Impact of the host immune response on the development of equine herpesvirus myeloencephalopathy in horses.

Herpesviruses establish a well-adapted balance with their host's immune system. Despite this co-evolutionary balance, infections can lead to severe disease including neurological disorders in their natural host. In horses, equine herpesvirus 1 (EHV-1) causes respiratory disease, abortions, neonatal foal death and myeloencephalopathy (EHM) in ~10 % of acute infections worldwide. Many aspects of EHM pathogenesis and protection from EHM are still poorly understood. However, it has been shown that the incidence of EHM increases to >70 % in female horses >20 years of age. In this study we used old mares as an experimental equine EHV-1 model of EHM to identify host-specific factors contributing to EHM. Following experimental infection with the neuropathogenic strain EHV-1 Ab4, old mares and yearling horses were studied for 21 days post-infection. Nasal viral shedding and cell-associated viremia were assessed by quantitative PCR. Cytokine/chemokine responses were evaluated in nasal secretions and cerebrospinal fluid (CSF) by Luminex assay and in whole blood by quantitative real-time PCR. EHV-1-specific IgG sub-isotype responses were measured by ELISA. All young horses developed respiratory disease and a bi-phasic fever post-infection, but only 1/9 horses exhibited ataxia. In contrast, respiratory disease was absent in old mares, but all old mares developed EHM that resulted in euthanasia in 6/9 old mares. Old mares also presented significantly decreased nasal viral shedding but higher viremia coinciding with a single fever peak at the onset of viremia. According to clinical disease manifestation, horses were sorted into an EHM group (nine old horses and one young horse) and a non-EHM group (eight young horses) for assessment of host immune responses. Non-EHM horses showed an early upregulation of IFN-α (nasal secretions), IRF7/IRF9, IL-1ß, CXCL10 and TBET (blood) in addition to an IFN-γ upregulation during viremia (blood). In contrast, IFN-α levels in nasal secretions of EHM horses were low and peak levels of IRF7, IRF9, CXCL10 and TGF-ß (blood) coincided with viremia. Moreover, EHM horses showed significantly higher IL-10 levels in nasal secretions, peripheral blood mononuclear cells and CSF and higher serum IgG3/5 antibody titres compared to non-EHM horses. These results suggest that protection from EHM depends on timely induction of type 1 IFN and upregulation cytokines and chemokines that are representative of cellular immunity. In contrast, induction of regulatory or TH-2 type immunity appeared to correlate with an increased risk for EHM. It is likely that future vaccine development for protection from EHM must target shifting this 'at-risk' immunophenotype.

Effect of age and dietary carbohydrate profiles on glucose and insulin dynamics in horses.

BACKGROUND: Glucose and insulin dynamics may be different in adult and aged horses. OBJECTIVES: To determine the effects of age and dietary carbohydrates on glucose and insulin dynamics in healthy horses. STUDY DESIGN: Balanced Latin square with four isocaloric diets: CONTROL (hay plus restricted-starch-and-sugar fortified pellets), STARCH (control plus kibbled corn), FIBER (control plus unmolassed sugar beet pulp/soybean hull pellets) and SUGAR (control plus dextrose powder). METHODS: A total of 16 healthy Thoroughbreds and Standardbreds divided into two age groups: ADULT (8.8 ± 2.9 years; n = 8) and AGED (20.6 ± 2.1 years; n = 8). Following dietary adaptation, horses underwent an insulin-modified frequently sampled intravenous glucose tolerance test (FSIGTT), modified oral sugar test (OST) and dietary meal challenge. Outcome variables included: insulin sensitivity (SI), disposition index (DI), glucose effectiveness (Sg) and acute insulin response to glucose (AIRg) from the FSIGTT; peak glucose, peak insulin, time to peak, area under the curve for glucose (AUCg) and insulin (AUCi) from the OST and dietary meal challenge. Data were analyzed using multivariable linear mixed regression modelling. RESULTS: AIRg was higher in AGED (mean [95% confidence interval]; 582.0 [455.0-709.0]) vs. ADULT (358.0 [224.0-491.0]; P = 0.03). ADULT and AGED horses had a higher SI on STARCH (adult: 3.3 [2.3-4.2]; aged: 2.8 [1.9-3.7]) and SUGAR (adult: 3.4 [2.5-4.3]; aged: 4.0 [3.1-4.9]) diets compared with CONTROL (adult: 2.0 [1.1-2.9], P = 0.029 (starch), P = 0.009 (sugar); aged: 1.4 [0.5-2.2], P = 0.009 (starch), P < 0.001 (sugar)). Feeding a STARCH (adult: 21581.0 [15029.0-28133.0]; aged: 35205.0 [29194.0-41216.0]) or SUGAR (adult: 26050.0 [19885.0-32215.0]; aged: 25720.0 [19770.0-31670.0]) meal resulted in postprandial hyperinsulinaemia (AUCi). MAIN LIMITATIONS: Study cohort contained two insulin-sensitive breeds and no insulin-resistant breeds. CONCLUSIONS: Age and diet should be considered when evaluating glucose and insulin dynamics.

