Dietary protein intake and stage of lactation differentially modulate amino acid transporter mRNA abundance in porcine mammary tissue.

To test the hypothesis that under restricted and surfeit protein intake the mammary gland undergoes adaptive regulation, changes in mammary tissue mRNA abundance of cationic amino acid (AA) transporter (CAT)-1, CAT-2B, alanine/serine/cysteine/threonine transporter 1 (ASCT1), and broad specificity transporter for neutral and cationic AA (ATB(0,+)), and CAT-1 protein abundance were investigated at 2 stages of lactation. Eighteen sows were allocated to a 2 x 3 randomized incomplete block design with 2 stages of lactation (early and peak) and 3 protein levels: deficient (D), adequate (A), or in excess (E) of lactation requirement. In early lactation, compared with A, sows fed E had lower (P = 0.05) piglet growth rate and sows fed D or E had lower (P < or = 0.05) casein yield. In early lactation, piglet growth rate and milk protein and casein yield increased from D to A and decreased from A to E (quadratic, P = 0.095, P < 0.05, and P < 0.01, respectively). Protein intake did not affect CAT-1, ASCT1, ATB(0,+) mRNA abundance, or CAT-1 protein level. Overall, CAT-2B mRNA abundance decreased linearly with increasing protein intake (P < 0.05). Compared with A, E decreased CAT-2B mRNA abundance (P < 0.05). Compared with early lactation, peak lactation did not increase CAT-1 mRNA abundance or relative CAT-1 protein content, but increased abundance of ASCT1 and ATB(0,+) mRNA (P < 0.01). Mammary CAT-2B appears to be adaptively regulated in vivo at the transcription level, whereas ASCT1 and ATB(0,+) mRNA abundances are associated only with stage of lactation. Neither protein intake nor stage of lactation affects porcine mammary CAT-1 gene expression in vivo.

Association of Escherichia coli J5-specific serum antibody responses with clinical mastitis outcome for J5 vaccinate and control dairy cattle.

Dairy cattle in two commercial Holstein herds were randomly selected to be vaccinated twice with J5, at approximately 60 days and 28 days before the expected calving date, or to be untreated controls. Based on whether milk production changed following clinical mastitis or whether cows were culled or died within 30 days after onset, 51 mastitis cases were classified as severe or mild. J5-specific antibody responses were evaluated by enzyme-linked immunosorbent assay of all 32 severe and 19 mild cases. The amounts of J5-specific immunoglobulin M (IgM), IgG1, and IgG2 antibodies in sera from the 27 J5 vaccinates were compared with those of the 24 controls. At drying off (before J5 vaccination), all cows had similar amounts of J5-specific antibody. Immediately after calving (approximately 28 days after the second vaccination), J5 vaccinates had significantly higher production of J5-specific IgG1 and IgG2 than controls. When cows were tested following clinical mastitis, none of the three antibody classes differed significantly between the controls and the vaccinates. Vaccinates that contracted Escherichia coli mastitis had 75% less milk loss than controls. The cows that contracted clinical mastitis later in lactation, the unvaccinated controls, and those infected with E. coli had more milk loss following mastitis. The hazards of being culled for all reasons and of being culled for mastitis were significantly lower for J5 vaccinates. Vaccination with J5 was associated with protection against milk production loss and culling following clinical mastitis, and it was also significantly associated with changes in J5-specific IgM, IgG1, and IgG2 antibodies in sera of vaccinated cows.

Effect of hyperimmunization with an Escherichia coli J5 bacterin in adult lactating dairy cows.

OBJECTIVE: To determine the effect of hyperimmunization with an Escherichia coli J5 bacterin on serum IgG2 concentration, incidence of clinical mastitis, and rate of survival to the end of the lactation period (ie, day 305) in adult lactating dairy cattle. DESIGN: Randomized controlled trial. ANIMALS: 1,012 Holstein cows in their second lactation and greater. PROCEDURES: All cows were given 3 doses of the J5 bacterin; cows in the hyperimmunization group were given an additional 3 doses during the first 3 months of lactation. Blood was collected from a small sample of cows to determine anti-J5 IgG2 concentrations. RESULTS: Cows in the hyperimmunization group had higher mean serum anti-J5 IgG2 concentrations than did control cows 28 days after administration of the fourth, fifth, and sixth doses of the J5 bacterin. However, mean serum anti-J5 concentrations during the subsequent lactation were not significantly different between groups. The proportions of cows that developed clinical mastitis were not significantly different between groups. However, control cows were more likely to have severe clinical mastitis than were cows in the hyperimmunization group. The percentage of control cows that remained in the herd to day 305 was significantly lower than the percentage of cows in the hyperimmunization group that did. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that hyperimmunization of mature lactating dairy cattle was associated with increased serum anti-J5 IgG2 concentrations and decreased incidence of severe clinical mastitis, but did not alter survival rate of cows that developed severe clinical mastitis.

