Jeanne L. Burton

Transportation of young beef bulls alters circulating physiological parameters that may be effective biomarkers of stress

Transportation causes stress in cattle that may alter numerous physiological variables with a negative impact on production and health. The objectives of the current study were to investigate the physiological effects of truck transportation and to characterize a pattern of phenotypes in the circulation that may aid in the early identification of stress-susceptible animals that often succumb to severe respiratory disease. Thirty-six young beef bulls (Aberdeen Angus, n = 12; Friesian, n = 12; and Belgian Blue x Friesian, n = 12) were subjected to a 9-h truck transportation by road. Blood (10 mL) was collected at -24, 0, 4.5, 9.75, 14.25, 24, and 48 h relative to the initiation of transportation (0 h). Plasma was collected for the assay of various metabolic, inflammatory, and steroid variables, and total leukocyte counts were determined in whole blood at each time point. Body weight and rectal temperature were recorded at -24, 9.75, and 48 h. Transportation decreased measures of protein metabolism in the plasma, including albumin (P = 0.002), globulin (P < 0.001), urea (P = 0.006), and total protein (P < 0.001), and increased creatine kinase (P < 0.001). The energy substrate beta-hydroxybutyrate was not changed (P = 0.27). Acute phase proteins haptoglobin and fibrinogen were both decreased (P < 0.001), whereas total leukocyte counts were elevated (P = 0.002). Circulating steroid concentrations were altered, because a classical acute increase in plasma cortisol was observed with the onset of transit (P < 0.001), in association with a decrease in dehydroepiandrosterone (P = 0.07), resulting in a profound increase in cortisol:dehydroepiandrosterone ratio (P < 0.001). Plasma testosterone was decreased, whereas plasma progesterone was increased (P < 0.001) in association with the increase in cortisol (P < 0.001). There was also an effect of breed for all variables except plasma urea, creatine kinase, and testosterone, perhaps indicating that a genetic component contributed to the physiological response to transportation stress, although without any clear trend. Taken together, this profile of physiological variables in the circulation of transportation-stressed bulls may aid in the future detection of disease-susceptible cattle after transportation. Further research to validate these potential biomarkers is necessary.

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.

An Immunogenomics Approach to Understanding Periparturient Immunosuppression and Mastitis Susceptibility in Dairy Cows

Studies comparing in vivo and in vitro functional capacities of leukocytes from non-parturient and periparturient dairy cows have provided substantial evidence that systemic and local mammary immune defenses are deficient around parturition. This evidence has lead to the reasonable hypothesis that immune deficiency underlies the heightened mastitis susceptibility of periparturient cows. Nutrition and vaccine studies substantiate this hypothesis, showing that dietary antioxidant supplementation and rigorous immunization regimes can bolster innate and humoral immunity to the point that mastitis severity and time for return to normal milk production are reduced. However, completely effective resolution of this significant production disease has not been achieved because so little is understood about its complex etiology. In particular, we possess almost no knowledge of how or why immune cells responding to parturient physiology end up with deficient functional capacities. Fluctuations in reproductive steroid hormones and chronic shifts in neuroendocrine hormones with roles in nutrient partitioning and appetite control may affect the expression of critical leukocyte genes in periparturient dairy cows. A thorough understanding of leukocyte biology during periparturition would seem a critical goal for future development of effective mastitis prevention strategies. Recently, our group has begun to use cDNA microarray technology to explore bovine leukocyte RNA for global gene expression changes occurring around parturition. We are working within the context of a hypothesis that the physiology of parturition negatively affects expression of critical genes in blood leukocytes. In the current study we initiated hypothesis testing using leukocyte RNA from a high producing Holstein cow collected at 14 days prepartum and 6 hours postpartum to interrogate a cDNA microarray spotted with >700 cDNAs representing unique bovine leukocyte genes. This analysis revealed 18 genes with ≥1.2-fold higher expression 14 days prepartum than 6 hours postpartum. BLASTN analysis of these genes revealed only one that can be considered a classical immune response gene. All other repressed genes were either unknown or putatively identified as encoding key proteins involved in normal growth and metabolism of cells. Given the critical roles of these repressed genes in normal cell functions, we may have identified good candidates to pursue with respect to periparturient immunosuppression and mastitis susceptibility.

Generation of EST and cDNA Microarray Resources for the Study of Bovine Immunobiology