Effect of dietary carbohydrates and time of year on ACTH and cortisol concentrations in adult and aged horses.

Diagnosis of equine pituitary pars intermedia dysfunction (PPID) remains a challenge as multiple factors (stress, exercise, and time of year) influence ACTH and cortisol concentrations. To assess endocrine status in a study designed to evaluate the effects of age and diet on glucose and insulin dynamics, we performed thyrotropin-releasing hormone (TRH) stimulation tests and overnight dexamethasone suppression tests in March, May, August, and October on 16 healthy Thoroughbred and Standardbred mares and geldings. Horses were grouped by age: adult (mean ± SD; 8.8 ± 2.9 yr; n = 8) and aged (20.6 ± 2.1 yr; n = 8). None of the horses showed clinical signs (hypertrichosis, regional adiposity, skeletal muscle atrophy, lethargy) of pituitary pars intermedia dysfunction. Horses were randomly assigned to groups of 4, blocked for age, and fed grass hay plus 4 isocaloric concentrate diets (control, starch-rich, fiber-rich, and sugar-rich) using a balanced Latin square design. Data were analyzed using a multivariable linear mixed regression model. Baseline ACTH was significantly higher in aged horses (mean ± standard error of the mean; 60.0 ± 10.7 pg/mL) adapted to the starch-rich diet compared to adult horses (15.7 ± 12.0 pg/mL) on the same diet (P = 0.017). After controlling for age and diet, baseline ACTH concentrations were significantly increased in October (57.7 ± 7.1 pg/mL) compared to March (13.2 ± 7.1 pg/mL; P < 0.001), May (12.4 ± 7.1 pg/mL; P < 0.001), and August (24.2 ± 7.1 pg/mL; P < 0.001), whereas post-TRH ACTH was higher in August (376.6 ± 57.6 pg/mL) and October (370.9 ± 57.5 pg/mL) compared to March (101.9 ± 57.3 pg/mL; P < 0.001) and May (74.5 ± 57.1 pg/mL; P < 0.001). Aged horses had significantly higher post-dexamethasone cortisol on the starch-rich diet (0.6 ± 0.1 µg/dL) compared to the sugar-rich diet (0.2 ± 0.1 µg/dL; P = 0.021). Post-dexamethasone cortisol was significantly higher in October (0.6 ± 0.1 µg/dL) compared to March (0.3 ± 0.1 µg/dL; P = 0.005), May (0.2 ± 0.1 µg/dL; P < 0.001), and August (0.3 ± 0.1 µg/dL; P = 0.004). Breed did not influence ACTH or cortisol measurements. In conclusion, in addition to age and time of year, diet is a potential confounder as animals on a starch-rich diet may be incorrectly diagnosed with pituitary pars intermedia dysfunction.

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.

Depressed DHEA and increased sickness response behaviors in lame dairy cows with inflammatory foot lesions.

Lameness is a multifactorial condition influenced by the environment, genetics, management and nutrition. Detection of lameness is subjective and currently limited to visual locomotion observations which lack reliability and sensitivity. The objective of this study was to search for potential biomarkers of inflammatory foot lesions that underlie most cases of lameness in dairy cows, with a focus on the sickness response and relevant endocrine, immune and behavioral changes. Serum and peripheral blood mononuclear cells (PBMC) were collected from eight sound and eight lame high-producing Holstein cows. Immune cell activation was investigated in PBMCs using a candidate gene approach in which the expression of pro-opiomelanocortin, interleukin-1beta, l-selectin, matrix metalloproteinase-9 and glucocorticoid receptor-alpha was measured via quantitative real time-RT-PCR. Endocrine changes were investigated by monitoring serum concentrations of cortisol and dehydroepiandrosterone (DHEA). Additionally, systematic behavioral observations were carried out to characterize a behavioral profile associated with a sickness response typical of this condition. Lame cows showed significantly lower eating (P=0.01) and ruminating (P=0.01) behaviors and higher incidence of self-grooming (P=0.04) compared to sound cows. Lame cows also showed a 23% decrease in serum DHEA (P=0.01) and 65% higher cortisol:DHEA ratio (P=0.06) compared to sound cows. However, no significant differences were found in candidate gene expression between lame and sound cows. In association with sickness behaviors, serum DHEA concentration and cortisol:DHEA ratio are promising objective indicators of inflammatory foot lesions in dairy cattle and may be useful as diagnostic targets for animals in need of treatment.