Gene expression profiling of peripheral mononuclear cells in lame dairy cows with foot lesions.

Lameness is a major health issue and likely the single most common cause of pain and discomfort in dairy cattle. Appropriate treatment is delayed or neglected due, in part, to lack of reliable detection. Assessment of cows with lameness is currently limited to subjective visual scoring systems based on locomotion and posture abnormalities. These systems are unreliable to detect lameness, and therefore, a large number of cows remain undiagnosed. The objective of this research was to search for potential biomarkers for lameness-associated painful inflammatory foot lesions in dairy cattle using microarray-based gene expression profiling of peripheral blood mononuclear cells (PBMC). BOTL5 microarrays spotted in duplicate with cDNA representing bovine immune response genes were interrogated with cDNA samples in an eight-array, balanced complete block design with dye swap. Samples from eight lame cows with inflammatory foot lesions and from eight sound cows were pair-matched by age, weight, days in lactation, and pregnancy status at time of PBMC collection and directly compared with each other on individual arrays. Statistical analysis of resulting fluorescence intensity data revealed 31 genes that were putatively differentially expressed in lame versus sound cows (P<0.05). Of these, BLASTn analysis and gene ontology information showed that 28 genes had high similarity or homology to known human and/or rodent genes. Validation of 15 of these genes known to be important in inflammation and pain was carried out using relative quantitative real-time RT-PCR, which confirmed the up-regulation of interleukin (IL)-2 (12.68+/-1.47-fold increase) and IL-10 (2.39+/-0.55-fold increase), matrix metalloproteinase-13 (MMP-13) (10.44+/-1.14-fold increase), and chemokine C-C motif receptor-5 (CCR5) (5.26+/-1.05-fold increase), in lame relative to sound cows (P< or =0.05). Similarly, granulocyte-macrophage colony-stimulating factor receptor alpha chain precursor (GM-CSF-R-alpha) (2.30+/-0.63-fold increase) and IL-4 (2.06+/-0.59-fold increase) showed a tendency (P=0.10) for up-regulation in lame compared to sound cows. PBMC co-expression of IL-2, MMP-13, CCR5 and IL-10, and potentially IL-4 and GM-CSF-R-alpha appears to be a promising, objective sign of lameness-related inflammatory foot lesions in dairy cattle. In conclusion, this study revealed potential biomarkers of the presence of foot lesions that could boost diagnostic accuracy of lameness and, ultimately, help identify animals in need of pain relief.

Milk and serum J5-specific antibody responses, milk production change, and clinical effects following intramammary Escherichia coli challenge for J5 vaccinate and control cows.

Holstein dairy cows (four J5 vaccinates and four controls) selected for no recorded intramammary disease and low somatic cell count (SCC) during the previous lactation were challenged by intramammary infusion of Escherichia coli. Vaccination with J5 was at 8 weeks and again 4 weeks before the anticipated calving date. Cows were challenged at 8 to 16 days in milk (DIM). Shedding of E. coli in milk was significantly higher among controls than vaccinates (no shedding) from 6 h to 21 h postchallenge. From 21 h to 132 h postchallenge, SCC in challenged quarters of controls (5,429,000/ml) was significantly higher than that of vaccinates (490,000/ml). On the day after challenge, milk production in control cows was 8 kg less, while vaccinates gained 0.5 kg, a significant difference. In serum immediately prior to challenge, J5-specific immunoglobulin G1 (IgG1) was significantly higher, IgG2 was nearly significantly higher, and IgM was the same in J5 vaccinates relative to controls. Vaccinates had proportionally more IgG2 in serum postcalving and in the first 12 h following challenge and less IgG2 in milk 24 h after challenge than the controls, approaching statistical significance. The ratio of J5-specific IgG1 and IgG2 combined compared to IgM was significantly higher in vaccinates than in controls in prechallenge serum (ratios of 15.8 and 3.2, respectively) and milk (5.0 and 1.3, respectively). Cows with higher IgM titers in milk 12 h postchallenge produced significantly less milk. Vaccination with J5 was significantly associated with higher production of J5-specific IgG1 and IgG2 in early lactation, reduced SCC, faster clearance of E. coli from milk, and less milk production loss following intramammary challenge.