Recent developments in expressed sequence tag (EST) and cDNA microarray technology have had a dramatic impact on the ability of scientists to study the responses of thousands of genes to external stimuli, such as infection, nutrient flux, and stress. To date however, these studies have largely been limited to human and rodent systems. Despite the tremendous potential benefit of EST and cDNA microarray technology to studies of complex problems in domestic animal species, a lack of integrated resources has precluded application of these technologies to domestic species. To address this problem, the Center for Animal Functional Genomics (CAFG) at Michigan State University has developed a normalized bovine total leukocyte (BOTL) cDNA library, generated EST clones from this library, and printed cDNA microarrays suitable for studying bovine immunobiology. Our data revealed that the normalization procedure successfully reduced highly abundant cDNA species while enhancing the relative percentage of clones representing rare transcripts. To date, a total of 932 EST sequences have been generated from this library (BOTL) and the sequence information plus BLAST results made available through a web-accessible database http://gowhite.ans.msu.edu. Cluster analysis of the data indicates that a total of 842 unique cDNAs are present in this collection, reflecting a low redundancy rate of 9.7%.For creation of first generation cDNA microarrays, inserts from 720 unique clones in this library were amplified and microarrays were produced by spotting each insert or amplicon 3 times on glass slides in a 48-patch arrangement with 64 total spots (including blanks and positive controls) per patch. To test our BOTL microarray, we compared gene expression patterns of concanavalin A stimulated and unstimulated peripheral blood mononuclear cells (PBMCs). In total, hybridization signals on over 90 amplicons showed upregulation (>3×) in response to Con A stimulation, relative to unstimulated cells. A second experiment with PBMCs from a different group of animals was performed to test reproducibility of microarray results. There was a high correlation between the 2 experiments (r = 0.72, P < 0.001). Resources described in this publication offer a highly efficient and integrated system to study gene expression changes in bovine leukocytes.

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.

Effects of stress on leukocyte trafficking and immune responses: implications for vaccination.

Increased susceptibility of animals to infectious disease during the periparturient period results in suffering and economic losses. Stress appears to delay inflammation by reducing efficiency of CD62L-mediated immune surveillance by phagocytes. It is important to note that the effects of stress are not limited to alteration of leukocyte trafficking patterns since various stressors (e.g., transport, parturition, and castration) also decrease IFN-gamma secretion by lymphocytes, and may decrease antigen presentation efficiency by down-regulating class II molecule expression on antigen presenting cells, and delay or impair immune responses to vaccination. Documented immunosuppression in periparturient animals, particularly the bias toward Th2 immune responses, and also changes in general leukocyte trafficking patterns suggest that vaccination intending to elicit cell-mediated immunity may not be efficacious at this point of the production cycle. Based on findings of numerous periparturient studies on immunosuppression in cattle, waiting at least 30 days after parturition before administering routine vaccinations is recommended.

Microarray analysis reveals difference in gene expression profiles of hair and wool sheep infected with Haemonchus contortus.

Sheep infected with the abomasal parasite, Haemonchus contortus, have reduced growth rates, decreased wool production, and anemia, and heavy infections may result in death. Anthelmintic treatment can remove worms, but the cost of treatment and prevalence of drug-resistant worms has led to greater focus on genetic resistance of the host to parasitism. Variation in parasite resistance exists within and among sheep breeds, and Caribbean hair sheep have greater resistance than most conventional wool breeds. Our objective was to investigate differences in gene expression between 24 parasite-resistant hair and 24 susceptible wool lambs to determine genetic mechanisms involved in resistance to H. contortus. Half of the animals of each breed were infected and sacrificed at 3 or 27 days post-infection; the remaining animals were uninfected controls. Breed differences in abomasum and abomasal lymph node tissue gene expression were assessed using bovine cDNA microarrays. Over 60 transcripts differed between breeds for each tissue and infection status. Genes differentially expressed between hair and wool sheep 3 days PI were assessed for gene function and mechanisms for greater immune cell infiltration, abomasal tissue repair, Th17 response, and anticoagulation were present in parasite-resistant hair sheep. By 27 days PI, hair sheep had greater expression of genes involved in gut motility, inflammatory cytokines, and cell proliferation and differentiation compared to wool sheep. Changes in these processes indicate Caribbean hair sheep have a stronger inflammatory response when infected with H. contortus which may facilitate the increased parasite resistance observed in these sheep.

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

ABSTRACT 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.

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.

Genetic Variation in Bovine Neutrophil Sensitivity to Glucocorticoid Challenge

Blood neutrophils use CD62L and CD18 adhesion molecules to contact and migrate rapidly through blood vessels in defense against infections in underlying tissues. Previous work showed that glucocorticoid hormones repress expression of CD62L and CD18, causing neutrophilia and increased mastitis susceptibility in dairy cows. The aim of this study was to determine whether bovine neutrophil sensitivity to glucocorticoids exhibits genetic variability. Test animals included 60 pedigreed Holstein bulls treated on 3 consecutive days with a synthetic glucocorticoid (dexamethasone) and five untreated control bulls. Five indicator traits of neutrophil glucocorticoid sensitivity were monitored, including circulating neutrophil counts and two measures on each of CD62L and CD18 expression. Random regression models with treatment-specific serial correlation were used to estimate genetic and non-genetic sources of variation before, during and after glucocorticoid administration. Significant genetic variation was observed for neutrophil CD18 expression, with longitudinal heritability estimates ranging from 0.10 to 0.54 and influenced by dexamethasone. Significant genetic variation was also observed for blood neutrophil counts (heritability estimates ranging from 0.11 to 0.24) but was not influenced by dexamethasone administration. Estimated genetic correlations between circulating neutrophil counts and various indicators of CD62L and CD18 expression were large and negative (-0.44 to - 0.78). These results imply significant genetic variability and pleiotropic effects for neutrophil traits that are important for stress-induced disease susceptibility in dairy cattle and might be exploited by genetic selection.