P.S.D. Weber

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.

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.

High titer growth of human and avian influenza viruses in an immortalized chick embryo cell line without the need for exogenous proteases.

The current method of growing influenza virus for vaccine production is through the use of embryonated chicken eggs. This manufacturing system yields a low concentration of virus per egg, requires significant downstream production for purification, and demands a considerable amount of time for production. We have demonstrated an immortalized chick embryo cell line, termed PBS-1, is capable of growing unmodified recent isolates of human and avian influenza A and B viruses to extremely high titers. In many cases, PBS-1 cells out perform primary chick embryo kidney (CEK) cells, Madin-Darby Canine Kidney (MDCK) cells and African green monkey kidney cells (Vero) in growth of recent influenza isolates. PBS-1 cells are free of any exogenous agents, are non-tumorigenic, and are readily adaptable to a variety of culture conditions, including growth on microcarrier beads. Influenza viruses grown in PBS-1 cells are released into the culture fluid without the need for exogenous proteases, thus simplifying downstream processing. In addition to offering a significant improvement in vaccine production, PBS-1 cells should prove valuable in diagnostics and as a cell line of choice for influenza virus research.

Distribution of insulin receptor and insulin-like growth factor-1 receptor in the digital laminae of mixed-breed ponies: an immunohistochemical study.

REASONS FOR PERFORMING STUDY Hyperinsulinaemia has been implicated in the pathogenesis of laminitis; however, laminar cell types responding to insulin remain poorly characterised. OBJECTIVES To identify laminar cell types expressing insulin receptor (IRc) and/or insulin-like growth factor-1 receptor (IGF-1R); and to evaluate the effect of dietary nonstructural carbohydrate (NSC) on their expression. METHODS Mixed-breed ponies (n = 22) received a conditioning hay chop diet (NSC ∼6%); following acclimation, ponies were stratified into lean (n = 11, body condition score [BCS]≤4) or obese (n = 11, BCS ≥7) groups and each group further stratified to remain on the low NSC diet (n = 5 each for obese and lean) or receive a high NSC diet (total diet ∼42% NSC; n = 6 each for obese and lean) for 7 days. Laminar samples were collected at the end of the feeding protocol and stained immunohistochemically for IRc and IGF-1R. The number of IRc(+) cells was quantified; distribution of IGF-1R was qualitatively described. Laminar IRc content was assessed via immunoblotting. RESULTS The number of IRc(+) cells was greater in the laminae of high NSC ponies than low NSC ponies (P = 0.001); there was a positive correlation between the change in serum insulin concentration and number of IRc(+) cells (r(2) = 0.74; P<0.0001). No epithelial IRc(+) cells were observed; IRc(+) cells were absent from the deep dermis. Analysis of serial sections identified IRc(+) cells as endothelial cells. The distribution of IGF-1R was more extensive than that of IRc, with signal in vascular elements, epithelial cells and fibroblasts. CONCLUSIONS Increased dietary NSC results in increased laminar endothelial IRc expression. Laminar keratinocytes do not express IRc, suggesting that insulin signalling in laminar epithelial cells must be mediated through other receptors (such as IGF-1R). POTENTIAL RELEVANCE Manipulation of signalling downstream of IRc and IGF-1R may aid in treatment and prevention of laminitis associated with hyperinsulinaemia.

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.

The effect of ractopamine hydrochloride on gene expression in adipose tissues of finishing pigs.

The long-term effect of feeding the catecholamine analog ractopamine (RAC; ractopamine hydrochloride, Elanco Animal Health, Indianapolis, IN) on the expression of genes involved in energy and lipid metabolism in subcutaneous adipose tissue was studied. Large White pigs (84 kg) were fed corn- and soybean meal-based diets supplemented with 0, 20, or 60 mg/kg of RAC for 14, 28, or 42 d. Expression (mRNA abundance) in adipose tissue of sterol regulatory binding protein-1 (SREBP-1), PPARα, PPARγ2, fatty acid synthase (FAS), glucose transporter 4 (GLUT4), and stearoyl-CoA desaturase was determined by Northern blotting. Feed intakes did not differ, and RAC (20 and 60 mg/kg) improved BW gain at d 14, 28, and 42 (P < 0.05) and increased loin eye area (measured on d 42 only; P < 0.05). Expression of SREBP-1 and PPARγ2 declined (P < 0.05) with RAC by d 28 and 42, whereas expression of PPARα was increased (P < 0.05) on d 14, 28, and 42. After 14 d, expression of FAS and GLUT4 was decreased (P < 0.05) with 60 mg/kg of RAC, whereas both RAC concentrations attenuated FAS expression on d 28 and 42. Overall, adipose tissue stearoyl-CoA desaturase expression was not affected by RAC but showed somewhat less expression (P < 0.15) on d 28 at 60 mg/kg of RAC. Although prolonged, chronic RAC feeding most likely downregulates adipose tissue membrane β-adrenergic receptors, mRNA abundances of anabolic lipid metabolism transcription factors, glucose transporters, and enzymes (SREBP-1, PPARγ2, FAS, GLUT4) were still attenuated up to d 42. Conversely, a transcription factor related to oxidative metabolism expression (PPARα) was enhanced. We conclude that even after 42 d, RAC still decreased expression of lipogenic genes in adipose tissue by yet undefined cyclic adenosine monophosphate-directed mechanisms, but in contemporary lean pigs, this effect is likely of limited practical significance.