Glucocorticoid modulation of Bcl-2 family members A1 and Bak during delayed spontaneous apoptosis of bovine blood neutrophils.

Neutrophils are critical for innate immune defense against microbial invasion but can also cause inflammatory tissue damage if their life span is not tightly regulated. Antiinflammatory glucocorticoids delay spontaneous apoptosis in human, rodent, and bovine neutrophils, but mechanisms involved are unknown. We hypothesized here that glucocorticoids delay neutrophil apoptosis by altering expression of key Bcl-2 apoptosis regulatory proteins, A1 and Bak, via activation of the cell's glucocorticoid receptors. To test this hypothesis, isolated bovine blood neutrophils were exposed to dexamethasone with and without glucocorticoid receptor antagonism (RU486) and aged ex vivo over 0-24 h for assessment of various spontaneous apoptosis pathway indicators and A1 and Bak abundance. Results show that dexamethasone preserved neutrophil mitochondrial membrane integrity, delayed caspase-9 activation, and reduced the rate of spontaneous apoptosis. Also, dexamethasone increased A1 and decreased Bak mRNA abundance. RU486 pretreatment of the cells abrogated each of these dexamethasone effects. Dexamethasone-induced increases in A1 mRNA were reflected in A1 protein increases, which also were observed in circulating neutrophils of dexamethasone-treated animals. Bak protein decreases were observed in neutrophils of the dexamethasone-treated animals but not in isolated neutrophils, suggesting that stimuli additional to (and perhaps regulated by) glucocorticoid are required to affect Bak protein expression changes in neutrophils. Collectively, our results are unique in demonstrating a mechanism behind glucocorticoid regulation of spontaneous apoptosis and implicate steroid receptor activation and subsequent regulation of A1 and Bak as contributors to mitochondrial membrane stability, reduced caspase-9 activity, and delayed apoptosis in bovine neutrophils exposed to glucocorticoids.

Gene expression signatures in neutrophils exposed to glucocorticoids: a new paradigm to help explain "neutrophil dysfunction" in parturient dairy cows.

Neutrophils are the first line of immunity against most pathogens that infect cattle. These normally short-lived white blood cells develop from myeloid-lineage cells in bone marrow. Upon maturation, bone marrow neutrophils are released into the circulation where they marginate on inflamed blood vessel endothelial cells and migrate through them into the area of infection. Once migrated, neutrophils do not reenter the circulation, but rather, perform their bactericidal functions and die by apoptosis in the tissue. The cytokine and hormonal milieu of the blood and extracellular tissue fluid can influence neutrophil development and immunity-related activities, but the molecular basis of these phenotypic changes and physiological benefits or drawbacks of them are poorly understood. In the current paper, we review new gene expression information that resulted from two of our functional genomics studies designed to evaluate effects of glucocorticoid hormones on bovine neutrophils. This work provides one model to describe complex changes that occur in neutrophils as the cells respond to glucocorticoids, which might act to alter the cells' functional priorities and tip the delicate balance between health and disease during stress, including at parturition. A bovine immunobiology microarray and real time RT-PCR were used to study blood neutrophils collected during the natural surge of endogenous glucocorticoid (cortisol) in parturient dairy cows and bone marrow neutrophils collected from glucocorticoid (dexamethasone)-treated dairy steers. The gene expression signatures we observed led us to perform additional phenotyping of the neutrophils and correlation analyses, which together painted a picture suggesting that glucocorticoids have key roles in modulating neutrophil development, life span, and tissue defense functions during parturition and hormone therapy. Based on these observations, we postulate that glucocorticoids orchestrate adaptive changes in the entire neutrophil system that support increased cell numbers and longevity in blood and heightened remodeling activity in tissues, while at the same time decreasing some important antimicrobial defense activities of the cells. Thus, our functional genomics studies have enabled us to elucidate multiple consequences of neutrophil exposure to glucocorticoids, highlighting a probable role for this interaction in the induction of parturition and partly explaining why some parturient dairy cows may experience heightened incidence and severity of inflammatory diseases like mastitis.

Effects of glucocorticoids on Fas gene expression in bovine blood neutrophils.