Analysis of the bovine neutrophil transcriptome during glucocorticoid treatment.

The objective of this study was to characterize a large portion of the bovine neutrophil transcriptome following treatment with the anti-inflammatory glucocorticoid dexamethasone (Dex). Total RNA was isolated from blood neutrophils of healthy cattle (5 castrated male Holsteins) immediately following cell purification (0 h) or after ex vivo aging for 4 h with or without added Dex. Additional neutrophils were cotreated with a glucocorticoid receptor (GR) antagonist (RU486) and Dex for 4 h. RNA was amplified, dye labeled (Cy3 or Cy5), and hybridized to a series of National Bovine Functional Genomics Consortium (NBFGC) microarrays. LOWESS data normalization followed by mixture model analyses showed that 11.15% of the spotted NBFGC cDNAs (2,036/18,263) were expressed in 4-h (untreated) neutrophils. Subsequent two-step mixed-model analysis detected (P < or = 0.05) 1,109 differentially expressed genes, of which contrast analysis indicated those that were independently responsive to aging (1,064), Dex (502), RU486 + Dex (141), or RU486 (357). In silico analysis revealed that 416 of the differentially expressed genes are unknown, 59 did not cluster well based on known function, and 634 clustered into 20 ontological categories. Independent validation of differential expression was done for 14 of the putatively Dex-responsive genes across these categories. Results showed that Dex induced rapid translocation of GR into the neutrophil nucleus and signaled dramatic alterations in expression of genes that delay apoptosis, enhance bactericidal activity, and promote tissue remodeling without inflammation or fibrosis. Thus these findings revealed hitherto unappreciated plasticity of blood neutrophils and potentially novel anti-inflammatory/wound-healing actions of glucocorticoids.

Short communication: amino acid transporters in porcine mammary gland during lactation.

The objective of this study was to determine whether mRNA transcripts for the amino acid (AA) transporter proteins CAT-1, CAT-2B, B(0,+), and ASCT1 are present in porcine mammary tissue (MT). Transcript abundance on d 7 and 17 of lactation was determined by Northern blot analysis, and absolute quantification was performed by real-time polymerase chain reaction. Porcine MT expresses CAT-1, CAT-2B, B(0,+), and ASCT1 during lactation. Preliminary findings indicate that B(0,+) mRNA abundance tended to decrease on d 17 compared with that on d 7.

Hyperimmunization of steers with J5 Escherichia coli bacterin: effects on isotype-specific serum antibody responses and cross reactivity with heterogeneous gram-negative bacteria.

Isotype-specific antibody responses and cross reactivity were profiled following hyperimmunization of steers with J5 Escherichia coli bacterin. The vaccine was administered at time 0, 30 d later, and every 2 wk for 10 subsequent immunizations. Blood was collected preimmunization and multiple times following each immunization. Isotype-specific anti-J5 Escherichia coli antibody response profiles in diluted sera harvested from each sample were assayed by ELISA and recorded as optical density. Selected sera were assayed for anti-J5 Escherichia coli antibody titers and used to determine cross reactivity against a variety of gram-negative bacteria. Immunization number and day postimmunization influenced response profiles for anti-J5 E. coli IgM, IgG(1)and IgG(2) antibodies. Two immunizations increased mean serum IgM and the IgG(1)antibody profiles above preimmunization levels, but 5 immunizations were required to detect significant IgG(2) antibody responses that were above preimmunization levels. Isotype-specific cross reactivity of the serum antibodies with a variety of heterologous gram-negative bacteria was also increased by hyperimmunization. However, no cross reactivity was observed for Staphylococcus aureus, purified lipopolysaccharide, or lipid A. Our results indicate that multiple booster doses of J5 E. coli bacterin may be required to elicit high levels of cross-reactive serum IgG(2) antibodies.