Transcript abundance of amino acid transporters, β-casein, and α-lactalbumin in mammary tissue of periparturient, lactating, and postweaned sows

The objective of these experiments was to test the hypothesis that transcript abundance of cationic AA transporter- and milk protein-encoding genes increase in the porcine mammary gland in response to higher lactation demand. Genes of interest included those encoding for the milk proteins α-lactalbumin (α-LA) and β-casein (β-CN; LALBA and CSN2, respectively), and AA transporter b(0,+)AT, y(+)LAT1, y(+)LAT2, ATB(0,+), CAT-1, and CAT-2b (SLC7A9, SLC7A7, SLC7A6, SLC6A14, SLC7A1, and SLC7A2, respectively). Mammary tissue was biopsied from 4 sows on d 110 of gestation (prepartum), on d 2 (early postpartum), on d 5 (early), and d 17 (peak) of lactation, and on d 5 after weaning (postweaning), and mRNA of target genes quantified by reverse transcription quantitative PCR. Compared with prepartum, CAT-1, ATB(0,+), y(+)LAT2, β-CN, and α-LA mRNA abundance was higher at early lactation, whereas compared with early lactation, only CAT-1 and α-LA mRNA abundance was higher at peak lactation. The CAT-2b, y(+)LAT1, and b(0,+)AT mRNA abundance did not differ when comparing either prepartum or peak lactation to early lactation. Compared with peak lactation, postweaning mRNA abundance of CAT-1, ATB(0,+), α-LA, and β-CN decreased, y(+)LAT2, CAT-2b, and b(0,+)AT remained unchanged, and y(+)LAT1 increased. The mRNA abundance of y(+)LAT2 increased from early postpartum to early lactation, and remained unchanged for CAT-1, ATB(0,+), α-LA, and β-CN. From prepartum to peak lactation, the mRNA abundance of CAT-1, y(+)LAT2, and ATB(0,+) was positively correlated with that of β-CN and α-LA. In conclusion, the expression of genes encoding for y(+)LAT1, CAT-2b, and b(0,+)AT remained unchanged in porcine mammary glands over prepartum to peak lactation period, whereas expression of genes encoding for CAT-1, ATB(0,+), and y(+)LAT2 was upregulated and positively correlated to expression of genes encoding for the mammary synthesized milk proteins β-CN and α-LA.

Laminar inflammatory events in lean and obese ponies subjected to high carbohydrate feeding: Implications for pasture-associated laminitis.

REASONS FOR PERFORMING STUDY Acute, massive enteral carbohydrate overload is associated with laminar inflammation in equids; it is unclear if the same is true for a more prolonged period of moderate dietary carbohydrate intake. OBJECTIVES To characterise laminar inflammation in ponies exposed to a dietary carbohydrate challenge meant to mimic acute pasture exposure. STUDY DESIGN In vivo experiment. METHODS Mixed-breed ponies (n = 22) received a diet of hay chop (nonstructural carbohydrate [NSC] ∼7% on a dry matter [DM] basis) for 4 weeks prior to initiation of the experimental feeding protocol. Following dietary acclimation, ponies were stratified into either Lean (n = 11, body condition score [BCS] ≤4) or Obese (n = 11, BCS ≥7) groups and each group further stratified to either remain on the control, low NSC diet (n = 5 each for Obese and Lean) or receive a high NSC diet (hay chop supplemented with sweet feed and oligofructose, total diet ∼42% NSC; n = 6 each for Obese and Lean) for a period of 7 days. Laminar samples were collected following euthanasia and sections stained immunohistochemically for CD163, MAC387/calprotectin and cyclo-oxygenase-2 (COX-2) using commercially available antibodies. The number of CD163 (+) and MAC387(+) cells was quantified for each section; the distribution of COX-2 expression was qualitatively assessed. Laminar mRNA concentrations of several proinflammatory molecules (interleukin-1β [IL-1β], IL-6, tumour necrosis factor-α [TNFα], IL-8, IL-10, monocyte chemoattractant protein-1 [MCP-1], MCP-2), inducible nitric oxide synthase (iNOS), intercellular adhesion molecule-1 (ICAM-1), E-selectin, plasminogen activator inhibitor-1 (PAI-1) and COX-2 were evaluated using real-time quantitative polymerase chain reaction (qPCR). RESULTS High carbohydrate feeding resulted in no increase in laminar proinflammatory cytokine expression; laminar COX-2 expression was increased by high carbohydrate feeding. No laminar leucocyte infiltration was observed in response to high carbohydrate feeding. CONCLUSIONS These results suggest that the marked laminar inflammation observed in models of sepsis-associated laminitis may not play a central role in the pathophysiology of pasture-associated laminitis.

Immortalized chick embryo cell line adapted to serum-free growth conditions and capable of replicating human and reassortant H5N1 influenza strains for vaccine production

The current process of influenza vaccine production can take 6-9 months and is dependent on the availability of embryonated eggs. Additionally, this process selects for receptor-binding variants with reduced antigenicity and requires significant downstream production for purification. We have established an immortalized chick embryo cell line, termed PBS-12SF, which is adapted to growth in serum free conditions, and is capable of replicating human and reassortant H5N1 influenza strains to high titers. In many cases, PBS-12SF cells produced higher growth titers of influenza virus than those of primary chick embryo kidney (CEK) cells, Madin-Darby Canine Kidney (MDCK) cells and African green monkey kidney cells (Vero). Additionally, in PBS-12SF cell cultures, influenza virus is released into the culture fluid without need for exogenous proteases, which can simplify downstream processing for vaccine production.