Blood neutrophils are extremely short-lived cells that are programmed for rapid apoptosis after differentiation in bone marrow. Recently, glucocorticoids have been shown to prolong survival of human and rodent neutrophils, but the mechanisms and implications for leukocyte homeostasis and health are unclear. In this study, we investigated the effects of endogenous and exogenous glucocorticoids on Fas expression in bovine neutrophils because Fas is a major death receptor that stimulates apoptosis in circulating cells. Our study subjects were four periparturient dairy cows whose blood concentrations of cortisol peaked at calving, 15 dexamethasone-treated steers and three untreated steers whose neutrophils were exposed to dexamethasone in vitro. Fas mRNA abundance changes in collected neutrophils were monitored numerous times relative to the in vivo glucocorticoid challenges, and the relationships between these data and circulating neutrophil counts were estimated by correlation analyses. Fas mRNA and protein abundance, caspase 8 activity, and survival of neutrophils in vitro were also monitored in the presence and absence of dexamethasone. In the periparturient cows, Fas mRNA abundance in circulating neutrophils showed a sharp decrease between calving and 12 h postpartum. Based on PROC CORR analysis (SAS), this correlated negatively with blood neutrophil count (r=-0.634; P=0.0009) and serum cortisol concentration (r=-0.659; P<0.0001), but showed no relationship with serum progesterone or estradiol concentrations (P > or =0.09). Administration of dexamethasone to steers also caused a pronounced reduction in neutrophil Fas mRNA abundance that persisted for 12 h and correlated negatively with blood neutrophil count (r=-0.748; P=0.0021). In vitro, dexamethasone caused dose-dependent loss of GR proteins from the cytosol of neutrophils concurrently with Fas mRNA downregulation, which was inhibited by the glucocorticoid receptor (GR) antagonist, RU486. Dexamethasone treatment of cultured neutrophils also reduced surface Fas expression, spontaneous and sFasL-induced caspase 8 activity, and rate of apoptosis in the cells. Taken together, these in vivo and in vitro results suggest that glucocorticoids inhibit Fas expression in bovine blood neutrophils via GR activation, possibly contributing to the cells' increased longevity in culture and the pronounced neutrophilia observed in parturient cows and hormone-treated steers. We thus conclude that glucocorticoid-activated GR may change the homeostasis of circulating neutrophils, in part through its negative effects on Fas gene expression and downstream apoptosis signaling pathways.

Mechanisms of glucocorticoid-induced down-regulation of neutrophil L-selectin in cattle: evidence for effects at the gene-expression level and primarily on blood neutrophils.

One anti-inflammatory action of glucocorticoids is down-regulation of surface L-selectin on circulating neutrophils. However, it is unclear if this is a result of release of affected bone marrow neutrophils or if the steroid has direct effects on L-selectin expression in existing blood neutrophils. We recently demonstrated that circulating neutrophils from cattle with high blood concentrations of endogenous glucocorticoid had reduced L-selectin mRNA, suggesting that the steroid interrupted L-selectin gene expression. In the current study, dexamethasone (DEX) was administered to cattle in vivo, and blood and bone marrow neutrophils were studied simultaneously within the animal to determine which pool of cells responds to glucocorticoids with inhibited L-selectin expression. Purified blood neutrophils were also treated with DEX +/- RU486 in vitro, and glucocorticoid effects on L-selectin expression were determined. Our results indicate that glucocorticoid-induced suppression of L-selectin, which accompanies neutrophilia, is likely mediated by direct effects of glucocorticoid receptor activation on intracellular reservoirs of L-selectin mRNA and protein in cattle, predominantly in blood neutrophils.

Microarray analysis of gene expression in blood neutrophils of parturient cows.

It is well documented that blood neutrophils from parturient dairy cows do not perform as well as neutrophils from nonparturient cows in laboratory assays of adhesion, migration, or phagocytosis-induced respiratory burst. However, little is known about the possible molecular basis for parturition-induced changes in neutrophils. cDNA microarray analysis was used in the current study to explore parturition-induced changes in gene expression profiles in bovine blood neutrophils. Total RNA from isolated blood neutrophils of four parturient Holstein cows was obtained before, during, and after parturition, reverse transcribed into cDNA, and sequentially labeled with Cy3 or Cy5 dyes prior to paired hybridizations to 1,056 member bovine total leukocyte (BOTL-3) microarrays in a loop design. Resulting gene expression data were LOWESS normalized by array and analyzed using a mixed model approach. Results showed that expression profiles for 302 BOTL-3 genes were influenced by parturition. BLASTn analysis and preliminary clustering of affected genes by biological function indicated that the largest proportion (14%) of changed genes encode proteins critical to regulation of apoptosis. Independent confirmation of altered expression for 16 of these genes was achieved using quantitative real-time RT-PCR (Q-RT-PCR). A predominantly survival phenotype inferred from the microarray and Q-RT-PCR results was substantiated by monitoring apoptosis status of blood neutrophils from castrated male cattle cultured in the presence of sera from parturient cows. Thus our combined gene expression and apoptosis phenotyping results suggest that bovine parturition may induce prolonged survival in normally short-lived blood neutrophils.

