHIL-1beta was augmented when the rHIL-1beta containing solution was preincubated with MAC-T monolayers for four hours. This indicated that MAC-T cells might generate other degranulating factors in response to the stimulation of rHIL-1beta. These MAC-T-derived degranulating factors did not have effect on the release of tertiary granule contents.

No description available.