Development and testing of a high-density cDNA microarray resource for cattle.

A cDNA microarray resource has been developed with the goal of providing integrated functional genomics resources for cattle. The National Bovine Functional Genomics Consortium's (NBFGC) expressed sequence tag (EST) collection was established in 2001 to develop resources for functional genomics research. The NBFGC EST collection and microarray contains 18,263 unique transcripts, derived from many different tissue types and various physiologically important states within these tissues. The NBFGC microarray has been tested for false-positive rates using self-self hybridizations and was shown to yield robust results in test microarray experiments. A web-accessible database has been established to provide pertinent data related to NBFGC clones, including sequence data, BLAST results, and ontology information. The NBFGC microarray represents the largest cDNA microarray for a livestock species prepared to date and should prove to be a valuable tool in studying genome-wide gene expression in cattle.

Immunity and mastitis. Some new ideas for an old disease.

The biggest challenge for host immune defense against mastitis-causing bacteria in dairy cows is to quickly recruit large enough numbers of opsonizing molecules and mature neutrophils into milk such that intramammary pathogens are cleared before they multiply significantly and the inflammatory response gets out of control. Currently, this challenge is best facilitated when established mastitis control procedures are practiced on the farm, including proper hygiene, milking procedures, and regular administration of approved mastitis vaccines. However, mastitis is still a significant problem. New animal functional genomics research is beginning to allow scientists to solve the puzzle of mastitis susceptibility. Results of this type of research offer the hope of giant leaps toward a clear identification of molecular genetic variation and potential gene targets for therapies and immune manipulations that could significantly reduce the risk of clinical mastitis in traditionally susceptible cows.

Altered expression of cellular genes in neutrophils of periparturient dairy cows.

The periparturient dairy cow undergoes a plethora of physiological changes, including changes in the immune system that lead to profound effects on animal health. Of the immune cells affected at parturition, the neutrophil has been of particular interest due to its primary role in innate immune defense against mastitis. Immune functions of bovine neutrophils are known to be depressed around parturition, but it has not been discerned at what level these alterations occur, including the possibility that parturition induces changes in expression of key genes. The hypothesis of the present study was that blood neutrophils respond to the physiology of parturition by altering the expression of genes needed for normal cellular functions. The main objectives of the study were to detect and characterize parturition induced changes in neutrophil gene expression, to determine if altered gene expression was significantly associated with the main steroid hormones of bovine parturition, and to obtain putative identities of differentially expressed neutrophil genes. Differential gene expression was detected and characterized through mRNA abundance changes in neutrophils, and steroid hormone concentrations by RIA assay of periparturient serum samples. Preliminary assessment of differential gene expression was done using differential display reverse transcription polymerase chain reaction (DDRT-PCR) followed by secondary screening using high throughput cDNA dot blot hybridization. Altered gene expression was confirmed using Northern blot hybridization and detailed expression patterns characterized using quantitative slot blot analysis. The identities of two fully characterized transcripts with clear parturition induced repression (P< or =0.02) in neutrophils had high DNA sequence homology with genes that encode bovine mitochondrial cytochrome b (cytb) and rig/ribosomal protein S15 (rig/RPS15). These proteins are critical for normal respiratory metabolism and translation in cells, respectively. The gene expression profiles for cytb were significantly related to serum progesterone concentration (r=0.44) and for rig/RPS15 to progesterone and estradiol concentrations (r=0.35, 0.36, respectively). Eleven additional transcripts showed evidence of parturition induced repression in neutrophils and were putatively identified as representing genes of the citric acid cycle and various DNA binding proteins. Results of this study show for the first time that genes regulating basic life functions of bovine neutrophils may be repressed by parturition, possibly due to influences of steroid hormones.